Biomedical Applications of Functionalized

Biomedical Applications of Functionalized Nanomaterials

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Biomedical Applications of Functionalized Nanomaterials Concepts, Development and Clinical Translation

Edited by

Bruno Sarmento José das Neves

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2018 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library

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Contents List of Contributors�� xv Preface��xxi

CHAPTER 1 F rom the “Magic Bullet” to Advanced Nanomaterials for Active Targeting in Diagnostics and Therapeutics��1 Alejandro Sosnik 1 Paul Ehrlich and the “Magic Bullet”�� 1 2 Passive Versus Active Targeting in Cancer as Model��2 3 Emerging Challenges and Perspectives�� 20 Acknowledgments�� 22 References�� 22

SECTION I LIGAND SELECTION AND FUNCTIONALIZATION OF NANOMATERIALS CHAPTER 2 C onjugation Chemistry Principles and Surface Functionalization of Nanomaterials��35 Victoria Leiro, Paula Parreira, Sidónio C. Freitas, Maria Cristina L. Martins and Ana Paula Pêgo 1 Conjugation Chemistry in the Context of Biomedical Nanomaterials�� 36 2 Conjugation Chemistry Principles�� 37 3 Self-Assembled Monolayers as a Powerful Tool for the Design of Surface-Engineered Nanomaterials�� 54 4 Challenges in (Bio)conjugation�� 60 References�� 60

CHAPTER 3 P hage Display Technology for Selection of Antibody Fragments��67 Daniela Teixeira and Maria Gonzalez-Pajuelo 1 Introduction�� 67 2 Antibody Phage Display Libraries�� 71 3 Selection and Screening of Antibody Phage Display Libraries�� 76

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4 Antibody Engineering�� 78 5 Conclusions and Future Perspectives��81 References�� 82

CHAPTER 4 R ibosome Display Technology for Selecting Peptide and Protein Ligands��89 Akira Wada 1 Introduction�� 89 2 Emergence of In Vitro Display Technologies�� 90 3 Basic Principles and Features of Ribosome Display Technology��92 4 Selection of Peptides Using Ribosome Display Technology�� 94 5 Selection of Antibody Fragments Using Ribosome Display Technology�� 96 6 Selection of Proteins Using Ribosome Display Technology�� 97 7 Conclusions and Future Perspectives��100 References�� 101

CHAPTER 5 Engineered Protein Variants for Bioconjugation�� 105 Cláudia S.M. Fernandes, Gonçalo D.G. Teixeira, Olga Iranzo and Ana C.A. Roque 1 Introduction�� 105 2 Bioconjugation on Natural Amino Acids�� 106 3 Bioconjugation on Unnatural Amino Acids�� 114 4 Affinity-Induced Bioconjugation�� 125 5 Conclusions and Future Perspectives��127 Acknowledgments�� 127 References�� 128

CHAPTER 6 B ioengineered Approaches for Site Orientation of Peptide-Based Ligands of Nanomaterials��139 Svetlana Avvakumova, Miriam Colombo, Elisabetta Galbiati, Serena Mazzucchelli, Rany Rotem and Davide Prosperi 1 Introduction�� 139 2 Control of Peptide Structure and Functionality�� 141 3 Impact of Bond Strength and Linker Length on Bioconjugation�� 148

Contents

4 Impact of Ligand Density on the Targeting Efficiency of Nanoconjugates�� 153 5 Protein Corona Effect and Minimization of Nonspecific Interactions�� 158 6 Conclusion and Future Perspectives�� 160 References�� 161

CHAPTER 7 N anozymes for Biomedical Sensing Applications: From In Vitro to Living Systems�� 171 Shichao Lin, Jiangjiexing Wu, Jia Yao, Wen Cao, Faheem Muhammad and Hui Wei 1 Introduction�� 171 2 Nanozymes for In Vitro Sensing�� 172 3 Nanozyme for Sensing in Living Systems�� 197 4 Conclusions and Perspectives�� 198 Abbreviations�� 200 Acknowledgments�� 201 References�� 201

CHAPTER 8 S ystematic Evolution of Ligands by Exponential Enrichment for Aptamer Selection�� 211 Meral Yüce, Hasan Kurt, Babar Hussain and Hikmet Budak 1 Introduction�� 212 2 Potential Aptamer Targets�� 212 3 Advantages of Aptamers�� 213 4 Random Oligonucleotide Libraries�� 214 5 Systematic Evolution of Ligands by Exponential Enrichment�� 214 6 Sequencing of the Enriched Aptamer Pools�� 224 7 Evaluation of Aptamer-Binding Kinetics��228 8 Post–Systematic Evolution of Ligands by Exponential Enrichment Modifications�� 233 9 Conclusion�� 236 Acknowledgment�� 237 References�� 237

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SECTION II SPECIFIC APPLICATIONS OF FUNCTIONALIZED NANOMATERIALS IN THERAPY AND DIAGNOSTICS CHAPTER 9 Graphene-Based Nanomaterials in Bioimaging�� 247 Jing Lin, Yan Huang and Peng Huang 1 Introduction�� 247 2 Synthesis of Graphene-Based Nanomaterials�� 249 3 Surface Functionalization of Graphene-Based Nanomaterials�� 251 4 Graphene-Based Nanomaterials in Bioimaging�� 251 5 Prospects and Challenges�� 277 6 Conclusions�� 280 Acknowledgments�� 280 References�� 280

CHAPTER 10 F unctionalized Transition Metal Dichalcogenide-Based Nanomaterials for Biomedical Applications��289 Priyadarshi Kumar, Zibiao Li and Swee Liang Wong 1 Introduction�� 289 2 Basic Properties of Transition Metal Dichalcogenides�� 293 3 Synthesis of Two-Dimensional Transition Metal Dichalcogenides�� 294 4 Functionalization of Transition Metal Dichalcogenides for Biomedical Applications�� 295 5 Conclusion and Outlook�� 310 References�� 312

CHAPTER 11 Intracellular Targeting Using Surface-Modified Gold Nanoparticles�� 315 Devika B. Chithrani 1 Introduction�� 315 2 Nuclear Targeting of Gold Nanoparticles�� 317 3 Structure of the Nuclear Pore Complex�� 318 4 Mechanism of Nuclear Entry and Transport�� 318 5 Different Surface Functionalizing Strategies for Nuclear Targeting of Nanoparticles�� 320

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6 Imaging Techniques for Probing Nuclear Targeting�� 325 7 Gold-Based Nanostructurmes for Improved Cancer Therapeutics�� 325 8 Radiation Therapy�� 326 9 Anticancer Drug Delivery�� 329 10 Conclusions and Future Direction�� 330 References�� 331

CHAPTER 12 M ultifunctional Magnetic Nanoparticles for Theranostic Applications��335 Cristina Tudisco, Maria T. Cambria and Guglielmo G. Condorelli 1 Introduction�� 336 2 Iron Oxide Nanoparticles: Magnetic Properties and Chemical Synthesis�� 336 3 Surface Modification Routes for the Preparation of Multifunctional Fe3O4 Magnetic Nanoparticles�� 341 4 Organic-Modified Magnetic Nanoparticles for Biomedical Applications�� 346 5 Concluding Remarks and Perspectives�� 360 References�� 361

CHAPTER 13 C ombinatorial Approach in Rationale Design of Polymeric Nanomedicines for Cancer�� 371 Amit Singh and Mansoor M. Amiji 1 Introduction�� 372 2 Challenges in Cancer Therapy and Motivation for Nanomedicines�� 372 3 Challenges With Developing Nanomedicines�� 376 4 Synthetic Approaches for Combinatorial Design�� 381 5 Illustrative Applications of Combinatorially Designed Polymeric Nanosystems�� 384 6 Microfluidic Technologies in Nanoparticle Formulation, Scale-Up, and Screening�� 389 7 Conclusions and Future Perspective�� 391 References�� 395

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CHAPTER 14 F unctional Moieties for Intracellular Traffic of Nanomaterials�� 399 Ana L. Silva, Liane I.F. Moura, Bárbara Carreira, João Conniot, Ana I. Matos, Carina Peres, Vanessa Sainz, Liana C. Silva, Rogério S. Gaspar and Helena F. Florindo 1 Introduction�� 400 2 Intracellular Delivery: Barriers and Challenges�� 401 3 Intracellular Delivery by Nanomaterials�� 406 4 Nanomaterial-Based Strategies to Target Subcellular Organelles�� 417 5 Conclusions and Future Perspectives��427 References�� 428

CHAPTER 15 F unctionalized Polymeric Nanostructures for Mucosal Drug Delivery��449 Lungile N. Thwala, Manuel J. Santander-Ortega, M. Victoria Lozano and Noemi S. Csaba 1 Introduction�� 450 2 Mucosal Barriers�� 456 3 Models for Studying Mucosal Drug Delivery�� 460 4 Formulation Strategies to Improve Mucosal Delivery�� 467 References�� 477

CHAPTER 16 B iofunctionalized Mesoporous Silica Nanomaterials for Targeted Drug Delivery�� 489 Antti Rahikkala, Jessica M. Rosenholm and Hélder A. Santos 1 Introduction�� 489 2 Mesoporous Silica Nanoparticles: From Fabrication to Applications�� 493 3 Future Perspectives�� 512 Acknowledgments�� 512 References�� 512

CHAPTER 17 N anoparticle-Mediated RNA Interference for Cancer Therapy�� 521 Tomohiro Asai, Leaf Huang and Naoto Oku 1 RNA Interference and Cancer Therapy�� 521 2 Systemic Delivery of RNA Interference Effectors to Tumors�� 524

Contents

3 Tumor Microenvironment as a Factor Influencing Nanoparticle-Mediated Delivery of RNA Interference Effectors�� 525 4 Noninvasive Pharmacokinetic Analysis of RNA Interference Effectors�� 527 5 Undesirable Effects Caused by RNA Interference Effectors and Delivery Vehicles�� 529 6 The Accelerated Blood Clearance Phenomenon of Delivery Vehicles�� 531 7 Therapeutic Studies of Anticancer RNA Interference Effectors Formulated in Nanoparticles�� 532 8 Concluding Remarks�� 534 Acknowledgments�� 534 References�� 534

CHAPTER 18 Biomolecular Therapeutics for HIV�� 541 Shasha Li and John C. Burnett 1 Introduction�� 541 2 Aptamer-siRNA Nanoparticles for Targeted Anti-HIV Therapeutics�� 543 3 Anti-HIV Vectors�� 545 4 Engineering HIV Resistance With Genome Editing��548 5 Chimeric Antigen Receptor T Cells�� 556 6 Concluding Remarks�� 557 Acknowledgments�� 558 References�� 559

CHAPTER 19 S elf-Assembled Peptide and Protein Nanofibers for Biomedical Applications��569 Dillon T. Seroski and Gregory A. Hudalla 1 Introduction�� 569 2 Classes of Self-Assembling Peptide and Protein Nanofibers�� 570 3 Applications of Self-Assembling Peptide and Protein Nanofibers for Biomedicine�� 580 4 Future Perspectives�� 592 References�� 593

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CHAPTER 20 P eptide-Modified Hydrogels for Therapeutic Vascularization��599 Tália Feijão, Ana L. Torres, Marco Araújo and Cristina C. Barrias 1 Introduction�� 599 2 Hydrogels in Vascular Tissue Engineering�� 604 3 Biofunctionalization of Hydrogels With Angiogenic Peptides�� 607 4 Conclusions and Future Perspectives��614 Acknowledgments�� 615 References�� 615

SECTION III MANUFACTURING, REGULATORY CHALLENGES AND CLINICAL TESTING OF FUNCTIONALIZED NANOMATERIAL-BASED PRODUCTS CHAPTER 21 M anufacturing and Safety Guidelines for Manufactured Functionalized Nanomaterials in Pharmaceutics��623 Matthias G. Wacker, Christine Janas, Fabrícia Saba Ferreira and Fernanda Pires Vieira 1 Introduction�� 624 2 Manufactured Nanomaterials in Pharmaceutics�� 624 3 Physicochemical Characterization of Nanomaterials�� 628 4 Critical Quality Attributes and Quality Control�� 637 5 Pharmacological Evaluation�� 642 6 Biopharmaceutical Characterization�� 644 7 Conclusion�� 646 Abbreviations�� 646 References�� 647

CHAPTER 22 R egulation of Biomedical Applications of Functionalized Nanomaterials in the European Union�� 653 Ruben Pita, Falk Ehmann and René Thürmer 1 Overview of European Union Legislation and Procedural Framework�� 654 2 Medicinal Products Developed with Nanotechnology�� 659

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3 Scientific Guidance�� 662 4 Medical Devices�� 671 5 International Convergence on Nanomedicines�� 674 6 Conclusions and Next Steps�� 676 Disclaimer�� 677 References�� 677

CHAPTER 23 T ranslational Exploration and Clinical Testing of Silica–Gold Nanoparticles in Development of Multifunctional Nanoplatform for Theranostics of Atherosclerosis�� 681 Alexander N. Kharlamov 1 Introduction�� 682 2 Silica–Gold Nanoparticles for Imaging and Therapy of Atherosclerosis�� 704 3 Future of Nanomedical Applications for Imaging and Therapy of Atherosclerosis�� 719 4 Conclusion�� 724 5 Future Perspectives�� 725 Abbreviations�� 727 Acknowledgments�� 728 References�� 728 Index�� 743

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List of Contributors Mansoor M. Amiji Northeastern University, Boston, MA, United States Marco Araújo Universidade do Porto, Porto, Portugal Tomohiro Asai University of Shizuoka, Shizuoka, Japan; University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Svetlana Avvakumova Università di Milano-Bicocca, Milano, Italy Cristina C. Barrias Universidade do Porto, Porto, Portugal Hikmet Budak Sabanci University, Istanbul, Turkey; Montana State University, Bozeman, MT, United States John C. Burnett Beckman Research Institute of City of Hope, Duarte, CA, United States Maria T. Cambria Università di Catania, Catania, Italy Wen Cao Nanjing University, Nanjing, China Bárbara Carreira Universidade de Lisboa, Lisbon, Portugal Devika B. Chithrani University of Victoria, Victoria, BC, Canada; Ryerson University, Toronto, ON, Canada; St Michaels’s Hospital, Toronto, ON, Canada Miriam Colombo Università di Milano-Bicocca, Milano, Italy Guglielmo G. Condorelli Università di Catania, Catania, Italy João Conniot Universidade de Lisboa, Lisbon, Portugal Noemi S. Csaba University of Santiago de Compostela, Santiago de Compostela, Spain Falk Ehmann European Medicines Agency, London, United Kingdom Tália Feijão Universidade do Porto, Porto, Portugal

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Cláudia S.M. Fernandes Universidade Nova de Lisboa, Caparica, Portugal Fabrícia Saba Ferreira National Health Surveillance Agency (ANVISA), Brasília, Brazil Helena F. Florindo Universidade de Lisboa, Lisbon, Portugal Sidónio C. Freitas Universidad Cooperativa de Colombia – Sede Medellín, Medellín, Colombia Elisabetta Galbiati Università di Milano-Bicocca, Milano, Italy Rogério S. Gaspar Universidade de Lisboa, Lisbon, Portugal Maria Gonzalez-Pajuelo FairJourney Biologics, Porto, Portugal Leaf Huang University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Peng Huang Shenzhen University, Shenzhen, China Yan Huang Shenzhen University, Shenzhen, China Gregory A. Hudalla University of Florida, Gainesville, FL, United States Babar Hussain Sabanci University, Istanbul, Turkey Olga Iranzo CNRS, Aix-Marseille Université, Marseille, France Christine Janas Goethe University, Frankfurt am Main, Germany Alexander N. Kharlamov De Haar Research Foundation, Rotterdam, The Netherlands; De Haar Research Foundation, New York, NY, United States Priyadarshi Kumar A*STAR (Agency for Science Technology and Research), Singapore, Singapore; Indian Institute of Science Education and Research, Pune, India Hasan Kurt Istanbul Medipol University, Istanbul, Turkey Victoria Leiro Universidade do Porto, Porto, Portugal Jing Lin Shenzhen University, Shenzhen, China


Shichao Lin Nanjing University, Nanjing, China Shasha Li Beckman Research Institute of City of Hope, Duarte, CA, United States Zibiao Li A*STAR (Agency for Science Technology and Research), Singapore, Singapore M. Victoria Lozano University of Castilla-La Mancha (UCLM), Albacete, Spain Maria Cristina L. Martins Universidade do Porto, Porto, Portugal Ana I. Matos Universidade de Lisboa, Lisbon, Portugal Serena Mazzucchelli Università di Milano, Milano, Italy Liane I.F. Moura Universidade de Lisboa, Lisbon, Portugal Faheem Muhammad Nanjing University, Nanjing, China Naoto Oku University of Shizuoka, Shizuoka, Japan Paula Parreira Universidade do Porto, Porto, Portugal Ana Paula Pêgo Universidade do Porto, Porto, Portugal; Universidad Cooperativa de Colombia – Sede Medellín, Medellín, Colombia Carina Peres Universidade de Lisboa, Lisbon, Portugal Ruben Pita European Medicines Agency, London, United Kingdom Davide Prosperi Università di Milano-Bicocca, Milano, Italy Antti Rahikkala University of Helsinki, Helsinki, Finland Ana C.A. Roque Universidade Nova de Lisboa, Caparica, Portugal Jessica M. Rosenholm Åbo Akademi University, Turku, Finland Rany Rotem Università di Milano-Bicocca, Milano, Italy

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Vanessa Sainz Universidade de Lisboa, Lisbon, Portugal Manuel J. Santander-Ortega University of Castilla-La Mancha (UCLM), Albacete, Spain Hélder A. Santos University of Helsinki, Helsinki, Finland Dillon T. Seroski University of Florida, Gainesville, FL, United States Ana L. Silva Universidade de Lisboa, Lisbon, Portugal Liana C. Silva Universidade de Lisboa, Lisbon, Portugal Amit Singh AllExcel Inc., West Haven, CT, United States Alejandro Sosnik Technion-Israel Institute of Technology, Haifa, Israel Daniela Teixeira FairJourney Biologics, Porto, Portugal Gonçalo D.G. Teixeira Universidade Nova de Lisboa, Caparica, Portugal; CNRS, Aix-Marseille Université, Marseille, France René Thürmer BfArM – Federal Institute for Drugs and Medical Devices, Bonn, Germany Lungile N. Thwala University of Santiago de Compostela, Santiago de Compostela, Spain; Wildlife Pharmaceuticals (Pty) Ltd., White River, South Africa Ana L. Torres Universidade do Porto, Porto, Portugal Cristina Tudisco Università di Catania, Catania, Italy Fernanda Pires Vieira National Health Surveillance Agency (ANVISA), Brasília, Brazil Matthias G. Wacker Fraunhofer-Institute for Molecular Biology and Applied Ecology, Frankfurt am Main, Germany; Goethe University, Frankfurt am Main, Germany Akira Wada RIKEN Center for Life Science Technologies, Yokohama, Japan Hui Wei Nanjing University, Nanjing, China


Swee Liang Wong A*STAR (Agency for Science Technology and Research), Singapore, Singapore Jiangjiexing Wu Nanjing University, Nanjing, China Jia Yao Nanjing University, Nanjing, China Meral Yüce Sabanci University Nanotechnology Research and Application Centre, Istanbul, Turkey

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Preface “There’s Plenty of Room at the Bottom,” the famous 1959 speech by Richard Feynman that is often quoted as the prelude to nanotechnology, provides an incredible voyage into the dimensions and possibilities at the nanoscale (and below!) (Feynman, 1960). The emergence of this new and incredibly wide field has led to an astonishing amount of fascinating developments in science and technology that would not otherwise allow us to live the wonders of modern days. Even now, the excitement and fresh possibilities do not seem to be fading as attention and investment are being set forward for continuing research and translation efforts. Among all the potential and actual applications of nanotechnology, the biomedical arena has been capitalizing on insights into cell and molecular biology, health and disease mechanisms, and processing and characterization of (nano)materials, to name a few, to develop novel diagnostic and therapeutic tools. The manipulation of materials at the nanoscale and use of nanomaterials are indeed frequent sources of innovation for health care-related products that have propelled in many ways nanomedicine into current (and hopefully prospective) clinical practice. In tandem with the “simpler” questions of scale and following on the principles already recognized in the original speech by Dr. Feynman, the use of nanomaterials for biomedical applications is now increasingly focused on providing function, often in multiple and complementary ways, for the rational design of precisely engineered systems (Araújo et al., 2017). Indeed, a wide array of advanced functional nanomaterials have been and are unceasingly being proposed, and a few are already set to start helping health-care professionals and, more importantly, patients. This book aims at providing a concise and up-to-date overview of the field of nanomaterials functionalized with diverse ligands, namely focusing on the most promising ones for biomedical applications. It starts with an introduction on the developments in the subject, from an historical perspective. The first section will be largely devoted to available strategies for identifying biological targets and screening of ligands regarding the optimization of anchoring to nanomaterials. Although standing as an individual section on its own, it provides the ground basis for the following content of the book. Specific applications of functionalized nanomaterials in therapy and diagnostics will be covered in second section. This part of the book conveys, in particular, practice-oriented contributions and is expected to address objective questions of the scientific community. In particular, extensive emphasis on case studies of successfully developed and some already marketed functionalized nanomaterials is provided. Finally, third section focuses on manufacturing, safety assessment, regulatory issues, and clinical translation pertinent to the subject of nanomaterials and nanomedicine, tentatively making of this book an indispensable compendium for worldwide drug and medical device policy makers and regulatory bodies.

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Research and development of nanomaterials, namely of those specifically functionalized for biomedical applications, has come a long way but many exciting opportunities and challenges remain. We intend with this book to provide academic, industrial, and health-care scientists interested on target drug delivery systems, tissue engineering and regenerative medicine, applied nanomaterial research and bioactive materials with a comprehensive, reference compendium that, above all, may be integrated into their daily practice, and continuing scientific efforts. Also, we envision that it may help researchers and professionals whose main topic is related with nanomedicine and personalized therapeutics to get familiarized with and exploit the synergic effect between functionalized nanomaterials and biomedical applications. Last but not least, we would like to express our deepest gratitude to all the scientists who accepted to share their valuable knowledge and expertise in this book. This is truly their book that we were honored to aid in its conception, organization, and overall edition. Finally, a word of appreciation is due to Elsevier and all of its staff for believing in our work and helping in the making of this book. Bruno Sarmento José das Neves July 2017 Porto, Portugal

REFERENCES Araújo, F., das Neves, J., Martins, J.P., Granja, P.L., Santos, H.A., Sarmento, B., 2017. Functionalized materials for multistage platforms in the oral delivery of biopharmaceuticals. Prog. Mater. Sci. 89, 306–344. Feynman, R.P., 1960. There’s plenty of room at the bottom. Eng. Sci. 23, 22–36. Available at: http://calteches.library.caltech.edu/1976/1/1960Bottom.pdf.

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From the “Magic Bullet” to Advanced Nanomaterials for Active Targeting in Diagnostics and Therapeutics

Alejandro Sosnik Technion-Israel Institute of Technology, Haifa, Israel

CHAPTER OUTLINE 1. Paul Ehrlich and the “Magic Bullet”��1 2. Passive Versus Active Targeting in Cancer as Model��2 2.1 Sugars�� 8 2.2 Transferrin and Lactoferrin�� 10 2.3 Folic Acid�� 13 2.4 Hyaluronic Acid�� 14 2.5 Antibodies�� 15 2.6 Aptamers�� 15 3. Emerging Challenges and Perspectives�� 20 Acknowledgments�� 22 References�� 22

1. PAUL EHRLICH AND THE “MAGIC BULLET” Paul Ehrlich, recipient of the Nobel Prize in Medicine in 1908 for his fundamental contributions to the understanding of the immune system, introduced the visionary concept of “magic bullet” (magische kugel in German) compounds more than one century ago (Winau et al., 2004; Schwartz, 2004). By “magic bullet,” he referred to an ideal therapeutic agent that selectively targets a pathogen, a cancer cell, or a toxin at sufficiently low concentrations that prevent any harm to the healthy cells of the patient. His research focused on the treatment of parasitic and bacterial infections and in the late 1900s led to the development of diamidodioxyarsenobenzol (also known as arsphenamine, Ehrlich 606, or Salvarsan), the first active agent for the treatment of syphilis, a bacterial infection caused by the spirochete Treponema pallidum (Winau et al., 2004). Intriguingly, the chemical structure of this drug remained under debate for almost 100 years and mass spectroscopy studies published in 2005 Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00001-X Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 1 From the “Magic Bullet” to Advanced Nanomaterials

FIGURE 1.1 Proposed chemical structures of Salvarsan. Recent research revealed that (A) and (B) are more likely than the originally proposed (C).

revealed that in fact it likely is the mixture of two small arsenic rings, a trimer (Fig. 1.1A) and a pentamer (Fig. 1.1B), and not the originally proposed noncyclic molecule (Fig. 1.1C) (Lloyd et al., 2005). Many years later and primarily motivated by the urgent need to improve the diagnosis and chemotherapy of cancer, the conceptual revolution introduced by Ehrlich that became the moto of drug designers was also embraced by the nanomedicine field (the application of nanotechnology tools in diagnosis, prophylaxis, and therapy of disease) and it paved the way for the design of a plethora of innovative nanomaterials that owing to their small size and uniquely fine-tuned shape and surface properties target specific cell populations by different passive and active pathways (Zhao, 2005; Datta et al., 2016). This chapter will overview the most outstanding hallmarks in the thrilling and, at the same time, struggling way of nanomedicine to realizing Ehrlich’s pioneering vision with special focus on cancer, a disease that owing to its broad incidence and high mortality rates worldwide led to remarkable breakthroughs that improved the efficacy of the diagnosis and the chemotherapy.

2. PASSIVE VERSUS ACTIVE TARGETING IN CANCER AS MODEL The rationale behind the “magic bullet” was to make selective the interaction between the diagnostic and therapeutic agent with molecular or cellular structures of the pathogen and thus to minimize toxic effects on the healthy cells of the host. This could be clearly exemplified for antibiotics where the different families target pathways that are exclusive in bacteria without interacting with counterpart ones (e.g., protein synthesis) in the host (Fig. 1.2) (Coates et al., 2002; Lewis, 2013). Antiviral (De Clercq, 2007), antiprotozoal (Horn and Duraisingh, 2014), and antifungal (Roemer and Krysan, 2014) drugs also inhibit mechanisms that are vital for the growth and proliferation of the pathogen with minimal or no effect on eukaryotic cells.

2. Passive Versus Active Targeting in Cancer as Model

FIGURE 1.2 Main targets of antibacterial drugs in bacteria: cell wall synthesis, DNA gyrase, metabolic enzymes, DNA-directed RNA polymerase, and protein synthesis. In the case of protein synthesis, aminoglycosides and tetracyclines target the 30S RNA, and macrolides, chloramphenicol, and clindamycin inhibit 50S RNA. Reproduced from Coates, A., Hu, Y., Bax, R., Page, C., 2002. The future challenges facing the development of new antimicrobial drugs. Nat. Rev. Drug Discov. 1, 895–910 with permission of Nature Publishing Group.

At the same time, it is worth remarking that regardless of their specificity, these drugs are accompanied by side effects that might range from negligible to severe. Cancer puts together different pathologies associated with abnormal and uncontrolled cell growth and displays the potential to spread to other body sites. Cancers claim 8.2 million lives every year, and with an expected increase of 70% of the cases in the next two decades, it represents one of the leading causes of death worldwide (Cancer - World Health Organization). Tremendous progresses have been made in the chemotherapy of cancer from the use of arsenicals in the early 1900s to moleculartargeting drugs that capitalize on the overexpression of specific receptors by tumor cells such as the tyrosine kinase inhibitors introduced in the mid-2000s (DeVita and Chu, 2008); note that Ehrlich also coined the term chemotherapy for the use of chemicals to treat disease. Regretfully, the specificity of anticancer drugs remains elusive and they display serious short- and long-term side effects that in many cases preclude the continuation of the treatment (Ahmad et al., 2016). Moreover, diagnosis

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in advanced stages of the disease and development of resistance reduces dramatically the therapeutic repertoire and the chance of cure (Zhou et al., 2017). Thus, investigation of more sensitive diagnostic tools became a field of equal impact as an effective chemotherapy (Kasahara and Tsukada, 2004). In this scenario, nanotechnology emerged as a phenomenal toolbox to make diagnosis more sensitive and efficacious and to overcome main pharmacokinetics, pharmacodynamics, and toxicological disadvantages of anticancer drugs through to the modification of fundamental features such as aqueous solubility and physicochemical stability in the biological milieu and pharmacokinetic parameters (e.g., increased biodistribution in the tumor with respect to off-target tissues and organs) (Heath and Davis, 2008; Ferrari, 2005; Schroeder et al., 2012). Another beneficial effect of nanomedicines would pertain to the ability to overcome resistance mechanisms such as efflux transporters of the adenosine triphosphate (ATP)-binding cassette superfamily, which reduce the effective intracellular concentration of the drug in the target cells (Sosnik, 2013). The ability to manipulate the matter at the atomic and molecular level and the invention of cutting-edge characterization methods (e.g., scanning tunnel microscope) led to the emergence of nanoscience and nanotechnology. More recently, the application of these tools to medicine gave birth to the field of nanomedicine and led to a revolution in the capabilities to diagnose and treat disease. For instance, the term “nanomedicine” was probably used for the first time in the book “Unbounding the Future: The Nanotechnology Revolution” authored by Drexler et al. (1991) and published by Morrow in 1991. First reports on the synthesis of nanoparticles for drug delivery date from the 1960s (Kreuter, 2007) and it was only in 1995 that an intravenous liposomal formulation of the anthracycline antibiotic doxorubicin commercialized as Doxil or Caelyx (Janssen) or the generic Myocet (Teva Pharmaceuticals) formally became the first US Food and Drug Administration (FDA)-approved nanomedicine (Barenholz, 2012). The most advantageous feature of this pioneering nanopharmaceutical was the ability to increase the accumulation of the cargo in highly vascularized tumors by the so-called enhanced permeability and retention (EPR) effect, a passive targeting pathway that relies on the presence of vascular imperfections (fenestrations) at the nanometer scale range in the endothelium that enables the extravasation of sufficiently small nanomaterials to the tumoral stroma and their increased accumulation with respect to the free drug (Fig. 1.3) (Fang et al., 2011). This phenomenon is accompanied by a lack of lymphatic drainage that disfavors clearance. Liposomal doxorubicin also reduces the exposure of cardiac muscle to the drug and its cardiotoxicity, confirming that the biodistribution is governed by the nanocarrier (Safra et al., 2000). However, in nonsolid (e.g., leukemia) or poorly vascularized tumors (e.g., bladder carcinoma), this mechanism cannot be exploited (Prabhakar et al., 2013). A similar principle of increased vascular permeability has been investigated in more recent years for the treatment of inflammatory diseases, among them infections (Fig. 1.4) (Azzopardi et al., 2013; Nehoff et al., 2014). Regardless of the remarkable breakthrough achieved with the EPR effect, the benefits of nanomedicines remained relatively limited because the increased


FIGURE 1.3 Nanomaterials extravasate into the tumor stroma through the fenestrations of the endothelium, a passive targeting pathway known as enhanced permeability and retention effect. Then, modification of the nanomaterial surface with specific ligands is exploited to make the cellular uptake more selective by an active targeting pathway. Reproduced from Ferrari, M., 2005. Cancer nanotechnology: opportunities and challenges. Nat. Rev. Cancer 5, 161–171 with permission of Nature Publishing Group.

accumulation in the tumor stroma did not ensure significantly higher intracellular delivery of the chemotherapy. Thus, the further modification of the nanomaterial surface with specific ligands that selectively bind cellular structures (e.g., receptors) overexpressed in the diseased (e.g., cancer) cells was attempted to favor the internalization of the drug-loaded nanocarrier by diverse endocytic pathways, a strategy known as active targeting (Fig. 1.3) (Ferrari, 2005; Byrne et al., 2008). A paradigmatic example of a nanomedicine involving both passive and active targeting is albumin-bound paclitaxel (nab-paclitaxel, Abraxane, Celgene Corp.) used in the therapy of metastatic breast, ovarian, and non–small cell lung cancer (Fig. 1.5) (Desai, 2012). This nanopharmaceutical product utilizes albumin transport pathways, including the glycoprotein 60 albumin receptor and subsequent caveolae-mediated endothelial transcytosis across the endothelium of the blood-tumor barrier and interaction with albumin-binding proteins in the tumor parenchyma such as secreted protein acidic and rich in cysteine (Fig. 1.6) (Desai, 2012; Hawkins et al., 2008; Yardle, 2013). So far, nab-paclitaxel remains a one-of-a-kind example of actively targeted nanomedicine that has been approved by the FDA in 2005 and by the European Medicines Agency in 2008 for metastatic breast cancer. Numerous attempts to translate actively targeted nanomedicines to the clinics failed in different stages or were abandoned because of economic considerations, while few products are still undergoing preclinical and early clinical trials (Xu et al., 2015).

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FIGURE 1.4 Effect of inflammation on the development of the enhanced permeability and retention (EPR) effect in inflammatory tissue. Inflammatory tissue will release a range of mediators that will induce the EPR effect. Inflammation will cause the vessel to dilate resulting in a higher blood flow. Furthermore, the contraction of endothelial cells will allow the penetration of nanoparticles into the tissue. The major difference between inflammatory tissue and tumor tissues in relation to macromolecular targeting is the presence of a functional lymphatic system in inflammation. Retention of nanomedicine in this case can be attributed to macrophage uptake. Reproduced from Nehoff, H., Parayath, N.N., Domanovitch, L., Taurin, S., Greish, K., 2014. Nanomedicine for drug targeting: strategies beyond the enhanced permeability and retention effect. Int. J. Nanomed. 9, 2539–2555 with permission of Dove Press.

An issue that remains elusive and controversial around active targeting is related to the fact that upon intravenous administration, nanoparticles usually undergo adsorption of plasma proteins (a process known as opsonization) such as albumins, fibronectins, complement proteins, immunoglobulins, and apolipoproteins, and thus the surface ligands could be partly or completely masked, precluding their


FIGURE 1.5 (A) Scheme of the nab-paclitaxel structure and (B) cryo-TEM microphotograph showing the spherical morphology of the nanoparticle. TEM, transmission electron microscope. Reproduced from Desai, N., 2012. Challenges in development of nanoparticle-based therapeutics. AAPS J. 14, 282–295 with permission of Springer.

FIGURE 1.6 Two routes of albumin to reach tumors. The left half of the figure shows albumin metabolism of a growing tumor, whereas the right half reflects the cytotoxic effect on tumor cells from the uptake of albumin-bound paclitaxel. Albumin and albumin-bound paclitaxel are hypothesized to reach the tumor stroma by both transcytosis and the enhanced permeability and retention effect. Reproduced from Yardle, D.A., 2013. nab-Paclitaxel mechanisms of action and delivery. J. Control. Release 170, 365–372 with permission of Elsevier.

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direct interaction and binding to the target (Nie, 2010). Although that in vitro studies of protein adsorption are often conducted to characterize this interaction, their ability to predict the performance in the complex in vivo environment is very low. Another constraint of active targeting with nanomedicines resides in the common use of expensive ligands (e.g., antibodies) and complex synthetic and purification pathways and production processes that are feasible in a laboratory scale, though that make scalability under an industrial setting cost-inviable and/or that have a strong impact on the final cost of the medication (Muthu and Wilson, 2012; Hare et al., 2017; Landesman-Milo and Peer, 2016). At the same time, there exists strong experimental evidence that if the nanocarrier is designed properly and using technologies that are more easily scaled up (e.g., spray drying) (Sosnik and Seremeta, 2015), active targeting might breakthrough the treatment of disease, especially considering the possibility of dramatically improving the efficacy of old (and usually cheaper) anticancer drugs as opposed to more innovative and expensive ones of controversial medical benefit (Siddiqui and Rajkumar, 2012). Another crucial issue to consider is that there exists strong evidence that active targeting can take place only when the ligand and the target are at a distance 7.5 cross-reactivity with amino groups can occur. Moreover, at higher pH other side reaction can occur—the

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CHAPTER 2 Principles and Surface Functionalization of Nanomaterials

SCHEME 2.5 (A) Thiol reactions: (a) thiol-maleimide Michael-type addition; (B) disulfide exchange reaction; and (C) disulfide reduction.

hydrolysis of the MAL group to an open ring and unreactive form. This hydrolysis is also dependent on the bulkiness and/or nature of the group next to the MAL, and thus linkers as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate with a cyclohexane ring as spacer provide increased stability of MAL to hydrolysis probably due to its steric effect.

2.2.2 Disulfide Bridge Disulfide bonds/bridges are usually generated from the oxidation of thiol groups, especially in biological contexts. Compounds containing a disulfide bridge are able to undergo disulfide exchange reactions (also called “interchange”) with thiols. This interchange supposes the thiol attack to the disulfide bond, breaking the SdS bridge, with the subsequent formation of a new mixed disulfide (Scheme 2.5B). The formation of this new SdS bond is reversible using reducing agents. Disulfide exchange reactions occur over a broad range of pH and buffer conditions, including physiological conditions. The most popular thiol–disulfide exchange reagents are pyridyl dithiol and 5-thio2-nitrobenzoic acid-thiol derivatives (Ellman, 1959; King et al., 1978). A particular case of the disulfide bridge interchange is its reduction when a thiolcontaining reducing agent (Scheme 2.5C) is used to break the SdS bond, such as dithiothreitol, 2-mercaptoethanol, or 2-mercaptoethylamine, or other nonthiol containing, such as tris(2-carboxyethyl)phosphine (Ruegg and Rudinger, 1977) to give origin to the corresponding thiolated compounds. These reductions can also occur over a broad range of pHs and buffers. Other common reactions for thiol groups are summarized in Table 2.1 (entries 8–13).

2. Conjugation Chemistry Principles

2.3 HYDROXYL REACTIONS Within this section, we will include not only those functional groups that are able to directly react with hydroxyl groups (-OH) but also those that can react with those functional groups resulting from the temporary -OH activation. The chemistry of the hydroxyl groups is very important for the modifications/functionalizations of compounds containing such groups, such as polysaccharides, glycoproteins, sugar of nucleic acids, or polymers as poly(ethylene glycol). One of the most common hydroxyl reactions are the ester and carbamate bond formation.

2.3.1 Ester Bond Formation: Strategies 2.3.1.1 Acyl Halides, Anhydrides, and O-Acylisoureas via Carbodiimide Coupling Despite the apparent simplicity of ester bond formation between small organic molecules under anhydrous conditions, the formation of ester linkages represents one synthetic challenge in complex natural products and/or macromolecules, in which electrophilic ester bonds must coexist with a great variety of functionalities, such as nucleophiles. Similarly to the amide bond formation, the direct coupling/condensation between the carboxylic acid and alcohol presents a high activation barrier; thus the proposed strategies seek the generation of the ester bond by indirect ways, previous activation of the carboxylic acid. Many of the methods previously mentioned for the activation of carboxylic acids for amide bond formation also allow the formation of ester bonds, such as the activation with acyl halides (Moulin et al., 2005; Antell, 1972), acylimidazoles (Kamijo et al., 1984), and carbodiimides/O-acylisoureas based on DCC, DIC, or EDC (Nakao et al., 1981; Leiro et al., 2005, 2008a,b). However, because alcohols are poorer nucleophiles than amines, the risk of formation of the nonreactive N-acylurea is higher. Nevertheless, the use of additives (mainly DMAP in catalytic amounts (Nakao et al., 1981) and HOBt or HOAt in stoichiometric amounts (Morales-Serna et al., 2010; Xu and Miller, 1998)) can minimize this side reaction by rapid reaction between the additives and the O-acylisourea. Next, the corresponding activated intermediates will react with the alcohol (alcoholysis) to form the desired ester in high yield and a short reaction time. The ester formation via alcoholysis of mixed anhydrides is another alternative strategy. The mixed anhydrides are normally obtained by reaction of 2,4,6-trichlorobenzoyl chloride with the corresponding carboxylic acid in the presence of an organic base (as Et3N) (Inanaga et al., 1979). As in the formation of amide bonds, the anhydrides can be performed and isolated, previous treatment with the alcohols in the presence of DMAP (Inanaga et al., 1979), or can be obtained in situ, accomplishing the esterification in one-pot reaction (Hikota et al., 1990).

2.3.1.2 Mitsunobu Coupling The Mitsunobu reaction (Mitsunobu, 1981) allows the conversion of alcohol groups into different functional groups, including esters. Contrary to the strategies discussed

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SCHEME 2.6 Hydroxyl reactions: (A) Mitsunobu coupling and (B) activation of hydroxyls with CDI and subsequent reaction with an amine generating a carbamate linkage. CDI, carbonyl diimidazole; DEAD, diethyl azodicarboxylate; DIAD, diisopropyl azodicarboxylate.

in the previous Section 2.3.1.1, in which the carboxylic acid is activated for nucleophilic attack by the hydroxyl, in the Mitsunobu reaction, the alcohol is activated toward nucleophilic attack from the carboxylic acid. This hydroxyl activation is achieved by reacting with a phosphine, typically triphenylphosphine, and a dialkyl azodicarboxylate, usually diisopropyl azodicarboxylate or diethyl azodicarboxylate (Scheme 2.6A).

2.3.2 Carbamate Linkage Formation: Strategies Using the suitable coupling or carbonylating reagents, hydroxyl groups are able to indirectly react with amines giving stable carbamate bonds (Beauchamp et al., 1983). Among the reagents used for these methods, one can find the previously mentioned CDI and also DSC and N-hydroxysuccinimidyl chloroformate. CDI is an active carbonylating agent because it can react with hydroxyl groups generating an imidazolyl carbamate active intermediate (Scheme 2.6B). This intermediate will react with the desired amine via a stable urethane (N-alkyl carbamate) linkage and releasing an imidazole molecule (Scheme 2.6B). In nonaqueous environments, DSC and N-hydroxysuccinimidyl chloroformate can also be used to activate a hydroxyl group to a succinimidyl carbonate derivative, which can be subsequently attacked by an amine giving a compound with a carbamate linkage. These reagents cannot be used in aqueous media because they rapidly hydrolyze. Other common reactions for hydroxyl groups are summarized in Table 2.1 (entries 14–17).


SCHEME 2.7 (A) Carboxylate reaction with diazoacetate giving an ester bond. (B) Formation of a hydrazone linkage by condensation of an aldehyde and hydrazide and subsequent reduction.

2.4 CARBOXYLIC ACID REACTIONS Besides the condensation reactions between carboxylic acids and amines, thiols, and hydroxyl groups, which require prior activation of the carboxylic acid as previously reviewed, diazoalkane and diazoacyl derivatives (as diazoacetyl compounds) can also react with carboxylate groups (Riehm and Scheraga, 1965) (Scheme 2.7A). In this case, the reactions will be spontaneous without addition of other reactants or catalysts. Because the reaction proceeds by the nucleophilic attack of the negatively charged oxygen of the carboxylate group to the diazoalkyl compound, this reaction can occur over a broader range of higher pHs (pH > 5, preferably). Unfortunately, the specificity of this reaction is very low at these pHs when other nucleophiles are present in the reaction milieu, such as thiols or amines. Because of this, selecting pH of 5.0 prevents, at least, the side reactions with amine. Moreover, in aqueous solution, the most likely side reaction is hydrolysis; therefore to conduct the reaction in a nonaqueous medium is highly recommended.

2.5 ALDEHYDES AND KETONES REACTIONS Aldehyde and ketone groups are also important functional groups to carry out chemistry conjugation. Among their possible reactions, the reaction between these groups and hydrazine derivatives (especially those derived from carboxylates), which renders a hydrazone linkage—a type of Schiff base, is of special interest (Scheme 2.7B). This reaction is usually faster with aldehydes than with ketones, providing a more stable hydrazone bond on the latter case. Interestingly, the higher lability, under slight acidic conditions, for hydrazone linkage from aldehydes is very useful to the design of pHresponsive drug delivery systems, which aims to release the cargo/drug in acidic environments, such as tumors (Etrych et al., 2014). However, if higher stability is needed for other applications, the hydrazone bond can be reduced with sodium cyanoborohydride, under nonaqueous conditions, rendering a highly stable CdN covalent linkage.

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2.6 ALKENES AND ALKYNES When thinking about the reactivity of unsaturated compounds as alkenes and alkynes, one immediately thinks about Michael or Michael-type additions, cycloaddition reactions (such as the classic Diels–Alder reaction), and, of course, the trendy click chemistry. As the most common Michael-type additions for (bio)conjugation and surface functionalization have been already discussed in Section 2.2.1, we will focus this section in cycloadditions, especially in click chemistry cycloadditions.

2.6.1 Diels–Alder Cycloaddition The Diels–Alder reaction is a well-known and established reaction in organic chemistry consisting of a highly selective [4+2] cycloaddition of a diene to an alkene (dienophile) to yield cyclohexene derivatives (Scheme 2.8A). This reaction provides an opportunity to surpass the limitations related to the coupling of chemically sensitive (bio)molecules (as antibodies), especially in aqueous environments where both the rate and stereoselectivity of Diels–Alder reactions are significantly increased (Otto and Engberts, 2003). Because of this, this cycloaddition has been recently applied to bioconjugation reactions as well, especially using MALs as the dienophile (Shi et al., 2007).

2.6.2 Click Chemistry In 2001, a period where the chemists looked for inspiration in the efficient processes with minimal production of waste from the biological systems, Sharpless et al. introduced the concept of “click chemistry” (Kolb et al., 2001). Click chemistry

SCHEME 2.8 (A) Diels–Alder Cycloaddition. (B) Huisgen 1,3-dipolar azide–alkyne cycloadditions (AAC) reactions (“click chemistry” reactions): (b.1) Cu(I)-catalyzed version (CuAAC) and (b.2) Cu-free or strain-promoted version (SPAAC).


philosophy is based on principles such as atom economy (no loss of atoms between steps), step economy or no activation steps, simple reaction conditions, orthogonality to avoid the use of protecting groups, catalysis to reduce the energy of activation while avoiding the use of stoichiometric reagents, purifications by simple procedures (nonchromatographic methods), high yields, and issues such as safety, toxicity, and more environmentally friendly processes (“green chemistry”) (Anastas and Eghbali, 2010). The reaction must also give origin to products with stable linkages under physiological conditions.

2.6.2.1 Huisgen 1,3-Dipolar Azide–Alkyne Cycloadditions Among the click reactions proposed, it is not surprising that Cu(I)-catalyzed azide– alkyne cycloaddition (CuAAC; Scheme 2.8b.1) has attracted special interest because of its incredibly high orthogonality (Rostovtsev et al., 2002), reliability, and experimental simplicity for nonspecialists. Particularly, it rapidly has found application in the bioconjugation, surface functionalization, and, in general, the biomedical field. In opposition to the thermal Huisgen 1,3-dipolar azide–alkyne cycloaddition (AAC), which requires high temperatures (∼100°C) and longer reaction times, Cu(I) catalysis notably decreases the activation energy, allowing very good rates of the reaction at RT, in both aqueous and organic solvents. Moreover, the ease with which a vast range of molecules and biomolecules are easily functionalizated with both azide and alkyne moieties (Debets et al., 2010b; Leiro et al., 2017), together with the small size and exceptional stability of these moieties in complex environments, has made CuAAC into the “upper crust” among click reactions. Nevertheless, in bioconjugation processes, the required Cu(I) catalyst could induce structural damage to biomolecules and, under the original conditions (CuSO4/ sodium ascorbate) (Rostovtsev et al., 2002), CuAAC can, sometimes, lack adequate kinetics at the micromolar range typically employed in bioconjugation. Fortunately, these possible drawbacks can be efficiently surpassed with the addition/use of a suitable Cu(I)-chelating ligand (Lewis et al., 2004). These stabilize Cu(I) oxidation state, increase the reaction rate, avoid side reactions, and reduce the structural Cu(I) damage to biomolecules, while facilitating the purification process. The ligands most usually employed in bioconjugation are tris(benzyltriazolylmethyl)amine (Chan et al., 2004), tris(hydroxypropyltriazolylmethyl)amine (Chan et al., 2004), and bathophenanthroline disulfonated disodium salt (Lewis et al., 2004). More recently, an electrochemically protected version of CuAAC, where Cu(II) is electrochemically reduced to Cu(I) in presence of a chelating ligand (Hong et al., 2008), has been reported. And, more interestingly, recently more benign Cu-free AAC bioconjugation strategies have been reported by Bertozzi et al. These strategies are based on the inherent ring strain of cyclooctynes as an efficient way to decrease the activation barrier of AAC (Agard et al., 2005) (Scheme 2.8b.2). Moreover, the optimization of the rate of this strain-promoted AAC variant (SPAAC) for faster bioconjugation under mild conditions has been achieved by means of use of higher reactive cyclooctynes (with electron-withdrawing groups and increased ring strain), such as difluorocyclooctyne

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(Baskin et al., 2007), dibenzocyclooctyne (Ning et al., 2008), dibenzozacyclooctyne (Debets et al., 2010a), biarylazacyclooctynone (Jewett et al., 2010), and bicyclononyne (Dommerholt et al., 2010) derivatives. SPAAC has been a crucial contribution when dealing with bioconjugation and macromolecular structures, especially for biomedical applications. To end, it is interesting to mention that the high impact of the “click chemistry” concept has originated in abusive use of this term. For example, well-known and classic reactions are now being “retagged” with the trendy “click” term (e.g., thioclick coupling, thio-bromo click, or Diels–Alder click reactions). Moreover, a number of reactions that do not fulfill the click principles aforementioned have been proposed as new examples of click reactions.

2.7 PHOTOCHEMICAL REACTIONS Photoreactive groups can be introduced on substrates to conjugate with other molecules (as target molecules) by exposure to UV radiation. Photosensitive functional groups are usually unreactive in typical thermal processes. Because of this, reagents designed with a photoreactive functionality can be used in highly controlled and orthogonal reactions. Therefore, photochemical reactions have become an important strategy for numerous (bio)conjugate applications. Some of the most popular photosensitive functionalities are aryl azides and halogenated aryl azides (Gilchrist and Rees, 1969), benzophenones (Walling and Gibian, 1965), anthraquinones (Koch et al., 2000), some diazo and diazirine derivative compounds (Bergmann et al., 1994), and psoralens compounds (derivatives of 9-methoxy-7 H-furo[3,2-g]chromen-7-one tricyclic ring structures) (Pathak, 1984).

3. SELF-ASSEMBLED MONOLAYERS AS A POWERFUL TOOL FOR THE DESIGN OF SURFACE-ENGINEERED NANOMATERIALS SAMs are well-ordered organic surfaces that are very useful for the screening of immobilized molecules because they are easy to prepare, easy to characterize, and enable surface control at the molecular scale (Love et al., 2005; Koepsel and Murphy, 2012; Ross and Lahann, 2015). SAMs are frequently used as proof-ofconcept model surfaces because the obtained knowledge in terms of strategies to maintain the bioactivity of immobilized biomolecules is easily translated to “real world” materials. One of the most used class of SAMs in biological studies is derived from the adsorption of alkanethiols on noble metals such as gold and silver (Love et al., 2005). A schematic view of the principal driving forces involved in SAMs formation is described in Fig. 2.1.

3. Self-Assembled Monolayers

FIGURE 2.1 Schematic representation of self-assembled monolayers of alkanethiols on gold. Alkanethiols are divided in three parts: the surface-active head group (thiol group) that binds strongly to the gold surface; the terminal group that is located at the monolayer surface and determines the interfacial properties of the monolayer; and the long-alkyl chain that facilitates the packing of the molecules in the monolayer and supports its stability due to Van der Waals interactions.

The high affinity of thiol groups to gold enables the correct alkanethiol orientation, independently of most terminal functional groups used. This fact allows SAMs preparation using alkanethiols that were previously conjugated with molecules such as ethylene glycol (EG), maleimide, biotin, and even peptides. Moreover, gold is a reasonably inert material, which makes sample manipulation simple, it is cell compatible, and it is the ideal substrate for the most commonly used surface characterization techniques, such as surface plasmon resonance spectroscopy, quartz crystal microbalance, infrared reflection absorption spectroscopy, and ellipsometry (Love et al., 2005). (Bio)molecules conjugation to alkanethiols can be performed before or after SAMs formation. An example of the first strategy is the utilization of RGD- or Cibacron Blue (CB)-terminated SAMs using RGD- or CB-terminated alkanethiols (Hansen et al., 2014; Martins et al., 2003). This allows preparing functionalized SAMs in one single step. However, problems concerning SAM organization, packing and surface exposure of the biomolecule make this first strategy less used. In addition, biomolecules conjugation after SAMs formation has the advantage of allowing the generation of multiple samples with distinct ligands in the same assay and in a short period (Love et al., 2005). For an effective biomolecule surface immobilization, biomolecules must be in adequate concentration, controlled orientation, and correct exposure from the surface without denaturation. Therefore, SAMs are usually prepared with a combination of two different thiols (mixed SAMs): one presenting the reactive functional group(s) for further biomolecules conjugation and the other presenting a nonfouling terminated group, such as an oligoEG, which avoids nonspecific adsorption (improving selectivity) and prevents biomolecules denaturation while maintaining their bioactivity (Castner and Ratner, 2002). The most commonly used strategies to achieve bioconjugation on SAMs will be briefly presented in the following sections.

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3.1 BIOMOLECULES CONJUGATION ONTO SELF-ASSEMBLED MONOLAYERS VIA COVALENT BINDING 3.1.1 Maleimide-Terminated Self-Assembled Monolayers Maleimide-terminated SAMs for bioconjugation of biomolecules containing thiol groups can be prepared using pure solutions of alkane disulfide maleimide terminated (R-S-S-R’) (note: since maleimide groups react with thiol groups, this strategy cannot be used with alkanes with free thiols) (Love et al., 2005). An example of this strategy can be observed in Fig. 2.2. For peptides/proteins immobilization in a controlled orientation, peptide/protein synthesis can be performed by incorporating an extra cysteine residue (-SH) at the N- or C-terminal. One application for this chemistry was performed by Wettero et al. (2008) to design surfaces able to trigger specific effects on cell behavior. For that, RGD peptides with an extra cysteine were immobilized onto SAMs in a controlled orientation.

3.1.2 Alkyne or Azide-Terminated Self-Assembled Monolayers  (“Click Chemistry”) “Click chemistry” is widely used in biomaterial science, namely in surface modification and functionalization, where pairs of functional groups can quickly and selectively react under mild aqueous conditions. This method requires that specific groups are added both to the monolayer and to the (bio)molecule prior to immobilization (Chelmowski et al., 2009). (A)

(B)

O

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Biomolec

ule

O

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OH

O

NH

OH

OH

OH

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NH

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OH

OH

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O

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NH

OH

Maleimide dissulfide OH

OH S

S

S

S

Gold S

S

S

S

S

S

S

S

S

Gold

S

EGn dissulfide

FIGURE 2.2 Schematic representation of self-assembled monolayers (SAMs) formation and bioconjugation through specific maleimide-thiol-chemistry (not to scale). (A) alkane dissulfide thiols (R-S-S-R′). (B) Maleimide/EGn-mixed SAMs. (C) biomolecule conjugation.


The most common “click” reaction is the one between an azide (R-N3) and a terminal or internal alkyne group (RdC^CdR) (Meldal and Tomoe, 2008) (Fig. 2.3). To develop biomaterials for cells substrates, Hudalla and Murphy performed proof-of-concept studies using mixed azide-terminated SAMs (N3-EG6/EG3-SAMs) to evaluate the bioactivity of an immobilized cell integrin-binding peptide (alkyneterminated RGDSP) via click chemistry (Hudalla and Murphy, 2009).

3.1.3 Carboxylic Acid-Terminated Self-Assembled Monolayers COOH-terminated SAMs are widely used to bind amine-containing biomolecules. In this case, the amide covalent is normally formed once the carboxylic acid group is activated by reaction with a carbodiimide, such as EDC and NHS. In a second step reaction, NH2-containing biomolecules will be covalently immobilized through their amine groups (NH2) (Lahiri et al., 1999; Palazon et al., 2014). Fig. 2.4 shows a schematic view of a peptide immobilization onto COOH/OH-mixed SAMs, prepared by a mixture of COOH-EG6- and EG3-terminated thiols (Freitas et al., 2014). This EDC/ NHS chemistry/strategy ensures that the biomolecule binding occurs on the longer chain (EG6-COOH-terminated) and thus allows a better biomolecule exposure from a nonfouling background (EG3) (Lahiri et al., 1999). (A)

N

N

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+

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+2

-N

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-N

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+N

+N

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N-

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N+

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N

N

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N R2

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N+

N

+2

R1

N

N

R2

S

R1

N N

R2

S

N

N

N

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Gold

FIGURE 2.3 Schematic representation of self-assembled monolayers (SAMs) formation and modification via “click” reaction for (A) alkyne or (B) azide-terminated SAMs to couple biomolecules-bearing azide or alkyne groups, respectively.

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For the development of anticoagulant surfaces, Freitas et al. (2014) used this chemistry to evaluate the effect of surface immobilization of a thrombin inhibitor peptide (Fig. 2.4). Another strategy to activate COOH-terminated SAMs is through CDI reaction. Generally, CDI activation is a simple and fast method to immobilize ligands through their terminal primary amine groups, but CDI can also react with OH groups (see Section 2.3.2) and, because of this, a selective COOH binding in the presence of OH groups is not possible (Hermanson et al., 2008).

3.1.4 Hydroxyl-Terminated Self-Assembled Monolayers As aforementioned, CDI can react with hydroxyl groups, creating an active imidazolyl carbamate intermediate that is capable of binding amine-containing biomolecules (Section 2.3.2) (Fig. 2.5) (Hermanson et al., 2008; Goncalves et al., 2009, 2010; Freitas et al., 2010). 2 1

2 &22+

2+

2 &

2+

2+

6

6

& 2+

2+

1+6('&

H XO HF RO RP %L 1

2

2

+ 2+

%LRPROHFXOH

6

6

6

*ROG

6

6

6

6

*ROG

*ROG

FIGURE 2.4 Schematic representation of a biomolecule (thrombin inhibitor peptide) immobilization via EDC/NHS chemistry. EDC, N-(3-dimethylaminopropyl)-N ′-ethylcarbodiimide; NHS, N-hydroxysuccinimide. 1

2+

2+

1

2+

2

2 1

58

&

1 1

2+

2+

2

&

2

1 2+

&

2+

2

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RP %L

H XO HF RO

+

%LRPROHFXOH

6

6

*ROG

6

6

6

*ROG

6

6

6

6

*ROG

FIGURE 2.5 Scheme for the covalent attachment of a ligand onto EG4-self-assembled monolayers (SAMs) via carbonyl diimidazole (CDI) chemistry. During the first step of the reaction, the terminal OH groups of the EG4-SAM react with CDI, forming imidazolyl-carbamate groups, which will react with the terminal primary amine of the ligand in a second reaction step.


Martins et al. (2009) used CDI chemistry to functionalize EG4-SAMs with a heparin-binding peptide (poly-l-lysine, l-Leucine; pKL) as a proof-of concept to develop heparin-binding filters for blood deheparinization.

3.2 BIOMOLECULES CONJUGATION ON SELF-ASSEMBLED MONOLAYERS VIA AFFINITY BINDING One of the strongest noncovalent interactions in nature is the biotin–avidin (or other biotin-binding proteins) system (Teulon et al., 2011). The high specificity and affinity to biotin (Kd = 10−13 M), the easiness of biomolecules functionalization with biotin, and the four symmetric equivalent biotin-binding sites, which enable biomolecule conjugation in a controlled orientation, make this system extremely useful. Biotin–SAMs are easily obtained by mixing biotin-terminated thiol (biotin–EG3) with an EG-terminated thiol. Biotin-containing biomolecules can be easily bound to these surfaces using avidin-based proteins as a bridge (Fig. 2.6) (Azzaroni et al., 2007; Wolny et al., 2010). Parreira et al. (2013, 2014) used this affinity binding strategy to bind biotinylated glycan structures (Gly-R), to understand if the resulting surfaces were able to attract and specifically bind the gastric pathogen Helicobacter pylori. These studies were then translated to create mucoadhesive microspheres for H. pylori infection treatment (Goncalves et al., 2016). This strategy was also applied for the development of anticoagulant biomaterials. The use of biotin–SAMs allowed biotinylated boophilin (thrombin inhibitor) immobilization with a correct density and orientation, demonstrating boophilin’s potential to improve biomaterials hemocompatibility (Freitas et al., 2012).

Biotinylated Biomolecule

Neutravidin

S

EG4–thiol

S

S

Biotin–EG3–thiol

S

S

S

S

S

S

Gold

FIGURE 2.6 Schematic drawing of the immobilization strategy of a biotinylated biomolecule onto biotin– self-assembled monolayers.

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4. CHALLENGES IN (BIO)CONJUGATION This chapter has sum up the main synthetic routes currently being explored to functionalize and/or modify a wide range of (bio)molecules or (bio)materials. Although not exhaustive, the list of proposed synthetic routes is rather broad and can be daunting for a new comer to the field. Nevertheless, the recent advances in (bio)conjugation have given even untrained users the possibility to conduct complex modification reactions in a simple and efficient manner. This is the ethos of click chemistry so explored in recent years. In many cases the synthetic routes have been previously established and tested in different settings, but in others optimization steps might be required as to improve selectivity of the reaction or improve conjugation yields. Sometimes, small pH adjustments can suffice, in others a deeper knowledge of the system might be required. Nonetheless, the rise in the number of commercially available coupling reagents and homo- and heterofunctional linkers has allowed the democratization of (bio)conjugation. But the field is in constant evolution and many challenges remain ahead. (Bio) conjugation is expected to be a key player in the future development of smart nano(bio)materials, continuing to contribute to the design of more sensitive and specific diagnostic agents; targeted and safer therapeutic molecules; controlled delivery systems of known therapeutics; novel implants with improved functionalities; and biocompatibility; among other examples. The pursuit of advanced intelligent biomaterials, responsive to biological triggers, is fueling the search for new linkers and moieties that are degraded or change conformation at a specific pH, temperature, or redox conditions. One is also observing the steady rise of reports on the development of new synthetic systems based on naturally occurring repeating units (e.g., nucleic acids, amino acids, and sugars). Working with biological building blocks assembled in a controlled fashion through (bio)conjugation has particularly resulted in novel (nano)materials with superior and controlled (biological) functionality. Here the developments observed in terms of solid-phase synthesis of peptides and nucleic acids have given a major contribution to the field in recent years and the development of orthogonal chemistries. So, the philosophy at the basis of click chemistry is expected to further permeate throughout the field. The quest is now focused on the expansion of the click chemistry toolbox by having additional orthogonal click reactions and on the development of greener chemistries. The ultimate goal is to achieve more efficient syntheses, simplify purification steps, and improve biocompatibility of the final products, the latter issue particularly relevant in the biomedical field.

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CHAPTER

Phage Display Technology for Selection of Antibody Fragments

3

Daniela Teixeira, Maria Gonzalez-Pajuelo FairJourney Biologics, Porto, Portugal

CHAPTER OUTLINE 1. Introduction�� 67 2. Antibody Phage Display Libraries�� 71 2.1 Antibodies From Naïve and Immune Phage Display Libraries�� 73 2.2 Antibodies From Synthetic and Semisynthetic Phage Display Libraries�� 74 3. Selection and Screening of Antibody Phage Display Libraries�� 76 4. Antibody Engineering�� 78 4.1 Affinity Maturation of Antibodies�� 79 4.2 Humanization of Antibodies�� 80 5. Conclusions and Future Perspectives�� 81 References�� 82

1. INTRODUCTION The technology of phage display described by Smith in 1985 was a key stepping stone for the development of new methods to generate monoclonal antibodies. In what is today widely acknowledged as a groundbreaking work, George Smith reported the linkage of genotype and phenotype of peptides by fusing peptide genes to the gene encoding for the minor coat protein III of filamentous phage. As a result, the gene encoding the peptide was packed within the same virion as a single-strained DNA, and the peptide-pIII fusion protein was displayed on the surface of filamentous phage allowing affinity purification of the peptide (Smith, 1985). The publication from Smith soon called the attention of scientists aiming to develop recombinant methods for generation of immunoglobulin (Ig)-based binding sites, bypassing the classical hybridoma technology. Consequently, new methods for cloning antibody genes and for expression of functional antibody fragments in the surface of filamentous phage were soon after reported (Huse et al., 1989; McCafferty et al., 1990; Orlandi et al., 1989; Ward et al., 1989). The successful combination of cloning of antibody genes in libraries via polymerase chain reaction technique (PCR) and phage display has resulted in a very reliable antibody discovery platform illustrated by the Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00003-3 Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 3 Phage Display Technology

number of phage display-derived antibodies under clinical development or already approved (Nixon et al., 2014). Because of very specific aspects of the biology of filamentous phage, the most frequent phage used for phage display is the filamentous phage of the Ff family (M13, Fd, f1) (Webster, 1996). These phage can only infect strains of Escherichia coli containing an F conjugative plasmid. The infection starts by specific interaction of the phage with the tip of the F-pilus produced by the bacteria. This F-pilus, assembled during the exponential growth phase of specific E. coli, consists of a protein tube formed by pilin subunits. The end of the phage attached to the pilus is taken to the membrane surface by depolymerization of the F-pilus. There, the major capsid proteins integrate the membrane and the phage DNA is translocated into the cytoplasm, where bacterial enzymes synthesize the complementary DNA strand and the resulting double-stranded replicative form functions as template for synthesis of phage proteins. The assembly of new phage particles occurs in bacteria at a site where the inner and outer membranes are in close contact culminating in the release of the particles to the media. During the assembly process the infected bacteria continues to grow, although with a generation time of around 50% longer than for uninfected bacteria. During the first generation one infected cell produces around 1000 phage particles, being 100–200 particles produced in the following generations (Webster, 1996). Because the F-pilus depolymerizes immediately on infection of one phage and because DNA replication and assembly of phage is not limited by the size of DNA, the Ff phages are excellent cloning vehicles. For the phage display of proteins, two different genetic systems have been developed. In the first system, foreign protein genes are inserted into the phage genome fused to a capsid protein gene (McCafferty et al., 1990). In a second and more commonly used and successful system, the expression of the protein is detached from the phage replication by using a separate plasmid (named phagemid) that contains the genes encoding the antibody fused to a capsid protein gene but do not contain any other phage protein-encoding gene (Barbas et al., 1991; Hoogenboom et al., 1991). By doing so, a higher genetic stability and a simplification of the amplification of the antibody genes are achieved. Phagemids contain replication origins of both E. coli and filamentous phage and a suitable selection marker. In the majority of cases, they also incorporate an amber codon between the C-terminus of the cloned gene and the start of the capsid protein gene, allowing the peptide to be made as a soluble fragment in appropriate nonsuppressor E. coli strains. Phagemids also include a peptide tag, allowing detection of the soluble peptide and, in some cases, a hexahistidine tag to enable rapid purification and concentration of the produced peptide by metal affinity chromatography. The rest of the elements necessary for assembly of phage are provided by a replication-defective helper phage on infection via F-pilus of E. coli containing a phagemid (Vieira and Messing, 1987). As a result, phage particles containing the DNA encoding for antibodies and displaying the antibody are secreted into the bacteria culture medium.

1. Introduction

FIGURE 3.1 Schematic representation of two different systems for the phage display of peptides by using protein III or protein VIII as fusion partners.

The most commonly used fusion partners for the phage display of proteins are pIII and pVIII capsid proteins, the products of the gene III and gene VIII of the Ff filamentous phage (Scott and Barbas, 2001) (Fig. 3.1). In general, unlike in the case of pVIII display, pIII display tolerates larger peptide fusions and performs better than pVIII display (Iannolo et al., 1995; Kretzschmar and Geiser, 1995; Malik et al., 1996). As pIII is a minor coat viral protein, phage particles produced from phagemid containing gene III would display 0–5 copies of the pIII fusion protein. This represents a very advantageous feature for eliminating avidity and for selecting high-affinity binders. On the other hand, in the case of the pVIII system several hundred copies of the fusion protein are displayed per particle. The increased valency of this system would be advantageous when selecting for low-affinity binders or in cases where avidity would increase the chances of finding specific target binders (Armstrong et al., 1996). Efficient expression in E. coli of full-length antibody, tetrameric structure consisting of two identical heavy chains and two identical light chains, is difficult because of limitations in the bacteria folding machinery, although it has been reported in rare cases (Mazor et al., 2007; Simmons et al., 2002). In fact, phage display of antibodies was first demonstrated using antibody variable domain fragments (McCafferty et al., 1990), and since then, isolation of many different antibody fragment formats with target-binding specificity and that can be readily reformatted to full-length antibodies have been described. Of the different antibody fragments that can be displayed on the phage surface, the single-chain variable fragment (scFv) format consisting of a variable domain of the antibody heavy chain (VH) and a variable domain of the antibody light chain (VL) connected by a flexible oligopeptide linker is the most popular. In part this might be because of the small size of scFvs that facilitates the cloning of the genes and is better tolerated by bacteria, thus resulting in an efficient display and in high expression levels (Skerra and Plückthun, 1991). Yet, it is important to refer that scFvs have the propensity to aggregate (Raag and Whitlow, 1995), which can complicate selection and characterization; in addition, scFvs are relatively unstable over long periods (Kramer et al., 2002). scFvs can spontaneously dimerize and oligomerize resulting in an avidity effect that might contribute to successful identification of binders against complex targets (Van der Woning et al., 2016). On the other hand, for that reason, affinity-based selection of these types of antibody fragments can result in unproductive selection of high-affinity binders.

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As an alternative, antigen-binding fragment (Fab) format consisting of the variable heavy chain along with its first constant domain (VHCH1) associated with a whole light chain (VLCL) also can be expressed on the phage surface. For that, phage and phagemid vectors have been designed, where the genes coding for VHCH1 or for VLCL are fused to gene III while the other chain is independently secreted resulting in the folding of the complete Fab on the phage surface and in the bacteria periplasm (de Haard et al., 1999; Hoet et al., 2005). One of the advantages of working with Fab molecules is related to the lack of tendency to multimerize the monovalent display achieved when using protein III-based phagemids. These two factors positively contribute to select high-affinity binders (de Haard et al., 1999). Although the use of Fab in phage display results in more stable antibody fragments, larger Fab molecules might result in some toxicity for expression in E. coli and might associate with lower yields as soluble fragments (Arndt et al., 2001). Single-domain antibody fragments (sdAb), consisting of only one variable domain from human IgG (VH or VL) or from the heavy chain-only antibodies of camelids (VHH), show high specificity and stability and have proven to be also very suitable antibody fragments for the generation of antibodies via phage display (Davies and Riechmann, 1996; Harmsen and de Haard, 2007; Maussang et al., 2013; Paz et al., 2005; Ward et al., 1989). Regardless the phage display vector, the fusion partner and the type of antibody fragment of choice, the selection of target-binding partners from a pool of antibodies is done through a process of in vitro repeated cycles typically named biopanning or phage display selections (Fig. 3.2). This process consists of cycles each including (1) incubation of the antibody repertoire and the target, (2) washing of nonspecific

Phage display repertoire (library)

Washing Phage are incubated with target

Screening

Amplificaon

Host bacteria

Eluon Infecon

FIGURE 3.2 Schematic representation of phage display process.

2. Antibody Phage Display Libraries

binders, and (3) elution and amplification of specific binders for further cycle or for screening. Presented here as a very general technology, it is the scope of the following sections to dig in further into the different types of antibody libraries, the different selection and screening strategies, and the use of phage display to engineer antibodies. Consequently, the flexibility and the power of the phage display for the generation and development of very different antibodies with very specific characteristic and for various purposes will be illustrated.

2. ANTIBODY PHAGE DISPLAY LIBRARIES Phage display libraries of different antibody fragments such as sdAb (human VH or VL and camelids VHH) or scFv or Fab offer great potential for the identification of specific high-affinity binding molecules with different effector functions against various targets. The success of this technology relies on the construction of highquality libraries containing large number of antibody fragment variants with high sequence diversity from natural repertoires (Berry and Popkov, 2005; Dobson et al., 2005) or synthetic repertoires (Fellouse and Sidhu, 2005), which can later be formatted to full antibodies. The process of constructing diverse phage display antibody libraries combines the amplification of the heavy chain and light chain variable domain genes, V-genes, from natural (i.e., immune and naïve) B cell repertoires or the generation of synthetic V-genes with the cloning of antibody fragments into phage/phagemid vectors. After transformation of appropriate E. coli strain (i.e., TG1) with the resulting phage/ phagemid vectors, bacteria are infected with helper phage for rescue of the phage particles expressing the antibody fragments (reviewed in Tohidkia et al., 2012) (Fig. 3.3). In comparison to sdAb display libraries that contain only a variable domain type (VH Natural B cell repertoires VH/VL

PCR

VH(CH1)

Cloning

gIII

Bacteria transformaon and rescue with helper phage

Phagemid DNA

Gene synthesis

VL(CL)

Anbody repertoire on phage (Phage Display Library)

Synthec repertoires

FIGURE 3.3 Schematic representation of generation of phage display antibody libraries. PCR, polymerase chain reaction.

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or VL), increased diversity in scFv and Fab display libraries result from the random combination of the pool of heavy and light chain V-genes. Different methods have been described for combining VH and VL genes in these libraries including sequential cloning (Eeckhout et al., 2004), overlap extension PCR (Söderlind et al., 2000), and recombination (Sblattero and Bradbury, 2000). The phage/phagemid vectors for construction of different antibody fragments libraries share several structural elements; however, for display of Fab antibody fragments these vectors are designed as bicistronic for expression of VLCL and VHCH1 genes from the same promoter. In this case, the VHCH1 is expressed fused to the coat protein and the VLCL chain is secreted into the periplasmic space where it associates with the other chain (Hoet et al., 2005). The most important aspects of phage display libraries are its size and diversity, which have the high impact in the successful isolation of antibody fragments with wanted specificity and affinity. In fact, it has been described that the larger the phage display library size, the higher the chance for isolation of a larger number of antibodies with higher affinities. For example, Marks et al. (1991) reported that a naïve library with a diversity of 1E+07 allows identification of antibody fragments with micromolar range affinities, whereas libraries with diversity above 1E+09 result in antibodies with subnanomolar range affinities (de Haard et al., 1999; Griffiths et al., 1994; Sheets et al., 1998). The library size is mainly limited by the low transformation efficiency of bacteria, which can be solved by increasing the number of transformations or by the accumulation of libraries generated from different donors. However, various factors can impact the quality of the library functional diversity including the process to generate the V-genes and their cloning in the library vectors (reviewed in Tohidkia et al., 2012). Therefore, quality control of phage libraries by different methods is key to evaluate its functional diversity. These include (1) determination of library size by tittering of transformants containing the phagemid for calculation of colony-forming units, (2) determination of the percentage of transformants containing the antibody fragment by PCR of antibody cloned genes, and (3) assessment of diversity by DNA fingerprinting analysis using enzymatic digestion (Schmitz et al., 2000) or by DNA sequencing, allowing not only precise determination of the library sequence diversity but also the percentage of frame shifts and/or stop codons that will prevent functional display of the antibody fragment on the phage (Zhao et al., 2016). Although the phage libraries contain billions of phage particles, not all antibody fragments represented in the library will be displayed because particular sequences may be sensitive to bacterial enzymes or be toxic to bacteria interfering with assembly of the phage. In addition, only a small percentage of phage particles will display the antibody fragment. For example, smaller antibody fragments such as sdAb are more easily displayed at high levels in bacteria (Davies and Riechmann, 1995; Ward et al., 1989) compared with larger size Fab (Arndt et al., 2001). Nevertheless, considerable optimization of growth conditions and phage rescue has been established for maximum display levels and yields of phage (reviewed in Tohidkia et al., 2012). Ultimately, the preferred antibody fragment format of the library should take into


consideration the source of V-genes, the physical properties of each antibody format, and the end application. Regardless of the similarities in the procedures to generate phage display libraries of different antibody formats, these can differ substantially regarding the sources of the V-genes, sources of samples for isolation of B cells, and the number of donors used for samples collection. In addition, the method for amplification, engineering, cloning of the V-genes, and library transformation methods also vary. Details of the different features of phage display libraries will be discussed in sections below.

2.1 ANTIBODIES FROM NAÏVE AND IMMUNE PHAGE DISPLAY LIBRARIES Both naïve and immune phage display libraries can contain V-genes from different natural repertoires such as human donors or other animals including mouse, rat, llama, chicken, rabbit, sheep, or nonhuman primates (Zhao et al., 2016). For construction of immune phage display libraries, on active immunization by administration of the immunogen to an animal, the B cells that can be isolated from different tissues (i.e., blood, spleen, or lymph nodes) are used for mRNA extraction, which is reverse transcribed to cDNA. From this cDNA, the VH- or VL-encoding genes are amplified from the IgG repertoires by PCR with sets of primers specific for more conserved regions of the antibodies genes (frameworks (FRs) or constant domains), allowing the amplification of different V-gene families as listed in the V BASE database (http://www2.mrc-lmb.cam.ac.uk/Vbase/). The used primers incorporate unique restriction endonuclease digestion sites permitting cloning of the amplified V-genes in the phage/phagemid vector between the coding sequence of the signal peptide and the N-terminus of the coat protein to construct the antibody library (reviewed in Tohidkia et al., 2012) (Fig. 3.3). A similar procedure is followed for generation of naïve phage display libraries, except that naïve B cells isolated from diverse lymphoid sources of nonimmunized animals (i.e., healthy human) are used for mRNA extraction and library construction. In addition, the V-genes repertoires of IgM isotype (primary immune response), IgG (secondary immune response), or all the five antibody isotypes have been used for generation of different naïve libraries (de Haard et al., 1999; Marks et al., 1991; Pansri et al., 2009; Vaughan et al., 1996). Naïve libraries have several advantages as they can be generated from an animal source that cannot be actively immunized because of ethical issues, i.e., to generate fully human antibodies from V-gene repertoires. In addition, they can be used for the isolation of antibodies against any target antigen of interest from a single library including self-antigens and target antigens that might be toxic or nonimmunogenic while bypassing the time-consuming immunization process. And, although discovery of antibodies from immune libraries enriched for V-genes encoding immunogen-specific antibodies that have been through the immune response affinity maturation process results in the identification of specific antibody fragments of higher affinity compared to naïve libraries, successful identification of high-affinity antibodies from large naïve

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phage libraries generated from large number of donors/samples and following optimized procedures for maximal coverage of all V-genes repertoires has been shown (de Haard et al., 1999; Pansri et al., 2009; Sheets et al., 1998; Vaughan et al., 1996). Interestingly, similar to a naïve library, the isolation of antibody fragments specific to several antigens different from the immunogen from a large immune library has been reported (Moon et al., 2011). Moreover, even though active immunization of human donors is unethical, phage display libraries have been generated from humans suffering from autoimmune diseases, cancer, or exposed to viral infections to allow identification of fully human antibodies with great potential as novel diagnostic and therapeutic agents (reviewed in Zhao et al., 2016). Furthermore, transgenic animals have been engineered to contain the majority of the human antibody V-genes repertoire instead of the host genes and therefore on immunization produce humanlike antibodies that can be cloned in natural phage display libraries (reviewed in Brüggemann et al., 2015).

2.2 ANTIBODIES FROM SYNTHETIC AND SEMISYNTHETIC PHAGE DISPLAY LIBRARIES In contrast to immune and naïve libraries that represent the natural diversity of antibody repertoires, synthetic and semisynthetic libraries represent nonnatural diversity of V-genes segments. These can be generated by introducing variability into antibody variable domains nucleotide sequences, more specifically in the complementary-determining regions (CDRs). Each V region consists of three CDRs that are hypervariable regions with great sequence diversity supported by more conserved regions named FRs. The approach to generate the diversity represented in the synthetic antibody library is both complex and critical for the successful identification of antibodies with superior properties. Indeed, synthetic libraries are designed to contain highly diverse nature-like variable sequences in combination with high levels of expression, stability, and solubility in the display system, while eliminating several biases represented in natural libraries (reviewed in Shim, 2015). Fully synthetic libraries have been designed in silico and synthetically generated based on the analysis of the sequence and structure of FRs and CDRs regions of antibodies. Synthetic libraries have been generated using as template only limited number of human germline V-genes selected based on their prevalence in human immune repertoires and high stability and expression (Arnaout et al., 2011; Silacci et al., 2005; Yang et al., 2009). In addition, synthetic libraries have been designed to include multiple variable heavy and light chain FRs regions (Knappik et al., 2000; Rauchenberger et al., 2003; Tiller et al., 2013). A major advantage of synthetic libraries with several FR sequences is that they can support different CDR canonical structures ensuring a high number of functional antibody fragments in the library. Diversification in the synthetic libraries can be introduced in few CDRs, commonly only CDR3, because of its central contribution for antigen binding (Knappik et al., 2000; Rauchenberger et al., 2003; Silacci et al., 2005) or by randomization of all CDRs and combination of all randomized CDRs from VH and VL resulting in higher conformational and sequence diversity in the library (Prassler et al., 2011;


Rothe et al., 2008; Yang et al., 2009). Regardless of the number of randomized CDRs, diversification takes into consideration the amino acid composition and length variation of germline CDR sequences represented in the natural repertoires. Different methods for CDR diversification have been applied including (1) PCR using degenerate oligonucleotides (Silacci et al., 2005; Yang et al., 2009) or (2) using the trinucleotide-directed mutagenesis technology (Knappik et al., 2000; Prassler et al., 2011; Rothe et al., 2008) or (3) using more novel solid-phase gene synthesis technology (Slonomics) (Tiller et al., 2013; Zhai et al., 2011). The latter results in the controlled incorporation of specific amino acids in a desired frequency in each position, and consequently, in higher proportion of functional clones in the library. This also avoids the presence of certain undesired amino acids prone to specific posttranslational modification in the antibodies (Tiller et al., 2013). An example of well-validated synthetic libraries is the human combinatorial antibody libraries (HuCAL, HuCAL GOLD, and HuCAL PLATINUM) that were sequentially improved regarding antibody fragment functional diversity and folding display, allowing identification of antibodies with picomolar affinities (Knappik et al., 2000; Prassler et al., 2011; Rauchenberger et al., 2003; Rothe et al., 2008). The Ylanthia is another very large fully synthetic human Fab library that was generated mainly focusing in improved biophysical characteristics of the antibody fragments for easy antibody development and manufacturing (Tiller et al., 2013). The development of next-generation synthetic libraries requires further work to improve the functional library size and affinity with optimization of synthetic binding sites and reduced immunogenicity. Semisynthetic phage display libraries result from a combination of both naïve and synthetic libraries where the V-genes are originally from natural antibody repertoires including germline V-genes and rearranged V-genes from both immune and nonimmune donors but synthetically randomized to introduce variability (Akamatsu et al., 1993; de Kruif et al., 1995a; Nissim et al., 1994). This allows the identification of antibodies with different specificities and higher affinities. Diversification of single or multiple FRs is mainly introduced by randomization of CDR3 of VH genes, as it has the highest loop sequence diversity and length and greatly contributes to antigen binding (Nissim et al., 1994). However, CDR3 of VL randomization has also been included to increase the library diversity (Akamatsu et al., 1993; Griffiths et al., 1994). As for synthetic libraries, to achieve highly diverse semisynthetic repertoires, similar approaches of CDR randomization have been applied using random degeneracy or more rational randomization based on CDR canonical structures and amino acid residues involved in antigen binding (de Kruif et al., 1995a; Hoet et al., 2005; Pini et al., 1998). Alternatively, Söderlind et al. (2000) reported the generation of a semisynthetic library by amplification of the CDR diversity from natural repertoires germline sequences and their assembly into a synthetic FR scaffold. Similar to naïve libraries, synthetic and semisynthetic libraries also allow the identification of antibody fragments against diverse target antigens and epitopes with high specificity and affinities in addition to optimal biochemical and biophysical properties.

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3. SELECTION AND SCREENING OF ANTIBODY PHAGE DISPLAY LIBRARIES The expression of different antibody fragments on the surface of filamentous phage, which are used in repetitive cycles of selections (i.e., biopanning) comprising (1) antigen binding, (2) washing, and (3) elution of phage binders, allows the selection of a subpopulation of specific phage antibody fragments with specific properties from large phage display libraries (approximately 1E+13 phage particles) (Fig. 3.2). During the first round of biopanning, the entire library is exposed to the antigen and specific binders can be enriched to higher or less degree. If several consecutive biopanning rounds are performed, considerable enrichment of specific phage binders can be achieved. Depending on the antibody fragment format and the type of phage display library, 2–4 rounds of selections are typically performed in short time to allow sufficient enrichment of specific phage. The success of each selection round, enrichment, can be evaluated by determining the phage titers applied and eluted after antigen incubation compared with selections performed in the absence of antigen (background). Alternatively, eluted pools of phage can be tested in enzyme-linked immunosorbent assay (ELISA) for positive binding to the target antigen. The outcome of the selection process largely depends on the phage display library quality; however, the biopanning strategy greatly influences the ability to select antibody fragments not only with the required specificity and affinity but also physical properties and functionality. In fact, phage displays selections are very flexible and different strategies can be designed to drive selection pressure in the desired direction by controlling specific parameters (reviewed in Bradbury and Marks, 2004). For example, selection of antibody fragments with unique specificities can be improved (1) by including subtraction/depletion steps before selections or (2) by counterselection with an irrelevant molecule during phage-antigen incubation to deplete the irrelevant binders present in the phage library. In contrast, if cross-reactivity to two different molecules (i.e., different species or different isoforms of the same target antigen) is needed, alternating rounds of selections using the different molecules increase the chances of identifying cross-reactive antibody fragments. Also, increasing the stringency of selections (1) by adjusting the antigen format and limiting its concentration or (2) by adjusting the time of phage incubation and washing results in the selection of a narrow panel of antigen-specific phage binders, potentially with lower off-rates and/or higher on-rates (Hawkins et al., 1992). To select antibody fragments with improved stability and solubility, as the phage particles are extremely resistant to harsh conditions, the selection conditions before, during, or after biopanning can be adjusted by treatment of phage at elevated temperatures, low pH, and enzymatic or detergent treatment (Jung et al., 1999). Internalization selection strategies have been used to identify internalizing antibodies where phage is recovered from within the cells after receptor-mediated endocytosis (Becerril et al., 1999). Finally, different elution conditions can be applied to recover the total pool of specific phage by low pH (HCl or glycine buffer), high pH

3. Selection and Screening of Antibody Phage Display Libraries

(triethylamine), or by enzymatic cleavage (trypsin) of a protease site present between the antibody fragment and the coat protein. To recover a pool of phage specific to a particular antigen epitope, a competitive elution strategy using an excess of a ligand or antibody binding to the antigen can be performed (Bradbury and Marks, 2004). Monitoring enrichment during selections allows not only to evaluate the efficiency of the selection but also enables the redesign and the fine tuning of selection strategies to improve the chances of isolating highly exquisite phage (Lou et al., 2001; Mutuberria et al., 1999). The result of selections can vary depending on the nature and quality of the antigen and on the method used to present the antigen during selections (Bradbury and Marks, 2004). The nature of the target antigen used in the biopanning can vary from (1) haptens, (2) peptides, proteins, and enzymes, (3) membrane fractions, viruslike particles, liposomes, and (4) whole cells to (5) tissue sections or whole tissues. The different antigen formats are generally used in solid phase selections where the target antigen is presented on a solid surface such as (1) immunotubes, microtiter plates, BIAcore sensor chips, (2) cellulose/poly(vinylidene) fluoride membranes (Liu et al., 2002), (3) magnetic beads, or (4) cells and tissues. Direct adsorption of the antigen to a surface involves antigen conformational changes, especially for small-size antigens, which may hinder relevant epitopes or drive selection on artificial epitopes not present in the native antigen. To prevent this, the antigen can be presented in solution in the biopanning. For example, selections using biotinylated antigen that is incubated in solution with the phage before capturing of the phage-antigen complex on a surface coated with streptavidin/neutravidin. These types of selections are typically used to present the maximum number of binding epitopes in the target antigen. In addition, they also allow to control the antigen concentration and the times of incubation, which is important to select the highest affinity antibodies. Similarly, capturing of the antigen by specific antibodies immobilized on a solid surface has been used for the selection of phage against specific conformational intact epitopes (Sanna et al., 1995). Using whole cells in solution selections is critical to isolate antibodies against cell surface antigens in its biological native state. In fact, cell selections allow isolation of antibodies against unknown antigens including various tumor antigens present on the cell surface (Mutuberria et al., 2004) or against antigens that are not readily available as purified protein (de Kruif et al., 1995b). To avoid selection of irrelevant binders against several proteins expressed on cells, negative selections can be performed with negative target cells before or after positive selection on target-expressing cells (Noronha et al., 2002). Alternatively, competition with an excess of negative target cells can be included during phage incubation with positive-target cells, as long as positive cells are labeled (i.e., biotin) to allow efficient separation by magnetically activated cell sorting (Siegel et al., 1997). Ultimately, in vivo phage display selections have been described where phage are directly injected into living animals and recovered from relevant tissue or cells collected, allowing selection of phage specific for an antigen in its natural environment (Johns et al., 2000).

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After several rounds of biopanning, a mixture of phage with different targetbinding properties is obtained, which needs to be individually screened for positive binding. Monoclonal preparation of phage from individual E. coli colonies picked randomly into 96-well plates can be tested for specificity using a standard antibodybinding screening assay, ELISA. Additionally, or alternatively, bacterial crude extracts containing the soluble antibody fragment can be expressed from the same phagemid in the periplasm of bacteria to be tested. The binding to the antigen of antibodies fragments displayed on phage can be detected with an antibody that recognizes a major coat protein, while soluble antibody fragments can be detected with an antibody that recognizes the peptide tags to which they are fused. For membrane targets, it is important to screen the binding of antibody fragments in solution to cells either by fluorescence-activated cell sorting or to whole cells fixed on a microtiter plate by cell ELISA. Additional screening assays can be performed for characterization of antibody fragments regarding specificity, affinity, functionality, and stability. These include, for example, homogeneous time-resolved fluorescence or fluorometric microvolume assay technology, which is very sensitive, and robust homogeneous mix-and-read assays to measure interactions that can be miniaturized from 96- to the 384- and 1536-well plate formats for high-throughput screens. In addition, the determination of dissociation rates (off-rates) of soluble antibody fragments from a target antigen attached to a sensor by surface plasmon resonance (SPR) (i.e., BIAcore, Octet or ProteOn) is used for discrimination between clones regarding affinity. After screening assays, the sequence of the variable domains of positive hits is determined by DNA sequencing. Recently, the development of high-throughput sequencing by next-generation sequencing technologies bypassed the limitation of Sanger sequencing that samples only a few hundred variants (Ravn et al., 2013). Therefore, the diversity of entire selection outputs can be determined before or in parallel with screening of monoclonal antibody fragments. This allows a further comprehensive analysis of unique sequences in the downstream screens and maximizes the chance of identifying specific antibodies. Automation of phage display selections and screening assays has been implemented mainly by adjusting procedures to robotics using high-throughput devices and workstations (Bradbury et al., 2003; Hallborn and Carlsson, 2002). For example, phage display selections integrated with high-throughput DNA sequencing allowed testing of thousands of antibodies (Edwards et al., 2003). Protein microarrays have also been applied for high-throughput screening of antibody specificities and affinities (Poetz et al., 2005).

4. ANTIBODY ENGINEERING Although antibodies with desirable specificities can be isolated from phage display libraries or via hybridoma, it is often the case that additional properties of the antibodies (i.e., affinity/efficacy and immunogenicity) are insufficient for the final

4. Antibody Engineering

application purpose. Acknowledgment of this hurdle has led to the development of several antibody engineering in vitro techniques to generate antibody lead variants. In this section the main antibody engineering in vitro techniques that might include the selection of variants from libraries via phage display are described.

4.1 AFFINITY MATURATION OF ANTIBODIES For therapeutic and research applications, antibodies with affinities in the low nanomolar or subnanomolar range are often required. These ranges of affinities are particularly difficult to obtain in antibodies selected from nonimmune or synthetic libraries, making necessary an antibody engineering process to deliver affinityimproved variants. In an approach to mimic the in vivo somatic hypermutation and selection process, a phage display library containing billions of antibody variants can be selected under conditions that favor antibodies with improved binding kinetics. Using such approach improved equilibrium dissociation constant (KD) values up to values of two orders of magnitudes when compared to the parental antibody are attainable (Thie et al., 2009). The antibody variants can be generated by (1) random mutagenesis of the V domain(s) or by (2) site-directed mutagenesis of CDRs in the V domains or by (3) antibody chain shuffling (Thie et al., 2009; Thie, 2010). In the case of the random mutagenesis process, the complete phagemid carrying the antibody fragment gene can be targeted by using an E. coli mutator strains (Irving et al., 1996) or the error-prone TempliPhi DNA amplification (Fujii et al., 2004). In both cases, the vector backbone is also mutated, so the recloning of the mutated antibody genes into an intact functional phagemid is needed. To avoid that, and in a more common used technique, random mutations can be introduced by error-prone PCR. In this reaction, the natural error rate of Taq polymerase is used during DNA amplification under suboptimal buffer conditions (Martineau, 2002; Tindall and Kunkel, 1988). When specific structural antibody information is available, site-directed mutagenesis of one or more CDRs residues that are key in antigen binding called “hotspots” is performed via PCR using specific randomized oligonucleotides. By using this strategy, the isolation of antibodies with picomolar range affinities has been reported (Li et al., 2014; Schier et al., 1996; Yang et al., 1995; Yau et al., 2005). In the absence of structural information, an antibody chain shuffling approach can be followed. In that scenario, one of the two antibody variable domains and/or even segments of the variable domains are replaced with a repertoire of naturally occurring variants (Klarenbeek et al., 2016; Lu et al., 2003; Thompson et al., 1996). Subsequent selection of affinity-improved variants can be achieved by applying different strategies at the phage display biopanning (Klarenbeek et al., 2016; Thie, 2010). For example, affinity-driven selections might include various cycles of selections where the antigen concentration and the number of input phage are decreased consecutively between rounds. To have a better control on the antigen concentration

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presented to the variants libraries and to avoid avidity, selections are normally performed in solution using biotinylated monomeric antigens captured via streptavidin beads. Moreover, selection of improved off-rate variants can be boosted by adding in solution an excess of nonbiotinylated antigen, and therefore, high off-rate variants are not captured and washed away during the selection process. To screen and rank the selected variants, monoclonal-soluble antibody fragments produced as crude extracts of bacteria grown in microtiter plates are produced. These are frequently tested for specificity via ELISA and ranked for off-rate via SPR. Identified off-rate improved variants can be formatted to full IgG or used as purified antibody fragment to determine kinetic parameters and affinity via SPR (Klarenbeek et al., 2016).

4.2 HUMANIZATION OF ANTIBODIES Despite the advance on technologies to generate fully human antibodies, there is still a vast majority of monoclonal antibodies derived from murine or other nonhuman sources. Initially, patient treated with rodent antibodies suffered from the development of human antimouse antibodies responses (Chatenoud et al., 1982), which showed a limitation in the clinical application of such antibodies. Nevertheless, this problem was quickly overcome by a breakthrough technology named “humanization” (Jones et al., 1986; Riechmann et al., 1988). In this process, commonly known as “CDR grafting,” the six CDRs of the parental murine monoclonal antibody are grafted onto a human germline FR of relevance. Some of the resultant humanized antibodies (with as low as 5% of nonhuman sequence) lead to commercially success drugs (i.e., Avastin, Herceptin) (Carter et al., 1992; Presta et al., 1997; Reichert, 2012) and many others are in clinical development (Reichert, 2014, 2016). Unfortunately, some of the antibodies humanized by this process have shown up to 100-fold lower affinities than the parental antibody (Carter et al., 1992; Makabe et al., 2008; Presta et al., 1997). To circumvent this issue, in a process called “back mutations,” the original nonhuman key FR residues involved in supporting the antigen-binding loops (CDRs) can be restored in the chosen human germline FR (Almagro and Fransson, 2008). Although this approach results in a recovery of affinity, it might also lead to an increase in immunogenicity (Hwang and Foote, 2005). Alternative humanization approaches in which libraries of humanized variants, with minimal nongermline FR and in some cases CDR residues, interrogated via phage display have been described (reviewed in Almagro and Fransson, 2008; Townsenda et al., 2015; Safdari et al., 2013). For example, Baca et al. (1997) reported the use of monovalent phage display-based method for optimizing the FR of humanized antibodies by random mutagenesis of important FR residues that support CDRs. As a result, an anti–vascular endothelial growth factor (VEGF) humanized antibody with an increased affinity of 125-fold compared to the original clone was selected. Using a similar method, a humanized highly potent anti-CD70 antibody

5. Conclusions and Future Perspectives

was obtained by Silence et al. (2014). In this case, deviating amino acids in the FR regions of a llama antibody were mutated by PCR using overlapping, degenerated oligonucleotides (encoding both the human and llama residues), and the resultant Fab phage display library was subjected to affinity-driven selections. In a similar case, combination of a phage display library containing human germline deviating amino acids in the region of a rat antibody with monovalent phage display affinity-driven selection lead to the isolation of variants with human identity and homology varying from 88% to 99% and with equal or lower off-rate values than the parental rat antibody (http://fjb.pt/wp-content/uploads/2015/05/IX_Humanizationof-a-rat-mAb-with-no-loss-of-affinity-1.pdf). In this last example, the closest human germline to be used as reference was chosen based on CDRs homology between human and murine antibodies as reported by Hwang et al. (2005). This method is based on the statement that if a nonhuman and a human antibody have similarly structured CDRs, the human FR will also support the nonhuman CDRs, with good retention of affinity. For that, the closest human FR sequence is chosen from the set of human germline genes based on the structural similarity of the human CDRs to those of the antibody to be humanized (same Chothia canonical structures). A very efficient method for antibody humanization, in which besides the FR residues, CDR amino acids are also targeted, has been recently described (Townsenda et al., 2015). In a method named the Augmented Binary Substitution (ABS) process, phage display libraries for three different species (rat, rabbit, and chicken) antibodies were generated in which FR and CDR residues (other than CDR3 of the heavy chain) are replaced in a binary substitution manner (residue is either the nonhuman or human residue). In addition, the CDR3 of the antibodies heavy chain was also augmented with 1 ± 1 random substitution per clone. After selecting and screening the resulting phage display libraries for target-binding capacity, highly humanized anti-RAGE, anti-pTau, and anti-Par-A33 antibodies with improved or equal affinity to the parental antibodies were identified. The ABS process also reduced the projected immunogenicity as the number of T cell epitopes were clearly decreased when compared to the parental antibodies.

5. CONCLUSIONS AND FUTURE PERSPECTIVES For more than 25 years now, phage display has been proved to be a very robust technology for the generation of monoclonal antibodies. In the therapeutic arena, this is illustrated by the number of phage display-derived antibodies under clinical development or already approved. The increasing number of people been trained in phage display of antibodies and the growing number of available reported protocols, together with the expiration of some of the core patents during the last years, might result in the establishment of this technology in areas where hybridoma has been by default the technology of choice for generation of monoclonal antibodies. With the sequencing of the human genome

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and the discovery of novel targets and new biology processes, more and more highly specific antibodies are required as basic research or as high-affinity reagents. At present, very few phage display-derived antibody reagents are commercially available (i.e., the anti-sulfotyrosine antibody, clone sulfo-1C-A2). Nevertheless, with the dissemination of phage display for the reasons mentioned above, more phage display antibodies can be expected in commercial catalogs of reagents suppliers in the future. After the establishment of phage display, other in vitro technologies for generation of monoclonal antibodies, as, for example, ribosome display, yeast display, and mammalian display, have been developed. The advantages of these over phage display are mainly related to the bypass of cell transformation in the case of the ribosome display and to the fact that full IgGs with eukaryotic glycosylation patterns are selected by yeast and mammalian display. On the other hand, these alternative in vitro systems show some disadvantages associated to reagent stability and to the high technological complexity in making and selecting large libraries. Rather than ruling out the phage display, these other younger display technologies can be considered complementary to the phage display technology. For example, large antibody fragments libraries could be explored by phage display and resulting antibody panels could be matured by yeast display to generate high-affinity antibodies. Similarly, human antibodies repertoires produced on immunization of transgenic animals developed during the last 30 years can also be explored by phage display instead of hybridoma technology. Despite the increasing number of emerging technologies and taking into account all the features and the perspectives described in this chapter, it seems that the generation of monoclonal antibodies by phage display is far from being abandoned and forgotten.

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Townsenda, S., Fennella, B.J., Apgarb, J.R., Lamberta, M., McDonnella, B., Granta, J., Wadea, J., Franklina, E., Foya, N., Shúilleabháina, D.N., Fieldsa, C., Darmanin-Sheehana, A., Kingb, A., Paulsenb, J.E., Hicklingc, T.P., Tchistiakovab, L., Cunninghama, O., Finlay, W.J.J., 2015. Proc. Natl. Acad. Sci. U.S.A. 112, 15354–15359. Van der Woning, B., De Boeck, G., Blanchetot, C., Bobkov, V., Klarenbeek, A., Saunders, M., Waelbroeck, M., Laeremans, T., Steyaert, J., Hultberg, A., de Haard, H., 2016. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops. mAbs 8, 1126–1135. Vaughan, T.J., Williams, A.J., Pritchard, K., Osbourn, J.K., Pope, A.R., Earnshaw, J.C., McCafferty, J., Hodits, R.A., Wilton, J., Johnson, K.S., 1996. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309–314. Vieira, J., Messing, J., 1987. Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3–11. Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.T., Winter, G., 1989. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544–546. Webster, E., 1996. Biology of filamentous bacteriophage. In: Kay, B.K., Winter, J., McCafferty, J. (Eds.), Phage Display of Peptides and Proteins: A Laboratory Manual. Academic Press, San Diego, pp. 1–20. Yang, H.Y., Kang, K.J., Chung, J.E., Shim, H., 2009. Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Mol. Cells 27, 225–235. Yang, W.-P., Green, K., Pinz-Sweeney, S., Briones, A.T., Burton, D.R., Barbas III, C.F., 1995. CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J. Mol. Biol. 254, 392–403. Yau, K.Y.F., Dubuc, G., Li, S., Hirama, T., Mackenzie, C.R., Jermutus, L., Hall, J.C., Tanha, J., 2005. Affinity maturation of a V(H)H by mutational hotspot randomization. J. Immunol. Methods 297, 213–224. Zhai, W., Glanville, J., Fuhrmann, M., et al., 2011. Synthetic antibodies designed on natural sequence landscapes. J. Mol. Biol. 412, 55–71. Zhao, A., Tohidkia, M.R., Siegel, D.L., Coukos, G., Omidi, Y., 2016. Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy. Crit. Rev. Biotechnol. 36, 276–289.

CHAPTER

Ribosome Display Technology for Selecting Peptide and Protein Ligands

4 Akira Wada

RIKEN Center for Life Science Technologies, Yokohama, Japan

CHAPTER OUTLINE 1. Introduction�� 89 2. Emergence of In Vitro Display Technologies�� 90 3. Basic Principles and Features of Ribosome Display Technology�� 92 4. Selection of Peptides Using Ribosome Display Technology�� 94 5. Selection of Antibody Fragments Using Ribosome Display Technology�� 96 6. Selection of Proteins Using Ribosome Display Technology�� 97 6.1 Creation of Structural Proteins With Target-Binding Affinity�� 97 6.2 Identification of Target Proteins That Bind to Bioactive Compounds�� 98 7. Conclusions and Future Perspectives�� 100 References�� 101

1. INTRODUCTION In the last decade, functionalized nanomaterials/nanoparticles have been applied in bioscience, biotechnological, and biomedical research. Especially, moleculartargeted nanoparticles have been powerful tools as biosensing devices for diagnosis and as carriers of drugs/bioactive compounds for therapy (Ho and Leong, 2010; Vatansever et al., 2012; Yu et al., 2012; Zhu et al., 2014; Hagemeyer et al., 2015; Safdari et al., 2016; Braz et al., 2017; Gomes et al., 2017). Therefore, the creation of target-binding ligands (e.g., compounds, peptides, proteins, and antibodies) and their integration into the internal or external portions or both of nanomaterials/nanoparticles has been indispensable in facilitating their multipurpose use in a wide range of biomedical applications (Jeon et al., 2013; Heu et al., 2014; Khoshnejad et al., 2016; Li et al., 2016; Fan et al., 2016; Tietze et al., 2017; Ta et al., 2017; Ranalli et al., 2017; Kennedy et al., 2017). Furthermore, in vitro display technologies (such as phage, ribosome, and mRNA display technologies) are known as powerful methods for selecting various peptides, proteins, and antibodies as ligands from combinatorial libraries (Li, 2000; Amstutz et al., 2001; Hosse et al., 2006; Rothe et al., 2006; Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00004-5 Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 4 Ribosome Display Technology

Murray and Baliga, 2013; Wada, 2013). Numerous ligands selected using these technologies have been reported to bind specifically to target molecules of interest (e.g., compounds, materials, metals, receptors, and enzymes). This chapter describes the principle, features, and utility of ribosome display technology for selecting targetbinding peptides, single-chain fragment variables (scFvs), and proteins and presents a perspective of ribosome display selection of new ligands with target-binding affinities that will be applicable to the fabrication of functionalized nanomaterials/ nanoparticles in biomedical applications.

2. EMERGENCE OF IN VITRO DISPLAY TECHNOLOGIES Recent research studies have proved that in vitro display selection of peptides, proteins, and antibodies is a useful approach for developing target-binding ligands. In fact, the selected ligands have been successfully utilized in a wide range of fields from bioscience to biomedicine. In the past two decades, phage display technology has been developed for the selection of artificial peptides as ligands with target-binding affinities (Fig. 4.1A). In vitro selection of phage-displayed peptides against the target molecules makes it possible to identify new peptide ligands that bind to the interfaces of proteins or cavities of enzymes. Occasionally, amino acid sequences of the selected peptide ligands are identical to those of the natural peptide ligands. For example, peptides with a His-Pro-Gln (HPQ) sequence were selected against streptavidin from a peptide library with a size of 107 by using phage display technology (Devlin et al., 1990). Interestingly, X-ray crystal structural analysis revealed that the HPQ motif was positioned at the ends of the β-barrels of streptavidin (Lam et al., 1991; Weber et al., 1992). The HPQ motif was also essential to maintain the affinity for the target protein, but the dissociation constant (KD) value was in the millimolar range. Hence, to enhance the affinity by stabilizing the peptide structure, a disulfideconstrained cyclic peptide library (CXnC; C: cysteine, X: natural amino acids,

FIGURE 4.1 Linking a combinatorial peptide/protein as phenotype and its corresponding gene as genotype using (A) a bacteriophage in phage display technology, (B) a peptide/protein– ribosome–mRNA complex in ribosome display technology, and (C) a peptide/protein– puromycin–mRNA complex in mRNA display technology. FPS, flexible protein spacer; PAL, puromycin-attached linker.

2. Emergence of In Vitro Display Technologies

n = 4, 5, or 6) was constructed and screened using phage display technology (Giebel et al., 1995). The result revealed that the selected cyclic peptides contained the HPQ sequence and had strong affinities with KD values in the nanomolar range. These results show that structural rigidity derived from the cyclic configuration could enhance the target-binding affinity. According to the above concept, a unique library consisting of bicyclic peptides with a CX6CX6C sequence, which were constrained using a cross-linking reaction between three cysteine residues and tris(bromomethyl) benzene, was synthesized (Heinis et al., 2009; Zorzi et al., 2017). Then, phage display screening of the bicyclic peptide library was performed against human plasma kallikrein. The identified bicyclic peptide had an inhibition constant of c.1.5 nM and effectively inhibited the coagulation pathway in human plasma. In addition to select peptide ligands exhibiting high affinities for target molecules, various libraries of structurally constrained peptides have been used in phage display selection (Henchey et al., 2008; Smeenk et al., 2012; Kim et al., 2015). These reports have revealed that structurally constrained peptide ligands have the potential to exhibit target-binding affinity and proteolytic stability. Such ligands would be useful in fabricating new molecular-targeted nanomaterials/nanoparticles suitable for diagnosis and therapy. However, affinities of the peptides selected by using phage display technology were frequently too low for them to act as target-binding moieties of drug candidates or bioprobes. This limitation is caused by the unavoidable problems associated with the use of both bacteriophages and live host cells (e.g., steric hindrance between bacteriophages and target molecules, the toxicity of peptides for Escherichia coli cells, and low efficiency in the infection of host cells with peptide-displayed bacteriophage) (Smith and Petrenko, 1997; Hoess, 2001). These issues limit the use of peptide libraries with small sizes of 107–9, which consequently excludes desired peptide ligands through repeated selections. Thus, cell-free display technologies, which are unaffected by these limitations, are expected to be devised. To overcome the difficulties associated with the use of living cells for in vitro selection, new display technologies (e.g., ribosome, mRNA, and cDNA display technologies) have been developed based on cell-free protein synthesis systems. A central feature of these technologies is the use of specific complexes linking combinatorial libraries of peptides/proteins of interest as phenotype and their corresponding mRNAs or DNAs as genotype. The transient complexes can be used as alternatives to bacteriophages during in vitro selection and are indispensable for reading out genetic information about the amino acid sequences of target-binding peptide/protein ligands. For example, in ribosome (polysome) display technology, peptide–ribosome(s)– mRNA complexes are transiently synthesized through the in vitro translation of mRNAs that are transcribed from a DNA library coding artificially randomized peptides (Fig. 4.1B). In selecting peptides against monoclonal antibodies using the ternary ribosomal complexes, peptides that contained an amino acid sequence identical to that of the epitope of the antibody were selected (Mattheakis et al., 1994). Furthermore, ribosome display technology was used for screening a peptide library with a size of 1013 against streptavidin as a target molecule (Lamla and Erdmann,

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2003). Interestingly, the identified peptides possessed the HPQ motif that is essential for target-binding affinity, and the shortened variants exhibited KD values at the low nanomolar level, indicating that these affinities were much higher than those of the phage-displayed peptides. On the other hand, in mRNA display technology, peptide–puromycin–mRNA complexes are constructed by covalently linking combinatorial peptides and their corresponding mRNAs (Fig. 4.1C). A covalent bond in the ternary complex is formed by a reaction between the C-terminus of a nascent peptide in the ribosome and a puromycin attached around the 3ʹ end of mRNA. Notably, although synthesis and purification of the complexes are usually executed using time-consuming multiple procedures, various peptide libraries with large sizes of 1012–14 can be constructed and applied for selecting desirable peptide ligands (Cho et al., 2000). Indeed, various mRNA display selections were performed to isolate the calmodulin binding (Huang and Liu, 2007; Cotten et al., 2011), murine double minute 2 homolog binding (Shiheido et al., 2011), and ligase-like peptides (Seelig and Szostak, 2007). Additionally, cDNA display technology has been devised using peptide–puromycin– mRNA–cDNA complexes formed by reverse transcription of the mRNA complexes as represented in Fig. 4.1C (Takahashi et al., 2003; Biyani et al., 2006; Ueno et al., 2012). Both mRNA and cDNA display technologies have been also used to create nonnatural amino acid-integrated macrocyclic peptides (Li and Roberts, 2003; Millward et al., 2007; Schlippe et al., 2012; Ito et al., 2013; Morioka et al., 2015). Considering the methods and protocols used for performing in vitro display selections, ribosome display is most likely the simplest and most manageable cellfree technology. Therefore, the next sections are focused on the basic principles and features of ribosome display technology and attempt to explain the various advantages of the technology for selecting specific peptides and proteins as target-binding ligands.

3. BASIC PRINCIPLES AND FEATURES OF RIBOSOME DISPLAY TECHNOLOGY In ribosome display selection, as described in Fig. 4.2 (upper center), DNA constructs should be synthesized to contain all elements needed for in vitro transcription and translation. The T7 promoter is necessary for in vitro transcription using T7 RNA polymerase. The ribosome-binding site and Kozak sequence are essential for promoting in vitro translation with eukaryotic and prokaryotic ribosomes, respectively. The sequence of the combinatorial peptide/protein library is inserted before that of a flexible protein spacer that is fused to the C-terminus of the library. The mRNAs generated from the DNA constructs can be directly used to form peptide/protein–ribosome– mRNA complexes through eukaryotic (Douthwaite et al., 2006; He and Taussig, 2007; He et al., 2012) or prokaryotic (Lipovsek and Plückthun, 2004; Zahnd et al., 2007; Dreier and Plückthun, 2012; Zawada, 2012) translation machinery. Moreover, all stop codons of the mRNAs were preliminarily removed to ensure that ribosome idling occurred around the 3ʹ end of the mRNAs during in vitro selection.

3. Basic Principles and Features of Ribosome Display Technology

FIGURE 4.2 A DNA construct used for ribosome display technology (ATG, start codon; CPL, combinatorial peptide/protein library; FPS, flexible protein spacer; KS, Kozak sequence; NS, no stop; RBS, ribosome-binding site; T7, T7 promoter), and a cycle scheme of ribosome display selection of target-binding peptides/proteins as ligands.

Similarly, in vitro selection of target-binding peptides/proteins using ribosome display technology can be carried out using the following cycle scheme (Fig. 4.2) regardless of the type of cell-free protein synthesis system (e.g., a rabbit reticulocyte lysate, wheat germ extract, and E. coli S30 extract). (1) DNA constructs coding the sequences of combinatorial peptides/proteins are transcribed for synthesizing an mRNA library. (2) The resulting mRNA library is translated to generate a library of ribosomal complexes that contain combinatorial peptides/proteins as phenotype and their mRNAs as genotype. By stalling the ribosomes on mRNAs without stop codons, nascent peptides/proteins fused to a flexible protein spacer can be displayed on the ribosomal complexes. (3) After mixing various peptide/protein–ribosome– mRNA complexes and target molecule–immobilized beads/plates, specific complexes are selected by affinity binding of the displayed peptides to target molecules. (4) mRNAs, which are recovered by dissecting the selected complexes, are reversetranscribed to generate cDNAs. (5) DNA constructs are synthesized again from the cDNA using polymerase chain reaction (PCR). These constructs are used to perform the next selection cycle, and the portions are analyzed by sequencing to identify target-binding peptides/proteins. According to the above principles and features of ribosome display technology (Fig. 4.2), ribosome display selection can be implemented using simplified operations, and the protocol can be modified to suit a wide range of research purposes.

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4. SELECTION OF PEPTIDES USING RIBOSOME DISPLAY TECHNOLOGY Based on the cycle scheme shown in Fig. 4.2, ribosome display technology allows the selection of new peptide ligands that bind to a wide range of target molecules. Particularly, selected peptides can be rapidly produced using automated synthesizers at reasonable costs and chemically modified to provide desirable functions, structures, or both. Moreover, in contrast to full antibodies, synthetic peptides can be preserved in both solid and solution states for a significant period. These advantages have made synthetic peptide ligands the focus of attention as target-binding parts of nanomaterials/nanoparticles. As described in Section 2, ribosome display technology enabled the selection of desired peptide variants with a high affinity for streptavidin as a target from a 1013-member peptide library (Lamla and Erdmann, 2003). This result demonstrates that ribosome display technology enables the successful creation of target-binding peptides through rapid and simplified operations compared with phage display and other cell-free display technologies. Moreover, a highly diverse combinatorial peptide library is important for the reliable identification of peptide ligands with high affinities for target molecules. Therefore, by developing a simplified scheme for producing nanomole quantities of the DNA elements essential for in vitro transcription and translation, large combinatorial peptide libraries of 1014 could be constructed and adapted to ribosome display selection coupled with the cell-free protein synthesis system of E. coli S30 lysate (Yang et al., 2008). Furthermore, ribosome display selection can also be carried out in combination with cell-free protein synthesis systems reconstituted with translational factors and ribosomes of E. coli (Ohashi et al., 2012; Kanamori et al., 2014). Such systems, referred to as PUREflex or PURE systems, contain only the quantity of proteins and enzymes needed for in vitro translation in a small test tube. Therefore, PURE ribosome display selection of target-binding peptides could be considered to minimize nonspecific interactions between peptides and biomolecules coexisting in solution. Ribosome display selection is used at temperatures below 4°C or on ice to suppress the decay of the peptide–ribosome–mRNA complexes that are simply formed through noncovalent interactions. Thus, an enhancement of the stability of ternary ribosomal complexes would make it possible to maintain the complexity of combinatorial peptide libraries and reliably select target-binding peptides at a high temperature. In fact, after the introduction of strong affinity interaction of a C-variant (Cv) RNA-associating protein (Cvap) with a Cv RNA motif at the 5′ terminus of the mRNA, the peptide–Cvap–ribosome–mRNA complexes were automatically stabilized and could be treated even at a prolonged ambient temperature. Then, a ribosome display selection using the stabilized complexes permitted the discovery of disulfide-constrained peptides with specific affinity for cobalt(II) complex and cobalt(II)-immobilized beads (Fig. 4.3A) (Wada et al., 2008). The features of stabilized ribosome display technology and characterization of the metal-binding peptidefused Cvap convinced us that the selected peptides could retain each target-binding

4. Selection of Peptides Using Ribosome Display Technology

FIGURE 4.3 Selection of a disulfide-constrained peptide–Cvap–ribosome–mRNA complex against Co(II) complexes (A). Selection of a peptide–bioactive protein–ribosome–mRNA complex against TiO2 nanoparticles (B-1), and self-adsorption of TiO2-binding peptide-fused hEGF onto TiO2 plate surfaces, which promotes the growth of mammalian cells (B-2). Cvap, C-variant (Cv) RNA-associating protein; hEGF, human epidermal growth factor; TiO2, titanium oxide.

affinity even if Cvap is replaced with other bioactive proteins. Based on the original concept, a combinatorial peptide library fused at the N-terminal of the human epidermal growth factor (hEGF) was constructed and screened against titanium oxide (TiO2) nanoparticles using ribosome display technology (Fig. 4.3B-1). The process was successful, and the identified peptides exhibited a strong affinity for the target TiO2 nanoparticles. Furthermore, the TiO2-binding peptide-fused hEGF spontaneously adsorbed onto TiO2 plate surfaces and significantly induced the growth of various mammalian cells (Fig. 4.3B-2) (Wada et al., 2014b; Tada et al., 2014). These results suggest that ribosome display selection using peptide–bioactive protein–ribosome– mRNA complexes against nanomaterials produced a diversity of nanomaterial surface-binding bioactive proteins. Probably, bioactive proteins with specific affinities for nanomaterial surfaces might contribute to the rapid fabrication of bioactive nanomaterials/nanoparticles suitable for objective biomedical applications.

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5. SELECTION OF ANTIBODY FRAGMENTS USING RIBOSOME DISPLAY TECHNOLOGY Antibodies have been used as indispensable tools in a wide variety of bioassays (e.g., enzyme-linked immunosorbent assay , western blotting, immunoprecipitation, and immunostaining). Recently, chimeric/humanized monoclonal antibodies have been in the limelight as molecularly targeted drugs for treating conditions such as inflammatory disease, cancer, and asthma (Clark, 2000; Presta, 2008; Pelaia et al., 2012). Antibody drugs (e.g., Remicade for psoriatic and rheumatoid arthritis and Herceptin for human epidermal growth factor receptor 2 [HER2]-positive metastatic breast and gastric cancers) have been considered as perfect pharmaceutical agents with therapeutic activities and extremely low adverse effects. However, several critical issues related to their prescription and production have emerged (e.g., elimination by the human immune and metabolic systems, side reactions through antibody-dependent cellular cytotoxicity, and antibody humanization–related patents). Based on the above-observed effects, the development of innovative methodologies for overcoming these problems or alternatives to antibody drugs has been encouraged. In particular, scFv, which was devised as a downsized antibody that maintains specific affinity for a target, has attracted much attention as a target-recognizing molecule for carrying drugs, bioactive compounds, siRNAs, and functionalized nanoparticles to disease-related cells or tissues. Phage display technology has been adapted to select scFvs that bind to target molecules or maturate their affinities and specificities (Eisenhardt et al., 2007). However, as mentioned in Section 2, unavoidable problems with the use of live cells and bacteriophages tend to disturb the accuracy of in vitro scFv selection. Therefore, cell-free display technologies should be suitable for the small-scale screening of large-sized scFv libraries (Hanes and Plückthun, 1997; He and Khan, 2005; He and Taussig, 2008; Bradbury et al., 2011; Fujino et al., 2012). In fact, ribosome display technology has been optimized to select scFvs bound to a bovine insulin (Hanes et al., 2000), cluster of differentiation 28 as a human T cell antigen (Rothe et al., 2007), Fas as a tumor necrosis factor (TNF) receptor (Chodorge et al., 2008), a rabies virus glycoprotein (Zhao et al., 2009), sulfadimidine as a veterinary medicine (Qi et al., 2009), diethylstilbestrol as a synthetic nonsteroidal estrogen (Sun et al., 2012), and citrinin as a mycotoxin (Cheng et al., 2015). Furthermore, the technology also enhanced the affinities of scFvs against a short peptide derived from the yeast transcription factor GCN4 (Zahnd et al., 2004), a bovine prion protein (Luginbühl et al., 2006), a receptor for advanced glycation end products (Finlay et al., 2009; Hufton, 2012), and an interleukin-13 as an inflammation regulating cytokine (Thom and Minter, 2012). Furthermore, to meet the need for large-scale generation of different scFvs, multiple scFv production strategies have been developed in combination with ribosome display technology. For example, a rationally designed DNA library consisting of complementarity determining regions (H2 and H3 of the heavy chain and L3 of the light chain of the antibody) was constructed and then cloned into the framework of scFv. The resulting library was screened against emerging cancer antigens using

6. Selection of Proteins Using Ribosome Display Technology

ribosome display technology, and multiple scFvs-recognizing target antigens were found by using comprehensive short-read deep sequencing (Larman et al., 2012). In addition, ribosome display technology was integrated with an array plate, which was spotted with individual antigens on the surface. The single process of ribosome display selection against the antigen array resulted in the discovery of scFvs bound to 16 different cancer biomarkers (Cong et al., 2016). The high-throughput ribosome display selections enable the simultaneous production of various scFvs, which could be used to promote proteomics research and synthesize prospective scFv-modified nanomaterials/nanoparticles.

6. SELECTION OF PROTEINS USING RIBOSOME DISPLAY TECHNOLOGY 6.1 CREATION OF STRUCTURAL PROTEINS WITH TARGET-BINDING AFFINITY Artificial peptides, which can interact with the target molecules of interest, have been selected from various types of combinatorial peptide libraries using in vitro display technologies. However, both the affinity and specificity of the selected peptides have occasionally been insufficient because of their low structural diversity or rigidity. Therefore, attempts have been made to create artificially designed structural proteins with target-binding affinities comparable to those of the antibodies for use in a wide range of fields such as bioscience research and biomedical applications (Binz et al., 2005; Hosse et al., 2006; Nuttall and Walsh, 2008; Löfblom et al., 2010; Helma et al., 2015; Škrlec et al., 2015). Particularly, the development of such target-binding proteins has been explored using ribosome display technology (Binz et al., 2005; Seeger et al., 2013; Mouratou et al., 2015). For example, ankyrin repeat modules were designed by randomizing amino acids positioned on the surfaces of a natural ankyrin repeat protein. Then, libraries of designed ankyrin repeat proteins (DARPins) were constructed by inserting various numbers of the ankyrin repeat module between N- and C-terminal capping repeat units. The result showed that the randomized amino acid positions on adjacent repeat modules provided a potential interaction surface on the rigid scaffold of DARPin (Binz et al., 2003). Ribosome display screening of the DARPins libraries successfully allowed the discovery of DARPins that could bind to eukaryotic protein kinases (Janus kinase 2, JNK2, and p38), E. coli maltose-binding protein (Binz et al., 2004), HER2 (Zahnd et al., 2006), LmrCD as a heterodimeric multidrug ABC exporter from Lactococcus lactis (Seeger et al., 2012), and both the non- and doublephosphorylated forms of extracellular signal-regulated kinase 2 (ERK2) (Kummer et al., 2012, 2013). Furthermore, after fusing the ERK2-binding DARPins with green fluorescent protein (GFP) and the ERK2 with Renilla luciferase, the intracellular complex formation could be observed based on energy transfer from luciferase to GFP in COS-7 cells.

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Interestingly, the DARPins inhibited the phosphorylation of cellular ERK2. These results indicate that DARPins could be a valuable biosensor and inhibitor for investigating the biological functions of proteins or enzymes in living cells. Moreover, the integration of a consensus loop structure into an internal module of DARPins afforded LoopDARPins that exhibit a higher structural diversity than that of conventional DARPins. After performing only a few rounds of ribosome display selection, the identified LoopDARPins showed high affinities at the low nanomolar to picomolar range against four types of antiapoptotic regulators (B cell lymphoma-2, BCL-W, BCL-extra large, and myeloid cell leukemia) and ERK2 (Schilling et al., 2014). In addition, using a protein engineering approach and X-ray crystal structure information, trimeric DARPin adapters against HER2 were successfully designed to develop a generic adenovirus delivery system (Dreier et al., 2013). Consequently, a series of DARPins has been used in a broad range of areas from basic biological research to therapeutics and diagnoses (Tamaskovic et al., 2012; Jost and Plückthun, 2014; Plückthun, 2015). Various structural proteins with target-binding affinities other than DARPins have also been developed using ribosome display selection. A dsDNA-binding protein, Sac7d, from the hyperthermophilic archaeon Sulfolobus acidocaldarius was engineered as a small and stable protein scaffold. The consecutive amino acid sequence located on the surface of Sac7d was randomized to construct a new structural protein library. Using ribosome display selection of the library, a specific Sac7d was generated with a high affinity for the bacterial outer membrane secretin, PulD, and inhibitory activity against the oligomerization of PulD (Mouratou et al., 2007, 2012). Furthermore, ribosome display selection enabled the production of nonantibody proteins that bind to a wide variety of target molecules such as GFP-Cκ fusion protein as an optical biosensing device that binds to a human IgE (Chen et al., 2008), affibodies that bind to a murine IgG1 Fab (Grimm et al., 2011), ubiquitin that binds to TNF-α (Hoffmann et al., 2012), and armadillo repeat proteins that bind to a neurotensin peptide (Varadamsetty et al., 2012). Thus, the above reports demonstrate that ribosome display selection combined with protein scaffold–based libraries has considerable potential to create numerous structural proteins with target-binding affinity. Therefore, nonantibody scaffold proteins, which specifically interact with target molecules, should be used in the functionalization of nanomaterials as next-generation candidates of molecular-targeted nanomedicines.

6.2 IDENTIFICATION OF TARGET PROTEINS THAT BIND TO BIOACTIVE COMPOUNDS Bioactive compounds, which specifically bind to target proteins or receptors associated with cancer or diseases, are promising drug candidates or bioprobes for inhibiting or inducing particular cellular functions. Such compounds could also be attractive target-binding ligands of nanomaterials/nanoparticles. Whenever new

6. Selection of Proteins Using Ribosome Display Technology

bioactive compounds are discovered, the identification of their target proteins is critical for understanding their mode of action and predicting adverse reactions. Until now, pull-down methods using compound-immobilized beads and mass spectrometric analysis have been widely used to isolate target proteins and determine their amino acid sequences. However, it is often challenging to differentiate nonspecific binding proteins from target proteins in cell extracts. Similarly, phage display technology has been used to identify target proteins from libraries constructed from cDNAs encoding parts of human proteins. Although various target proteins have already been identified using phage display selection (Jin et al., 2002; Shim et al., 2004; Kuroiwa et al., 2013), several problems remain such as the cell toxicity of proteins for host cells, the effect of codon use bias, and lack of protein expression. To address the difficulties related to both the pull-down method and phage display technology, in vitro selection using cell-free protein synthesis system is thought to be effective. Therefore, innovative approaches to the detection and identification of target proteins have been established using ribosome display technology. For instance, a library consisting of human protein–ribosome–mRNA complexes was constructed and screened based on specific affinity for target molecules. Then, mRNAs recovered from target-binding ribosomal complexes were analyzed using parallel deep sequencing. The approach based on the parallel analysis of translated open reading frames (PLATO) allowed the identification of unknown protein partners of LYN kinase, autoantibodies, and the molecularly targeted drugs gefitinib and dasatinib (Zhu et al., 2013). Additionally, PLATO was used to determine immune targets of antibodies from the serum of patients (Larman et al., 2014) and human immunodeficiency virus type 1 (HIV-1) Tatassociated proteins responsible for the transcription and latency of HIV-1 (Jean et al., 2017). Unfortunately, massive DNA sequencing followed by multistep cloning to identify target proteins requires a considerable length of time and is costly. These procedures should be avoided to expand the versatility of ribosome display identification. Therefore, based on the basic principles of ribosome display selection (Fig. 4.4), a model pool consisting of full-length human proteins was synthesized and displayed on ribosomal complexes. Then, after affinity selections against photocrosslinked compound beads, reversed transcription (RT)-PCR products reflecting the quantity of recovered mRNAs were quantified, and they demonstrated the specific enrichment of target proteins bound to the immune-suppressing drugs, FK506, and cyclosporine A (Wada et al., 2014a). Taken together, these reports suggest that ribosome display is a powerful technology for precisely selecting and identifying target proteins that interact with bioactive compounds or protein partners of interest. Presumably, the approaches for ribosome display identification might be applicable to the comprehensive determination of undesirable proteins that nonspecifically bind to nanoparticle medicines to predict their main and side reactions after administration.

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FIGURE 4.4 A cycle scheme of in vitro identification of target proteins of bioactive compounds using ribosome display technology. (1) DNA constructs coding full-length human proteins (FHPs) are transcribed in vitro for generating a pool of mRNA. (2) The resultant mRNA pool is translated in vitro for constructing a pool of ternary complexes that contain FHP, ribosomes, and mRNA. (3) Specific ternary complexes are selected on the basis of the affinity for photocrosslinked compound beads. (4) mRNAs encoding target protein candidates are recovered through dissociation of the selected complexes. (5) RT-PCR of the recovered mRNAs is carried out for synthesizing DNA constructs again. The synthesized DNA constructs can be used for performing the next selection or identifying target FHPs.

7. CONCLUSIONS AND FUTURE PERSPECTIVES Currently, in vitro display technology is increasingly expected to be used for the selection of peptides, proteins, and antibodies that could be used in the tailored functionalization of nanomaterials/nanoparticles. As described in this chapter, ribosome display technology is a useful method for selecting artificial peptides and structural proteins as ligands with target-binding affinities and specificities. In particular, this technology enables the selection of desirable peptide/protein ligands simply and quickly at the benchtop level. Hence, ribosome display selection could successfully produce diverse target-binding moieties for synthesizing nanomaterials/nanoparticles that are applicable for diagnosis and therapy. However, in considering the clinical applicability of target-binding ligand- modified nanomaterials/nanoparticles, several limitations such as in vivo targeting ability, stability, retention, and potential immunogenicity need to be resolved. Therefore, to overcome these difficulties, it would be necessary to develop chemically improved ligands with protease resistance and metabolic stability, to enhance targetbinding affinities based on structural information. In addition, these developments would enable the performance of theoretical calculations for predicting the molecular and cellular modes of action. In the near feature, a preferable integration of these

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approaches would allow the fabrication of next-generation functionalized nanomaterials/nanoparticles that could contribute to the prevention, diagnosis, and therapy of a wide variety of diseases.

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CHAPTER

5

Engineered Protein Variants for Bioconjugation

Cláudia S.M. Fernandes1,a, Gonçalo D.G. Teixeira1,2,a, Olga Iranzo2, Ana C.A. Roque1 1Universidade

Nova de Lisboa, Caparica, Portugal; Université, Marseille, France

2CNRS, Aix-Marseille

CHAPTER OUTLINE 1. Introduction�� 105 2. Bioconjugation on Natural Amino Acids�� 106 2.1 Lysine and Amine-Targeted Strategies�� 107 2.2 Cysteine/Thiol-Targeted Strategies�� 110 2.3 Tyrosine�� 112 2.4 Other Natural Amino Acids�� 113 3. Bioconjugation on Unnatural Amino Acids�� 114 3.1 Unnatural Amino Acids Used for Bioconjugation and Types of Chemistry Involved�� 115 3.1.1 Ketone/Aldehyde�� 115 3.1.2 Azides�� 117 3.1.3 Alkynes�� 120 3.1.4 Alkenes/Tetrazines�� 121 3.2 Incorporation of Unnatural Amino Acids in Peptides/Proteins�� 122 4. Affinity-Induced Bioconjugation�� 125 5. Conclusions and Future Perspectives�� 127 Acknowledgments�� 127 References�� 128

1. INTRODUCTION Bioconjugation is a tool at the interface between chemistry and biology. It deals with the establishment of covalent bonds between a biomolecule and another molecule or material. This chapter will uniquely focus in cases where the biomolecule is a peptide or a protein; however, bioconjugation can also occur in other biological moieties (e.g., nucleic acids, sugars) and particles (e.g., viral particles). There is a wide range of entities a Authors

contributed equally.

Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00005-7 Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 5 Engineered Protein Variants for Bioconjugation

FIGURE 5.1 Bioconjugation strategies can target natural or unnatural amino acids of a protein. (A) Targeting natural amino acids of a protein allows a simple and low-cost means of bioconjugation. (B) Unnatural amino acids can be introduced within a protein sequence rendering a unique site for bioconjugation. represents molecules that can be bioconjugated with proteins: peptides, proteins, DNA or RNA, labeling agents, drugs, biomaterials (e.g., polyethylene glycol), and matrices (e.g., nanoparticles, patterned surfaces).

that can be conjugated to the peptide/protein. These include other biological molecules (e.g., peptide), small or large synthetic molecules (e.g., drugs or polymers), particulates, or surfaces (e.g., nanoparticles or patterned surfaces). Such bioconjugates present a combination of properties and functions inherent to each of its individual components, and find several applications, namely for diagnostics and therapeutics both in vitro and in vivo or for technological ends (e.g., adsorbents for purification purposes). For bioconjugation to occur, there must be suitable chemical handles at the peptide or protein’s side. It is therefore possible to take advantage of naturally occurring functionalities, namely the chemical groups of amino acid side chains, or to endow new properties through the introduction of artificial chemical groups for further conjugation (Fig. 5.1).

2. BIOCONJUGATION ON NATURAL AMINO ACIDS The most common methodologies for bioconjugation take advantage of the reactivity of the different functional groups inherent to amino acids side chains. Conventional bioconjugation strategies based on natural amino acid (NAA) are generally

2. Bioconjugation on Natural Amino Acids

straightforward and easy to perform as there is no need for specialized techniques (Spicer and Davis, 2014). Amine and thiol groups are good nucleophiles (Steen Redeker et al., 2013), making lysines and cysteines the amino acids most targeted by bioconjugation strategies (Spicer and Davis, 2014; Boutureira et al., 2015). The amine terminal of proteins can too display reactivity (Spicer and Davis, 2014). Also, carboxylic acid groups can be activated to render them reactive toward nucleophiles. Therefore these chemical groups are the most used for nonspecific covalent bioconjugation strategies (Steen Redeker et al., 2013). However, strategies targeting other amino acids side chains have been reported by several authors with their respective advantages and disadvantages. Yet, the array of NAAs and their functional groups is limited and their abundance may be a critical factor when searching for selectivity and precision in a bioconjugation strategy (Spicer and Davis, 2014). Therefore, there is a search for tools that overcome the drawbacks of the more conventional bioconjugation strategies. Chemical modification of proteins and the use of nonnatural functionalities can be used for a higher diversity of approaches to bioconjugation. The chemical modification of an amino acid side chain offers a unique opportunity for the increase of available strategies; these examples, however, will be scarcely discussed within this topic. For a more complete review on the topic, please consult Boutureira et al. (2015) and references within. Examples, advantages, and disadvantages of different bioconjugation strategies will be further discussed.

2.1 LYSINE AND AMINE-TARGETED STRATEGIES Lysine is the most common amino acid targeted in bioconjugation protocols (Spicer and Davis, 2014). Lysines are present in most proteins and can represent 6% of the overall amino acid content (Gauthier and Klok, 2008). Its high prevalence can offer 20 or more sites for attachment of the conjugate molecule (Stephanopoulos and Francis, 2011). The reason for its popularity is the highly nucleophilic primary amine in the side chain (Sletten and Bertozzi, 2009), which is very reactive toward electrophilic reagents without needing to be activated (Brady and Jordaan, 2009; Jonkheijm et al., 2008). The reactivity of lysine is particularly advantageous when selectivity is not required or when multiple conjugations are vital (Spicer and Davis, 2014). This is particularly useful when a higher signal readout or molecule density is desired. There is available a plethora of electrophilic reagents available for primary amine modification, such as activated acids, N-hydroxysuccinimide (NHS) esters, sulfonyl chlorides, vinyl sulfones, iso(thio)cyanates, and squaric acids (Boutureira et al., 2015). A collection of NHS esters and iso(thio)cyanates are commonly available from commercial suppliers (Stephanopoulos and Francis, 2011). A summary of the strategies described below is available in Fig. 5.2A. NHS esters are the most commonly used agents to react with primary amines, leading to the formation of stable peptide bonds (Steen Redeker et al., 2013; Kalkhof and Sinz, 2008). This allows the bioconjugation of proteins via lysine residues and has been used by several authors (e.g., the bioconjugation of ricin to gold surfaces

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FIGURE 5.2 Lysine and amine-targeted bioconjugation strategies. (A) Reactivity of the lysines’ side chain. (B) Reactivity of the N-terminus of the protein. NHS, N-hydroxysuccinimide.

(Wang et al., 2012) or the antibody trastuzumab to the drug tubulysin (Cohen et al., 2014)). NHS esters exhibit half-lives of few hours at physiological pH conditions, with hydrolysis and reactivity increasing with the pH values (Kalkhof and Sinz, 2008). The conjugation of lysines with activated acids is usually achieved using NHS esters as well. However, NHS esters’ reactivity is pH-dependent and cross-reaction with other amino acids has been reported, including serine, tyrosine, arginine (Miller et al., 1997), and cysteine (Antolovic et al., 1995). A systematic study based on mass spectrometry on amino acid reactivities with NHS-activated cross-linkers has been published (Mädler et al., 2009). Preferential conjugation with amines over other nucleophiles can be, in principle, achieved by using more electrophilic molecules, as activated esters, sulfonyl chlorides (Kengne-Momo et al., 2010), or iso(thio)cyanates (Tu et al., 2016).


Vinyl sulfones can be used to target lysines at higher pH values (9.5) (Steen Redeker et al., 2013; Masri and Friedman, 1988); however, it is more commonly used to target the thiol group of cysteines, as discussed below. Vinyl sulfones are stable in aqueous solutions and do not form by-products on reaction (Morales-Sanfrutos et al., 2010). For example, vinyl sulfones derivatives have been used to conjugate lysozyme with glucose via the lysine residue (Morales-Sanfrutos et al., 2010). However, at basic pH conditions it will react preferentially with histidines, as described by Del Castillo et al. (2014). Squaric acids allow an amine-selective bioconjugation strategy. Squaric acid esters undergo a selective and sequential amidation (Steinbach et al., 2014). It is compatible with aqueous media, with a very slow hydrolysis and allows the modification of proteins with water-insoluble amines (Wurm et al., 2013). It has been used, for example, to create bovine serum albumin (BSA)-polymer systems (Steinbach et al., 2014). The disadvantage of these compounds is its tolerance for other chemical groups, as hydroxyl groups present in other amino acid side chains (Wurm et al., 2013). The functional groups of glyoxyl and glutaraldehyde also react with primary amines (Steen Redeker et al., 2013). Glyoxyl-functionalized molecules offer aldehyde groups (Steen Redeker et al., 2013), which react with amine groups to form a Schiff’s base at pH 10.0, which then needs to be reduced to form a stable covalent bond (Mendes et al., 2011). Glutaraldehyde-functionalized molecules perform similar to the previous example, with the advantage of being able to react with terminal amines at neutral pH (pH 7.0–8.5) (Mendes et al., 2011). The high abundance of lysine residues can also be perceived as a disadvantage. On the one hand, it will be impossible to obtain a site- or regioselective conjugation using these amine-based strategies (Spicer and Davis, 2014; Boutureira et al., 2015). This was experimentally observed by Wang et al. (2012), who immobilized ricin on a gold surface using NHS as the electrophile. These authors used an antiricin aptamer-modified atomic force microscopy tip and observed different orientation of the immobilized ricin because of the coupling with different lysine residues. Another problem arising from such heterogeneous labeling can result in the conjugation with a lysine in, or near, the active site of the protein of interest, damaging its function. On the other hand, a high abundance of a certain reactive group will result in a low probability of full reaction, rendering a mixture of many products with a variable number of modifications (Spicer and Davis, 2014; Stephanopoulos and Francis, 2011). The chemical modification of lysine is often the first step for a second modification by a bioorthogonal reaction, recently reviewed in detail (Boutureira et al., 2015). Examples include Edman degradation (modification of the N-terminal with phenyl isothiocyanates), reductive alkylation using cyanoborohydride (Jentoft and Dearborn, 1979), and cyclohexene sulfonamide derivates (Asano et al., 2014). The amine group at the N-terminus of a protein is less reactive when compared with the strong nucleophilic character of lysine’s side chain (Spicer and Davis, 2014). However, many of the amine-directed strategies discussed above can be optimized to target the N-terminus of a protein (Fig. 5.2B). The different pKa of the N-terminus

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can be used to explore a pH-dependent strategy (Steen Redeker et al., 2013) as it has a lower basicity (and therefore more deprotonated groups at neutral pH (Baker et al., 2006)). However, this strategy is usually limited to a minor number of proteins with no or only few lysines in its amino acid sequence (Stephanopoulos and Francis, 2011). Cyanate esters are molecules commonly used at neutral pH values to target the amino terminal (Steen Redeker et al., 2013). They activate foreign hydroxyl groups that will further react with the amine groups (Palsson et al., 2003). This technique has been first described with the coupling of peptides and proteins to polysaccharides (Axén et al., 1967). At pH 6.0 and under alkaline conditions (pH 8.4), NHS esters react preferably with the N-terminus (Kalkhof and Sinz, 2008). A recent successful example includes the bioconjugation with tumor necrosis factor-α using aminereactive NHS chemistry (Sur et al., 2015). Contrary to the strategies described before, the authors reported a site-specific bioconjugation strategy named PRINT, which relies on the combination of a reversible side chain blocking and cleavage by a protease. The N-terminal specificity was confirmed by mass spectral analysis. The N-terminus has also been target for the generation of reactive ketones via transamination mediated by the cofactor pyridoxal-50-phosphate (Gilmore et al., 2006), serine oxidation (Geoghegan and Stroh, 1992), transamination (Witus et al., 2010), aldehyde reaction with serine through oxazolidine (Tam et al., 1999), aldehyde reaction with cysteine through thiazolidine (Tam et al., 1999), or through Pictet–Spengler reaction with N-terminal tryptophan (Li et al., 2000). For these strategies to occur it is vital that the N-terminal is not acylated (Stephanopoulos and Francis, 2011).

2.2 CYSTEINE/THIOL-TARGETED STRATEGIES Cysteine residues contain a thiol group in their side chain. At pH below 9.0 this group is a stronger nucleophile than the primary amine of lysine, which is protonated at this pH (pKa ∼ 8 for cysteine, pKA ∼ 10.9 for lysine) (Steen Redeker et al., 2013). Although the presence of lysines can interfere with the common methodologies for bioconjugation via cysteine, its overall low abundance in proteins (cysteines are the second least abundant amino acid, with a prevalence of 1.36% (Morales-Sanfrutos et al., 2010)), combined with the fine tuning of reaction conditions, can confer a certain degree of site selectivity (Boutureira et al., 2015). If no cysteine is available in the protein of interest, it can be easily inserted by site-directed mutagenesis (Steen Redeker et al., 2013). Cysteine easily forms mixed disulfides (Hemantha et al., 2014) or can be alkylated with suitable electrophiles, such as α-halocarbonyls (e.g., iodoacetamide) (Hemantha et al., 2014) and Michael acceptors (e.g., maleimides or vinyl sulfones) (Stephanopoulos and Francis, 2011; Massa et al., 2014) (Fig. 5.3A). Some of these compounds are commercially available (Stephanopoulos and Francis, 2011). Thiol groups can form disulfide bonds with other disulfide-containing molecules via thiol–disulfide exchange. This reaction is selective for cysteines residues, however, must be performed under oxidative conditions, and the bond formed is not resistant to reducing agents (e.g., mercaptoethanol or dithiothreitol) (Steen Redeker et al., 2013). This can be an advantage if the goal is to release the partner in a reducing


FIGURE 5.3 Cysteine and thiol-targeted bioconjugation strategies. (A) Reactivity of the cysteines’ side chain. (B) Reactivity of the cysteine located at the N-terminus of a protein.

environment (e.g., the release of rhodamine from green fluorescent protein in the mammalian cell cytosol (Moody et al., 2012)). One of the most commonly used chemical groups for the covalent bioconjugation via free thiols are maleimides, which react stoichiometrically with cysteines (Steen Redeker et al., 2013). This coupling reaction is highly efficient and specific, although the protein must be maintained in a reduced form (Steen Redeker et al., 2013). A recent example includes the preparation of an antibody–drug conjugate, where a polyethylene glycol molecule functionalized with the drug doxorubicin and a free maleimide group reacted with the free thiol groups of the Fab portion after treatment with β-mercaptoethylamine (Zhou et al., 2016). Bromo-substituted maleimides have been used to allow the addition on multiple thiols to a single maleimide group (Smith et al., 2010). The major disadvantage of these protocols is that reducing agents are usually used prior to the protocol to ensure the reduced state of the cysteine. However, these must be removed before bioconjugation to avoid competition between the thiol of the reducing agent and the thiol of the protein and performed right after (Steen Redeker et al., 2013). Vinyl sulfone reacts with thiol groups in aqueous solutions and under mild conditions (Masri and Friedman, 1988). These groups are stable for long periods and are not prone to hydrolysis in aqueous solutions (Lopez-Jaramillo et al., 2012).

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The proteins avidin, concanavalin A, lysozyme, and BSA have been successfully conjugated with different vinyl sulfone–functionalized fluorescent molecules (MoralesSanfrutos et al., 2010). The drawback of these strategies is their cross-reaction with amine and hydroxyl groups at more basic pH (Steen Redeker et al., 2013). It is possible to couple an N-terminal cysteine and a C-terminal thioester by “native chemical ligation” (NCL) (Fig. 5.3B) (Stephanopoulos and Francis, 2011), a reaction important for the synthesis of polypeptides (Spicer and Davis, 2014). This will be discussed in Section 3. Cysteines have been chemically modified to allow bioconjugation with a second molecule. The classical radical thiol–ene reaction between thiols and olefins is used to modify proteins under ultraviolet irradiation (Wittrock et al., 2007). A similar reaction with alkynes (thiol–yne reaction) has also been reported (Lo Conte et al., 2011). Cysteines have also been alkylated to generate site-selective sites for modification (Chalker et al., 2009). The thiols of cysteines have been eliminated to yield dehydroalanine groups, which function as acceptors for exogenous thiol (Bernardes et al., 2008) and have been alkylated to serve as efficient substrates for cross-metathesis reactions (Lin et al., 2008). For further examples, please consult Boutureira et al. (2015) and references within. The drawbacks of most of thiol-targeted bioconjugation protocols are related with the cross-reactivity with other side chains, including the primary amine of lysine (Stephanopoulos and Francis, 2011), related with the presence of free cysteine at enzymes’ active site, which may be targeted during the bioconjugation protocol and render the enzyme inactive (Stephanopoulos and Francis, 2011). Furthermore, cysteines that naturally appear in proteins are usually paired with a second cysteine, together forming an oxidized disulphide bridge (Steen Redeker et al., 2013). Thus, its reduction for bioconjugation purposes may disrupt this interaction and have adverse consequences on the stability of the protein of interest (Kanje et al., 2016). This is the case of most antibodies. The stability of the protein can also be disrupted if an additional cysteine is introduced by site-directed mutagenesis (Stephanopoulos and Francis, 2011). To overcome this drawback, Junutula et al. (2008) developed a method to predict suitable conjugation sites, which they used to engineer cysteine substitutions without perturbing the immunoglobulins’ folding or binding. They have used this approach to successfully conjugate an antibody with the drug monomethyl auristatin E.

2.3 TYROSINE Tyrosine residues possess a phenolic hydroxyl group (Seim et al., 2011), which confer unique reactivity. This amino acid is usually found buried within the protein structure because of its nonpolar character (Spicer and Davis, 2014). It is often represented near the active site of proteins (Gauthier and Klok, 2008), therefore its targeting for bioconjugation protocols must be performed with caution (Steen Redeker et al., 2013). Because of this characteristic tyrosine residues can be genetically introduced without destabilizing the protein structure or function, nor the overall charge state (Joshi et al., 2004).


FIGURE 5.4 Examples of other endogenous amino acid side chain–targeted bioconjugation strategies. (A) An example of the tyrosine’s side chain. (B) Reactivity of the glutamate’s and aspartate’s side chain.

Several chemical approaches are adopted when targeting tyrosines. Tyrosines can be targeted using three-component Mannich reactions with aldehyde and aniline reagents (Fig. 5.4A) (Joshi et al., 2004; Romanini and Francis, 2008; McFarland et al., 2008), palladium-catalyzed alkylation (Tilley and Francis, 2006; Antos and Francis, 2006), nickel(II)-catalyzed radical coupling with magnesium monoperoxyphthalate (Kodadek et al., 2005; Meunier et al., 2004), cerium(IV)ammonium nitrate-catalyzed oxidative coupling (174 Spicer and Davis, 2014) and diazonium salts for diazo arylation in the ortho-position (Schlick et al., 2005; Hooker et al., 2004). Barluenga’s reagent can be used to introduce an ortho-iodide on tyrosine; however, side reactions with other amino acids may occur if stoichiometry and pH are not carefully controlled (Espuña et al., 2006). Such reactions are often limited to unique tyrosines expressed at the surface (either naturally or genetically introduced) and careful control of the reactions must be assumed, which limits the use of tyrosines for a more widespread use as target for bioconjugation (Spicer and Davis, 2014).

2.4 OTHER NATURAL AMINO ACIDS The bioconjugation of proteins via the carboxylic side chain of glutamic and aspartic acids is quite common because carboxyl moieties constitute a major fraction of

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protein surface (Rusmini et al., 2007). The carboxylic function can be activated using N,N-dicyclohexyl carbodiimide or (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and reacted with amines to form a peptide bond (Steen Redeker et al., 2013) (Fig. 5.4B). The EDC chemistry is rather unstable in aqueous solution, representing both a disadvantage (careful control of conditions) and an advantage (deactivation of the cross-linker is possible prior to subsequent reactions) (Steen Redeker et al., 2013). Chemical modification of the carboxylic moieties using diazo compounds is also possible, resulting in reversible alkylation (McGrath et al., 2015). Other scarce examples of using other amino acid side chains can be found in the literature. Histidine also has a nucleophilic character, which has been used in classical alkylation/acylation methods (Boutureira et al., 2015). It has successfully been targeted because of its preferential reaction with vinyl sulfones over lysine by Del Castillo et al. (2014). Methionine alkylation of thioether groups allows the reversible introduction of a broad range of functional groups to render stable sulfonium derivates (Kramer and Deming, 2013) (e.g., alkenes, alkynes, carbohydrate probes, boronic acids, azides, and PEG chains—more examples can be found in Boutureira et al. (2015) and references within). Tryptophan is the rarest amino acid in proteins (Steen Redeker et al., 2013), making it an attractive target for site-specific bioconjugation. The chemical modification of the indole side chain group has been performed using in situ generated rhodium carbenoid reagents, resulting in alkylated indoles (Antos and Francis, 2006). The disadvantage of this reaction is that it must be performed under very acidic pH conditions, which may affect the protein’s structure (Steen Redeker et al., 2013). Tryptophans have also been modified with metallocarbenoid reagents (Brady and Jordaan, 2009; Novick and Rozzell, 2005).

3. BIOCONJUGATION ON UNNATURAL AMINO ACIDS The incorporation of unnatural amino acids (UAA) plays an important role in sitespecific covalent bioconjugation. Indeed, UAA can be considered as chemical handles for the introduction of orthogonal functional groups into proteins allowing a precise control of the conjugation site and stoichiometry (Spicer and Davis, 2014). It usually involves the replacement of a NAA by an analog bearing a unique chemical handle (e.g., azide, aldehyde, or ketone), followed by a second bioconjugation reaction that targets this new chemical group. This bioorthogonal group is introduced at a position in the protein that it is strategically chosen to have a minimal influence on the conformation of the target-binding site and therefore without interfering with the protein’s activity (Steen Redeker et al., 2013). The introduction of these reactive functional groups is crucial to dress proteins with new properties, such as enhanced fluorescence, photocrosslinking ability, and photochemical switching behavior (Stephanopoulos and Francis, 2011).

3. Bioconjugation on Unnatural Amino Acids

The different UAA used, their type of chemistry and linkages, as well as their incorporation by solid-phase peptide synthesis (SPPS) or in vivo, are addressed in the following sections.

3.1 UNNATURAL AMINO ACIDS USED FOR BIOCONJUGATION AND TYPES OF CHEMISTRY INVOLVED There are several bioorthogonal chemistries that are used for the labeling and the sitespecific immobilization of proteins (biomolecules) on surfaces. For instance, “click” reactions, which have become significantly popular among academic and industrial research (Steen Redeker et al., 2013). The click chemistry concept was defined as an ideal set of efficient and highly selective chemical reactions in organic chemistry (Steen Redeker et al., 2013; Kolb et al., 2001). The objective was to develop a set of selective and modular “blocks” by joining small units together with heteroatom links (C-X-C) (Kolb et al., 2001). For this, click reactions occur in mild reaction conditions, are insensitive to oxygen and water, have the ability to use water as a reaction medium, and generate stable products under physiological conditions (Steen Redeker et al., 2013). In addition, they have to be modular, wide in scope, stereospecific, give high chemical yields, and generate inoffensive by-products (Kolb et al., 2001). The most common reactions include cycloadditions of unsaturated species, nucleophilic substitutions, nonaldol carbonyl chemistry, and additions to carbon– carbon multiple bonds.

3.1.1 Ketone/Aldehyde Aldehydes and ketones are two very versatile groups in synthetic organic chemistry and were among the first functionalities to be explored as bioorthogonal chemical reporters (Lang and Chin, 2014). The carbonyl group of aldehydes and ketones, considered as mild electrophiles, reacts with amines under acidic conditions (pH 4–6) to form a reversible Schiff base (Lang and Chin, 2014; Jencks, 1959). The use of strong α-effect nucleophiles, such as hydrazines or alkoxyamines, can shift the equilibrium in favor of the hydrazone and oxime-ligated products, respectively (Fig. 5.5A and B) (Lang and Chin, 2014; Sander and Jencks, 1968). This increases the reaction rate becoming more attractive to use in biological systems (Steen Redeker et al., 2013). The slow rate of this type of reactions can be also overcome by using excess of the conjugation partners. However, this may lead to off-target reactivity and also to some problems of toxicity if applied, for instance, for labeling within living cells (Lang and Chin, 2014). Because of the fact that acidic conditions will be difficult to be obtained inside most of the cellular compartments, the ketone/aldehyde reactions with nucleophiles are best suited for in vitro or cell surface labeling (Mahal et al., 1997; Sadamoto et al., 2004; Chen et al., 2005; Zhang et al., 2003). Nonetheless, this disadvantage and the slow kinetics of the ketone/aldehyde–hydrazine/alkoxyamine condensation can be overcome by the use of aniline as a nucleophilic catalyst (Dirksen et al., 2006a,b). Aniline accelerates substantially the reaction at neutral pH by forming a highly reactive protonated electrophile with the carbonyl group, which then undergoes rapid

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FIGURE 5.5 Examples of bioconjugation strategies for unnatural amino acids (UAAs) involving carbonyl groups. (A) Oxime ligation; (B) hydrazone ligation; (C) Pictet–Spengler ligation; (D) thiazolidine ligation.

transamination to form the hydrazone or oxime product (Dirksen et al., 2006a,b). These reactions have been successfully applied for biomolecule labeling on the cell surface and of an intracellular bacterial receptor (Dirksen et al., 2008; Zeng et al., 2009; Rayo et al., 2011). The wide use of the hydrazine and oxime bioconjugation approach relays on the fact that aldehyde and keto groups can be introduced into proteins efficiently, protein functionality and stability has minimal perturbations, the process is simple (i.e., no auxiliary reagents are needed), and the mild conjugation conditions provide homogeneous products in almost quantitative yields. However, the resulting C]N linkages are still susceptible to hydrolysis under physiologically conditions. Therefore, biological applications where long-term stability at physiological temperatures and at low concentrations will be needed will require covalent linkages that are more stable than the oxime bonds. In this regard, Agarwal et al. (2013b) have recently developed a strategy based on the Pictet– Spengler ligation to generate more stable bioconjugates by forming stable CdC bonds (Fig. 5.5C). The first approach explored the bioorthogonal reaction of aldehydes and aminooxy-functionalized indoles to generate an intermediate oxyiminium ion that undergoes intramolecular CdC bond formation with the indole nucleophile to form a hydrolytically stable oxacarboline product. The drawback was still the


acidic conditions needed for optimal performance because this ligation reaction used an aminooxy nucleophile to form the C]N intermediate. The subsequent replacement of the aminooxy nucleophile by an alkylhydrazine nucleophile improved the kinetics of the reaction near to neutral pH (Agarwal et al., 2013a,b). Carbonyl groups can also react with cysteine-1,2-aminothiol groups (e.g., cystamine), producing a stable thiazolidine ring at pH 4–5 (Fig. 5.5D) (King and Wagner, 2014; Shao and Tam, 1995). In addition, Shao et al. showed that thiazolidine ligation was a faster reaction than oxime and hydrazone ligations. The higher reaction rate and the formation of a stable product make thiazolidine ligation an attractive reaction for bioconjugation. Although carbonyl-based ligation reactions are interesting and widely used for their selectivity and their minimal perturbations of the protein functionality and stability, they appear to be limited to mainly cell surface reaction as their acidic conditions or catalysts are only applicable outside living cells.

3.1.2 Azides Azide is considered as one of the most suited groups for bioorthogonal reactions and click chemistry purpose. In contrast to ketone and aldehyde, there are hardly any azides occurring in biological systems. Azides possess highly intrinsic energy but no natural reaction partner (King and Wagner, 2014), have small sizes and neutral overall charge, and finally they are kinetically stable under physiological conditions. The Staudinger ligation (Staudinger and Hauser, 1921) appears to be a good candidate for bioconjugation reactions using azides. In this reaction, azides react with triphenylphosphine reagents containing an electrophilic trap to produce an aza-ylide intermediate that reacts with the electrophilic ester carbonyl group forming a fivemembered ring that undergoes hydrolysis to generate a final stable amide bond (Fig. 5.6A) (Steen Redeker et al., 2013; Staudinger and Hauser, 1921). A new variant of this reaction was described shortly after (King and Wagner, 2014; Saxon and Bertozzi, 2000; Saxon et al., 2000; Nilsson et al., 2000), referred to as “traceless Staudinger ligation,” where the final amide-linked product is liberated from the phosphine oxide moiety. The Staudinger ligation has been employed in a variety of applications. For instance, Raines et al. applied this reaction as cysteine-free alternative to NCL for peptide ligation (Nilsson et al., 2001) and in combination with NCL in the assembly of artificial RNAase A (Nilsson et al., 2005). In addition, it was used in other applications, such as protein immobilization on solid support for in vitro and in vivo imaging (Saxon and Bertozzi, 2000; Prescher et al., 2004), biomolecules labeling in vitro and in vivo (Saxon and Bertozzi, 2000; Prescher et al., 2004; Vocadlo et al., 2003), protein enrichment (Vocadlo et al., 2003) and detection (Charron et al., 2009), as well as (Lemieux et al., 2003) protein modification. Nonetheless, the Staudinger ligation has some drawbacks. Namely, its slow kinetics (second-order rate constant in the low 10−3 M−1s−1 range) (Lin et al., 2005), the oxidation lability of the phosphine compounds (need to use relatively high concentrations of the phosphine reagent), and the potential of phosphines cross-reactivity with disulfides (Lang and Chin, 2014; King and Wagner, 2014).

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FIGURE 5.6 Examples of bioconjugation reactions with azides. (A) Staudinger ligation; (B) coppercatalyzed azide–alkyne cycloaddition; (C) strain-promoted azide–alkyne cycloaddition; (D) [3 + 2] cycloaddition with oxanorbornadienes. UAA, unnatural amino acids.

Azides can react with alkynes in a Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) or 1,3-dipolar [3 + 2] cycloaddition (Fig. 5.6B), one of the most standard examples of click chemistry reactions (Rostovtsev et al., 2002; Tornøe et al., 2002). This reaction presents high interest in biological sciences because of its good selectivity, high yield, and mild reaction conditions (room temperature in various solvents). Additionally, both azides and alkynes are introduced in proteins without affecting the protein structure and function (Steen Redeker et al., 2013). The azide and the alkyne can form very fast a stable 1,2,3-triazole linkage under physiological conditions, in the presence of Cu(I). The mechanism proposed by Sharpless et al. (Rostovtsev et al., 2002) describes firstly the introduction of the terminal alkyne into a copper acetylide and a subsequent attack of the azide (King and Wagner, 2014). Recently, this mechanism has been further refined and a dicopper intermediate has been proposed (Worrell et al., 2013). The major drawbacks of this reaction are the


Cu(I)-dependent side reactions and the Cu(I) cytotoxicity (Baskin et al., 2007; Plass et al., 2011), which have limited its application mainly for labeling in the extracellular space (King and Wagner, 2014). CuAAC has been widely used in many different biological studies, for example, to label phospholipids for their imaging in vivo (Jao et al., 2009) and in vitro (Neef and Schultz, 2009), to virus surface remodeling (Steinmetz et al., 2009), to modify/label proteins in vitro and in vivo (Link and Tirrell, 2003; Ngo and Tirrell, 2011; Liu and Schultz, 2010; Deiters et al., 2003), to label nucleic acids (Weisbrod and Marx, 2008), and affinity-based probe profiling (Speers et al., 2003). Different approaches have been developed to overcome the copper cytotoxicity. Namely, the use of water-soluble ligands for Cu(I) coordination, the use of copperchelating organic azides, and the introduction of ring strain into the alkyne moiety. In the first case, the water-soluble ligands coordinate Cu(I) to form an activated copper catalyst capable of promoting the CuAAC at low micromolar concentrations of metal reducing at the same time the potential toxicity of Cu(I) (Besanceney-Webler et al., 2011; Del Amo and Wang, 2010; Hong et al., 2009; Kennedy et al., 2011). In the second case, the effective Cu(I) concentration is raised at the reaction site by using azide ligands containing an internal copper-chelating moiety (Brotherton et al., 2009; Kuang et al., 2010; Uttamapinant et al., 2012). The final strategy involves the use of alkynes, which have been activated to react with improved kinetics in the absence of catalyst. In this regard, the use of cyclooctyne moieties increases reactivity as a result of the ring-strain release (Steen Redeker et al., 2013; Baskin et al., 2007; Plass et al., 2011). The strain-promoted azide–alkyne cycloaddition (SPAAC) (Fig. 5.6C) has become a powerful tool not only for protein and antibody labeling but also for other applications such as antibody-free Western Blot analysis (Boutureira et al., 2015) because additional reagents or toxic metals that may damage biomolecules are not required. For instance, Bertozzi et al. proved its applicability on modification of purified proteins (Baskin et al., 2007). In further experiments, the reaction was successfully applied in vitro to fibroblast cells (Baskin et al., 2007). Furthermore, SPAAC was used to image tumors in living mice with the help of nanoparticles (Koo et al., 2012) and 18F PET where the fluorine was attached to both azide and cycloalkyne (Jeon et al., 2012). Other fields of application were found in virus modification and DNA labeling (Qiu et al., 2013). However, the complex synthesis of cyclooctynes and the facts that their increased bulkiness and hydrophobicity can affect the protein structure and stability (Kim et al., 2013), and their increased activation can promote side reactions with naturally occurring thiols can be considered as disadvantages. Electron-deficient sulfonyl azides can also react with activated alkenes (oxanorbornadienes or norbornenes) in a metal-free [3 + 2] cycloaddition (Fig. 5.6D), similar to the SPAAC (Alder, 1930; Huisgen et al., 1980). However, the product of azide– alkene cycloadditions is a relatively unstable triazoline unlike the aromatic triazoles formed in the classic click cycloaddition. An oxanorbornadiene that is both strained and electron-deficient was used as a dipolarophile in a reaction with azides (van Berkel et al., 2008). In this case, the strained double bond in oxanorbornadiene reacts with azides to form an intermediate triazoline that spontaneously undergoes a retro

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Diels–Alder reaction, with release of furan, leading to stable 1,2,3- or 1,4,5-triazoles. This reaction was used to selectively bioconjugate an oxanorbornadiene-functionalized protein and an azide-modified cyclic peptide in aqueous buffers. Although oxanorbornadienes are easier to synthesize than their cyclooctyne counterparts, this cycloaddition reaction is quite slow and not entirely chemoselective with respect to other functional groups found in proteins, which may have limited its widespread use (Lang and Chin, 2014; van Berkel et al., 2008).

3.1.3 Alkynes Alkynes can also be used for other cycloaddition reactions besides those already described in the azide-based UAAs section (Fig. 5.7A). For instance, cyclooctynes can react with more reactive 1,3-dipoles than azides, such as nitrones and nitrile oxides, to achieve faster rates (Boutureira et al., 2015; Lang and Chin, 2014). Namely, benzannulated cyclooctynes react with substituted nitrones ∼30 times faster than azides through a strain-promoted alkyne–nitrone cycloaddition (SPANC) reaction yielding N-alkylated isoxazolines (Fig. 5.7B). The utility of SPANC strategy for cellular imaging was demonstrated by labeling epidermal growth factor receptors that were overexpressed on the surface of human breast cancer cells (McKay et al., 2011). Cellular labeling was obtained by the reaction of the cyclic nitrone-modified EGF with a DIBO-biotin and secondary labeling with a streptavidin-coupled fluorophore. Other interesting examples included the modification of the chemokine interleukin-8 (Ning

FIGURE 5.7 Bioconjugation reactions with alkynes. (A) Strain-promoted azide–alkyne cycloaddition; (B) strain-promoted nitrone–alkyne cycloaddition. UAA, unnatural amino acids.


et al., 2010) and the site-selective conjugation of scFvs antibodies to nanoparticles (Colombo et al., 2012). For in vivo applications, the stability of nitrones, prone to hydrolysis, will need to be optimized. A useful variant of the SPAAC reaction is the so-called strain-promoted alkyne–nitrile oxide cycloaddition, which uses nitrile oxides as reactive handles (Kirshenbaum et al., 2002). The drawback of nitrile oxides for strain-promoted cycloaddition is their tendency to dimerize (Feuer, 2007), which can be overcome by producing in situ the nitrixic oxide.

3.1.4 Alkenes/Tetrazines Alkenes are suitable functional groups to carry out bioorthogonal ligations because there are no naturally occurring functional groups; they have good compatibility with water and high selectivity. Besides the reactions already described in previous sections, [4 + 2] Diels–Alder cycloaddition reactions, both the normal and the inverse electron demand types, have been explored as bioconjugation reactions. The normal electron-demand Diels–Alder reactions have been though scarcely used in biological applications because of their sluggish kinetics and side reactions of the primary dienophile maleimide employed (King and Wagner, 2014; Arumugam and Popik, 2011; Nguyen et al., 2007). One decade ago, the inverse electron-demand Diels–Alder reactions (Fig. 5.8) were introduced to the bioorthogonal ligation pool (Blackman et al., 2008; Devaraj et al., 2008). Unlike the classic Diels–Alder reaction where an electron-rich diene reacts with an electron-poor dienophile in the inverse electron-demand reaction, an electron-rich dienophile reacts with an electronpoor azadiene, i.e., the tetrazine. It was demonstrated that highly strained alkenes (electron-rich dienophile), such as transcyclooctene and norbornene, can react rapidly with tetrazines (Fig. 5.7). This approach was successfully employed to functionalize thioredoxin (Blackman et al., 2008) and to label the cell surface of living cells (Devaraj et al., 2008). Since their introduction, these types of cycloaddition reactions have been applied in the bioconjugation of biomolecules with different objectives. Namely, these reactions have been used to modify proteins in vivo both in bacteria and in mammalian cells (Lang et al., 2012), to image chemotherapeutics inside living cells (Devaraj et al., 2010), to label biomarkers on cells with magneto-fluorescent nanoparticles

FIGURE 5.8 Bioconjugation reaction between a transcyclooctene and a tetrazine derivative—inverse electron-demand Diels–Alder cycloaddition. UAA, unnatural amino acids.

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(Haun et al., 2010), and to conjugate quantum dots (Han et al., 2010). Potential biomedical applications include tetrazine-based PET imaging (Zeglis et al., 2011) and 18F-scintigraphic imaging (Reiner et al., 2011). One advantage of this reaction is the possibility of forming fluorogenic compounds by conjugating tetrazines to red fluorophores, (Lang and Chin, 2014; Devaraj et al., 2010). Additionally, isonitriles can react with tetrazines in a [4 + 1] cycloaddition in aqueous media (Boutureira et al., 2015), as demonstrated by the introduction of a fluorophore into the tertiary isonitrile-labeled C2A domain of synaptotagmin-I (Stöckmann et al., 2011).

3.2 INCORPORATION OF UNNATURAL AMINO ACIDS IN PEPTIDES/PROTEINS The site-specific incorporation of UAAs with orthogonal chemical reactivities can be nowadays achieved through different methodologies encompassing chemical or/ and recombinant processes. In this section we give a general overview of different approaches currently used and the references given should be consulted for more detailed descriptions. SPPS represents a straightforward strategy to incorporate UAAs into peptides and proteins. Nowadays is a well stablished and technically easy to perform methodology that can profit of automatization through the use of peptide synthesizers. However, it is limited by the size of the peptide/protein. SPPS becomes a less-efficient strategy for peptides/proteins with more than 50 amino acids mainly due to a decrease in the final yield, the accumulation of truncated sequences arising from incomplete deprotection and/or coupling reactions and the difficulties to purify and characterized the final product because of the closely related impurities (truncated sequences) (Park and Cochran, 2009; Kimmerlin and Seebach, 2005; Kent, 1988, 2003). Although different strategies have been developed to obtain more pure crudes, (e.g., more reactive coupling reagents, acetylation of unreacted amine terminal, longer coupling and deprotection reaction times, and double coupling steps), the accumulation of truncated peptides of similar sizes is still a major disadvantage. To overcome the size limitation, sequential and convergent fragment synthetic protocols have been applied. These strategies rely on the SPPS of fully protected segments and subsequent coupling either in solid (when one of the fragments is still attached to the resin) or in solution (Lloyd-Williams et al., 1997; Chan and White, 2000). An important step forward in this field was the introduction of the NCL (Dawson et al., 1994). This strategy involves the chemoselective reaction of an unprotected peptide containing a C-terminal thioester with another unprotected peptide that has an N-terminal cysteine in aqueous solution. Namely, the thiolate of the N-terminal cysteine residue of one peptide attacks the C-terminal thioester of the second peptide (transthioesterification) and then a rapid intramolecular S → N acyl transfer reaction takes place to produce an amide bond between the two peptides. NCL has allowed construction of proteins up to ∼200 amino acids (Dawson et al., 1994; Park and Cochran, 2009), including also UAAs. The limitation of the initial NCL is the need of an N-terminal cysteine residue and/or C-terminal thioester group


in the peptide segments, which leads to a Cys residue at the ligation site. Alternatives strategies for the NCL have been developed that are mediated by other amino acids overcoming the need of Cys (Kimmerlin and Seebach, 2005; Kent, 2003; LloydWilliams et al., 1997). Another useful strategy is the expressed protein ligation (EPL), a variant of the NCL approach. It is a protein semisynthetic technique that allows segments of recombinant and synthetic origin to be linked by a native peptide bond, readily enabling the addition of unnatural functionalities both in vitro and in vivo (Blaschke et al., 2000; Severinov and Muir, 1998; Muir et al., 1998; Ottesen et al., 2004; Muir, 2003; Hahn and Muir, 2005). As well as NCL, EPL needs the presence of N-terminal cysteine residues and/or C-terminal thioester groups. The recombinant C-terminal thioester fragment can be produced by expressing the protein fused to a mutant intein. After a self-catalyzed intramolecular rearrangement (protein splicing), an intermediate thioester is generated that is ready to react with the N-terminal Cys fragment. The recombinant thiol fragment can be produced by proteolytic processing of a fusion protein. Both fragments can be also prepared chemically using SPPS and equivalent synthetic methodologies and combined with the required recombinant segment to generate the final semisynthetic protein (Fig. 5.9). This strategy allows synthetic

FIGURE 5.9 Expressed protein ligation representative scheme. SPPS, solid-phase peptide synthesis. Adapted from Hahn, M.E., Muir, T.W., 2005. Manipulating proteins with chemistry: a cross-section of chemical biology. Trends Biochem. Sci. 30, 26–34. https://doi.org/10.1016/j.tibs.2004.10.010.

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peptides to be attached to large recombinant proteins and also the simultaneous inclusion of many UAAs. Both strategies, NCL and EPL, are very powerful methodologies that have changed the landscape of the protein sizes attainable by synthesis allowing at the same time the incorporation of UAAs. Lastly, UAAs can also be genetically incorporated into proteins (Lang and Chin, 2014; Kim et al., 2013; Liu et al., 2007). UAAs structurally similar to NAAs are recognized by wild-type or engineered aminoacyl-tRNA synthetase (aaRS) and then incorporated into proteins. These methods rely on the reassignment of codons to a given UAA and use bacterial strains auxotrophic for the structural-related canonical amino acid. Such strains cannot biosynthesize the native amino acids and require uptake from the growth media (UAAs). For instance, this strategy has been commonly employed to incorporate azide- and alkyne-UAAs into methionine auxotrophic Escherichia coli strains (Kiick et al., 2002). Reactive ketone functional group was also introduced by using an engineered phenylalanyl-tRNA synthetase to replace phenylalanine with pacetylphenylalanine (Datta et al., 2002). Although many UAAs have been successfully incorporated using this methodology, the approach has several drawbacks: the global replacement of NAAs with structural analogues is often too toxic to support exponential growth of cells; wild-type aaRSs typically only misacylate close structural analogues of the canonical amino acid; and the reassignment of sense codons results in the global incorporation of the UAA instead of the required site-specific incorporation, therefore limiting its application in vivo. To overcome this, an alternative approach was developed that uses the reassignment of nonsense (stop) codons, such as the amber codon UAG, to incorporate UAAs (Wang et al., 2001). Since then, several groups have reprogrammed the cell’s translational machinery by evolving additional tRNA/tRNA synthetase (tRNA/aaRS) pairs that site-specifically incorporate UAAs in response to the nonsense codons (Liu and Schultz, 2010). This strategy relays on an orthogonal suppressor tRNA/aaRS pair capable of loading the desired UAA to a tRNA specific for the codon while being invisible to any of the endogenous cellular tRNA machinery. This has been mainly achieved by transferring tRNA/aaRS from phylogenetically distant organisms into the target organism (e.g., from archaea to E. coli, from E. coli to yeast, from archaea to mammalian cells, etc.). For example, an engineered tRNATyr/TyrRS pair derived from the archaebacteria Methanococcus jannaschii has been used to incorporate a large number of UAAs into proteins in E. coli in response to the amber codon TAG (Wang et al., 2001). Another commonly used system is the pyrrolysine tRNA/aaRS of Methanosarcina barkeri/mazei (Liu and Schultz, 2010). Recently, orthogonal tRNA/ aaRS pairs derived from leucyl (Wu et al., 2004), tryptophanyl (Zhang et al., 2004), lysyl (Niu et al., 2013), and prolyl (Chatterjee et al., 2012) pairs have been developed. These methods have been employed to incorporate over 150 UAAs, having different structures and reactive functional group, in bacteria, yeast, and mammalian cells (Liu and Schultz, 2010; Kim et al., 2013). Thus, it is possible to effectively reprogram the translational machinery to efficiently incorporate UAAs with novel chemical functionality at a single site of a specific protein within a cell (Kim et al., 2013).

4. Affinity-Induced Bioconjugation

4. AFFINITY-INDUCED BIOCONJUGATION Another challenge arises when one tries to bioconjugate a specific protein that is present in a complex mixture, as living cells or their lysates (Stephanopoulos and Francis, 2011). Targeting the side chain of one amino acid will lead to the targeting of most proteins. To tackle this issue, the bioconjugation strategy must target only the protein of interest, which means it must be site-specific. A different set of conjugation strategies have arisen to tackle this assignment, which rely on the enzymatic modification of a specific set of amino acids, self-labeling tags, or the use of proteins or peptides that naturally display affinity toward a certain target. Biotin-based affinity pairs can also be used as a bioconjugation tool. Although the interaction between biotin and its affinity partners (streptavidin and analogues) is not covalent, it is the interaction with the highest affinity constant known in the scientific community (1015 M) (Miller et al., 1997). Therefore, streptavidin can be conjugated with other biotin-functionalized molecules. For example, Lackey et al. (1999) have studied the hemolytic activity of a streptavidin-biotin polymer system, which responds to pH variation. The conjugation of biotin with a second molecule can be done chemically or can be achieved enzymatically, using biotin ligase (BirA) (Chen and Ting, 2005). BirA biotinylates a lysine residue within a 15-residue target peptide (GLNDIFEAQKIEWHE), commonly known as AviTag (Fairhead and Howarth, 2015). The biotin ligation reaction occurs independently of its surroundings (Steen Redeker et al., 2013). The tolerance of the enzyme isolated from E. coli is limited to biotin isosters; however, BirA from Pyrococcus horikoshii and yeast are able to transfer azido- and alkynyl biotin, allowing further conjugation (Slavoff et al., 2008). Another example of enzymatic modification is based on the protein sortase A (SrtA), a transpeptidase from Staphlococcus aureus (Steen Redeker et al., 2013; Tsukiji and Nagamune, 2009). This protein catalyzes a cell wall sorting reaction that attaches the surface proteins to the cell wall envelope. SrtA recognizes the motif LPXTG (X, any amino acid) and cleaves the protein between threonine and glycine, creating a covalent enzyme intermediate. A covalent bond is formed when the N-terminus of an oligoglycine nucleophile attacks the threonine carbonyl group of the thioester intermediate (Proft, 2009). The major advantages of this strategy are that it is highly specific and it is possible to do this reaction at physiological conditions (Tsukiji and Nagamune, 2009). Recently, Kornberger et al. used this strategy to functionalize an antibody with the toxin gelonin (Kornberger and Skerra, 2014). Jiang et al. (2012) reported on a C-terminal modification and immobilization via SrtA-mediated ligation of recombinant human thrombomodulin. Other examples can be consulted elsewhere (Tsukiji and Nagamune, 2009). The protein farnesyltransferase (PFTase) catalyzes the transfer of a farnesyl isoprenoid group from farnesyl diphosphate to the cysteine in the motif CAAX (Duckworth et al., 2006; Gauchet et al., 2006). PFTase can be used to modify proteins with groups that incorporate a biorthogonal functionality, as alkynes and azides (Rashidian et al., 2012).

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Rashidian et al. (2012) have used this strategy to yield aldehyde-functionalized proteins for further immobilization of a fluorescent label or a polyethylene molecule. Other enzymatic-based bioconjugation strategies include lipoic acid ligase (LplA catalyzes the attachment of an alkyl or aryl azido-lipoic acid derivative to proteins containing the correct peptide substrate) (Fernández-Suárez et al., 2007), phosphopantetheinyl transferases (AcpS or Sfp attaches a coenzyme A-activated phosphopantetheine group to proteins containing the appropriate protein/peptide substrate) (Wang et al., 2016), transglutaminase (TGase allows the attachment of primary amine-containing probes to proteins tagged with polyglutamine sequences) (Lin and Ting, 2006), and formylglycine-generating enzyme (FGE catalyzes the transformation of a cysteine to a formylglycine when recognize the motif CXPXR) (Carrico et al., 2007)—see Sletten and Bertozzi (2009) and references therein for examples. Self-labeling protein tags can be used for bioconjugation strategies where the tag itself is linked directly to the protein of interest and the protein acts as the enzyme that catalyzes the modification (Milles and Lemke, 2013). This facilitates the covalent modification of a protein of interest with a small-molecule substrate (Stephanopoulos and Francis, 2011). This is the example of ReAsH (red florescent) and FlAsH (green fluorescent) labeling systems, where the protein of interest is fused to a tetracysteine motif (CCXXCC), which strongly binds a range of organic arsenicals fluorescent dyes (Sletten and Bertozzi, 2009; Irtegun et al., 2011). This strategy has found application on the production of sensor proteins for imaging, representing an alternative to the conjugation of the protein of interest to the larger and naturally fluorescent green fluorescent protein (Irtegun et al., 2011). It has the advantage of causing minimal disruption of the protein’s amino acid sequence and structure (Adams et al., 2002). Other examples include CLIP tags (Gautier et al., 2008), SNAP tags (Engin et al., 2013; Keppler et al., 2003), HaloTags (Los et al., 2008), and acyl carrier protein domains (Meier et al., 2006). Their mechanism of bioconjugation has been reviewed elsewhere (Steen Redeker et al., 2013). These strategies are very selective and can be used with living cells if the substrate is membrane permeable (Stephanopoulos and Francis, 2011). The introduction of a reactive tag into the protein of interest renders them specific substrates for the enzymatic modification, without labeling other proteins present in the mixture (Stephanopoulos and Francis, 2011). Another type of affinity-induced bioconjugation is based on the natural affinity between the target protein and its natural target. This strategy has been explored by Alves et al. (2013), where antibodies were labeled with biotin, fluorescent molecules, small peptides, and drugs. For this, these molecules were conjugated with indole3-butyric acid, which displays natural affinity toward the nucleotide binding site present in the Fab region. Other natural affinity partners of antibodies have also been explored, as a photoactivable Z domain from protein A (Konrad et al., 2011; Yu et al., 2013) and the C2 domain from protein G (Kanje et al., 2016). These approaches can also be combined with UAAs for further bioconjugation possibilities (Kanje and Hober, 2015).

5. Conclusions and Future Perspectives

5. CONCLUSIONS AND FUTURE PERSPECTIVES Bioconjugation on proteins and peptides is becoming an increasingly relevant area because of the rise of advanced medicinal products, as antibody drug conjugates or biobetters (e.g., PEGylated versions of biopharmaceuticals) and the need for better diagnostic tools, as targeted imaging agents. Proteins usually contain several copies of a specific amino acid, therefore bioconjugation strategies targeting NAAs typically result in multiple conjugation sites (Steen Redeker et al., 2013). In addition, it becomes difficult to control the level and/ or position of bioconjugation, resulting in random protein labeling (Steen Redeker et al., 2013). These strategies are useful for a nonspecific covalent bioconjugation. Nonetheless, if site selectivity is indeed required, site-directed gene mutagenesis and recombinant protein expression are powerful tools that can be used to introduce unique reactive amino acid groups (Spicer and Davis, 2014; Stephanopoulos and Francis, 2011). It is also possible to create amino acid-specific depletion mutants (all but one amino acid of a specific type are removed); however, this strategy can have adverse effects on a protein’s structure and function (Steen Redeker et al., 2013). NAAs can also be targeted through transition metal-mediated approaches (Boutureira et al., 2015), through the use of iridium, palladium, rhodium, and gold complexes. Bioconjugation on UAAs is an approach that overcomes most disadvantages of NAAs, mainly site-specific conjugation and control over the extent of conjugation. However, it also adds other problems and promotes new challenges. In particular, introduction of UAAs through SPSS is often limited to relatively short peptides and protein sequences, which do not require complex posttranslational modifications. Despite the enormous advances made on the biological introduction of UAAs onto proteins, the methods are still to be developed for large-scale implementation and for proteins produced in mammalian systems. In view of the complexity of biological systems, methods that can be employed in cells or that take advantage of the natural selectivity of binding partners toward target peptides and proteins are also promising approaches that with due tuning can represent a viable and mild option to sitespecifically modify peptides and proteins.

ACKNOWLEDGMENTS This work was supported by the European Research Council through the grant reference SCENT-ERC-2014-STG-639123 (2015–20) and by the Unidade de Ciências Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and cofinanced by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The authors thank Fundação para a Ciência e a Tecnologia, Portugal, for the research fellowship PD/BD/105871/2014 for C. S. F. OI and ACAR acknowledge the support from CNRS and Fundação para a Ciência e a Tecnologia through the Programme International de Coopération Scientifique—Project PICS-147340.

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CHAPTER

6

Bioengineered Approaches for Site Orientation of PeptideBased Ligands of Nanomaterials

Svetlana Avvakumova1, Miriam Colombo1, Elisabetta Galbiati1, Serena Mazzucchelli2, Rany Rotem1, Davide Prosperi1 1Università

di Milano-Bicocca, Milano, Italy; 2Università di Milano, Milano, Italy

CHAPTER OUTLINE 1. Introduction�� 139 2. Control of Peptide Structure and Functionality�� 141 2.1 Nonspecific Adsorption Versus Covalent Conjugation�� 141 2.2 Biotechnological Approaches�� 142 2.3 Chemical Ligation Strategies�� 144 2.4 Nonclassical Conjugation Strategies�� 147 3. Impact of Bond Strength and Linker Length on Bioconjugation�� 148 3.1 Streptavidin–Biotin�� 149 3.2 His6-Tag—Metal Coordination�� 149 3.3 Thiol-Disulfide/Thioether/Metal Coordination: The Native Chemical Ligation�� 151 3.4 Azide–Alkene/Alkyne Cycloaddition�� 151 3.5 Hydrazine/Amine–Aldehyde Ligation�� 152 3.6 Staudinger Ligation�� 152 4. Impact of Ligand Density on the Targeting Efficiency of Nanoconjugates�� 153 5. Protein Corona Effect and Minimization of Nonspecific Interactions�� 158 6. Conclusion and Future Perspectives�� 160 References�� 161

1. INTRODUCTION The growing impact of nanotechnology on biomedical science in view of a potential routine clinical application of nanomaterials—with the aim to improve the available diagnostic and therapeutic approaches—has raised numerous problems, which often require the joint efforts of interdisciplinary research groups to be adequately faced. Thus a need for new technologies to produce bioactive organic–inorganic hybrid nanomaterials with control on physical and biochemical properties is now Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00006-9 Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 6 Bioengineered Approaches for Site Orientation

widespread in fields such as material science, biophysics, biotechnology, molecular biology, pharmacology, and molecular medicine (Nano on reflection, 2016). The design of ideal targeted nanoparticles needs careful optimization of fundamental features including uniform size and shape, surface charge, optical and magnetic properties, and efficient functionalization with suitable homing ligands to improve the signal amplification and target selectivity toward malignant cells (Blanco et al., 2015). One of the greatest challenges in designing targeted nanoparticles functionalized with homing peptides and proteins to optimize molecular recognition resides in the possibility to finely control the ligand orientation on the nanoparticle surface (Avvakumova et al., 2014). So far, three main approaches have been followed to reach this goal: (1) native proteins having domains with high affinity for small peptides can be captured via ligand immobilization on surface-modified nanoparticles; (2) proteins containing affinity tags recognized by small molecules or complexes commonly utilized for protein purification can be genetically modified to introduce a recognition sequence specific for protein immobilization; and (3) site-specific conjugation can occur via chemoselective ligation exploiting genetically engineered residues on the polypeptide sequence. In this chapter, we highlight the fundamental criteria required for an optimal biofunctionalization of nanoparticles and provide an overview of recent approaches allowing for the efficient and orientation-controlled immobilization of complex molecules, including peptides and proteins, for biomedical application. The reader will find that there are a few elements that cooperate with ligand orientation in affecting the biological activity of nanoconjugates (Sapsford et al., 2013). As a matter of fact, the impact of the control of ligand orientation on the nanoparticle surface in determining the actual affinity of the immobilized biomolecules toward the biological target can be appreciated individually, but it is even strongly influenced by the contribution of further correlated items, including multivalency due to ligand density and organization; unfolding and misfolding processes that could potentially occur on binding of ligands to the nanoparticle; thermodynamic and kinetic features related to the interaction of the ligand with the surface of nanoparticles; and the importance of the nanoparticle interaction with the environment, namely the formation of the so-called “protein corona” in biological fluids. The intricate network of effects due to interdependence of these five issues governs the biological activity of the synthesized bionanoconjugates and, as a consequence, its potential utility for biomedical application. Although at present it remains challenging to accurately predict such a biological activity when designing targeted nanoparticles, a control on the abovementioned “magic five” contributions is expected to improve the quality standard of the next-generation nanomedicines. Besides illustrating the strategies that have been developed to achieve optimal control on ligand positioning on the surface of nanoparticles, here we suggest that of all these features need to be carefully taken into account when assessing the biological effects of synthetic nanoparticles designed for biomedical purposes.

2. Control of Peptide Structure and Functionality

2. CONTROL OF PEPTIDE STRUCTURE AND FUNCTIONALITY The choice of the appropriate strategy to achieve an optimal protein or peptide conjugation to nanomaterials is crucial in view of their functional efficacy because the method adopted for bioconjugation strongly influences the bioactivity of nanoconjugates, affecting the orientation, accessibility, and stability of the ligands on their surface (Medintz, 2006). For example, the impact of proper positioning of targeting ligands on the affinity toward specific receptors was demonstrated using recombinant scFv antibody molecules, in which two distinct ligation sites were introduced in the peptide sequence. The scFv molecule was immobilized on identical colloidal nanoparticles exploiting these two different anchors, resulting in two groups of nanoparticles having the same physical properties but different orientation of the scFv. The resulting nanoconjugates exhibited very different targeting efficiency toward the relevant molecular receptor (Mazzucchelli et al., 2013b). Thereby, the development of a biologically active nanoconjugate requires mild conjugation conditions to preserve the peptide structure and an accurate control of the molecular organization on the nanoparticle surface. Indeed, unsuitable ligand positioning and limited accessibility could strongly hamper the correct ligand functionality (Avvakumova et al., 2014). In this scenario, three general approaches could be considered (1) the biomolecule immobilization by adsorption; (2) the nonspecific covalent conjugation; and (3) the site-directed controlled conjugation.

2.1 NONSPECIFIC ADSORPTION VERSUS COVALENT CONJUGATION In principle, the first strategy could be applicable to a broad range of biomolecules and seems to take place under mild condition, but it suffers from very poor control of ligand orientation and accessibility. In addition, protein adsorption seldom affords sufficient retention of the molecular 3D structure, which is essential prerequisite to preserve the nanoconjugate functionality (Occhipinti et al., 2011). Actually, the interactions involved in protein adsorption processes are the same required for the structural stabilization of the biomolecule with the result that adsorption is generally associated to instability, partial unfolding, or even protein denaturation (Avvakumova et al., 2014; Wong et al., 2009). In the second approach, protein immobilization occurs; exploiting functional groups previously introduced on the surface of nanoparticles. This strategy could be applied to a lot of nanomaterials, and the conjugation reaction is generally assisted by coupling agents, including 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or glutaraldehyde. In contrast to direct adsorption, the coupling-mediated immobilization assures the stable association between protein and nanomaterials and prevents protein instability because of the loss of stabilizing interactions between amino acids. In this approach, the native protein conformation is normally maintained. In an Fourier-transform infrared spectroscopy (FTIR) study conducted on antibodyconjugated iron oxide nanoparticles, the effect of the immobilization strategy on protein folding was assessed comparing direct adsorption of human immunoglobulins G

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(IgG) molecules on colloidal nanoparticles with covalent coupling via a short polyethylene glycol (PEG) linker. Evidence was provided that direct adsorption results in a time-dependent loss of secondary structure while covalent coupling was much more stable (Occhipinti et al., 2011). However, this strategy lacks specificity and is not suitable to allow the precise control of ligand orientation. Therefore, also this approach does not have all the necessary features to be effectively applied to nanoconjugates (Wong et al., 2009).

2.2 BIOTECHNOLOGICAL APPROACHES In contrast, the site-directed controlled conjugation approaches are able to solve a number of drawbacks related to the nonspecific nature of the other two approaches. In addition, they maximize protein stability because they are based on biological reactions, which take place under highly selective and mild conditions (Avvakumova et al., 2014). These strategies could be split into two distinct classes, namely covalent and noncovalent methods, characterized by a few common features (Fig. 6.1 and Table 6.1). First, the attachment sequence is specific for each protein and it is inserted ad hoc in a well-defined place, sometimes by engineering with DNA recombinant technology (Avvakumova et al., 2014). This has the advantage to avoid interference with protein stability and to allow full control of spatial orientation of the biomolecule, to make all ligands virtually active. In addition, the conjugation reaction

FIGURE 6.1 Schematic representation of (A) noncovalent and (B) covalent site-directed controlled conjugation approaches.

Table 6.1 Advantages and Disadvantages of Covalent and Noncovalent Conjugation Strategies Noncovalent

Advantages

Disadvantages

References

• Polyhistidine/Ni-NTA • Cobalt/Ni-NTA • Glutathione S transferase/glutathione • Maltose-binding protein/ maltose Streptavidin/biotin

Peptide/protein could easy modified with this tag using recombinant technique

Low affinity between target peptide and molecules; not suitable for in vivo studies

Yang et al. (2009), Xu et al. (2004), Pan et al. (2011) and Zhou et al. (2012)

Too big molecule

Protein A or protein G or their variants

Highly stable interaction; it could be suitable for in vivo studies Stable and highly specific bond

Thiol chemistry

Fast and easy to assess

Copper-catalyzed azide– alkyne cycloaddition (“click” reaction)

Fast, high yielded without temperature control; bioproduct free

Bioclick reaction

Robust and mild approach; modification by recombinant technique Catalysts are not required Mild conditions; stable; site-selective

Staudinger reaction Nonclassical covalent

Enzyme-mediated ligation

Li et al. (2010), Härmä et al. (2000) and Choi et al. (2008) Potentially immunogenic; exploit- Avvakumova et al. (2014), able only with antibody Mazzucchelli et al. (2010), Lim et al. (2009), Corsi et al. (2011) and Colombo et al. (2012) Potentially undesirable interacAvvakumova et al. (2014), tions with other peptide/protein Giovanelli et al. (2012) and Cys Tasso et al. (2015) Instability and toxicity of reagents Jewett and Bertozzi required (2010), Changa et al. (2010), Li et al. (2016), Brantley et al. (2011) and Ning et al. (2010) Difficult to quantify the immobiTang and Becker (2014) lized proteins and New and Brechbiel (2009) Slow and does not run to full conversion Difficult to quantify the immobilized proteins

143

Soellner et al. (2003) and Watzke et al. (2006) Ta et al. (2012), Walper et al. (2015), Parthasarathy et al. (2007), Galbiati et al. (2015), Leung et al. (2012), Colombo et al. (2012), Mazzucchelli et al. (2013) and Aslan et al. (2004)


Classical covalent

Conjugation Strategy

144


generally takes place under physiological conditions allowing optimal ligand stability. Among noncovalent methods of site-directed controlled conjugation (Fig. 6.1A), there are those approaches borrowed from classical biochemistry and cellular biology that take advantage of specific interactions between affinity tags and their natural ligands, including polyhistidine/Ni-nitriloacetic acid (NTA) (Medintz, 2006; Yang et al., 2009; Xu et al., 2004), Cobalt-NTA (Occhipinti et al., 2011), glutathione S-transferase/glutathione (Pan et al., 2011), and maltose-binding protein/maltose (Zhou et al., 2012). Although these approaches are reported to maintain the ligand bioactivity allowing the precise control of ligand orientation and stability, there is still an important drawback relating to the nature of the linkage between the biomolecule and the nanomaterial. This bond usually consists in an affinity interaction that could be broken using an excess of free competitor and makes this nanoconjugates generally unsuitable for in vivo applications. Exception to the rule is represented by the streptavidin/biotin pair: although the interaction between streptavidin and the biotin ligand is not covalent, this bond is strong enough (Kd = 10−15 M) to be considered as long-term stable also in vivo (Li et al., 2010; Härmä et al., 2000). However, biotin-streptavidin technology is broadly exploited for the development of in vitro diagnostic assays as well, while poor in vivo application is mainly due to the large size of streptavidin, which negatively influences the nanoconjugate dimensions and thus the in vivo performance (Li et al., 2010; Härmä et al., 2000; Choi et al., 2008). Alternative noncovalent conjugation strategies could be used in particular to drive nanomaterial conjugation with antibodies, exploiting short fragments or fulllength Staphylococcus aureus protein A or G as universal tool for the well-oriented immobilization of IgGs (Avvakumova et al., 2014). These proteins bind in a strong (Kd = 10−9 M), yet reversible, manner to the Fc portion of IgGs leading to highly stable bond that could be broken only under harsh conditions, including extreme pHs and/or denaturing agents (Mazzucchelli et al., 2010). For example, protein G was produced in fusion with cell-penetrating peptides, such as TAT, to facilitate the intracellular penetration of IgG–nanoparticle conjugates (Lim et al., 2009). A singledomain protein A was engineered with a cysteine tripod at the C-terminus to mediate a tight anchor on thiol-reactive nanoparticles (Mazzucchelli et al., 2010; Corsi et al., 2011), and it was further exploited as a biotemplate for the in situ generation of sizecontrolled gold nanoparticles (Colombo et al., 2012b). This conjugation strategy was explored for the conjugation of superparamagnetic iron oxide nanoparticles with the anti-HER2 antibody trastuzumab, showing favorable tumor-targeting efficiency and good biodistribution profile in vivo (Corsi et al., 2011).

2.3 CHEMICAL LIGATION STRATEGIES On the other hand, covalent conjugation methods seem to exhibit higher control on ligand bioactivity. Some of these methods exploit specific chemical reactions, including thiol chemistry, “click” reaction and relative bioorthogonal variants, and the Staudinger ligation (Fig. 6.1B; Table 6.1) (Avvakumova et al., 2014; Tang and Becker, 2014; New and Brechbiel, 2009). All of these strategies make use of a reactive


residue naturally present in the polypeptide sequence of the ligand or intentionally inserted ad hoc by site-directed mutagenesis. The prerequisite to maintain ligand and hence nanoconjugate functionality is that the inserted residue does not affect the ligand folding, especially in proximity of the active site of the molecule. Moreover, the homogenous orientation of the ligand, which is a fundamental requirement for nanoconjugate bioactivity, should be preserved by the presence of only one reactive residue in the protein primary sequence (Fiandra et al., 2013; Baniukevic et al., 2013; Polito et al., 2008a; Jewett and Bertozzi, 2010; Changa et al., 2010; Haun et al., 2010; Saxon and Bertozzi, 2000; Parkhouse, 2008). In addition, the immobilization strategy used should be fast, specific, and high yielding. The use of multidentate dithiol/zwitterion polymers is an example to develop a universal and tunable platform for the oriented immobilization of whole antibodies to nanoparticles (Giovanelli et al., 2012). The approach consists of a controlled substitution of some thiol groups in the polymer chain to enable the covalent binding of an intermediate protein A layer that subsequently supports the oriented antibody immobilization (Tasso et al., 2015). In this case, protein A is immobilized taking advantage of sulfhydryl–maleimide chemistry, where surface-accessible lysine residues in the protein are first modified with the heterobifunctional (amine-to-sulfhydryl) linker SMCC thereby introducing maleimide groups on the protein, and then reacted with sulfhydryl groups of the polymer. This approach has shown several advantages compared to other covalent conjugation techniques: (1) no need for ligand activation (e.g., EDC/NHS chemistry); (2) no undesired cross-linking between nanoparticles and proteins (e.g., bis-NHS linkers); (3) no side reactions; (4) all modifications take place on the protein intermediate layer; and, finally, (5) modification is orthogonal, which excludes linker-induced protein cross-linking. Another chemical method involved in antibody-oriented conjugation takes advantage of the use of the nucleotide binding site (NBS), a highly conserved binding pocket located in the region between the variable light (VL) and the variable heavy (VH) domain of the antibody isotypes. In a recent example, a UV photocrosslinking reaction between antibody NBS and a photocrosslinkable NBS ligand conjugated to inorganic surfaces was successfully achieved (Alves et al., 2012). Another approach for site-specific conjugation of proteins relies on an inteinmediated expressed protein ligation (EPL) that allows azide- and fluorescently labeled peptides to be efficiently ligated to the carboxyl terminus of recombinant proteins (Elias et al., 2010). EPL refers to a native chemical ligation between a recombinant protein with a C-terminal thioester and a second agent bearing an N-terminal cysteine. The C-terminal thioester can be readily introduced onto any recombinant protein (i.e., the targeting ligand) through the use of auto-processing proteins called inteins. Specifically, when an intein is cloned downstream of the targeting ligand, thiols (using 2-mercaptoethanesulfonic acid, MESNA) can be used to induce the site-specific cleavage of the intein, resulting in the formation of a reactive thioester. The thioester will then react with any agent via the N-terminal cysteine. Thus, combining EPL and click chemistry conjugations allows to produce a highly efficient,

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site-specific conjugation strategy leading to expanded application in the functionalization of nanoparticle carriers (Hui et al., 2014). Copper-catalyzed azide–alkyne cycloaddition is referred to as a “click” reaction that is extensively used for nanoparticle functionalization with several benefits, including fast reactivity, high product yielding, no need of temperature control, and avoidance of by-products formation (Li et al., 2016). This reaction consists in the formation of a triazole ring, which can be reversed only under ultrasounds (Brantley et al., 2011). Also in this case, to make the conjugation oriented, it is possible to genetically modify the involved peptides with the insertion of nonnatural amino acids bearing an alkyne or an azido group. However, this method presents some limitations, including instability and toxicity of the required Cu(I) catalyst, which can be overcome by introducing strained alkynes (Jewett and Bertozzi, 2010), e.g., cyclooctyne derivatives. “Click” reaction exploiting such strained alkynes to promote a catalyst-free cycloaddition is usually referred to as “strain-promoted alkyne–azide cycloaddition” (SPAAC) and works properly even in living animals (Changa et al., 2010). For this reason, this class of strained cycloaddition reactions belongs to the so-called bioorthogonal reactions. As an alternative to the azido group, a localized reactive site could be generated by mild oxidation of an N-terminal serine residue that was genetically encoded in the peptide sequence, giving rise to a transient nitrone group that is even more reactive than the azido functionality (Ning et al., 2010). This variant of SPAAC reaction, termed strain-promoted alkyne–nitrone cycloaddition, was successfully applied to the site-directed conjugation of scFv antibodies to superparamagnetic iron oxide nanoparticles for the targeting of breast cancer cells (Colombo et al., 2012b). Oxime ligation is a highly efficient click-type bioorthogonal condensation reaction between an oxyamine and a carbonyl group of an aldehyde or a ketone. The formed oxime is highly stable toward hydrolysis (Kd ≤ 10−8 M) compared with the corresponding imines or hydrazones. Thus, oxime ligation is compatible with most biomolecule functionalities including amines and ideally suited for application in living system (Tang et al., 2015). A site-specific modification of viral nanoparticles by introducing a small aldehyde tag onto the virus capsid was exploited for covalent attachment of hydrazide-functionalized molecules, including antibodies and peptides, in a site-specific manner. A short peptide containing 13-amino acid sequence was inserted into the viral capsid. On expression, the encoded cysteine was modified to an aldehyde tag, which in turn was covalently conjugated with hydrazide- or hydroxylamine-functionalized molecules (Liu et al., 2012). Staudinger ligation is one of the most efficacious methods known for the sitespecific, uniform, and covalent immobilization of peptides and proteins. It consists in the formation of an amide bond between an arylphosphine and an azido group. This reaction occurs in high yields at room temperature in aqueous or wet organic solvents, and it allows to preserve the biological activity of the protein (Soellner et al., 2003; Watzke et al., 2006).


2.4 NONCLASSICAL CONJUGATION STRATEGIES There are a lot of nonclassical covalent protein conjugation strategies, which have been explored to drive nanomaterials functionalization with biologically active molecules keeping control on the chemical features of the nanoconjugates. These approaches are characterized by the exploitation of the natural aptitude of an enzyme to promote the covalent binding of specific molecules and have been summarized in Fig. 6.2 and Table 6.1. This process usually occurs under mild conditions and in a stable and site-directed manner taking advantage of the highmolecular selectivity of the enzyme-binding site (Ta et al., 2012; Walper et al., 2015). An interesting example is represented by the S. aureus sortase, which was used to catalyze the attachment of a tagged model protein substrate to polystyrene beads chemically modified with the Sortase A ligand Gly3 (Fig. 6.2A) (Parthasarathy et al., 2007). A general strategy involves the insertion of the functional sequence of a selected enzyme at the N or C-terminal position of the targeting ligand by DNA engineering. Hence, within this “bimodular” approach, the enzyme sequence could be expressed as a fusion protein with the bioactive molecule which, in turn, behaves like a “targeting module” (Galbiati et al., 2015; Mazzucchelli et al., 2013a; Colombo et al., 2012a). As a result, the enzyme sequence works as a “capture module” to mediate the formation of a site-directed, specific, oriented, and covalent bond with the

FIGURE 6.2 Schematic representation of nonclassical covalent conjugation approaches: (A) Sortasemediated conjugation and (B) Capture module–mediated conjugation strategies.

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nanomaterial, whereas the other fusion partner (the targeting module) affords the biological activity to the nanoconjugate (Fig. 6.2B). To minimize the overall hindrance of the fusion ligand, the capture module should be selected among naturally available small enzymes or even engineered fragments containing the enzyme active site. The capture module is designed to specifically bind a little molecule previously immobilized on the nanoparticle. This molecule, in turn, could be a suicide inhibitor or a physiological substrate, which interacts with the enzyme active site in a covalent and irreversible manner (Galbiati et al., 2015). Examples of small enzymes (i.e., ≤30 kDa) that have been explored as capture module are the O6-alkylguanine transferase (Leung et al., 2012; Colombo et al., 2012a), the haloalkane dehalogenase (Mazzucchelli et al., 2013a), and the serine protease cutinase (Galbiati et al., 2015). In these systems, the orientation of the bioactive molecule is uniform and dependent on the 3D structure of the fusion protein (Galbiati et al., 2015; Leung et al., 2012; Colombo et al., 2012a; Mazzucchelli et al., 2013a). They display advantages related to the size of the ligand to be immobilized on the nanomaterial surface; the rate and the conditions of binding; the specificity and irreversibility of the bond; and the monovalent nature of both capture module and ligand, which eludes the cross-linking effects usually occurring with biotin/streptavidin (Aslan et al., 2004). Moreover, nonclassical strategies may overcome the formation of nonspecific linkages, which take place fatally in classical covalent conjugation methods. Additional benefits have been reported in the case of short peptide measuring between 5 and 30 amino acids (Mazzucchelli et al., 2013a). Here, the presence of the capture module introduces a sort of protein “spacer” between the peptide and the nanoparticle, which improves the ligand accessibility preventing undesired interactions (Mazzucchelli et al., 2013a).

3. IMPACT OF BOND STRENGTH AND LINKER LENGTH ON BIOCONJUGATION It is a common experience that when we stick two things together, we expect them to stay together. That is most often the case in bio–nanoconjugation. The linker is expected to produce a stable bond and not interfere in any other way. In some cases the linker role is more demanding, for example, the use of “smart” linkers, able to release the cargo on specific cues (Clark and Davis, 2015; Koo et al., 2008) or creating the bond in vivo (Saxon and Bertozzi, 2000; Boeneman et al., 2010). Another important issue to be considered is the linker length and volume. Length might be important in several applications, such as fluorescence resonance energy transfer (Avvakumova et al., 2014) and where access to surface ligands needs to be modulated (Ghaghada et al., 2005). A bulky linker might have reduced flexibility and have a substantial impact on surface hydrophobicity, hydrodynamic diameter, and electrostatic charge of the nanoconjugate. Different chemical conjugation methods differ regarding these parameters and need to match the envisioned holistic construct/application. For example, a drug

3. Impact of Bond Strength and Linker Length on Bioconjugation

molecule bound to a nanocarrier needs to be released at some point, thus requiring a reversible bond and excluding other binding techniques. Another example is NP-bound ligands for cell receptor binding and activation, this interaction requires high accessibility of the ligand and might be hindered by a hydrophobic linker (Stefanick et al., 2013). Lastly, some methods require premodification of the ligand (e.g., through its genetic sequence), whereas others might be applicable using commercially available proteins. We will try to illustrate the impact of this concept taking some examples of general utility or selected from the strategies listed in the paragraph above. These examples are summarized in Table 6.2.

3.1 STREPTAVIDIN–BIOTIN Streptavidin is broadly utilized as linker in biotechnology because the conjugation scheme using the wild-type (WT) streptavidin provides a very stable bond under well-preserved biological conditions (Kd ∼ 10−15, pH 3–13) (Weber et al., 1989; Katz, 1997). Streptavidin is a 60 KDa tetrameric protein with a globular subunit organization of 5 nm in size (Kuzuya et al., 2008)—a large and bulky linker compared to alternative techniques. Modified versions of this conjugation scheme exist, e.g., the biotin analog 2-iminobiotin (Katz, 1997), which shows a weaker affinity and a pH dependence (5 × 10−8 M at pH 9; 1 × 10−4 M at pH 3) or the monomeric streptavidin mutant that also shows a weaker bond (Kd = 38 nM) (Lim et al., 2011). Some techniques allow production of a tetrameric protein with only one functional binding site that exhibits similar biotin affinity as the WT protein (Howarth et al., 2006).

3.2 HIS6-TAG—METAL COORDINATION This technique is especially useful as many commercially available recombinant proteins have the histidine tag already incorporated in them. The His6 tag has a dissociation constant Kd = 10−13 M at pH 8 to NTA-immobilized Ni2+ ion. This affinity can be varied by the ligand immobilizing the metal ion and the metal ion itself. The interaction comes from the HIS imidazole coordinative binding to the metal ion, and thus a function of pH. The pKa of the histidine imidazole is around 6.1 (Bornhorst and Falke, 2000; Sundberg and Martin, 1974), which makes this bond suitable for many biological relevant conditions and microenvironments, although not for all. The utility of this strategy for nanoconjugation was demonstrated using both aqueous transition metal ions (e.g., Ni2+-NTA) and binding of solid phases, such as ZnS nanoparticles with a 10−9 M dissociation constant (Sapsford et al., 2007). The linker length would be a function of the His6-tag position in the amino acid sequence of the polypeptide and of the 3D folding of the protein. In addition, the immobilization method of the metal ion can play a role. However, a minimum length of a few nanometers should be expected (Boeneman et al., 2010; Bornhorst and Falke, 2000; Guignet et al., 2004).

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Model Technique

Linker Characteristics

Streptavidin–biotin

• Kd ∼ 10−15, pH 3–13 • 5 nm in diameter

His tag—metal coordination

• Kd = 10−13M to NTA-Ni at pH 8 • Kd = 10−9 M to ZnS NPs • Length of a few nanometers • Stable, as function of redox potential • Subnanometer linkers • Stable • Subnanometer • Keq = 10−6 M, lifetime of minutes • Subnanometer • Stable • Subnanometer

Thiol-disulfide Thiol-thioether Thiol-metal coordination Native chemical ligation

Azide–alkene/alkyne cycloaddition Hydrazine/amine– aldehyde ligation

Staudinger ligation

Applicability to Oriented Conjugation

Notable Modifications/ Alternatives

• Streptavidin protein fusion (Albarran et al., 2005) • Biotin acceptor peptide (Predonzani et al., 2008) • Biotinylation of synthetic biopolymers (Geahlen et al., 1992) • Protein-His6 fusion

• 2-iminobiotin, monomeric streptavidin, tetrameric monovalent streptavidin • HALO tag, SNAP tag

• Cysteines • Cysteines • Cysteines • N-terminal cysteine

• Stable • N-terminal serine, glycine • Few nanometers, depending on catalysis method • Kd = 10−4−10−6, lifetime of days • N-terminal serine, glycine • Subnanometers • Modification of synthetic biopolymers

• Stable • Subnanometer

• Different metals and metalcoordinating ligands. • FLASH + Cys4 tag

• Metabolic labeling (Saxon and Bertozzi, 2000) • Synthetic biopolymers

• Enzyme-mediated ligation (e.g., sortase) • Traceless Staudinger ligation

• Oxime formation from N-terminal serine, glycine • Oxidation of sugars to aldehyde


Table 6.2 Summary of Conjugation Methods: Their Bond Strength and Linker Length Characteristics

3. Impact of Bond Strength and Linker Length on Bioconjugation

3.3 THIOL-DISULFIDE/THIOETHER/METAL COORDINATION: THE NATIVE CHEMICAL LIGATION Disulfide bonds are formed and broken in the body as a function of oxidant/reductant concentration and catalysis (Thorpe et al., 2002; Zavialov et al., 1998; Zakharchuk et al., 2008). The disulfide bond stability is thus dependent on its microenvironment. For example, these bonds are relatively stable in the blood, while they are rapidly disrupted in the reducing environment of the cell cytoplasm (Koo et al., 2008). The disulfide bonds can be readily introduced with subnanometer linkers, including succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and cysteine residues. Thioethers formed using maleimides or iodoacetamides are very stable and uncleavable under physiological conditions. Similar to disulfide, this linkage can be designed to be shorter than 1 nm (Fang et al., 2009; Brinkley, 1992; Mattson et al., 1993). Reversible metal coordination bonds form directly between thiols, as well as disulfides and other sulfur functions, and a variety of metals, metal oxide, semiconductors, and soluble ions (Love et al., 2005; Langeland et al., 1999). The kinetics and thermodynamics of such coordinative interaction depend greatly on the specific participants. However, they are generally much less stable than the other mentioned methods of conjugation. For example, in the functionalization of a gold surface with monothiolated DNA (using the Langmuir model) (Chen and Frank, 1989), an equilibrium dissociation constant in the order of Keq = 10−6 M was found, corresponding C to the equation: ϑeq = , where θeq is the fraction of occupied binding sites on C + Keq the surface and C is the thiolated compound concentration in solution. The dissociation kinetics constant was found to be in the order of Kdiss = 10−3 s−1, corresponding to dϑ = − Kdiss * ϑ , where θ is the fraction of occupied binding sites. This the equation dt means a lifetime of minutes after excess DNA is removed (Yang et al., 1998). These bonds can become more stable by using multidentate ligands (Uyeda et al., 2005). This conjugation method is unique because it allows direct binding to the inorganic surface, thus achieving high proximity between the core and the ligand. Native chemical ligation yields a native peptide bond through the thiol group of an N-terminal cysteine residue of an unprotected peptide and the C-terminal thioester of a second unprotected peptide (Johnson and Kent, 2006; Helms et al., 2007), which rearranges to a native amide bond at the ligation site. This reaction is chemoselective and regioselective and the new bond formed is very (kinetically) stable in physiological solutions, with hydrolysis half-life in the order of tens or even hundreds of years (Smith and Hansen, 1998; Radzicka and Wolfenden, 1996). The minimum linker length in this case could be two amino acid long.

3.4 AZIDE–ALKENE/ALKYNE CYCLOADDITION This reaction yields a stable product under biological conditions (Meldal and Tomøe, 2008). The basic versions of these linkers are very small and rigid structures, forming a single five-membered ring after conjugation. Some methods enable deliberate

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reversal or further functionalization of this ring (Brantley et al., 2011; Hein et al., 2009). As mentioned previously, bioorthogonal variations of this method using strained alkenes/alkynes to replace Cu(I) catalysis also produce stable conjugates (Debets et al., 2011). However, in these cases, linker size is often much larger, sometimes adding 6–14 more carbon atoms to the conventional hydrophobic linker structure. Especially important application of this method uses an azides alternative—a nitrone that can be generated from an N-terminal serine (Ning et al., 2010), yielding an isoxazoline ring (Colombo et al., 2012b; Ning et al., 2010). Isoxazoline functional groups are known to go through thermal and catalytic ring opening (Kalgutkar et al., 2003; MacKenzie et al., 2014). This does not, however, result in link cleavage. This technique might be relevant also to other N-terminal amino acids (Gilmore et al., 2006).

3.5 HYDRAZINE/AMINE–ALDEHYDE LIGATION Using a hydrazine function, this ligation method yields a new hydrazone bond (Prasuhn et al., 2010). This bond can undergo reversible hydrolysis, with Kd = 10−4−10−6 dissociation constant (Dirksen and Dawson, 2008). The rate constants are pH-dependent (accelerating at acidic conditions) and can be catalyzed by aniline. At pH 7 (and no catalysis) the hydrolysis of half-life would be ∼8 days (Dirksen and Dawson, 2008; Kalia and Raines, 2008). Higher stability can be achieved through reduction of the newly formed bond. This also allows stable conjugation through amine-aldehyde functions (Colombo et al., 2016). Such linkers can be in the 1 nm order in length.

3.6 STAUDINGER LIGATION This reaction yields an amide bond (Saxon and Bertozzi, 2000; Köhn and Breinbauer, 2004) (Fig. 6.3A) described as stable in biological environment (Smith and Hansen, 1998; Radzicka and Wolfenden, 1996). The binding is done through a single benzene ring but include a bulky triphenylphosphine oxide. A modified version of this method can produce a traceless conjugation (Fig. 6.3B), forming a zero-length conjugation and releasing the bulky triphenylphosphine oxide.

FIGURE 6.3 (A) Staudinger ligation product conjugate. (B) Traceless Staudinger ligation product conjugate.

4. Impact of Ligand Density on the Targeting Efficiency of Nanoconjugates

4. IMPACT OF LIGAND DENSITY ON THE TARGETING EFFICIENCY OF NANOCONJUGATES It is now widely accepted that the targeting efficiency of ligand–nanoparticle conjugates is dependent not only on particle concentration, dosing time, and cell surface receptor expression level but also on the number of ligands present on the nanoparticle surface, i.e., the ligand density. In other words, ligand density is defined as the ratio of ligand number conjugated or adsorbed on the surface of each nanoparticle. The ligands can be organized on the nanoparticle surface in two distinct distributions— namely, randomly attached ligands or uniformly attached ligands—and this distribution directly influences the nanoparticle interaction with cell membrane, and, consequently, cell uptake kinetics (Junshi et al., 2016). In these terms, nanoparticle internalization kinetics can be related to a first-order reaction in which the ligands are irreversibly converted into ligand–receptor complex via the binding reaction. Consequently, nanoparticles with a higher ligand density induce a faster diffusion of receptors toward the binding site, which, in turn, accelerates the wrapping kinetics. Ligand density can be also related to multivalent interactions between nanoparticles and receptors. Nanoparticles functionalized with one or more molecules (e.g., antibodies) can have different interaction profiles with biological membranes. Table 6.3 provides a list of examples on how the ligand density variation can influence on targeting ability of the final nanoconjugate. Colombo et al. have developed a nanovector based on gold nanoparticles conjugated with a controlled number of targeting antibodies—namely, one or two molecules of trastuzumab—to prove how the number of antibodies affects in vitro and in vivo targeting efficacy of nanoparticles in a mouse model of breast cancer. Unexpectedly, the authors found that the tumor homing and protracted therapeutic efficacy were best achieved with just one antibody attached per nanoparticle rather than two, in contrast to an intuitive belief that in vivo targeting efficacy rises proportionally to the number of targeting antibodies. Hypothetically, a combination of the enhanced permeability and retention (EPR) effect and active targeting takes place for monoconjugated nanoparticles, in which the active targeting contribution becomes more important in proximity to the cancer cells. In contrast, the EPR effect would be dominant for bis-conjugated nanoparticles. In addition, in vivo results suggest that long-term intratumor retention of monoconjugated nanoparticles contributes to a sustained therapeutic effect over time as compared with both bis-conjugated nanoparticles and trastuzumab in standard HER2-positive breast cancer treatment. In this way, controlled conjugation of nanoparticles with a controlled ligand density, in particular monoconjugated nanoparticles, may lead to more efficient targeting strategies (Colombo et al., 2016). We have mentioned that a strict correlation between the number of surface ligands and the targeting efficiency in vivo of nanoconjugates is not easily predictable. However, in vitro, it has been demonstrated that increasing surface ligand density often results in enhanced cellular internalization (Fakhari et al.,

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2011; Pirollo and Chang, 2008; Gu et al., 2008). It should be noted that in this case fine control is required to determine the ideal density of targeting ligand to optimize multivalent effects while maintaining stealth properties. The ligand density, in turn, is highly dependent on different factors, including ligand size, length and flexibility, and its packing structure. In addition, variation in surface ligand density has been shown to impact particle toxicity, binding coefficient to the target receptor, and tumor accumulation (Reuter et al., 2015). It has been shown that the cell uptake mechanism of nanoparticles can be controlled by tuning their multivalency, i.e., the ligand density. For example, Dalal et al. showed that cellular internalization mechanism of folate-functionalized quantum dots shifts from caveolae-to clathrin-mediated endocytosis because the nanoparticle multivalency increases from 10 to 40 ligands/nanoparticles. Although caveolaemediated trafficking allocates the nanoparticles into the perinuclear region, clathrinmediated endocytosis guides them to the lysosomes, suggesting the importance of multivalent interaction in cellular endocytosis and intracellular trafficking for subcellular targeting applications (Dalal et al., 2016). The authors proposed an explanation to this endocytosis “switch.” They expected that low multivalency (ligand density 20) of nanoparticles caused receptor clustering on the cell surface via cross-linking. Stronger binding of nanoconjugate with the cell surface via multiple receptors reduced the receptor mobility and restricted nanoparticle localization at a fixed position of the membrane, thus initiating an additional signaling pathway for clathrin-mediated endocytosis. Consequently, the faster kinetics of clathrin-mediated endocytosis over caveolae-mediated endocytosis led to a predominant effect of clathrin-mediated endocytosis at high ligand density (>40) (Dalal et al., 2016) (Fig. 6.4). A compromise between ligand amount on the nanoparticle surface and targeting efficiency is essential in the design of optimized drug delivery vehicles. Although the total number of ligands per nanoparticle depends on the nanoparticle size (larger particles can accommodate more ligands), it is the surface density (ligands/mm2) rather than the number of molecules per particle that determines the targeting affinity of the nanoconjugate. This was demonstrated in a study by Xiang et al. Liposomes of different sizes containing different number of antibodies per liposome were used to study the binding affinity to antigens in vitro using surface plasmon resonance (SPR). For this purpose, a gold chip was coated with antigene molecules and a flow of antibodyconjugated liposomes was run on the chip surface. The authors showed a significant increase of the binding affinity at low ligand density on liposomes. However, when the number of antibodies increased, the binding affinity reached a plateau, and further addition of molecules on the liposomes did not significantly affect the binding affinity, resulting in signal saturation. The increase in binding affinity was attributed to the reduction of the dissociation rate and to the increase in the association rate. In addition, even if the threshold of the antibody amount per liposome for larger

References

Size

Ligand Density

Target/Cell

Hydrogel PRINT NPs/ZEGFR affibody

80 × 320 nm (rod-shaped)

0.65−4.5 × 10−3

EGFRoverexpressing epidermoid carcinoma (A431) and alveolar macrophage (MH-S) cells

Hydrogel PRINT NPs/ZEGFR affibody

50 × 60 nm (spherical)

1.2−3.0 × 10−3 ligands/nm2 corresponding to 20–40 ligands/NP

Dalal et al. (2016)

CdSe/ZnS quantum dots (QD)/folate

35–50 nm

10–110 ligands/NP

Xiang et al. (2015)

PEGylated liposomes/ Antimouse IgG

280 nm

10.7–73.4 ligands/NP

Reuter et al. (2015)

NP/Ligand

ligands/nm2 corresponding to 100–700 ligands/NP

Comments

Continued

155

The optimum ligand density was observed at ∼1.8 × 10−3 LG/nm2 (280 ligands/NP) in the cell population that only exhibited internalized NPs, and with a further increase in ligand density, the majority of cells displayed NPs both internalized and bound to the outer membrane. EGFRCellular association independent of overexpressing epi- ligand density, which is attributed to dermoid carcinoma the particle size, as there are multiple (A431) and alveolar accounts stating that optimal particle macrophage (MH-S) diameter for receptor-mediated endocells cytosis is 50 nm. Folate receptor over- For ligand density 10, additional clathrin-mediated endocytosis is initiated, and for ligand density >40, clathrin-mediated endocytosis dominates. Gold sensor chips/ As ligand density increased, the antigen coated binding affinity increased dramatically passing from 10.7 Ab/liposome to 21.6 Ab/liposome. At ligand density about 40 Ab/liposome-binding affinity reached a saturation value.


Table 6.3 List of Examples on How the Ligand Density Variation can Influence Targeting Efficiency of Nanoconjugates In Vitro

156

References

NP/Ligand

Size

Ligand Density

Target/Cell

PEGylated liposomes/ Antimouse IgG

130 nm

3.8–14.2 ligands/NP

Comments

Lee et al. (2015)

Graphene oxide/ folic acid

50 nm

0–0.190 ligands/ nm2 corresponding to 7.1−61.4 × 1016 ligands/mg GO

Calderon et al. (2011)

Polystyrene/ R6.5 or YN1 antibody

1 μm

0–6650 ligands/μm2

With increasing ligand density, the binding affinity increased significantly at low surface coverage (3–5 Ab/ liposome). It reached a plateau at ∼9 Ab/liposome. After that point, further addition of the targeting antibody on the liposomes did not significantly increase the binding affinity. Folate receptor over- In vitro, increasing ligand density expressing human increased the cellular uptake of GO; cervical cancer KB in vivo, below a critical ligand density cells ligand conjugation does not show significant improvement in the tumor accumulation, whereas, above the critical ligand density tumor targeting efficacy increased significantly. ICAM/Human Increasing the Ab density enhanced umbilical vein ECs lung accumulation with minimally (HUVECs) reduced liver and spleen uptake. Binding to activated HUVEC reached similar saturation with all tested Ab densities. In quiescent cells, carriers reached approximately threefold lower binding saturation, even at high Ab density, and carriers with low Ab density did not reach saturation, reflecting avidity below threshold. Binding kinetics was positively regulated by Ab density.


Table 6.3 List of Examples on How the Ligand Density Variation can Influence Targeting Efficiency of Nanoconjugates In Vitro—cont’d


FIGURE 6.4 The effect of folate multivalency on the folate receptor–mediated endocytosis mechanism and subcellular trafficking. The proposed mechanism shows that higher multivalency and faster kinetics of clathrin-mediated endocytosis can induce preferential clathrin-mediated endocytosis. As commonly used nanoparticles have high multivalency, they often mask the caveolae-mediated endocytosis of folate functionalized nanoparticles (Dalal et al., 2016).

liposomes was much larger than that for smaller liposomes (∼40 antibodies/liposome vs. ∼9 antibodies/liposome, respectively), saturation occurred at approximately the same antibody surface density of ∼1.5 × 108 antibodies/mm2, independent of the size of the liposome (Xiang et al., 2015). Lee et al. investigated how the amount of folate ligands on the surface of nanographene oxide (nGO) could affect folate receptor targeting both in vitro and in vivo. The uptake of nGOs by KB cells in vitro exhibited almost linear enhancement by increasing folate concentration on the nGO surface. In contrast, the in vivo data on tumor accumulation suggested that below a critical ligand density, nGO ligand conjugation did not show statistically significant improvement in the tumor accumulation, whereas ligand conjugation above the critical concentration resulted in a significant enhancement of tumor targeting. The biodistribution results confirmed the critical concentration of ligand density on the nGO surface: below this concentration, the ligand conjugation was not only unable to improve the tumor targeting of nGO but also increased the uptake of

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nGO in some major organs expressing folate receptor, including liver and lung, compared with nontargeted nGO, thus resulting in rather adverse effect. On the other hand, above the critical concentration, a significant benefit of ligand conjugation was obtained by delivering more nGOs to the tumor and by lowering nonspecific accumulation to the liver and the lung. Notably, too high ligand density, i.e., much above the critical concentration did not provide any additional benefit (Lee et al., 2015). Varying ligand surface density can be a useful approach for improving carrier delivery to specific organs while reducing nonspecific uptake by the body clearance system. Calderon et al. explored how antibody surface density affects accumulation of anti-ICAM polystyrene carriers in lung versus liver and spleen. Anti-ICAM carriers bearing 6935 antibodies/μm2 rapidly disappeared from the circulation and specifically accumulated in the lung vasculature. On the contrary, at high anti-ICAM ligand density, the carriers accumulated at a much lower extent in the liver and spleen. Yet, at low anti-ICAM surface density, considerable lung targeting still occurred, while the accumulation of the carriers in the liver and spleen increased. In vitro, the authors compared the binding kinetics of carriers coated by antibodies at different surface densities (1100 or 4100 antibodies/μm2) in quiescent endothelial cells expressing a basal level of ICAM-1, like in healthy endothelium, and TNF-α activated cells overexpressing ICAM-1 similar to pathologically altered endothelium. As expected, the number of anti-ICAM coated carriers bound per cell was higher in activated versus quiescent cells, for both antibodies surface densities. This high binding level is related to the receptor–ligand pairing where a minimal receptor density threshold must be exceeded for a ligand to bind linearly to the receptor, in accordance with upregulated ICAM-1 expression in activated endothelial cells. The binding kinetics of either types of carrier was faster for quiescent versus activated cells. The faster kinetics in quiescent cells can be explained in terms of earlier saturation of fewer ICAM-1 receptors available for binding anti-ICAM carriers (Calderon et al., 2011).

5. PROTEIN CORONA EFFECT AND MINIMIZATION OF NONSPECIFIC INTERACTIONS As above mentioned, the protection of ligand structure and arrangement on the nanoparticle surface is of primary importance for the preservation of the efficacy of biofunctionalized nanoparticles in vivo. When designing a targeted nanoconjugate, it should be taken into account that nonspecific protein adsorption from biological fluids invariably affects the targeting selectivity of nanoparticles to a specific organ or tissue (Zanganeha et al., 2016; Monopoli et al., 2012). The nanoparticle surface properties, particularly the surface charge and the kind and amount of associated targeting molecules, affect the composition, thickness, and conformation of protein corona influencing the biological identity of the functionalized nanoparticle and thus its distribution, fate, therapeutic efficiency, and toxicity (Monopoli et al., 2012; Salvati et al., 2013; Wang et al., 2011).

5. Protein Corona Effect and Minimization of Nonspecific Interactions

Several materials exhibit suitable properties that confer significant reduction of the adsorption of serum proteins on nanoparticles. A few examples, including dextran, polyoxazolines, glycoderivates, polymers, and self-assembled monolayers consisting of PEG, have been explored so far with different outcomes. PEG is the principal reagent used by nanoparticles designers, but complete inhibition of corona formation remains challenging (Sapsford et al., 2013). The character and the composition of corona proteins are crucial to make a prediction on how nanoparticles interact with cells because the corona proteins govern the specific cellular receptors used by the protein–nanoparticle complex (Fleischer and Payne, 2012; Doorley and Payne, 2012), the cellular internalization pathways (Doorley and Payne, 2012), and the immune response as well (Tenzer et al., 2013; Yan et al., 2013). Protein corona could be schematically represented as a double-layer coating wrapping the nanoparticle, consisting of a first tightly bound monolayer, usually referred to as “hard” corona, which is composed of proteins with high affinity for the nanoparticle surface (Rocker et al., 2009), whereas the outer layer, termed “soft” corona, consists of a loose protein layer that reflects the abundance of serum proteins, predominantly comprising albumin and its derivatives (Walczyk et al., 2010; Casals et al., 2010). In contrast, the composition of the “hard” corona does not necessarily reflect the abundance of proteins in serum. Indeed, proteins of lower abundance, including immunoglobulins, apolipoproteins, and fibrinogen, can be found in the “hard” corona because of their higher affinity to the nanoparticle surface compared to albumins (Jedlovszky-Hajdu et al., 2012). The protein corona is a universal phenomenon; however, it is unique for each kind of nanoparticles. The protein corona–nanoparticle complex has been detected on different classes of nanoparticles, including metallic (Casals et al., 2010; JedlovszkyHajdu et al., 2012; Hirsch et al., 2013; Shang et al., 2012; Lacerda et al., 2010), polymeric (Cedervall et al., 2007), silica (Lesniak et al., 2012), and semiconductor nanoparticles, so-called quantum dots (Zanganeha et al., 2016). It has been reported that the protein adsorption increases by increasing the surface charge of nanoparticles; in particular, neutral and negatively charged nanoparticles exhibit lower interaction with plasma proteins compared with positively charged ones that interact strongly with blood components (Aillon et al., 2009; Reddy et al., 2012). As the protein corona defines biological identity of nanoparticles in a biological environment, the interaction with the cell surface receptors is eventually driven by the corona–nanoparticle complex thus mediating the internalization in cells (Fleischer and Payne, 2014). The protein corona not only impacts the targeting and delivery properties of nanoparticles, but also it influences their toxicity and pathophysiology (Tenzer et al., 2013). For this reason, in the last few years, new synthetic “stealth” polymers have been investigated with the aim to improve the long-term prevention of nonspecific protein adsorption. Potential alternatives to PEG include polyoxazolines, dendrons, polypeptoids, and zwitterionic polymers. These macromolecules proved to be an excellent substitute to the PEGylated surfaces showing high protein resistance

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(Wyszogrodzka and Haag, 2009). In early 2000, “self” peptides derived from CD47, an integrin-associated protein “marker of self” on red blood, were discovered (Oldenborg et al., 2000). More recently, such peptides were successfully applied to the surface modification of colloidal nanoparticles, which resulted in much stronger reduction of protein adsorption and nanoparticle recognition by macrophages leading to potentially safe treatments (Rodriguez et al., 2013). An alternative approach to preventing the adsorption of plasma proteins is to intentionally associate them to the nanoparticles as targeting molecules, for example, by isolating selected proteins of interest from plasma and attaching the purified molecules to the nanoparticles ex vivo. Various cellular membrane markers have been identified, transferrin being one of the most largely used. Indeed, transferrin can be covalently linked to the NPs to target cells overexpressing transferrin receptors (Milani et al., 2012). In a conceptual evolution of this strategy, several research groups have recently explored a new class of biomimetic nanoparticles exploiting biological membranes harvested and purified from entire cells to coat the nanoparticles resulting in smart “biofriendly” nanodelivery systems. Example of cell types utilized for this purpose was erythrocytes, which offer the advantage of extremely long circulating half-lives (Hu et al., 2011), or leukocytes, which use their cell surface interactions to bind to inflamed endothelium and respond to tissue infections (Parodi et al., 2013).

6. CONCLUSION AND FUTURE PERSPECTIVES For many years, the chemical strategies exploited to achieve the immobilization of bioactive molecules on colloidal nanoparticles have been limited to generic and straightforward approaches that could allow to achieve stable linkages, irrespective of the way in which those molecules were positioned on the surface of nanoparticles. The rationale behind this rough conjugation approach relied on a statistical distribution of ligands in which one should expect that at least a small fraction of active molecules was allocated in a configuration suitable to exert its biological function. This assumption, however, could not be given for granted in several cases because it was often difficult to assess both the actual number and arrangement of the immobilized ligands. Nowadays, numerous studies have demonstrated that there are a few crucial issues that play a role in determining the biological activity (e.g., the targeting efficiency) and the fate of nanoconjugates both in vitro and in vivo. These concerns become particularly relevant in view of a next clinical translation of nanomedicines. In this chapter, we have addressed some of these major issues related to the strategy chosen for the immobilization of targeting biomolecules, which in turn affects the biomedical potential of colloidal nanoparticles. In particular, we highlighted the importance of controlling the ligand orientation and density on the nanoparticle surface as well as the strict interdependence among them, although controversial evidence has been provided on the possibility to actually predict the effects of modulating independently these two parameters.

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At the end of this chapter, in the light of the relevance of choosing the appropriate conjugation strategy for a specific experiment, we should point out that a major restriction, which strongly limits the real potential of improving and optimizing such strategies, resides in the lack of adequate methods for the accurate characterization of the organic component of nanoconjugates. So far, conventional methods have mainly exploited either indirect approaches—including flow cytometry and confocal laser scanning microscopy (Fiandra et al., 2013), SPR (Schneider et al., 2015), and microcalorimetry (Huang and Lau, 2016), which provide evidence of the biological affinity of nanoconjugates for specific cellular or molecular receptors—or direct methods—including comparative FTIR (Occhipinti et al., 2011), binding energy analysis (XPS) (Azhdarzadeh et al., 2016), vibrating sample magnetometry, thermogravimetric analysis, and circular dichroism (Ahmad et al., 2016). However, all of these methods are not adequate to offer compelling confirmation of the structural features of the conjugated organic ligands. Unfortunately, the most reliable method for the characterization of organic molecules, i.e., the nuclear magnetic resonance (NMR), is seldom applicable to nanoconjugates due to spectral signal broadening deriving from the constrained mobility of ligands on the nanoparticle surface combined with field interferences, especially occurring with paramagnetic nanomaterials (Sillerud et al., 2006). Because of such an urgent need, great advances are expected in the next years. Recently, a few seminal works have suggested that high-resolution magic angle spinning (HRMAS) NMR may solve the problems related to field inhomogeneity and might open a new frontier establishing HRMAS NMR as an election technique for the structural characterization of organic ligands bound to the surface of nanoparticles (Xu et al., 2004; Polito et al., 2008b; El-Boubbou et al., 2010). Considering the impressive progresses attained in the last 10 years in the field of nanobioconjugate chemistry, we foresee that in the near future we will assist to a renewed spur in the development of a new generation of targeted nanomaterials leading to enhanced molecular selectivity and improved biological activity, with great potential in biotechnology and medicine.

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CHAPTER

7

Nanozymes for Biomedical Sensing Applications: From In Vitro to Living Systems

Shichao Lina, Jiangjiexing Wua, Jia Yao, Wen Cao, Faheem Muhammad, Hui Wei Nanjing University, Nanjing, China

CHAPTER OUTLINE 1. Introduction�� 171 2. Nanozymes for In Vitro Sensing�� 172 2.1 Nanozyme as Peroxidase Mimic for Colorimetric Sensing�� 172 2.2 Nanozyme as Oxidase Mimic for Colorimetric Sensing�� 181 2.3 Nanozyme Combines Peroxidase and Oxidase Mimics for Colorimetric Sensing�� 193 2.4 Others Based on Fluorometric, Chemiluminescent, and Electrochemical Sensing�� 195 3. Nanozyme for Sensing in Living Systems�� 197 4. Conclusions and Perspectives�� 198 Abbreviations�� 200 Acknowledgments�� 201 References�� 201

1. INTRODUCTION In the field of artificial enzymes, the functional nanomaterials with enzyme-like characteristics, termed as nanozymes, are currently garnering immense attention (Wei and Wang, 2013). Previously, inorganic materials were considered biocatalytically inert. However, with the development of nanotechnology, researchers found that some nanomaterials, such as fullerene derivatives, metal, and metal oxides, exhibited unexpected intrinsic enzyme-like activities, which has ignited intensive research activity in the field of nanozymes (Gao and Yan, 2013, 2016; Lin et al., 2014d; Xie et al., 2012; Nakamura and Isobe, 2003; Mancin et al., 2016; Karakoti et al., 2010; Wang et al., 2016b). Compared with natural enzymes, nanozymes a Both

authors are contributed equally.


171

172

Nanozymes for Biomedical Sensing Applications

have various advantages, such as robustness to environmental conditions, low cost, ease of mass production, and so on. Besides, nanozymes are also endowed with unique properties in terms of their physical responses toward external stimuli, size- (shape-, structure-, composition-) dependent enzyme-mimicking activities, large surface area, etc. Engineering and bioconjugation of nanozymes can introduce more functionalities and as a result diversify their great potential. To date, numerous nanomaterials have been explored, which mimic the activities of various natural enzymes, such as horseradish peroxidase (HRP), haloperoxidase, superoxide dismutase, catalase, oxidase, esterase, nuclease, phosphatase, protease, ferroxidase, and sulfite oxidase (Fig. 7.1) (Wang et al., 2016a; Vernekar et al., 2014; Xue et al., 2014; Kim et al., 2012a; Chen et al., 2006, 2012a; Gao et al., 2007; Manea et al., 2004; Dugan et al., 1997; Cai et al., 2015; Tonga et al., 2015; Natalio et al., 2012). Since the first application of nanozyme in sensitive H2O2 and glucose detection reported by Wei and Wang (2008), great efforts have been devoted to exploring the applications of nanozymes in biomedical sensing and therapeutics. Various bioactive small molecules, nucleic acids, metal ions, cancer cells, and even bacteria have been detected by using nanozyme-based biosensing strategies (Wang et al., 2016a; Fan et al., 2012). In this chapter, the recent exciting progress of nanozymes will be discussed, focusing on engineering nanozymes for in vitro and in vivo sensing. Here, we do not attempt to cover all the related publications. Instead, we use representative examples to illustrate various nanozyme-based biosensing strategies for in vitro and in living systems. Finally, the perspectives on current challenges facing nanozyme technology and future directions will also be included.

2. NANOZYMES FOR IN VITRO SENSING For in vitro sensing, various nanozyme-based biosensors have so far been reported, such as colorimetric, fluorescent, chemiluminescent, and electrochemical biosensors (Wang et al., 2016a). Among these biosensing techniques, colorimetric biosensors are the most promising ones because of the ease of sample preparation, simplicity, portability, and low cost. Colorimetric biosensors depend on visual observations of color changes or spectrometric measurements of the bioanalytes. In some cases such as dipstick test strips, the targets can even be detected by naked eyes.

2.1 NANOZYME AS PEROXIDASE MIMIC FOR COLORIMETRIC SENSING Like natural peroxidase, nanozymes-based peroxidase mimics catalyze the oxidation of the substrate with peroxide (hydrogen peroxide in most cases). Thus, H2O2 can be detected by monitoring the production of oxidized substrate. H2O2 detection is of great interest because of its important roles in biology, medicine, food industry, and environmental protection. Wei and Wang (2008) developed a colorimetric

A brief timeline for the development of artificial enzymes (natural enzymes are also listed for comparison). NPs, nanoparticles; SOD, s uperoxide dismutase. Reprinted with permission from Wang, X., Hu, Y., Wei, H., 2016a. Inorg. Chem. Front. 3, 41–60. Copyright (2016) Royal Society of Chemistry.

2. Nanozymes for In Vitro Sensing

FIGURE 7.1

173

174


method for H2O2 detection. They used Fe3O4 magnetic nanoparticles (MNPs) as peroxidase mimic and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as signaling substrate (Fig. 7.2A). Green colored ABTS+ was generated in the presence of H2O2, which could be quantified by absorption spectra or even visualized by naked eyes. Since then, a considerable number of studies have been devoted to H2O2 detection by using the peroxidase-mimicking activities of various nanomaterials (see Table 7.1 for more examples). Nanozymes are endowed with unique properties in terms of their physical responses toward external stimuli and size- (shape-, structure-, composition-) dependent enzyme-mimicking activities (Wei and Wang, 2013). Thus, by

FIGURE 7.2 (A) Nanozyme as peroxidase mimic for colorimetric sensing of H2O2 and glucose when combined with glucose oxidase (GOx). (B) The sensing format in (A) could be extended to other targets (substrate 1 here) when combined with a suitable oxidase. (C) Target of interest as substrate 0 could be determined if it could be converted into an oxidase substrate. Numerous transduction signals can be adopted for sensing (such as colorimetric, fluorometric, chemiluminescent, and surface-enhanced Raman scattering signals when the corresponding substrates are used; and electrochemical signals when a nanozyme is immobilized on an electrode). ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Reprinted with permission from Wang, X., Hu, Y., Wei, H., 2016a. Inorg. Chem. Front. 3, 41–60. Copyright (2016) Royal Society of Chemistry.

Table 7.1 H2O2 Detection With Peroxidase Mimics Methods

Linear Range

Limit of Detection

Fe3O4 MNPs Fe3O4 MNPs

Color. Color.

5–100 μM 0.5–150.0 μM

3 μM 0.25 μM

Fe3O4 MNPs

Color.

1–100 μM

0.5 μM

Fe3O4 graphene oxide composites Fe-substituted SBA-15 microparticles Iron phosphate microflowers [Fe(III)(biuret-amide)] on mesoporous silica FeTe nanorods Fe(III)-based coordination polymer Fe3O4 nanocomposites

Color.

1–50 μM

Color.

Protein-Fe3O4 and GOx nanocomposites GOx/Fe3O4/GO magnetic nanocomposite

Comments

References Wei and Wang (2008) Chang et al. (2009)

0.32 μM

Substrate: ABTS Substrate: DPD H2O2 in rainwater, honey, and milk was tested. Substrate: TMB Fe3O4 was encapsulated in mesoporous silica. Substrate: TMB

0.4–15 μM

0.2 μM

Substrate: TMB

Liu et al. (2011b)

Color.

10–50 μM

10 nM

Substrate: TMB

Wang et al. (2012a)

Color.

0.1–5 mM

10 μM

Substrate: TMB

Malvi et al. (2012)

Color. Color.

0.1–5 μM 1–50 μM

55 nM 0.4 μM

Substrate: ABTS Substrate: TMB

Roy et al. (2012) Tian et al. (2012)

Color.

5–80 μM

1.07 μM

Liu et al. (2014a)

Color.

0.5–200 μM

0.2 μM

Substrate: TMB Fe3O4 was functionalized by 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin. Substrate: TMB

Color.

0.1–100 μM

0.04 μM

Substrate: DPD

Chang and Tang (2014)

Kim et al. (2011b)

Dong et al. (2012)

Liu et al. (2014b)


Nanozymes

Continued

175

176

Nanozymes

Methods

Linear Range

Limit of Detection

Comments

References

Iron(III) hydrogen phosphate hydrate crystals MIL-53(Fe)

Color.

57.4–525.8 μM

1 μM

Substrate: TMB

Zhang et al. (2014c)

Color.

0.95–19 μM

0.13 μM

Ai et al. (2013)

CuO NPs AuNPs

Color. Color.

0.01–1 mM 18–1100 μM

N/A 4 μM

AuNC@BSA Au@Pt core/shell nanorods Nickel telluride nanowires Graphene oxide Hemin–graphene hybrid nanosheets Carbon nanodots Carbon nitride dots Tungsten carbide nanorods CoFe LDH nanoplates CoxFe3-xO4 nanocubes Porphyrin-functionalized Co3O4 Nanostructures Carboxyl-functionalized mesoporous polymer

Color. Color.

0.5–20 μM 45–1000 μM

20 nM 45 μM

Substrate: TMB MIL-53(Fe): a metal-organic framework Substrate: 4-AAP and phenol Substrate: TMB Cysteamine was the ligand for AuNPs. Substrate: TMB Substrate: OPD

Color. Color. Color.

0.1–0.5 μM 0.05–100 μM 0.05–500 μM

25 nM 50 nM 20 nM

Substrate: ABTS Substrate: TMB Substrate: TMB

Wan et al. (2014) Song et al. (2010) Guo et al. (2011)


1–100 μM 1–100 μM 0.2–80 μM

0.2 μM 0.4 μM 60 nM

Substrate: TMB Substrate: TMB Substrate: TMB

Shi et al. (2011) Liu et al. (2012b) Li et al. (2014)


1–20 μM 1–60 μM 1–75 μM

0.4 μM 0.36 μM 0.4 μM

Substrate: TMB Substrate: TMB Substrate: TMB

Zhang et al. (2012) Yang et al. (2014b) Liu et al. (2014c)

Color.

1–8 μM

0.4 μM

Substrate: TMB

Liu et al. (2011c)

Chen et al. (2012b) Jv et al. (2010) Wang et al. (2011) Liu et al. (2012a)


Table 7.1 H2O2 Detection With Peroxidase Mimics—cont’d

PtPd nanodendrites on graphene nanosheets (PtPdNDs/GNs) Pt-DNA complexes

Color.

0.5–150 μM

0.1 μM

Substrate: TMB

Chen et al. (2014)

Color.

0.979–17.6 mM

0.392 mM

Chen et al. (2012c)

Manganese selenide nanoparticles Prussian blue nanoparticles MWCNTs-Prussian blue nanoparticles

Color.

0.17–10 μM

0.085 μM

Substrate: TMB 3.92 μM was detected with PVDF membrane. Substrate: TMB

Color.

0.05–50 μM

0.031 μM

Substrate: ABTS

Zhang et al. (2014d)

Color.

1 μM −1.5 mM

100 nM

Wang et al. (2014b)

Polypyrrole (PPy) nanoparticles

Color.

5–100 μM

Polyoxometalate Polyoxometalate Fe3O4 MNPs

Color. Color. Fluor.

1–20 μM 0.134–67 μM 10–200 nM

0.4 μM 0.134 μM 5.8 nM

BiFeO3 NPs

Fluor.

20 nM-20 μM

4.5 nM

Fe3O4 MNPs

Fluor.

0.18–900 μM

0.18 μM

Fe3O4 MNPs

Fluor.

0.04–8 μM

0.008 μM

Substrate: TMB Carbon nanotubes were filled with Prussian blue nanoparticles. Substrate: TMB PPy has been successfully employed to quantitatively monitor the H2O2 generated by macrophages. Substrate: TMB Substrate: TMB Substrate: rhodamine B Fluorescence of rhodamine B was quenched. Substrate: BA Oxidation of BA gave fluorescence. H2O2 in rainwater was tested. Fluorescence of CdTe quantum dot (QD) was quenched. Substrate: BA Oxidation of BA gave fluorescence.

Qiao et al. (2014)

Tao et al. (2014)

Luo et al. (2010)

Gao et al. (2011) Shi et al. (2014) Continued


Liu et al. (2012c) Wang et al. (2012b) Jiang et al. (2011)

177

178

Nanozymes

Methods

Linear Range

Limit of Detection

Cupric oxide Nanoparticles

Fluor.

5–200 μM

0.34 μM

Fe(III)–TAML activator CoFe2O4 NPs

CL CL

0.06–1 μM 0.1–4 μM

0.05 μM 0.02 μM

CoFe2O4 NPs CoFe2O4 NPs with chitosan coating

CL CL

0.1–10 μM 1 nM–4 μM

10 nM 0.5 nM

Fe3O4 MNPs Fe3O4 microspheresAgNP hybrids Fe3O4 MNPs Fe3O4 MNPs

E-chem E-chem

4.2–800 μM 1.2–3500 μM

1.4 μM 1.2 μM

E-chem E-chem

0–16 nM 1–10 mM

1.6 nM N/A

Fe3O4 MNPs

E-chem

20–6250 μM

2.5 μM

Fe3O4 Nanofilms on TiN substrate Fe3O4 MNPs Fe3O4 MNPs

E-chem

1–700 μM

1 μM

E-chem E-chem

0.2–2 mM 0.1–6 mM

0.01 mM 3.2 μM

Comments

References

Substrate: terephthalic acid Hu et al. (2014b) Terephthalic acid was oxidized by hydroxyl radical to form a highly fluorescent product. Vdovenko et al. (2014) CoFe2O4 NPs form complexes with He et al. (2010) beta-CD. H2O2 in natural water was tested. Shi et al. (2011) CoFe2O4 NPs was coated with chitosan. Fan and Huang (2012) H2O2 in natural water was tested. Zhang et al. (2008) H2O2 in disinfected FBS samples was Liu et al. (2010) tested. Fe3O4 was loaded on CNT. Kang et al. (2011) Fe3O4 was entrapped in mesoporous Kim et al. (2011a) carbon foam, and the composite was used to construct a carbon paste electrode. Not a linear response. Fe3O4 MNPs and PDDA-graphene formed Liu et al. (2011a) multilayer via layer-by-layer assembly. H2O2 in toothpaste was tested. H2O2 in walgreens antiseptic/oral debriding Yang et al. (2011) agent, crest whitening mouthwash solution, diet coke, and Gatorade was tested. Fe3O4 was on reduced graphene oxide.

Zhang et al. (2011) Ye et al. (2012)


Table 7.1 H2O2 Detection With Peroxidase Mimics—cont’d

Fe2O3 NPs Fe2O3 NPs Iron oxide NPs/CNT Fe3O4/self-reduced graphene nanocomposites

E-chem E-chem E-chem E-chem

20–140 μM 20–300 μM 0.099–6.54 mM 0.001–20 mM

11 μM 7 μM 53.6 μM 0.17 μM

Dutta et al. (2012a)

FeS nanosheet FeS needle FeSe NPs FeS Co3O4 NPs Hemin–graphene hybrid nanosheets Layered double hydroxide-hemin nanocomposite Helical CNT LDH nanoflakes Calcined LDH CdS

E-chem E-chem E-chem E-chem E-chem E-chem

0.5–150 μM 5–140 μM 5–100 μM 10–130 μM 0.05–25 mM 0.5–400 μM

92 nM 4.3 μM 3.0 μM 4.03 μM 0.01 mM 0.2 μM

Dai et al. (2009) Dutta et al. (2012b)

E-chem

1–240 μM

0.3 μM

Zhang et al. (2014a)

E-chem E-chem E-chem E-chem

0.5–115 μM 12–254 μM 1–100 μM 1–1900 μM

0.12 μM 2.3 μM 0.5 μM 0.28 μM

Cui et al. (2011a) Wang et al. (2009) Cui et al. (2011b) Maji et al. (2012)

Fe2O3 was modified with Prussian blue. Extracellular H2O2 released from HeLa cells stimulated by CdTe QDs was established by this approach.

Miao et al. (2009) Fang et al. (2014)

Maji et al. (2012) Mu et al. (2012) Guo et al. (2011)


4-AAP, 4-aminoantipyrine; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); AuNPs, gold nanoparticles; BA, benzoic acid; BSA, bovine serum albumin; CD, cyclodextrin; CNT, carbon nanotube; Color., colorimetric; DPD, N,N-diethyl-p-phenylenediamine sulfate; E-chem, electrochemical; FBS, fetal bovine serum; Fluor., fluorometric; GOx, glucose oxidase; LDH, layered double hydroxide; MNPs, magnetic nanoparticles; MWCNTs, multiwalled carbon nanotubes; OPD, o-phenylenediamine; PDDA, poly(diallyldimethylammonium chloride); PVDF, polyvinylidene difluoride; SBA-15, Santa Barbara amorphous type material; TAML, tetraamidomacrocyclic ligand; TMB, 3,3′,5,5′-tetramethylbenzidine. Adapted with permission from Liu, Q.Y., Li, H., Zhao, Q.R., Zhu, R.R., Yang, Y.T., Jia, Q.Y., Bian, B., Zhuo, L.H., 2014a. Glucose-sensitive colorimetric sensor based on peroxidase mimics activity of porphyrin-Fe3O4 nanocomposites. Mater. Sci. Eng. C Mater. Biol. Appl. 41, 142–151. Copyright (2016) Royal Society of Chemistry.

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modulating the peroxidase mimics catalyzed reactions, analytes of interest have been detected through numerous strategies (Zheng et al., 2013; Shamsipur et al., 2014; Ni et al., 2014; Niu et al., 2014; Tan et al., 2014; Sun et al., 2015; Lin et al., 2015; Chen et al., 2015; Zhang et al., 2015a; Wang et al., 2015; Yang et al., 2016). For example, a colorimetric method for kanamycin detection has been developed by Sharma et al. (2014). Kanamycin is an aminoglycoside antibiotic and widely used in veterinary medicine. Monitoring the kanamycin contamination in food is of great importance considering the potential transfer of kanamycin to humans in the food chain and its serious side effects. They used a kanamycin-binding aptamer (called Ky2 aptamer) to block the surface of gold nanoparticles (AuNPs), which would then inhibit the peroxidase-like activity of AuNP. In the presence of kanamycin, the aptamer molecules were desorbed from the surface of AuNP because of the high affinity and specificity of Ky2 aptamer toward kanamycin. Thus the nanozyme activity of AuNPs was recovered (Fig. 7.3). Through such a switchable strategy, kanamycin could be detected within 3–8 min with high selectivity, and

FIGURE 7.3 Schematic representation of the “turn-off/turn-on” nanozyme activity of aptamer-functionalized gold nanoparticle (AuNP) for the detection of kanamycin. Step A shows intrinsic peroxidase-like activity of pristine AuNP that gets “turned-off” after functionalization with the Ky2 aptamer and remains “turned-off” in the absence of kanamycin (Step B); however, it gets “turned-on” again in the presence of kanamycin (Step C). TMB, 3,3′,5,5′-tetramethylbenzidine. Reprinted with permission from Sharma, T.K., Ramanathan, R., Weerathunge, P., Mohammadtaheri, M., Daima, H.K., Shukla, R., Bansal, V., 2014. Aptamer-mediated ‘turn-off/turn-on’ nanozyme activity of gold nanoparticles for kanamycin detection. Chem. Commun. 50, 15856–15859. Copyright (2014) Royal Society of Chemistry.


the detection limit was 1.49 nM. In another study, because of the specific Hg2+–Pt0 metallophilic interactions, Li et al. (2015a) found that Hg2+ could inhibit the peroxidase-mimicking activity of bovine serum albumin-templated platinum nanoparticles (PtNPs), and thus a colorimetric sensor for Hg2+ with a low detection limit of 7.2 nM was fabricated. The simple and direct colorimetric sensing based on peroxidase mimic has also been employed for immunoassays. Many research groups combined the classic sandwich immunoassay and nanozymes together for sensing analytes of interest (see Table 7.3 for more information). Kim et al. (2014) developed an ultrafast colorimetric immunoassay with a new synergistically integrated nanocomposite (Fig. 7.4). A highly active peroxidase-mimicking nanocomposite was fabricated by encapsulating Fe3O4 MNPs and PtNPs within porous carbon. Owing to the high catalytic activity of PtNPs, the nanocomposite showed a 50 times higher catalytic efficiency compared with that of MNPs alone. Thus, the color generation took place very rapidly for the new assay system. Clinically important target molecules, such as human epidermal growth factor receptor 2 (HER2) and diarrhea-causing rotavirus, were detected in only 3 min at room temperature with high specificity and sensitivity.

2.2 NANOZYME AS OXIDASE MIMIC FOR COLORIMETRIC SENSING Oxidase uses oxygen to oxidize its substrates. Colorimetric sensors based on nanozymes with oxidase-mimicking activities can be established as the peroxidase-mimicking ones demonstrated in Section 2.1. Andreescu Group evaluated the oxidase mimetic properties of nanoceria and developed colorimetric assay for dopamine and catechol (Hayat et al., 2015). They also found that the sensitivity of this method varied significantly with the type of nanoparticle, buffer composition, pH, and so on. Recommendations of the experimental conditions were provided to achieve a high sensitivity and maximize the oxidase-like activity of nanoceria. Recently, Wei et al. developed a self-regulated sensing strategy for sensitive and selective in vitro bioassays (Fig. 7.5) (Cheng et al., 2016a). They demonstrated an in situ rational modulation of the oxidase-mimicking activity of nanoceria via proton-producing (or proton-consuming) enzyme-catalyzed bioreactions. For example, acetylcholinesterase (AChE), a proton-producing enzyme, could catalyze the hydrolysis of acetylcholine to produce choline and acetic acid (i.e., the source of proton), resulting in an enhancement of the nanoceria’s catalytic activity. Based on this phenomenon, some important targets such as AChE and urease were detected with good selectivity and sensitivity. Moreover, on adding a secondary modulator such as F−, an inhibitor of urease, a color gradient from colorless to blue could be observed. Therefore, the detection of F− and other bioactive targets (i.e., nerve agents, drugs, and ions) has also been achieved through cooperatively modulating the oxidase-like activity of nanoceria (Cheng et al., 2016a). Using F−-enhancement effect on oxidase-mimicking activity of nanoceria, an ultrasensitive F− detection was achieved with a detection limit of 0.64 μM in water and in toothpastes by Liu et al. (2016).

181

182

Methods

Linear Range

Limit of Detection

Fe3O4 MNPs

Color.

50–1000 μM

30 μM

Fe3O4 MNPs with PDDA coating

Color.

39–100 μM

30 μM

Fe3O4 MNPs

Color.

30–1000 μM

3 μM

Fe3O4 GO composites

Color.

2–200 μM

0.74 μM

Fe3O4 nanocomposites

Color.

5–25 μM

2.21 μM

3–1000 μM

1.0 μM

Nanozymes

Comments

References

Substrate: ABTS Selectivity against sugars: fructose, lactose, and maltose. Substrate: ABTS GOx was electrostatically assembled onto the Fe3O4@PDDA. Glucose in serum samples was tested. Compared with glucometer. Selectivity against sugars: galactose, lactose, mannose, maltose, arabinose, cellobiose, raffinose, and xylose. Substrate: TMB Fe3O4 was encapsulated in mesoporous silica with GOx. Showing the recycle capability. Comparison between free MNPs vs. encapsulated MNPs. Substrate: TMB Glucose in urine was tested. Substrate: TMB Fe3O4 was functionalized by 5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrin. Substrate: TMB

Wei and Wang (2008)

Glucose

Protein-Fe3O4 and GOx Color. nanocomposites

Yu et al. (2010)

Kim et al. (2011b)

Dong et al. (2012) Liu et al. (2014a)

Liu et al. (2014b)


Table 7.2 Targets Detection Combining Oxidases and Peroxidase Mimics

Color.

1–80 μM

0.21 μM

GOx/Fe3O4/GO magnetic nanocomposite Graphite-like carbon nitrides

Color.

0.5–600 μM

0.2 μM

Color.

5–100 μM

0.1 μM

Iron oxide NPs

Color.

31.2–250 μM

8.5 μM

Iron oxide NPs

Color.

31.2–250 μM

15.8 μM

Iron oxide NPs

Color.

0.12–4 μM

0.5 μM

ZnFe2O4

Color.

1.25–18.75 μM

0.3 μM

[Fe(III)(biuret-amide)] on mesoporous silica

Color.

20–300 μM

10 μM

FeTe nanorods

Color.

1–100 μM

0.38 μM

Fe(III)-based coordination polymer

Color.

2–20 μM

1 μM

Mesoporous Fe2O3–graphene nanostructures CuO NPs

Color.

0.5–10 μM

0.5 μM

Color.

0.1–8 mM

N/A

Substrate: TMB Glucose in blood and urine was tested. Substrate: DPD Substrate: TMB Glucose in serum was tested. Substrate: ABTS Iron oxide NPs was coated with glycine. More robust than HRP toward NaN3 inhibition. Substrate: ABTS Iron oxide NPs was coated with heparin. More robust than HRP toward NaN3 inhibition. Substrate: ABTS Iron oxide NPs was coated with APTES and MPTES. Substrate: TMB Glucose in urine was tested. Substrate: TMB Glucose in mice blood plasma was tested. Substrate: ABTS Glucose in spiked blood was tested. Substrate: TMB Glucose in serum was tested. Substrate: TMB Glucose in serum was tested. Substrate: 4-AAP and phenol

Mitra et al. (2014) Chang and Tang (2014) Lin et al. (2014a) Yu et al. (2009)

Liu and Yu (2011)

Su et al. (2012) Malvi et al. (2012) Roy et al. (2012) Tian et al. (2012) Xing et al. (2014)

Chen et al. (2012b) Continued


γ-Fe2O3 nanoparticles

183

184

Nanozymes

Methods

Linear Range

Limit of Detection

Comments

References

V2O5 nanowires and AuNPs nanocomposite AuNPs

Color.

0–10 μM

0.5 μM

Substrate: ABTS

Qu et al. (2014)

Color.

2.0–200 μM

0.5 μM

Jv et al. (2010)

Au@Pt core/shell nanorods Nickel telluride nanowires Manganese selenide nanoparticles Graphene oxide

Color.

45–400 μM

45 μM

Substrate: TMB Cysteamine was the ligand for AuNPs. Substrate: OPD

Color.

1–50 μM

0.42 μM

Substrate: ABTS

Wan et al. (2014)

Color.

8–50 μM

1.6 μM

Substrate: TMB

Qiao et al. (2014)

Color.

1–20 μM

1 μM

Song et al. (2010)

Graphene oxide

Color.

2.5–5 mM

0.5 μM

Hemin–graphene hybrid nanosheets Carbon nanodots

Color.

0.05–500 μM

30 nM

Substrate: TMB Glucose in blood and fruit juice was tested. Substrate: TMB Graphene oxide was functionalized by chitosan. Substrate: TMB

Color.

1–500 μM

1 μM

Shi et al. (2011)

Carbon nitride dots MWCNTs-Prussian blue nanoparticles

Color. Color.

1–5 μM 1 μM −1 mM

0.5 μM 200 nM

CoFe LDH nanoplates

Color.

1–10 mM

0.6 μM

Substrate: TMB Glucose in serum was tested. Substrate: TMB Substrate: TMB Carbon nanotubes were filled with Prussian blue nanoparticles. Substrate: TMB

Liu et al. (2012a)

Wang et al. (2014a) Guo et al. (2011)

Liu et al. (2012b) Wang et al. (2014b) Zhang et al. (2012)


Table 7.2 Targets Detection Combining Oxidases and Peroxidase Mimics—cont’d

Color.

8–90 μM

2.47 μM

Substrate: TMB

MoS2 Nanosheets Tungsten disulfide nanosheets

Color.

5–150 μM

1.2 μM

Color.

5–300 μM

2.9 μM

Prussian blue nanoparticles Fe3O4 MNPs

Color.

0.1–50 μM

0.03 μM

Substrate: TMB Glucose in serum was tested. Substrate: TMB Glucose in serum of normal persons and diabetes persons was tested. Substrate: ABTS

Fluor.

1.6–160 μM

1.0 μM

Fe3O4 MNPs

Fluor.

0.05–10 μM

0.025 μM


Fluor.

3–9 μM

3 μM

BiFeO3 NPs

Fluor.

1–100 μM

0.5 μM

CoFe2O4 NPs CoFe2O4 NPs

CL CL

0.1–10 μM 0.05–10 μM

0.024 μM 10 nM

Hemin–graphene hybrid nanosheets

E-chem

0.5–400 μM

0.3 μM

Fluorescence of CdTe quantum dot was quenched. Glucose in serum was tested. Substrate: benzoic acid (BA) Oxidation of BA gave fluorescence. Glucose in serum was tested. GOx was electrostatically assembled onto the Fe3O4@PDDA. Oxidation of AU gave fluorescence. Glucose in serum was tested. Selectivity against sugars: arabinose, cellobiose, galactose, lactose, maltose, raffinose, and xylose. Oxidation of BA gave fluorescence. Glucose in serum was tested. Other sugars CoFe2O4 NPs were coated with chitosan. Glucose in serum was tested.

Yang et al. (2014b) Lin et al. (2014b) Lin et al. (2014c)

Zhang et al. (2014d) Gao et al. (2011)

Shi et al. (2014)

Liu and Tseng (2011)

Luo et al. (2010) Shi et al. (2011) Fan and Huang (2012) Guo et al. (2011)


CoxFe3-xO4 nanocubes

Continued

185

186

Nanozymes

Methods

Linear Range

Limit of Detection

Fe3O4 MNPs

E-chem

6–2200 μM

6 μM

Fe3O4 MNPs

E-chem

0.5–10 mM

0.2 mM

Fe3O4–enzyme–polypyrrole nanoparticles

E-chem

0.5 μM-34 mM

0.3 μM

Color.

28.6–190.5 μM

Color.

Color.

Comments

References

Glucose in serum was tested. Compared with clinical analyzer. Nafion for high selectivity against AA, UA, sucrose, and lactose. Fe3O4 was encapsulated in mesoporous carbon with GOx, and the composite was used to construct a carbon paste electrode. Comparison between free MNPs vs. encapsulated MNPs. Glucose in serum was tested.

Yang et al. (2009)

15 μM

Substrate: TMB MIL-53(Fe): a metal-organic framework (MOF).

Ai et al. (2013)

0.6–8 μM

0.13 μM

Substrate: TMB Dopamine in serum was tested.

Niu et al. (2014)

1–50 nM

2.6 nM

Ag/Pt bimetallic nanoclusters were produced through a DNA-templated method.

Zheng et al. (2014)

Kim et al. (2011a)

Yang et al. (2014a)

Ascorbic Acid MIL-53(Fe) Dopamine CoxFe3-xO4 nanoparticles Thrombin Ag/Pt bimetallic nanoclusters



Glutathione Fe-MIL-88NH2 MOF

Color.

1–100 μM

0.45 μM

Substrate: TMB

Jiang et al. (2014)

Color.

1–80 μM

0.39 μM

Substrate: TMB

Jiang et al. (2014)

Color.

1–80 μM

0.40 μM

Substrate: TMB

Jiang et al. (2014)

Fe3O4

Fluor.

20–100 μM

20 μM


MNPs with PDDA coating Fe3O4 MNPs

Choline oxidase was electrostatically assembled onto the Fe3O4@PDDA. Oxidation of AU gave fluorescence.

E-chem

1 nM–10 mM (log)

0.1 nM

Zhang et al. (2011)

Platinum nanoparticles

Color.

6–400 μM

2.5 μM

Fe3O4 and choline oxidase were immobilized together on electrode. Selectivity against AA and UA. Substrate: N-ethyl-N-(3-sulfopropyl)-3methylaniline sodium salt and 4-amino-antipyrine

Color.

100 nM-10 mM

39 nM

Substrate: TMB

Qian et al. (2014)

Color.

10–200 μM

2.84 μM

Substrate: N-ethyl-N-(3-sulfopropyl)-3methylaniline sodium salt and 4-amino-antipyrine

He et al. (2014)

Color.

0–7 μM

0.3 μM

Substrate: TMB

Shamsipur et al. (2014)

Cysteine Fe-MIL-88NH2 MOF Homocysteine Fe-MIL-88NH2 MOF Choline

Acetylcholine Fe3O4 nanospheres/ reduced graphene oxide Platinum nanoparticles Glutathione Carbon nanodots


He et al. (2014)

Continued

187

188

Methods

Linear Range

Limit of Detection

Fe3O4 MNPs

Color.

10–250 μM

5 μM

Au@Pt core/shell nanorods

Color.

30–300 μM

30 μM

Fe3O4 MNPs

Color.

10–200 mg/L

5 mg/L


Fluor.

2–80 μM

2 μM

Color.

1–800 nM

0.2 nM

Nanozymes

Comments

References

Substrate: TMB Fe3O4 was encapsulated in mesoporous silica with cholesterol oxidase. Showing the recycle capability. Comparison between free MNPs vs. encapsulated MNPs. Substrate: OPD

Kim et al. (2011b)

Substrate: ABTS Galactose in dried blood samples from normal persons and patients was tested. Plates were used for sensing. Galactose oxidase was electrostatically assembled onto the Fe3O4@PDDA. Oxidation of AU gave fluorescence.

Kim et al. (2012b)

Substrate: TMB

Ni et al. (2014)

Cholesterol

Liu et al. (2012a)

Galactose


Melamine Bare AuNPs



Kanamycin AuNPs

Color.

1–100 nM

4.52 nM

Substrate: TMB AuNPs were modified by kanamycin aptamer.

Sharma et al. (2014)

Color.

1–200 μM

0.5 μM

Substrate: TMB Xanthine in serum and urine samples was tested.

Wang et al. (2011)

Ag nanoparticles

Color.

0.5–800 nM

0.125 nm

Sun et al. (2014)

Carbon nanodots

Color.

0–0.46 μM

23 nM

Substrate: TMB Mercury(II) in blood and wastewater was tested. Substrate: TMB

Platinum nanoparticle

Color.

0.01–4 nM

8.5 pM

Substrate: TMB

Mohammadpour et al. (2014) Wu et al. (2014)

E-chem

0.1–1 mM

4 μM

The calcium ion in a milk sample was tested.

Mu et al. (2014)

Xanthine AuNC@BSA Mercury(II)

Co3O4 nanomaterials

4-AAP, 4-aminoantipyrine; AA, ascorbic acid; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); APTES, 3-aminopropyltriethoxysilane; AU, amplex ultrared; AuNPs, gold nanoparticles; BSA, bovine serum albumin; Color., colorimetric; DPD, N,N-diethyl-p-phenylenediamine sulfate; E-chem, electrochemical; Fluor., fluorometric; GOx, glucose oxidase; HRP, horseradish peroxidase; MNPs, magnetic nanoparticles; MPTES, 3-mercaptopropyltriethoxysilane; MWCNTs, multiwalled carbon nanotubes; OPD, o-phenylenediamine; PDDA, poly(diallyldimethylammonium chloride); TMB, 3,3′,5,5′-tetramethylbenzidine; UA, uric acid. Adapted with permission from Liu, Y., Yuan, M., Qiao, L.J., Guo, R., 2014b. An efficient colorimetric biosensor for glucose based on peroxidase-like proteinFe3O4 and glucose oxidase nanocomposites. Biosens. Bioelectron. 52, 391–396. Copyright (2016) Royal Society of Chemistry.


Calcium Ion

189

190

Nanozyme

Target

Format

Fe3O4 nanoparticles (NPs) with dextran coating

preS1 TnI

Fe3O4 NPs with chitosan coating

Mouse IgG CEA

Antigen-down immunoassay Capture–detection sandwich immunoassay Antigen-down immunoassay Capture–detection sandwich immunoassay Sandwich immunoassay Antigen-down immunoassay

Fe2O3 NPs with Prussian blue coating Ferric nanocore residing in ferritin Fe(1-x)MnxFe2O4 NPs with PMIDA coating MnFe2O4 NPs with citric acid coating Fe-TAML

Co3O4 nanoparticles

CEA IgG

Comments

References Gao et al. (2007)

Gao et al. (2008)

Zhang et al. (2010)

Avidin Nitrated human ceruloplasmin Mouse IgG

Antigen-down immunoassay Sandwich immunoassay

Avidin–biotin interaction

Tang et al. (2011)

Antigen-down immunoassay

Both direct and indirect assay.

Sticholysin II


Human IgG


Bhattacharya et al. (2011) Figueroa−Espi et al. (2011) Kumari et al. (2014)

Vascular Endothelial growth factor


Fe-TAML was encapsulated inside mesoporous silica nanoparticles.

Dong et al. (2014)


Table 7.3 Nanozyme as Peroxidase Mimics for Immunoassay

Platinum nanoparticles Platinum nanoparticles on graphene oxide Gold

Cytokeratin 19 fragments Folate receptors

Sandwich immunoassay Antigen-down immunoassay

Respiratory syncytial virus

Sandwich immunoassay

Rod-shaped Au@PtCu

Human IgG


Au@Pt nanorods with PSS coating Graphene oxide

Mouse IL-2


PSA


Nanoparticles–graphene oxide hybrids

Song et al. (2014) Zhang et al. (2014b) The peroxidase-like activ- Zhan et al. (2014) ity of gold. Nanoparticles–graphene oxide hybrids could be enhanced by mercury(II). The detection limit can Hu et al. (2014a) be as low as 90 pg/mL. He et al. (2011) Clinical samples were tested.

Qu et al. (2011)


CEA, carcinoembryonic antigen; PMIDA, N-(phosphonomethyl)iminodiacetic acid; PSA, prostate-specific antigen; PSS, poly(styrenesulfonate). Adapted with permission from Liu, Q.Y., Zhu, R.R., Du, H., Li, H., Yang, Y.T., Jia, Q.Y., Bian, B., 2014c. Higher catalytic activity of porphyrin functionalized Co3O4 nanostructures for visual and colorimetric detection of H2O2 and glucose. Mater. Sci. Eng. C Mater. Biol. Appl. 43, 321–329. Copyright (2016) Royal Society of Chemistry.

191

192


FIGURE 7.4 Immunoassay based on a nanocomposite entrapping both magnetic nanoparticles (MNPs) and platinum nanoparticles (PtNPs) in mesoporous carbon. Reprinted with permission from Kim, M.I., Ye, Y., Woo, M.-A., Lee, J., Park, H.G., 2014. Adv. Healthc. Mater. 3, 36–41. Copyright (2013) John Wiley and Sons.

FIGURE 7.5 Rationally modulate the oxidase-like activity of nanoceria for self-regulated bioassays. AChe, acetylcholinesterase; TMB, 3,3′,5,5′-tetramethylbenzidine. Reprinted with permission from Cheng, H., Lin, S., Muhammad, F., Lin, Y.-W., Wei, H., 2016a. ACS Sens. 1, 1336–1343. Copyright (2016) American Chemical Society.


2.3 NANOZYME COMBINES PEROXIDASE AND OXIDASE MIMICS FOR COLORIMETRIC SENSING When an oxidase is combined with a peroxidase mimic, the corresponding oxidase substrate can be determined, as shown in Fig. 7.2B. According to the previous studies, glucose, choline, d-alanine, uric acid, and xanthine have been detected using peroxidase mimics and natural oxidase combinations (see Table 7.2 for more examples). Wei and Wang (2008) developed a sensitive and selective colorimetric approach to detect glucose by combining GOx with Fe3O4 MNPs as the peroxidase mimic (Fig. 7.6). In this direction, Qu et al. also designed a nanosystem to mimic enzyme cascade reactions for selective and sensitive colorimetric detection of glucose. They used V2O5 nanowires as peroxidase mimic and AuNP as glucose oxidase (GOx) mimic, respectively (Fig. 7.7) (Qu et al., 2014). Besides the glucose detection in diluted

FIGURE 7.6 Colorimetric detection of glucose by combining glucose oxidase with Fe3O4 magnetic nanoparticles as a peroxidase mimic. (A) A dose-response curve for glucose detection using glucose oxidase (GOx) and Fe3O4 magnetic nanoparticles (MNPs). (B) The linear calibration plot for glucose. The error bars represent the standard deviation of three measurements. (C) Typical photographs for glucose detection with the colorimetric method developed using GOx and Fe3O4 MNPs. Reprinted with permission from Wei, H., Wang, E., 2008. Fe3O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2O2 and glucose detection. Anal. Chem. 80, 2250–2254. Copyright (2008) American Chemical Society.

193

194


FIGURE 7.7 (A) Schematic representation of the preparation of V2O5–PDA–AuNP composite material and its mimicking of an enzyme cascade reaction process. (B) Illustration of the glucose oxidase-like catalytic activity of AuNPs regulated by DNA hybridization, resulting in suppression and restoration of mimicking of the enzyme cascade reaction by the V2O5–PDA– AuNP composite material. This mechanism underpins the colorimetric sensing of target DNA. ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); AuNP, gold nanoparticle; PDA, polydopamine; ssDNA, single-stranded DNA. Reprinted with permission from Qu, K.G., Shi, P., Ren, J.S., Qu, X.G., 2014. Nanocomposite incorporating V2O5 nanowires and gold nanoparticles for mimicking an enzyme cascade reaction and its application in the detection of biomolecules. Chem. A Eur. J. 20, 7501–7506. Copyright (2014) John Wiley and Sons.

blood and apple juice, the nanocomposite with dual enzyme-mimetic activities was also used for the colorimetric sensing of target DNA. It is well known that singlestranded DNA (ssDNA) and double-stranded DNA (dsDNA) have different affinities toward AuNPs. The ssDNA strongly binds to AuNPs, leading to effective surface passivation and suppression of the AuNP’s catalytic activity. In the presence of target DNA, it can hybridize and form dsDNA, which actually has a weaker affinity toward AuNPs and thus leads to the recovery of GOx activity of AuNPs. By taking advantage of this phenomenon, as low as 2.3 nM of target DNA was detected.


2.4 OTHERS BASED ON FLUOROMETRIC, CHEMILUMINESCENT, AND ELECTROCHEMICAL SENSING Besides colorimetric sensing, numerous other transduction signals such as fluorescence, chemiluminescence (CL), and electrochemistry have also been employed for constructing nanozymes-based biosensors (Fig. 7.2C and Tables 7.1–7.3). Fang et al. (2014) fabricated Fe3O4/reduced graphene oxide nanocomposites as peroxidase mimic to modify glassy carbon electrode for the reliable detection of extracellular H2O2 released from living cells, which may be useful for understanding biological effects of nanomaterials. In a recent work by Zhang et al. (2015b), 3D Fe- and N-doped carbon nanostructures as peroxidase mimic were used for fluorescence detection of H2O2. The nanostructure’s high peroxidase-mimicking activity was attributed to the presence of highly active Fe–N and doped N species as well as the large surface area of carbon, which in turn led to highly sensitive detection of H2O2 with a detection limit of 68 nM. Li et al. (2015b) combined the peroxidase-mimicking activity of nanozyme and surface-enhanced Raman scattering (SERS) together to detect melamine in milk powder. They synthesized bifunctional chitosan-modified popcorn-like Au–Ag nanoparticles with both high peroxidase-like activity and SERS properties. The charge transfer complex produced via the oxidation of 3,3′,5,5′-tetramethylbenzidine by H2O2 would give strong SERS signals; while in the presence of melamine, the intensities of signals would be decreased because of the melamine-mediated consumption of H2O2. Based on this method, trace melamine was sensitively detected with a limit as low as 8.51 nM. An innovative concept for chemiluminescent detection of sulfite was reported by Zhang et al. (2013). It was demonstrated that CoFe2O4, as an oxidase mimic, could catalyze luminol oxidation to produce intensified CL, and the CL intensity was affected by sulfite. The role of sulfite in the luminol–CoFe2O4 NP–sulfite system was found to be concentration dependent. At low sulfite concentration, CL inhibition was observed because of the consumption of dissolved O2 by sulfite; however, at high sulfite levels, CL enhancement occurred because of the generation of stronger oxidative radicals, which could react with luminol. Based on this, they constructed a flow injection CL assay to determine the trace sulfite level in white wine samples with satisfactory results. Fan, Li and et al. reported the modulation of GOx-like catalytic activities of AuNPs with DNA (Luo et al., 2010a; Zheng et al., 2011). As mentioned in Section 2.2, ssDNA and dsDNA have different binding affinities toward AuNPs. Based on this phenomenon, a general sensing platform was developed (Fig. 7.8). The probe ssDNA would interact with AuNPs and thus inhibited their catalytic activities. In the presence of targets (such as a complementary ssDNA, a complementary miRNA, or an analyte for an aptamer if the probe ssDNA was an aptamer), the probe ssDNA would form a complex with its target, which could lead to the recovery of the catalytic activities of AuNPs nanozymes. The colorimetric, chemiluminescent, and AuNPs’ intrinsic plasmonic signals have been used for the signaling. Recently, nanozymes have also been explored for point-of-care. For instance, Ebola virus and human chorionic gonadotropin (hCG) were efficiently and rapidly detected with portable devices such as a dipstick strip or a smart phone, as shown in Fig. 7.9 (Duan et al., 2015; Kim et al., 2015; Zhu et al., 2014). Catalase catalyzes

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FIGURE 7.8 Illustration of the glucose oxidase-like catalytic activity of gold nanoparticles (AuNPs) regulated by DNA hybridization, which can be either amplified by horseradish peroxidase (HRP) cascaded color or chemiluminescence variations (path a) or lead to nanoplasmonic changes owing to size enlargement (path b). Orange strand = target; green strand = adsorption probe. ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Reprinted with permission from Luo, W., Zhu, C., Su, S., Li, D., He, Y., Huang, Q., Fan, C., 2010a. ACS Nano 4, 7451–7458. Copyright (2010) American Chemical Society.

Absorbent pad

Control line

Nanozyme

(A)

Nitrocellulose membrane

AuNPs

Test line

(B)

FIGURE 7.9 (A) Nanozyme-based strips for Ebola detection using Fe3O4 magnetic nanoparticles as peroxidase mimic and (B) for human chorionic gonadotropin detection using hierarchically structured platinum nanoparticles as peroxidase mimic. AuNPs, gold nanoparticles. (A) Reprinted with permission from Duan, D., Fan, K., Zhang, D., Tan, S., Liang, M., Liu, Y., Zhang, J., Zhang, P., Liu, W., Qiu, X., Kobinger, G.P., Fu Gao, G., Yan, X., 2015. Biosens. Bioelectron. 74, 134–141. Copyright (2015) Elsevier. (B) Reprinted with permission from Kim, M., Kim, M.S., Kweon, S.H., Jeong, S., Kang, M.H., Kim, M.I., Lee, J., Doh, J., 2015. Adv. Healthc. Mater. 4, 1311–1316. Copyright (2015) John Wiley and Sons.

3. Nanozyme for Sensing in Living Systems

FIGURE 7.10 Pt nanoparticles (PtNPs) as catalase mimics for immunoassay on a volumetric bar-chart chip. Reprinted with permission from Song, Y.J., Xia, X.F., Wu, X.F., Wang, P., Qin, L.D., 2014. Integration of platinum nanoparticles with a volumetric bar-chart chip for biomarker assays. Angew. Chem. Int. Ed. 53, 12451–12455. Copyright (2014) John Wiley and Sons.

the decomposition of H2O2 into H2O and O2 gas, and by exploiting the generation of O2 gas, Song et al. (2014) developed a microfluidics platform named volumetric barchart chip, which could easily measure the volume of produced O2 gas with colored solution in the chip channel (as shown in Fig. 7.10). They used PtNPs as the catalase mimic and then labeled PtNPs with detection antibody for diagnosis. With such a device, they have detected cancer biomarker cytokeratin 19 fragment (CYFRA 21-1) in serum and HER2 expressed on three breast cancer cell surfaces.

3. NANOZYME FOR SENSING IN LIVING SYSTEMS Despite the substantial progress in nanozymes for in vitro sensing of important biotargets, limited efforts have been devoted to developing nanozymes for biosensing in living systems. As we know, the catalytic activity of currently developed nanozymes is still far lower compared with their natural counterparts. What’s worse, these inorganic nanomaterial-based enzyme mimics fail to offer exquisite three-dimensional conformations in the active sites with selectivity toward specific substrates. Hence, these drawbacks have limited their practical applications in living systems (Wei and Wang, 2013; Larsen et al., 2011). Recent progress showed that the selectivity problems could be partially solved by combining a nanozyme with a natural enzyme (Wei and Wang, 2008). However, the catalytic reactions catalyzed by a nanozyme and a natural enzyme were split into single isolated reactions rather than coupled tandem reactions. Such a separation would inevitably induce lower local concentrations of catalysts and substrates, diffusion barriers,

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and decomposition of unstable intermediates, therefore resulting in poor performance of the overall enzymatic reactions. To tackle these challenges, Wei et al. recently pioneered in engineering an integrated nanozymes (INAzymes), which was inspired by biological systems using multiple enzymes within subcellular compartments to enhance the cascade catalytic activities, for in vivo neurochemical monitoring in living brains (Cheng et al., 2016b). They fabricated this highly effective INAzymes by coassembling molecular catalysts (e.g., hemin) and natural enzymes (e.g., GOx) inside zeolite imidazolate framework (i.e. ZIF-8) nanostructures (Fig. 7.11A). Because of the nanoscale proximity effect, the obtained INAzyme of hemin and GOx exhibited more than 600% enhancement of the catalytic activity when compared with the mixture of hemin@ZIF-8 and GOx@ZIF-8 (Fig. 7.11C). After establishing that the INAzyme could be used for sensitive and selective detection of glucose in vitro, they went on to construct an analytical platform by immobilizing the INAzyme into the channel of a microfluidics chip. When assisted with in vivo microdialysis, the dynamic changes of cerebral biomolecules (i.e. glucose) following ischemia and perfusion could be successfully monitored with the help of the INAzyme-based sensing platform (Fig. 7.11D and E). This strategy was general and applicable to other combination of catalyst guests (such as hemin/lactate oxidase and hemin/GOx/invertase).

4. CONCLUSIONS AND PERSPECTIVES This chapter highlights the recent exciting developments in nanozymes for biomedical sensing, especially engineering nanozymes for in vitro and in vivo sensing. Nanozymes as peroxidase mimic, oxidase mimic, catalase mimic, and even the combination of two enzymes have been used for sensing various bioactive small molecules (such as H2O2 and glucose), nucleic acids, metal ions, cancer cells, and so on. The most common sensing methods such as colorimetric sensors, fluorescent biosensors, chemiluminescent biosensors, and electrochemical biosensors have been discussed here in details. While the development of nanozymes in biomedical sensing has been entirely evidenced from the above-described examples, there are still some remaining challenges to be tackled. Here, we speculate a few such directions that should be considered in future studies. 1. The catalytic activity and efficiency of most nanozymes are still lower than that of the natural enzymes. Although engineering the nanozymes through surface modification or coassembly of multinanozymes within confined space may increase the stability and efficiency of nanozymes, yet the effective surface area is blocked. Therefore, better experimental techniques and detailed theoretical mechanisms are required to achieve a balance between the stability and activity, which would help develop higher performance nanozymes. 2. Most of the currently developed nanozymes are not able to offer exquisite threedimensional conformations in the active sites for selectively recognizing specific substrates. Recently, Yan, Gao and et al. have reported a new strategy to mimic

Adapted with permission from Cheng, H., Zhang, L., He, J., Guo, W., Zhou, Z., Zhang, X., Nie, S., Wei, H., 2016b. Anal. Chem. 88, 5489–5497. Copyright (2016) American Chemical Society.

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Integrated nanozymes (INAzymes) for monitoring the dynamic changes of brain glucose following ischemia/reperfusion. (A) Schematic and transmission electron microscopy image of the INAzymes. (B) Reactions catalyzed by the INAzymes. (C) Normalized cascade catalytic activity of the INAzyme (1) and the mixture of hemin@ZIF-8 and GOx@ZIF-8 (2) showing a more than 600% enhancement for the INAzyme when compared with the mixture of hemin@ZIF-8 and GOx@ZIF-8. (D) Schematic illustration of the global cerebral ischemia. (E) Continuously monitoring the dynamic changes of glucose level in the striatum of a living rat brain following global ischemia/reperfusion with the INAzyme-based sensing platform.

4. Conclusions and Perspectives

FIGURE 7.11

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the enzymatic microenvironment of natural HRP through modifying the Fe3O4 with single amino acid, which would broaden the future studies for preparing nanozymes with substrate selectivity (Fan et al., 2017). 3. For the future biomedical sensing applications, more broad and diverse bioactive targets should also be considered. As evidenced by the above-mentioned examples, biosensors have mostly been fabricated using peroxidase mimic, oxidase mimic, and the combination of these two nanozymes. To broaden this field, new materials should be explored for enzyme-mimicking activities for biosensing, rather than only mimicking redox enzymes (such as peroxidase, oxidase, and catalase). Moreover, DNA-nanozyme system has recently been reported for chiral recognition of L-/d-glucose, and such regulation strategies could be another future research direction in nanozymes (Zhan et al., 2015). . Because the biomedical sensing applications are evolved from in vitro sens4 ing to in vivo, the biosafety and translational promise of nanozymes should be considered and systematically studied. Although nanozymes are more stable and robust than natural enzymes, the in vivo complex environment may still result in some unexpected interactions with nanozymes. Although iron oxide nanoparticle-based reagents, such as Resovist, have already been approved for clinical use (Huang et al., 2009), the toxicities of various promising nanozymes are still needed to be tested before clinical trials.

ABBREVIATIONS 4-AAP 4-Aminoantipyrine AA Ascorbic acid ABTS 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) AgNP Silver nanoparticle APTES 3-Aminopropyltriethoxysilane AU Amplex ultrared AuNC Gold nanocluster AuNP Gold nanoparticle BA Benzoic acid BSA Bovine serum albumin CD Cyclodextrin CEA Carcinoembryonic antigen CNT Carbon nanotube Color. Colorimetric DAB Diazoaminobenzene DOPA Dopamine DPD N,N-Diethyl-p-phenylenediamine sulfate dsDNA Double-stranded DNA E-chem Electrochemical ELISA Enzyme-linked immunosorbent assay EPR Electron paramagnetic resonance FBS Fetal bovine serum

References

Fluor. Fluorometric hCG Human chorionic gonadotropin HPNP 2-Hydroxypropyl-4-nitrophenylphosphate HRP Horseradish peroxidase LDH Layered double hydroxide LOD Limit of detection Meth Methods MNPs Magnetic nanoparticles MPTES 3-Mercaptopropyltriethoxysilane NMDA N-Methyl-d-aspartate NPs Nanoparticles OPD o-Phenylenediamine PDDA Poly(diallyldimethylammonium chloride) PLGA Poly(d, l-lactic-co-glycolic acid) PMIDA N-(phosphonomethyl)iminodiacetic acid PSA Prostate-specific antigen PSS Poly(styrenesulfonate) PVDF Polyvinylidene difluoride Ref References SBA-15 Santa Barbara amorphous type material SOD Superoxide dismutase ssDNA Single-stranded DNA TAML Tetraamidomacrocyclic ligand TMB 3,3′,5,5′-Tetramethylbenzidine UA Uric acid

ACKNOWLEDGMENTS We thank National Natural Science Foundation of China (21722503, 21405081, and 21550110189), 973 Program (2015CB659400), Natural Science Foundation of Jiangsu Province (BK20160615 and BK20130561), Shuangchuang Program of Jiangsu Province, PAPD program, Fundamental Research Funds for Central Universities, Six Talents Summit Program of Jiangsu Province, Open Funds of the State Key Laboratory of Coordination Chemistry (SKLCC1619), Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201501), Open Funds of the State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1704), China Postdoctoral Science Foundation (2016M590437 and 2015M581770), and Thousand Talents Program for Young Researchers for financial support.

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Systematic Evolution of Ligands by Exponential Enrichment for Aptamer Selection

8

Meral Yüce1, Hasan Kurt2, Babar Hussain3, Hikmet Budak3,4 1Sabanci

University Nanotechnology Research and Application Centre, Istanbul, Turkey; Medipol University, Istanbul, Turkey; 3Sabanci University, Istanbul, Turkey; 4Montana State University, Bozeman, MT, United States

2Istanbul

CHAPTER OUTLINE 1. Introduction�� 212 2. Potential Aptamer Targets�� 212 3. Advantages of Aptamers�� 213 4. Random Oligonucleotide Libraries�� 214 5. Systematic Evolution of Ligands by Exponential Enrichment�� 214 5.1 Incubation of Random Oligonucleotide Pool With Target of Interest�� 215 5.2 Separation of Unreacted Oligonucleotides and Elution of Target-Bound Oligonucleotides�� 217 5.3 Amplification of the Eluted Aptamer Candidates�� 218 5.3.1 Single-Stranded DNA Production Methods�� 221 5.3.2 Enrichment of the Random Oligonucleotide Library Through Iteration�� 223 6. Sequencing of the Enriched Aptamer Pools�� 224 6.1 Evaluation of Sequencing Data�� 225 7. Evaluation of Aptamer-Binding Kinetics�� 228 8. Post–Systematic Evolution of Ligands by Exponential Enrichment Modifications�� 233 8.1 Length Modification: Truncation�� 233 8.2 Backbone Modification: Sugar Ring Alteration�� 234 8.3 Tail Modification: 3’ or 5’ Modification�� 235 9. Conclusion�� 236 Acknowledgment�� 237 References�� 237


211

212

CHAPTER 8 Systematic Evolution of Ligands

1. INTRODUCTION Systematic evolution of ligands by exponential enrichment abbreviated as SELEX is an in vitro method of ligand screening for a selected analyte from random DNA, RNA, or peptide libraries. The method was developed independently by two research groups in 1990. In the study of Ellington and Szostak, a set of RNA molecules that specifically bind to a variety of organic dyes were isolated from a random sequence RNA library and they were termed as aptamers (Ellington and Szostak, 1990). The randomized RNA pool used in that study was developed through the solid-phase phosphoramidite chemistry, which displayed an effective complexity of ∼1013 individual molecules. In the study of Tuerk and Gold, a similar approach was applied by coincidence for selection of RNA-binding motifs against bacteriophage T4 DNA polymerase (gp43) from a pool that was randomized at specific positions and theoretically included 65,536 individual sequences (Tuerk and Gold, 1990). The iteration of the evolution (variation), selection, and replication steps in the procedure was named as SELEX by Craig Tuerk, who was a PhD student of Larry Gold at the time (Gold, 2015). In fact, those highly degenerate or random oligonucleotide libraries had been previously synthesized and used in different ways (Kaiser et al., 1987; Oliphant and Struhl, 1988, 1989; Oliphant et al., 1989); however, the rounds of incubation, selection, and amplification steps were initially applied in the aforementioned two core studies. The method of SELEX has attracted considerable attention from the worldwide scientific community and related industrial sector since the inception. Thereafter, aptamers are perceived as small DNA, RNA, or peptide-based antibody alternatives, with target-specific three-dimensional structures selected from large-scale random libraries through various in vitro iteration processes. In this chapter, the key steps of an iteration cycle for DNA and RNA pools were described in a comprehensive manner, which could be of interest for investigators entering the field.

2. POTENTIAL APTAMER TARGETS In spite of the fact that proteins, which bind to nucleic acids as a part of their function, are initially supposed to be the main targets of the aptamers, SELEX strategy has been successfully applied to ions, small chemical moieties, nanomaterials, cell surface proteins, growth factors, organelles, and even whole cells as the targets (Yüce et al., 2015). The flexibility of the method and diversity of the random oligonucleotide libraries have enabled the generation of hundreds of target-specific aptamers so far, which have been employed in sensing (Akki et al., 2015; Kurt et al., 2016), imaging (Fan et al., 2016), biomarker discovery (Jin et al., 2016), targeted drug delivery (Liu et al., 2016), and pharmaceutical applications (Kimoto et al., 2016), as a synthetic and cost-effective alternative to the conventional affinity probes. Aptamers can be screened for molecules that cannot be targeted by antibodies due to steric hindrance, structural properties, toxicity, or lack of immune response (Bruno et al., 2012; Lauridsen et al., 2012). Besides, they are nonimmunogenic themselves (Eyetech and Group, 2002).

3. Advantages of Aptamers

3. ADVANTAGES OF APTAMERS Nucleic acid-based aptamers offer substantial advantages over antibodies. First of all, in vitro selection provides convenience during the selection, which is independent of animals. Aptamer development process can be tuned to a broad range of environmental conditions, mimicking the desired application media. Economic feasibility of aptamer development is fairly compelling as a result of the appropriateness of aptamers (in the range of around 40–80 bases) for highyield chemical synthesis, allowing modifications and conjugations for various applications. In contrast, antibody development faces the high cost of labor, animal sacrifice, and low yields. As mentioned earlier, aptamers cover a wider range of target molecules as compared to the antibodies. They can bind to chemical compounds (Zimmermann et al., 2000), ions (Wu et al., 2014), proteins (Sarell et al., 2014), toxic agents (Ma et al., 2015), pathogens (Huang et al., 2015), nonimmunogenic targets (Dausse et al., 2005), and whole cells (Sefah et al., 2010). Also, the relatively small molecular weight of the aptamers (∼10–30 kDa) allows them to reach the epitopes that antibodies (∼150 kDa) cannot capture on small targets, reducing the possible risk of steric hindrance. On the other hand, target repertoire of antibodies is limited to the immune response of the host cells. Antibodies have relatively low chemical, physical, and thermal stability that limits their storage and on-site applications. For example, they could lose their unique binding properties arising from their tertiary structure under slightly elevated temperatures or pH conditions. On the other hand, chemical synthesis suitability of aptamers offers a broad range of modification options to improve the ultimate stability without compromising binding properties. Depending on the application conditions, an aptamer can be chemically engineered for higher base stacking, chain flexibility, or resistance to enzymatic activity over a range of biological media, before or after the selection (Tolle et al., 2015; Gao et al., 2016). In fact, chemical functionalization provides a significant opportunity to compensate the features that aptamers naturally lack. Especially, aptamers with low molecular weights experience issues in therapeutic applications because of renal filtration and enzymatic degradation. The versatility of chemical aptamer modification remedies these problems with high-molecular-weight polymer conjugates or modified nucleotides, which is discussed later in this chapter. On the other hand, binding affinities of the aptamers are not always as high as their counterpart, antibodies. Thus, aptamer employment in practical, analytical, and therapeutic applications is less frequent (Tombelli et al., 2005; Thiel and Giangrande, 2009). Considering the welldocumented research and clinical studies in the antibody field, aptamer research, which has been only developed since the 1990s, still has a long way to go from laboratory benches to the practical implementation in biotechnology market. Fortunately, the first aptamer-based therapeutic called as Pegaptanib or Macugen for the treatment of age-related macular degeneration was approved by FDA in 2004 (Ng et al., 2006), and many others are under clinical trials (Ni et al., 2011; Sundaram et al., 2013), showing signs of future excellence.

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4. RANDOM OLIGONUCLEOTIDE LIBRARIES Aptamers are selected from random oligonucleotide libraries that contain as many individual sequences as possible to guarantee the pool diversity, from which the strongest binders can be isolated through iteration. Combinatorial nucleic acid synthesis chemistry is used to produce a large number of different sequences, called library or pool, which allows modified bases, unnatural bases, or variety of labeling moieties to be incorporated into the oligonucleotide at the desired positions (Ecker et al., 1993; Pollard, 1998). Combinatorial chemistry is described as “the systematic and repetitive, covalent connection of a set of different ‘building blocks’ of varying structures to each other to yield a large array of diverse molecular entities” (Gallop et al., 1994). The pools produced by combinatorial chemistry may have a random distribution of the bases or a distribution centered on a known (wild-type) sequence. During the design of the pools, the caution should be taken to avoid mispriming or complementary ends, which can severely affect the pool amplification. Currently, there are several companies worldwide providing ready to use or customized random libraries and primers with modifications for specific applications, including the aptamer selection. DNA aptamers are selected from random DNA pools that contain a random region of 20–80 bases flanked by primer-binding regions for amplification. The short libraries are easier to produce and manage, whereas longer libraries enhance the pool diversity (Dihua et al., 2015). The ratio and type of the individual bases in the pool are tunable in most cases, which could be 1:1:1:1 or GC-rich or unproportional with natural or modified nucleotides. For RNA aptamer selection, random DNA pool includes a T7 promoter region at the beginning of one primer site for in vitro transcription (Milligan et al., 1987). Amplification of the RNA aptamers is performed after cDNA production through the reverse transcription technique. RNA pools offer more diverse secondary structures as compared with DNA pools, as a result of the 2′-OH group of the ribose sugar. The primer could also be modified with various molecules such as biotin for single-stranded DNA (ssDNA) production in the workflow or fluorescent tag for tracking over the course of SELEX rounds. The pools can be synthesized as partially or entirely randomized. The partially degenerate pools are named as doped pools that contain derivatives of a known sequence region or an even known aptamer sequence. Degeneration level of the doped pool is arranged in a way that it could yield the desired level of diversity and free from much of those nonfunctional sequences.

5. SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT Once the selection pool is designed and synthesized, it is incubated with the target of interest, which is considered as the first step of a SELEX cycle. The second step of the process is the separation of the unbound oligonucleotides from the aptamer–target

5. Systematic Evolution of Ligands by Exponential Enrichment

complex and elution of the bound oligonucleotides from the target. The eluted oligonucleotides are amplified and converted into ssDNA for DNA aptamer selection while they are reverse transcribed into cDNAs for amplification in case of RNA aptamer selection. The recovered single-stranded oligonucleotides are used as the input library for the next iteration round. The cycles are repeated until the random pool gets dominated or enriched by the sequences that have a noticeable binding affinity toward the target. Once the desired level of affinity is obtained, the enriched pool is sequenced and analyzed with bioinformatics tools for consensus sequences or motif regions that are thought to be in charge of selective binding to the target of interest. Bioinformatics analyses are followed by the chemical synthesis of the selected aptamer candidates and assays for the confirmation of affinity and selectivity. The aptamers with high affinity toward their targets are used in various fields, ranging from sensing, imaging, function blocking, drug delivery, and nanotechnology to therapeutic applications. The major steps of a magnetic bead-based SELEX are illustrated in Fig. 8.1 and explained further below with examples from the literature.

5.1 INCUBATION OF RANDOM OLIGONUCLEOTIDE POOL WITH TARGET OF INTEREST Incubation of the initial random pool with the target molecule can be directly in solution where the molecules are free in their 3D structures, or it can proceed on a support surface, which is coated with the target molecule. Based on the applications, target molecules, and sources, a broad range of incubation strategies have been reported in the literature (Yüce et al., 2015). Solution-based incubations are conducted in a tube that includes a portion of the pool (∼1013−14 individual sequences) dissolved in a buffer of selection and the target molecule preferentially prepared in the same buffer as the pool. The pool is usually denatured (for example, heating at 95°C) and renatured (leaving at room temperature) prior to the incubation, allowing individual sequences to take their unique secondary structures. On the other side, the target molecule is favored to be in its purest form to eliminate the evolution of oligonucleotides against the residual targets. The incubation solution can be various buffers such as phosphate-buffered saline (PBS), tris-buffered saline (TRIS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 3-(N-morpholino) propanesulfonic acid (MOBS), water, and diluted serum, supplemented with additional ions (Mg, Na, and K) or surfactants (Tween) to reduce nonspecific binding and keep the oligonucleotides stable. The temperature of the incubation can also vary from room temperature to 37°C. Following the incubation step, centrifugation, electrophoresis, or commercial filter units (Millipore, USA) can be applied to remove unreacted or weakly bound oligonucleotides from the aptamer–target complex. In the case of immobilization-based incubation, the target molecule is immobilized on a surface through either with a specific tag such as polyhistidine (Tok and Fischer, 2008), biotin, and thiol or N-hydroxysuccinimide (NHS)–1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) chemistry for covalent attachment

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FIGURE 8.1 The steps of a magnetic bead-based DNA aptamer selection. (A) Modification of magnetic beads with the target of interest. (B) Preparation of the random oligonucleotide pool. (C) Incubation of the target modified magnetic beads with the random pool. (D) Separation of the target-bound oligonucleotides from unreacted oligonucleotides with a magnet. (E) Elution of the target-bound oligonucleotides from the carrier surface. (F) Amplification of the eluted sequences for the enrichment of the pool. (G) Single-stranded DNA production step to eliminate antisense strands. (H) Sequencing and data analyses of the enriched final pool. PCR, polymerase chain reaction.

(Nadal et al., 2012). The literature suggests using specific tags instead of covalent binding in which target molecules are distributed in a random orientation. In both cases, the target molecules are not allowed to move freely in their 3D structure, and thus the selection is constrained with the preferred orientation of the molecules on the surface. Although the magnetic beads are among the most used immobilization surfaces for aptamer selection (Mann et al., 2005; Nadal et al., 2012; Hünniger et al., 2014; Lin et al., 2015), there are also gold chips (Ngubane et al., 2014; Dausse et al., 2016), affinity columns (Vianini et al., 2001), microfluidics (Hybarger et al., 2006;


Lin et al., 2015), ELISA plates (Arnold et al., 2012), coverslips (Lauridsen et al., 2012), and nanoporous sol–gel microarray droplets (Lee et al., 2013). Besides, these methods can offer contamination-free selection, parallel selection, amplification-free selection, or fast selection options, all enabling the best of selection in different conditions. Ultimately, the incubation step is followed by the separation and elution steps that are described below.

5.2 SEPARATION OF UNREACTED OLIGONUCLEOTIDES AND ELUTION OF TARGET-BOUND OLIGONUCLEOTIDES In each SELEX iteration, there will be a vast number of oligonucleotides that do not bind to the target and freely suspended in the incubation solution. Thus separation of the unreacted oligonucleotides is required before the elution of target-bound oligonucleotides (Darmostuk et al., 2015). To identify the oligonucleotides that bind to an analyte, their elution from the complex is also necessary so that the sequences can be amplified, enriched, and utilized in the following steps. The separation step may vary according to the immobilization method. If the pool and target are incubated freely in a solution, a centrifuge or filtering step could be used to eliminate the unreacted oligonucleotides. In electrophoresis-based selection methods, the separation can be visualized naturally on the gel by the small size of free nucleotides compared to the target–aptamer complexes. If magnetic beads are used for immobilization, a magnet will be practical to remove the weakly bound or unrelated oligonucleotides. The chip platforms are also practical for the purpose in which the unreacted nucleotides can be isolated through the integrated microfluidics. The elution step, on the other hand, usually includes a step of heating the target and oligonucleotide complex at around 95–100°C to elute the oligonucleotide sequences by weakening the hydrogen bonding. Yang et al. (2013) reported the separation of unbound and nonspecifically bound ssDNA sequences with three consecutive washes with 500 μL of 0.2% bovine serum albumin (BSA) and centrifugation at 8000 × g for 10 min. For elution of the bound ssDNA, nanoparticle–ssDNA conjugate solution in 50 μL ddH2O was heated at 100°C for 5 min and eluted sequences were collected in the supernatant by centrifugation at 22,000 × g for 15 min. Bawazer et al. (2014) described a similar procedure for the elution of bound DNA from the targeted ZnO nanoparticles after the incubation step. For separation of the unbound DNA sequences, nanoparticles were washed four times each with 1 mL of binding buffer (300 mM NaCl, 5 mM MgCl2, 20 mM Tris–HCl, pH 7.6) and water by centrifugation and the supernatant was removed by aspiration. For elution of the bound sequences, nanoparticle–DNA conjugates were suspended in 100 μL elution buffer (1 mM ethylenediaminetetraacetic acid [EDTA], 10 mM Tris–HCl, pH 8.0), which was heated at 95°C for 5 min followed by several seconds of centrifugation at 20,000 × g, and the supernatant containing the eluted DNA was shifted to a new test tube for polymerase chain reaction (PCR) amplification. Centrifugation is generally applied in the SELEX methods where the target molecule is not immobilized on a carrier surface, thus free in the incubation media. Another simplified elution process was

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reported by Lauridsen et al. (2012) who linked the target α-bungarotoxin molecule over a PEGylated glass coverslip by a covalent bond. The coverslip was incubated with fluorescent ssDNA library and washing step was applied to remove the unbound oligonucleotides. The fluorescent signal of the aptamer–target conjugate was monitored by fluorescence microscopy. For elution, the coverslip was crushed, and bound sequences were eluted by the heating again. This procedure did not require any sophisticated equipment, so it could be a convenient method among other single-step aptamer selection procedures. Another interesting elution method is based on enhanced salt concentration. In one study, the target Kallikrein-related peptidase 6 was immobilized on an ELISA plate and incubated with the random oligonucleotide library overnight (Arnold et al., 2012). For the separation of unbound and weakly bound sequences, protein–ssDNA complex was incubated with 100 μL of PBS (5 mM MgCl2 and 150 mM NaCl) for 5 min followed by solution collection. For the elution, protein-ssDNA complex was treated with 0.5, 1.0, 1.2, 1.4, and 1.5 M NaCl solution for 5 min each. Afterward, 1.4 and 1.5 M salt solution were desalted by 10 kDa MW microcon filters for further amplification. It was found that higher NaCl concentrations eluted more tightly bound sequences from the target as NaCl is capable of breaking the electrostatic interactions, the main interaction responsible for binding of proteins and DNA aptamers (Perez-Jimenez et al., 2004). Park et al. (2009) described an interesting protocol where a chip-based selection and elution of aptamers were achieved using a microfluidic system. The reported system utilizes the sol–gel arrays of proteins combined with a microfluidic chip for selecting RNA aptamers. The microfluidic chip consisted of five sol–gel binding droplets embedded with the target proteins. After the incubation of the oligonucleotide library with the target protein, microheaters attached on top of each sol–gel binding droplets triggered the elution of protein-bound sequences. This microheating-based SELEX system enhanced the selection efficiency and reduced the number of selection cycles required for the screening of high-affinity aptamers, enabling fast, high-throughput, and cost-effective screening methodology. It should also be noted that elution step might be skipped in the case that targets are immobilized on magnetics beads, which can be directly used in PCR as the template (Kojima et al., 2005). A good separation of the unreacted or weakly bound oligonucleotides from the incubation media and elution of the target-bound sequences without significant loss allow strong aptamer candidates to enrich and dominate the library over the course of SELEX cycles.

5.3 AMPLIFICATION OF THE ELUTED APTAMER CANDIDATES Amplification is the last major step of the pool iteration. The eluted DNA sequences are subjected to amplification and strand separation steps that are followed by the next round of selection. A direct PCR method is usually applied to amplify the eluted DNA sequences and that is followed by ssDNA production step where the positive


strand is separated and purified from the amplification media for the next incubation cycle (Mann et al., 2005; Woo et al., 2015). Additional steps of reverse transcription and in vitro transcription are applied in RNA aptamer selection where an RNA library transcribed from chemically synthesized random DNA library is used for the aptamer selection. After the elution, RNA sequences are reversely transcribed into DNAs for PCR amplification. The amplified DNA is transcribed into RNA for the next selection cycle during which all sequences become single-stranded, thereby ready for the next incubation cycle (Cho et al., 2004; Sarell et al., 2014; Thiel et al., 2015). The amplification is done by PCR, which is a technique to amplify a single or few template DNA molecules into thousands to million copies. PCR components, in addition to the template molecule, include deoxynucleotide triphosphates (dNTPs), forward, and reverse primers that are partially complementary to the sense and antisense DNA strands, polymerase enzyme (Taq, Pfu), MgCl2 for enzyme efficiency and specificity, and buffer solution to maintain a specific pH during the reaction. A simple PCR reaction consists of three steps and starts with denaturation of both strands at 95°C followed by annealing of primers at 55–62°C. In the last phase, DNA polymerase enzyme binds the complementary dNTPs from the dNTPs mixture and produces the new strand. The last process is called as elongation or extension and leads to the production of copies of the template DNA along with the repetition (5–25) of previous two steps (Song et al., 2012; Darmostuk et al., 2015). There are several types of PCR procedure including standard PCR, real-time PCR (RT-PCR), emulsion PCR (e-PCR), and digital PCR (d-PCR), and utilization of these PCR types in SELEX has been briefly discussed below. Bawazer et al. (2014) reported the successful use of standard PCR for identification of highly specific biomineralizing DNA aptamers, which had the specific binding ability to ZnO. The denaturation, annealing, and elongation steps of the PCR were performed at 95, 62, and 70°C, respectively. Similarly, Hamula et al. (2016) reported the amplification of eluted sequences by using polyA20/5Sp9-modified 40-nt forward primer and 20-nt reverse primer. The forward primer modified with a polyadenine tail (A20) at 5′ (polyA20/5Sp9) was linked to the original primer sequence through a triethylene glycol spacer (IDT Spacer 9). This addition was for the identification of forward strand from the reverse strand of the PCR product. The PCR products were converted into ssDNA by utilizing denaturing polyacrylamide gel electrophoresis (PAGE) where the designed primer enabled a high resolution for the separation of sense and antisense strands. RT-PCR is a type of PCR, which monitors the progress of amplification in realtime end of amplification, thus making it a potential procedure to reduce the number of SELEX cycles. Ruff et al. (2012) reported that capillary electrophoresis-SELEX combined with indirect measurement of aptamer–ligand complexes through RT-PCR identified aptamers highly specific to BSA just after 3 selection cycles, instead of 10–15 cycles in conventional SELEX. Ouellet et al. (2015) reported a new aptamer selection platform called high-fidelity SELEX that can block fixed regions of the pool, thus ensuring the functional diversity in the genome library. Amplification of

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the eluted DNA was done through d-PCR that not only removes the amplification artifacts but also stoichiometrically converts the amplicons into ssDNA, which is a prerequisite for the next selection rounds, making the selection process easy and simpler. Similarly, e-PCR is another amplification method, which significantly enhances the efficiency of SELEX cycle. For instance, e-PCR was used for identification of ABH2 protein-specific aptamers within first 3 SELEX rounds unlike 8–10 rounds while using standard PCR (Yufa et al., 2015). The entire elution solution can be utilized as a template for the amplification reaction as long as the final oligonucleotide concentration of the eluted pool is not too high, which might lead the production of nonspecific by-products (Tolle et al., 2014). Alternatively, the eluted pool can be divided into several small portions, and each part could be amplified separately under identical conditions (Bawazer et al., 2014). Optimization of the amplification conditions is essential to prevent nonspecific by-products (Tolle et al., 2014), which could compromise the successful selection of the target-specific oligonucleotides by dominating the pool content. Recurrent PCR reactions in SELEX are prone to artificial by-product formation. Tolle et al. (2014) reported that such by-products do appear in repetitive amplification process in SELEX and these are of two types: ladder- and nonladder type. Such by-products formed during the in vitro selection experiments prohibit the enrichment of the target-specific aptamers. Therefore, efforts have been put to reduce the by-product formation during SELEX. The standard PCR products of a 94 mer random DNA oligonucleotide library from two different PCR conditions were shown in Fig. 8.2. $

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FIGURE 8.2 Standard polymerase chain reaction (PCR) results of a 94 mer random DNA library. (A) Products obtained with 5–8 cycles of PCR reaction under optimized conditions. (B) Products obtained with excess amounts of template DNA in the reaction. Polyacrylamide gel electrophoresis (6%–8%) was run at 100–120 V. 5–6 μL of sample was mixed with 1–2 μL of loading buffer. Blue arrows (dark gray in print version) indicate the specific DNA bands while the yellow arrows (light gray in print version) refer to the unreacted primer or primer dimers.


As can be seen, the amplicons of the reaction A are entirely reasonable and bear almost no by-products. However, the amplicons from the reaction B contain a significant number of nonspecific products at different sizes that could interfere with the enrichment process of the library. Shao et al. (2011) have shown that e-PCR is one of the promising procedures that reduced the by-product formation during recurrent PCR cycles significantly to an undetectable level. In e-PCR, it was possible to completely eliminate product– product hybridization and most of the primer-product hybridization by optimization of the conditions, both of which are responsible for by-product formation in conventional PCR. The effectiveness of e-PCR for the inhibition of nonspecific by-products has also been established in another study (Yufa et al., 2015). Similarly, adding nonequal amounts of forward and reverse primers during amplification also helps in reducing the production of nonspecific by-products and ssDNA production, which is called as asymmetric PCR (He et al., 2013).

5.3.1 Single-Stranded DNA Production Methods In DNA aptamer selection process, obtaining quality ssDNA after each PCR step is vital because the produced ssDNA serves as the input for the next selection cycle. Therefore, care should be taken so that ssDNA is obtained with none or minimum sequence diversity loss. Various methods have been utilized for the production of ssDNA production. For example, asymmetric PCR is such method in which amplification is carried out with an excess amount of the forward primer and a small amount of the reverse primer, which results in excess production of the desired strand. However, purification of ssDNA from double-stranded DNA (dsDNA) molecules and other leftover reaction materials are required in this method (Svobodová et al., 2012). This approach has also been termed as single-primer-limited amplification and is based on the limited supply of reverse primer (∼5-fold molar quantity of the template) and was followed by plus-stranded DNA amplification with a forward primer (10-fold molar quantity). Plus-stranded ssDNA was obtained by gel excision. This procedure not only produced ssDNA but also optimization resulted in contamination-free products (He et al., 2013). Another commonly used and simple method is to use biotinylated reverse primers in the PCR, which yields biotinylated dsDNA duplex, followed by binding of biotinylated PCR products to the streptavidin-coated magnetic beads. Finally, thermal/chemical denaturation releases nonbiotinylated ssDNA from the magnetic beads, which is used for the next step (Espelund et al., 1990; Svobodová et al., 2012). Another method for the purpose is the enzyme-based degradation of the nondesired strand during PCR, e.g., lambda exonuclease and T7 gene 6 exonuclease enzymes degrade the specific DNA strand (Citartan et al., 2011; Avci-Adali et al., 2010; Nadal et al., 2012). During PCR, lambda exonuclease completely digests the 5’-phosphorylated DNA strand, but this enzyme has a negligible effect on the nonphosphorylated and ssDNA strands (Avci-Adali et al., 2010). An example of ssDNA pool obtained from double-stranded PCR products using lambda exonuclease digestion reaction was shown in Fig. 8.3. The ssDNA band is slightly faded compared with dsDNA band,

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FIGURE 8.3 Polyacrylamide gel electrophoresis results of polymerase chain reaction (PCR) and lambda exonuclease digestion reaction products. (A) Double-stranded DNA (dsDNA) product of 94 bp random DNA pool obtained with standard PCR. (B) Single-stranded DNA (ssDNA) pool obtained from the product in (A) using lambda exonuclease digestion reaction. The gel was stained with ethidium bromide and visualized under a standard UV light.

as expected. The visualization process can be improved by commercial fluorescent dyes for single-strand products. On the contrary, T7 gene 6 exonuclease protects the DNA strand containing several phosphorothioates at its 5′-end and digests the strand without phosphorylation. After the denaturation, strands are liberated on the basis of retention time (Nadal et al., 2012). A conventional method for ssDNA production from amplified dsDNA is based on denaturing PAGE, which uses a urea polyacrylamide gel (Hamula et al., 2016). In this approach, the specific longer primer is used to produce amplification strands of different lengths and is separated on denaturing PAGE based on the size. Fluorescence or UV shadowing is used for identification of the desired strand, which is excised from the gel for purification. Svobodová et al. (2012) have compared the three methods for ssDNA production, which are asymmetric PCR, enzymatic digestion, and nonbiotinylated strand separation with streptavidin beads. They found that all these methods produce good quality and quantity of ssDNA, which was evident as clear and well-defined bands in gel electrophoresis and enzyme-linked oligonucleotide assay–based ssDNA quantification data. However, they found that asymmetric PCR was the most efficient method followed by enzymatic digestion, which produced higher amounts of ssDNA. Conclusively, these methods can be employed for ssDNA generation once their limitations are considered and adapted accordingly. In the case of RNA aptamer selection, the elution process is followed by reverse transcription that produces cDNAs, which is used as the template for the amplification reaction. The amplified products are converted into RNAs, which are naturally


single-stranded, with transcription and utilized for the next incubation cycle after purification.

5.3.2 Enrichment of the Random Oligonucleotide Library Through Iteration Once the ssDNA or RNA library is obtained as discussed in the previous section, they are used in the incubation step of the second cycle. These processes of incubation, separation, elution, amplification, and ssDNA production are iterated or repeated until the initial random oligonucleotide pool is significantly dominated by the target-specific sequences, which is considered as enrichment (Yüce et al., 2015). After performing 10–15 iterations, the subsequently enriched pool could have one to several aptamers specific to the target. Iteration for an RNA aptamer takes more time as RNA has to be reverse transcribed to cDNA for amplification and then amplified DNA has to be transcribed back to RNA (Szeto et al., 2013). The enrichment can be monitored for each SELEX cycle or only for the final pool through fluorescent-based methods such as spectroscopy, microscopy, flow cytometer, or other methods such as surface plasmon resonance (SPR), microscale thermophoresis (MST), biolayer interferometry (BLI), backscattering interferometry (BSI), or absorbance spectroscopy. The affinity of the enriched pools obtained over the course of SELEX rounds should be increasing compared with the initial random pool, and also they should present a distinct affinity level toward the target as compared with the negative or counter targets. To monitor the enrichment of candidate aptamers during selection rounds and to verify the affinity of aptamer candidates to a particular analyte, flow cytometer has routinely been utilized to measure the fluorescence signal from the binding interaction of an aptamer with its target. It was shown that target cells that had a higher binding affinity with fluorophore-labeled DNA probes emitted higher fluorescence intensities compared with the cells that did not have specifically bound DNA probes. In this way, the DNA aptamers with higher affinity to target cancer cells were identified after rounds of enrichment on the basis of fluorescence signal measured by a flow cytometer (Shangguan et al., 2007; Zhu et al., 2012). Similarly, Yang et al. (2013) used the flow cytometer to monitor whether higher affinity aptamers were being enriched in the selected ssDNA pool and to check the binding capacity of the candidate aptamers to their target analyte. For this purpose, candidate aptamers (300 nM) or fluorescein isothiocyanate (FITC)-labeled ssDNA pool (100 nM) was incubated for 45 min at 37°C with Salmonella paratyphi bacteria (108 cfu/mL) with moderate shaking. Flow cytometer was used to measure fluorescence based on 10,000 events, and imaging was performed by confocal microscopy, following the fixation of the cells. The ssDNA aptamers with high-binding affinity to the bacterial cells were enriched during first 10 selection rounds. It was measured in terms of increased fluorescence intensity on bacteria by flow cytometry (FC), and it showed a remarkable change in the 14th round, thus indicating the strong enrichment of ssDNA pool for cell-specific aptamers. The fate of enrichment depends on the initial oligonucleotide library and applied SELEX steps. Specified initial libraries with increased diversity can be utilized to

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enhance the chance of obtaining tight-binding sequences. A negative SELEX or negative selection step is typically applied to remove oligonucleotides with affinity to the immobilization surface (Murata and Sato, 2014; Kolovskaya et al., 2013; Park et al., 2014; Szeitner et al., 2014). On the other side, a counter-SELEX step is operated to remove the sequences with affinity to the structurally similar targets (Kim et al., 2013; McKeague et al., 2010; Chumphukam, 2013; Darmostuk et al., 2015). PCR cycles are optimized and reduced to prevent the formation of nonspecific byproducts. Gel extraction procedures are optimized for the best yield. Binding kinetics of the SELEX pools, so the enrichment, can also be observed over the course of selection to avoid unrelated oligonucleotide authority in the pool. The enriched pool is finally sequenced to retrieve the randomized sequence data; it is processed by bioinformatics tools to find the sequence motifs responsible for the specific binding (Moreno et al., 2003; Schütze et al., 2011). Contrary to the general perception, the most abundant sequences of the final selection round do not always have the highest target affinity. Rather, performing round-to-round enrichment of sequences can help in identification of high-affinity aptamers that could reduce the PCR bias and number of cycles (Dupont et al., 2015).

6. SEQUENCING OF THE ENRICHED APTAMER POOLS In conventional SELEX, cloning of enriched pools was followed by conventional sequencing but the major limitation with conventional sequencing is that it might miss the high-affinity aptamers during screening and insufficient information is received about the pool enrichment. The emergence of next-generation sequencing (NGS) platforms has enabled the deep coverage and comprehensive analysis of the enriched pools. NGS enables the characterization and quantification of aptamers, identification, and comparison of the functional motifs in a given pool or among other pools. It also helps to identify the rare motifs of aptamers (Darmostuk et al., 2015). Now, we can process hundreds of thousands of aptamer candidates or all SELEX cycles simultaneously; which could be considered as a significant improvement in the field. NGS platforms can yield hundreds of thousand sequences of an enriched pool contrary to few dozens of the traditional sequencing procedures. This high coverage of read number and length provides great information not only about enrichment level of the pool but also about the structural features of the sequences (Yüce et al., 2015). On the other side, to avoid high costs of the deep sequencing, to check possible contamination, and to confirm that enriched pool is diverse, some of the pool could still be cloned and sequenced conventionally over the course of initial SELEX cycles (Zimmermann et al., 2010a). However, the continuous reduction in NGS costs enables the wider usage of NGS sequencing in aptamer development recently. High-throughput sequencing has also been used as the main tool for library screening as an alternative SELEX method, called as HT-SELEX. This method has become popular for selection of genomic aptamers that refer to functional genomic

6. Sequencing of the Enriched Aptamer Pools

domains in DNA, which recognize and bind to the specific molecules. For example, Zimmermann et al. (2010b) obtained thousands of full-length sequences of the enriched pool by 454 NGS platform. Data analysis of the sequencing helped in identification of RNA aptamers with high affinity to the Escherichia coli regulator protein Hfq. Wilson et al. (2014) have reported that by using 454 GS FLX NGS platform, they were able to identify aptamers specific to thrombin in a single cycle compared with several iteration steps in other methodologies. Bawazer et al. (2014) utilized AB SOLID sequencing platform for the selection of a biomineralizing aptamer specific to ZnO. This aptamer sequence was selected from a diverse DNA library by using a newly developed clustering method called AutoSOME after only one selection cycle against 5–15 cycles in conventional SELEX method. This discussion shows how NGS-based SELEX has reduced the number of selection rounds in a significant manner, thus saving the time and resource. Nowadays, the Illumina sequencing is one of the most frequently used platforms in SELEX as it generates a comparatively higher number of sequence reads and read lengths. Dupont et al. (2015) used Illumina HiSeq 2000 sequencing platform for sequencing of the enriched pool, and this yielded 1.58 × 106 sequence reads for 69,945 unique RNA sequences. The most recurring sequence (SEQ ID 2202) was found 8975 times. This shows how deep sequencing helped in identification of a vast number of reads, thus providing the quantitative assessment of changes in the pool structure during the SELEX process. RNA sequencing also provided the information about the aptamer secondary structure, binding sites of aptamer, functional effects of aptamers, and the likelihood of any specific target conformations. In another study by Valenzano et al. (2016), Illumina HiSeq 2000 platform was employed to identify 15 aptamers for tyramine, an amino acid found in protein-rich foods, which could be used as a determinant for food freshness. The sequencing data were analyzed through AptaIT GmBH software on the basis of sequence families (consensus regions) and family similarities. These aptamers had 51% of G-base showing typical aptamer feature and yielded affinities within 0.2–152 μM range. From above discussion, it is evident that when combined with SELEX, highthroughput sequencing drastically reduced the number of selection cycles. However, NGS platforms generate billions of reads whose accurate and efficient analysis is a prerequisite for the identification of high-affinity aptamers. For the purpose, identification of the secondary structure of aptamers is the most valuable step (Hoinka et al., 2012). But, most of the software programs analyze the frequency of a particular sequence in oligonucleotide pools and are unable to provide the structural information about aptamers.

6.1 EVALUATION OF SEQUENCING DATA In comparison to conventional sequencing techniques such as Sanger sequencing, NGS provides a wealth of information about aptamer sequences, variations, and frequencies within the enriched oligonucleotide pool. NGS can be applied only to the final enriched pool and the pools from the previous SELEX cycles. Thus, it enables

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the observation of in vitro evolution for a selected sequence or a motif family over the course of SELEX cycles. Individual sequences and sequence frequencies can be ranked and filtered with a set of predefined parameters for analysis. The filtered data can be processed for common motif structures and secondary structure stability. Evaluation of such large-scale NGS data could be cumbersome for those with limited bioinformatics experience. However, there are a number of readily available bioinformatics tools for the process of the SELEX data received from NGS, with user-friendly interfaces. Below, a set of NGS data assessment tools used in aptamer research has been summarized. CLC Workbench (CLC bio, Aarhus, Denmark) is a licensed software for genomics and transcriptomics analysis of high-throughput sequencing data. In a study for thrombin-specific DNA aptamer selection, CLC was utilized to assess the sequence frequency by extract and count function (Riley et al., 2015). In another DNA aptamer selection report by Kimoto et al. (2013), the tool was used to extract recognition probes from the total reads, which was 150,364 and 51,461 clones for VEGF-165 and IFN-γ targets, respectively. CLC tools were also utilized for the evaluation of results from conventional sequencing techniques, for aptamer sequence alignment purposes (Nadal et al., 2012). Below, in Fig. 8.4 we presented two alignment data using CLC

FIGURE 8.4 Alignment of a small portion of NGS data obtained from an enriched aptamer pool. The initial pool was 94 mer from which 44 mer of primer sequences were trimmed. (A) Successful alignment of some sequences in the pool showing important sequence similarity. (B) Another part of sequences from the same pool, which is indicating a smaller amount of sequence homology. The alignment data were achieved with CLC Genomics Workbench trial version (v.8.0.1).

6. Sequencing of the Enriched Aptamer Pools

Genomics Workbench (v.8.0.1) trial version in which a small part of a sequenced aptamer pool raised against a protein target was used for illustration purposes. Clustal is an online multiple sequence alignment platform for protein and nucleic acid research, which is usually operated for sequence alignment, phylogenetic tree construction, and motif position exploration purposes in aptamer studies (Shigdar et al., 2011; Wilson et al., 2014). The Clustal web server provides different versions, currently named as Clustal Omega, Clustal W, and Clustal X (Thompson et al., 2002; Sievers et al., 2014). AptaCluster (Hoinka et al., 2014) and FASTAptamer (Alam et al., 2015) are open-source tools for identifying the overwhelming NGS data of SELEX pools. They provide a ranking of sequences by frequencies, cluster motifs, and round-byround enrichment of each sequence in SELEX rounds. Commercially available COMPAS tools (Blank, 2016) provide quality control assessment of starting oligonucleotide libraries and clustering of motif families in early SELEX rounds based on covariance models, which reduce the overall number of iteration cycles and thus the labor. AptaTRACE is another approach developed to evaluate high-throughput NGS data from all cycles of the selection at once, which reveals sequence-structure motifs within a large data set independent of the target properties (Phuong et al., 2016). An arbitrary number of binding spots together with corresponding structural preferences can be discovered by AptaTRACE, unlike to its counterparts. This software is capable of identifying motifs even when these are in a trace amount of sequenced pool making it powerful software that can help to reduce the number of selection cycles, time, and expenditures of SELEX experiments. APTANI (Caroli et al., 2015) implements the same procedure of an earlier aptamer analyses tool called AptaMotif (Hoinka et al., 2012) that was an ensemble-based procedure for effective extraction of sequence-structure motifs from small-scale SELEX data. APTANI, however, is a version with improved data process abilities, which can find target-specific sequences and secondary structure information from HT-SELEX data, along with visual presentations such as graphical and tabular abstracts. Therefore, it allows determining the high-affinity aptamers and their putative binding motifs in a practical manner. AptaGUI software is a graphical user interface which can visualize the HT-SELEX data (Hoinka et al., 2015). This software is capable of preprocessing of data, and measuring the enrichment of individual aptamers and aptamer families during selection rounds in the form of graphical display. It can also provide information for nucleotide distributions, sequence clustering and diversity analysis of aptamer families across different selection rounds. There are also motif-based analysis tools available online, such as MEME suite that provides common motif families in a given data set with a limited number of DNA aptamer (Moore et al., 2015) or RNA aptamer (Reinholt et al., 2016) sequences. A motif sequence obtained with MEME motif discovery tool was shown in Fig. 8.5, in which the data in Fig. 8.4A were used as input. Such an approach is quite convenient to reduce the complexity of NGS data set and to find the smallest consensus sequences that are in charge of the specific binding.

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FIGURE 8.5 An example of motif obtained with MEME online motif discovery tool. The data from Fig. 8.4A were used as the input. The motif yielded a very low E-value of 4.0e-245 that shows the statistical significance of the motif (E-value 2 layers) TMDCs have broken out-of-plane symmetry and inplane inversion symmetry (Huang et al., 2012b) with chemical formula MX2, where M and X represent the central transition metal atom and the surrounding chalcogen atoms (Mo, S, Se), respectively, as shown in Fig. 10.1(A–E) (Chhowalla et al., 2013; Radisavljevic et al., 2011). Atoms of the same layer are covalently bonded in a hexagonal lattice while adjacent layers are held by weak Van der Waals forces, allowing thinning of the stack to easily take place. MoS2, WS2, WSe2, and MoSe2 are some examples of TMDCs, which are actively researched (Chhowalla et al., 2013). Depending on the elements involved and the structural arrangement between different physical phases, their electronic properties will vary, allowing flexibility in a wide range of electronic, optoelectronic, and photonic applications. Recent studies on mesoporous nanoparticles have shown that the large surface area of mesopores favors loading of guest drug molecules (Fig. 10.2) (Chen et al., 2010) Therefore, 2D materials with high surface-to-volume ratio are appealing candidates for biomedical purposes (Kurapati et al., 2016; Chimene et al., 2015) such as biosensors (Wang et al., 2015a; Liu et al., 2014a, 2015; Cheng et al., 2014; Yin et al., 2014; Yong et al., 2014; Qian et al., 2015; Huang et al., 2014a,b; Lee et al., 2014; Lin et al., 2014; Pumera and Loo, 2014; Wang et al., 2014a,b; Yang et al., 2015b; Chao et al., 2015;

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Feng et al., 2014; Huang et al., 2013a; Loan et al., 2014; Song et al., 2014; Tan et al., 2015; Tan and Zhang, 2015; Xi et al., 2014; Zhang et al., 2015), drug delivery (Chao et al., 2015; Tan et al., 2015; Wang et al., 2015b; Zhu et al., 2013; Liu et al., 2014b), bioimaging (Wang et al., 2015a; Liu et al., 2015; Cheng et al., 2014; Yin et al., 2014; Yong et al., 2014; Qian et al., 2015; Anbazhagan et al., 2014; Wang et al., 2014c), and therapeutic agent (Wang et al., 2015a; Liu et al., 2014a, 2015; Cheng et al., 2014; Yin et al., 2014; Yong et al., 2014; Qian et al., 2015). It is essential to understand the various functionalization approaches that enable TMDCs to fulfill different purposes. In this chapter, we will summarize the different types of functionalization of TMDCs for biomedical uses. The structural and electronic properties of TMDCs, which make them compelling candidates for biorelated systems, will be discussed and the various synthesis methods of the material will be touched on. An in-depth look on the different functionalization strategies or approaches of TMDCs and their associated applications will also be presented. We aim to provide the readers an overview of the latest advances in functionalization of TMDCs for biomedical purposes and an outlook for future research.

FIGURE 10.2 Schematic showing conventional spherical mesoporous nanoparticles versus twodimensional materials in drug delivery applications. Reproduced with permission from Chen, Y., Tan, C., Zhang, H., Wang, L., 2015. Chem. Soc. Rev. 44 (9), 2681–2701 with permission from The Royal Society of Chemistry. Copyright 2015, Royal Society of Chemistry.

2. Basic Properties of Transition Metal Dichalcogenides

2. BASIC PROPERTIES OF TRANSITION METAL DICHALCOGENIDES Owing to weak interlayer Van der Waals forces, TMDCs can be easily thinned down to a single molecular plane made up of three atoms along the out-of-plane axis (∼0.7 nm height; Fig. 10.1E). Because of its planar characteristic, TMDCs exhibit a very large surface-to-volume ratio, which provides straightforward access for large area functionalization. The relatively large surface area also allows maximal interaction with the target biomaterial for increased efficiency and sensitivity in the case of sensing and imaging (Fig. 10.2). Furthermore, lack of dangling bonds on its intrinsic basal surface allows high stability in both air and liquids as it does not readily react with ambient chemicals (Chhowalla et al., 2013). This stability is retained even when their lateral dimensions are reduced to the order of tens of nanometers (Tan et al., 2015). Such stability allows these materials to be easily introduced into biological systems for biomedical purposes or exposed to their associated environments for external diagnostics. Semiconducting TMDCs possess an electronic band gap that increases with decreasing thicknesses (Wang et al., 2012). Coupled with considerable charge carrier mobility, it enables TMDCs to be used in field-effect transistors as biosensors where changes in conductance when exposed to the target analytes allow monitoring of their concentrations. Compared with graphene, which has no electronic band gap, higher sensitivities can be reached because of a larger current on–off ratio, which enhances the signal-to-noise ratio (SNR). In addition, as the layered crystal is thinned down to a single monolayer, it experiences a transition from an indirect electronic band gap to a direct electronic band gap (Wang et al., 2012). This results in a significant increase in photoluminescence (PL) yield of monolayer TMDC as compared to its thicker counterparts. As 2D TMDCs are atomically thick, they are highly sensitive to external elements, and the variation in PL strength in the presence of specific environments can be exploited for these materials to act as biosensors (Kalantar-zadeh and Ou, 2016; Kalantar-zadeh et al., 2015; Yang et al., 2015a; Chen et al., 2015; Pumera and Loo, 2014; Wang et al., 2014c; He and Tian, 2016). Electrochemical responses of these materials are also very sensitive to external perturbations and therefore can also function as a parameter for biosensing (Huang et al., 2014a,b; Wang et al., 2014b; Huang et al., 2013a). Because of their unique electronic band structure, TMDCs exhibit strong optical absorption in the near-infrared (NIR) region (≈800 nm), higher than that of graphene oxide, and its extinction coefficient is also better than that of gold nanorods (Chou et al., 2013). These properties allow TMDCs to be efficient photothermal agents for use in drug delivery or therapeutic purposes as NIR lasers can efficiently penetrate through tissues with depth of several centimeters while having low tissue absorption. Presence of such photothermal agents enhances the selectivity of the excitation laser, allowing generation of heat in targeted areas (e.g., at tumor tissue) for therapeutic or drug release purposes while lowering the power density of laser required (Chen et al., 2015). Suspensions of TMDCs such as MoS2 with low concentrations of 100 s

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of ppm can reach temperatures greater than 80°C when exposed for several minutes to NIR radiation (≈800 nm, 0.8 W/cm2) (Chen et al., 2015).

3. SYNTHESIS OF TWO-DIMENSIONAL TRANSITION METAL DICHALCOGENIDES 2D TMDCs can be produced through both top-down and bottom-up methods. When 3D bulk counterpart is thinned down to its constituent layers using various means, it is called top-down approach, and when single layers are formed using chemical precursors, these are called bottom-up approach. In this section we will talk in detail about various methods of synthesizing TMDCs, and specific pros and cons of each approach will be described.

3.1 TOP-DOWN APPROACH The top-down approach is subjected to the direct exfoliation of layered bulk crystals by applying different kinds of forces. Mechanical exfoliation is the most extensively used method for this purpose, where an adhesive tape is used to peel off thin layers of TMDCs and deposit them on the desired surface (Radisavljevic et al., 2011). Although crystal quality of the material produced by this method is the highest compared with other methods, it is greatly limited by the randomness in thicknesses of the flakes that can be obtained and its low yield, which prevents potential scaling up. Thus they cannot be used for biomedical purposes, which require larger quantities. However, 2D TMDCs obtained in this manner are well suited as on-chip sensors because of their enhanced sensitivity to environmental perturbations by virtue of their good electronic and physical quality (Lee et al., 2014; Wang et al., 2014a; Sarkar et al., 2014). An alternative liquid/chemical-based exfoliation method has been demonstrated to achieve much larger quantities from bulk TMDCs crystals or powders, albeit with much smaller physical dimensions ( 30 > 110 > 280 > 170 nm, where the uptake of the 50 nm particles was 2.5 times higher than for the 30 nm particles (Lu et al., 2009). This corroborated other studies with other types of NPs, where it has been shown that 50 nm particles tend to have the highest cellular uptake (Chithrani et al., 2006; Osaki et al., 2004). Most of the intravenously administered MSNs of every size have been shown to accumulate in the liver and spleen, less in the lung, and a few in the heart and kidney (He et al., 2011b). Furthermore, particles of smaller size showed longer circulation time. Exposure to crystalline silica may lead to silicosis and chronic inflammation, although the former is probably more related to more traditional causes than possible future pharmaceutical applications, which only concern the amorphous form of silica. Zhang et al. (2012a) studied the effect of amorphous silica NPs of 16 nm diameter that were produced either by low-temperature colloidal (Stöber silica) or by hightemperature pyrolysis (fumed silica) routes. They performed hemolytic assays on erythrocytes and assessed viability and ATP levels in epithelial cells and macrophages, finding the colloidal silicas essentially nontoxic, whereas fumed silicas had toxicity related to postsynthetic thermal annealing or environmental exposure. They proposed that the toxicity of fumed silicas correlate with hydroxyl concentration, which generates reactive oxygen species and causes erythrocyte hemolysis (Fig. 16.3). They found that aggregation of fumed silica NPs can have toxicity levels that are comparable or exceed those of crystalline silica NPs. Furthermore, there was a strong interaction between the silica NP surface silanols and the cell membranes, which

FIGURE 16.3 Radicals that can exist on the amorphous silica surface. Reprinted with permission from Zhang, H., Dunphy, D.R., Jiang, X., Meng, H., Sun, B., Tarn, D., Xue, M., Wang, X., Lin, S., Ji, Z., 2012a. Processing pathway dependence of amorphous silica nanoparticle toxicity: colloidal vs pyrolytic. J. Am. Chem. Soc. 134, 15790–15804.

2. Mesoporous Silica Nanoparticles: From Fabrication to Applications

resulted in modest to robust hemolysis of RBCs. Fumed silica was found to aggregate on the cell membrane, whereas Stöber silica NPs were internalized within the cells. Reducing the surface area normalized hydroxyl concentration by heat treatments was found to reduce the toxicity due to the interactions between the NPs and the cells. The mesoporosity also reduces the interfacial contact of hydroxyls with cells, which can lead to lower cytotoxicity (Kettiger et al., 2015). The toxicity can also be reduced by coating the NPs with polymers (Lin and Haynes, 2010) and lipid bilayers (LBs) (Ashley et al., 2011) that shield cells from hydroxyl-related interactions. The measured toxicities also vary between different cells. Yu et al. (2011) studied the effect of Stöber silica NPs (115 nm), mesoporous silica NPs (120 nm), and mesoporous silica nanorods with aspect ratio 2 (77 nm × 198 nm), 4 (159 nm × 594 nm), and 8 (136 nm × 1028 nm), all of these with (positive surface charge) and without (negative surface charge) (3-aminopropyl)triethoxysilane amine surface modifications, on macrophages (RAW 264.7), cancer epithelial cells (A549), and erythrocytes. The cancer cells were shown to be highly resistant to the NPs with only 1000 μg/mL concentration of the NPs causing moderate cytotoxicity (60%–70% cell viability). The cell viability of the macrophages was dependent on the surface charge of the NPs with 50% proliferation inhibition (IC50) observed at 50–100 μg/mL for bare negatively surface charged silica. On the contrary, the IC50 values increased multifold with values ranging from 180 to 470 μg/mL when the MSNs were amine-modified. The observed effects on the macrophages were decreased density and rounded cells with bare NPs and swollen vacuoles with amine-modified NPs. The toxicity of the silica particles was not due to the degradation or any associated contaminants, but due to the interactions between the cells and the MSNs. The hemolytic activity on RBC was porosity- and geometry-dependent with the bare NPs, where mesoporous silica with high aspect ratio had reduced hemolytic activity. With the positively charged amine-modified NPs there seemed to be a threshold at 30 mV below which they induced reduced hemolytic activity. The optimal NP size regarding passive targeting has been determined to be between 50 and 300 nm: NPs with size less than 50 nm can pass through intercellular gaps in normal blood vessels, which results in nonspecific distribution in the body, while the maximum hydrodynamic stability is achieved with NPs with size up to 300 nm (Barbe et al., 2004). On the other hand, in rats ∼25% of silica NPs larger than 600 nm have shown to accumulate in the lungs and ∼63% in the liver 24 h after injection (Borchardt et al., 1994). Larger NPs also take longer time to dissolve, which, together with particle agglomeration, can lead to organ accumulation and long-term toxicity (He et al., 2011b). The effect of silica NPs on the cells of the human immune system is also very important regarding successful applications on immunotherapy or drug delivery. Vallhov et al. (2007) studied the effect of mesoporous silica nano- (270 nm, AMS-6) and microparticles (2.5 μm, AMS-8) on monocyte-derived dendritic cells (MDDCs). The MDDCs were incubated for 24 h with different concentrations of the nanoand microparticles. Subsequently, the degree of apoptosis and necrosis of the MDDCs was examined by measuring the binding of Annexin V-fluorescein and

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FIGURE 16.4 The viability of monocyte-derived dendritic cell (MDDC) was decreased by AMS-6 and AMS-8 at 24 h. The total sum of necrotic, early, and late apoptotic cells at 24 h using propidium iodide and Annexin-V staining was quantified. Results represent the mean ± the standard error of the mean from four independent experiments. MDDC cultured in medium only was included as a negative control and bacterial lipopolysaccharide (LPS) was included as a positive control (0.1 μg/mL) in both methods. *P 500, and CCR5Δ32 heterozygotes, ATI Not on ART, CD4 counts>500 On ART, aviremic, CD4 counts >500, ATI On ART, aviremic, CD4 counts ≥500, ATI On ART, aviremic, CD4 counts ≥450, ATI

CD4 T cells

Adenovirus

None

CD4 T cells

Adenovirus

None

CD4 T cells

Adenovirus

None

CD4 T cells and CD4/CD8 T cells CD4/CD8 T cells

Adenovirus mRNA

Cytoxan (Dose escalation) Cytoxan (1 g/m2)

CD4 T cells

mRNA

Cytoxan (1 g/m2)

On ART, aviremic, CD4 counts 200–750, ATI

CD34 HSPC

mRNA

Busulfan (Dose escalation)

ART, antiretroviral therapy; HIV, human immunodeficiency virus; HSPC, hematopoietic stem/progenitor cell, ZFN, zinc-finger nuclease. Information obtained from website Clinicaltrials.gov and publically reported by sponsors.

4. Engineering HIV Resistance With Genome Editing

Clinical Trial

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to individual bases in a target-binding site (Schmid-Burgk et al., 2013; Reyon et al., 2012). TALEN have shown similar DNA cleavage efficiency with ZFN when targeted to the same genes (Reyon et al., 2012; Sander et al., 2011; Tesson et al., 2011; Hockemeyer et al., 2011). The advantages of TALEN such as high cleavage efficiency, easy designing, and limitless targeting make them a powerful tool for geneediting application. Another novel gene-editing enzyme, known as a megaTAL, is a hybrid nuclease that combines a reprogrammed homing endonuclease with a TALE DNA binding (Boissel et al., 2014). A custom designed megaTAL against CCR5 has been demonstrated to achieve targeted gene editing at CCR5 via NHEJ in primary human T cells (Sather et al., 2015). In a later study, this megaTAL was used to successfully disrupt CCR5 and achieve HIV-infection resistance in CD4+ T cells in vitro and in humanized mice (Romano Ibarra et al., 2016).

4.3 CRISPR/CAS9 GENOME EDITING The CRISPR/Cas (CRISPR-associated proteins) is a microbial adaptive immune system against viruses and plasmids. CRISPR/Cas system uses RNA-guided nucleases to cleave foreign genetic elements (Garneau et al., 2010; Deveau et al., 2010; Makarova et al., 2011; Chylinski et al., 2014). Three main types (I–III) of CRISPR systems have been categorized across a wide range of bacterial and archaeal hosts, based on core elements content and sequences (Makarova et al., 2011). In the type II system, Cas9–crRNA complex of the Streptococcus thermophilus CRISPR/Cas system has been demonstrated that can introduce in vitro a DSB at a specific site in DNA containing a sequence complementary to crRNA (Gasiunas et al., 2012) (Fig. 18.4). The RNA-guided nuclease function of CRISPR/Cas9 is reconstituted in mammalian cells through the heterologous expression of human codon–optimized Cas9 and the crRNA: tracrRNA (Cong et al., 2013; Mali et al., 2013; Jinek et al., 2013). Furthermore, there is another form of guide RNA: the crRNA and tracrRNA can be fused together to create a chimeric, single-guide RNA (sgRNA). Cas9 can thus be redirected toward almost any target of interest in immediate vicinity of the PAM sequence by altering the 20-nt guide sequence within the sgRNA (Jinek et al., 2012). Streptococcus pyogenes Cas9 (SpCas9) makes a blunt cut between the 17th and 18th bases in the target sequence (3 bp 5′ of the PAM) (Jinek et al., 2012). Mutating catalytic residues in either the RuvC or the HNH nuclease domain of SpCas9 converts the enzyme into a DNA nicking enzyme (Jinek et al., 2012). On cleavage by Cas9, the target locus typically undergoes one of two major pathways for DNA damage repair: the error-prone NHEJ or the high-fidelity HDR pathway, both of which can be used to achieve a desired editing outcome. In the absence of a repair template, DSBs are religated through the NHEJ process, which leaves scars in the form of indel mutations. NHEJ can be harnessed to mediate gene knockouts because indels occurring within a coding exon can lead to frameshift mutations and premature stop codons (Perez et al., 2008). Multiple DSBs can additionally be exploited to mediate larger deletions in the genome (Chen et al., 2011).

4. Engineering HIV Resistance With Genome Editing

4.4 CRISPR/CAS9 TARGETING HIV 1 GENOME Because of the simplicity of designing a CRISPR sgRNA against for any DNA target, it is now well established that CRISPR/Cas9 can readily be adapted to specifically cleave regions within the HIV-1 genome (White et al., 2016). In theory, CRISPRmediated gene disruption of proviral DNA in infected cells could potentially purge the latent reservoir and ultimately cure HIV infection (Zhu et al., 2015; Ebina et al., 2013). Indeed, profound suppression of HIV-1 production and infection was reported in different cell types including latently infected CD4+ T cell lines, primary CD4+ T cells, and induced human pluripotent stem cells (Kaminski et al., 2016; Zhu et al., 2015; Liao et al., 2015; Ebina et al., 2013; Yin et al., 2016; Wang et al., 2016c,d). However, recent studies have revealed that targeting the proviral HIV genome with CRISPR/Cas9 will ultimately select for escape mutants, thereby driving viral evolution and escape (White et al., 2016; Wang et al., 2016c,d; Yoder and Bundschuh, 2016). A similar study using ZFN against the HIV-1 pol gene also revealed mutational escape (De Silva Feelixge et al., 2016). Developing new strategies overcome HIV-1 viral escape mechanism is urgently needed. One strategy is to program Cas9 with multiple sgRNAs that target proviral DNA. Liao et al. (2015) showed that multiplexing sgRNAs to target multiple HIV-1 DNA regions increased suppression of HIV-1 infection. This study also showed that CRISPR/ Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression, and replication in human cells (Liao et al., 2015). Another strategy involves engineering of novel Cas9 variants that are able to cleave DNA outside of the target sequence, so that the mutations arising from NHEJ repair will not prevent Cas9/sgRNA binding and DNA cleavage, thus not leading to viral resistance (Liang et al., 2016). Different Cas9 variants have been developed, which can recognize different PAMs to increase the flexibility of the CRISPR system (Kleinstiver et al., 2015; Hirano et al., 2016; Anders et al., 2016). Moreover, Cpf1 protein is discovered from a Class 2 CRISPR-Cas System, which cleaves in the more distal region of the target sequence that is less critical for sgRNA binding (Zetsche et al., 2015). Those explorations imply that CRISPR/Cas9 is a viable technology for disrupting HIV-1 genes.

4.5 CRISPR/CAS9 TARGETING CORZECEPTORS The chemokine receptors CXCR4 and CCR5 play critical roles as coreceptors for viral entry during infection with T cell tropic X4 and macrophage tropic R5 HIV-1 viral strains, respectively (Bieniasz and Cullen, 1998). Thus, knockout or knockdown of CCR5 or CXCR4 or both receptors by using CRISPR/Cas9 could inhibit HIV-1 infection. Moreover, endogenous genes are less susceptible to mutational escape than viral genes, thus offering a more predictable and potentially effective antiviral strategy than targeting the proviral genome (White et al., 2016). Because of these factors, as well as the excitement from the “Berlin patient” case study and encouraging progress of the CCR5 ZFN approach, it is not surprising that numerous groups have focused on harnessing the CRISPR/Cas9 genome-editing technology to disrupt CCR5 (Ye et al., 2014; Mandal et al., 2014; Wang et al., 2014a; Cho et al., 2013).

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In one example of CCR5 gene editing, a combination of double-strand cleavage with TALEN or CRISPR/Cas9 and piggyBac technology, with positive and negative selections, generated biallelic Δ32 mutations precisely matching the naturally occurring homozygous CCR5Δ32 genotype (Ye et al., 2014). This study demonstrated high efficiency of TALEN and RNA-guided CRISPR-Cas9–induced homologous recombination in induced pluripotent stem cells (iPSCs). The resulting genemodified iPSCs were then differentiated into monocytes and macrophages that were resistant to HIV-1 infection (Ye et al., 2014). Wang et al. constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs and transduced human CD4+ T cells. The CCR5 gene is disrupted, and cells are not only resistant to R5-tropic HIV-1, including transmitted/founder HIV-1 isolates but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection (Wang et al., 2014a). CXCR4 has also been by CRISPR/Cas9-mediated genome editing in human primary CD4+ T cells by to achieve HIV-1 resistance (Hou et al., 2015). This study also showed that the Cas9-mediated ablation of CXCR4 demonstrated high specificity and negligible off-target effects without affecting cell division and propagation.

5. CHIMERIC ANTIGEN RECEPTOR T CELLS T lymphocytes have advantages as a therapy for HIV-1 because they are able to migrate to sites of infection where they lyse targeted cells. In addition, infused T cells recruit other immune cells and provide a safe and efficacious long-lasting immunity against HIV-1. Adoptive T cell therapy has been investigated as an attractive strategy for controlling or eliminating HIV and restoring T cell immunity in HIV patients (Romeo and Seed, 1991; Yang et al., 1997; Masiero et al., 2005; Zhen et al., 2015). Adoptive transfer of CD8+ T cells genetically engineered to express “chimeric antigen receptors” (CARs) represents a potential approach toward a functional cure of HIV infection. The “first-generation” CARs contain only a single intracellular signaling motif (Fig. 18.6). The extracellular and transmembrane domains of CD4 linked to the zeta chain intracellular domain of the CD3/T cell receptor complex. In clinical studies of these first-generation CARs, it was demonstrated CARs targeted by single-chain variable-region Ab constructs directed against the gp120 or gp41 subunits of HIV-1 Env (Masiero et al., 2005; Roberts et al., 1994). Roberts et al. have developed two classes of HIV-specific chimeric receptors. The antigen-binding domain of the receptors is comprised of either CD4 or a single chain specific for the envelope glycoprotein gp41. The potency and safety of adoptive therapy with CD4+ (Yang et al., 1997; Walker et al., 2000) or CD4+ and CD8+ T cells modified with chimeric T cell receptor containing the human CD4 molecule as the extracellular domain (Mitsuyasu et al., 2000; Deeks et al., 2002). In a clinic study, stable levels of CD4z T cells engraftment have been verified and achieved long-term persistence of gene-modified T cells (Scholler et al., 2012). The improved “second-generation” and “third-generation” CARs could potentially enhance clinical efficacy against HIV-infected cells. The improved CARs contain

6. Concluding Remarks

FIGURE 18.6 Human immunodeficiency virus (HIV)-specific T cells engineered to express CARs are programmed to bind a particular antigen (such as an epitope unique to HIV) on the surface of HIV-infected cells and induce cytotoxic killing of the targeted cells. The ectodomain contains the antigen-recognition domain, whereas the endodomain contains the cellsignaling domain. The generation of CAR T cells differs by its signaling domains, with third generation combining multiple signaling domains such as CD3z-CD28-41BB or CD3zCD28-OX40 to enhance the signaling potency.

additional intracellular motifs from costimulatory molecules such as CD28, 4-1BB (CD137), and OX40 (CD134) have shown promise in the cancer field (Hombach et al., 2013). These CARs also provide potency for curing HIV-1. In a study focusing on the design of a novel extracellular targeting domain for an anti-HIV CAR, a bispecific CAR was created by linking the CD4 segment to a single-chain variable fragment of the 17b human monoclonal antibody. This provided enhanced potency comparable to the CD4 CAR, presumably due to higher affinity with HIV-1 (Liu et al., 2015).

6. CONCLUDING REMARKS Emerging biomolecular therapies against HIV-1 offer improved specificity and efficacy over combinatorial ART. Such therapies also may be capable of eliminating or significantly lowering viremia levels, which could alleviate the requirements of daily ART compliance and the toxic effects associated with these drugs. However, many of these approaches are still in preclinical or early clinical stages, so there are still a lot of unanswered questions about their anti-HIV efficacy and overall safety profile in humans.

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Aptamer-siRNA chimera or conjugate strategies are capable of targeted delivery to HIV-infected or HIV-susceptible cells, thereby reducing off-target effects. These RNA-based therapies have been demonstrated to provide robust antiviral activity on weekly dosing in HIV-infected humanized mice (Zhou et al, 2011, 2013) and protecting female mice from mucosal HIV transmission for up to four days after intravaginal dosing of the HIV microbicide (Wheeler et al, 2011, 2013). The apparent favorable pharmacokinetics of anti-HIV AsiCs could extend the duration of therapy and thus prolong the intervals between treatments beyond what is currently possible with ART. However, dosage intervals are still in the range of days-to-weeks, rather than weeks-to-months, and drug administration by injection would not offer the convenience of an oral pill. Furthermore, translating these therapies to the clinic will require manufacturing of synthetic RNAs, which is currently expensive and difficult to scale up. Thus, aptamer–siRNA therapeutics must overcome several hurdles before they become attractive alternatives to ART. A commonality between anti-HIV approaches that use expression vectors genome-editing modalities and CAR T cells is that each is typically administered as a cell-based therapy. Another essential feature of each of these strategies is that they all aim to create genetic resistance to HIV-1, thus potentially establishing a curative approach for HIV/AIDs. However, for each of these approaches, cells are modified ex vivo and then transfused back into the patient. Although the process of ex vivo cell manipulation and transplantation is technically complicated and expensive, it typically can ensure efficient, or at least consistent, delivery of the therapeutic into the cells. Nevertheless, these approaches would become much more expansive with the development of in vivo delivery approaches that would circumvent the need for cell-based delivery. One potential strategy is direct injection of lentiviral gene therapy vectors, such as the intraosseous infusion for the in vivo transduction of HSPCs (Wang et al., 2014b, 2015b). With the development of nanoparticles of the in vivo delivery of CRISPR/Cas9 (Jiang et al., 2017; Mout et al., 2017; Yin et al., 2016), it is also conceivable that a genome-editing system could be delivered by systemic injection to create genetic HIV resistance in T cells, macrophages, and HSPCs. It is clear that biomolecular therapies hold promise as novel treatments strategies for HIV/AIDS but are largely dependent on advances in manufacturing and delivery technologies. The recent explosion of applications with CRISPR/Cas9 genome editing highlights how quickly technological advances can occur, but it is paramount that this and similar technologies undergo comprehensive optimization and thorough preclinical evaluation before proceeding into the clinic. Nevertheless, with each technological breakthrough, the prospect of creating a long-lasting HIV prophylactic or an HIV-resistant immune system grows stronger.

ACKNOWLEDGMENTS This work was funded by National Institutes of Health grants R01MH113407 and R01AI42552.

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CHAPTER

Self-Assembled Peptide and Protein Nanofibers for Biomedical Applications

19

Dillon T. Seroski, Gregory A. Hudalla University of Florida, Gainesville, FL, United States

CHAPTER OUTLINE 1. Introduction�� 569 2. Classes of Self-Assembling Peptide and Protein Nanofibers�� 570 2.1 α-Helical Peptide Nanofibers�� 570 2.1.1 Sequence Designs and Material Characteristics of α-Helical Peptides�� 572 2.2 Collagen Mimetic Peptides�� 574 2.3 β-Sheet Peptide Nanofibers�� 574 2.4 Peptide Amphiphiles�� 578 2.5 Recombinant Protein Nanofibers�� 579 3. Applications of Self-Assembling Peptide and Protein Nanofibers for Biomedicine�� 580 3.1 Scaffolds for Cellular and Tissue Engineering�� 580 3.1.1 “Bare” Scaffolds�� 581 3.1.2 Cell-Adhesive Scaffolds�� 581 3.2 Drug Delivery�� 584 3.2.1 Small-Molecule Delivery�� 585 3.2.2 Therapeutic Protein Delivery�� 586 3.3 Preventing and Treating Disease�� 589 3.3.1 Membrane-Disrupting Peptides�� 589 3.3.2 Vaccines�� 591 3.3.3 Immunomodulatory Materials�� 591 4. Future Perspectives�� 592 References�� 593

1. INTRODUCTION Spontaneous organization of small-molecule building blocks into highly ordered structures, commonly referred to as “self-assembly” (Whitesides and Grzybowski, 2002), is receiving increasing interest for fabricating functional biomaterials. Self-assembly is Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00019-7 Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 19 Self-Assembled Peptide and Protein Nanofibers

FIGURE 19.1 (A) Scanning electron microscopy of protein nanofibers and (B) transmission electron microscopy of peptide nanofibers. (A) Reprinted with permission of Elsevier Li, C., Vapari, C., Jin, H., Kim, H., Kaplan, D.L., 2006. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 27 (16), 3115–3125. (B) Reprinted with permission of Springer Seroski, D.T., Restuccia, A., Sorrentino, A.D., Knox, K.R., Hagen, S.J., Hudalla, G.A., 2016. Co-assembly tags based on charge complementarity (CATCH) for installing functional protein ligands into supramolecular biomaterials. Cell. Biomol. Eng.

ubiquitous in nature, with common examples including protein folding (intramolecular assembly), protein tertiary structures and DNA hybridization (intermolecular assembly), and membranes formed via amphipathic phospholipid bilayer packing. Understanding the molecular interactions that mediate self-assembly in nature enables rational design of synthetic biomolecules, such as peptides and proteins, which can organize into biomaterials with specific nanoscale architectures. In turn, synthetic self-assembling peptides and proteins can be modified with “functional ligands,” such as peptides, proteins, carbohydrates, or small-molecule drugs, which endow the resultant biomaterials with functional properties. In this chapter, we survey recent advances in designing peptides and proteins that self-assemble into high-aspect ratio fibers with nanoscale features (i.e., “nanofibers”) as demonstrated in Fig. 19.1 and the use of functionalized nanofibers as biomaterials for tissue regeneration, drug delivery, vaccines, and immunomodulation.

2. CLASSES OF SELF-ASSEMBLING PEPTIDE AND PROTEIN NANOFIBERS 2.1 α-HELICAL PEPTIDE NANOFIBERS

The discovery of the α-helix by Linus Pauling and Corey in 1950 opened the door to understanding one of the major structural components of peptides and proteins. Since then, numerous naturally occurring peptides and proteins have been discovered with α-helix domains, leading researchers to design and model peptides after sequences found in nature. A common structural motif that has a strong propensity for forming

2. Classes of Self-Assembling Peptide and Protein Nanofibers

FIGURE 19.2 (A) Wheel diagram for the naturally occurring alpha-helix sequence in GCN4-p1. (B) Schematic of a “staggered” assembly between two peptides. (C) Primary amino acid sequence for SAF-P1 and SAF-P2 showing the “staggered” amino acid interactions. (D) Schematic representation of the “staggered” assembly of the αFFP peptide to form pentameric coiled-coil structures. (C) Reproduced with permission of the American Chemical Society Pandya, M.J., Spooner, G.M., Sunde, M., Thorpe, J.R., Rodger, A., Woolfson, D.N., 2000. Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis. Biochemistry 39 (30), 8728–8734. (D) Adapted with permission of Elsevier Potekhin, S.A., Melnik, T., Popov, V., Lanina, N., Vazina, A.A., Rigler, P., Verdini, A.S., Corradin, G., Kajava, A.V., 2001. De novo design of fibrils made of short α-helical coiled coil peptides. Chem. Biol. 8 (11), 1025–1032.

α-helices is the heptad repeat (HPPHPPP)n, where H denotes a hydrophobic residue and P denotes a polar residue. One of the most studied and well-understood examples of natural heptad repeats is the leucine zipper motif, such as that found in the yeast transcription factor GCN4, which has been used to rationally design peptides that assemble into coiled coils with tunable strand numbers (Harbury et al., 1993). A wheel diagram uses a seven-residue repeat notation, (abcdefg)n, to visually represent amino acid sequence, ordering, and spatial distribution within an α-helix (Fig. 19.2A). In the wheel diagram, a is the first residue in the heptad repeat and

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g is the last. Moving from residue a to g places each sequential residue further down the helix (into the page), as represented by decreasing circle diameter. When designing α-helices, understanding the arrangement of residues in the molecule is crucial for intentionally creating residue–residue interactions. For example, a and d residues are often hydrophobic, such that their colocalization on one face of the helix establishes a hydrophobic surface that stabilizes helix–helix interactions. As we will discuss below, understanding helix design and the ability to precisely place amino acids at particular locations within peptides are necessary for programming α-helical peptide self-assembly into highly elongated nanofibers with widths ranging from tens to hundreds of nanometers.

2.1.1 Sequence Designs and Material Characteristics of α-Helical Peptides

In 1997, Kojima et al. developed a peptide containing three-heptad repeats (LETLAKA)3, now called α3, with enough α-helical structure to form extended nanofibrils with widths between 5 and 10 nm (Kojima et al., 1997). The α3 peptide deviates from the classic heptad repeat with a structural motif HPPPHPH, where the fourth residue is polar instead of hydrophobic. The leucine and alanine residues form a hydrophobic face of the α-helix in positions a, d, e, and g. This hydrophobic face is responsible for the formation of bundle structures that lead to the fibrillization of the peptide. To further understand self-assembling α-helical peptides, in 2000, Woolfson et al. created a heterodimeric pair of α-helical peptides, referred to as “self-assembling fiber” (SAF) peptides, SAF-P1 (KIAALKQKIASLKQEIDALEYENDALEQ) and SAF-P2 (KIRALKAKNAHLKQEIAALEQEIAALEQ) (Pandya et al., 2000). These peptides assemble via a “staggered” interaction (Fig. 19.2B), wherein the peptides associate together in an offset pattern (Fig. 19.2C). This staggered orientation creates “sticky ends” to encourage favorable interactions between the two peptides. This structural design allows the SAF peptides to assemble and form elongated nanofibers when together; however, they do not assemble when alone. Following in the footsteps of Woolfson, in 2001, Kajava developed the self-assembling α-helical peptide, αFFP (Potekhin et al., 2001). αFFP (QLAREL(QQLAREL)4) self-assembles at acidic pH into a staggered fivestranded coiled-coil structure (Fig. 19.2D), reminiscent of tropocollagen assembly. The sequence design of αFFP uses four key principles to obtain the desired five-stranded coiled-coil structure. First, from known pentameric structures, it was determined that apolar residues placed at positions a and d do not disrupt the coiled-coil secondary structures. Thus, leucine was chosen because it occurs often in natural coiled coils. Second, alanine is placed at position e because apolar residues at e and g promote the formation of multistranded coils due to the formation of a hydrophobic cluster. Third, glutamic acid and arginine were placed at positions f and g, respectively, to promote salt-bridge formation and favor a parallel orientation of the coiled-coil strands. Lastly, glutamine was placed at positions b and c to encourage interhelical hydrogen bonds between strands. Using these loose rules, these interactions yielded a peptide that forms a pentameric


coiled-coil assembly with a well-controlled assembly. Continuing with staggered assemblies, in 2004, Conticello et al. created the YZ1 peptide, a homodimer based off of the loosely translated principles used in the structural design of αFFP (Zimenkov et al., 2004). YZ1 forms a homodimer because of the placement of isoleucine and leucine residues at the a and d positions. Parallel aligned dimers were rendered energetically unfavorable and electrostatically repulsive by placement of glutamic acid residues in the first three e and g positions in the heptad repeat and lysine residues in the last three e and g positions. This placement of charged residues is what encourages the “staggered” dimerization of the peptide to form long nanofibers. Precisely and systematically varying the primary sequence of established peptides can provide molecular-level insights into their self-assembly characteristics. For example, in 2005, Kojima et al. studied a de novo designed peptide, R3 (AKALTEL)3, which is the reverse sequence of α3 (LETLAKA)3. Unexpectedly, the R3 peptide demonstrated greater α-helical structure and stability than its predecessor. It is assumed that the greater propensity for forming an α-helix arose from salt-bridge formation between the reversed glutamic acid and lysine residues. More recently, in 2008 Woolfson et al. designed the self-assembling peptide MagicWand1 (MW1) (KIKALKYEIAALEQEIAALEQKIAALKQ). The sequence design of MW1 is similar to the SAF peptides with a few variations (Gribbon et al., 2008). The first alteration was the removal of asparagine at the a position to increase the thermal stability of the peptide. Then, the first and last heptad of the sequence contain positively charged lysines at the g and e positions, whereas the middle two heptads instead have glutamic acid residues at the g and e positions. Lastly, the solubility was increased by placing a lysine and tyrosine at the first b and f locations, respectively. A list of the collagen mimetic peptides (CMPs) mentioned above can be found in Table 19.1.

Table 19.1 List of α-Helical Peptides, Their Sequences, Developers, and Year Published Peptide

Sequence

Developers Year

A3

(LETLAKA)3

1997

SAF-P1 SAF-P2 αFFP YZ1 R3

KIAALKQKIASLKQEIDALEYENDALEQ KIRALKAKNAHLKQEIAALEQEIAALEQ QLAREL(QQLAREL)4 EIAQLEKEIQALEKENAQLEKKIQALRYKIAQLREKNQALRE (AKALTEL)3

Kojima/ Miura Woolfson

2001 2004 2005

MW1

KIKALKYEIAALEQEIAALEQKIAALKQ

Kajava Conticello Kojima/ Mirua Woolfson

2000

2008

573

574


2.2 COLLAGEN MIMETIC PEPTIDES Collagen is the most abundant protein in the human body and provides structural support in connective tissues (Holmgren et al., 1998). Collagens are characterized by a primary sequence rich in repeats of the amino acid triad, (X-Y-Gly)n (where X and Y are usually proline and/or hydroxyproline), which favors self-assembly of a triplehelix coiled-coil structure (Josse and Harrington, 1964). Because of collagen’s wide range of functions and its high stability, there is great interest in creating assemblies that mimic its structure as the basis for new biomaterials; however, collagen cannot be directly expressed using microbial hosts, as mammalian collagen requires posttranslational modifications to convert proline into hydroxyproline. Thus CMPs, in which hydroxyproline can be directly incorporated using conventional peptide synthesis techniques, are receiving increasing interest. The CMP (Pro-Hyp-Gly)10 is based on the most abundant three amino acid sequence found in collagen (Persikov et al., 2000). This repeat in particular forms stable triple helices, whereas substitutions of either the proline or hydroxyproline residue were found to decrease the triple-helix thermal stability or prevent assembly into a helix altogether (Gauba and Hartgerink, 2007; Fallas et al., 2009). In 2007, Conticello and Chaikof reported a 36 amino acid long CMP called CPII, (Pro-ArgGly)4-(Pro-Hyp-Gly)4-(Glu-Hyp-Gly)4, which readily self-assembles into a triple helix (Fig. 19.3A) (Rele et al., 2007). Here the central (Pro-Hyp-Gly) is responsible for creating the hydrophobic core required for spontaneous assembly. The flanking regions of the core contain either a positively charged arginine residue or a negatively charged glutamic acid residue to promote electrostatic attraction and repulsion, which allow for a staggered assembly (similarly to collagen and MW1 previously described). More recently, in 2011 Hartgerink produced a variant of CPII, known as KOD, where the Arg and Glu are swapped for Lys and Asp, respectively (O’Leary et al., 2011). Because of the arrangement of residues within the CMP, the spatial orientation between Lys and Asp provides stronger interactions, as seen in Fig. 19.3B and allows for more stable fibers that form hydrogels at higher concentrations. A list of the CMPs mentioned above can be found in Table 19.2.

2.3 β-SHEET PEPTIDE NANOFIBERS In 1992 Shuguang Zhang identified an amphipathic domain, (AEAEAKAK)2 (EAK16), in the yeast protein zuotin (Zhang et al., 1993). Synthesis of the EAK16 domain yielded a peptide that readily self-assembled into robust β-sheet nanofibers. EAK16 is thought to assemble due to two factors: the ionic bonding between the positive lysine and negative glutamic acid residues and the hydrophobic core formed from the “interlocking” of the alanine methyl groups. At relatively low concentrations EAK16 was found to closely mimic the natural structure of the extracellular matrix as visualized by SEM (Zhang et al., 2005). This property, which was unique to EAK16 at the time, has since sparked interest in creating synthetic peptides that can self-assemble to form synthetic scaffolds that closely mimic the fibrillar structure of natural extracellular matrices. Using EAK16 as a model, Zhang created the


FIGURE 19.3 (A) Schematic of CPII repeat unit showing the individual domains responsible for triple helix formation. (B) Assembly of KOD CMP into a triple helix that demonstrates Lys–Asp salt bridges and unpaired Lys and Asp residues. (C) Fiber packing. CMP, collagen mimetic peptides. (A) Reproduced with permission from American Chemical Society Rele, S., Song, Y., Apkarian, R.P., Qu, Z., Conticello, V.P., Chaikof, E.L., 2007. D-periodic collagen-mimetic microfibers. J. Am. Chem. Soc. 129 (47), 14750–14787. (B) Reproduced with permission from Nature Publishing Group O’leary, L.E., Fallas, J.A., Bakota, E.L., Kang, M.K., Hartgerink, J.D., 2011. Multi-hierarchical self-assembly of a collagen mimetic peptide from triple helix to nanofibre and hydrogel. Nat. Chem. 3, 821–828.

self-assembling de novo peptide RADA16 (RARADADA)2 in 1993 (Zhang et al., 1995). RADA16 differs from EAK16 in swapping the negatively charged glutamic acid for aspartic acid and the positively charged lysine to arginine. Like EAK16, the alternating hydrophobic alanine residues provide excellent water solubility and Table 19.2 Collagen Mimetic Peptides, Their Sequences, Developers, and Year Published Peptide

Sequence

Developers

Year

(POG)10 CPII

(Pro-Hyp-Gly)10 (Pro-Arg-Gly)4—(pro-Hyp-Gly)4—(Glu-Hyp-Gly)4

2000 2007

KOD

(Pro-Lys-Gly)4—(pro-Hyp-Gly)4—(Asp-Hyp-Gly)4

Brodsky Conticello/ Chaikof Hartgerink

2011

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solution stability at physiological conditions. At high concentrations RADA16 forms stable hydrogels and retains an extracellular matrix–like structure, similar to EAK16. Because of the versatile nature of RADA16, it is commercially available under the name PuraMatrix and is widely used in regenerative medicine, cellular/tissue engineering, and drug delivery. Although RADA16 was based on EAK16, the peptides P11-2 (QQRFQWQFEQQ) and Q11 (QQKFQFQFEQQ) were rationally designed by Aggeli and Boden and Messersmith and Collier, respectively (Aggeli et al., 1997; Collier and Messersmith, 2003). The individual peptide molecules form elongated β-sheet fibers through the hydrogen bonding between carbonyls and amides along the backbone of each peptide molecule. Secondly, P11-2 and Q11 have positive and negative residues placed at opposite ends of the peptide sequence to promote antiparallel alignment and create favorable electrostatic interactions. Next the motif designs have six glutamine residues spaced across the molecule to encourage hydrophobic interactions between adjacent molecules. To further strengthen interstrand interactions three hydrophobic residues are placed in the core of both peptide designs, which creates a “hydrophobic core” that mediates thermodynamically favorable aromatic pidpi stacking between molecules, and in turn, collapse of β-sheet nanofibers into ribbons and tapes (Fig. 19.4A) (Aggeli et al., 2001). In addition to single-peptide assemblies, pairs of peptides can be designed to selectively coassemble into β-sheet nanofibers via complementary interactions. For example, P11-13/P11-14 developed by Aggeli/Ingham and CATCH(+)/CATCH(−) developed by Hudalla coassemble via electrostatic complementarity (Kyle et al., 2012; Seroski et al., 2016). The parent molecules for P11-13/P11-14 and CATCH(+)/ CATCH(−) are based on P11-2 and Q11, thus the coassembling peptides use the same guiding principles for molecular assembly as the formerly established self-assembling peptides; however, it is noted the minimum concentration required for CATCH peptide assembly is more than an order of magnitude higher than that of its parent peptide Q11. The increase in the critical assembly concentration is likely due to the high charge density on each individual peptide unit and the β-sheet nanofibers themselves. In addition, nanofiber propagation depends on alternating association of CATCH(+) and CATCH(−), thus the statistical probability of interaction between a free peptide and growing fiber end is half of that for single-molecule self-assembling peptide systems. In 2004, Pochan and Schneider developed the β-hairpin self-assembling peptide MAX1 (Ozbas et al., 2004). A unique property of the β-hairpin peptides is the placement of a d-proline residue at the center of the MAX peptide, which causes a type II β-turn that allows for intramolecular hydrogen bonding, as seen in Fig. 19.4B. The alternating lysine and valine residues form a hydrophilic face in the interior of the molecule and a hydrophobic face on the exterior of the molecule, respectively, which allows for self-assembly as shown in Fig. 19.4C. By swapping single residues in the MAX1 sequence, β-hairpin structures can be designed to assemble under certain conditions or to have specific assembly properties (De Leon Rodriguez et al., 2016). For example, MAX6 (V16 → E) self-assembles under different physical conditions as


FIGURE 19.4 (A) Model of hierarchical self-assembly from monomer to fiber. (B) β-hairpin sequence of MAX1 and (C) Random-coil MAX1 peptide undergoing intramolecular folding followed by self-assembly. (D) “Missing tooth” MDP assembly demonstrating the gap formed during the packing of the fibers. MDP, multidomain peptides. (A) Adapted with permission of PNAS Aggeli, A., Nyrkova, I.A., Bell, M., Harding, R., Carrick, L., McLeish, C.B., Semenov, A.N., Boden, N., 2001. Hierarchical self-assembly of chiral rod-like molecules as a model for peptide β-sheet tapes, ribbons, fibrils, and fibers. PNAS 98 (21), 11857–11862. (C) Reproduced with permission of the American Chemical Society Ozbas, B., Kretsinger, J., Rajagopal, K., Schneider, J.P., Pochan, D.J., 2004. Salt-triggered peptide folding and consequent self-assembly into hydrogels with tunable modulus. Macromolecules 37, 7331–7337. (D)Adapted with permission of the American Chemical Society Li, I., Moore, A.N., Hartgerink, J.D., 2016. “Missing tooth” multidomain peptide nanofibers for delivery of small molecule drugs. Biomacromolecules 17, 2087–2095.

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MAX1 due to the negative and hydrophilic glutamic acid residue (Haines et al., 2005). MAX7 (V16 → C) was found to increase the stiffness of MAX hydrogels because of cysteine residue cross-linking. More recently, MAX8 (L15 → E) was found to have increased assembly kinetics and an increased thermal stability due to the creation of intramolecular attractive electrostatic interactions (Branco et al., 2009). Multidomain peptides (MDP) designed by Hartgerink use a block ABA pattern for self-assembly (Dong et al., 2007; Kumar et al., 2015). The B block consists of an alternating hydrophilic–hydrophobic pattern, whereas the A block consists of hydrophilic lysine residues that act to overcome water insolubility due to the hydrophobic core. This alternating B block motif leads to hydrophilic residues presented on one face and hydrophobic residues on the opposing face, thereby establishing a “hydrophobic driving force” for collapse of MDPs into extended nanofibers. An example of an MDP is the “missing tooth” assembly with a schematic (Fig. 19.4D) demonstrating the drug loading “cavity” formed in the middle of the nanofiber structure. A list of the β-sheet fibrillizing peptides mentioned above can be found in Table 19.3.

2.4 PEPTIDE AMPHIPHILES Peptide amphiphiles (PA) are a class of self-assembling peptide analogues developed by Matthew Tirrell and Gregg Fields in 1995 (Berndt et al., 1995; Yi et al., 1996). In 2001, Stupp et al. developed a self-assembling PA with a bioactive molecule component (Hartgerink et al., 2001), which is based on a molecular design with four domains: (1) A hydrophobic alkyl tail, (2) a β-sheet forming peptide segment, Table 19.3 β-Sheet Fibrillizing Peptides, Their Sequences, Developers, and Year Published Peptide

Sequence

Developers

Year

EAK16 RADA16-I RADA16-II P11-2 Q11 MAX1 MAX8 P11-13 P11-14 SLac CATCH(+) CATCH(−) Missing-tooth

Ac-AEAEAKAKAEAEAKAK-Am Ac-RADARADARADARADA-Am Ac-RARADADARARADADA-Am Ac-QQRFQWQFEQQ-Am Ac-QQKFQFQFEQQ-Am VKVKVKVKV DPLP TKVKVKVKV VKVKVKVKV DPLPTKVEVKVKV Ac-EQEFEWEFEQE-Am Ac-QQOFOWOFOQQ-Am KSLSLSLRGSLSLSLKGRGDS Ac-QQKFKFKFKQQ-Am Ac-EQEFEFEFEQE-Am K2(SL)2SA(SL)2K2 K2(SL)2(SA)2(SL)2K2

N/A Zhang

1993 1995

Aggeli/Semenov Collier Schneider/Pochan Schneider Aggeli/Ingham

1997 2003 2004 2010 2012

Hartgerink Hudalla

2015 2016

Hartgerink

2016


FIGURE 19.5 Molecular structure of PA1 demonstrating the different regions required for self-assembly. PA, Peptide amphiphiles. Adapted with permission of Elsevier Niece, K.L., Czeisler, C., Sahni, V., Tysseling-Mattiace, V., Pashuck, E.T., Kessler, J.A., Stupp, S.I., 2008. Modification of gelation kinetics in bioactive peptide amphiphiles. Biomaterials 29 (34), 4501–4509.

(3) charged amino acids to increase solubility, and (4) a small bioactive molecule (Fig. 19.5) (Cui et al., 2010). The alkyl tails in region 1 pack into a hydrophobic core within the interior of the nanofiber. Region 2 consists of a short peptide segment that promotes hydrogen bonding between molecules for enhanced polymerization and often contains cysteines to form covalent disulfide bridges. Region 3 contains glycine residues and charged residues, where the former act as a flexible “linker” to separate the bioactive components in region 4 from the self-assembly domains, whereas the latter determine PA solubility and the pH responsiveness of nanofiber assembly. Lastly, region 4 is usually a bioactive molecule that is “presented” on the exterior of the PA nanofiber. These regions all play a pivotal role in the self-assembly and functionality of PAs, thus there has been a strong emphasis to date on establishing relationships between the chemical composition of each individual region, PA self-assembly properties, and nanofiber functionality (Hartgerink et al., 2002; Niece et al., 2008).

2.5 RECOMBINANT PROTEIN NANOFIBERS Protein self-assembly is integral to the formation of highly ordered protein architectures that are widely found in cellular structural support elements and connective tissues. Some examples of naturally occurring proteins that form “nanofibers” include collagens, elastin, silk, keratin, amyloid beta, and prions (Scheibel, 2005; Petkova et al., 2005; Wang and Goodson, 2007). Many self-assembling proteins have been designed based on these naturally occurring proteins to date, and herein we will highlight examples of the use of self-assembling protein nanofibers as hydrogel scaffolds, biomimetic cellular support structures, and drug delivery vehicles. Silks, one of the most widely used recombinant proteins, have been utilized by humans as textiles for thousands of years because of their unique material properties. More recently, recombinant silks have gained interest as materials for biomedical applications (Vepari and Kaplan, 2007; Wang et al., 2006; Zhang et al., 2009). Recombinant silk proteins are typically based on protein sequences derived from silk

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of spiders and silkworms. Recombinant silk proteins are copolymers of hydrophobic and hydrophilic blocks consisting primarily of glycine, alanine, and serine residues. The hydrophobic blocks typically form β-sheets through hydrogen bond interactions and hydrophobic effects. Recombinant silk proteins have a very high elastic modulus and are extremely light relative to other materials with comparable moduli, thus making them broadly useful for biomedical engineering applications. Another class of recombinant protein forming nanofibers is the elastin-mimetic polypeptides and the silk-elastin-like protein polymers (SELPs). Elastin-mimetic polypeptides are modeled after the human elastin protein and primarily consist of glycine, valine, and proline residues (Hwang et al., 2009). An example of a fibrillizing elastin-mimetic polypeptide, (Val-Pro-Gly-Val-Gly)4(Val-Pro-Gly-Lys-Gly), was developed by Conticello and Chaikof in 1999 (Huang et al., 2000). This assembly uses valine and proline residues to create a hydrophobic face, which mediates assembly of the proteins into large fiber bundles. SELPs, such as elastin-mimetic polypeptides, are also block copolymers, but they have variable and tunable ratios of silklike (S) and elastin-like (E) regions (Megeed et al., 2002; Ner et al., 2009). For example, the SELP protein SELP0 is made up of eight elastin-like regions followed by two silklike regions, repeated 18 times, denoted as [(E)8(S)2]18. SELPs with specific physical properties can be created by varying the ratio of elastin-like and silklike regions. The final class of self-assembling protein subunits described here is commonly found in and derived from prions and amyloid fibers. These proteins are tandem repeats of a single domain where the repeat segments combine to form β-sheet nanofibers. Two examples of these self-assembling protein domains are sup35 and SH3 (Serio et al., 2000; Bader et al., 2006). These assembling domains can be modified via fusion to secondary protein domains, which provides a simple route to generate nanofibers with functional properties, such as catalysis or fluorescence (Baldwin et al., 2006; Men et al., 2009; Baxa et al., 2002).

3. APPLICATIONS OF SELF-ASSEMBLING PEPTIDE AND PROTEIN NANOFIBERS FOR BIOMEDICINE 3.1 SCAFFOLDS FOR CELLULAR AND TISSUE ENGINEERING The current state-of-the-art scaffolds for cell and tissue culture are glass, tissue culture (TC) plates, isolated ECM proteins (e.g., collagen, gelatin, and laminin), and intact ECM (e.g., Matrigel). TC plates and glass are relatively cheap and are widely used for cell culture, but the cell culture environment on their surfaces is vastly different from in vivo conditions (Gelain et al., 2007). In particular, planar glass and TC plates limit cell growth to two dimensions, which contrasts with 3D environments that cells reside in nature, and have elastic moduli orders of magnitude above the native cell environment in the body. ECM proteins and Matrigel provide cells a more “natural” scaffold because they mimic the three-dimensional (3D) environment cells develop in.

3. Applications of Self-Assembling Peptide and Protein Nanofibers

However, these products are often animal-derived, have ill-defined composition, and may contain biological impurities, such as growth factors that create batch-tobatch variability. These challenges with TC plates and animal-derived scaffolds have increased interest in self-assembled peptide scaffolds in the regenerative medicine and tissue engineering fields.

3.1.1 “Bare” Scaffolds

The discovery of the β-sheet fibrillizing peptide EAK16 and its remarkably similar nanoscale structure to natural ECM fostered movement toward the development of peptide-based scaffolds for cell/tissue engineering and regenerative medicine. Synthetic peptide scaffolds have several advantages over the current “gold” standard scaffolds as they allow for 3D growth, avoid biological impurities, are usually nonimmunogenic, are biodegradable, and have tunable moduli that are closer to that of native cellular environments. RADA16-I and RADA16-II were used as a “bare” scaffold in the culture of rat hippocampal neurons (Holmes et al., 2000). Cell growth on the RADA16 scaffold was almost indistinguishable from hippocampal neurons grown on Matrigel. Another β-fibrillizing peptide, KLD-12 (KLDLKLDLKLDL), was also effective as an ECMmimicking scaffold for culturing chondrocytes (Kisiday et al., 2002). The KLD-12 peptide scaffold induced chondrocyte proliferation, as well as production and deposition of natural ECM over the artificial ECM-mimicking scaffold.

3.1.2 Cell-Adhesive Scaffolds A key feature of self-assembling peptides is the ability to append functional domains, such as cell adhesion peptides, onto their termini via standard peptide synthesis methods without perturbing the assembly properties of the parent peptide (Fig. 19.6A). Thus, self-assembling peptides with appended functional domains can provide scaffolds that not only mimic the native ECM structure but also can elicit or promote specific cellular responses. The first PA cell-adhesive scaffold incorporated the laminin-derived peptide IKVAV to promote neurite sprouting and direct cell growth (Silva et al., 2004). IKVAV was placed in the “bioactive” region 4 of the PA that is presented on the exterior surface of the nanofibers. IKVAV-PA molecules retain their self-assembly properties and form hydrogels at low weight/volume percentages (wt%). Murine neural progenitor cells remained viable when cultured on IKVAV-PA hydrogel scaffolds to a similar extent as cells cultured on poly(d-lysine) (PDL), a cell culture control. Cells on IKVAV-PA hydrogels expressed more β-tubulin, with a lower overall percentage of astrocytes, than cells cultured on laminin and PDL. Overall, this study demonstrated that IKVAV-PA hydrogels induce excellent neurite outgrowth, in part because of incorporating a “bioactive” component in the PA scaffold. The self-assembling peptide Q11 enhanced human umbilical vein endothelial cell (HUVEC) response when IKVAV or RGDS (a fibronectin cell-binding domain) domains were appended onto its N-terminus (Jung et al., 2009). HUVEC proliferation rate on Q11 + RGDS-Q11 was similar to that of cells on fibronectin-adsorbed Q11.

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FIGURE 19.6 (A) Self-assembly schematic with a self-assembling peptide and a self-assembling peptide with an appended peptide ligand. (B) Phase contrast images of human aortic endothelial cell cultured on different gel scaffolds: (a) Collagen I, (c) Matrigel, (e) RADA16-I, (g) 90% RADA16, 10% YIG, (i) 90% RADA16, 10% TAG, (k) 90% RADA16, RYV. Fluorescent staining with TRITC–phalloidin and DAPI to visualize actin fibers (yellow [white in print version]) and nucleus (blue [black in print version]): (b) Collagen I, (d) Matrigel, (f) RADA16-I, (h) 90% RADA16, 10% YIG, (j) 90% RADA16, 10% TAG, (l) 90% RADA16, 10% RYV. (B) Adapted with permission of Elsevier Genové, E., Shen, C., Zhang, S., Semino, C.E., 2005. The effect of functionalized self-assembling peptide scaffolds on human aortic endothelial cell function. Biomaterials 26, 3341–3351.


IKVAV-Q11 did not demonstrate an increase in cell proliferation, but a spindle-like phenotype was observed, possibly suggesting a switch to a more migratory phenotype of HUVEC. The interest in creating other peptide scaffolds with cell-specific recognition sites increased dramatically following the IKVAV-PA, IKVAV-Q11, and RGDS-Q11 cellular responsive scaffolds. Most notable is the functionalization of the β-sheet fibrillizing peptide RADA16, which has been modified with numerous cellular responsive peptides (see Table 19.4). Modified RADA16 can possess functional ligands on either the C or N-terminus without perturbing self-assembly. In an early example, RADA16 was functionalized with two peptide ligands derived from laminin (YIG and RYV) and one peptide ligand derived from collagen (TAG). These bioactive peptides increased the growth of human aortic endothelial cells (HAEC) when compared to cells grown on bare RADA16 and maintained a similar phenotype to cells grown on collagen I or Matrigel scaffolds as seen in Fig. 19.6B (Genové et al., 2005). A subsequent study developed RADA16 variants modified with “bone marrow homing peptides” (BMHP), BMHP1 (SKPPGTSS), or BMHP2 (PFSSTKT) to induce differentiation of adult mouse neural stem cells. After 7 days, cells cultured on BMHP1 or BMHP2 scaffolds had qualitatively similar expression of β-tubulin (red, neuron marker) and nestin (green, neural progenitor marker), when compared with cells cultured on Matrigel, whereas expression of these markers was lower for cells cultured on bare RADA16. Together, these data suggest that BMHP1-functionalized RADA16 scaffolds provide the necessary stimuli for NSC differentiation into neurons and neural progenitors, whereas bare RADA16 lacks these stimuli. Table 19.4 RADA16 Variants Used as Cell Adhesion/Responsive Scaffolds and Their Biological Source Peptide

Sequence

Functional Peptide Source

RADA16-I YIG RYV TAG BMHP1 BMHP2 ALK DGR PRG SVV LKK VGV RED KLT

Ac–(RADA)4–Am YIGSR–GG–(RADA)4 RYVVLPR–GG–(RADA)4 TAGSCLRKFSTM–GG–(RADA)4 (RADA)4–GG–SKPPGTSS (RADA)4–GG–PFSSTKT (RADA)4–GG–ALKRQGRTLYGFF (RADA)4–GG–DGRGDSVAYG (RADA)4–G–PRGDSGYRGDS (RADA)4–Linker–SVVYGLR (RADA)4–Linker–LKKTETQ (RADA)4–Linker–VGVAPG (RADA)4–Linker–REDV (RADA)4–Linker–KLTWQELYQLKYKG

None Laminin 1 Laminin 1 Collagen IV Bone marrow homing peptides Bone marrow homing peptides Osteogenic growth peptide Osteopontin RGD repeat from fibronectin Osteopontin Thymosin b4 Elastin Fibronectin VEGF mimicking peptide

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RADA16 can also provide peptide scaffolds that enhance osteoblast proliferation and differentiation via presentation of cell adhesion motifs, DGR, and PGR, as well as an osteogenic growth peptide, ALK (Horii et al., 2007). Alkaline phosphatase activity of osteoblasts grown on ALK, DGR, and PRG hydrogel scaffolds increased when compared to cells on bare RADA16. Interestingly, cells on PRG scaffolds also secreted more osteocalcin than cells on ALK, DGR, or bare RADA16 scaffolds. The last example discussed here is RADA16 variants modified with peptides having pro-angiogenic properties: SVV (SVVYGLR), LKK (LKKTETQ), VGV (VGVAPG), RED (REDV), KLT (KLTWQELYQLKYKGI), and PRG (PRGDSGYRDGS) (Wang et al., 2011). RADA16 scaffolds functionalized with these peptides promoted HUVEC growth, and cells on KLT- and PRG-modified RADA16 scaffolds adopted a different morphology than cells on other scaffolds. It was hypothesized that SVV, LKK, VGV, and RED scaffolds did not induce any changes in morphology because of the excess of hydrophobic residues in the peptide sequence causing the decorated face to be inaccessible to the cells. Notably, the KLTand PRG-modified scaffolds increased HUVEC survival and attachment when compared to pure RADA16 and induced morphologies similar to Matrigel and collagen. In 2015, Woolfson et al. developed scaffolds for murine embryonic neural stem cell proliferation and differentiation based on hydrogelating SAF (hSAF) peptides modified with the cell adhesion sequence, RGDS (Nehrban et al., 2015). After 14 days of culture, NSCs grown on unmodified hSAF and modified hSAF scaffolds clumped together to form what is termed a “neurosphere.” RGDS-modified hSAF saw a twofold increase in the average number of cells and the size of neurospheres, when compared with the unmodified hSAF. Notably, neurospheres on RGDSmodified hSAFs differed greatly from cells on laminin controls, on which NSCs were planar and heavily interconnected. NSC differentiation was studied through the neuronal differentiation markers MAP2 and GFAP. The expression levels of these markers are directly correlated to the NSC phenotype, where MAP2 is indicative of neuronal cells and GFAP is indicative of astrocytes. NSCs grown on RGDS-modified hSAF expressed the neuronal marker MAP2 at comparable levels as cells on laminin controls, whereas NSCs on unmodified hSAF expressed lower levels of MAP2. However, neither the undecorated or decorated hSAF saw a significant change in the astrocyte marker GFAP when compared to laminin.

3.2 DRUG DELIVERY Hydrogels are 3D networks of water-soluble polymers that are rendered insoluble via covalent or noncovalent cross-links. Hydrogels offer a widely used and effective method for delivery of small-molecule drugs and biological therapeutics (e.g., proteins and DNA) because their physical and chemical properties can be modified to tailor the release profile of encapsulated cargoes and they are fabricated under mild conditions that are favorable for maintaining cargo activity (Hoare and Kohane, 2008; Peppas, 1997). β-sheet peptide and protein nanofibers can laterally associate


and entangle into physically cross-linked hydrogels, and here we will survey recent advances in hydrogels of self-assembling peptides and proteins for drug delivery.

3.2.1 Small-Molecule Delivery Hydrogels based on silk proteins can be used for delivery of small-molecule drugs. For example, hydrogels consisting of either a high-molecular weight silk protein (SPH) or a composite of SPH and a low-molecular weight silk protein (SPL) release the drug, buprenorphine, at rates that correlated with SPH concentration (Fang. et al., 2006). It was reasoned that increasing SPH concentration led to a more densely packed nanofiber network, and in turn a more tortuous path that buprenorphine must travel through to escape the hydrogel. This was further supported with composite SPH/SPL hydrogels, for which 10% SPH with 6% SPL had a slower diffusion rate than 10% SPH with 2% SPL. Hydrogels based on β-sheet peptide nanofibers can also be used for smallmolecule drug delivery. For example, RADA16 gels released encapsulated small-molecule dyes, such as phenol red, bromophenol blue, 3-PSA, 4-PSA, and Coomassie Brilliant Blue G-250 (CBBG), at rates that correlated with chemical features of the dyes (Nagai et al., 2006). In particular, bromophenol blue and CBBG did not elute from the gels after 7 days, suggesting that they nonspecifically adsorbed onto RADA16 nanofibers. Furthermore, 3-PSA eluted at a faster rate that the more electrostatically charged 4-PSA, suggesting that the net charge of drugs can influence their release kinetics. Similarly to the SPH/SPL gels discussed above, the diffusivity of phenol red, 3-PSA, and 4-PSA decreased as RADA16 concentration increased. Curcumin is a promising drug with antiinflammatory and antitumorigenic properties; however, its clinical use is challenged by its hydrophobicity. Encapsulating curcumin in shear-thinning MAX8 hydrogels can enhance its therapeutic efficacy (Altunbas et al., 2011). In particular, 1% MAX8 hydrogels loaded with 2 mM curcumin-induced death of DAOY cells, whereas 2% MAX8 hydrogels were largely ineffective for delivery, likely due to decreasing curcumin release kinetics with increasing peptide concentration. MDPs also allow for delivery of small hydrophobic drugs. For example, “missing tooth” MDPs discussed briefly above are designed to have a cavity in the hydrophobic core that can be loaded with hydrophobic drugs, such as SN-38, daunorubicin, diflunisal, etodolac, levofloxacin, and norfloxacin (Li et al., 2016). Encapsulation efficiency of drugs within the missing tooth cavity correlated with drug hydrophobicity, such that highly hydrophobic drugs (e.g., SN-38, diflunisal, and etodolac) had higher encapsulation efficiencies than less hydrophobic drugs (e.g., daunorubicin, levofloxacin, and norfloxacin). Interestingly, the release of the more hydrophobic molecules from “missing tooth” hydrogels was delayed when compared with hydrophilic molecules, suggesting that the hydrophobic pocket may mediate retention of hydrophobic drugs through nonspecific interactions, thereby providing a nonspecific mechanism for controlled release.

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3.2.2 Therapeutic Protein Delivery 3.2.2.1 Modulating Protein Diffusion Protein activity is often sensitive to changes in pH, temperature, or presence of organic solvents, which challenges efforts to encapsulate and deliver active therapeutic protein payloads from many conventional polymeric drug delivery vehicles. Hydrogels, and especially those based on self-assembled peptide and protein nanofibers, are therefore ideal delivery systems for therapeutic proteins. In particular, most peptide and protein nanofibers undergo gelation at or near physiological temperature, pH, and ionic strength and do not require the use of cross-linking agents, which are ideal fabrication conditions for preserving therapeutic protein activity. Self-assembly and nanofiber formation can occur in the presence of proteins, which facilitates efficient entrapment of therapeutic protein cargoes within the resultant 3D hydrogel network at well-defined and reproducible doses. Additionally, chemical and physical properties of the hydrogels, such as porosity, net charge, and presentation of protein-binding ligands, can be systemically modified to tailor therapeutic protein release kinetics. This section will survey examples of tailoring self-assembled peptide nanofiber chemical or physical features to tune therapeutic protein diffusion and release kinetics. In general, protein release from hydrogels is dependent on the chemical and physical properties of both the hydrogel and the protein (Koutsopoulos et al., 2008; Branco et al., 2010). For example, diffusivity of lysozyme (14.3 kDa), trypsin inhibitor (20.1 kDa), bovine serum albumin (BSA) (66 kDa), and IgG (∼150 kDa) through RADA16 hydrogels decreased with increasing protein molecular weight likely because the coefficient of diffusion is lower for larger proteins and the hydrogel network creates a tortuous network. In addition, protein net charge influences rate of release from self-assembled peptide hydrogels (Table 19.5). As demonstrated by the release profile of these proteins in Fig. 19.7A, the negatively charged α-lactalbumin Table 19.5 Proteins Used in the Release Studies From MAX8 Hydrogels

Protein Α-Lactalbumin Lysozyme Myoglobin Bovine serum albumin Lactoferrin Human IgG

Molecular Weight (kDa)

Hydrodynamic Diameter (nm)

Daq (37°C) (10–8 cm2 s)

Charge Isoelectric at pH Point, pI 7.4

14.1 14.7 17.4 66

3.2 4.1 3.9 7.2

142 111 113 63

4.2–4.5 11.0 7.0 4.6–4.8

– + = –

77 146

6.1 10.7

74 41

8.4–9.0 5.8–8.0

+ =

Adapted with permission from Elsevier Branco, M.C., Pochan, D.J., Wagner, N.J., Schneider, J.P., 2010. The effect of protein structure on their controlled release from an injectable peptide hydrogel. Biomaterials 31, 9527–9534.


and BSA were found to adsorb onto the surface of the positively charged MAX8 peptide fibers, thereby inhibiting protein release, whereas positive and neutral proteins released rapidly at rates that depended on the charge and size of the protein. For example, the positively charged protein lactoferrin had a release profile comparable to the much lighter protein myoglobin. A small, net positive protein, lysozyme, had the fastest release profile, whereas a heavy, neutral charged protein, IgG, had the slowest release profile excluding the adsorbed proteins.

FIGURE 19.7 (A) Release profile of proteins of different sizes and charges from 1% MAX8 hydrogels. (B) Burst release profiles of WGA from microgels fabricated from admixtures of WGA and Q11 nanofibers with 0% GlcNAc-Q11 (circles), 5% GlcNAc-Q11 (squares), 10% GlcNAc-Q11 (triangles), or 25% GlcNAc-Q11 (diamonds) (C) Blue-light transilluminated digital still images assessing time-dependent GFP retention within CATCH hydrogels fabricated CATCH-GFP (left, black bars) or the control protein mCATCH-GFP (right, gray bars). (A) Reproduced by permission of Elsevier Branco, M.C., Pochan, D.J., Wagner, N.J., Schneider, J.P., 2010. The effect of protein structure on their controlled release from an injectable peptide hydrogel. Biomaterials 31, 9527–9534. (B) Reproduced by permission of The Royal Society of Chemistry Fettis, M.M., Wei, Y., Restuccia, A., Kurian, J.J., Wallet, S.M., Hudalla, G.A., 2016. Microgels with tunable affinity-controlled protein release via desolvation of self-assembled peptide nanofibers. J. Mater. Chem. B 4, 3054–3064. (C) Adapted with permission of Springer Seroski, D.T., Restuccia, A., Sorrentino, A.D., Knox, K.R., Hagen, S.J., Hudalla, G.A., 2016. Co-assembly tags based on charge complementarity (CATCH) for installing functional protein ligands into supramolecular biomaterials. Cell. Biomol. Eng.

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Together, the preceding observations suggest that, in general, the net charge of a protein can inform the design of self-assembled peptide hydrogels with tunable protein release kinetics. Toward biomedical applications, variants of RADA16-I with C-terminal extensions that endow peptide hydrogels with net negative or positive charge, such as GGDGEA (RADA16-DGE) or GGPFSSTKT (RADA16-PTS), provided tunable release of positively and negatively charged cytokines (Gelain et al., 2010). Unexpectedly, negatively charged vascular endothelial growth factor (VEGF) released more slowly from net neutral RADA16 gels than the positively charged cytokines, basic fibroblast growth factor, and brain-derived neurotrophic factor, likely due to charge separation along the RADA16 nanofibers from the guanidinium group in arginine. The negatively charged RADA16-DGE continued the trend of more rapid release of the more negatively charged VEGF when compared to release of positively charged cytokines, whereas positively charged RADA16PFS demonstrated attenuated VEGF release kinetics. The effective release of βFGF was examined through the proliferation of neural stem cells seeded on RADA16, RADA16-DGE, and RADA16-PFS hydrogels. It is important to note that in this study a slow release of βFGF was desired for stimulating the NSCs over multiple weeks. Cells cultured within RADA16-DGE proliferated to the greatest extent, followed by cells within neutral RADA16 gels, whereas proliferation of cells within RADA16-PFS was not different from the negative control. The increase in proliferation within the net neutral and net negative hydrogels was likely due to hydrogel electrostatic retention of βFGF, which established an extended release profile that was sufficient to stimulate NSCs.

3.2.2.2 Stable Integration of Protein Domains Into Hydrogels Although most therapeutic delivery systems seek controlled release of protein from a hydrogel, certain applications may benefit from retaining proteins within the hydrogel as a functional component. CATCH peptides developed by our group can act as recombinant self-assembly tags that integrate folded protein domains into β-sheet nanofibers, thereby immobilizing the protein within the resultant hydrogel (Seroski et al., 2016). A proof-of-concept study used CATCH tags to integrate GFP into CATCH hydrogels. The GFP domain was retained within hydrogels over multiple days when fused to a CATCH tag, whereas GFP lacking a CATCH tag was rapidly released from the hydrogel as demonstrated in Fig. 19.7C. We envision that this approach will be broadly useful for integrating folded protein domains into selfassembled hydrogels for various applications.

3.2.2.3 Affinity-Controlled Release Heparin is a glycosaminoglycan that binds to various growth factors, including those that enhance angiogenesis. To mediate local growth factor presentation to cells, Stupp et al. developed PAs with a heparin-binding domain (LRKKLGKA) that can capture free heparin, and in turn heparin-binding growth factors that promote angiogenesis, such as FGF-2 and VEGF (Rajangam et al., 2006, 2008). Heparin-binding PA nanofibers increased rat corneal angiogenesis and increased blood vessel density in murine


subcutaneous tissue and omentum postimplantation and postislet transplantation, respectively, by binding heparin and activating FGF-2 and VEGF (Stendahl et al., 2008, Ghanaati et al., 2009). More recently, this general approach to capture proteins and locally enhance their signaling activity has been adapted to enhance bone and tooth regeneration (Lee et al., 2009, 2015). As an alternative to macroscopic hydrogels, our group recently developed micron-sized Q11 hydrogels (i.e., microgels) formed through a desolvation process (Fettis et al., 2016). Admixtures of proteins and Q11 nanofibers codesolvated into protein-loaded microgels with encapsulation efficiencies of ∼85%. Proteins encapsulated within Q11 were rapidly released because the peptide is neutrally charged and therefore does not influence protein release kinetics via electrostatic interactions. Thus, we endowed microgels with affinity-controlled release properties by incorporating Q11 variants modified with moieties that bind to proteins via specific noncovalent interactions. For example, a Q11 variant modified with the monosaccharide n-acetylglucosamine (GlcNAc-Q11) provided affinity-controlled release of a GlcNAc-binding protein, wheat germ agglutinin, with kinetic profiles that could be tuned by varying GlcNAc content of the microgels (Fig. 19.7B).

3.3 PREVENTING AND TREATING DISEASE 3.3.1 Membrane-Disrupting Peptides Polycationic materials can induce cell lysis via membrane disruption, which has led to increasing interest in membrane-disrupting self-assembling peptides as antimicrobial and anticancer agents (Li et al., 2010). For example, in 2012 Schneider et al. developed self-assembling peptides with antimicrobial characteristics (Veiga et al., 2012). The PEPXR peptides are variants of the MAX8 peptide where the X (2, 4, 6, 8) denotes the number of lysine residues changed to arginine residues. The PEPXR peptides all demonstrate high antimicrobial activity against Escherichia coli (Fig. 19.8A) and Staphylococcus aureus (Fig. 19.8B) with cell death exceeding ∼95% for all peptides except PEP2R. The antimicrobial activity is proposed to result from interactions between the charged arginine residues and the cellular membrane that cause membrane rupture. Importantly, in contrast to their antimicrobial activity, PEP8R caused hemolysis in ∼25% of human red blood cells (hRBCs), whereas PEP6R, PEP4R, and PEP2R caused hemolysis in only ∼15% of hRBCs, which together suggest the potential of these peptides as selective, highly effective antimicrobials. Another example of a modified self-assembling peptide with cell membrane disrupting properties is the PA-(KLAKLAK)2 (Standley et al., 2010). (KLAKLAK)2 is a cationic α-helical peptide that induces cancer cell death by disrupting the cell membrane when fused to cell-penetrating molecules. The peptide (KLAKLAK)2 alone cannot be transported across the cell membrane because of its high polarity and hydrophilicity, however, once fused to the hydrophobic PA it is readily internalized by cancer cells. PA-(KLAKLAK)2 causes roughly 75% cell death in breast cancer lines, whereas the (KLAKLAK)2 peptide alone does not induce cell death. This study

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FIGURE 19.8 Antimicrobial activity of 0.5, 1, 1.5, and 2 wt% hydrogels against (A) Escherichia coli and (B) Staphylococcus aureus after 24 h at 37°C. (C) Brightfield photomicrographs of Jurkat T cell agglutination in culture media with or without Gal-1 in the presence of 1 mM Q11 nanofibers with 0, 100, or 250 μM LacNAc, or 250 μM b-lactose. (B) Reproduced with permission of Elsevier Veiga, A.S., Sinthuvanich, C., Gaspar, D., Franquelim, H.G., Castanho, M.A., Schneider, J.P., 2012. Arginine-rich self-assembling peptides as potent antibacterial gels. Biomaterials 33 (35), 8907–8916. (C) Reproduced with permission of Springer Restuccia, A., Tian, Y.F., Collier, J.H., Hudalla, G.A., 2015. Self-assembled glycopeptide nanofibers as modulators of Galectin-1 bioactivity. Cell. Mol. Bioeng. 8 (3), 471–487.


further demonstrates the biomedical versatility of PAs that is afforded by their ability to “present” bioactive molecules on the surface of high-aspect ratio nanofibers.

3.3.2 Vaccines Peptide vaccines are challenged by both poorly controlled antigen display and low immunogenicity that often necessitates the use of immunostimulatory adjuvants with ill-defined modes of action and variable efficacy. Collier et al. demonstrated that Q11 peptide nanofibers modified with peptide antigens act as “self-adjuvanting” vaccines that elicit robust immune responses without any additional immunostimulatory molecules. For example, the Q11 peptide itself is largely nonimmunogenic, yet when a 17 amino acid peptide epitope from chicken egg ovalbumin (OVA) is attached to Q11, herein called O-Q11, strong anti-OVA antibody titers are raised (Rudra et al., 2009). Notably, O-Q11 elicited antibody titers at levels comparable to OVA in complete Freund’s adjuvant (CFA), which is a potent experimental adjuvant, composed of inactivated mycobacteria. However, CFA elicits strong inflammatory responses that are difficult to attenuate because it is biologically derived and has a poorly defined composition. Thus, the robust adjuvanticity of chemically defined Q11 nanofibers presents a significant advantage over using biologically derived adjuvants. Further work demonstrated that Q11 also adjuvanted a malaria epitope (NANP)3, invoking a persistent antibody response without frequent boosts or use of other adjuvants (Rudra et al., 2012). In addition to raising antibodies, Q11 nanofibers can also elicit an adjuvant-free CD8 T cell response (Chen et al., 2013), and act as a carrier for folded protein antigens that elicits strong antiprotein antibody responses similar to those produced by OVA-Q11 and (NANP)3-Q11 (Hudalla et al., 2013, 2014). Interestingly, Q11-based vaccines are effective without inducing local inflammation that is normally associated with vaccines, through mechanisms that are currently undefined (Chesson et al., 2014).

3.3.3 Immunomodulatory Materials

Functional molecules displayed on the surface of β-sheet peptide nanofibers, such as cell adhesion peptides, can mediate specific interactions between cells and nanofibrillar hydrogels, as discussed above in the context of tissue regeneration. Alternatively, nanofibers can be used to display other functional molecules, such as immunomodulatory proteins and carbohydrates. For example, a variant of the zuotin peptide EAK16-II modified with a hexahistidine tag (EAK-H6) enabled noncovalent antibody immobilization on nanofiber surfaces (Zheng et al., 2011). In particular, EAK-H6 enabled selective binding of an antihexahistidine antibody, which in turn bound to protein A/G through the antibody Fc domain. Immobilized protein A/G then bound an anti-CD4 antibody through its Fc domain, which mediated selective capture of T cells on the “surface” of EAK peptide nanofibers. A subsequent study with this platform demonstrated localized IgG in vivo for up to 5 days (Wen et al., 2013). Toward immunomodulation, this strategy was used to hinder the migration of donor antigen-presenting cells (dAPCs) away from an allograft skin transplant site (Wen et al., 2014), which can initiate a robust immune response against the

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graft. dAPCs were selectively targeted through I-Ad, a class II major histocompatibility complex molecule that is uniquely expressed by the donor cells. EAK-H6 membranes with bound antibodies were retained under skin grafts for nearly 1 week in vivo. EAK-H6 membranes with bound anti-I-Ad antibodies recognized I-Ad on the surface of dAPCs in vitro and in vivo, impeded dAPC migration away from the transplantation site, and suppressed graft-recipient T cell expression of interferongamma ex vivo. Together, these data suggest the potential of self-assembled peptide hydrogels modified with antibodies to enhance transplant tolerance by locally suppressing allogeneic T cell responses. Galectins are carbohydrate-binding proteins that act as extracellular signals in immunological tolerance, inflammation, and antigen-specific T cell activation, which has led to increasing interest in galectin–carbohydrate interactions as therapeutic targets (Farhadi and Hudalla, 2016). An important physical feature of galectin– carbohydrate interactions in nature is the “glycocluster effect,” in which binding affinity is enhanced via high density, polyvalent carbohydrate display on the cell surface, and extracellular matrix. Self-assembled peptide nanofibers offer a unique material method for creating biomaterials that can interact with galectins by recreating the glycocluster effect. For example, we created variants of Q11 modified with the galectin-binding disaccharide, n-acetyllactosamine (LacNAc), by treating GlcNAc-Q11 nanofibers with a glycosyltransferase enzyme in the presence of a sugar donor. LacNAc-Q11 nanofibers bound galectin-1 and galectin-3 with tunable affinities that correlated with LacNAc concentration and inhibited galectin-mediated T cell apoptosis more effectively than a soluble small-molecule galectin inhibitor as demonstrated in Fig. 19.8C (Restuccia et al., 2015).

4. FUTURE PERSPECTIVES Interest in peptides and proteins that form nanofibers, and the use of these nanofibers as the basis for materials for biomedical applications, is likely to grow and evolve as our understanding of the self-assembly process and methods to manipulate it increase. In part, the continued interest in self-assembly stems from its simple, well defined, and reproducible generation of higher-ordered structures, which provides many advantages over top-down approaches used to create materials with nanoscale features. To date, “bottom-up” self-assembly approaches have led to new materials for various biomedical applications, including cell scaffolding, drug delivery, selective cell lysis, and vaccines, as discussed above; however, many emerging applications, such as 3D bioprinting, scaffolds with cell adhesion/signaling domains with integrated cell-responsive proteins, photoelectronics and fiber optics, and wound healing, have received limited attention to date (Loo et al., 2015; Djalali et al., 2002; Schneider et al., 2008). The customizability of peptide and protein self-assembly units allows for the creation of specific and controllable microenvironments that may well exceed or augment the current systems and their applications. By simply appending a functional

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Standley, S.M., Toft, D.J., Cheng, H., Soukasene, S., Chen, J., Raja, S.M., Band, V., Band, H., Cryns, V.L., Stupp, S.I., 2010. Induction of cancer cell death by self-assembling nanostructures incorporating a cytotoxic peptide. Cancer Res. 70 (8), 3020–3026. Stendahl, J.C., Wang, L., Chow, L.W., Kaufman, D.B., Stupp, S.I., 2008. Growth factor delivery from self-assembling nanofibers to facilitate islet transplantation. Transplantation 86 (3), 478–481. Veiga, A.S., Sinthuvanich, C., Gaspar, D., Franquelim, H.G., Castanho, M.A., Schneider, J.P., 2012. Arginine-rich self-assembling peptides as potent antibacterial gels. Biomaterials 33 (35), 8907–8916. Vepari, C., Kaplan, D.L., 2007. Silk as a biomaterial. Prog. Polym. Sci. 32 (8–9), 991–1007. Wang, Y., Goodson, T., 2007. Early aggregation in prion peptide nanostructures investigated by nonlinear and ultrafast time-resolved fluorescence spectroscopy. J. Phys. Chem. B 111, 327–330. Wang, Y., Kim, H., Vunjak-Novakovic, G., Kaplan, D.L., 2006. Stem cell-based tissue engineering with silk biomaterials. Biomaterials 27 (36), 6064–6082. Wang, X., Qiao, L., Horii, A., 2011. Screening of functionalized self-assembling peptide nanofiber scaffolds with angiogenic activity for endothelial cell growth. Prog. Nat. Sci. Mat. Int. 21 (2), 111–116. Wen, Y., Kolonich, H.R., Kruszewski, K.M., Giannoukakis, N., Gawalt, E.S., Meng, W.S., 2013. Retaining antibodies in tumors with a self-assembling injectable system. Mol. Pharm. 10 (3), 1035–1044. Wen, Y., Liu, W., Bagia, C., Shaojuan, Z., Bai, M., Janjic, J., Giannoukakis, N., Gawalt, E.S., Meng, W.S., 2014. Antibody-functionalized peptidic membranes for neutralization of allogeneic skin antigen-presenting cells. Acta Biomater. 10 (11), 4759–4767. Whitesides, G.M., Grzybowski, B., 2002. Self-assembly at all scales. Science 295 (5564), 2418–2421. Yi, C., Berndt, P., Tirrell, M., Fields, G.B., 1996. Self-assembling amphiphiles for construction of protein molecule architecture. J. Am. Chem. Soc. 118 (50), 12515–15520. Zhang, S., Holmes, T., Lockshin, C., Rich, A., 1993. Spontaneous assembly of a selfcomplementary oligopeptide to form a stable macroscopic membrane. PNAS 90 (8), 3334–3338. Zhang, S., Holmes, T., DiPersio, M., Hynes, R., Su, X., Rich, A., 1995. Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials 16, 1385–1393. Zhang, S., Gelain, F., Zhao, X., 2005. Designer self-assembling peptide nanofiber scaffolds. Semin. Cancer Biol. 15 (5), 413–420. Zhang, X., Reagan, M.R., Kaplan, D.L., 2009. Electrospun silk biomaterial scaffolds for regenerative medicine. Adv. Drug Deliv. Rev. 61 (12), 988–1006. Zheng, Y., Wen, Y., George, A.M., Steinbach, A.M., Phillips, B.E., Giannoukakis, N., Gawalt, E.S., Meng, W.S., 2011. A peptide-based material platform for displaying antibodies to engage T cells. Biomaterials 32, 249–257. Zimenkov, Y., Conticello, V.P., Guo, L., Thiyagarajan, P., 2004. Rational design of a nanoscale helical scaffold derived from self-assembly of a dimeric coiled coil motif. Tetrahedron 60 (34), 7237–7246.

CHAPTER

Peptide-Modified Hydrogels for Therapeutic Vascularization

20

Tália Feijão, Ana L. Torres, Marco Araújo, Cristina C. Barrias Universidade do Porto, Porto, Portugal

CHAPTER OUTLINE 1. Introduction�� 599 1.1 The Need for Provascularization Strategies�� 599 1.2 Mechanisms Involved in Microvasculature Formation�� 601 1.3 The Role of Biomaterials in Provascularization Approaches�� 602 2. Hydrogels in Vascular Tissue Engineering�� 604 2.1 Protein- and Polypeptide-Based Hydrogels�� 605 2.2 Polysaccharide-Based Hydrogels�� 605 2.3 Synthetic Hydrogels�� 606 3. Biofunctionalization of Hydrogels With Angiogenic Peptides�� 607 3.1 Hydrogel Immobilization of Peptides by Affinity Binding�� 607 3.2 Hydrogel Immobilization of Peptides by Chemical Conjugation�� 608 3.2.1 Chemical Conjugation With Growth Factor–Mimicking Peptides�� 610 3.2.2 Chemical Conjugation With Matrix Protein–Mimicking Peptides�� 611 3.2.3 Chemical Conjugation With Other Angiogenic Peptides�� 613 4. Conclusions and Future Perspectives�� 614 Acknowledgments�� 615 References�� 615

1. INTRODUCTION 1.1 THE NEED FOR PROVASCULARIZATION STRATEGIES Tissue engineering (TE) strategies aim at restoring the structure and function of damaged tissues, and often rely on the implantation of biomaterial-based scaffolds laden with cells or neotissues. Frequently, the distance between cells located at deeper areas of the scaffold and the local capillary network exceeds a limit of a few hundred microns, creating hypoxic regions (Moon and West, 2008; Novosel et al., 2011). Therefore, cell survival within constructs of clinically relevant dimensions is highly dependent on the establishment of a neovasculature, not only at the periphery Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00020-3 Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 20 Peptide-Modified Hydrogels for Therapeutic Vascularization

Vascular diseases

Tissue engineering

peripheral artery disease

Scaffold implantation

Lack of neovascularization

neovascularization

Ischemia Muscle below blockage begins to die

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FIGURE 20.1 Provascularization strategies are key for successful tissue engineering (TE) applications for the regeneration of vascularized tissues, namely to scale up TE constructs, and can also be of great value in the clinical management of ischemic vascular diseases. Right image: scheme of arterial system was adapted from Servier Medical Art.

but also inside the scaffold. In the absence of an appropriate vascular network, gradients of oxygen and nutrients are established, creating deficiencies at the innermost regions of the scaffold, where metabolic waste products will also tend to accumulate (Fig. 20.1). Such events have a negative impact on cells viability and function and ultimately result in deficient scaffold–host integration and implant failure (Moon and West, 2008; Novosel et al., 2011). Although the need for provascularization strategies in TE has been clearly identified, they still represent a huge challenge in the development of complex artificial tissues, and successful approaches are still limited. Therapeutic vascularization through regenerative medicine also provides a promising avenue for the clinical management of ischemic vascular diseases. Atherosclerosis, the primary cause of these diseases, is characterized by the narrowing of arteries and small blood vessels due to plaque deposition, commonly affecting the brain, heart, and lower limbs. Vessel occlusion and blockage of blood flow leads to ischemia, followed by tissue damage and dysfunction. Peripheral arterial disease (PAD) (Fig. 20.1) is a quite debilitating condition, where occlusion of atherosclerotic arteries, frequently located in lower limbs, results in hypoxia and tissue necrosis, causing pain. In the most severe cases, it can result in gangrene, which may ultimately lead to member amputation or even death (Fowkes et al., 2016; Olin et al., 2016). PAD is also a major risk factor for heart attack and stroke, two of the leading causes of death in developed countries (Go et al., 2014). Currently, the prevalence of PAD is increasing and it has become a global problem in the 21st century (Fowkes et al., 2016; Olin et al., 2016). Symptoms or signs of PAD are frequently observed

1. Introduction

in patients with diabetes. Ischemic diabetic foot ulcers are generally difficult to treat, being a major cause of lower leg amputation. The relatively poor outcome of these chronic wounds probably results from a combination of factors, including the anatomic distribution of the vascular lesions that makes them difficult to treat, problems associated with microvascular dysfunction and impaired formation of collateral vessels, and also the presence of multiple comorbidities, such as infection and neuropathy. The particular characteristics of diabetes-associated PAD pose significant technical challenges associated with revascularization using standard clinical procedures (Hinchliffe et al., 2016). Although there are currently a number of established treatments for occluded blood vessels, such as angioplasty, stenting, thrombolysis, and surgical bypass, all of them present inherent limitations and complications (Hinchliffe et al., 2016). Moreover, there are currently no FDA-approved treatments to reproducibly enhance vascularization (Van Hove and Benoit, 2015). Clearly, the ability to promote neovascularization processes in a controlled way, and its therapeutic exploitation, will be of enormous value to a broad range of medical and TE applications (Fig. 20.1).

1.2 MECHANISMS INVOLVED IN MICROVASCULATURE FORMATION Understanding the mechanisms associated with new vessels formation is essential for developing successful strategies to induce and control vascularization, under both physiological and pathological conditions. In adult life, neovascularization was once thought to be limited to sprouting of new capillaries from preexisting vessels, involving activation, migration, and proliferation of mature endothelial cells (EC), followed by vascular stabilization, a process known as angiogenesis (Risau, 1997). However, in 1997 it was reported for the first time that circulating endothelial progenitor cells (EPC) from adult peripheral blood (PB) and derived from bone marrow were able to participate in the de novo vascular formation consistent with the process of vasculogenesis (Asahara et al., 1997). In fact, three processes coexist, contributing to the remodeling of vascular network and reposition of normal vascularization: vasculogenesis, angiogenesis, and arteriogenesis (Semenza, 2007). This was a true paradigm shift from the original thinking, which limited adult neovascularization processes to angiogenesis. Further studies have shown that EPC may home to sites of vascular remodeling, such as wound healing, limb ischemia, postmyocardial infarction, or tumor growth, where they differentiate into EC (Asahara et al., 1999a,b; Assmus et al., 2002; Gill et al., 2001; Lyden et al., 2001; Shintani et al., 2001). Different in vitro studies with EPC derived from PB or umbilical cord blood showed the existence of at least two EPC subpopulations, with distinct morphology, proliferative capacity, and vasculogenic or angiogenic activity: early and late EPC. Early EPC, with spindle-like shape morphology and myeloid/hematopoietic properties share features with immune cells, particularly monocytes/macrophages. These cells are characterized by low proliferative capacity, acting in a paracrine fashion by releasing several proangiogenic cytokines. Late EPC, also known as outgrowth EC,

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with cobblestone-shape reminiscent of EC, have a more mature EC-like phenotype with high proliferative capacity. These cells have lower cytokine release capacity but physically contribute to formation of new capillaries by inosculating into preexisting vascular networks (Hirschi et al., 2008; Sieveking et al., 2008). Recruitment and homing of EPC into active remodeling sites require a coordinated multistep process, including chemoattraction, adhesion, transendothelial migration, and tissue invasion. Different cytokines and growth factors (GF) are involved in these processes. Stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) are key players in EPC recruitment (Asahara et al., 1999a,b; Ceradini and Gurtner, 2005; Tepper et al., 2005), but other factors are also involved (Dimmeler et al., 1999; Heissig et al., 2002). EPC may contribute to neovascularization through direct incorporation into neovessels, differentiation into mature EC, and paracrine/juxtacrine signaling (Balaji et al., 2013). Postnatal vascular remodeling is mainly triggered by low oxygen levels (hypoxia) (Rey and Semenza, 2010). Formation of new capillaries from preexisting blood vessels occurs primarily by EC sprouting, but bridging and intussusceptive growth can also occur (Fig. 20.2) (Jain, 2003). VEGF is a pivotal regulator of new blood vessel formation. VEGF signaling promotes EC chemotaxis, activation, proliferation, and matrix degradation, inducing the motile and invasive behavior that drives EC sprouting (Galiano et al., 2004). During neovascularization, the surrounding extracellular matrix (ECM) is subjected to dramatic changes in terms of structure and composition, which provide important guiding cues along the process (Hangai et al., 2002; Hynes, 2002). For the formation of a functional microvascular network, nascent vessels have to undergo maturation, a process tight regulated by different cytokines and GF. New EC sprouts connect with adjacent vessels for anastomosis, pericytes are recruited to stabilize vessels, and a basal membrane is deposited at the abluminal surface (Carmeliet, 2003). Finally, cell– cell junctions are strengthened and a mature quiescent phenotype is reestablished.

1.3 THE ROLE OF BIOMATERIALS IN PROVASCULARIZATION APPROACHES Inspired by the natural mechanisms and processes involved in microvasculature formation, classical provascularization approaches are commonly based on the delivery of angiogenic drugs, frequently GF, and vascular cells (Phelps et al., 2010). Unfortunately, proangiogenic therapies have so far resulted in disappointing outcomes in clinical trials, showing inconsistent and often transient effects (Chu and Wang, 2012). It is nowadays clear that some of these failures might be related to the way these bioactive agents are administrated, rather than to a lack of therapeutic potential. Low stability (in the case of drugs) or viability (in the case of cells), low persistence at lesion sites, and undesirable effects at off-target tissues are classical challenges of drug/cell delivery that can be tackled using biomaterials. In fact, conjugation with biomaterial-based carriers can significantly improve the effectiveness of biological agents, by providing protection from the harsh in vivo environment, physicochemical

Vasculogenesis involves the differentiation of EC from angioblasts or hemangioblasts and their organization into a primary capillary plexus; whereas angiogenesis refers to the formation of new capillaries from preexisting blood vessels via alternative processes of sprouting, intussusception, and bridging. New capillaries become stabilized by pericytes.

1. Introduction

FIGURE 20.2

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stabilization, and regulation of spatial–temporal availability (Bader and Putnam, 2014; Van Hove and Benoit, 2015). Different types of biomaterials have been tested as drug/cell carriers in angiogenic therapies, with hydrogels being among the most widely used. In terms of therapeutic drugs, hydrogels have long been used as GF reservoirs. The majority of available studies are based on the use of single GF (García et al., 2016). Significantly, since angiogenic processes depend on the tightly coordinated spatial and temporal presentation of various factors, more complex hydrogel systems providing sequential release of multiple GF have also been described (Chen et al., 2007; Zieris et al., 2011). Instead of full-length GF and other relevant proteins, alternative strategies explored the use of small peptidic fragments. Given their high biospecificity, low immunogenicity, small size, and relative ease of synthesis, the use of peptides as bioactive moieties may result in less expensive and more easily tuneable formulations, and thus advance the design of functional biomaterials. Recently, there has been an increased interest in peptide drugs and numerous sequences have been identified for different applications, including proangiogenic therapies. Several therapeutic angiogenic peptides are currently in preclinical testing, as recently reviewed in Van Hove and Benoit (2015). In this chapter, we will present an overview of strategies involving the biofunctionalization of hydrogels with different types of angiogenic peptides for therapeutic vascularization.

2. HYDROGELS IN VASCULAR TISSUE ENGINEERING Hydrogels consist of highly hydrated polymeric 3D networks and have been extensively used to deliver angiogenic compounds and cells. Depending on the type of polymer, hydrogels may be obtained by mild physical or chemical cross-linking, allowing the immobilization of drugs and cells without compromising their bioactivity and viability, respectively. Hydrogels share some structural features with the native ECM, thus recreating physiologic microenvironments that support drug stability and cellular activities, which makes them ideal reservoirs for these therapeutic agents. The high permeability of hydrogel networks is an important trait in the context of drug/cell immobilization. It not only allows drugs to diffuse across the polymeric mesh, but also preserves the viability of entrapped cells, by facilitating the in-and-out transport of nutrients, bioactive molecules, oxygen, and metabolic products. Hydrogels are versatile materials that generally present tuneable biophysical and biochemical properties, which can be used to control the drug/cell immobilization and release processes (Wang et al., 2017). In addition, hydrogels can be designed for minimally invasive administration, and their degradability can be tuned as to occur in a timely fashion that coincides with the pace of tissue regeneration and vascularization processes (Bidarra et al., 2016; Fonseca et al., 2011). In provascularization strategies, hydrogels have been used in many different approaches, which range from

2. Hydrogels in Vascular Tissue Engineering

more simple reservoirs of proangiogenic molecules and cells to more complex cellinstructive matrices where de novo 3D microvascular networks can develop (Chen et al., 2012; Liu et al., 2015; Phelps et al., 2010). A wide range of hydrogel forming natural and synthetic polymers is currently available. Their main properties are briefly discussed.

2.1 PROTEIN- AND POLYPEPTIDE-BASED HYDROGELS Protein-based hydrogels are very popular, and the most commonly used include collagen, gelatin, fibrin, and Matrigel. Their structural properties, combined with the intrinsic presentation of cell-recognition domains, make them useful ECM mimics. In fact, these hydrogels inherently present specific recognition sites for key cellular process, such as cell adhesion and proteolytic degradation, providing physiological cellular microenvironments. They can be formed with appropriate matrix density, under mild conditions, being thus suitable for drugs and cells entrapment. However, batchto-batch variability between distinct protein isolations may be reflected on hydrogel properties, making it difficult to repeatedly maintain consistency. This is particularly relevant for Matrigel, a commercially available 3D matrix widely used in vascular research. Matrigel is a solubilized basement membrane preparation, derived from mouse sarcoma, rich in ECM proteins and GF, commonly used for analyzing the tubulogenic ability of EC. Yet, while presenting attractive properties, concerns regarding the unknown composition and large variability of Matrigel largely limit its applicability as 3D in vitro model, while its animal origin makes it unsuitable for clinical use. Another potential disadvantage of protein-based hydrogels is related to their mechanical properties that can be difficult to tune and decouple from other factors. Mechanical properties can only be varied over a limited range, which is generally achieved by adjusting the total protein concentration, but this concomitantly affects the density of presented ligands. Also, as these hydrogels are susceptible to enzymatic degradation and remodeling, they are often unsuited for long-term applications. The properties of protein-based hydrogels can be potentiated through chemical modification of native proteins with different types of biological moieties, including peptides. Self-assembling angiogenic peptides (SAPs) are a specific class of polypeptides that are able to form nanostructures through intermolecular hydrogen bonding. Further interactions between those structures under specific conditions, such as high ionic concentrations, can lead to the formation of hydrogel-like 3D networks that emulate ECM architecture (Hartgerink et al., 2001). For vascularization strategies, angiogenic motifs can be incorporated into SAPs, usually by extending or intercalating the original SAP sequence with short bioactive amino acid (Kumar et al., 2015, 2016).

2.2 POLYSACCHARIDE-BASED HYDROGELS There are many different types of hydrogel-forming polysaccharides, including alginate, agarose, gellan gum, pectin, hyaluronic acid, and chitosan, among others. These natural polymers, derived from different plant or animal sources,

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generally present low toxicity and good biocompatibility. Polysaccharide-based hydrogels combine some of the properties of protein-based and synthetic hydrogels, as they can range from highly bioactive to bioinert hydrogels. Hyaluronic acid, for example, is a natural glycosaminoglycan that plays important roles not only as structural but also as functional ECM component, being able to specifically interact with different cell surface receptors, namely CD44 and CD168. Also, some polysaccharide-based hydrogels, namely hyaluronic acid and chitosan, are susceptible to degradation by mammalian enzymes. In contrast, other polysaccharides, such as alginate and agarose, are essentially bioinert, as they lack specific cell/enzyme-recognition sites, at least as far as it is currently known. The composition of some polysaccharides gels can be considered as relatively well defined, but adequate purification of raw materials is a fundamental step. Fortunately, for some of these polymers, biomedical-grade products are currently commercially available, allowing customers to select products with quite consistent properties. The biomechanical properties of polysaccharide-based hydrogels are relatively easy to tune, generally by changing the polymer mass and/or the cross-linking density. Moreover, polysaccharides are chemically versatile, presenting different types of functional groups that offer the potential for covalent modification by multiple chemical routes.

2.3 SYNTHETIC HYDROGELS Different types of synthetic hydrogels are currently available for TE applications, namely those based in poly(ethylene glycol) (PEG), poly(vinyl alcohol), and poly(2-hydroxyethyl methacrylate). Synthetic hydrogels can be fine-tuned to provide a wide range of material structural/mechanical properties, but their chemical 3D networks are devoided of biofunctionalities for cell recognition. However, such bioinertness can be regarded as an advantage when it comes to artificial ECM design because synthetic hydrogels can play the role of “blank slates” for further modification with well-defined cues, to elicit specific cell responses. This way, controlled cellular microenvironments can be established, where the level of complexity can be additively or synergistically increased. The simplest settings generally capture basic structural and mechanical features of the ECM and try to direct essential cellular responses, namely cell adhesion (Kyburz and Anseth, 2015). The design of more complex microenvironments may allow the manipulation of other cellular processes, such as guiding cell morphogenesis or directing cell differentiation, for example (Hanjaya-Putra et al., 2011). To allow spatial control over matrix biofunctionality, hydrogels conjugated with multiple bioactive moieties, often arranged in complex gradients or patterns, have been developed (DeForest and Anseth, 2011; Lee et al., 2015). Also, orthogonal bioconjugation reactions have been recently described, which can be used for sequential modifications under cytocompatible conditions, providing a means to achieve dynamic tuning and temporal control over matrix properties (DeForest and Anseth, 2011; Lee et al., 2015).

3. Biofunctionalization of Hydrogels With Angiogenic Peptides

3. BIOFUNCTIONALIZATION OF HYDROGELS WITH ANGIOGENIC PEPTIDES From a technical and economic standpoint, the functionalization of hydrogels with small synthetic peptides presents several advantages over the use of full-length proteins, which are much more sensitive and complex to use, as well as more expensive (Maia et al., 2013). In terms of therapeutic effect, similar or even superior angiogenic ability of bioactive peptides, as compared to parental proteins, has already been demonstrated in a few studies (Mulyasasmita et al., 2014; Santulli et al., 2009). For example, the stability of soluble QK, a 15-amino acid VEGF-mimicking peptide originally developed by D’Andrea et al. (2005) has been reported to be higher than that of the whole protein. While VEGF-15 has a half-life of less than 15 min, being afterwards rapidly degraded, QK has been shown to degrade only after 24 h in 50% human serum (Mulyasasmita et al., 2014). Most of the therapeutic peptides have been tested in their soluble form, but wellknown limitations such as poor intestinal permeability and short circulating half-lives in the blood stream may reduce their efficacy. Thus, combining bioactive peptides with hydrogel carriers for controlled delivery appears as a promising strategy to overcome these limitations and maintain efficacious therapeutic dosages at target sites. Drug loading in hydrogels can be achieved either via noncovalent physical approaches, such as entrapment/encapsulation and affinity-based immobilization, or by covalent immobilization. Drug immobilization by simple entrapment/encapsulation, which will not be discussed herein, may present some disadvantages because these systems often exhibit poor drug-loading capacity and fail to provide predictable and controllable patterns of drug release. They are particularly unfitted for the immobilization of small drugs, such as peptides, as diffusion-controlled release kinetics of these compounds is generally too fast to provide sustained delivery over adequate periods, frequently resulting in burst release and rapid clearance (Lustig and Peppas, 1988). In this chapter, only peptide immobilization strategies based on affinity binding and covalent binding will be described. Some proangiogenic peptides consist on de novo designed sequences, whereas others are synthetic analogues of peptide sequences present in angiogenic proteins (Finetti et al., 2012; Van Hove and Benoit, 2015). Although much emphasis has been placed on the role of angiogenic cytokines and GF in vascular development, current knowledge suggests that ECM components are equally important. These are particularly relevant when designing hydrogels as 3D microenvironments for vascular cells. Therefore, grafting hydrogel with matrix protein–mimicking peptides has been a prevalent strategy to build ECM analogues, which will also be briefly discussed herein.

3.1 HYDROGEL IMMOBILIZATION OF PEPTIDES BY AFFINITY BINDING Molecular recognition hydrogels may provide an interesting alternative to passive physical entrapment, namely for immobilization of small compounds that

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would otherwise be rapidly released from the 3D network. A mixing-induced two-component hydrogel has been reported, consisting on two engineered protein components containing tryptophan-rich (C) and proline-rich (P) peptide domains (Mulyasasmita et al., 2014). These domains rapidly associate on mixing, forming physical hydrogels under physiological conditions, which allows mild immobilization of bioactive agents and injectability. Affinity immobilization of QK peptides was achieved by synthesizing QK fused to a single proline-rich domain (P1) that acted as the affinity tag. When protein components and P1–QK conjugates were mixed, peptides become entrapped in the 3D network and docked onto preexisting C domains. Release kinetics of P1–QK from the hydrogel was shown to be significantly slower than that of unmodified QK. The use of a proline dimer (P2) as an alternative affinity tag resulted in higher affinity immobilization strength and slower release kinetics. Both fusion peptides (P1–QK and P2–QK) maintained in vitro angiogenic activity.

3.2 HYDROGEL IMMOBILIZATION OF PEPTIDES BY CHEMICAL CONJUGATION Compared to other immobilization strategies, chemical conjugation of bioactive factors may present additional advantages, namely by affording increased stability and greater control over spatial/temporal presentation in the extracellular environment. For chemical conjugation, functional groups natively present in peptide amino acid residues are coupled to polymers exhibiting complementary functionalities. Among natural amino acids, commonly targeted conjugation sites are amino groups and thiol groups of cysteine residues (Cobo et al., 2015). To expand the conjugation toolbox, noncanonical amino acids or other appropriate external chemical moieties can be introduced in the peptide, prior to immobilization. Similarly, carrier polymers can be chemically modified to yield different types of analogues containing acrylate, azide, alkyne, olefin, thiol, aldehyde, N-hydroxysuccinimide, or maleimide functionalities, among others (Krishna and Kiick, 2010; Zhu and Marchant, 2011). This way, derivatized polymers can be coupled to native or chemically modified peptides for building polymer–peptide hybrids via a variety of coupling procedures, including carbodiimide, azide–alkyne cycloaddition, Michael addition, disulphide coupling, amidation of activated esters, oxime ligation, etc. (Cobo et al., 2015; Jabbari, 2011; Jonker et al., 2012; Jung and Theato, 2013). Although a more detailed description of bioconjugation strategies is out of the scope of this chapter (for a detailed review see Jabbari, 2011; Cobo et al., 2015), some common examples are depicted in Fig. 20.3. Preserving the bioactivity of the immobilized peptide is a key requirement for its successful application, and it can generally be easily achieved by selecting an adequate ligation approach. For example, it has been shown that the QK peptide remains bioactive over a wide range of concentrations when presented in immobilized form, bound to different types of substrates (Chan et al., 2011; Finetti et al., 2012; Koepsel et al., 2012; Lee et al., 2010).


FIGURE 20.3 Examples of bioconjugation reactions used in the preparation of polymer–peptide hybrid hydrogels (Jabbari, 2011; Cobo et al., 2015).

It is not always clear whether angiogenic peptides can exert their biological function in the immobilized state, or whether they need to be first released to interact with their receptors. Notably, since angiogenesis depends on the concerted actions of soluble and insoluble bioactive compounds, different peptides are likely to have different modes of activity. Several strategies can be used to promote peptide release, whenever desirable (Fig. 20.4). For instance, hydrolytically or enzymatically degradable amino acid sequences can be incorporated in the designed peptide, flanking the bioactive region (Maia et al., 2013). In alternative, peptides can be grafted to carrier hydrogels via degradable linkers or can be released on hydrogel degradation (Chandrawati, 2016; Mauri et al., 2017). Stimulus-responsive hydrogels are particularly appealing, allowing for unique levels of control over drug release in response to external cues (Koetting et al., 2015).

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FIGURE 20.4 Mechanisms of drug release from hydrogel matrices. (A) Passive diffusion of entrapped drug; (B) drug diffusion from affinity-binding matrix; (C) diffusion of covalently bound drug from hydrogel with degradable (hydrolytically or enzymatically) sites; (D) diffusion of drug covalently bound via degradable (hydrolytically or enzymatically) linkers; (E) diffusion of covalently bound drug from degradable hydrogel.

3.2.1 Chemical Conjugation With Growth Factor–Mimicking Peptides Proangiogenic approaches using peptides have been largely focused on the use of VEGF mimics, owing to the key role of this GF in the early stages of angiogenesis. The above-mentioned QK peptide, which mimics the receptor binding α-helix region of VEGF, is probably the most widely studied. Based on VEGF 17–25 amino acid region, QK was rationally designed to preserve the corresponding α-helix secondary structure of the parental protein and the 3D presentation that determines ligandreceptor binding (D’Andrea et al., 2005; Van Hove and Benoit, 2015). In its soluble form, it has been shown to promote EC proliferation and tubulogenesis (Van Hove and Benoit, 2015). QK stands as an excellent candidate for the functionalization of provascularization biomaterials, not only for its well-documented angiogenic properties but also for its short peptide sequence, which makes it easy to synthesize and conjugate to polymeric carriers. Leslie-Barbick et al. (2011), studied the effect of sequestering QK in a PEG matrix. Peptide terminal amino groups were first modified with a PEG-succinimidyl ester linker and then covalently bound to a PEG hydrogel by photocrosslinking. The PEGylated peptide showed increased solubility and bioactivity, promoting EC


tubulogenesis when immobilized on the surface of hydrogels or in bulk collagenasedegradable hydrogels. In a mouse cornea micropocket angiogenesis assay, acellular PEG-QK hydrogels combined with soluble VEGF were shown to support a higher level of vessel coverage, as compared with unbound VEGF and PEG-VEGF hydrogels. In this case, soluble VEGF was added to all groups to induce an external angiogenic response into the normally avascular cornea. Chan et al. (2011) proposed a different version of the QK peptide, aimed at mimicking the matrix-bound VEGF189 isoform, for incorporation in collagen scaffolds, as these are widely used for vascular engineering. The bifunctional peptide QKCMP combines the EC-specific QK domain with a collagen-binding domain that binds collagen, in a similar way that VEGF binds heparin via its heparin-binding domain. Collagen-bound QKCMP was shown to promote network formation in human umbilical vein endothelial cells (HUVEC) seeded on modified collagen and to activate the canonical pathway in VEGF signaling, by promoting ERK1/2 phosphorylation. In 3D, both QKCMP-modified and plain collagen induced sprouting of entrapped EC spheroids, possibly in response to soluble VEGF present in the media, but increased tubulogenic behavior was observed in the presence of QKCMP. Unfortunately, a control without soluble VEGF was not tested. Given that the soluble form of QKCMP showed no morphogenic effects on EC, VEGF addition was meant to simulate the synergistic action of insoluble and soluble VEGF isoforms during angiogenesis. An elegant approach has been recently described, where a VEGF-mimicking peptide [K-(SL)3(RG)(SL)3-K-G-KLTWQELYQLKYKGI] was engineered to contain the angiogenic motif QK combined with a self-assembling domain, to promote the formation of a nanofibrous, thixotropic hydrogel (Kumar et al., 2016). This biodegradable hydrogel has been shown to induce a mature angiogenic response when injected into mice with induced hind limb ischemia, promoting tissue recovery. A number of other GF-derived proangiogenic peptides have shown promising results, but unfortunately their application in hydrogel-immobilized form has not yet been reported, as far as we known. An interesting example is the PAB2-1c peptide (Lin et al., 2007), which was designed to mimic platelet-derived growth factor and could be used to stimulate pericyte recruitment and vessel maturation, particularly when combined with VEGF-mimicking peptides.

3.2.2 Chemical Conjugation With Matrix Protein–Mimicking Peptides The cross talk between EC and the ECM plays an important role in the processes of blood vessel formation. Several ECM components, including collagens, laminin, and fibronectin, provide important signaling cues that regulate different stages of neovascularization processes, modulating the behavior of vascular cells, namely in terms of survival, migration, proliferation, morphogenesis, and differentiation (Davis and Senger, 2005; Rhodes and Simons, 2007). In particular, adhesion to the ECM is required for EC sprouting, morphogenesis, and vessel stabilization. In blood vessels, EC are attached to a basal lamina, rich in laminin and collagen type IV. During sprouting angiogenesis, this basement membrane is degraded and cells become exposed to interstitial and provisional ECM components, such as collagen I and fibrin, which

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activate EC and drive sprout formation until reestablishment of a continuous basement membrane. In this context, a common approach to guide neovascularization in TE constructs relies on the use of instructive scaffolds that mimic vascular extracellular microenvironments. Such scaffolds are often based in hydrogels decorated with matrix protein–mimicking peptides. Several cell-binding motifs based on ECM-derived peptides have been investigated in approaches to modulate cell adhesion and spreading. The prototypical cell adhesion peptide RGD is among the most widely used for the biofunctionalization of hydrogels (Hersel et al., 2003; Rowley et al., 1999). RGD is the minimal essential cell adhesion sequence of fibronectin and has been identified more than two decades ago by Pierschbacher and Ruoslahti (1984). Shortly after, it has been tested in a covalently immobilized form (Brandley and Schnaar, 1988). Modification with RGD peptides promotes integrin-mediated cell binding to otherwise nonadhesive hydrogels, being often recurrent because cell–matrix adhesion is a strict requirement for the survival of anchorage-dependent cell types. Different studies have addressed the effect of hydrogel-immobilized RGD on vascular cells. HUVEC entrapped in RGD-alginate 3D matrix were shown to proliferate, express EC-phenotypic markers, and organize into multicellular 3D networks, while they remained round and dispersed within unmodified alginate hydrogels (Bidarra et al., 2011). Also, when HUVEC-laden hydrogels were placed in a basal membrane hydrogel, outward migration and formation of tubular-like structures were only observed in the presence of RGD. More recently, dynamic systems have been described, where presentation of bioligands can be triggered “on-demand,” both in vitro and in vivo, and be harnessed to direct specific processes. Light-triggered in vivo activation of caged RGD peptides has been used to regulate cell adhesion, inflammation, and vascularization of implanted biomaterials (Lee et al., 2015). A cyclic RGD peptide was modified with a photolabile caging group (DMNPB), which can be removed via transdermal light exposure resulting in a fully active peptide. To demonstrate control over in vivo vascularization using this strategy, PEG hydrogels modified with caged RGD, protease-degradable cross-links, and VEGF were polymerized into subcutaneous pockets in mice. Hydrogels presenting caged RGD peptides but non-UV exposed had few blood vessels, whereas those that were UV-activated showed robust functional blood vessel formation and had three times more vessels. Besides RGD, other cell-binding motifs have been investigated in proangiogenic approaches, namely laminin-derived peptides based in IKVAV and YIGSR amino acid sequences. Laminins belong to a family of heterotrimeric glycoproteins, each containing one α, β, and γ chain. The different chains characterize protein functions and properties, determining integrin ligand specificity and mediating multiple signaling pathways. The peptide IKVAV derives from laminin α-chains, whereas YIGSR is present in a cysteine-rich site of the laminin β-chains. YIGSR, IKVAV, and RGD peptides covalently incorporated in degradable PEG hydrogels showed different abilities to modulate tubule formation and stabilization when presented, individually or in combinations, to endothelial and pericyte precursor cells (Ali et al., 2013). All peptides promoted the assembly of entrapped HUVEC


in tubular-like structures, which were stabilized by pericytes and expressed collagen type IV and laminin. Yet, best results were obtained with the RGD/YIGSR combination, both in vitro and in vivo. The proangiogenic effects of two hydrogel-immobilized peptides derived from the matricellular protein secreted protein acidic and rich in cysteine (SPARC, also known as osteonectin) have been recently reported (Van Hove et al., 2015). SPARC113 and SPARC118 were grafted to PEG hydrogels as cross-linking peptides flanked by matrix metalloproteinase (MMP)-degradable sequences (Van Hove et al., 2015). Both peptides contained the tripeptide GHK, which was shown to positively affect wound healing, namely by regulating EC function and promoting angiogenesis (Pickart, 2008). Bioactive peptide fragments present in MMP2–degraded gel solutions promoted HUVEC tube formation, with SPARC118 showing increased tube length, for all tested doses. After subcutaneous implantation, the two hydrogels presented similar degradation rates and were able to promote angiogenesis, which was significantly increased as compared with free peptides and gels modified with scrambled sequences. As hydrogel degradation, and consequently peptide release, is expected to be highly dependent on the local proteolytic environment, future studies should focus on the assessment of these systems using appropriate diseased models, where the type and concentration of MMPs can be quite different. The same bioactive sequence GHK was covalently coupled to alginate by carbodiimide chemistry, as a strategy to enhance the proangiogenic capacity of mesenchymal stem cells (MSC) (Jose et al., 2014). In vitro, human MSC entrapped in GHK-functionalized alginate hydrogels secreted increased amounts of VEGF and bFGF, as compared with unmodified gels. GF secretion was nearly abrogated when MSC were pretreated with anti-α6 and anti-β1 antibodies, suggesting that SPARC may interact with these integrins. It would be interesting to further investigate the precise role of integrins on GHK-mediated angiogenesis, as signaling pathways for SPARC peptides are still not well understood. The osteopontin-derived peptide sequence SVVYGLR binds to α4β1, α4β7, and α9β1 integrins and has been described as a potent angiogenic factor (Egusa et al., 2009; Hamada et al., 2004). This peptide sequence has been conjugated to phenol residues of gelatin–PEG–tyramine, via enzyme-mediated reaction, yielding injectable hydrogels with tuneable physicochemical properties and peptide concentration (Park et al., 2012). The peptide was reported to enhance spreading and tube formation of HUVEC seeded on hydrogel surfaces and to promote neovascularization when implanted subcutaneously in mice, although no quantitative data have been provided.

3.2.3 Chemical Conjugation With Other Angiogenic Peptides The soluble form of Ac-SDKP peptide, derived from thymosin beta-4, has been described as a natural inhibitor of pluripotent hematopoietic stem cell proliferation and as a stimulator of angiogenesis, both in vitro and in vivo (Koutrafouri et al., 2001; Wang et al., 2004). This peptide has been selectively bound to acrylated hyaluronic acid hydrogels via thiol groups from cysteine residues (Song et al., 2014). Unfortunately, the immobilization process was poorly characterized and the effect of

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hydrogels on EC function was not tested in vitro. In a mouse model of chronic myocardial infarction, hydrogels with immobilized Ac-SDKP did not show improved regeneration potential. Yet, Ac-SDKP-HA hydrogels with entrapped stem cell homing factor SDF-1 showed a significant increase of myocardial regeneration and recovery of heart function, as compared to groups with only one or none of these factors, suggesting a potentially interesting synergistic effect.

4. CONCLUSIONS AND FUTURE PERSPECTIVES The use of biofunctional biomaterials to promote vascularization is growing, supported by progresses in our understanding of vascular cell biology and key elements of neovascularization processes. In particular, the design of increasingly sophisticated biomaterial systems for therapeutic vascularization has benefited from a greater knowledge on the dynamic interaction of multiple GF, acting in different time windows and spatial locations, and on the ECM–vascular cells cross talk. Technological advances in the field of biomaterial science, solid-phase peptide synthesis, and bioconjugation strategies have also significantly contributed to advancements in this field. Polymer–peptide hybrids have emerged as a promising class of biomaterials, where the ECM-like structural properties of hydrogels can be combined with the bioactivity, specificity, and design flexibility of peptide moieties. In what concerns to the use of biofunctional hydrogels as 3D microenvironments for vascular cells, ambitious goals have been fulfilled along the past decade, and it is nowadays possible to build advanced hydrogels where de novo vascular networks can be induced to grow. These systems stand out as promising tools for diverse applications, not only as vehicles for therapeutic vascularization but also as diagnostic platforms for screening angiogenic and antiangiogenic compounds. They also provide biologically meaningful 3D models to study vascular mechanisms in physiological and pathological processes, especially if combined with microfluidics to promote perfusion and mimic blood flow conditions. Another exciting development in this area is the combination of biofunctional hydrogels with bioprinting technologies, to fabricate well-defined vascular-like structures with a precise spatial arrangement of cells. More functional and “smarter” hydrogel-based reservoirs for proangiogenic drugs are also being currently produced, which have been shown to induce robust proangiogenic responses in vivo. Tuning these systems for specific therapeutic applications still represents a main challenge, due to the difficulty in selecting the most effective GF or combinations of GF, while controlling the respective doses and release kinetics. Physiologically relevant drug delivery might eventually be achieved using stimuli-responsive systems activated by local- and/or time-specific environmental triggers. Importantly, off-target delivery of angiogenic drugs should be reduced as much as possible, to avoid unwanted secondary effects such as encouragement of tumor development or other pathologies associated with excessive angiogenesis. Clearly, future research should strongly focus on the evaluation of biofunctional hydrogels using relevant in vivo in animal models and human clinical trials.

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ACKNOWLEDGMENTS T. Feijão, A.L. Torres, and M. Araújo contributed equally to this chapter. This work was supported by the European Regional Development Fund (ERDF) through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and by Portuguese funds through Portuguese Foundation for Science and Technology (FCT) in the framework of the project Ref. PTDC/BBB-ECT/2518/2014. A.L. Torres and C.C. Barrias thank FCT for the doctoral grant SFRH/BD/94306/2013 and research position IF/00296/2015 (FCT and POPH/ESF), respectively.

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Semenza, G.L., 2007. Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms of blood vessel formation and remodeling. J. Cell. Biochem. 102 (4), 840–847. https://doi. org/10.1002/jcb.21523. Shintani, S., Murohara, T., Ikeda, H., Ueno, T., Honma, T., Katoh, A., et al., 2001. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation. 103 (23), 2776–2779. https://doi.org/10.1161/hc2301.092122. Sieveking, D.P., Buckle, A., Celermajer, D.S., Ng, M.K.C., 2008. Strikingly different angiogenic properties of endothelial progenitor cell subpopulations. Insights from a novel human angiogenesis assay. J. Am. Coll. Cardiol. 51 (6), 660–668. https://doi.org/10.1016/j. jacc.2007.09.059. Song, M., Jang, H., Lee, J., Kim, J.H., Kim, S.H., Sun, K., Park, Y., 2014. Regeneration of chronic myocardial infarction by injectable hydrogels containing stem cell homing factor SDF-1 and angiogenic peptide Ac-SDKP. Biomaterials. 35 (8), 2436–2445. https://doi. org/10.1016/j.biomaterials.2013.12.011. Tepper, O.M., Capla, J.M., Galiano, R.D., Ceradini, D.J., Callaghan, M.J., Kleinman, M.E., Gurtner, G.C., 2005. Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells. Blood. 105 (3), 1068–1077. https://doi.org/10.1182/blood-2004-03-1051. Van Hove, A.H., Benoit, D.S.W., 2015. Depot-based delivery systems for pro-angiogenic peptides: a review. Front. Bioeng. Biotechnol. 3, 1–18. https://doi.org/10.3389/ fbioe.2015.00102. Van Hove, A.H., Burke, K., Antonienko, E., Brown, E., Benoit, D.S.W., 2015. Enzymaticallyresponsive pro-angiogenic peptide-releasing poly(ethylene glycol) hydrogels promote vascularization in vivo. J. Control. Release. 217, 191–201. https://doi.org/10.1016/j. jconrel.2015.09.005. Wang, D., Carretero, O.A., Yang, X.-Y., Rhaleb, N.-E., Liu, Y.-H., Liao, T.-D., Yang, X.-P., 2004. N-acetyl-seryl-aspartyl-lysyl-proline stimulates angiogenesis in vitro and in vivo. Am. J. Physiol. Heart Circ. Physiol. 287 (5), H2099–H2105. https://doi.org/10.1152/ ajpheart.00592.2004. Wang, J., Kaplan, J.A., Colson, Y.L., Grinstaff, M.W., 2017. Mechanoresponsive materials for drug delivery: harnessing forces for controlled release. Adv. Drug Deliv. Rev. 108, 68–82. https://doi.org/10.1016/j.addr.2016.11.001. Zhu, J., Marchant, R.E., 2011. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev. Med. Devices. 8 (5), 607–626. https://doi.org/10.1586/erd.11.27. Zieris, A., Chwalek, K., Prokoph, S., Levental, K.R., Welzel, P.B., Freudenberg, U., Werner, C., 2011. Dual independent delivery of pro-angiogenic growth factors from starPEGheparin hydrogels. J. Control. Release. 156 (1), 28–36. https://doi.org/10.1016/j. jconrel.2011.06.042.

SECTION

III

Manufacturing, Regulatory Challenges and Clinical Testing of Functionalized Nanomaterial-based Products


CHAPTER

21

Manufacturing and Safety Guidelines for Manufactured Functionalized Nanomaterials in Pharmaceutics

Matthias G. Wacker1,2, Christine Janas2, Fabrícia Saba Ferreira3, Fernanda Pires Vieira3 1Fraunhofer-Institute 2Goethe

for Molecular Biology and Applied Ecology, Frankfurt am Main, Germany; University, Frankfurt am Main, Germany; 3National Health Surveillance Agency (ANVISA), Brasília, Brazil

CHAPTER OUTLINE 1. Introduction�� 624 2. Manufactured Nanomaterials in Pharmaceutics�� 624 2.1 Medicinal Products�� 625 2.2 Medical Devices�� 627 3. Physicochemical Characterization of Nanomaterials�� 628 3.1 Chemical Characterization of Excipients�� 629 3.2 Morphology and Shape�� 629 3.3 Particle Size and Size Distribution�� 630 3.4 Surface Charge�� 630 3.5 Surface Properties and Reactivity�� 632 3.6 Drug Load�� 633 3.7 In Vitro Stability and Degradation�� 633 3.8 In Vitro Drug Release�� 634 4. Critical Quality Attributes and Quality Control�� 637 4.1 Critical Quality Attributes�� 637 4.1.1 Particle Morphology, Size, and Size Distribution�� 637 4.1.2 Net Charge�� 637 4.1.3 Encapsulation Rate/Drug Binding�� 638 4.1.4 Drug Release Rate�� 638 4.1.5 Polymorphic Form�� 638 4.1.6 Purity Profile�� 639 4.1.7 Sterility and Endotoxin Levels�� 639 4.1.8 Functional Testing of Binding and Activity�� 639 Biomedical Applications of Functionalized Nanomaterials. https://doi.org/10.1016/B978-0-323-50878-0.00021-5 Copyright © 2018 Elsevier Inc. All rights reserved.

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CHAPTER 21 Manufacturing and Safety Guidelines

4.2 In Process Control and Quality Control�� 639 4.3 Specification Ranges�� 640 5. Pharmacological Evaluation�� 642 5.1 Preclinical Evaluation�� 642 5.2 Clinical Evaluation�� 643 6. Biopharmaceutical Characterization�� 644 6.1 Biorelevant In Vitro Release Studies�� 644 6.2 In Vitro–In Vivo Correlation�� 645 6.3 Pharmacokinetic Studies and Biodistribution�� 645 7. Conclusion�� 646 Abbreviations�� 646 References�� 647

1. INTRODUCTION Over the past decade, a variety of manufactured nanomaterials have been designed for pharmaceutical applications. The emerging number of products registered by regulatory agencies all over the world highlighted the need for a “timely development of a transparent, consistent, and predictable regulatory pathway” (FDA, 2007). Novel nanoscale biomaterials were used in bioimaging, biomedical, and theranostic applications. For medicinal products, the manufacture and safety of such nanomaterials is carefully evaluated during drug approval on a case-by-case basis. Essentially, most relevant regulatory agencies do not have a procedure in place to register or to approve novel excipients for use in medical devices or medicinal products (Wacker et al., 2016). Every substance used in a medicinal product must be introduced to the authorities in an application requesting market authorization for the pharmacologically active substance, e.g., a new drug application, or an abbreviated new drug application. Medical devices have to comply with the safety guidelines issued by the regulatory agency (SCENIHR, 2015; FDA, 2014a; Proposal for a Regulation, 2012). In any case, a detailed physicochemical characterization, the definition of critical quality attributes (CQAs), and a safety assessment have to be undertaken. It was pointed out that, in contrast to other pharmaceutical products, more scientific considerations should be taken into account because of the unique properties of nanomaterials from a quality perspective and regarding the regulatory review (Tyner et al., 2015). With a better understanding of their characteristics and latest trends in material development further testing requirements will be defined (FDA, 2007). In the following, the requirements for design and manufacture of products involving the use of functionalized nanomaterials will be discussed.

2. MANUFACTURED NANOMATERIALS IN PHARMACEUTICS At present, there is only a limited number of pharmaceuticals in the market containing a certain amount of highly advanced functionalized manufactured nanomaterials.

2. Manufactured Nanomaterials in Pharmaceutics

Nanocarrier devices for drug delivery have been designed for the parenteral (ECHA, 2012), the peroral, the dermal, or the inhalation route of administration. However, there is also a multitude of excipients, which fall under the existing legislation on “nanomaterials” (Wacker et al., 2016). Great efforts were made by regulatory bodies all over the world to develop procedures for characterization and safety assessment of this new family of products. A legal definition of “nanomaterial” was issued by the European Commission (EC) in 2011 and has been implemented into the safety guidelines for cosmetics, food, and medical devices (Wacker et al., 2016). It is explicitly limited to particulate matter considering that human and environmental exposure is more likely for particulate materials than for embedded nanomaterials. Thus, some materials described as nanomaterials by other organizations are not covered. For illustration, the International Organization for Standardization (ISO) definition of nanomaterial also includes materials with larger external dimensions, if they have internal or surface structures in the nanoscale (ISO, 2010). The Food and Drug Administration of the United States (US-FDA) published a guidance for industry entitled “Considering whether an FDA-regulated product involves the application of nanotechnology” but this guidance does not establish regulatory definitions of “nanomaterial,” “nanotechnology,” or other related terms (FDA, 2014a). It identifies two key aspects to be evaluated, the particle dimensions and dimension-related properties or phenomena, and provides an initial screening tool that can be broadly applied to a wide range of products (FDA, 2014a). Furthermore, the US-FDA excluded nanomaterials formed during conventional production processes from a more detailed characterization. Differently, the European Union decided to request a detailed characterization of all nanomaterials (ECHA, 2012). This also applies to a number of excipients that have been used in medicinal products or medical devices for many years and puts more pressure on the pharmaceutical industry to deal with the challenges in nanomaterial characterization (Wacker et al., 2016). The specifics in these definitions (see Table 21.1) also reveal some of the difficulties in nanomaterial characterization. While the EC advised by its scientific committees (EC, 2011) recommended to use the number concentration of particles as a key parameter for size distribution, the US-FDA suggested to provide particle number, weight, or surface area (FDA, 2014a). Both authorities are aware that different technologies will be needed for the great variety of materials that are covered by these definitions.

2.1 MEDICINAL PRODUCTS Among other applications, nanotechnology has been used to improve the properties of medicinal products. Generally speaking, there are three different strategies that were successful in the pharma market: the nanocarrier technology, nanomolecular drugs and drug conjugates, and the nanocrystal or micelle technology. Most nanocarriers in the market are liposomes and were manufactured for the delivery of poorly soluble compounds (Wacker, 2013). They are composed of

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Table 21.1 Definitions of “Nanomaterial” in the European Union and the United States With Most Important Differences Highlighted

“‘Nanomaterial’ means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm - 100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%.”

“…a material or end product is engineered to have at least one external dimension, or an internal or surface structure, in the nanoscale range (approximately 1 nm to 100 nm) or […] a material or end product is engineered to exhibit properties or phenomena, including physical or chemical properties or biological effects, that are attributable to its dimension(s), even if these dimensions fall outside the nanoscale range, up to one micrometer (1000 nm).”

phospholipids that assemble into nanosized carriers and have a certain affinity to one or more active pharmaceutical ingredients (APIs). More often, the chemical identity of the drug molecule is not impacted by the manufacturing process. In some cases, the surface of such nanocarriers was modified with targeting ligands, e.g., antibodies (Low et al., 2011) or proteins (Zensi et al., 2009, 2010), to actively target the nanocarriers to the desired site of action. Furthermore, the decoration of the carrier surface with hydrophilic polymer chains resulted in a reduced protein adsorption and a prolonged circulation time of nanoparticles and liposomes (Wacker, 2013). BIND Therapeutics uses block copolymers composed of poly lactic acid and polyethylene glycol (PEG) in products based on the Accurin technology. Several pharmaceutical manufacturers applied this technology to deliver poorly soluble cytostatic agents and entered clinical trials. Importantly, the physicochemical properties and surface chemistry of the excipient strongly impact biodistribution in physiological environment. Several guidelines and reflection papers deal with the safety aspects of such drug delivery devices (EMA, 2013a,b,c; FDA, 2015a). Another strategy has been followed in the manufacturing of nanomolecular drugs and drug conjugates (Luhmann and Meinel, 2016). The engineering of modified macrobiomolecules has emerged from the growing biopharmaceuticals market and differs from the abovementioned formulation approaches. Biomolecules undergo the same safety and efficacy evaluations as any other API. Because of their small size in the lower nanometer range (1–20 nm) biodistribution also differs from most other nanocarriers, which tend to accumulate in lungs, liver, spleen, and kidney. Several biomolecules (e.g., interferon β) have been modified on their surface with PEG chains to prolong their circulation time compared with the native protein. More recent approaches are aiming to control the site of covalent modification more appropriately, e.g., by introducing nonnatural amino acids into the protein structure

2. Manufactured Nanomaterials in Pharmaceutics

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FIGURE 21.1 Strategies applied with nanotechnology products and major characteristics of each group.

of carrier proteins (Wandrey et al., 2016). In future, these technologies may allow distinct control of the surface characteristics and the biopharmaceutical properties. From a regulatory perspective, the guidelines for biopharmaceuticals and biosimilars may be applied (FDA, 2015b). Producing these nanomolecular drugs in bioreactors, manufacturers face the same difficulties in the definition of quality standards. On the one hand, the slight differences in material design make it more difficult to exclude such products from a detailed biopharmaceutical and pharmacological characterization. On the other hand, this also protects the innovator product more efficiently from competitors developing a generic product (Fig. 21.1). The third group of products is nanocrystals. They are composed of small particles of poorly soluble API stabilized with emulsifying agents or polymers. At an increased excipient concentration some of the formulations also form micelles, complexes, or micelle-like nanoparticles (Janas et al., 2016). Most nanocrystal formulations are often manufactured to improve the dissolution rate without serving the targeted delivery of API. For this reason, they are not involving the use of functionalized materials but have been widely applied in pharmaceutical formulation development. The health risks arising from such a formulation approach are due to the higher plasma concentration of the drug substance. In this regard, the toxicity is more predictable and not nanospecific. However, the ability of such nanoparticles to pass filter membranes and to increase the bioavailability in many biota may pose a threat to the environment and requires specific treatments for waste water.

2.2 MEDICAL DEVICES Nanotechnology has also been applied to the development of medical devices. In contrast to pharmaceuticals, a guidance was set up by the European Scientific

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Committee on Emerging and Newly Identified Health Risks (SCENIHR) to define the characterization steps to be conducted with such products (SCENIHR, 2015). The European guideline covers the following products: • Noninvasive surface contacting medical devices, which come into contact only with the intact skin • Invasive surface contacting medical devices, which come into contact with compromised skin (e.g., textiles to cover patients in operating theater) • Invasive external communication devices, which come into contact with the blood path, either indirectly or with circulating blood, and devices in contact with tissue, bone, and dentin (e.g., surgical and dental instruments) • Invasive implantable medical devices, which remain in the human body after the procedure (e.g., dental composite filler materials) • Specific types of medical devices, which are injectable (e.g., diagnostic iron oxide particles) Although most medical devices are not explicitly evaluated case by case, an approach based on different phases has been proposed for products involving the use of nanotechnology. The requirements for each group of medical devices are selected regarding their potential to release free nanoparticles during the time of exposure. They may not only involve a detailed physicochemical characterization but also a number of biological assays or even in vivo studies. During phase 1, an analysis of particle release from the device is undertaken. In phase 2, the expected distribution of particles and their potential to persist must be evaluated. The hazard potential is investigated in phase 3. For this purpose, toxicity is studied in vitro and/or in vivo. Finally, in phase 4, risks are defined based on the information obtained during these investigations (SCENIHR, 2015). In the United States, medical devices that involve the application of nanotechnology are described by the US-FDA guidance on “nanotechnology in FDA-regulated products” (FDA, 2014a). This guidance gives an overall framework for US-FDA’s approach to the regulation of nanotechnology products.

3. PHYSICOCHEMICAL CHARACTERIZATION OF NANOMATERIALS Today, most regulatory authorities have established procedures for the characterization of nanomaterials in medicinal products or medical devices (Wacker et al., 2016). Contrasting the European situation, the US-FDA applies similar regulations to nanomaterials as to all other substances. Strategies for their physicochemical characterization were discussed in a report of the nanotechnology task force dealing with the challenges of this emerging technology (FDA, 2007). A number of key parameters have been identified, e.g., the particle size and size distribution, surface area, chemical composition, surface reactivity, coating, solubility, shape, and aggregation.

3. Physicochemical Characterization of Nanomaterials

Generally speaking, their characterization requires additional care not only regarding the selection of reference materials but also the applicability of a certain method to the nanomaterial. The optimal technique depends on the type of the nanomaterial, the intended route of administration, and the matrices from which the nanomaterial must be extracted. Despite all efforts, there was no “gold standard” established applying to all nanosized particles (Crist et al., 2013).

3.1 CHEMICAL CHARACTERIZATION OF EXCIPIENTS As the purity of the starting materials used for manufacture of nanomaterials (e.g., lipids, polymers) is essential to the quality of the drug product, these aspects have to be considered in the selection of analytical technologies and specification ranges. The European Pharmacopoeia monograph “Functionality-related characteristics of excipients” proposes to identify characteristics relevant to the function of a substance. Commonly, the level of information to be provided with the relevant submission depends on complexity of the excipients. The “guideline on excipients in the dossier for application for marketing authorisation of a medicinal product” should be considered (EMEA, 2006). Using materials from multiple sources (e.g., animal, plant, synthetic sources) or suppliers requires additional characterization and comparability studies (EMA, 2013a). According to the US-FDA guidance on liposomal drug products, substances applied in parenteral drug delivery (e.g., modified lipids) require a characterization at the same level of detail as the drug substance (FDA, 2015c). Accordingly, for welldefined synthetic or semisynthetic materials, the exact chemical structure should be provided but also a high level of purity should be achieved. For lipids, this characterization includes not only the composition of fatty acids but also the exact location in the molecule. In the case of naturally sourced lipid mixtures (e.g., egg lecithin), the lipid composition as a range of percentages for each lipid and its fatty acid composition are described. Similarly, the exact chemical structure of polymeric materials and the in vitro stability of the chemical bonds have be studied. A guideline issued by the European SCENIHR listed many of the analytical technologies that may be applied to the characterization of nanomaterials (SCENIHR, 2015).

3.2 MORPHOLOGY AND SHAPE Exploring the surface properties of solid powders, electron microscopy (EM) has been used in the characterization of biomaterials. In addition to the differences in batches or qualities, the surface structure of nanomaterials also impacts the in vivo biodistribution. For this reason, the SCENIHR proposed the use of EM technologies to characterize substances to identify properties relevant to the toxicokinetic profile (SCENIHR, 2015). An effect of particle shape on cellular uptake and cell damage has been reported earlier (Albanese et al., 2012). Furthermore, the morphology also plays a pivotal role in the evaluation of particle size measurements. Many of the technologies applied in the routine measurement are based on the intensity fluctuations detected for light scattered at the surface of spherical particles (Tscharnuter, 2000).

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Therefore, spherical particles are required for an accurate measurement (Beyer et al., 2015a). Additionally, the formation of agglomerates may occur, which disintegrate into primary particles on administration. Not only transmission and scanning electron microscopy (TEM and SEM) but also atomic force microscopy (AFM) are widely applied in the characterization of nanomaterials. Other alternatives are scanning transmission electron microscopy and scanning tunneling electron microscopy. A disadvantage of the TEM and SEM is that material morphology may undergo changes during the drying process during sample preparation. Therefore, the OECD noticed that AFM measurements can be performed in solution and dispersion. Thus, samples may be protected from alteration during preparation and the measurement procedure. Unfortunately this technique was reported to be difficult to apply under this condition (OECD, 2016). Cryo-TEM of the frozen samples is also an alternative that applies to particles in liquid dispersion.

3.3 PARTICLE SIZE AND SIZE DISTRIBUTION For functional manufactured nanomaterials used in medical applications, the particle size and size distribution plays a pivotal role for biodistribution (Crist et al., 2013). Importantly, the quantitative determination becomes more difficult when applying different standards to size distribution. Most technologies based on light scattering or laser diffraction (LD) patterns calculate the particle size from the intensity fluctuations measured under defined conditions. In this area, dynamic light scattering (DLS) and LD are the most common technologies. The particle size distribution is often provided as a size distribution by particle mass, by particle number, or by intensity. From the analytical perspective, the intensity-based profile uses the raw data without further processing, e.g., by calculating the particle mass from the material density. For this reason, it should also be described in the dossier. The European regulations use the number size distribution in their definition of nanomaterials. It may be calculated from the total mass concentration and the density of the material under the assumption that a spherical particle shape is given. Nanoparticle tracking analysis is a relatively new technology that combines the measurement of intensity fluctuations with imaging data and provides the number concentration. For the characterization of nanomaterials in medical devices, the SCENIHR recommends the use of two different methods based on two different principles. Similar recommendations have been made by European Food Safety Authority (2011) and SCCS (2012). According to the OECD guidelines, the characterization by TEM or SEM has to be made before further quantification if the particle shape is not known yet (OECD, 2016). The methods applied to particle characterization and also some of the relevant ISO guidelines are listed below (see Tables 21.2 and 21.3).

3.4 SURFACE CHARGE Most particles exhibit a positive or negative surface charge contributing to the stability of dispersion (Berg et al., 2009). Unfortunately, there is no technique in place to


Table 21.2 Methods for Particle Size Determination Method Dynamic light scattering

Scanning electron microscopy Transmission electron microscopy Atomic force microscopy

Measurement Range

Resolution

5–500 nm (JRC E, 2012) 1–2000 nm (SCENIHR, 2015) >1 nm (JRC E, 2012) 0.4 nm (SCENIHR, 2015) >50–100 nm (SCENIHR, 2015) >1 nm (JRC E, 2012) 0.05 nm (SCENIHR, 2015) >1 nm (JRC E, 2012) Atomic

Atomic force microscopy

>1 nm (JRC E, 2012) Atomic

Atomic force microscopy

>1 nm (JRC E, 2012) Atomic

Nanoparticle tracking analysis

Centrifugal liquid sedimentation

>25 nm (JRC E, 2012) 10–2000 nm (Malvern) 10–15000 nm (SCENIHR, 2015) >20 nm (JRC E, 2012)

Laser diffraction

0.1 μm–3 mm

Guidelines and Standards… NIST-NCL PCC-1 ISO 22412:2008 ASTM E2490-09(2015) NIST-NCL Joint Assay Protocol, PCC-7 ASTM WK54615 NIST-NCL Joint Assay Protocol, PCC-6 E2859-11 NIST-NCL Joint Assay Protocol, PCC-6 E2859-11 NIST-NCL Joint Assay Protocol, PCC-6 E2859-11 ISO/DIS 19430.2 E2834-12

ISO 13318-1:2001 ISO 13318-2:2007 ISO 13318-3:2004 ISO 13320:2009 Ph. Eur. 2.9.31

quantify this property because of the electric double layer on the nanoparticle surface formed by counter ions in the solution (MHLW, 2016). In a liquid environment, the zeta potential is often applied to the characterization of solid surfaces and colloidal particles (SCENIHR, 2015; FDA, 2015c; MHLW, 2016). It is defined as the potential difference between the stationary solvent layer around the nanoparticles in solution and the dispersion medium (Hunter, 1981). Most regulatory authorities propose the use of electrophoretic light scattering method

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(Wacker, 2013; MHLW, 2016) but there are also some other techniques available (Xu, 2008). In many cases, variations in zeta potential measurements occur at different pH values, ionic strength, or conductivity of the solvent. For this reason, the test conditions should be specified (Hunter, 1981; Předota et al., 2016; Wacker et al., 2011). The surface charge was considered as a CQA of nanocarrier formulations because it impacts the in vivo clearance, tissue distribution, and intracellular uptake of liposomes (MHLW, 2016). For nanoparticles composed of human serum albumin, the zeta potential profile was recorded in a pH range between 3 and 10. A pH value higher than 6.5 resulted in a constant zeta potential of approximately −40 mV indicating high stability of the dispersion (Wacker et al., 2011). Investigating pH-responsive liposomes, high stability was reported at pH 7.4, but a rapid aggregation occurred at lower pH values (Vila-Caballer et al., 2016). Furthermore, the cellular uptake was significantly higher (Vila-Caballer et al., 2016). When particles are exposed to physiological media a change in the surface charge occurs, which also impacts the interaction with biological surfaces. For this reason, the zeta potential should be determined also in presence of proteins and ions present at the site of administration (see Section 3.7). A zeta potential profile (e.g., at different pH values, ion, or protein concentrations) may support the interpretation regarding the tolerance of the material to changes in the microenvironment and the manufacturing process (Wacker et al., 2011).

3.5 SURFACE PROPERTIES AND REACTIVITY The surface properties and reactivity of nanoparticles are relevant characteristics regarding their interactions with biological surfaces in vivo. Research thus far highlights that size, shape, and surface characteristics of nanoparticles may impact protein adsorption and also have the capability to modify the structure of the adsorbed protein molecules. In the area of nanomedicines, this surface interaction contributes to nanoparticle uptake (Saptarshi et al., 2013), biodistribution, and side effects associated with immunogenicity and allergic or pseudoallergic reactions to the material. Modifying the surface characteristics of nanoparticles may be used to passively or actively target drug delivery devices to specific tissues to increase their stability and structural integrity in physiological environment. The conformational surface structure, the specific (for actively targeted systems) or unspecific, (for actively and passively targeted systems) binding affinity to target and nontarget cells should be investigated (Bamrungsap et al., 2012). For liposomes, several tissues with major importance for biodistribution have been discussed. They include liver, kidney, lung tissue, the macrophages, and the target tissue of the drug substance (EMA, 2013a,c). From a safety perspective, the linker chemistry is of special interest and should be carefully investigated. In the past, parenteral drug delivery systems were modified on their surface with PEG units or hydroxyethyl starch to protect them from clearance mechanisms of the human body. By decorating the outer surface of the nanoparticles with stealth-imparting polymeric substances, specific protein adsorption patterns


prolong the in vivo circulation time. Later approaches were focused on the decoration of the nanoparticle surface with drug targeting ligands. Such systems require further investigation of the binding affinity (see Sections 4.1.8 and 5.1).

3.6 DRUG LOAD For most nanocrystal formulations, the drug load is defined as the highest amount of the compound forming stable nanoparticle dispersion over the intended shelf life at a certain excipient concentration. Other formulations such as nanomolecular drugs and drug conjugates exhibit a chemically defined drug load (e.g., the ratio of a small-molecular API bound to a macromolecular carrier), which also affects the biodistribution pattern. The covalent modification of biopharmaceuticals with drugs or diagnostics may be quantified by chemical analysis, e.g., using high-performance liquid chromatography, size exclusion chromatography (SEC), ultra performance liquid chromatography, or mass spectrometry (MS) (Miksinski, 2013). Different to this nanomolecular therapeutics, the solubility of the API in the storage medium and the unspecific interactions between the API and the carrier material are responsible for the entrapment of a compound into nanocarriers. For the quantification of the encapsulation rate, several separation technologies may be applied, e.g., centrifugation or filtration (Beyer et al., 2015a; Villa Nova et al., 2015). For liposomes, the US-FDA recommended to quantify the leakage rate of drug from the liposomes throughout shelf life (FDA, 2015a). The amount and ratio (molar and weight-by-weight percentage) of the lipid compared with the drug substance and the drug amount expressed as “mg/mL” or “mg/Vial” for liquid products and “mg” for dry products according to this guidance have to be declared (FDA, 2015a). Similarly, the EMA discussed the quantification of the fraction of encapsulated active substance (amount of free/entrapped API) for liposomal formulations (FDA, 2015a) and the ratio of iron to carbohydrates for diagnostic iron oxide particles (EMA, 2013c).

3.7 IN VITRO STABILITY AND DEGRADATION The evaluation of physical, chemical, photochemical, biological, and microbiological parameters affecting the quality, safety, and efficacy of drug products is recommended by the ICH guidelines (ICH, 2003). This involves not only a number of physicochemical attributes but also the functionality of a product after a defined period and under defined conditions of storage. The covalent modifications of drug molecules such as nanomolecular drugs or drug conjugates can be analyzed by using liquid chromatography in combination with photometric detection or MS. They may also be used for the evaluation of plasma stability as recommended by the US-FDA (Saber and Leighton, 2015). For nanocarrier formulations detecting the physical stability in presence of physiologically relevant media is required (Zolnik and Sadrieh, 2009). Because the particle size and charge of nanomaterials is responsive to a variety of mechanisms (e.g., ionization, adsorption, dissolution, erosion, degradation),

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a standardization of test conditions must be undertaken. An OECD guideline recommends to provide the following sample information (OECD, 2012): • The method of dispersion (e.g., stirring, sonication) • Mass concentrations of the excipients • Type of water and solvents, pH of buffers, buffer composition • Shape, volume, and material type of the container during dispersion preparation • Size distribution, surface area, surface charge, aspect ratio for fibers, length of nanotubes, surface functionality • For metal nanoparticles the concentration of dissolved metal ions in the supernatant • Impurities (e.g., metal ions, organic solvents, endotoxins) • For sampling from stock dispersions: consistent sampling point (depth on vessel) and documentation about homogenization prior to the sampling For some of the analytical technologies involved in the characterization of nanocarriers, difficulties in the detection of particle size, size distribution, and zeta potential are to be expected. Commonly, methods based on the detection of light scattered at the particle surface require a difference in the scattering intensities between medium and formulation (e.g., DLS, laser Doppler electrophoresis). Furthermore, the background signal of the physiological fluid should be recorded. In vitro stability tests involving particle size and zeta potential measurements in presence of human serum have been conducted with particles in a size range between 50 and 250 nm earlier (Janas et al., 2016; Villa Nova et al., 2015). In some cases, the measurement of a charge profile (e.g., in response to pH, protein concentration, ionic strength) may support extrapolation to in vivo conditions. The US-FDA recommends the use of electron microscopic techniques, inductively coupled plasmaMS, particle size, and surface measurements for stability testing of nanosized drug products in bulk, dosing solutions, and test media (FDA, 2012). In cases when a nanoformulation is diluted prior to the administration, studies on the in-use have to be investigated. For nanomaterials sensitive to temperature, accelerated test conditions may not be applicable (Tyner et al., 2015). Furthermore, the ISO has drafted a guideline for analysis of aggregates and agglomerates of nanomaterials in the environment (ISO, 2016).

3.8 IN VITRO DRUG RELEASE Currently, in vitro release testing is widely applied by the pharmaceutical industry in the quality control of peroral dosage forms. The existing technologies sensitively discriminate between variations in batch quality, e.g., resulting from postapproval changes in the composition or the manufacturing process. For immediate release formulations one-point specifications are sufficient to characterize the release profile. Sustained release formulations are described by using a three-point specification. In quality control, nonphysiological conditions may be applied to a certain degree if there is still enough evidence for a relationship between the


release profile and the in vivo efficacy (e.g., determined as the pharmacokinetic profile) of the medicinal product. A short release time and the detection of relevant changes between the formulations are major criteria for method development. Essentially, US-FDA and the EMA have described the drug release to be an essential parameter in the regulatory procedures for liposomes (EMA, 2013a; FDA, 2015c). Biorelevant release studies are conducted to support the preclinical evaluation of pharmacodynamic responses and the biopharmaceutical characterization of nanoformulations (see Sections 5.1 and 6.1). For nanoformulations, specification ranges should address the intended in vivo release profile and the type of the nanoformulation (see Section 4.3). After years of development, there is still no gold standard established. Commonly, “sample and separate” procedures or dialysis-based techniques are applied. The most important methodologies are listed in Table 21.3. To compare different release profiles in the quality control, the US-FDA proposes to use the difference factor (f1) and similarity factor (f2), which have also been applied in the evaluation of postapproval changes of immediate release solid oral dosage forms (FDA, 1997). In specific, for nanocarrier formulations such as liposomes, other mathematical models may be applied to provide a more appropriate description of the raw data (Xie et al., 2015; Janas et al., 2017). Furthermore, the evaluation of a release test may involve the use of more complex mathematical models.

Table 21.3 Important Technologies in the Drug Release Testing of Nanomedicines Name

Manufacturer

Setup

References

Dispersion releaser

Pharma Test Apparatebau AG (Germany)

Villa Nova et al. (2015), Wacker and Janas (2013) and Janas et al. (2017)

Dialysis adapter “A4D”

Sotax AG, (Switzerland)

Dialysis cell mounted into USP dissolution apparatus 2 Advantages • Accelerated forced dialysis • Robust setup • Various media applicable Limitations • Evaporation in long-term experiments (>200 h) Dialysis cell mounted into USP dissolution apparatus 4 Advantages • Variable in volume (continuous flow) Limitations • Delayed release from donor compartment • High variability at slow pumping rates

Bhardwaj and Burgess (2010)

Continued

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Table 21.3 Important Technologies in the Drug Release Testing of Nanomedicines—cont’d Name

Manufacturer

Setup

Dialysis sac, e.g., Float-A-Lyzer

Spectrum Labs

Dialysis adapter mounted into USP dissolution apparatus 2 or 4 Advantages • Low cost Limitations • Delayed release from donor compartment • High variability at slow pumping rates and sedimentation of particles • Poorly standardized Filtration after sample collection Advantages • Robust setup • Various media applicable Limitations • Strong mechanical forces applied • Effect on release rate likely • Poor sensitivity Centrifugation after sample collection Advantages • Robust setup • Various media applicable Limitations • Mechanical forces applied • Effect on release rate likely • Poorly effective for lowdensity materials Extraction after sample collection Advantages • Robust setup • Various media applicable Limitations • Column adsorption • Effect on release rate likely

Syringe filtration

Centrifugation

Solid-phase extraction

References

Beyer et al. (2015b) and Juenemann et al. (2010)

Fugit and Anderson (2014)

Guillot et al. (2015)

4. Critical Quality Attributes and Quality Control

4. CRITICAL QUALITY ATTRIBUTES AND QUALITY CONTROL During the development of each medicinal product, manufacturers need to identify the chemical, physical, biological, and microbiological attributes that can be defined and measured to ensure that the final product remains within the acceptable quality limits. These CQAs are carefully monitored over the whole life cycle of the product and will also be requested by the regulatory authorities. For each product group, there are several CQAs under discussion.

4.1 CRITICAL QUALITY ATTRIBUTES 4.1.1 Particle Morphology, Size, and Size Distribution Nanocarrier formulations are designed to target the API to a certain site of action. Their properties must be controlled precisely to facilitate specific interactions with biological surfaces. In this context, the particle morphology, size, and size distribution play a pivotal role in the deposition. All three parameters are strongly affected by the fabrication process, which is often based on mechanical disintegration or selfassembly of the carrier structure. In many cases, nanomilling, high-pressure homogenization, or nanoprecipitation processes are applied (Wacker, 2013). Because of the limitations of the analytical technology, a careful evaluation of the routine procedures is required (Beyer et al., 2015a). Currently, there is no full validation procedure requested but a confirmation of properties with a second technology. Most guidelines propose the characterization of particle size with at least two techniques. One of them should be based on an imaging method to confirm the morphology (e.g., EM) (SCENIHR, 2015; EFSA, 2011; SCCS, 2012). Similarly, nanocrystal formulations are manufactured by mechanical disintegration, self-assembly, or nanoprecipitation. Importantly, the properties of particle size and size distribution of these orally or subcutaneously administered formulations are only of minor interest to the biodistribution compared to intravenous nanocarriers. An appropriate setup for quantification of the release rate is required. Stability and release are the most essential characteristics. In this regard, not only the particle size and size distribution but also other parameters such as the polymorphic form may support the long-term stability studies. The size and size distribution of nanomolecular drugs and drug conjugates is more a chemical than a physical property and may be determined by SEC, MS, or similar technologies of chemical analysis. When using high concentrations, the formation of aggregates and the chemical reactions between different molecules have to be considered. A molecular volume may be designed by selecting various polymeric materials for conjugation of biopharmaceuticals.

4.1.2 Net Charge The net charge of nanoformulations may trigger some specific interactions with cellular surfaces. For nanocarriers, it is an essential parameter determining the time of persistence (e.g., circulation time, aggregation, and accumulation in physiological environment)

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within the human body. It is highly affected by the formulation design and must be optimized for each site of administration, e.g., by selection of the appropriate polymer or lipid composition or the use of ions to decorate the particle surface. The quantification in presence of body fluids is more difficult than during manufacture, but it also affects other parameters such as the release rate that may change because of the aggregation of particles. For most nanocarrier formulations, the determination of the net charge is requested by the authorities (EMA, 2013a; FDA, 2015c; MHLW, 2016). For nanocrystal formulations, the effect on the in vitro stability and the release rate under biorelevant conditions are more important than structure of agglomerates formed in the human body.

4.1.3 Encapsulation Rate/Drug Binding The encapsulation rate of nanoformulations is essential to the in vivo performance of targeted drug delivery devices and determines the amount of the drug substance that may be redirected by the carrier to the target site. A change in the encapsulation has been reported for liposomes and nanoparticles during the time of storage. Furthermore, the covalent attachment of drugs to drug conjugates may be hydrolyzed. For this reason, the encapsulation or drug binding rate is monitored over the time of storage. Importantly, the parameter may dramatically change on administration. Biorelevant release studies provide an improved marker for the in vivo performance.

4.1.4 Drug Release Rate More than for other quality attributes, many physical and chemical parameters have a strong influence on the release rate. For this reason, it has been proposed for the detection of quality changes of liposomes by the US-FDA and other regulatory authorities (EMA, 2013a; FDA, 2015c; MHLW, 2016; Xie et al., 2015). Currently, there is no “gold standard” established for testing the drug release from nanosized carriers. A number of methods were described in the literature some of which were also used for the characterization of the existing drug products. However, the use of a harmonized setup between different countries is required to find a basis for further improvements in the quality of nanomedicines. Currently, commercial products for the release testing of nanoformulations based on dialysis procedures are provided by Sotax AG and Pharma Test Apparatebau AG (see Table 21.3). The technology may also allow the testing of biopharmaceuticals for the degradation and release of conjugated drug substance, e.g., in presence of serum proteins. The drug release is a strong marker for product quality but requires a very sensitive measurement when applied to nanomedicines.

4.1.5 Polymorphic Form Changes in the crystal structure occurring during the time of storage may impact the dissolution rate of drug substances. For this reason, the polymorphic form is essential to the in vivo performance of nanocrystals. Furthermore, polymorphism of lipids has been used to induce a certain release behavior and drug–carrier interactions. For most polymeric carriers, nanomolecular drugs, or drug conjugates, the parameter can be an indicator for quality changes occurring over the time of storage but has only minor impact on the in vivo performance. Although most nanomolecular drugs and drug conjugates are highly soluble, high excipient concentrations used in nanocarrier


devices make the drug substance remain in the amorphous state. However, a crystallization of the drug substance changes the in vivo performance.

4.1.6 Purity Profile As for most other formulations, the purity of the raw materials is an essential safety aspect in the manufacture of nanomedicines. Products manufactured by mechanical disintegration could be contaminated with residues of metal or plastics from the milling or homogenization procedures. But also drugs and excipients may contain impurities, which have to be identified (EMA, 2013a; FDA, 2015c; MHLW, 2016). For those substances with influence on biodistribution (e.g., lipid materials in liposomes), characterization must have the same level of detail as the drug substance itself (see Section 6.1). Modified biopharmaceuticals such as drug conjugates have a much more difficult impurity profile because they are partly fabricated in living organisms and undergo a multistep purification process. Additionally, small-molecular entities and linker molecules are bound to these biopharmaceuticals. They are manufactured by chemical synthesis from a number of precursor molecules. For this reason, drug conjugates require a characterization of biological and chemical impurities from the two types of synthesis that are involved in the production process.

4.1.7 Sterility and Endotoxin Levels During the manufacture of parenterals low endotoxin levels and sterility of the product have to be maintained. For most dispersed formulations this is difficult to achieve because of the challenges in applying filtration technology. For nanoparticles significant membrane adsorption and clogging of the membrane during the separation process or the disruption of the carrier structure when subjecting it to strong mechanical forces are likely. Also, many of the endotoxin tests use turbidimetric quantification, which interferes with the formulation design. This applies not only to most nanoformulations but also to microparticles and emulsions. Drug conjugates and nanomolecular drugs are very often small enough to be filtrated efficiently. For such formulations, the total membrane adsorption is of interest to the process performance.

4.1.8 Functional Testing of Binding and Activity The functional testing of nanocarrier devices is very specific for a certain drug molecule. Many of the assays applied in their characterization are also used for the testing of the API. However, this preclinical evaluation is more challenging for nanomaterials (see Section 5.1).

4.2 IN PROCESS CONTROL AND QUALITY CONTROL Producing nanomedicines in a well-defined manufacturing process with all associated in process controls is the basis for high-quality products. The guidelines of the US-FDA recommend a detailed process flow diagram and a description of unit operations. Furthermore, the ranges have to be defined for all monitored process parameters and process controls. It is highlighted that nanomedicines are sensitive to changes in the manufacturing conditions, including changes in the scale of production (batch size).

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For this reason, sensitive detection of the process performance must be undertaken. The risk levels are defined during formulation development where the impact of each process parameter (e.g., shear force, pressure, temperature) on the quality of the final product is carefully investigated (FDA, 2015a). Nanomaterials exhibit unique properties that depend on the CQA discussed previously. More often, these characteristics may change over time. Therefore, nanomaterials require precise characterization and identification at all stages of design, development, and final production (SCENIHR, 2015). Furthermore, the deviations of these process controls should be sufficiently understood by accumulating knowledge about the manufacturing process (e.g., process parameters and variability of raw materials) during process development (MHLW, 2016). The process should be planned to result in a homogeneous product that can be robustly manufactured (MHLW, 2016). The process parameters that have an impact on the structure of the nanoparticle should be identified. In quality control, the CQA of nanomedicines are tested by using a wide range of analytical techniques, but it is limited to defined time points. Although process controls should provide sufficient information about the process performance, the quality control allows a more detailed characterization. Both aspects have to be considered to result in a safe and effective manufacturing process.

4.3 SPECIFICATION RANGES For most nanomedicines, the definition of specification ranges is difficult because of the challenges in their characterization and the complexity of their interactions with the physiological environment. For each of the three types of products, a specific combination of CQA has to be considered. Nanocarrier devices and modified biopharmaceuticals require a more careful selection of these specifications due to the accumulation and biodistribution mechanism, which is partly based on the size but also on the surface properties. A combination of different distribution patterns in one formulation design may impact the efficacy and toxicity of such a product. In this context, a higher encapsulation rate may coincidentally occur in a small fraction of particles and lead to the accumulation in specific tissues due to particle size or surface properties (see Fig. 21.2). Because it is more difficult to control such interactions between formulation parameters, the selection of more narrow specification ranges is advised (see Table 21.4).

FIGURE 21.2 Illustration of the drug and size distribution in a dispersed system where 71% of the particles are in the specification range for particle size but only 30% of the drug is encapsulated in this size fraction.


Table 21.4 Critical Quality Attributes (CQAs) Applied to Different Formulation Technologies and the Proposed Specification Scheme Formulation Technology Nanocarrier technology

Important CQA

Selection of a Specification Range

Particle size

Selection of size range based on physiological target site. Monodisperse size distribution confirmed. Changes in the net charge should be observed. Encapsulation rate in the formulation should be determined after manufacture and over the time of storage. At least three early time points should be selected to characterize the burst release. Two later time points can be used for the remaining part of the profile. Purity profile depending on the function and nature of excipient and API. Comparable to other parenteral formulations but more careful validation of the assays applied because of the interference of dispersed units with the assay design. Function of each active component of the drug delivery system (e.g., SME and ligand or conjugated biopharmaceutics) and accumulation and uptake assays for tissues exposed to the nanoformulation (e.g., mucin interaction, uptake into macrophages). Exact chemical composition or a range of molecules synthesized by the conjugation procedure using characterization technologies for biological and chemical molecules. Drug binding rate and site of the modification in the biopharmaceutical molecule. Bound and unbound fraction of the drug. Purity of the conjugate taking into account biological (e.g., proteins from manufacturing process of biopharmaceutical compound) and chemical impurities (e.g., drug substance, linker molecules). Comparable to other formulations but more careful validation of the assays applied because of the interference of dispersed units with the assay design. Function of each active component of the drug delivery system (e.g., SME and ligand or conjugated biopharmaceutics) and accumulation and uptake assays for tissues exposed to the nanoformulation (e.g., mucin interaction, uptake into macrophages).

Net charge Encapsulation rate/ Drug binding Drug release rate

Purity profile Sterility and endotoxin levels

Function-related assays

Drug conjugates

Chemical structure

Drug binding

Purity profile

Sterility and endotoxin levels

Function-related assays

Continued

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Table 21.4 Critical Quality Attributes (CQAs) Applied to Different Formulation Technologies and the Proposed Specification Scheme—cont’d Formulation Technology Nanocrystal formulations

Important CQA

Selection of a Specification Range

Particle size

Selected specification range based on the intended release rate and stability of the product. Immediate release, one-point specification may be sufficient to describe oral drug products. Other formulations (e.g., subcutaneous similar) should be fully characterized. Polymorphic form may change over the time of storage and is essential to the drug release. Purity of the SME and the excipients. Comparable to other formulations but more careful validation of the assays applied because of the interference of dispersed units with the assay design.

Drug release rate

Polymorphic form Purity profile Sterility and endotoxin levels

API, active pharmaceutical ingredient; SME, small-molecular entities.

5. PHARMACOLOGICAL EVALUATION The pharmacological evaluation of nanomedicines requires a number of preclinical and clinical studies. The preclinical development is also supporting formulation development in the selection of promising formulation candidates and provides first information about toxicological issues or the proposed dose range of the medicinal product. It is part of the regulatory documentation for development and registration of new pharmaceutical products. The application of marketing authorization requires detailed data on efficacy, quality, and safety (Dobrovolskaia et al., 2008). It involves animal (in vivo) studies and in vitro studies.

5.1 PRECLINICAL EVALUATION The in vitro tests applied to nanomedicines must be carefully validated because of the interaction between particles and the setup used for their characterization. They support the in vivo experiments by identifying the mechanisms of specific pharmacodynamic responses and by helping to thoroughly understand and control their nanobiointeractions (Wacker et al., 2016; Bigdeli et al., 2016). Many of the drug formulations rapidly release the drug substance in the in vitro setup (Beyer et al., 2015b). A biorelevant release study may be conducted to determine the drug content of the nanocarrier when reaching the site of action (Beyer et al., 2015b). During the preclinical evaluation, the characteristics and quality of the test product should be comparable to the drug product for clinical use (MHLW, 2016). The pure drug substance should be used as a reference (MHLW, 2016). The US-FDA

5. Pharmacological Evaluation

addresses the preclinical evaluation of nanocarriers in their guideline on the liposomal products. The following aspects should be considered (FDA, 2015c): • Expected clinical application of the nanoparticle drug product. • Nanoparticle formulations. • Properties of the active substance. • Blood concentration and tissue distribution including the accumulation and retention in the target organ and/or tissue of both the active substance and nanoparticle drug product. • The location and biorelevant release of the active substance. • The binding of the nanoparticle to the target cells if a ligand (targeting moiety) or antibody is conjugated to the nanoparticle surface to provide targeting delivery. • The animal species and model should be selected considering the differences in the expression and distribution of the receptor or epitope between the selected animal species and humans, if a ligand (targeting moiety) or antibody is conjugated to the nanoparticle surface to provide targeting delivery. • The intracellular fate of the nanoparticles (including lipids or other components) following cellular entry by endocytosis or other mechanism, if the intracellular release of the active substance plays an important role in exhibiting the pharmacodynamic effect. Nanomaterials are applied in a broad spectrum of medicinal products. At present, there is no harmonized procedure or regulatory guidance document available for the preclinical evaluation of this therapeutics. Even though there are several standards defined for the biological evaluation of nanomaterials and devices, they are not sufficient to describe the complex interactions that may arise during drug therapy.

5.2 CLINICAL EVALUATION The clinical trials should be designed, conducted, and analyzed according to sound scientific principles to achieve their objectives and should be reported appropriately. Before any clinical trial is carried out, results of nonclinical investigations or previous human studies should indicate that the drug is acceptably safe for the proposed investigation in humans (ICH, 1997). The first-in-human studies require detailed knowledge of the pharmacokinetic parameters and preclinical pharmacodynamic studies. For nanomedicines, the total active substance, the free drug substance, and all metabolites should be quantified (MHLW, 2016). The deposition pathways and elimination (including metabolism and excretion) as well as several important pharmacokinetic measures (cmax, AUC) and parameters (e.g., clearance, volume, half-life) of a nanoformulation may differ from the nonnanoformulation. Because the interplay between drug release from the drug product and the tissue uptake is very complex, a quantification of the total drug substance in the plasma may not reflect the bioavailability at the site of action. For this reason, the evaluation of pharmacokinetics must also involve the biodistribution

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pattern of a formulation prior to the clinical evaluation (MHLW, 2016). For definition of an appropriate study design, the following aspects should be considered: • Route of administration (e.g., oral, topical, sublingual, inhalation, or injection) • Noninvasive and invasive use • Location of tissue contact (e.g., skin, mucosal membrane, breached or compromised surface, blood, tissue, bone, dentin) • Contact time (limited ≤24 h, prolonged >24 h to 30 days, permanent >30 days) If there is a nonnanoformulation in the market, a comparison with the new dosage form is recommended.

6. BIOPHARMACEUTICAL CHARACTERIZATION 6.1 BIORELEVANT IN VITRO RELEASE STUDIES As the “quality by design” paradigm has been described in the ICH guideline on “pharmaceutical development,” the industry has given more attention to the topic of in vitro–in vivo correlation (IVIVC) (see also Section 6.2). Biorelevant in vitro release studies are applied in the prediction of the in vivo performance of peroral tablets (Juenemann et al., 2011) but were also proposed for nonoral dosage forms. The liberation of a drug substance from the carrier is of major importance for the pharmacokinetics and biodistribution pattern (see Section 6.2). The therapeutic outcome achieved not only with nanocrystal and micelle technology but also with nanocarriers is very sensitive to changes in the release rate. Nanocrystals and micelles are more often “immediate release” formulations. Over the shelf life the dissolution rate may decrease on crystal growth of the particles. A great variety of nanocarrier formulations have been evaluated for several administration routes. Not only most of the liposomal carriers are administered intravenously but also the subcutaneous route, the peroral route, the dermal route, or the inhalation route of administration are of special interest. Most dialysis-based technologies allow the testing of such nanoformulations without disrupting the carrier structure, but the membrane permeation is limiting the sensitivity of the release experiment. A compromise has been found in the use of buffer solutions, which do not contain physiological components such as proteins or enzymes. They were used in the release testing of liposomes and nanoparticles earlier (Bhardwaj and Burgess, 2010). Unfortunately, the protein binding to nanomaterials plays a pivotal role in the biodistribution by altering the particle size, surface properties, and release mechanism (Zolnik and Sadrieh, 2009). For this reason, the dispersion releaser technology has been applied to increase the membrane permeation by subjecting sensitive nanocarriers to mild agitation (Janas et al., 2017; Villa Nova et al., 2015). For nanoparticle formulations and nanocrystals, which exhibit a certain stability to shear forces, “sample and separate” technologies were successful (Juenemann et al., 2011). When using buffer media during the release experiments, they may be combined with the

6. Biopharmaceutical Characterization

in vitro blood partitioning assay (NCL, 2013). In this experiment, the equilibrium concentration between plasma and blood cells is determined.

6.2 IN VITRO–IN VIVO CORRELATION Establishing an IVIVC at an early time point in formulation development may help to understand the in vivo fate of a drug product based on the in vitro data. In many cases, IVIVC supported the planning of further animal or human studies reducing the number of experiments. Especially for highly permeable drug substances (BCS class 1 and 2), the dissolution data have been successfully used to predict the in vivo performance (FDA, 1997). Similarly, the US-FDA recommends to establish an IVIVC for liposomal products (FDA, 2015a). Unfortunately, only few examples may be found in literature (FDA, 2015a; Juenemann et al., 2011; Cao et al., 2013; Jiang et al., 2011). A reliable IVIVC is obtained by using the appropriate setup and conditions reflecting some aspects of human physiology. Recently, a number of novel technologies with increased sensitivity have been applied to the release testing of nanoformulations. Providing substantial information about release properties of a drug product, they may help to understand the influences of formulation properties to pharmacokinetics and biodistribution. This need for highly sensitive and validated in vitro release tests was also discussed by US-FDA and SCCS (FDA, 2015a; SCCS, 2012). At present, the absence of relevant data and appropriate pharmacokinetic models is limiting the predictive power of the current in vitro tests.

6.3 PHARMACOKINETIC STUDIES AND BIODISTRIBUTION Generally speaking, the pharmacokinetic characterization of drug products is one of the basic requirements in formulation development and the process of drug approval (FDA, 1999, 2014b). The absorption, distribution, metabolism, and excretion of the drug substance must be determined to estimate the pharmacokinetic profile of invasively applied medicinal products (SCENIHR, 2015). Furthermore, the liberation of the drug from the delivery system has to be evaluated for liposomes, polymeric nanoparticles, and drug conjugates. The biodistribution of nanocarriers and drug conjugates differs from the pure drug substance because of the differences in absorption, volume of distribution, clearance, interaction with plasma or serum protein, adhesion to blood cells, or vascular endothelium. For this reason, a detailed in vivo characterization in more than one species is recommended by several regulatory agencies (EMA, 2013a; FDA, 2015c; MHLW, 2016). Nanocrystal formulations are appropriately characterized by their dissolution rate that is also responsible for the more rapid uptake of the drug substance. Many studies have been conducted to investigate the direct uptake of nanoparticles from several tissues (e.g., lungs, GI tract, skin). In most cases, particles in the nanosize range do not pass these barriers to a relevant extent. Commonly, pharmacokinetic parameters include area under the plasma concentration versus time curve (AUC), the peak plasma concentration, the time to peak

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plasma concentration, elimination half-life, volume of distribution, total clearance, renal clearance, and accumulation for both free and total drug. For mass balance studies, blood, urine, and fecal samples should be collected for the radiolabeled moiety (FDA, 2015c). For all administration routes resulting in systemic circulation of the drug delivery system, the accumulation in liver, spleen, kidney, and lungs should be investigated (SCENIHR, 2015). Studying the uptake into macrophages, in vitro stability tests and investigation of the altering protein corona may support the understanding of the elimination and accumulation pathways. Evidently, the pharmacokinetic profile and biodistribution of drug delivery devices is not sufficiently reflected by the abovementioned pharmacokinetic parameters as reported by EMA and US-FDA (EMA, 2013a; FDA, 2015c). It is also affected by the particle size, the surface characteristics, and the drug release rate (Lankveld et al., 2010; De Jong et al., 2008; Owens and Peppas, 2006) of the carrier system. Because of their size, renal elimination is not the preferred route of elimination for nanomedicines, but it has to be taken into account particularly for degradation products and the drug substance released from the carrier. SCENIHR suggested single and repeated dose kinetic studies and an extended follow-up period (SCENIHR, 2015).

7. CONCLUSION Even after years of development, there are only few products in the market that take advantage of functionalized manufactured nanomaterials. Because of the high safety levels in the production of pharmaceuticals and medical devices, for each of these products, extensive characterization of the physicochemical, pharmacological, and biopharmaceutical properties has been undertaken. More research will be required to understand the interplay between the manufacture of nanomaterials, the therapeutic efficacy of the drug product, and the specific toxicological aspects that may arise from the use of nanotechnology. Designing nanoformulations from these nextgeneration excipients and translating them into high-quality products will be one of the greatest challenges in the future of nanomedicines.

ABBREVIATIONS AAS Atomic absorption spectrometry AFM Atomic force microscopy ANDA Abbreviated new drug application ANVISA Brazilian Health Surveillance Agency API Active pharmaceutical ingredient CDER Center for Drug Evaluation and Research CHMP Committee for Human Medicinal Products CQA Critical quality attribute DLS Dynamic light scattering EC European Commission

References

EFSA European Food Safety Authority EMA European Medicines Agency FTIR Fourier transformation infrared spectrometry GC Gas chromatography GRAS Generally recognized as safe HPLC High-performance liquid chromatography ICP-MS Inductively coupled plasma mass spectrometry IVIVC In vitro–in vivo correlation LC Liquid chromatography LD Laser diffraction MS Mass spectrometry NDA New drug application NMR Nuclear magnetic resonance NTA Nanoparticle tracking analysis OECD Organisation for Economic Co-operation and Development PBPK model Physiologically based pharmacokinetic model PEG Polyethylene glycol Ph. Eur. European Pharmacopoeia SCENIHR Scientific Committee on Emerging and Newly Identified Health Risks SEC Size exclusion chromatography SEM Scanning electron microscopy SIMS Secondary ion mass spectrometry STEM Scanning transmission electron microscopy STM Scanning tunneling electron microscopy TEM Transmission electron microscopy UPLC Ultra performance liquid chromatography US-FDA Food and drug administration of the United States XPS X-ray photoelectron spectroscopy XRD X-ray diffraction

REFERENCES Albanese, A., Tang, P.S., Chan, W.C.W., 2012. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu. Rev. Biomed. Eng. 14 (1), 1–16. Bamrungsap, S., Zhao, Z., Chen, T., Wang, L., Li, C., Fu, T., et al., 2012. Nanotechnology in therapeutics: a focus on nanoparticles as a drug delivery system. Nanomed. (Lond.) 7 (8), 1253–1271. Berg, J.M., Romoser, A., Banerjee, N., Zebda, R., Sayes, C.M., 2009. The relationship between pH and zeta potential of ∼ 30 nm metal oxide nanoparticle suspensions relevant to in vitro toxicological evaluations. Nanotoxicology 3 (4), 276–283. Beyer, S., Xie, L., Grafe, S., Vogel, V., Dietrich, K., Wiehe, A., et al., 2015a. Bridging laboratory and large scale production: preparation and in vitro-evaluation of photosensitizer-loaded nanocarrier devices for targeted drug delivery. Pharm. Res. 32 (5), 1714–1726. Beyer, S., Moosmann, A., Kahnt, A.S., Ulshofer, T., Parnham, M.J., Ferreiros, N., et al., 2015b. Drug release and targeting: the versatility of polymethacrylate nanoparticles for peroral administration revealed by using an optimized in vitro-toolbox. Pharm. Res. 32 (12), 3986–3998. https://doi.org/10.1007/s11095-015-1759-2. Epub July 28, 2015.

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Bhardwaj, U., Burgess, D.J., 2010. Physicochemical properties of extruded and non-extruded liposomes containing the hydrophobic drug dexamethasone. Int. J. Pharm. 388 (1–2), 181–189. Bigdeli, A., Palchetti, S., Pozzi, D., Hormozi-Nezhad, M.R., Baldelli Bombelli, F., Caracciolo, G., et al., 2016. Exploring cellular interactions of liposomes using protein corona fingerprints and physicochemical properties. ACS Nano 10 (3), 3723–3737. Cao, X., Deng, W.W., Fu, M., Zhu, Y., Liu, H.F., Wang, L., et al., 2013. Seventy-two-hour release formulation of the poorly soluble drug silybin based on porous silica nanoparticles: in vitro release kinetics and in vitro/in vivo correlations in beagle dogs. Eur. J. Pharm. Sci. 48 (1–2), 64–71. Crist, R.M., Grossman, J.H., Patri, A.K., Stern, S.T., Dobrovolskaia, M.A., Adiseshaiah, P.P., et al., 2013. Common pitfalls in nanotechnology: lessons learned from NCI’s Nanotechnology Characterization Laboratory. Integr. Biol. U.K. 5 (1), 66–73. De Jong, W.H., Hagens, W.I., Krystek, P., Burger, M.C., Sips, A.J.A.M., Geertsma, R.E., 2008. Particle size-dependent organ distribution of gold nanoparticles after intravenous administration. Biomaterials 29 (12), 1912–1919. Dobrovolskaia, M.A., Aggarwal, P., Hall, J.B., McNeil, S.E., 2008. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Mol. Pharm. 5 (4), 487–495. EC, 2011. Commission Recommendation of 18 October 2011 on the Definition of Nanomaterial (2011/696/EU). Brussels. ECHA, 2012. Guidance on Information Requirements and Chemical Safety Assessment. EFSA, 2011. Guidance on the Risk Assessment of the Application of Nanoscience and Nanotechnologies in the Food and Feed Chain. Parma. EMA, 2013a. In: Reflection Paper on the Data Requirements for Intravenous Liposomal Products Developed with Reference to an Innovator Liposomal Product (EMA/ CHMP/806058/2009/Rev. 02). Committee for Medicinal Products for Human Use (CHMP). EMA, 2013b. In: Reflection Paper on Surface Coatings: General Issues for Consideration Regarding Parenteral Administration of Coated Nanomedicine Products (EMA/325027/2013). Committee for Medicinal Products for Human Use (CHMP). EMA, 2013c. In: Reflection Paper on the Data Requirements for Intravenous Iron-based Nano-colloidal Products Developed with Reference to an Innovator Medicinal Product. Committee for Medicinal Products for Human Use (CHMP). EMEA, 2006. In: Guideline on Excipients in the Dossier for Application for Marketing Authorisation of a Medicinal Product (EMEA/CHMP/QWP/396951/2006). Committee for Medicinal Products for Human Use (CHMP). FDA, 1997. Rockville. In: Guidance for Industry: Dissolution Testing of Immediate Release Solid Oral Dosage Forms. CDER. FDA, 1999. Guidance for Industry: Population Pharmacokinetics. Rockville. FDA, 2007. A Report of the U.S. Food and Drug Administration: Nanotechnology Task Force. Rockville. FDA, 2012. FDA’s Regulatory Science Programm in Nanotechnology (Science Board Update). FDA, 2014a. Final Guidance for Industry: Considering Whether an FDA-Regulated Product Involves the Application of Nanotechnology. Silver Spring. FDA, 2014b. Guidance for Industry: Bioavailability and Bioequivalence Studies Submitted in NDAs or INDs. Rockville. FDA, 2015a. Rockville. In: Guidance for Industry, Liposome Drug Products. CDER.

References

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Miksinski, S.P. (Ed.), 2013. Regulatory Considerations for Antibody Drug Conjugates Drug Information Association (DIA) 2013-49th Annual Meeting. NCL, 2013. Method GTA-13: In Vitro Blood Partitioning Assay. OECD, 2012. Guidance on Sample Preparation and Dosimetry for the Safety Testing of Manufactured Nanomaterials. Paris. OECD, 2016. Physical-chemical Properties of Nanomaterials: Evaluation of Methods Applied in the OECD-WPMN Testing Programme. Paris. Owens, D.E., Peppas, N.A., 2006. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 307 (1), 93–102. Předota, M., Machesky, M.L., Wesolowski, D.J., 2016. Molecular origins of the zeta potential. Langmuir 32 (40), 10189–10198. Proposal for a Regulation of the European Parliament and of the Council on medical devices, and amending Directive 2001/83/EC, Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009, 2012. Saber, H., Leighton, J.K., 2015. An FDA oncology analysis of antibody-drug conjugates. Regul. Toxicol. Pharmacol. 71 (3), 444–452. Saptarshi, S.R., Duschl, A., Lopata, A.L., 2013. Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle. J. Nanobiotechnol. 11, 26. SCCS, 2012. Guidance on the Saftey Assessment of Nanomaterials in Cosmetics. Brussels. SCENIHR, 2015. Guidance on the Determination of Potential Health Effects of Nanomaterials Used in Medical Devices. Brussels. Tscharnuter, W., 2000. Photon correlation spectroscopy in particle sizing. In: Meyers, R.A. (Ed.), Encyclopedia of Analytical Chemistry. John Wiley & Sons Ltd, Chinchester, pp. 5469–5485. Tyner, K.M., Zou, P., Yang, X.C., Zhang, H.L., Cruz, C.N., Lee, S.L., 2015. Product quality for nanomaterials: current US experience and perspective. Wires Nanomed. Nanobiotechnol. 7 (5), 640–654. Vila-Caballer, M., Codolo, G., Munari, F., Malfanti, A., Fassan, M., Rugge, M., et al., 2016. A pH-sensitive stearoyl-PEG-poly(methacryloyl sulfadimethoxine)-decorated liposome system for protein delivery: an application for bladder cancer treatment. J. Control. Release 238, 31–42. Villa Nova, M., Janas, C., Schmidt, M., Ulshoefer, T., Grafe, S., Schiffmann, S., et al., 2015. Nanocarriers for photodynamic therapy-rational formulation design and medium-scale manufacture. Int. J. Pharm. 491 (1–2), 250–260. Wacker, M., Janas, C., 2013. Adapter für die Freisetzung disperser Arzneizubereitungen (Dispersion Releaser) inventors. Patent no. DE102013015522.3. Germany. Wacker, M., Zensi, A., Kufleitner, J., Ruff, A., Schutz, J., Stockburger, T., et al., 2011. A toolbox for the upscaling of ethanolic human serum albumin (HSA) desolvation. Int. J. Pharm. 414 (1–2), 225–232. Wacker, M.G., Proykova, A., Santos, G.M., 2016. Dealing with nanosafety around the globeRegulation vs. innovation. Int. J. Pharm. 509 (1–2), 95–106. Wacker, M., 2013. Nanocarriers for intravenous injection–the long hard road to the market. Int. J. Pharm. 457 (1), 50–62. Wandrey, G., Wurzel, J., Hoffmann, K., Ladner, T., Buchs, J., Meinel, L., et al., 2016. Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform. J. Biol. Eng. 10, 11.

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Xie, L., Beyer, S., Vogel, V., Wacker, M.G., Mantele, W., 2015. Assessing the drug release from nanoparticles: overcoming the shortcomings of dialysis by using novel optical techniques and a mathematical model. Int. J. Pharm. 488 (1–2), 108–119. Xu, R., 2008. Progress in nanoparticles characterization: sizing and zeta potential measurement. Particuology 6 (2), 112–115. Zensi, A., Begley, D., Pontikis, C., Legros, C., Mihoreanu, L., Wagner, S., et al., 2009. Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones. J. Control. Release 137 (1), 78–86. Zensi, A., Begley, D., Pontikis, C., Legros, C., Mihoreanu, L., Buchel, C., et al., 2010. Human serum albumin nanoparticles modified with apolipoprotein A-I cross the blood–brain barrier and enter the rodent brain. J. Drug Target 18 (10), 842–848. Zolnik, B.S., Sadrieh, N., 2009. Regulatory perspective on the importance of ADME assessment of nanoscale material containing drugs. Adv. Drug Deliv. Rev. 61 (6), 422–427.

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CHAPTER

Regulation of Biomedical Applications of Functionalized Nanomaterials in the European Union

22

Ruben Pita1, Falk Ehmann1, René Thürmer2 1European

Medicines Agency, London, United Kingdom; 2BfArM – Federal Institute for Drugs and Medical Devices, Bonn, Germany

CHAPTER OUTLINE 1. Overview of European Union Legislation and Procedural Framework�� 654 2. Medicinal Products Developed with Nanotechnology�� 659 3. Scientific Guidance�� 662 3.1 Considerations on Pharmaceutical Quality of Nanomedicines�� 662 3.2 European Medicines Agency Reflection Papers�� 669 3.3 Scientific Advice�� 670 3.4 Innovation Task Force�� 670 3.5 Combination Products/Drug–Device Combination Products�� 671 3.6 Ancillary Medicinal Substances�� 671 4. Medical Devices�� 671 4.1 Borderline Products�� 671 4.2 Medical Devices Regulations�� 672 4.3 Scientific Committee on Emerging and Newly Identified Health Risks Guidance�� 673 5. International Convergence on Nanomedicines�� 674 5.1 Joint Research Center and EU-NCL�� 675 5.2 GSRS16�� 676 6. Conclusions and Next Steps�� 676 Disclaimer�� 677 References�� 677


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CHAPTER 22 Regulation of Biomedical Applications

1. OVERVIEW OF EUROPEAN UNION LEGISLATION AND PROCEDURAL FRAMEWORK The first European legal act in the pharmaceutical sector—Council Directive 65/65/ EC of January 26, 1965—was fundamentally shaped by the thalidomide tragedy in the mid-20th century. To safeguard public health in European Member States (MS), the Council Directive introduced the imperative requirement of having a marketing authorization issued by a competent authority before a medicinal product could be placed in the market of a MS. Since then pharmaceutical legislation of the European Union (EU) has changed considerably with the adoption of new and revised legislation (e.g., regulation and directives) to better protect public health pre- and postmarketing authorization, the need for the grant of a marketing authorization remains, however, as a paramount condition. The current version of this legal provision, Article 6 of Directive 2001/83/EC of the European Parliament and the Council of the [Union] Code Relating to Medicinal Products for Human Use, states that No medicinal product may be placed on the market of a Member State unless a marketing authorisation has been issued by the competent authorities of that Member State in accordance with this Directive or an authorisation has been granted in accordance with Regulation (EC) No 726/2004, read in conjunction with Regulation (EC) No 1901/2006 of the European Parliament and of the Council of 12 December 2006 on medicinal products for paediatric use (and Regulation (EC) No 1394/2007.

This principle goal of protection of public health must nevertheless be achieved without hindering the development of pharmaceutical industry or trade in medicinal products in the EU. To achieve the intended good functioning of the internal market, MS must agree to the harmonization of national provisions and promotion of a uniform application of technical standards regarding quality, safety, and efficacy. For a comprehensive overview of the development of EU pharmaceutical legislation over the last 50 years it is recommended to consult the information available at the European Commission (EC) Website (European Commission, 2015) purposely prepared in 2015 for its half a century celebration. All EU legislation in the area of medicinal products for human use is contained in volume 1 of “The Rules Governing Medicinal Products in the EU” (European Commission EudraLex). The main EU regulations and directives of particular relevance to developers of biomedical application in nanomaterials are • Directive 2001/83/EC of the European Parliament and of the Council of November 6, 2001 on the Community code relating to medicinal products for human use; • Regulation (EC) No 726/2004 of the European Parliament and of the Council of March 31, 2004 laying down Community procedures for the authorization and supervision of medicinal products for human and veterinary use and establishing a European Medicines Agency (EMA);

1. Overview of European Union Legislation and Procedural Framework

• Directive 2001/20/EC of the European Parliament and of the Council of April 4, 2001 on the approximation of the laws, regulations, and administrative provisions of the MS relating to the implementation of good clinical practice in the conduct of clinical trials on medicinal products for human use (to be replaced by the new Clinical Trials Regulation [CTR] EU No 536/2014); • Regulation (EC) No 141/2000 of the European Parliament and of the Council of December 16, 1999 on orphan medicinal products; • Commission Regulation (EC) No 847/2000 of April 27, 2000 laying down the provisions for implementation of the criteria for designation of a medicinal product as an orphan medicinal product and definitions of the concepts “similar medicinal product” and “clinical superiority”; • Regulation (EC) No 1901/2006 of the European Parliament and of the Council of December 12, 2006 on medicinal products for pediatric use and amending Regulation (EEC) No 1768/92, Directive 2001/20/EC, Directive 2001/83/EC and Regulation (EC) No 726/2004; • Regulation (EC) No 1394/2007 of the European Parliament and of the Council of November 13, 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004; • Commission Regulation (EC) No 2049/2005, of December 15, 2005, laying down, pursuant to Regulation (EC) No 726/2004 of the European Parliament and of the Council, rules regarding the payment of fees to, and the receipt of administrative assistance from, the EMA by micro, small- and medium-sized enterprises (European Medicines Agency Supporting); • Commission Regulation (EC) No 507/2006 of March 29, 2006 on the conditional marketing authorization for medicinal products for human use falling within the scope of Regulation (EC) No 726/2004 of the European Parliament and of the Council; • Commission Directive 2003/94/EC of October 8, 2003 laying down the principles and guidelines of good manufacturing practice (GMP) in respect of medicinal products for human use and investigational medicinal products for human use; • Commission Regulation (EC) No 1234/2008 of November 24, 2008 concerning the examination of variations to the terms of marketing authorizations for medicinal products for human use and veterinary medicinal products; • Commission Regulation (EC) No 668/2009 of July 24, 2009 implementing Regulation (EC) No 1394/2007 of the European Parliament and of the Council regarding the evaluation and certification of quality and nonclinical data relating to advanced therapy medicinal products developed by micro, small, and medium-sized enterprises; • Commission Implementing Regulation (EU) No 520/2012 of June 19, 2012 on the performance of pharmacovigilance activities provided for in Regulation (EC) No 726/2004 of the European Parliament and of the Council and Directive 2001/83/EC of the European Parliament and of the Council; • Commission Delegated Regulation (EU) No 357/2014 of February 3, 2014 supplementing Directive 2001/83/EC of the European Parliament and of the

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Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council as regards situations in which postauthorization efficacy studies may be required; • Commission Delegated Regulation (EU) No 1252/2014 of May 28, 2014 supplementing Directive 2001/83/EC of the European Parliament and of the Council regarding principles and guidelines of GMP for active substances for medicinal products for human use; • Regulation (EU) 2017/745 of the European Parliament and of the Council of April 5, 2017 on medical devices, amending Directive 2001/83/EC, Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009 and repealing Council Directives 90/385/EEC and 93/42/EEC (not included in volume 1 referenced above); • Regulation (EU) 2017/746 of the European Parliament and of the Council of April 5, 2017 on in vitro diagnostic medical devices and repealing Directive 98/79/EC and Commission Decision 2010/227/EU (not included in volume 1 referenced above). The legal acts are complemented by a series of notices and guidelines (European Commission, 2014): • Volume 2—Notice to applicants and regulatory guidelines for medicinal products for human use; • Volume 3—Scientific guidelines for medicinal products for human use; • Volume 4—Guidelines for GMPs for medicinal products for human and veterinary use; • Volume 6—Notice to applicants and regulatory guidelines for medicinal products for veterinary use; • Volume 7—Scientific guidelines for medicinal products for veterinary use; • Volume 8—Maximum residue limits; • Volume 9—Guidelines for pharmacovigilance for medicinal products for human and veterinary use; • Volume 10—Guidelines for clinical trial. The Notices to Applicants referenced above (Volume 2 and Volume 6) have been prepared by the EC, in consultation with the competent authorities of the MS and the EMA. Although they do not have legal force in the sense that they are not legally binding, they have some weight in the interpretation of EU law and offer helpful guidance. These notices are thus equally important to both new and more established developers of biomedical applications using nanomaterials as they detail the particular consideration to be held for the different legal basis of a marketing authorization application (namely Articles 8(3), 10(1), 10(3), 10(4), 10(a), 10(b), and 10(c) of the Directive), authorization procedures, legal and scientific requirements, and applicable periods of data and market protection or exclusivity. It cannot be emphasized enough the importance of being familiar with these documents already at an early stage of development of a new product.

1. Overview of European Union Legislation and Procedural Framework

There are two main types of marketing authorization procedures in the EU, the national procedures, i.e., marketing authorization granted by the competent authority of an MS, and the European procedure, i.e., marketing authorization granted by the EC. Regardless of the submission procedure, the same general requirements and scientific principles apply to all applications. These procedures benefit from the same pool of experts, to the extent that assessors from the same national competent authorities are responsible for their scientific assessment, and that scientific discussions may be brought by a particular MS to a committee or working party of the EMA when the need for further scientific discussion is identified at national level. The three national procedures are 1. Purely national procedure, where an applicant submits its application in one MS only; 2. Mutual recognition procedure (MRP), where an applicant submits its application to new MS (Concerned MS) to recognize the marketing authorization previously granted in one MS (Reference Member State); 3. Decentralized procedure (DCP), where an applicant submits its application to more than one MS, and no previous marketing authorization has been exists. For more information on national procedures and a list of all EU national competent authorities for human and veterinary medicinal products consult the webpage of the Heads of Medicines Agencies (www.hma.eu). The European procedure is managed by the EMA, not by MS contrary to national procedures, and is referred to as the centralized procedure (CP). For a comparative analysis of the procedure steps of MRP, DCP, and CP, consult the chapter on EU regulation in Kilcoyne et al. (2013). The CP is mandatory for medicinal products that fall in the following classes: • recombinant DNA, controlled expression of genes coding for biotechnologically active proteins in prokaryotes and eukaryotes including transformed mammalian cells, hybridoma, and monoclonal antibody methods; • advanced therapy medicinal products (as defined in Article 2 of Regulation (EC) No 1394/2007 (European Parliament & Council of the European Union, 2007)); • medicinal products for human use containing a new active substance for the treatment of acquired immune deficiency syndrome, cancer, neurodegenerative disorder, diabetes, autoimmune diseases and other immune dysfunctions, and viral diseases; • Medicinal products for veterinary use intended primarily for use as performance enhancers to promote the growth of treated animals or to increase yields from treated animals; • And, designated orphan medicinal products. An optional scope of the CP is also available and is open for applicants showing that their medicinal product constitutes a significant therapeutic, scientific, or technical innovation or that the granting of the authorization is in the interest of patient health at EU level.

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The EMA was established in the 1995 in accordance with Council Regulation No. 2309/93, now repealed by Regulation (EC) No 726/2004. EMA’s responsibilities are defined in EU legislation and include the scientific evaluation of applications for EU marketing authorizations for human and veterinary medicines in the CP, coordination of the EU’s safety monitoring or “pharmacovigilance” system for medicines, coordination of referral procedures, coordination of inspections, implementation of the EU telematics programme, and stimulating innovation and research in the pharmaceutical sector. The scientific work of the EMA is conducted by the following committees: • Committee for Medicinal Products for Human Use; • Pharmacovigilance Risk Assessment Committee; • Committee for Medicinal Products for Veterinary Use; • Committee for Orphan Medicinal Products; • Committee on Herbal Medicinal Products; • Committee for Advanced Therapies; • Pediatric Committee. The EMA has a number of working parties and related groups (European Medicines Agency Working Parties), which can be consulted by its scientific committees on scientific issues relating to their particular field of expertise. A network of more than 4500 experts appointed by EU MS guarantees the highstandard scientific assessment of medicines by the EMA (European Medicines Agency European Experts). These experts serve as members of the Agency’s scientific committees, working parties, or scientific assessment teams and are responsible for addressing questions raised during the assessment of the benefit risk of a medicinal product. These EMA experts are also responsible for providing scientific advice (see below information on scientific advice), as well as for drafting reflection papers and guidance documents with the objective of mitigating scientific and regulatory gaps resulting from the emergence of innovative technologies, such as nanotechnology. The EU medicines regulatory framework is constantly evolving to address public health needs, taking into account the medical progresses in understanding the pathophysiology of a disease, changing clinical patterns and epidemiology of diseases, globalization (disease outbreaks and pandemics), lifestyle (obesity and diabetes epidemic), and impact that authorized medicines themselves have on diseases. To successfully fulfill its tasks, it is essential that the EMA fosters the collaboration of the European Medicines Regulatory Network with civil society (e.g., healthcare professionals, patients, academia), other EU bodies and international institutions (e.g., EC, European Chemicals Agency [ECHA], European Food Safety Authority [EFSA], European Center for Disease Prevention and Control, World Health Organization, Organization for Economic Cooperation and Development), and regulatory authorities from other regions (e.g., US Food and Drug Administration, Japanese Medical Products Agency, Health Canada, etc.) so that innovative products addressing the health needs of the European citizens can be optimally developed and made accessible as early as possible.

2. Medicinal Products Developed with Nanotechnology

Many of the organizations named above have published guidance documents relevant for their specific stakeholders. For companies or organizations developing new medicinal products using nanotechnology it is recommended to look beyond the boundaries of their own professional disciplines as helpful advice can also be identified in these guidance documents.

2. MEDICINAL PRODUCTS DEVELOPED WITH NANOTECHNOLOGY Article 1 of Directive 2001/83/EC defines a medicinal product as (a) Any substance or combination of substances presented as having properties for treating or preventing disease in human beings; or (b) Any substance or combination of substances which may be used in or administered to human beings either with a view to restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action, or to making a medical diagnosis.

The same Directive classifies specific classes of medicinal products, such as chemical, biological, and herbal. These three main classes are further distributed into subgroups, such as plasma-derived medicinal products, vaccines, radiopharmaceuticals and precursors, homeopathic medicinal products, and advanced therapy medicinal products, as defined in Annex I of Directive 2001/83/EC. To each of these classes specific requirements apply regarding, for example, data to be included in the marketing authorization application, GMPs, labeling, or environmental risk assessment. Medicinal products are thus mainly defined by the origin of their active substance(s); this is justified by the particular considerations to be held regarding their physicochemical characteristics and consequently potential risks and associated uncertainties. The origin of an active substance in a medicinal product will therefore be a major driver to establish, which specific requirements are relevant when submitting an application for marketing authorization. Also, it is the subsequent complexity inherent to, for example, the active substance(s), delivery system, mechanism of action, pharmacodynamics, pharmacokinetics, intended use and target population that will define the additional volume of quality, and nonclinical and clinical data that will be necessary to establish its benefit risk. The above considerations are equally important to define in which legislative framework the product under development would be captured (e.g., medicinal product vs. medical devices—see below section on medical devices). Because of the complexity, diversity, and constant advances of nanotechnologies applied in medicinal products (approved and under development), it became a very challenging task to adopt a widely accepted definition for nanomedicines, to the extent that there is currently no universally accepted or formal definition of

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“nanomedicine.” Although no legal definition for a nanomedicine exists, it will nonetheless be presented the current recommended and working definitions in this field. In October 2011 the EC published a recommendation on the definition of nanomaterial (European Commission, 2011): ‘nanomaterial’ as a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%.

The Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) has nonetheless acknowledged that the 100 nm threshold is not scientifically justified and noted that the special circumstances prevailing in the pharmaceutical sector, stating that this definition “should not prejudice the use of the term ‘nano’ when defining certain pharmaceuticals and medical devices” (SCENIHR, 2010). Ongoing assessments are being performed by the Joint Research Center (JRC, https://ec.europa.eu/jrc/en) of the EC toward the review of the above recommendations (for further reading consult European Commission, 2016). The definition of nanotechnology as provided by the EMA on its Website reads “Nanotechnology is defined as the use of tiny structures – less than 1000 nm across – that are designed to have specific properties” (European Medicines Agency Nanotechnology). In this simple statement there are embedded three concepts, which represent the basis for the Agency’s reflection on the description of nanomedicines: • There is mention of structures, and not just of substances, at a scale of up to 1000 nm; • The structures are designed on purpose and not incidentally at nanoscale; • The structures have specific properties not obtainable with the isolated individual components of the nanostructure. These specific properties are related to the nanotechnology applied, its intended use, and expected clinical advantages. As seen above while discussing the specific requirements for the currently defined classes, a definition is critical as it increases the certainty to both industry and regulators of which requirements are applicable. In any case, the expertise, flexibility, and robustness of the EU regulatory framework have so far been able to safely accommodate new nanomedicines into the market by understanding their individual nature and proportionally adapting the available regulatory provisions to each application for marketing authorization. Table 22.1 below includes a nonexhaustive list of medicinal products applying nanotechnology approved through the CP. It is also worth noting, as an example of the recognition by COMP and adaptability of EU regulation to the potential benefits of nanomedicines, a list of known active substances repurposed with the use of liposomal technology to improve their

2. Medicinal Products Developed with Nanotechnology

Table 22.1 Nonexhaustive List of Nanomedicines Approved Following Assessment via the Centralized Procedure by European Medicines Agency Tradename/ Active Substance

Platform/ Technology

Therapeutic Area

Market Authorization Holder

Approval

Liposomes Caelyx doxorubi- Sterically stacin hydrochloride bilized (Stealth) pegylated liposomes

Myocet doxorubicin

Visudyne verteporfin

DepoCyte cytarabine

Mepact mifamurtide

Onivyde irinotecan

Liposomeencapsulated doxorubicin– citrate complex Liposomal formulation of semisynthetic mixture of porphyrins Multivesicular liposomes with unique structure of multiple nonconcentric aqueous chambers (DepoFoam) Fully synthetic analogue of a component of Mycobacterium sp. cell wall encapsulated in multilamellar liposomes Pegylated liposomes

Multiple myeloma, ovarian neoplasms, breast neoplasms, Kaposi sarcoma Breast neoplasms

Janssen-Cilag International N.V.

21/06/1996

Cephalon Europe

13/07/2000

Degenerative myopia, agerelated macular degeneration Meningeal neoplasms

Novartis Europharm Ltd.

27/07/2000

Pacira Limited

11/07/2001

High-grade resectable nonmetastatic osteosarcoma

IDM PHARMA SAS

06/03/2009

Pancreatic neoplasms

Baxalta Innovations GmbH

14/10/2016

Prophylaxis of organ rejection in renal transplant

Wyeth Europa Ltd.

13/03/2001

Nausea and vomiting

Merck Sharp & Dohme Ltd.

11/11/2003

Nanoparticles Rapamune sirolimus

Emend aprepitant

API particles in nanocrystal colloidal nanodispersion stabilized with poloxamer Colloidal dispersion of nanocrystals

Continued

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Table 22.1 Nonexhaustive List of Nanomedicines Approved Following Assessment via the Centralized Procedure by European Medicines Agency—cont’d Tradename/ Active Substance Abraxane paclitaxel

Cholib fenofibrate/ simvastatin

Market Authorization Holder

Platform/ Technology

Therapeutic Area

Solvent-free colloidal suspension of albuminbound spherical nanoparticles Nanocrystal colloidal dispersion

Metastatic breast cancer

Abraxis BioSciences Ltd.

11/01/2008

Dyslipidemias

Abbott Healthcare Products Ltd.

26/08/2013

Wet macular degeneration

Pfizer Limited

31/01/2006

Bracco International BV

26/03/2001

Approval

Polymer Conjugates Macugen pegaptanib

Pegylatedmodified oligonucleotide

Gas Dispersions SonoVue sulfur hexafluoride

Sulfur hexafluoride Contrast agent gas as “microbub- for echocarbles” dispersion diography and ultrasonography

clinical profile, and hence achieve a significant benefit in the context of the orphan legislation, is presented in Table 22.2. Would there ever be identified that it is necessary to revise the current pharmaceutical legislation to accommodate new products developed with nanotechnology, it will be necessary to carefully reflect on the broad range of platforms resorting to nanotechnology (medicinal products, medical devices, food cosmetics, chemicals, etc.), and the need to ensure consistency between platforms, that the right products are covered by the right legislative framework, and proportionally regulated so as to guarantee public health while enabling access and industrial development.

3. SCIENTIFIC GUIDANCE 3.1 CONSIDERATIONS ON PHARMACEUTICAL QUALITY OF NANOMEDICINES Physicochemical properties can significantly affect the clinical performance of a product and its safety. Because of their particular physicochemical properties, nanotechnology-based medicinal products generally require more complex strategies than

3. Scientific Guidance

Table 22.2 Applications With Orphan Designation Using Nanotechnology Product and Designated Orphan Indication

Designation Date

Grounds for Significant Benefit as Claimed by Sponsor

Doxorubicine polyisohexylcyanoacrylate nanoparticles Treatment of hepatocellular carcinomas

21/10/2004

Amikacin sulfate (liposomal) Treatment of Pseudomonas aeruginosa lung infection in cystic fibrosis

25/07/2006

Doxorubicin hydrochloride (liposomal) Treatment of soft tissue sarcoma

27/10/2006

Paclitaxel (liposomal) Treatment of pancreatic cancer

31/10/2006

Cisplatin (liposomal) Treatment of pancreatic cancer

08/06/2007

Vincristine sulfate liposomes

08/07/2008

In the designated product, doxorubicin is entrapped in nanoparticles of polyisohexylcyanoacrylate. Once administered to the patient, the nanoparticles are adsorbed to the surface of tumor cells and doxorubicin entrapped in these particles is subsequently released on the cancer cell. The nanoparticles degrade and release also polyisohexylcyanoacrylate, which may contribute to delivering doxorubicin into the cancer cells. This may increase the presence of the drug in the tumor cells and may enhance its activity. Amikacin sulfate (liposomal) might be of potential significant benefit for the treatment of cystic fibrosis mainly because it may provide a major contribution to patient care. Doxorubicin hydrochloride (liposomal) might be of potential significant benefit for the treatment of soft tissue sarcoma mainly because it might improve the long-term outcome of the patients. Paclitaxel (liposomal) might be of potential significant benefit for the treatment of pancreatic cancer because it may improve the long-term outcome of the patients. According to the sponsor, paclitaxel (liposomal) will, by binding specifically to the cells that line blood vessels it will inhibit the growth of newly formed blood vessels, contribute to the destruction of the tumor. The inclusion of cisplatin in liposomes is expected to increase the concentration of the drug in the cancer cells, compared with normal cells, and decrease the adverse effects of the active substance. In this medicine, vincristine is contained in liposomes. This is expected to improve the way the medicine works compared with the conventional form of the medicine, by slowing down the clearance of the medicine from the body.

Treatment of acute lymphoblastic leukemia

Continued

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Table 22.2 Applications With Orphan Designation Using Nanotechnology—cont’d Product and Designated Orphan Indication

Designation Date


Topotecan hydrochloride (liposomal) Treatment of glioma

05/09/2008

Gadodiamide (liposomal) Treatment of glioma

03/12/2008

Daunorubicin (liposomal) Treatment of acute myeloid leukemia

03/12/2008

Ciprofloxacin (liposomal) Treatment of cystic fibrosis

24/07/2009

Doxorubicin hydrochloride (in heat-sensitive liposomes)

23/02/2011

The liposomal delivery system improve the way the medicine works compared with the conventional form of the medicine because it can be injected directly into the brain, concentrating the medicine at the site of the tumor and reducing side effects by reducing the amount of free active substance in the blood. Gadodiamide (liposomal) is expected to show how much of the tumor the medicine has reached and to indicate where more treatment may be needed. It is also expected to help to see that any catheters (small tubes inserted into the veins) are correctly placed. When it is contained within liposomes, it is expected to remain in the patient’s body for longer than “free” daunorubicin, which might lead to the improved treatment of patients with this condition The liposomal formulation for inhalation may allow delivery of higher amounts of ciprofloxacin directly into the lungs compared with existing formulations of ciprofloxacin. Also, the liposomal formulation is expected to reduce the bitterness of the medicine, allowing it to be given by inhalation and it is expected to remain in the patient’s lungs for longer than free ciprofloxacin. The doxorubicin is contained within liposomes, which allow the active substance to accumulate mainly in the liver. The liposomes used in this medicine are heat-sensitive particles that release the doxorubicin when their temperature reaches 39.5°C. When this medicine is used together with radiofrequency ablation, the heat from the probe provokes the release of doxorubicin within the tumor, which is expected to improve the antitumor effects of this treatment.

Treatment of hepatocellular carcinoma


Table 22.2 Applications With Orphan Designation Using Nanotechnology—cont’d Product and Designated Orphan Indication

Designation Date


Nanoliposomal irinotecan Treatment of pancreatic cancer

09/12/2011

Liposomal combination of cytarabine and daunorubicin Treatment of acute myeloid leukemia

11/01/2012

Liposomal daunorubicin Treatment of acute myeloid leukemia

10/10/2012

Recombinant human transglutaminase-1 encapsulated into liposomes Treatment of transglutaminase1-deficient autosomal recessive congenital ichthyosis

07/06/2013

Modified messenger ribonucleic acid encoding human ornithine transcarbamylase enzyme encapsulated into lipid nanoparticles Treatment of ornithine transcarbamylase deficiency

20/04/2017

The nanoliposomes are expected to accumulate within the tumor and release the medicine slowly over time, thereby decreasing the rate at which the irinotecan is removed from the body and allowing it to act for longer; this is expected to improve the treatment of patients with this condition. The liposomes are expected to remain in the patient’s body for longer than free cytarabine and daunorubicin and accumulate in the patient’s bone marrow. The liposomes protect the anticancer medicines from being metabolized early, the extended residence time at the site of action is expected to enhance their effect on cancer cells. The liposomes are expected to keep the daunorubicin in the patient’s body for longer than “free” daunorubicin and to accumulate in the patient’s bone marrow. The liposomes protect the anticancer medicine from being broken down early and enhance its effect on cancer cells. This medicine is expected to be available as a cream that contains the transglutaminase-1 enzyme as its active substance. Transglutaminase-1 is contained within tiny fatty particles called liposomes, which are expected to carry the enzyme through the skin. This is expected to help restore the normal formation of the outer layer of the skin. The genetic material is enclosed in fatty particles to protect it and it is injected with another substance to help it enter liver cells. Giving the medicine by infusion (drip) into a vein every 7–14 days is expected to enable liver cells to produce ornithine transcarbamylase and so reduce the symptoms caused by its deficiency.

Information collected in May 2017 from the Register of designated Orphan Medicinal Products European Commission. Register of Designated Orphan Medicinal Products. Available at: http:// ec.europa.eu/health/documents/community-register/html/orphreg.htm.

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conventional pharmaceutical dosage forms regarding their manufacture and quality control. It is recognized that the unique properties of nanomaterial formulations may require greater regulatory scrutiny to ensure quality, safety, and efficacy. The regulatory requirements for pharmaceutical development are described in the Guideline on Pharmaceutical Development (ICH Q8(R2)) (ICH, 2009) published by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH, available at http://www.ich.org/). ICH Q8 defines that the aim of pharmaceutical development is to design a quality product and its manufacturing process to consistently deliver the intended performance of the product. Pharmaceutical development should include, at a minimum, the following elements: • Defining the quality target product profile (QTPP) as it relates to quality, safety, and efficacy, considering, e.g., the route of administration, dosage form, bioavailability, strength, and stability; • Identifying potential critical quality attributes (CQAs) of the drug product, so that those product characteristics having an impact on product quality can be studied and controlled; • Determining the CQAs of the drug substance, excipients, etc., and selecting the type and amount of excipients to deliver drug product of the desired quality; • Selecting an appropriate manufacturing process; • Defining a control strategy. It is important to recognize that quality cannot be tested into products but should instead be built into the product design. A CQA is a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. CQAs are generally associated with the drug substance, excipients, intermediates (in-process materials), and finished product. CQAs for nanomedicines normally include more product-specific aspects than for other dosage forms. Potential drug product, CQAs, derived from the QTPP and/or prior knowledge is used to guide the product and process development. The list of potential CQAs can be modified when the formulation and manufacturing process are selected and as product knowledge and process understanding increase. A control strategy is designed to ensure that a product of required quality will be produced consistently. The controls can include parameters and attributes related to active substance and finished product materials and components, facility and equipment operating conditions, in-process controls, finished product specifications, and the associated methods and frequency of monitoring and control (ICH Q8). A comprehensive pharmaceutical development approach will generate process and product understanding and identify sources of variability. Sources of variability that can impact product quality should be identified, appropriately understood, and subsequently controlled. Product and process understanding, in combination with quality risk management (see ICH Q9), will support the control of the process such


that the variability (e.g., of raw materials) can be compensated for in an adaptable manner to deliver consistent product quality. Process development studies should provide the basis for process validation. Process validation should confirm that the control strategy is adequate to the process design and the quality of the product. Defining the control strategy can start with existing knowledge evaluation, risk assessments, and process characterization planning followed by execution of process characterization studies. The next steps are the development of commercial control strategy and the evaluation of commercial control strategy. The control strategy also addresses testing of materials (raw materials, excipients, and active ingredient). Ensuring the appropriate quality of materials is essential for nanoformulations, and the qualification of suitable suppliers is vital. Because of the large variety of different nanomedicinal applications, it is impossible to define a list of CQAs applicable for all nanoformulations. A wide range of nanoparticle systems exists; in some systems the active substance is in nanoform, in other systems nanoadditives are incorporated or alternatively the active substance delivery system or nanocarrier is in the nanosize. Some attributes are relevant irrespective of whether the formulation is a nanomedicinal product or not (e.g., appearance, assay, purity, particulate matter, sterility, bacterial endotoxins, deliverable volume, pH, osmolality for a parenteral dosage form, residual solvents, or elemental impurities). Different attributes are relevant for liposomes, pegylated liposomes, iron sucrose complexes nanoemulsions, nanocrystals, etc. It is therefore important to define the nano-related attributes, which will be established on a case-by-case decision dependent on the nanosystem in question and the final dosage form of the medicinal product. Cellular binding studies and cellular uptake studies or other functional assays or in vitro techniques should also be considered during characterization. Current considerations in EMA guidance documents are driven by the experience with active substance delivery systems using nanotechnology that are under development, under regulatory review, or have received marketing authorization in Europe. The following list shows some attributes, which are relevant for nanoformulations: • Size; • Size distribution or polydispersity index; • Shape/morphology; • Surface charge; • Surface composition (e.g., pegylated liposome); • Encapsulation efficiency; • Nonencapsulated active pharmaceutical ingredient; • Release (in vitro dissolution test); • Polymer molecular weight; • Polymer biodegradability; • Interaction with biological medium; • Conjugation chemistry; • Functionality of targeting moieties;

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• Aggregation/Agglomeration; • Polymorphism; • In-use stability/compatibility with, e.g., devices. Particle detection methods are becoming more sensitive and able to provide significantly more information on nanoformulations. Numerous methods are available to characterize the particle profile. These methods differ by size and concentration limits, as well as value of information. A complete particle landscape can only be obtained when different orthogonal methods are used together. Those analytical methods, which will be included in the release and/or shelf-life specification of the nanoformulation, need to be validated to demonstrate that they are suitable for their intended purpose. Equally important is the fact that testing of conventional attributes could also be challenging for nanoformulations. For example, some nanoparticles interfere with tests to quantify bacterial endotoxins; this can generate misleading results by either over or under underestimating results, and specific solutions are needed case by case. It should also be assessed if traditional analytical methods are adequate for characterization of nanomaterials or whether some may need to be applied differently. Development of analytical methods should be undertaken early during product development to ensure the adequate characterization of nonclinical and clinical batches to avoid the need to perform any additional in vitro or in vivo bridging studies. In the case that novel excipients are used in a nanoformulation, specific requirements apply not only concerning pharmaceutical quality but also toxicological evaluations. Directive 2001/83/EC defines a novel excipient is an excipient that is being used for the first time in a medicinal product or by a new route of administration. It may be a new chemical entity or a well-established one, which has not yet been used for human administration and/or for a particular human administration pathway in the EU and/or outside the EU. These requirements are described in the Guideline on Excipients in the Dossier for Application for Marketing Authorization of a Medicinal Product. Full details of manufacture, characterization, and controls, with crossreferences to supporting safety data (nonclinical and/or clinical), should be provided. Packaging of nanoformulations is often an issue and the suitability of packaging materials, containers, and container closure systems need to be addressed. A possible interaction between product and container, including migration and sorption, should be considered because the presence of leachables could not only be safety relevant but also negatively impact the quality of the nanoformulation. As for all medicinal products stability testing plays an essential role, this is also the case for nanoformulations. Any observed trends need to be further evaluated also with respect to potential changes and impact on the overall performance (safety and efficacy) of the nanoformulation. In addition to the characterization studies conducted under normal conditions, stress-test studies should be conducted to compare physical and chemical degradation and to gain a better understanding of the degradation profile of a particular nanoformulation.


The stability-indicating properties of the analytical methods should be evaluated to provide assurance that changes, which may be critical for the formulation, would be detected. Another important aspect is the preparation of the medicinal product prior to administration because for some nanoformulations complex reconstitution, dilution, or mixing steps are required, which should be well investigated and validated to avoid misapplications or errors. As with conventional formulations, it should be documented that the combination of the intended nanoformulation and packaging material permits correct dosing and is suitable for the administration to the intended patient population. From a pharmaceutical quality point of view, for products developed with reference to an innovator product—so-called Article 10 marketing authorization applications of Directive 2001/83/EC (European Parliament & Council of the European Union, 2001) (e.g., liposomes or intravenous iron-based nanocolloidal products)— tremendous knowledge has been gained over the last few years. Although initially limited regulatory experience was available, the assessment of a high number of scientific advice procedures, and the first of such abbreviated marketing authorization applications, allowed regulators to significantly improve their understanding on nanomedicines. However, there still remain some scientific challenges. In some cases, it continues to be challenging to define the extent to which characterization/ comparability testing will be required. It is also a question how similar is sufficiently similar, what is the relevance of “small differences,” and which differences in numerical values for certain attributes are acceptable. Future applications and international exchange will allow regulators to differentiate between “need to know” and “nice to know.”

3.2 EUROPEAN MEDICINES AGENCY REFLECTION PAPERS Based on the experience developed in the context of Scientific Advice and evaluation of Marketing Authorization Applications, the following reflection papers (European Medicines Agency Multidisciplinary) have been drafted addressing specific technical and regulatory principles applicable to liposomal formulations, iron oxide nanoparticles for the treatment of iron-deficient anemia, surface coating of nanomedicines, and block copolymer micelles: • Data requirements for intravenous iron-based nanocolloidal products developed with reference to an innovator medicinal product; • Data requirements for intravenous liposomal products developed with reference to an innovator liposomal product; • Surface coatings: general issues for consideration regarding parenteral administration of coated nanomedicine products; • Development of block copolymer micelle medicinal products. Besides the reflection papers mentioned above, a technical paper from 2013 coauthored by experts from different regulatory authorities (Ehmann et al., 2013) presented

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further scientific and regulatory considerations on the regulatory requirements for the marketing authorization of, what has been nicknamed as, “nanosimilar” medicinal products. The level of complexity of nanomedicines (e.g., different types and molecular arrangements of liposomal formulations and iron nanoparticles) clearly indicates that such products cannot be characterized by physicochemical means only, and demands for additional data to be generated by the sponsors to prove that such Article 10 marketing authorization applications (e.g., generic and hybrid) have the same, or similar, safety and efficacy of the innovator product. Conceptually, the approach is similar to the one successfully implemented for biosimilars with a stepwise methodology for determining the extent and need for quality, nonclinical, and clinical comparative tests.

3.3 SCIENTIFIC ADVICE The EMA provides scientific advice (European Medicines Agency Scientific Advice) by addressing questions made by developers, with the objective of facilitating the development and availability of high-quality, effective, and acceptably safe medicines, for the benefit of patients. The advice is given in the light of the current scientific knowledge and based on the documentation submitted by developers. Scientific advice is prospective in nature, i.e., it focuses on development strategies rather than preevaluation of data to support a marketing authorization application. Scientific advice helps to ensure that developers perform the appropriate tests and studies (Hofer et al., 2015). Scientific advice received from the Agency is not legally binding on the Agency or on the medicine developer regarding any future marketing authorization applications for the medicine concerned. Resort to EMA or national scientific advice by developers of medicinal products using new technologies, such as nanotechnology, is highly recommended.

3.4 INNOVATION TASK FORCE Sponsors of nanomedicines are encouraged to seek the support available via the Innovation Task Force (ITF) and the newly established EU Innovation Offices Network (European Union Medicines Agencies Network Strategy to 2020— Working together to improve health), at an early stage of their research. The ITF is a multidisciplinary group that includes scientific, regulatory, and legal competences. It was set up to ensure coordination across the Agency and to provide a forum for early dialogue with applicants on innovative aspects in medicines development. The Agency’s tools for opinion-making are proven to be the main avenues to development success and patients’ access. Early dialogue is crucial to support innovation not only in Europe but also in the context of global medicines development for the benefit of global health and to inform international convergence in policies and regulatory requirements. More information on how to request an ITF briefing meeting is available in a dedicated EMA webpage (European Medicines Agency Innovation in Medicine).

4. Medical Devices

3.5 COMBINATION PRODUCTS/DRUG–DEVICE COMBINATION PRODUCTS In February 2017, the EMA started a public consultation on a new “Concept paper on developing a guideline on quality requirements of medicinal products containing a device component for delivery or use of the medicinal product” (European Medicines Agency, 2016). The guideline, which will eventually result from this concept paper, will consider the data requirements with respect to quality aspects in relation to the safety and performance of the medical device, whether it is an integral component of the medicinal product or a stand-alone device, including usability studies in target patient populations with the relevant clinical conditions. In the EU, there is no legal definition for a product where a medicinal product and a medical device are presented together either as an integral combination or presented separately for use together. The terminology in the context to this concept paper is restricted to medicinal product as defined by Directive 2001/83/EC. Drug–device combination product (DDC) suitability for the intended purpose (e.g., administration of a medicinal product) should take into account the quality aspects of the device in itself, its use with the particular medicinal product, the complexity of the device component, the patient population, the caregiver characteristics where relevant, and the clinical situation in which the DDC is to be used. It was therefore considered to be appropriate to draft guidance on quality data requirements for medicinal products incorporating, or used with, medical devices (i.e., DDCs) to both assessors and pharmaceutical industry.

3.6 ANCILLARY MEDICINAL SUBSTANCES EMA provides scientific opinions on the quality and safety of active substances incorporated in medical devices that are liable to act on a patient’s body with an action that is ancillary, (i.e., supports) the action of the device. Also procedural guidance is published by the EMA (European Medicines Agency Ancillary Medicinal). If the active substance is derived from human blood or human plasma—or falls under the scope of the CP—the notified body must consult EMA as the competent authority to issue a scientific opinion. For other substances, the notified body and device manufacturer must choose a competent authority in a Member State of the EU to issue a scientific opinion. Notified bodies may also consult EMA in cases, for example, where the Agency has already evaluated a medicine containing the same active substance. The notified body should give due consideration to the scientific opinion when making its certification decision.

4. MEDICAL DEVICES 4.1 BORDERLINE PRODUCTS The increasing complexity of nanomedicines may lead to borderline cases, where the distinction between medical device and medicinal product may be challenging to

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define, e.g., devices for administering medicinal products where the device and the medicinal product form a single integral product designed to be used exclusively in the given combination and which are not reusable or refillable (Medicines and Healthcare Products Regulatory Agency, 2015), or where there is a combination of multiple modes of action with which neither of the mode is secondary to the other (Lebourgeois, 2008). The differences in the rules and regulatory requirements between medicinal products and medical devices are significant (European Commission Medical Devices; Parvizi and Woods, 2014; Chowdhury, 2010). Developers of novel applications should be at an early stage of development define which requirements would be applicable. For example, iron oxide nanoparticles injected into tumor cells to be heated up by radiation or an external magnetic field have been previously classified as a medical device because the immediate effect is mechanical as the tumor cells burst. On the other hand there were discussions that, one might regard the legislation on medicinal products to be applicable as the burst cells are metabolized at a later point in time. Another nanoparticle-enhanced radiotherapy technology was also classified a medical device while in the United States it is considered a medicinal product.

4.2 MEDICAL DEVICES REGULATIONS On April 2017 new Regulations on medical devices and in vitro diagnostics were adopted, namely Regulation (EU) 2017/745 and Regulation (EU) 2017/746 (see above). These Regulations replace the existing Directives and will have the force of law throughout the EU when they come into effect. The new Regulations on medical and in vitro diagnostic medical devices will help to ensure that all medical devices— from heart valves to sticking plasters to artificial hips—are safe and perform well. The new rules will improve market surveillance and traceability and make sure that all medical and in vitro diagnostic devices are designed to reflect the latest scientific and technological state of the art. The rules will also provide more transparency and legal certainty for producers, manufacturers, and importers and help to strengthen international competitiveness and innovation in this strategic sector. Once devices are available for use on the market, manufacturers will be obliged to collect data about their performance and EU countries will coordinate more closely in the field of market surveillance. To allow manufacturers and authorities to adapt to the new rules governing medical devices, a transitional period is provided of 3 years after publication for the Regulation on medical devices and 5 years after publication for in the Regulation on vitro diagnostic medical devices. Medical devices are classified in four classes ranging from low risk to high risk (I, IIa, IIb, and III). Nanomaterials are addressed in the new Regulations and it is stated that There is scientific uncertainty about the risks and benefits of nanomaterials used for devices. In order to ensure a high level of health protection, free movement of goods and legal certainty for manufacturers, it is necessary to introduce a uniform definition for nanomaterials based on Commission Recommendation 2011/696/EU, with the necessary flexibility to adapt that definition to scientific

4. Medical Devices

and technical progress and subsequent regulatory development at Union and international level. In the design and manufacture of devices, manufacturers should take special care when using nanoparticles for which there is a high or medium potential for internal exposure. Such devices should be subject to the most stringent conformity assessment procedures. In preparation of implementing acts regulating the practical and uniform application of the corresponding requirements laid down in this Regulation, the relevant scientific opinions of the relevant scientific committees should be taken into account.

Article 2 of the Regulation (EU) 2017/745 contains definitions of “nanomaterials,” “particle,” “agglomerate,” and “aggregate” for the purposes of the definition of “nanomaterials.” As provided by Article 2, “‘nanomaterial’ means a natural, incidental or manufactured material containing particles in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1–100 nm” The EC is empowered to adopt delegated acts to amend the definition of “nanomaterial” in the light of technical and scientific progress and taking into account definitions agreed at Union and international level. Regulation (EU) 2017/745 also states that “Devices shall be designed and manufactured in such a way as to reduce as far as possible the risks linked to the size and the properties of particles which are or can be released into the patient’s or user’s body, unless they come into contact with intact skin only. Special attention shall be given to nanomaterials.” Rule 19 addresses the classification of medical devices incorporating or consisting of nanomaterials, it states that Rule 19: All devices incorporating or consisting of nanomaterial are classified as:

• class III if they present a high or medium potential for internal exposure; • class IIb if they present a low potential for internal exposure; and • class IIa if they present a negligible potential for internal exposure.

4.3 SCIENTIFIC COMMITTEE ON EMERGING AND NEWLY IDENTIFIED HEALTH RISKS GUIDANCE A guidance document titled “Guidance on the Determination of Potential Health Effects of Nanomaterials Used in Medical Devices” (SCENIHR, 2015) has been adopted by the SCENIHR in January 2015. It addresses the use of nanomaterials in medical devices and provides information for risk assessors regarding specific aspects that need to be considered in the safety evaluation of nanomaterials. A phased approach is recommended for evaluating the risk of the use of nanomaterials in medical devices based on potential release and characteristics of the nanomaterials to avoid unnecessary testing. The phases cover particle release (phase 1), particle distribution and persistence (phase 2), hazard assessment (toxicological

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evaluations) (phase 3), and risk characterization/risk assessment (phase 4). In phase 1 an evaluation of the potential for the device to release nanoparticles either directly or due to wear of the device during use should be carried out. In phase 2 the aim is to determine the distribution of the particles released and also their persistence potential. In phase 3 the hazard is assessed using appropriate toxicity tests taking account of the exposure characteristics and potential for persistence in specific organs. This will provide input for the final risk characterization (phase 4). In summary, the potential risk from the use of nanomaterials in medical devices is mainly associated with the possibility for release of free nanoparticles from the device and the duration of exposure. The guidance document has been drafted for medical devices, and not all aspects are relevant for medicinal products. Nevertheless, following the spirit of this guidance could be helpful for companies developing nanomedicines.

5. INTERNATIONAL CONVERGENCE ON NANOMEDICINES Collaborations between the EMA and European Institutions, such as the EC Directorates and its scientific bodies (e.g., ECHA, EFSA, and JRC) have been important to develop a consistent approach to products using nanotechnology, supporting policies, which considered the progress and experience accumulated in related frameworks. Independent initiatives, including conferences organized by the European Technology Platform on Nanomedicine, the Products Quality Research Institute, the FDA in 2014 (Bartlett et al., 2015), and the CLINAM annual conferences—offer yearly dedicated open regulatory sessions for discussion with the scientific community on the opportunities and challenges arising from the clinical application of nanotechnologies. In 2015, the International Pharmaceuticals Regulatory Forum (IPRF) was given the responsibility of the establishment and governance of the international nanomedicines expert group. This group has the support of many authorities from different regions, including FDA, Health Canada, Japan, Swissmedic, the Association of Southeast Asian Nations (ASEAN), and the Brazilian regulatory Agency (National Health Surveillance Agency or ANVISA [Agência Nacional de Vigilância Sanitária]), among others (International Pharmaceutical Regulators Forum, 2015), and was given the mandate of sharing nonconfidential information, promoting consensus findings on regulatory standards and convergence focused on nanomedicines/nanomaterial in medicinal products, and borderline and combination products. In 2016, for the first time information and mapping (International Pharmaceutical Regulators Forum IPRF Nanomedicines) of the activity on nanomedicines in the participating regions was made available, providing a valuable starting point for understanding current regulations and points of potential convergence on critical issues. Deliverables for the 2015/2016 work plan include

5. International Convergence on Nanomedicines

• Information sharing and mapping (annual regulatory updates); • Compilation, mapping and discussion on terminology, and definitions with focus on the classification of nanomedicines/nanotechnology in drug products; • Compilation of information for understanding synergies between the nano(medicine) safety and nano(medicine) toxicology fields; • Compilation of investigations required for “generic” nanomedicines/nanotechnology in drug products used in regulatory procedures; • Mapping and exchange of requirements for nanomedicine/nanotechnology in drug product class specific guidance (e.g., liposomal formulations); • Exchange and mapping of general CQA principles for nanomedicines/nanotechnology in drug products.

5.1 JOINT RESEARCH CENTER AND EU-NCL The EC (JRC) in 2016 published the results of a survey titled “Identification of regulatory needs for nanomedicines.” This first survey, of a series of three, was performed within the nanomedicine working group of the IPRF with the aim to get a general overview on the status and regulatory needs of nanomedicines. The EU’s Horizon 2020 research and innovation programme launched in 2015 the first transdisciplinary testing infrastructure for the characterization of medicinal products involving nanotechnology—EU-NCL Nanomedicine Characterization Laboratory (www.euncl.eu). The EU-NCL is a cooperative arrangement between six European Laboratories and the Nanotechnology Characterization Laboratory (NCINCL) of the United States. The facilities will offer access to their existing analytical services for public and private developers to characterize the quality and safety of nanomedicines that are aiming to enter into clinical trials or seeking to apply for marketing authorization. To ensure that methods developed/validated in the EU-NCL are relevant for regulatory purposes, and the obtained information can support the regulatory decision-making, the questions in the survey to 18 regulatory agencies have been defined according to recommendations of the SCENIHR, the NCI-NCL, the expert team of the EU-NCL, and recommendations include in EMA’s reflection papers related on nanomedicine. A similar questionnaire, performed within the EU project “NanoReg” on manufactured nanoparticles, was also taken into account. The obtained information can be classified into three categories: (1) regulatory experience with nanomedicines, (2) information needs of regulators for the characterization of nanomaterials (toxicology and physicochemical) and (3) further steps that can support the acceptance of nanotechnology-based products in healthcare. The respondents of the survey confirmed the need for the harmonization of information requirements on nanospecific properties. In addition, the relevance for regulatory decisions of a number of critical physicochemical properties proposed in the scientific literature was questioned within the survey. Finally, there was consensus among the regulatory agencies for a need of an independent nanocharacterization testing facility that can support regulators in the evaluation of sophisticated material and the performance of new test methods before entering into clinical applications.

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5.2 GSRS16 The Global Summit on Regulatory Science (GSRS) is an international conference for discussion of innovative technologies and partnerships to enhance translation of basic science into regulatory applications. GSRS organizes an annual conference providing a platform where regulators, policy makers, and bench scientists from various countries can exchange views and harmonize strategies via global collaboration. To engage the global community to address regulatory science research and training needs, GSRS is held in different countries on an annual basis. The GSRS annual conference in 2016 (GSRS16) was held in Maryland (USA) and was dedicated to nanotechnology standards and applications. The goals of the GSRS16 were to • Educate a broad group of stakeholders on the state of the art in nanotechnology science, measurement methods, and standards for regulatory applications; • Identify the most immediate needs in nanotechnology science, measurement methods, and standards relevant to regulatory applications; • Facilitate greater coordination between stakeholders in the development of standards. The discussions at GSRS16 focused on different nanomaterial-containing applications, including drugs, medical devices, food and food contact materials, and personal care products, and benefited from the participation of regulators and international organizations from different regions, academia, industry, and standardization bodies. A summary of the presentations, panel discussions, challenges, needs, and considerations are included in the GSRS16 report available on the webpage of the National Institute for Public Health and the Environment http://www.rivm. nl/dsresource?objectid=644647ac-3ca5-4bb5-b6b0-8d742e359c8b&type=PDF. The report provides a valuable insight into the current discussions and priorities of industry, academia, and regulators.

6. CONCLUSIONS AND NEXT STEPS As extensively reflected in previous publications (Ehmann and Pita, 2016; Papaluca et al., 2017), despite the challenges presented by the complex and ever-evolving applications of nanotechnology, the European regulatory framework is sufficiently robust to evaluate, authorize, and ensure the oversight of current nanomedicines. Regulatory science shall evolve so it is ready for the technical and regulatory challenges of future nanomedicines, and to answer the fundamental question of how the established principles of assessing quality, safety, and efficacy, can be enriched by the advances in the understanding of nanotechnology properties, especially considering the potential of nanomedicines to address with a wide range of different,

6. References

efficient, and innovative approaches to the many unmet medical needs (Papaluca et al., 2015). As explained above, it is important to understand early on in the development what are the applicable requirements of a particular legislative framework, and indeed which legislative framework(s) would be relevant to consider according to the foreseen uses of a particular nanomaterial or nanosystem. Sponsors of novel nanomedicines are strongly encouraged to seek the support available via the ITF and the EU Innovation Offices Network to discuss and plan for Regulatory and Scientific advice at individual national or EU level on the strategy for their research and development. The recognized transversal use of nanotechnology has led to the collaboration between regulators and experts from different disciplines. The joint effort between the various partners in different regions is already resulting in an increased awareness of the need to develop international guidance and technical standards for nanomaterials. Nanotechnology is an important and promising platform for new medicines, and while recognizing the challenges encountered by new technologies, the EU will continue promoting the development and timely access of safe and effective nanomedicines.

DISCLAIMER The views expressed in this article are the personal views of the authors and may not be understood or quoted as being made on behalf of or reflecting the position of the European Medicines Agency, its committees or working parties, or the Federal Institute for Drugs and Medical Devices (BfArM).

REFERENCES Bartlett, J.A., Brewster, M., Brown, P., Cabral-Lilly, D., Cruz, C.N., David, R., Eickhoff, W.M., Haubenreisser, S., Jacobs, A., Malinoski, F., Morefield, E., Nalubola, R., Prud’homme, R.K., Sadrieh, N., Sayes, C.M., Shahbazian, H., Subbarao, N., Tamarkin, L., Tyner, K., Uppoor, R., Whittaker-Caulk, M., Zamboni, W., 2015. Summary report of PQRI Workshop on Nanomaterial in Drug Products: current experience and management of potential risks. AAPS J. 17, 44–64. Chowdhury, N., 2010. Regulation of nanomedicines in the EU: distilling lessons from the pediatric and the advanced therapy medicinal products approaches. Nanomed. (Lond.) 5, 135–142. Ehmann, F., Pita, R., 2016. The EU is ready for non-biological complex medicinal products. GaBI J. 5, 30–35. Ehmann, F., Sakai-Kato, K., Duncan, R., Hernán Pérez de la Ossa, D., Pita, R., Vidal, J.M., Kohli, A., Tothfalusi, L., Sanh, A., Tinton, S., Rober, J.L., Silva Lima, B., Amati, M.P., 2013. Next-generation nanomedicines and nanosimilars: EU regulators’ initiatives relating to the development and evaluation of nanomedicines. Nanomed. (Lond.) 8, 849–856.

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European Commission, 2011. Commission Recommendation of 18 October, 2011 on the Definition of Nanomaterial, pp. 38–40 Official J. Eur. Union, L 275. European Commission, 2014. EudraLex – EU Legislation. Available at: https://ec.europa.eu/ health/documents/eudralex_en. European Commission, 2015. 50 Years of EU Pharmaceutical Legislation. Available at: http:// ec.europa.eu/health/human-use/50years_en. European Commission, 2016. Towards a Review of the EC Recommendation for a Definition of the Term “nanomaterial”: Part 3: Scientific-technical Evaluation of Options to Clarify the Definition and to Facilitate its Implementation. Available at: https://ec.europa.eu/ jrc/en/publication/eur-scientific-and-technical-research-reports/towards-review-ecrecommendation-definition-term-nanomaterial-part-3-scientific-technical. European Commission. EudraLex – Volume 1-Pharmaceutical Legislation for Medicinal Products for Human Use. Available at: https://ec.europa.eu/health/documents/eudralex/ vol-1_en. European Commission. Medical Devices. Available at: https://ec.europa.eu/growth/sectors/ medical-devices_en. European Commission. Register of Designated Orphan Medicinal Products. Available at: http://ec.europa.eu/health/documents/community-register/html/orphreg.htm. European Medicines Agency, 2016. Concept Paper on Developing a Guideline on Quality Requirements of Medicinal Products Containing a Device Component for Delivery or Use of the Medicinal Product. Available at: http://www.ema.europa.eu/docs/en_GB/ document_library/Scientific_guideline/2017/02/WC500221747.pdf. European Medicines Agency. Ancillary Medicinal Substances. Available at: http://www. ema.europa.eu/ema/index.jsp?curl=pages/regulation/q_and_a/q_and_a_detail_000160. jsp&mid=WC0b01ac05800265cf. European Medicines Agency. European Experts. Available at: http://www.ema.europa.eu/ema/ index.jsp?curl=pages/about_us/landing/experts.jsp&mid=WC0b01ac058043244a. European Medicines Agency. Innovation in Medicines. Available at: http://www.ema. europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000334. jsp&mid=WC0b01ac05800ba1d9. European Medicines Agency. Multidisciplinary: Nanomedicines. Available at: http://www. ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000564. jsp&mid=WC0b01ac05806403e0. European Medicines Agency. Nanotechnology. Available at: http://www.ema.europa. eu/ema/index.jsp?curl=pages/special_topics/general/general_content_000345. jsp&mid=WC0b01ac05800baed9. European Medicines Agency. Scientific Advice and Protocol Assistance. Available from: http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_ content_000049.jsp&mid=WC0b01ac05800229b9. European Medicines Agency. Supporting SMEs. Available at: http://www.ema.europa.eu/ema/ index.jsp?curl=pages/regulation/general/general_content_000059.jsp. European Medicines Agency. Working Parties and Other Groups. Available at: http://www. ema.europa.eu/ema/index.jsp?curl=pages/about_us/general/general_content_000240. jsp&mid=WC0b01ac0580028d2b. European Parliament & Council of the European Union, 2001. Directive 2001/83/ec of the European Parliament and of the Council of 6 November, 2001 on the Community Code Relating to Medicinal Products for Human Use, pp. 67–128 Official J. Eur. Union, L 311.

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European Parliament & Council of the European Union, 2007. Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November, 2007 on Advanced Therapy Medicinal Products and Amending Directive 2001/83/EC and Regulation (EC) No 726/2004, pp. 121–137 Official J. Eur. Union, L 324. Hofer, M.P., Jakobsson, C., Zafiropoulos, N., Vamvakas, S., Vetter, T., Regnstrom, J., Hemmings, R.J., 2015. Regulatory watch: impact of scientific advice from the European medicines agency. Nat. Rev. Drug Discov. 14, 302–303. International Conference on Harmonisation (ICH), 2009. Pharmaceutical Development Q8(R2). Available at: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Quality/Q8_R1/Step4/Q8_R2_Guideline.pdf. International Pharmaceutical Regulators Forum, 2015. IPRF Nanomedicines Working Group. Available at: https://www.i-p-r-f.org/index.php/en/working-groups/nanomedicinesworking-group/. International Pharmaceutical Regulators Forum. IPRF Nanomedicines Working Group – Information Sharing and Mapping (as of January 2016). Available at: https://www.i-p-r-f. org/files/7414/5387/7778/IPRF_Nanomedicines_WG_-_info_mapping_Jan2016.pdf. Kilcoyne, A., Ambery, P., O’Connor, D., 2013. Pharmaceutical Medicine. Oxford University Press, Oxford, UK. Lebourgeois, C., 2008. Device and drug combination products: a new regulatory, reimbursement and marketing challenge. Lifescience Online. Medicines and Healthcare Products Regulatory Agency, 2015. Guidance: Borderlines between Medical Devices and Medicinal Products. Available at: https://www.gov.uk/government/ publications/borderlines-between-medical-devices-and-medicinal-products. Papaluca, M., Greco, M., Tognana, E., Ehmann, F., Saint-Raymond, A., 2015. White spots in pharmaceutical pipelines-EMA identifies potential areas of unmet medical needs. Expert Rev. Clin. Pharmacol. 8, 353–360. Papaluca, M., Ehmann, F., Pita, R., Hernan, D., 2017. Regulatory issues in nanomedicines. In: Cornier, J., Owen, A., Kwade, A., Van de Voorde, M. (Eds.), Pharmaceutical Nanotechnology: Innovation and Production. Wiley-VCH, Weinheim, Germany, pp. 497–520. Parvizi, N., Woods, K., 2014. Regulation of medicines and medical devices: contrasts and similarities. Clin. Med. (Lond.) 14, 6–12. Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR), 2010. Scientific Basis for the Definition of the Term “Nanomaterial”. Available at: http:// ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_030.pdf. Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR). Guidance on the Determination of Potential Health Effects of Nanomaterials Used in Medical Devices. 2015, Available at: https://ec.europa.eu/health/scientific_committees/emerging/ docs/scenihr_o_045.pdf.

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CHAPTER

23

Translational Exploration and Clinical Testing of Silica–Gold Nanoparticles in Development of Multifunctional Nanoplatform for Theranostics of Atherosclerosis

Alexander N. Kharlamov1,2 1De

Haar Research Foundation, Rotterdam, The Netherlands;

2De

Haar Research Foundation, New York, NY, United States

CHAPTER OUTLINE 1. Introduction�� 682 2. Silica–Gold Nanoparticles for Imaging and Therapy of Atherosclerosis�� 704 2.1 Noble Metal Nanoparticles for Needs of Cardiology�� 704 2.2 Medical Applications of Plasmonic Photothermal Therapy�� 705 2.3 Gold Nanoparticles in Oncology and Infectious Diseases�� 714 2.4 Plasmonic Photothermal Therapy of Atherosclerosis�� 715 2.5 Mechanism of Plasmonics�� 715 2.6 A Concept of Multifunctional Uniform Nanoparticle and Near-Infrared Laser for Theranostics of Atherosclerosis�� 718 3. Future of Nanomedical Applications for Imaging and Therapy of Atherosclerosis�� 719 3.1 Biodegradable Nanoparticles for Management of Atherosclerosis�� 719 3.2 Nanoparticles for Targeting�� 721 3.3 Prevention and Therapy of Thrombosis With Nanoparticles�� 722 3.4 Medical Devices in Hands of Nanotechnologies�� 723 4. Conclusion�� 724 5. Future Perspectives�� 725 Abbreviations�� 727 Acknowledgments�� 728 References�� 728


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CHAPTER 23 Translational Exploration and Clinical Testing

1. INTRODUCTION The complications of atherosclerosis remain the main cause of the mortality across the globe. The nanomedicine provides a cardiology with the tools for imaging and treatment of atherosclerosis, which are able to replace conventional approaches and revolutionize medicine chasing the ultimate goal to conquer atheroinflammatory disorder. The achievements of nanomedicine in cardiology are starting to be quantitatively characterized through the prism of the systems biology and biomedicine (Cormode et al., 2010; Kharlamov and Gabinsky, 2012; Kharlamov et al., 2015; Yeager et al., 2013; Son et al., 2015; Zhu et al., 2015; Singh et al., 2016; Chnari et al., 2006; Lewis et al., 2015, 2016; Cormode et al., 2008a,b; Duivenvoorden et al., 2014; SanchezGaytan et al., 2015; Tang et al., 2015; Pérez-Medina et al., 2016; Herbst et al., 2010; Danila et al., 2009; Paulis et al., 2012; Muro et al., 2006, 2008; Khondee et al., 2011; Zhang et al., 2008; Garnacho et al., 2012; Kelly et al., 2005; Nahrendorf et al., 2006; Burtea et al., 2009; Homem de Bittencourt et al., 2007; Kowalski et al., 2013; Dziubla et al., 2008; Nakamura et al., 2013; Kusunose et al., 2013, 2014; Southworth et al., 2009; Pan et al., 2013; Kheirolomoom et al., 2015; Michalska et al., 2012; Iverson et al., 2011; Maiseyeu et al., 2009; Sharma et al., 2010; Luehmann et al., 2014; Uchida et al., 2011; Zhao et al., 2013, 2014; Marrache and Dhar, 2013; van Tilborg et al., 2010; Nie et al., 2015; Broz et al., 2008; Peters et al., 2009; Kamaly et al., 2013; Winter et al., 2006; Joner et al., 2008; Cyrus et al., 2008; Almer et al., 2011, 2013; Park et al., 2008; Chen et al., 2013; Rogers and Basu, 2005; Lobatto et al., 2015; Kuo et al., 2014; Fredman et al., 2015; Kamaly et al., 2016; Matuszak et al., 2016; Feng et al., 2016; Dou et al., 2016; Yu et al., 2000; Marsh et al., 2007; Bi et al., 2009; Mei et al., 2010; Kempe and Kempe, 2010; Karagkiozaki et al., 2010; Myerson et al., 2011; Korin et al., 2012; Wang et al., 2012a,b; Kawata et al., 2012; Uesugi et al., 2012; Cheng et al., 2014; Duan et al., 2015; Oumzil et al., 2016; Banai et al., 1998; Bhargava et al., 2006; Chorny et al., 2006, 2010; Nakano et al., 2009; Zhu et al., 2010; Masuda et al., 2011; Acharya et al., 2012; Paul et al., 2012; Luderer et al., 2011; Yang et al., 2013; Lemos et al., 2013; Deng et al., 2013; Oh and Lee, 2013; Lee et al., 2014; Granada et al., 2016; Che et al., 2016; Bahnson et al., 2016; Mulder et al., 2008; Boisselier and Astruc, 2009; Jayagopal et al., 2010; Skajaa et al., 2010; Lobatto et al., 2011; Lewis et al., 2011; Yu et al., 2011; McDowell et al., 2011; Godin and Ferrari, 2012; Rhee and Wu, 2013; Kharlamov, 2013; Mieszawska et al., 2013; Wildgruber et al., 2013; Tan et al., 2013; Agoston-Coldea, 2013; Mulder et al., 2014; Yin et al., 2014; Schiener et al., 2014; Weissig and Guzman-Villanueva, 2015; Sengel, 2015; Korin et al., 2015; Stendahl and Sinusas, 2015a,b; Allen et al., 2016; Bobo et al., 2016; Chung, 2016; Calcagno et al., 2016; Bhullar et al., 2016; Karimi et al., 2016; Carnovale et al., 2016; Bucharskaya et al., 2016; Dweck et al., 2016; Kratz et al., 2016; Cicha et al., 2016; Karagkiozaki et al., 2016; Zhang et al., 2016a,b; Gadde and Rayner, 2016; Bagheri et al., 2016; Bietenbeck et al., 2016; Park et al., 2016; Feiner et al., 2016; Granada et al., 2011; Felfoul et al., 2016; Browne and Feringa, 2006; Mostafalu et al., 2016; Manjunath and Kishore, 2014; Saadeh and

1. Introduction

Vyas, 2014; Wehner et al., 2016; Smilowitz, 2012; Shademan et al., 2016; Xiao et al., 2014; Liu et al., 2016; Oldenburg et al., 1999; Hirsch et al., 2003; Rockson et al., 2000; Tucker – Schwartz et al., 2012; Kumar et al., 2016; Chinetti-Gbaguidi et al., 2014; Franzén et al., 2016; Hu et al., 2016; Raibert, 2010; Buss et al., 2016; Lemos et al., 2013; Koene, 2013; Wang et al., 2004; Park et al., 2014; Chen et al., 2012; Au et al., 2011; Douma et al., 2011; Nahrendorf et al., 2009; Pissuwan et al., 2006; Owen et al., 2011; Cromer Berman and Walczak, 2011; Faulkner and Long, 2011; Qu et al., 2011a,b; Wang and Emelianov, 2011; Bayer et al., 2011; Lapotko, 2009a,b; Zharov, 2005; Godin et al., 2010; Khlebtsov et al., 2008; Li et al., 2013; Appelman et al., 1996; Chen et al., 2011; Tietze et al., 2011; van Bochove et al., 2011; Choi et al., 2011; Ji et al., 2007; ; Vader et al., 2012; Citaddino et al., 2012; Chan et al., 2011; Yia-Herttuala et al., 2011; Al-Jamal and Kostarelos, 2011; Kunjachan et al., 2012; Nissen et al., 2003; Chang et al., 2012; Nam et al., 2012; Lapotko, 2011). Furthermore, the knowledge about optimal strategies for the research and development in this field, which might be claimed by the real-world cardiology becomes of high value. According to the systematic review (Kharlamov, 2016) of the nanomedical tools for imaging, therapy, and theranostics of atherosclerosis, just few hundreds of studies and bedside trials were explored in PubMed between 1980 and 2016 with a clinical relevance to the main three targets: (1) lumen stenosis, (2) (athero)thrombosis, and (3) atherogenesis (metabolism of cholesterol and immune response in both stable and progressive plaques). The number of papers testing any approaches for the management of atherosclerosis achieved 109,801 during 36 overviewed years. The keyword “nanoparticle (NP)” was revealed in 133,016 articles since 1980, which is a 21.14% higher than in case of “atherosclerosis.” In fact, the age of nanomedicine started in 2000 with a further soaring pattern of publications up to 14,010 manuscripts totally by Sep., 2016. Regretfully, the progress of nanomedicine for needs of cardiology and particularly atherosclerosis was not that bright and characterized by only 512 papers since 2005, which weighs just about 3.65% of nanomedicine and a 0.38% of the entire nanoindustry. Among 135 specially assigned for analysis papers with 120 studies introducing particular technologies (Cormode et al., 2010; Kharlamov and Gabinsky, 2012; Kharlamov et al., 2015; Yeager et al., 2013; Son et al., 2015; Zhu et al., 2015; Singh et al., 2016; Chnari et al., 2006; Lewis et al., 2015, 2016; Cormode et al., 2008a; Duivenvoorden et al., 2014; SanchezGaytan et al., 2015; Tang et al., 2015; Pérez-Medina et al., 2016; Herbst et al., 2010; Danila et al., 2009; Paulis et al., 2012; Muro et al., 2006, 2008; Khondee et al., 2011; Zhang et al., 2008; Garnacho et al., 2012; Kelly et al., 2005; Nahrendorf et al., 2006; Burtea et al., 2009; Homem de Bittencourt et al., 2007; Kowalski et al., 2013; Dziubla et al., 2008; Nakamura et al., 2013; Kusunose et al., 2013, 2014; Southworth et al., 2009; Pan et al., 2013; Kheirolomoom et al., 2015; Michalska et al., 2012; Iverson et al., 2011; Maiseyeu et al., 2009; Sharma et al., 2010; Luehmann et al., 2014; Uchida et al., 2011; Zhao et al., 2013, 2014; Marrache and Dhar, 2013; van Tilborg et al., 2010; Nie et al., 2015; Broz et al., 2008; Peters et al., 2009; Kamaly et al., 2013; Winter et al., 2006; Joner et al., 2008; Cyrus et al., 2008; Almer et al., 2011, 2013; Park et al., 2008; Chen et al., 2013; Rogers and Basu, 2005; Lobatto et al.,

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684


2015; Kuo et al., 2014; Fredman et al., 2015; Kamaly et al., 2016; Matuszak et al., 2016; Feng et al., 2016; Dou et al., 2016; Yu et al., 2000; Marsh et al., 2007; Bi et al., 2009; Mei et al., 2010; Kempe and Kempe, 2010; Karagkiozaki et al., 2010; Myerson et al., 2011; Korin et al., 2012; Wang et al., 2012a; Kawata et al., 2012; Uesugi et al., 2012; Cheng et al., 2014; Duan et al., 2015; Oumzil et al., 2016; Banai et al., 1998; Bhargava et al., 2006; Chorny et al., 2006, 2010; Nakano et al., 2009; Zhu et al., 2010; Masuda et al., 2011; Acharya et al., 2012; Paul et al., 2012; Luderer et al., 2011; Yang et al., 2013; Lemos et al., 2013; Deng et al., 2013; Oh and Lee, 2013; Lee et al., 2014; Granada et al., 2016; Che et al., 2016; Bahnson et al., 2016; Mulder et al., 2008; Boisselier and Astruc, 2009; Jayagopal et al., 2010; Skajaa et al., 2010; Lobatto et al., 2011; Lewis et al., 2011; Yu et al., 2011; McDowell et al., 2011; Godin and Ferrari, 2012; Rhee and Wu, 2013; Kharlamov, 2013; Mieszawska et al., 2013; Wildgruber et al., 2013; Tan et al., 2013; Agoston-Coldea, 2013; Mulder et al., 2014; Yin et al., 2014; Schiener et al., 2014; Weissig and Guzman-Villanueva, 2015; Sengel, 2015; Korin et al., 2015; Stendahl and Sinusas, 2015a,b; Allen et al., 2016; Bobo et al., 2016; Chung, 2016; Calcagno et al., 2016; Bhullar et al., 2016; Karimi et al., 2016; Carnovale et al., 2016; Bucharskaya et al., 2016; Dweck et al., 2016; Kratz et al., 2016; Cicha et al., 2016; Karagkiozaki et al., 2016; Zhang et al., 2016b; Gadde and Rayner, 2016; Bagheri et al., 2016; Bietenbeck et al., 2016; Hu et al., 2016) (see Fig. 23.1 and Table 23.1), there were identified 79 studies (65.83%) of nanotechnologies with a focus on atherogenesis (cholesterol/lipid metabolism and immune response) (Cormode et al., 2010; Kharlamov and Gabinsky, 2012; Kharlamov et al., 2015; Yeager et al., 2013; Son et al., 2015; Zhu et al., 2015; Singh et al., 2016; Chnari et al., 2006; Lewis et al., 2015, 2016; Cormode et al., 2008a; Duivenvoorden et al., 2014; Sanchez-Gaytan et al., 2015; Tang et al., 2015; Pérez-Medina et al., 2016; Herbst et al., 2010; Danila et al., 2009; Paulis et al., 2012; Muro et al., 2006, 2008; Khondee et al., 2011; Zhang et al., 2008; Garnacho et al., 2012; Kelly et al., 2005; Nahrendorf et al., 2006; Burtea et al., 2009; Homem de Bittencourt et al., 2007; Kowalski et al., 2013; Dziubla et al., 2008; Nakamura et al., 2013; Kusunose et al., 2013, 2014; Southworth et al., 2009; Pan et al., 2013; Kheirolomoom et al., 2015; Michalska et al., 2012; Iverson et al., 2011; Maiseyeu et al., 2009; Sharma et al., 2010; Luehmann et al., 2014; Uchida et al., 2011; Zhao et al., 2013, 2014; Marrache and Dhar, 2013; van Tilborg et al., 2010; van Tilborg et al., 2010; Nie et al., 2015; Broz et al., 2008; Peters et al., 2009; Kamaly et al., 2013; Winter et al., 2006; Joner et al., 2008; Cyrus et al., 2008; Almer et al., 2011, 2013; Park et al., 2008; Chen et al., 2013; Rogers and Basu, 2005; Lobatto et al., 2015; Kuo et al., 2014; Fredman et al., 2015; Kamaly et al., 2016; Matuszak et al., 2016; Feng et al., 2016; Dou et al., 2016; Mulder et al., 2008; Boisselier and Astruc, 2009; Jayagopal et al., 2010; Skajaa et al., 2010; Lobatto et al., 2011; Lewis et al., 2011; Yu et al., 2011; McDowell et al., 2011; Godin and Ferrari, 2012; Rhee and Wu, 2013; Kharlamov, 2013; Mieszawska et al., 2013; Wildgruber et al., 2013; Tan et al., 2013; Agoston-Coldea, 2013; Mulder et al., 2014; Yin et al., 2014; Schiener et al., 2014; Weissig and Guzman-Villanueva, 2015; Sengel, 2015; Korin et al., 2015; Stendahl and Sinusas, 2015a,b; Allen et al., 2016; Bobo et al., 2016; Chung, 2016; Calcagno et al., 2016; Bhullar et al., 2016;

1. Introduction

FIGURE 23.1 The publishing activity in the field of nanomedicine for the management of atherosclerosis. Panel performs a schematic representation of the synergy between different research efforts chasing the main clinical targets (modular networking): (1) atherogenesis, (2) lumen stenosis, (3) thrombosis. Numbers reflect quantity of the published manuscripts for imaging, therapy, and imaging-and-therapy (I&T)/theranostic objectives. In case of both lumen stenosis and thrombosis nanotechnologies mostly focused on therapy with total ignorance of the imaging merely because of the high availability of the wide-range imaging techniques for coronary arteries such as CT, MRI, CT/PET, angiography/QCA, and intravascular imaging (IVUS, OCT, and others). The dark purple (dark gray in print version) lines demonstrate integrative approaches between the listed strategies with original studies, but the blue (light gray in print version) lines—for review articles as a result of the analytic activity. The only technology that met all three clinical targets identified as a BVS on chip (Son et al., 2015). CT, computed tomography; ICAM, intercellular adhesion molecule; IVUS, intravascular ultrasound; MRI, magnetic resonance imaging; PET, positron emission tomography; QCA, quantitative coronary angiography; VCAM, vascular cell adhesion molecule.

Karimi et al., 2016; Carnovale et al., 2016; Bucharskaya et al., 2016; Dweck et al., 2016; Kratz et al., 2016; Cicha et al., 2016; Karagkiozaki et al., 2016; Zhang et al., 2016b; Gadde and Rayner, 2016; Cormode et al., 2010), 23 studies (19.17%) targeting lumen stenosis (mostly, medical devices) (Banai et al., 1998; Bhargava et al., 2006; Chorny et al., 2006, 2010; Nakano et al., 2009; Zhu et al., 2010; Masuda et al., 2011; Acharya et al., 2012; Paul et al., 2012; Luderer et al., 2011; Yang et al., 2013;

685

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Table 23.1 Translational Achievements of Nanotechnologies for Imaging, Therapy, and Multifunctional Theranostics of Atherosclerosis

References

Study (No. of Tested Technologies if Applicable)

Application

Tested Technology

Translational Studies With Noble Metal Nanoparticles for Needs of Cardiology Cormode et al. (2010)

Bench

Imaging

Multicolor CT with targeted gold nanoparticles

Kharlamov and Gabinsky (2012) (PLASMONICS)

Bench

Therapy

Plasmonic photothermal therapy with noble metal (silica–gold vs iron-bearing vs stenting control) nanoparticles

Kharlamov et al. (2015) (NANOM-FIM trial, NCT01270139; first released in 2013)

Bedside

Therapy

Plasmonic photothermal therapy with noble metal (silica–gold vs iron-bearing vs stenting control) nanoparticles

Yeager et al. (2013)

Bench

Imaging, multifunctional theranostics

IVUS/IVPA (intravascular ultrasound/photoacoustics) imaging as a tool for localized temperature monitoring during laser heating

Son et al. (2015)

Bench

Multifunctional theranostics

Bioresorbable electronic scaffold integrated with therapeutic nanoparticles

Zhu et al. (2015)

Bench

Therapy

Upconversion nanoparticlemediated photodynamic therapy (PDT) induces THP-1 macrophage apoptosis

Singh et al. (2016)

Bench

Therapy

Photothermal therapy of thrombus with gold nanorods and near-infrared laser

1. Introduction

Brief Methodology

Brief Results

Au-HDL, an iodine-based contrast agent with further macrophage targeting, and calcium phosphate were imaged and evaluated in a variety of phantoms in apolipoprotein E–knockout (apo E-KO) mice. 101 Yucatan miniature swine with a number of the NP delivery techniques: (1) intracoronary-infused circulating stem progenitor cells (SPC), (2) intracoronary-infused, ultrasoundmediated, albumin-coated, gas-filled microbubbles, (3) CD73+105+ SPC in the composition of a bioengineered on-artery patch (cardiac surgery), (4) CD73 + CD105 + SPC engrafted by manual subadventitial injection (cardiac surgery). Patients were assigned to receive either (1) nanointervention with delivery of silica–gold NP in a bioengineered on-artery patch (n = 60) or (2) nanointervention with delivery of silica– gold iron-bearing NP with targeted microbubbles and stem cells using a magnetic navigation system (n = 60) versus (3) stent implantation (n = 60).

Spectral CT may yield valuable information about atherosclerotic plaque composition. Reduction of the total atheroma volume at 6 months in the groups achieved −22.92% (P

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