Biomedical Reviews

BMR

Biomedical Reviews

Volume 26, 2015

An International Journal of Cell Biology of Disease

Official Journal of the Bulgarian Society for Cell Biology and the Medical University, Varna, Bulgaria

Biomedical Reviews Volume 26, 2015

© Bul­garian Society for Cell Biology ISSN 1314-1929 (online)

Editor-in-Chief

Editors

Editorial Board

George N. Chaldakov Laboratory of Cell Biology Department of Anatomy and Histology Medical University of Varna (MUV) BG-9002 Varna Bulgaria Tel.: 359 52 754 394 [email protected]

Luigi Aloe Institute of Neurobiology and Molecular Medicine National Research Council Eurpean Brain Research Institute Rome, Italy [email protected]

Jerzy Beltowsky (Lublin, Poland)

John Heuser Electron Microscopy Center WPI Institute for Cell and Material Sciences Kyoto University Kyoto, Japan [email protected]

Marco Fiore (Rome, Italy)

Associate Editors Danko D. Georgiev Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA Peter I. Ghenev Department of General and Clinical Pathology MUV, BG-9002 Varna Bulgaria Wale A.R. Sulaiman Department of Neurosurgery/ Spine Center Ochsner Clinic Foundation New Orleans, LA 70121, USA Dimiter T. Tomov Department of Health Economics and Management Faculty of Public Health MUV, BG-9002 Varna Bulgaria

Krassimir Ivanov Rector Medical University, Varna, Bulgaria [email protected] Ronald Mathison Department of Physiology and Biophysics The Faculty of Medicine The University of Calgary Calgary, Alberta Canada [email protected] Hiroshi Yamamoto Department of Biochemistry and Molecular Vascular Biology Graduate School of Medical Science Kanazawa University Kanazawa, Japan [email protected]

Gheorghe Benga (Cluj-Napoca, Romania) Trifon Chervenkov (Varna, bulgaria) Michail Davidoff (Hamburg, Germany) Krikor Dikranian (St. Louis, MO, USA) Takashi Fujiwara (Shigenobu, Japan) Rosa Gomariz (Madrid, Spain) Rama Gopalan (Chennai, India) Alexander Hinev (Varna, Bulgaria) Bhanu Jena (Detroit, MI, USA) Anna Kadar (Budapest, Hungary) Peirong Lu (Suzhou, China) Stephen Manning (Lyndon, West Bromwich, UK) Jiradej Manosroi (Chiang Mai, Thailand) Arto Palkama (Helsinki, Finland) C. Anthony Poole (Auckland, New Zealand) Gorana Rančič (Niš, Serbia) Christos Stournaras (Heraklion, Greece) Nikolai Temnyalov (Varna, Bulgaria) Neşe Tunçel (Eskişehir, Turkey) Kamen Valchanov (Cambridge, UK) Andreas Wree (Rostock, Germany) Stanislav Yanev (Sofia, Bulgaria)

Anton B. Tonchev Department of Anatomy, Histology and Embryology MUV, BG-9002 Varna Bulgaria

Medical University Varna

“Prof. Dr Paraskev Stoyanov”

Medical University, Varna, Bulgaria

Copyright © 2015 by the Bulgarian Society for Cell Biology www.bgscb.org/BMR.htm http:/press.mu-varna.bg/ojs/ Copyright © 2015 by the Medical University, Varna, Bulgaria All rights reserved. The Publisher gives consent for individual copies of the articles to be reprinted for personal or internal use only. This consent does not extend to other copyrights, such as copyright for general distribution, creating new collective works, or resale. Inquiries concerning reproduction outside of these terms should be directed to the Editors. Computer design Svetlana Koeva Abstracted in CAB Abstracts/Global Health databases, Chemical Abstracts, Excerpta Medica database (EMBASE), Compendex, EMBiology, Elsevier BIOBASE, Index Copernicus International, Index Scholar and Scopus.

BIOMEDICAL REVIEWS An International Journal of Cell Biology of Disease

Volume 26, 2015

Editor-in-Chief George N. Chaldakov, MD, PhD, FIACS

Scope and Purpose Biomedical Reviews (ISSN 1314-1929 ; online) is an official journal of the Bulgarian Society for Cell Biology and the Medical University, Varna, Bulgaria. The Journal is published annually, and includes state-of-the-science (SOS) Reviews and Dance Round (a form of short, position papers) focused on disease-oriented molecular cell biology, presented in concise form.

Editorial Policy Contributors to Reviews as well as Dance Rounds are, in general, invited by the Editors and the Editorial Board, but idea proposals for Reviews and Dance Rounds are welcome. Prospective authors should send a brief summary, citing key references, including their own, to the Editors or a member of the Editorial Board. Submission of full-length articles without prior consultation is not prefered. Manuscripts are peer-reviewed by the Editors, Editorial Board members, and/ or external experts before final decisions regarding publication are made. All material in Biomedical Reviews represents the opinions of the authors and does not reflect opinions of the Bulgarian Society for Cell Biology, the Medical University, Varna, Bulgaria, the Editors, the Editorial Board, or the institutions with which the authors are affiliated. Publication of Biomedical Reviews is truly a collaborative process. We appreciate the brain-and-heart partnership having with our authors and are committed to further maintaining the excellence of the Journal.

BMR Biomedical Reviews

Contents



Editor's foreword

III

Sousei (live together)



Spotlight

IV

Luigi Aloe: Son of Calabria, aristocrat of world neuroscience



Announcement

VI

The First International Rita Levi-Montalcini’s Scientific Meeting Nerve Growth Factor: Neuroscience and Therapy



Reviews

1

The amazing brain Krikor Dikranian (USA)

13

Resveratrol: more than a phytochemical Parichehr Hassanzadeh, Fatemeh Atyabi, and Rassoul Dinarvand (Iran)

23

Gold nanoparticles: A promising therapeutic approach Harsharan Pal Singh, Ashmeet Kaur, Ishpreet Kaur, Harpal Singh Buttar, and Sukhwinder Kaur Bhullar (India, Canada, Turkey)

37

Nanofiber devices for the targeted-delivery of therapeutically active plant and herbal ingredients Sukhwinder K. Bhullar and Harpal S. Buttar (Turkey, Canada)



Dance Round

43

Serum cholesterol and triglyserides in Parkinson’s disease and essential tremor Borislav Ivanov, Ara Kaprelyan, Ivan Dimitrov, Margarita Grudkova, Natalia Usheva, Vesselina Nestorova, and Nadezhda Deleva (Bulgaria)

47

Selected abstracts of 4th International Symposium on Adipobiology and Adipopharmacology (ISAA)

73 I n t e r v i e w 83

Instructions to authors

Front cover: Resting-state functional connectivity from the Human Connectome Project (HCP) data. Columns 1, 2 and 3: functional connectivity from a “seed” in parietal cortex (black disk), based on a group average of 468 subjects; 52 left-hemisphere contiguous parcels from the resting-state networks (RSN); parcellated connectivity map for a default-mode network parcel containing the selected seed, based on 447 HCP subjects. Column 4: groupaverage parcellated connectome showing relative connection strength between regions. Image courtesy of the HCP consortium - http://humanconnectome.org. From Dikranian’s review, pp 1-12, Figure 6.

Biomed Rev 26, 2015

II

BMR B i o m e d i c a l R e vi e w s

Editor's foreword

Dear Colleagues, In October 2015, Professor Maria Staykova at The John Curtin College of Medicine, Biology and Environment, Australian National University, Canberra, Australia sent to me a verse entitled Brain created by child from fourth class: With the brain man thinks that thinks. Moreover the brain is used for headache. Located in the head, behind the nose. When someone sneezes, it drips. The brain is very sensitive organ. For this reason people use it rarely.

*Sousei expressed by two kanji (Chinese characters) written by Hiro Yamamoto

In December 2015, Professor Hiroshi Yamamoto at Department of Biochemistry and Vascular Biology, Kanazawa University, Kanazawa, Japan wrote to me: Dear George, I wish you health, happiness and something fantastic in 2016. Enclosed is my hope for this year - it‘s called „Sousei“, meaning „live together“.* Hiro I immediately replied: Hiro, after the wishes for health, your „Sousei“ - to live together in mutual understanding – is the most important message to all people in the world. Because: Brain and heart are very sensitive organs. For this reason people should use them more often - together. George, BHF-ly yours George N. Chaldakov

Biomed Rev 26, 2015

III

BMR Biomedical Reviews

Spotlight

LUIGI ALOE: SON OF CALABRIA, ARISTOCRAT OF WORLD NEUROSCIENCE The ancient Greek word ἀρετή (arete), in its basic sense, means „excellence of any kind“, and, respectively, aristocrat (aristos - best, kratos - power) – power of the best, of the excellence, of the moral virtue, of the nobility, of the knowledge and study. The man of arete is a person of the highest effectiveness, using all own creative faculties to achieve real results. Dr Luigi Aloe, a perfect example of arete, was born in 1943 in Amantea - 600 km south of Rome. We leave in the morning with his Lancia and passed through Campania and Calabria to get in Amantea, moving along orange and olive trees and vineyards of Southern Italy - Enotria (from Greek, the land of vine) as it was called in the time of Magna Graecia. - Southern Italy is not just wine, it is a land of philosophers - said Luigi. Here in Elea were Xenophanes, Parmenides and Zeno and his paradoxes. And as a student recited a verse of Parmenides‘ “fast flying chariot, and girls pointing my way”. Pythagoras and his students were also Southerners - lived in Croton and drank Ciro during the symposium. In Calabria was born philosopher Tommaso Campanella and Nobel Prize winner in medicine Renato Dulbecco.- Hopos esti (that is) - all roads lead to Enotria first, and then to Rome - summed up his story Luigi. Amantea is a relatively small city, located between the Tyrrhenian Sea and the mountain Celia, in the province of Cosenza, Calabria region. Since they were born, sea and mountain are entwined in plenty of sun, wine and fish.

Biomed Rev 26, 2015

During the winter population of about 15,000 people, while in the summer - more than a million visitors. They ate tons of fish and drank thousands of liters of wine. Here you can feel better the sense of Italian bel paese (beautiful country) and dolce vita. Luigi reminds frequently of his grandmother Rosaria who very diligently cared for him and his brothers Rocco, Franco, Alfredo and sister Rosaria. The sun was rising when we left Amantea and move 600 kilometers north to continue our research in Rome. When returned to Varna, I wrote to Luigi: Absent already in Amantea, Signora Rosaria is drinking wine with God in Paradise.

IV

Spotlight And delightedly smiles seeing that Luigi Aloe is a force and a soul of nature, who strives to always be arete in studying the multiple biology of NGF and related molecules in health and disease. A selected list of his research as Visiting Scientist includes: Department of Biology, Washington University, St. Louis, Missouri, USA; Chicago Faculty of Medicine, Chicago University, Chicago, Illinois, USA; Department of Anatomy, Harvard University, Boston, Massachusetts, USA; Deparment of Biobehavior Science, Oxford University, Oxford, England; Department of Anatomy, Cambridge University, Cambridge, England; Department of Pharmacology, McGill University, Montréal, Canada; Nency Institute of Warsavia, Warsaw, Poland; Laboratory of Cell Biology, Medical University, Varna,

Biomed Rev 26, 2015

Bulgaria; Faculty of Medicine, University Arcavacata, Cosenza, Italy. Starting his scientific carrier in 1967 with Rita LeviMontalcini in Washington University in St. Louis, Missouri, Luigi Aloe in 1973 returned to bel paese at Institute of Neurobiology, Consiglio Nazionale delle Ricerche (CNR) in Rome, from 1995 being Research Director of the Institute. Sicut matribus sit Deus nobis (As to our mothers may God be to us)*, Luigi. George N. Chaldakov A paraphrase of the Latin proverb Sicut patribus sit Deus nobis (As to our fathers may God be to us). *

V

THE FIRST INTERNATIONAL RITA LEVI-MONTALCINI’S SCIENTIFIC MEETING THE FIRST INTERNATIONAL RITA LEVI-MONTALCINI’S SCIENTIFIC MEETING NERVE GROWTH FACTOR: NEUROSCIENCE AND THERAPY NERVE GROWTH FACTOR: NEUROSCIENCE AND THERAPY April 22-23, 2016, Bologna, Italy April 22-23, 2016, Bologna, Italy WWW.FIRST-NGF-RL-MONTALCINI-CONFERENCE.ORG WWW.FIRST-NGF-RL-MONTALCINI-CONFERENCE.ORG KEYNOTE AND INVITED SPEAKERS KEYNOTE AND INVITED SPEAKERS L. Aloe, ICBN, CNR Roma; and L. Calzà, L. Aloe, ICBN, CNR Roma; University Bologna, Italy and L. Calzà, L. Aloe, ICBN, CNR Roma; and L. Calzà, University Bologna, Italy Opening ceremony and welcome. University Bologna, Italy and Opening ceremony welcome. P. Levi-Montalcini, Torino, Italy Opening ceremony and welcome P. Levi-Montalcini, Torino, the Italy Rita Levi-Montalcini outside lab. P. Levi-Montalcini, Torino, Italy Rita Levi-Montalcini outside the lab. Rita Levi-Montalcini outside UK. the lab P. Anand, London, P. Anand, London, UK. P. Anand, London, UK NGF and anti-NGF mechanisms and treatments and anti-NGF and treatments NGF NGF andneuropathy anti-NGF mechanisms andpain. treatments for for and mechanisms chronic neuropathy andBologna, chronic forCalzà, neuropathy andpain chronic L. Italy. pain. L. Calzà, Bologna, Italy.in neurodegenerative L. Calzà, Bologna, Italy Neurovascular coupling Neurovascular coupling in neurodegenerative Neurovascular in neurodegenerative diseases diseases coupling diseases E. Cattaneo, E. Cattaneo, Milano,Milano, Italy Italy. Translating the natural E. Cattaneo, Milano, Italy. Translating the natural history of human striatal Translating the natural history ofdevelopment human striatalinto history ofinto human striatal development into development pluripotent stem cells differentiation pluripotent stem cells differentiation. pluripotent stem cells differentiation. Chaldakov, Varna, BG. G. N.G. Chaldakov, Varna, Bulgaria G. Chaldakov, brain diabetes to BG. adipose Alzheimer. FromFrom brain diabetes toVarna, adipose Alzheimer From brain diabetes to adipose Alzheimer. C. Emanueli, London, UK. C. Emanueli, London, UK C. Emanueli, London, UK. for cardiovascular protection and NGF NGF for cardiovascular protection and regeneration NGF for cardiovascular protection and regeneration. M. Eriksdotter, Stockholm, Sweden regeneration. M. Eriksdotter, Stockholm, Cell therapy with NGF in Alzheimer´s Sweden. disease M. Eriksdotter, Sweden.disease. Cell therapy withStockholm, NGF in Alzheimer´s B. Falsini, Roma, Italy CellFalsini, therapyRoma, with NGF in Alzheimer´s disease. B. Italy. Role B. of Nerve Growth Factor in Retinal Degenerations: Falsini, Roma, Italy. Role of Nerve Growth Factor in Retinal Neuro-enhancement and Neuroprotection Role of Nerve Growth Factor in Retinal Degenerations: Neuro-enhancement and M. Hiriart, Mexico City, Mexico Degenerations: Neuro-enhancement and Neuroprotection. NGF Neuroprotection. and pancreatic beta-cells T. Hokfelt, Stockholm, Sweden. T. Hokfelt, Stockholm, Swedenunderlying T. Hokfelt, Stockholm, Sweden. neuropathic Exploring mechanisms Exploring mechanisms underlying neuropathic pain Exploring mechanisms underlying neuropathic pain pain A. Iannitelli, Roma,Roma, Italy Italy. A. Iannitelli, A. Iannitelli, Roma, Italy.growth Searching a role a ofrole nerve factor infactor psychiatric Searching ofgrowth nerve in disorders Searching adisorders. role of nerve growth factor in psychiatric disorders. Jian psychiatric Ge, Beijing, China Jian Ge, Beijing, China. Jian Ge, Beijing, The application of nerve growth factor infactor optic in nerve injury The application of China. nerve growth optic The application of nerve growth factor in optic nerve injury. Jizong Zhao, Beijing, China nerve Jizonginjury. Zhao, Beijing, China. The function and mechanisms of related neural circuits for Jizong Zhao,and Beijing, China. of related neural disorders of emotion and memory The function mechanisms The function and mechanisms related neural circuits for disorders of emotionofand memory. circuits for disorders of emotion and memory.

A. Lambiase, Roma, Italy. A. Lambiase,human Roma,Nerve Italy. Growth Factor Recombinant A. Lambiase, Roma, Italy Recombinant human Nervecorneal Growthulcer: Factor treatment for neurotrophic from Recombinant human Nerve Growth ulcer: Factor from treatment for treatment for neurotrophic corneal basic science to clinical trial. neurotrophic corneal ulcer: from basic science to clinical trial basic science toJerusalem, clinical trial.Israel. P. Lazarovici, P. Lazarovici, Jerusalem, Israel P. Lazarovici, Jerusalem, Israel. Cross talk between neurotrophin receptors Crosstalk talk between neurotrophin receptors p75NTR and α9β1 Cross between neurotrophin receptors p75NTR and α9β1 integrin in glioma angiogenesis integrin in glioma and invasiveness p75NTR and α9β1angiogenesis integrin in glioma angiogenesis and invasiveness. and invasiveness. Matsuda, Tokyo, Japan H.H.Matsuda, Tokyo, Japan. Atopic dermatitis andJapan. nerve growth H. Matsuda, Tokyo, Atopic dermatitis and nerve growthfactor factor. Atopic dermatitis and Italy nerve J.Meldolesi, Meldolesi, Milano, J. Milano, Italy. growth factor. J. Meldolesi, Milano, Italy.their NGF receptors: news activation and role NGF receptors: newsabout about theirexpression, expression, NGF receptors: news about their expression, in PC12 cells activation and role in PC12 cells. activation roleItaly in PC12 cells. A. Roma, Italy. A.Micera, Micera,and Roma, A. Micera, Roma, Italy.potential NGF retinal disorders: insights from NGF ininretinal disorders: potentialtherapeutic therapeutic NGF in retinal disorders: potential therapeutic ocular biological fluids insights from ocular biological fluids. insights fromSan ocular biological W.W.Mobley, Diego, USA. fluids. Mobley, San Diego, USA W. Mobley,Alzheimer San Diego, USA.ininDown Preventing Disease Down Preventing Alzheimer Disease Syndrome: The NGF Preventing Alzheimer Disease in Down Connection Syndrome: The NGF Connection. Syndrome: The NGFDavis, Connection. S.S.K.K.Raychaudhuri, Davis, Raychaudhuri, CA,CA, USAUSA. S. K. Raychaudhuri, CA,immunity USA. Regulatory role oninnate innate immunity. Regulatory role of of NGF NGFDavis, on Regulatory role of NGF on innate immunity. S.S.P.P. Raychaudhuri, Davis, Raychaudhuri, Davis, CA.CA. USAUSA. S. P. Raychaudhuri, Davis, CA. USA. NGF/TrkA system in autoimmune diseases: From NGF/TrkA system in autoimmune diseases: From bench to beside NGF/TrkA system in autoimmune diseases: From bench to beside D. Ribatti, Bari, Italy bench to beside Italy. D.The Ribatti, role ofBari, nerve growth factor in angiogenesis D. Ribatti, The role of Bari, nerveItaly. growth factor in angiogenesis. D. Santucci, Roma, Italy factor in angiogenesis. The role of nerve growth D. Santucci, Roma, Italy. Altered expression ofItaly. NGF and BDNF human saliva and plasma D. Santucci, Roma, Altered expression of NGF and BDNF human levels in humans exposed to space or extreme environment Alteredand expression of NGF BDNF human saliva plasma levels in and humans exposed to S.D. Skaper, Padova, Italy saliva and plasma levels in humans exposed to space or extreme environment. Nerveorgrowth factor: feeling the pain space extreme environment. S.D. Skaper, Padova, Italy. M.G. Spillantini, Cambridge, UK pain. S.D. Skaper, Italy.the Nerve growth Padova, factor: feeling Mechanisms toxicity in neurodegenerative diseases with Nerve growth of factor: feeling theUK. pain. M.G. Spillantini, Cambridge, protein aggregation M.G. Spillantini, Cambridge, UK. Mechanisms of toxicity in neurodegenerative Mechanisms ofprotein toxicity in neurodegenerative P. Tirassa,with Roma, Italy aggregation. diseases communication and NGF eye drops diseases with protein aggregation. P.Eye-brain Tirassa, Roma, Italy P. Tirassa, Roma, Italy M.H. Tuszynski, San Diego,and CA, NGF USA eye drops Eye-brain communication Eye-brain communication andfor NGF eye drops NGFTuszynski, and BDNF Gene Alzheimer’s Disease M.H. SanTherapy Diego, CA, USA. M.H. San Diego, CA, NGF Tuszynski, and BDNF Gene Therapy for USA. Alzheimer’s NGF and BDNF Gene Therapy for Alzheimer’s Disease. Disease. Poster Presentation and Discussion. Poster Presentation and Discussion. Announcing the winner of the “Rita Levi-Montalcini Award”.

Announcing the winnermeeting of the “Rita Award”. Closing andLevi-Montalcini farewell

Closing meeting and farewell

Organizers: Luigi Aloe (CNR, Roma, [email protected]) and Laura Calzà (University of Bologna, Organizers: Luigi Aloe (CNR, Roma,[email protected]) [email protected]) and Laura Calzà (University of Bologna, [email protected]) Scientific Secretariat: L. Giardino (IRET-ONLUS, [email protected]), M.L. Scientific Secretariat: L.Rocco Giardino (IRET-ONLUS, [email protected]), M.L. (ICBN, CNR, [email protected]) Rocco (ICBN, CNR, [email protected])

Biomed Rev 26, 2015

Biomedical Reviews 2015; 26: 1-12

© Bul­garian Society for Cell Biology ISSN 1314-1929 (online)

THE AMAZING BRAIN

Krikor Dikranian Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, MO, USA

It is in the human nature to be curious about how we feel pain, see the world, hear bird’s songs, remember, forget, reason. We want to understand the nature of love, anger, satisfaction, desire and madness. This is a short story about the evolution of the science on the human brain and about major brain discoveries. It gives a concise historic perspective of the understanding of the nervous system - from ancient Egypt to the birth of Renaissance, with the works of Vesalius and his esteemed contemporaries. The contributions of 17th century neuroanatomists such as Tomas Willis followed by the pre-modern neuroscience researchers Camillo Golgi and especially Santiago Ramon y Cajal are highlighted. The contribution of transgenic mouse models and the application of modern noninvasive imaging methods such as positron emission tomography (PET) and magnetic resonance imaging (MRI) for ground braking functional studies on the human brain are briefly reviewed. Important 21st century projects such as the Human and Mouse Connectome projects and the White House Brain Initiative are also presented. Biomed Rev 2015; 26: 1-12. Key words: psychikon pneuma, census communis, NGF, PET, fMRI, connectome

THE BRAIN is wider than the sky, … The brain is deeper than the sea, …The brain is just the weight of God… Emily Dickinson. Part One: Life. CXXVI In: Complete Poems, 1924 PREHISTORIC TIMES, ANCIENT EGYPT AND THE GRECO-ROMAN PERIOD Evidence suggests that our prehistoric ancestors appreciated that the brain was vital to life. About 7000 years ago people started boring holes in living skulls. Surprisingly many times it was done with the aim not to kill but cure (1). Ancient

Egyptian medicine was a compound of rational, magical and religious elements (2) Soldiers, embalmers and even cooks knew about the brain as a tissue. Open skull fractures allowed Egyptian surgeons to observe the brain, which they called the “the marrow of the skull” or “ais“ (3). They had a second word for the brain – “amen”, they described hemispheric convolutions and saw the living brain pulsating. However there

Received 10 November 2015, revised 22 November 2015, accepted 23 November 2015. Correspondence to Krikor Dikranian, MD, PhD, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Ave, Saint Louis, MO, 63119, USA. Tel: 314 362 3548, Fax: 314 362 3446 E-mail: [email protected]

Dikranian

2 is no evidence that a concept of the brain’s function has been articulated even if there were words to name this anatomical structure. Egyptians thought so little of the brain that they mostly discarded it when mummifying the body in preparation for eternal life. The heart, not the brain, was considered to be the seat of the soul and the repository of memories. Nevertheless, Egyptian doctors made careful observations of illness and injury (3, 4). Recovered papyri indicate that physicians were aware of the symptoms of brain damage. In a patient with an open skull fracture, a surgeon describes the cerebrospinal fluid and meninges. Imhotep was an important figure in ancient Egyptian medicine. He is considered to be the author of the famous “Edwin Smith papyrus” (1600 B.C.E). This document was buried with its owner in a rock tomb at Thebes. Unearthed in 1862 by grave robbers it was sold to the British egyptologist Edwin Smith. The papyrus is 15 feet long with writings on both sides, it consists of 500 lines of text, contains 48 cases - traumatic head injuries, spinal column injuries and injuries to other parts of the body. The Greco-Roman world stretches from the first written Greek texts to the fall of the West Roman Empire lasting for almost 1500 years (5). Early Greek anatomists viewed the human brain as an empty shell and the origin of emotions being located in the heart (6). Aristotle (382-322 B.C.E) thought of the brain to be a secondary organ - a cooling agent for the heart and a place where spirits circulated freely. Hippocrates (460-379 B.C.E) saw the brain as the seat of emotion, pain and anxiety, sensation and the seat of intelligence. He wrote “…. it ought to be generally known that the source of our pleasure, merriment, laughter, amusement, as of our grief, pain, anxiety and tears, is none other than the brain. It is specifically the organ, which enables to think, see and hear, and to distinguish the ugly and the beautiful, the bad and the good, pleasant and unpleasant. It is the brain too which is the seat of madness and delirium, of the fears and frights which assail us, often by night, but sometimes even by day…” (4). Erasistratus (304– 250 B.C.E) was anatomist and royal physician. Along with Herophilus (335–280 B.C.E), he founded a school of anatomy in Alexandria, where they carried out anatomical research. He is credited for his description of the valves of the heart. He also concluded that the heart functioned as a pump, that the arteries were full of air and carried the “animal spirit”. Herophilus distinguished between motor and sensory nerves and described several cranial nerves. He identified the meninges and ventricles and recognized the division between cerebellum (paraenkephalis) and cerebrum (enkephalos). He is “the first Biomed Rev 26, 2015

man to search into the causes of disease” through dissection (7). After Herophilos and Erazistratus there was a rapid decline in Alexandrian anatomical sciences and medicine. Dissection virtually disappears in the west until the rise of medieval universities in the 12th century (4). Galen was the first physician to produce an accurate description of the anatomy of the brain He discovered three liquid filled ventricles and hypothesized that they were the sites of storage of the vital breath; he assigned the soul and higher cognitive functions in the cerebellum. Galen was born in 129 C.E. in Pergamos. His ethics and his comprehensive works embody the philosophical, scientific, and medical thinking of Greek antiquity and Greco-Roman culture. Galen began his medical studies, at age 17, as a ” therapeutes” at the renowned Asklepieion of Pergamos. From his earliest youth he was familiar with classic reasoning and ideals embodied in the teachings of Hippocrates, Plato and Aristotle. Galen spent some time at Smyrna, Corinth, and Alexandria, where he studied anatomy. At age 30, Galen moved to Rome and built an enviable medical practice (4, 8). He also lectured in public amphitheaters and performed animal experiments before lay audiences. He became private physician to the emperor Marcus Aurelius. Galen placed the overseeing soul and its functions, in the “psychikon pneuma” (psychic spirit) in the brain. The heart did play a secondary role: through the carotid arteries, it supplied the brain with the aeriferous blood that contributed to the formation of the psychikon pneuma. Pneuma reaches the brain mixed with arterial blood and via the nostrils. In the brain it is processed into psychikon pneuma, giving rise to thought. It was guided as a signal via the nerves to give rise to the senses and move the voluntary muscles. From this came Galen’s idea of ligating or sectioning the nerves to see what impairment(s) resulted, he further wrote “…the brain in man was indeed bipartite. It had a ventricle placed longitudinally on each side . . . this third one extended to the so called cerebellum; for the cerebellum was set off by itself, as well as the cerebrum, and was like the jejunum and very much folded. From this the observer may learn that as in those animals that surpass the others in speed of running, such as the stag and hare, well-constructed with muscles and nerves for this, so also, since man greatly surpasses other beings in intelligence, his brain is greatly convoluted”. EUROPE’S DARK AGES, LATE ANTIQUITY AND THE RENAISSANCE The historic period following Galen’s body of work is

The amazing brain considered a time when medicine declined and anatomical knowledge was stagnant (9). Scientific curiosity was put on ice only to be thawed by Andreas Vesalius. However starting late antiquity (4th century C.E.) scholars in Europe followed by the Islamic civilization were introducing important changes. All the available Greek and Hellenistic works were translated into Syriac, Hebrew and Arabic. During the 9th century C.E. knowledge of the brain, nerves and senses was brought together in the unified Arabic medical compendia, their Latin versions became standard text at the early European medical schools (10). Physicians in medieval Europe weren’t as idle as it may seem, as a new analysis of the oldest-known  preserved human dissection in Europe reveals. The specimen consists of a human head and shoulders with the top of the skull and brain removed. The arteries are filled with a red “metal wax” compound that helped preserve the body. During this period the anatomy of the brain consolidated around three principle ventricles.  Traditionally imagination was located in the anterior ventricle, memory in the posterior ventricle, and reason located in between.  Avicenna (c. 980 –1037), the great Persian physician, philosopher and anatomist wrote that “sensus communis” (common sense) was housed in the “faculty of fantasy”, receiving “all the forms which are imprinted on the five senses”.  Memory preserved what common sense has received.  By contrast, the famous medieval anatomist Mondino de’ Liuzzi wrote in his Anatomy in 1316 that common sense lay in the middle of the brain.  Late antiquity also marks the emergence of Galenism. At the same time the attempt by many scholars and translators to present a succinct account of particular anatomical or medical topics has also contributed to the disappearance of the ambiguities and qualifications expressed by Galen in his original works, replacing their practical and empirical side with the dogmatic. Among other things the Renaissance marks the introduction of anatomical structure into printed books. For centuries, anatomy that had relied solely on textual description and the authority of the written word was transformed. Leonardo da Vinci was born on the 15th of April 1452. For Leonardo (1452-1519) the study of Anatomy became a science (11-13). He began to examine the relationship between the brain and the olfactory and optical nerves through experimenting with wax injections that helped him to model the ventricles.  He sketched the brain from many different perspectives, looking closely at the ventricles and the origins of the nerves from the medulla.  Leonardo’s images were considerably more anatomical (14). His brain sketch represents Biomed Rev 26, 2015

3 a sagittal section through the skull and is influenced by a description by Avicenna. Nothing of the structure of the brain is shown except the ventricles represented as three cavities separated by constrictions and placed in a row, one behind the other. A prolongation of the anterior one extends into the eye and probably represents the optic nerve. The more Leonardo looked, the less he was sure about the function of each ventricle.  His goal was to find the location of “sensus communis” but most importantly he tried to locate the seat of soul, as did most of brain investigators at this time.  Of the parts of the brain other than the ventricles, he gives little information. Leonardo and later Descartes in the 15th century defined the hydraulic fluid theory of brain function, implying that the cerebrospinal fluid was pumped up through the ventricles producing limbs movements. The last of his written notes reads: “June 24, 1518, Saint John’s Day, at Amboise, in the palazzo of Cloux; I shall go on”. It is believed that his last spoken words were “I have offended God and mankind because my work did not reach the quality it should have”. Charles Estienne (1504-1564) studied medicine in Paris and earned his degree in 1542. Contemporary of Vesalius, began his anatomical book prior to the Fabrica. His principal anatomic work was called “De Dissection Partum Corporis Humani Libri Tres” and was published in 1545 with about 60 woodcuts (14, 15). One of his remarkable observations was the central canal of the spinal cord. In 1561 Estienne became bankrupt and is said to have died in debtor’s prison. At the onset of the 16th century, much more was known about the peripheral nerves than the brain. Galen continued to be influential into the 16th century, when a young and rebellious physician Andreas Vesalius (1514-1564) began the practice of using real human bodies to study anatomy. At age 18, he entered the University of Paris (16). There, professors adhered to the works of Hippocrates and Galen, and thought it below themselves to perform dissections. Young Vesalius and fellow colleagues raided the gallows of Paris for bodies and skeletons to dissect. During one of his anatomical lessons in the medical school, he took the scalpel away from the barber-surgeon, and started dissecting himself. By the age of 22 Vesalius was giving his own dissection centered anatomical lectures. The publication of his De Humani Corporis Fabrica is a monument in the history of science and medicine (Fig. 1). It is almost symbolic that the year Vesalius died at the age of 49 in 1564, the great Galileo was born. Two of the seven books in the Fabrica are dedicated to neuroscience (13, 16). Vesalius ridiculed the ventricular doctrine of brain

4

Dikranian Figure 1. Andreas Vesalius, title page of Humani Corporis Fabrica. Libri Septem, 1543. Vesalius is seen surrounded by representatives of the university, the city, the church, nobility, doctors and student. The bearded man to the right of the central figure of the skeleton is perhaps Dr. Lazarus de Frieis, a friend of Vesalius. Some of the decorations on the top represent the lion of Venice, the ox head of the University of Padua and the monogram of the publisher, Johannes Oporinus. There is still debate about the illustrator of the Fabrica, with speculation that the artist Stephan van Calcar came from the studio of Titian. The publication of this book was a turning point in the history of modern medicine. Washington University Bernard Becker Library, Rare Book Collection, with permission.

function. His principle argument against placing the soul in the ventricle was that many animals have ventricles similar to humans and yet they are denied a “soul”. He believed the true function of the brain’s gyri were to allow blood vessels to bring nutrients to the deeper in brain tissue. One of his brain illustrations shows a horizontal dissection of the human brain (17). Vesalius revered Galen highly but very often his studies of the human form did not fit with Galen’s descriptions. These often matched the anatomies of dogs, apes, sheep or other farm animals. He found 200 discrepancies and publicly broke from the Galenic tradition (14, 18). Sixteenth and early seventeenth-century anatomists contributed a great deal to the physical description of the brain but made few significant advances in their understanding of its Biomed Rev 26, 2015

function (19). Bartolomeo Eustachi (1500-1574) was a brilliant anatomist. In his book “Tabulae Anatomicae” tables 17 and 18 are dedicated to the brain. Eustachi was a contemporary of Andreas Vesalius. He did not embrace Vesalius’ reformed anatomy and instead remained a staunch defender of Galen. Eustachi’s Galenism did not, however, prevent him from being a skilled and observant anatomist. He is credited with discovering the Eustachian tube and describing the cochlea among other structures. The copperplate engravings he created are remarkable for their clarity and detail. Some of them are more accurate than Vesalius’ woodcuts. Unfortunately, the majority were not published until 1714, almost 200 years after Eustachi’s death (Fig 2). Varolio (1543-1575) worked in Bologna and then in Rome and served as a personal physician

The amazing brain

Figure 2. Bartolomeo Eustachi. Tabulae Anatomicae. Plate XVI, Cranial nerves peripheral nerves and plexuses are illustrated, Amstelaedami: Apud R. & G. Wetstenios, Rome, 1722. Washington University Bernard Becker Library, Rare Book Collection, with permission. to the pope. He started the examination of the brain from the base and his illustrations show the optic nerves and the chiasm. Not until the 1660s did the anatomy of the brain change significantly.  Within a few years of each other, the English physician Thomas Willis published his Anatomy of the Brain in 1664 and the Danish anatomist Nicolaus Steno published his “Lecture on the Anatomy of the Brain” in 1669.  Both launched powerful criticisms of Galen’s idea of animal spirits which Steno wrote, were “words without any meaning.”  He further argued for a more careful exploration of the cortex and the ventricles, writing about sensus communis:  “that beautifully arched cavity does not exist.”  Willis brought this point further home by arguing that the ventricles were not formed as part of God’s design to house the spirits but Biomed Rev 26, 2015

5 “accidentally from the complication of the brain.”  Given that, “the supreme seat of the Soul” could hardly be there.  Nor could it be in the pineal gland, as Descartes had proposed. Tomas Willis (1621-1675) was born on 27 January 1621 in Wiltshire, England. Young Thomas was educated in Oxford. He obtained his medical degree in 1646. He became a member of an informal group of experimental scientists “The Virtuosi” who, together with the Virtuosi of London were the forerunners of the Royal Society. He was professor of natural philosophy at Oxford in 1660 and, on moving to London in 1666, acquired the largest fashionable practice of his day. Dr. Willis performed necropsies on his patients and made extensive anatomic dissections on the brain. In his images the anatomy of brain convolutions, sulci a fissures are much more clearly defined than Vesalius. His co-workers included the physicists Robert Hooke and Robert Boyle, Richard Lower, an anatomist, physiologist, and clinician, who administered the first blood transfusion, and Sir Christopher Wren, the renowned architect (of Saint Pauls’ cathedral in London) and artist who was responsible for the engraved plates from which the illustrations are derived in his book Cerebri Anatome Nervorum Descriptio et Usus published in 1664 in Oxford (13, 14) (Fig. 3). This work included Willis’s classification of the cranial nerves and his description of the arterial pattern at the base of the brain - the circle of Willis. His most important contribution, a discussion of cerebral circulation, was based on ingenious use of india ink injections and inspired by Harvey’s ideas of the circulation of the blood.  He emphasized the capability for collateral circulation if an artery becomes blocked. He comments on the dissection he made of a patient who has died of abdominal illness: “When his skull was opened we noted amongst the usual intracranial findings, the right carotid artery, in its intracranial part, bony or even hard, its lumen being almost totally occluded; so that the influx of the blood being denied by this route, it seemed remarkable that this person had not died previously of an apoplexy: which indeed he was so far from, that he enjoyed to the last moments of his life, the free exercise of his mental and bodily functions. For indeed, nature had provided a sufficient remedy against the risk of apoplexy in the vertebral artery of the same side in which the carotid was wanting, since the size of this vessel was enlarged, becoming thrice that of the contralateral vessel.” (14). Willis thought that the cerebral cortex covered many subcortical centers, that cortical gray matter was responsible for animal spirits, while the white matter distributed the spirits to the body, governing movement

6

Dikranian as Erasistratus suggested: as coils of the small intestine. Willis died in London at age 54 and was buried in Westminster Abbey. THE CAJAL ERA AND ITS FAMOUS CONTEMPORARIES

Figure 3. Tomas Willis (1621-1675). Cerebri Anatome: Cui Accessit, Nervorum Descriptio Et Usus, page 256. This volume of “The Anatomy of the Brain” was published in London in 1664 and was translated from Latin to English a few years later. The original illustrations were made by Sir. Christopher Wren. The brain was approached from below and removed from the skull before being dissected. In this image the famous “circle of Willis”, a collateral arterial network at the base of the brain, is illustrated. Washington University Bernard Becker Library, Rare Book Collection, with permission.

and sensation. He implicated the “cortical and grey part of the cerebellum” in the functions of memory and movements. The cortex initiates voluntary movements whereas the cerebellum is involved only in involuntary movements. These statements were obviously supported not only by his dissections, but also from his experiments on animals and from analyzing his patients. Interestingly, despite the importance of the cortex for Dr. Willis, his work contains no separate drawing of the cortex; he apparently never asked Wren or anybody in his team to produce one. For the next 150 years the cortex will be drawn Biomed Rev 26, 2015

Camillo Golgi (1843–1926) graduated medicine in 1865 at the University of Pavia (20). He believed that mental diseases could be due to organic lesions of the neural centers. In 1872 financial problems forced him to interrupt his academic career and he accepted the post of chief resident physician to the Hospital for the chronically ill in a small town near Milano and Pavia. Here in his kitchen, made into a rudimentary laboratory, working mainly at night by candlelight, he discovered in 1873 a silver chromate method for staining nerve tissue, the so-called black reaction “la reazione nera”. In 1881 he was appointed to the chair of General Pathology at the University of Pavia. In the minds of most neuroscientists the name of Camillo Golgi is associated with the theory that nerve cells communicate with one another by means of an intricate network of anastomosing axonal branches contained (13). In 1875 Golgi published, in an article on the olfactory bulbs, the first drawings of neural structures as visualized by this technique. The discoveries of Golgi led Wilhelm von Waldeyer-Hartz to postulate in 1891 that the nerve cell is the basic structural unit of the nervous system, which is a critical point in the development of modern neurology. In 1906 Golgi shared the Nobel Prize with Santiago Ramón y Cajal “in recognition of their work on the structure of the nervous system”. He remained as professor emeritus at the University of Pavia until his death in 1926. Santiago Ramón y Cajal (1852-1934) took his Licentiate in Medicine at Saragossa in 1873 and as an army doctor took part in an expedition to Cuba in 1874-75. Back in Spain he became an assistant in the School of Anatomy in the Faculty of Medicine at Saragossa and then, at his own request, Director of the Saragossa Museum. In 1883 he was appointed Professor of Descriptive and General Anatomy at Valencia. In 1887 he was appointed Professor of Histology and Pathological Anatomy at Barcelona and in 1892 he was appointed to the same Chair at Madrid. In 1900-1901 he was appointed Director of the «Instituto Nacional de Higiene». Cajal was a painter, artist, and gymnast. Applying the Golgi stain with virtuoso’s dexterity, he presented the world for the first time with visible populations of individual neurons, Santiago Ramòn y Cajal wrote the Neuron Theory in 1887 (21) It is considered to be one of the principle conquests of the 20th century. The formulation of the Neuron doctrine was achieved between 1888 and 1889. In fact Cajal admits that 1888 is his “fortunate year’, he is then

The amazing brain at the University of Barcelona. When Cajal began his studies the reticular theory, supported by Golgi was the prevailing theory among the scientific community. Cajal was the first to provide fundamental scientific data by publishing his studies on basket cells of the cerebellum of birds, which establish nest-like terminals around the body of a Purkije cell. In 1888 he publishes another paper on the parallel and climbing fibers terminating freely on the dendrites of Purkinje cells. In his words “… having arrived at the level of the first arms of the mentioned dendritic stems, they split up into snaking parallel plexuses that ascend all along the protoplasmic branches, hugging their form, like ivy or lianas that cling to the trunks”. He thus proves that there is in fact contiguity but not continuity among nerve terminals. He establishes that “the nerve cells are independent elements that are never anastomosed” and “nervous propagation is verified by contacts at the level of certain apparatus or cogs devices”. In 1906 he shared the Nobel Prize with Camillo Golgi. Before the decision there had been some severe controversies between the two scientists on the one hand, and the members of the jury on the other hand, because Golgi’s discovery was older, and because the works of Ramon y Cajal were so dependent on that of Golgi, without which Ramon y Cajal would probably never have arrived at his results. However, many consider Ramón y Cajal the greater of the two. Golgi described their relationship as that of “two Siamese brothers attached to the back”. RITA LEVI-MONATALCINI AND NGF: THE “GROWTH FACTOR” ERA OF NEUROBIOLOGY The first cell growth factor, nerve growth factor (NGF), was discovered by Rita Levi-Montalcini in the early 1950’s in Washington University in Saint Louis, Missouri, USA (reviewed in 22). Originally identified as neurite outgrowthstimulating factor, later studies revealed that non-neuronal cells, including immune cells, endothelial cells, cardiomyocytes, pancreatic beta cells, testicular Leydig cells, prostate epithelial cells and adipose tissue cells, are also targets for and/or sources of NGF. Nerve growth factor is well recognized at present to mediate multiple biological phenomena, ranging from the neurotrophic through immunotrophic and epitheliotrophic to metabotrophic effects. Consequently, NGF and other members of the neurotrophin family (pro-NGF, brain-derived neurotrophic factor, neurotrophin-3, -4/5, -6) are implicated in the pathogenesis of a large number of neurological and nonneurological diseases, ranging from Alzheimer’s and other neurodegenerative diseases to ocular, cutaneous, cancer, and Biomed Rev 26, 2015

7 cardiometabolic diseases such as atherosclerosis, obesity, type 2 diabetes, metabolic syndrome (23). THE BRAIN FUNCTION AND ITS MAPS The very first to describe the structure of a human brain in regard to its integral function and pathways were Pierre Broca, Carl Wernicke, who analyzed the effect of the different localization of the brain lesions over the limbs motility and speech function. Pierre Paul Broca (1824–1880) provided compelling evidence for the concept of “localization of function,” which holds that different parts of the brain do different things (24). In 1861 Dr. Broca encountered two patients. One was an epileptic man named Leborgne known as “Tan” nicknamed after the only syllable he was capable of uttering (25). He was able to understand spoken language but couldn’t articulate his thoughts in speech – something that perplexed Broca enormously, given that one of Leborgne’s first symptoms was a weakening of function in the right side of his body, which progressed to more loss of motor control and eventually the loss of sight and some of his mental faculties. When Leborgne died, Broca dissected his brain and found a massive lesion in the left frontal cortex and concluded that this must somehow be related to the loss of speech. Then came Lelong, who after a fall was only able to utter a few words. When Lelong died, Broca discovered a similarly dramatic lesion in the left side of his brain. Broca also coined the term “Limbic lobe” which is related to emotions and emotional expression. After Broca published his findings damage-related language deficits, Carl Wernicke (1848-1905) researched the effects of brain disease on speech and language. He discovered that not all language deficits were the result of damage to Broca’s area. Damage to the left posterior, superior temporal gyrus resulted in deficits in language comprehension. This region is now referred to as Wernicke’s area, and the associated syndrome is known as  receptive aphasia. Korbinian Brodmann’s (1868-1918) integrated evolutionary ideas and histological analysis of the cortex with functional localization (13, 26). He studied the cytoarchiteacture of more than 52 areas in the human brain, a huge body of work which he did in the Neurobiologisches Laboratorium in Berlin and published his results in a 1909 monograph “Localization in the cerebral Cortex”. Brodmann defined these functional areas distinguished by their different cellular organization and neuronal populations.  Thus, several functional cortical areas such as primary motor cortex, premotor cortex, sensory

8 cortex and visual cortex have been identified, each having their morphologic particularities (27). Wilder Penfield (18911976) graduated Oxford University in 1916 and his exposure to Dr. Charles Sherrington inspired him to study the nervous system (28). He completed his medical degree at John Hopkins School of Medicine and later became a neurosurgeon. As a surgical intern in Peter Bent Brigham Hospital in Boston he was inspired by Prof. Cushing’s surgical techniques. Working with a succession of neurosurgical associates, performing groundbreaking surgeries for epilepsy, he studied the responses of the human cerebral cortex to electrical stimulation. He characterized topographical distribution of the primary and secondary sensory and motor areas, the representation of language function in the inferior frontal, superior frontal and temporo-parietal cortex. He defined the role of the hippocampi and the lateral temporal cortex in memory.  XX AND XXI CENTURY ADVANCES IN NONINVASIVE BRAIN RESEARCH In 1971, Godfrey Hounsfield first introduced X-ray computed tomography (CT) in London. He created 3-dimensional transaxial tomographic images of an intact object with data arising from a large number of projections through the object. Hounsfield’s invention literally changed the practice of medicine. He and Alan Cormack, received the 1979 Nobel Prize for Physiology or Medicine. Although these noninvasive imaging techniques were very informative about the central nervous system, information on the brain function was to be the province of Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI)(29, 30). PET works by detecting gamma rays so as to allow the site of radioactive decay using “tomographic” methods analogous to those used for CAT scans. PET can be used as a quantitative probe for a broad range of metabolic, biochemical, and pharmacological measurements. It is used to study brain activity and also to reveal mechanisms of “neurovascular coupling” - the relationship between electrical brain activity and the vascular responses observed by PET and MRI. PET derives its name and fundamental properties from a group of radionuclides (15O,  11C,  18F and  13N) with short half-lives, a unique decay scheme, and chemical properties. The first cyclotron dedicated to biomedical research and radiation therapy was installed at the Hammersmith Hospital in London. A second cyclotron was installed at the Mallinckrodt Institute of Radiology of the Washington University School of Medicine. Michel Ter-Pogossian will be remembered as the “father of PET.” Biomed Rev 26, 2015

Dikranian Ter-Pogossianhe came to the United States in 1946. He was drawn to Washington University Department of Physics by the reputation of Arthur Holly Compton (a physicist and a Nobel laureate) and worked as research assistant. He joined the faculty of Mallinckrodt Institute in 1950 and was appointed Professor of radiation sciences in 1961. In 1973 as a head of Mallinckrodt Institute’s Division of Radiation Sciences and together with his team of physical scientists, chemists, and physicians – Drs Phelps, Cox and Donald developed the concept and the design of the first PET scanner. MRI has better resolution in space and time compared to PET, and offers a wide range of scan types that are informative about brain structure, function, and connectivity. Mechanistically, MRI involves complex quantum-mechanical phenomena. Protons (hydrogen atoms, mainly in water molecules) act like tiny magnets that are aligned to the strong magnetic field of the scanner. These protons can be transiently knocked out of alignment by brief radio frequency ‘pulses’ generated by the scanner. As the protons gradually realign to the main magnetic field (‘relax’), they emit faint radio frequency signals detected by sensitive electrical receivers within the scanner. These signals are reconstructed to form images whose appearance depends upon the particulars of the pulse sequence as well as the individual brain being scanned (Fig. 4A). Over the past two decades, there have been dramatic advances in the methods of acquisition, analysis, and visualization of MRI data such as functional MRI (fMRI), and diffusion MRI (dMRI). Structural MRI enables highresolution visualization of cortical and subcortical structures in individual subjects. Structural MRI pulse sequences reveal contrast based on tissue type (e.g., gray vs white matter). They include ‘T1-weighted’ (T1w) and ‘T2-weighted’ (T2w) scans. Functional MRI relies on the Blood Oxygenation Level Dependent (BOLD) contrast mechanism. Brain electrical leads to increased blood flow and hence elevated oxyhemoglobin levels relative to what is metabolically required by the active tissue. Because oxyhemoglobin and deoxyhemoglobin have different magnetic properties, regions of increased oxyhemoglobin (higher activity) can be differentiated from those with higher deoxyhemoglobin (lower activity) using appropriate MRI pulse sequences - a T2* scan. BOLD signal likely reflects local synaptic as well as neuronal action potentials. FMRI has greater sensitivity and higher resolution in both space and time. The brain is always active (even when not doing an overt task, and even while asleep), and this is

The amazing brain

9

Figure 4. A. High resolution axial slice from the brain volume of an individual Human Connectome Project (HCP) subject obtained with structural MRI, showing the high resolution of the imaging data being collected. Image courtesy of the HCP consortium - http://humanconnectome.org. B. Functional Connectivity: A map of the average “functional connectivity” in the human cerebral cortex collected on healthy subjects while “at rest” in the MRI scanner. Regions in yellow/red are functionally connected to the “seed” location in the right frontal cortex (black circle, arrow), whereas regions in green and blue are weakly connected or not connected at all.  Image courtesy M. F. Glasser and S. M. Smith for the HCP consortium.

reflected in spontaneous fluctuations in the BOLD signal that exceed the modulation caused by specific tasks. The spontaneous BOLD fluctuations are not random, but tend to be correlated in time with the fluctuations at many other locations, both nearby and at long distances. The pattern of strong rfMRI correlations is similar to the pattern of strong anatomical connectivity - the correlation patterns are often referred to as ‘functional connectivity’ even though they are imperfect surrogate for genuine anatomical connectivity (Fig. 4B). TaskfMRI typically reveals a spatially complex pattern of activation and deactivation when comparing different tasks. Multiple cortical areas are activated by any given task or task contrast; different tasks may show overlapping activation patterns. The brain contains spatially distributed networks and subnetworks serving diverse brain functions. Diffusion MRI (dMRI) uses pulse sequences that are sensitive to the rate at which molecules Biomed Rev 26, 2015

diffuse in different directions. Because diffusion is faster along the length of axons compared to transversely, across axonal and myelin membranes, dMRI reveals the orientation of fiber bundles in regions where they course in parallel (Fig. 5A). Tractography is a method for estimating the long-distance trajectories of major pathways (Fig. 5B). THE MOUSE AND THE HUMAN CONNECTOMES Understanding how the precise interconnections of neurons account for brain functions has been a preoccupation of neuroscientists for over a century. Conceptually, a “connectome” is a “comprehensive” map of neural connectivity. Neuroscientists have used three main sets of anatomical approaches to study neural connectivity in experimental animals, predominantly mice and primates: single-cell impregnation, optically based tract-tracing and later electron

Dikranian

10

Figure 5. A. Diffusion Fractional Anisotropy. Principal diffusion directions images from the Human Connectome Project (HCP) dMRI data provide a measure of how water diffuses in the brain. Diffusion directions are RGB-color encoded - red: left–right, green: anterior–posterior, blue: inferior–superior. Image courtesy of the HCP consortium - http://humanconnectome.org. B. Structural Connectivity: 3D probabilistic trajectories of white matter fibers arising from a seed in the left frontal cortex. The orientation vectors at each voxel are RGB color-coded coded (red: Left-right, green: anterior posterior, blue: inferior-superior). Image courtesy S. Sotiropoulos and T. E. J. Behrens for the HCP consortium.

microscopy. Serial section electron microscopy has been the method of choice for overcoming the limited resolution of light microscopy and the insufficiencies of tract-tracing. Nonetheless, large-scale reconstruction, especially over long distances, remains a distant hope. The green fluorescent protein (GFP) revolution has led to a technical renaissance in brain imaging. The so-called “Brainbow” transgenic mice were engineered in Dr. Jeff Lichtman’s and Dr. Joshua Sane’s lab (31, 32). The role of Brainbow mice in connectomics was huge. It not only generated strikingly beautiful images but contributed to the discovery and mapping of many brain neuronal circuits. Dr. Lichtman went further using serial electron microscopy to reconstruct various axonal processes, their synapses on dendrites and their spines. For the living human brain, connectivity can only be estimated for the ‘macro-connectome’, using noninvasive neuroimaging. MRI provides two complementary methods for inferring connectivity. Diffusion MRI is used to infer structural connectivity, and resting-state fMRI is used to infer functional connectivity. The Human Connectome Project (HCP) is an effort centered at Washington University School of Medicine in Saint Louis and University of Minnesota Medical School to study brain circuits in a large population of healthy Biomed Rev 26, 2015

12 000 adults. Its primary goal is to delineate the typical patterns of structural and functional connectivity in the healthy adult human brain (31). HCP is also using task-fMRI to help delineate the relationships between individual differences in the neurobiological substrates of mental processing and both functional and structural connectivity. The results from the HCP will be published in the public domain and will offer a critical stepping-off point for future studies that will examine how variation in human structural and functional connectivity play a role in neurological and psychiatric disorders. The Human Connectome Project is collecting behavioral measures of a range of motor, sensory, cognitive and emotional processes that will delineate a core set of functions relevant to understanding the relationship between brain connectivity and human behavior Altogether, the HCP will lead to major advances in our understanding of what makes us uniquely human and will set the stage for future studies of abnormal brain circuits in many neurological and psychiatric disorders (Fig. 6). THE BRAIN INITIATIVE The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) initiative was announced in 2013 by the White House. This multiagency Initiative is led by the

The amazing brain

11

Figure 6. Resting-state functional connectivity from the Human Connectome Project (HCP) data. Columns 1, 2 and 3: functional connectivity from a “seed” in parietal cortex (black disk), based on a group average of 468 subjects; 52 left-hemisphere contiguous parcels from the resting-state networks (RSN); parcellated connectivity map for a default-mode network parcel containing the selected seed, based on 447 HCP subjects. Column 4: group-average parcellated connectome showing relative connection strength between regions. Image courtesy of the HCP consortium - http://humanconnectome.org.

National Science Foundation along with the National Institutes of Health and the Defense Advanced Research Projects Agency and includes private partners (34). It holds great promise for addressing fundamental questions about healthy brain function, advancing treatments for brain disorders or traumatic brain injury, and for generating brain-inspired “smart” technologies to meet our future needs as a society. BRAIN Initiative is committing approximately $200 million in 2015. It will sponsor studies related to quantitative and predictive theories of brain function, development of innovative technologies for understand brain function and treating brain disorders. It will help the development of cyber tools and standards for data acquisition, analysis and integration, foster multi-scale and multimodal modeling to relate dynamic brain activity to cognition and behavior and at the same time apply comparative analyses across species. Taken together these important scientific initiatives and modern imaging technology will bring us closer to understanding the mysteries of the central nervous system and help us understand and cure neurological diseases.

Biomed Rev 26, 2015

As it can be seen by this brief synopsis, the research on the brain has gone through a remarkable historic development and transformation. This journey has been lead by great human individuals many of whom have not been mentioned here in the interest of space. There are countless many who deserve mention. These renaissance men and women left their illuminating mark on the progress of neuroscience. Many of their original ideas, concepts and beliefs have endured the test of time. Knowledge of the anatomy and function of the nervous system are the pillars of contemporary clinical neurology. While the long itinerary of this knowledge dates back to antiquity, current studies on the amazing and mysteriously beautiful human brain is perhaps the most dynamic field of modern human endeavor. REFERENCES 1. Verano O, Finger S. Ancient trepanation. In: Finger S, Boller K, Tyler L, editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier, 2010.

12 2. Brandt-Rauf P, Brandt-Rauf S. History of occupational medicine: relevance of Imhotep and the Edwin Smith papyrus, Br J Med 1987; 44: 68-70. 3. York G, Steinberg D. Neurology in ancient Egypt. In: Finger F, Boller K, Tyler L, editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier 2010. 4. Gross C. From Imhotep to Hubel and Wiesel. The story of visual cortex. In: Cerebral Cortex, vol. 12, Rockland et al., editors, Plenum Press, NY, 1997. 5. Karenberg A, The Greco-Roman world, In: Finger F, Boller K, Tyler L, editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier 2010. 6. Crivellato E, Ribatti D. History of Neuroscience: Soul, mind, brain: Greek philosophy and the birth of neuroscience. Brain Res Bull 2007; 71: 327–336. 7. Adrian W. Herophilus, Erasistratus, and the birth of neuroscience. Lancet 1999; 354: 1719–1720. 8. Pasipoularides A. Galen, father of systematic medicine. An essay on the evolution of modern medicine and cardiology. Int J Cardiol 2014; 172: 47–58. 9. Russell G. After Galen: Late Antiquity and the Islamic world. In: Finger F, Boller K, Tyler L, editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier 2010. 10. Schalick III, W. Neurological conditions in the European Middle Ages. In: Finger F, Boller K, Tyler L, editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier 2010. 11. Kickhofel, E. Sine ars scientia nihil est: Leonardo da Vinci and beyond. Epilepsy Behav 2009; 14: 5-11. 12. Pevsner J. Leonardo da Vinci’s contributions to neuroscience. Trends Neurosci 2002; 25: 217-220. 13. Bentivoglio M, Mazzarello P. The anatomical foundations of clinical neurology. In: Finger F, Boller K, Tyler L, editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier 2010. 14. Ione A. Visual images and neurological illustration, In: Finger F, Boller K, Tyler L, editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier 2010. 15. Tubbs R, Salter E. Charles Estienne (Carolus Stephanus) (ca.1504–1564): Physician and Anatomist. Clin Anat 2006; 19: 4–7. 16. Roberts M. Human dissection – From Galen to the great revelations of Andreas Vesalius. http://brainblogger. com/2011/08/20/human-dissection-from-galen-to-thegreat-revelations-of-andreas-vesalius/. Biomed Rev 26, 2015

Dikranian 17. Linden D. Five hundred years of brain images. Arch Neurol 2002; 59: 308-313. 18. Abbot A. The man who bared the brain. Nature 2015; 521: 160. 19. Isler H. The development of neurology and the neurological sciences in the 17th century, In: Finger F. Boller K, Tyler L. editors.Handbook of Clinical Neurology, Vol. 95 History of Neurology. Elsevier, 2010. 20. Mazzarello P, Garbarino C, Calligaro, A. How Camillo Golgi became ‘‘the Golgi”. FEBS Lett 2009; 583: 3732–3737. 21. Lopez-Munoz F, Boya J, Cecilio, A. Neuron theory, the cornerstone of neuroscience, on the centenary of the Nobel Prize award to Santiago Ramon y Cajal. Brain Res Bull 2006; 70: 391–405. 22. Levi-Montalcini R. The nerve growth factor: 35 years later. Science 1987; 237: 1154-1162. 23. Chaldakov GN, Aloe L. Homage to Rita Levi-Montalcini, the Queen of modern neuroscience. Biomed Rev 2012; 23: 1-7. 24. Roxo M. Franceschini P, Zubaran C, Kleber F, Sander J. The limbic system conception and its historical evolution. The Scientific World Journal 2011; 11: 2428–2441, 25. Lazar R, Mohr J. Revisiting the Contributions of Paul Broca to the Study of Aphasia. Neuropsychol Rev 2011; 21:236–239.  26. Zilles K, Amunts K. Centenary of Brodmann’s map — conception and fate, Nature Reviews, Neuroscience 2010; 11: 139-145. 27. Brodmann K. Vergleichende Lokalisationslehre der Großhirnrinde in ihren Prinzipien dargestellt auf Grund des Zellenbaues, Barth, Leipzig, 1909. 28. Ward, Z. Letter to the editor: Reexamining Penfield’s homunculus. J History Neurosci 2014; 23: 198-203. 29. Raichle M. Positron emission tomography. Ann Rev Neurosci 1983; 6: 249-267. 30. Raichle M. The origins of functional brain imaging in humans, In: Finger F, Boller K, Tyler L. editors, Handbook of Clinical Neurology, Vol. 95, History of Neurology, Elsevier, 2010. 31. Lichtman J, Sanes J, Livet J. A technicolour approach to the connectome. Nat Rev Neurosci 2008; 9: 417–422. 32. https://www.youtube.com/watch?v=F37kuXObIBU). 33. http://www.humanconnectome.org/. 34. http://braininitiative.nih.gov/.

Biomedical Reviews 2015; 26: 13-21

© Bul­garian Society for Cell Biology ISSN 1314-1929 (online)

RESVERATROL: MORE THAN A PHYTOCHEMICAL

Parichehr Hassanzadeh1*, Fatemeh Atyabi1,2, and Rassoul Dinarvand1,2 1 Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, 2Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Teheran, Iran

In recent years, alternative and complementary medicine including the plant-based drugs with antioxidant and neuroptotective effects has attracted a growing interest. Resveratrol, a polyphenolic compound which is found in various plant species, has emerged as a promising nutraceutical with therapeutic potentials in neuropsychiatric, cardiometabolic and cancer diseases, also aging. The abundance of research providing promising findings about the multi-spectrum therapeutic applications of resveratrol and its encouraging potential to treat or prevent chronic and age-related disorders has raised a considerable number of clinical trials. Recently, resveratrol is implicated the biology of nerve growth factor (NGF), a critical player in the maintenance of neuronal growth and function. Furthermore, resveratrol affects the endocannabinoid signalling (eCBs) which exerts modulatory effects in the survival signalling pathways, neural plasticity, and a variety of neuroinflammatory and neurodegenerative processes. The therapeutic effects of this ubiquitous signalling system in Alzheimer’s disease, epilepsy, multiple sclerosis, mood and movement disorders, spinal cord injury, and stroke have been well-documented. In the present review, the implication of NGF and eCBs in the mechanism of action of resveratrol, that may be of therapeutic significance in neurological and non-neurological disorders, is highlighted. Biomed Rev 2015; 26: 13-21. Key words: polyphenol, nerve growth factor, endocannabinoid system, neurological disorders

INTRODUCTION Over the last few decades, studies on the health benefits of herbs have shown the ability of plants to synthesize a variety of compounds which interact with biological pathways (1). Resveratrol (3,5,4′-trihydroxy-trans-stilbene; C14H12O3, Fig. 1), a polyphenolic compound which is found in various

plant species, has shown a wide range of pharmacological activities. This phytochemical (nutraceutical) inhibits the cyclooxygenase-1 and 2 expression and nuclear factor-κB (NFκB) signalling and reduces the production of proinflammatory cytokines leading to the antiinflammatory and analgesic effects (2). Resveratrol via the induction of apoptosis of cancer cells and metabolism of carcinogens as well as

Received 5 December 2015, revised 12 December 2015, accepted 12 December 2015. Correspondence to Dr Parichehr Hassanzadeh, Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Tel.: +98 21 66959095, Fax: +98 21 66581558, Cell phone: +98 912 1887745 E-mail: [email protected]

14

Figure 1. The chemical structure of resveratrol.

inhibition of cancer initiation and progression signalling exerts its anticancer effects (3). Since NF-κB signalling is a well-characterized target of cancers (4), inhibition of this signalling pathway by resveratrol (2) may be implicated in the anticarcinogenic effects of this natural polyphenol. Regarding the cardiometabolic protection, resveratrol inhibits inducible nitric oxide synthase (iNOS) and platelets aggregation, elevates high-density lipoprotein, and reduces low-density lipoprotein, trigycerides, and reactive oxygen species (ROS) accumulation (5). Resveratrol acts against the oxidative stress via the up-regulation of antioxidant enzymes synthesis and enhancement of the anti-oxidant capacity, free radical scavenging activity, as well as the inhibition of lipid peroxidation, and regeneration of α-tocopherol (6). Obesity, because of its complexity and multi-factorial nature, has remained as a challenging medical problem (7). Resveratrol by inhibition of the adipogenesis and down-regulation of lipogenic genes, and enhancement of fat oxidation has shown promising anti-obesity effects (8). In type 2 diabetes mellitus, resveratrol, by activating sirtuin 1, elevates the glucose uptake by muscles and modulates the β-cells insulin secretion (9). Regarding the neuroprotective properties of resveratrol, there are reports indicating its inhibitory effects against the memory deterioration, neuroinflammation, and oxidative stress in the hippocampus. Resveratrol improves the sensorimotor function, modulates the cholinergic neurotransmission, and prevents the oxidative damage of astroglial cells and cognitive decline. This natural compound downregulates the glutamateinduced extracellular signal-regulated kinase activation and interleukin-1β (IL-β) expression in the hippocampus (10-12). Study on the primary cortical neuron cultures has revealed Biomed Rev 26, 2015

Hassanzadeh, Atyabi and Dinarvand that resveratrol through the inhibition of intracellular calcium increase and production of ROS exerts the protective effect against N-Methyl-D-aspartate-induced neuronal death (13). Furthermore, this natural polyphenol by antioxidant activities protects the hippocampal neurons against the NO-induced toxicity (14). In Alzheimer’s disease, resveratrol has been shown to reduce the intracellular amyloid-β peptides, NO production, iNOS expression, prostaglandin E2 accumulation, and NF-κB translocation (15). Resveratrol exhibits therapeutic potential in other neurodegenerative diseases such as Parkinson’s disease (16) and amyotrophic lateral sclerosis (17) that may be due to its antioxidant and anti-inflammatory properties. This polyphenolic compound prevents the spinal cord and brain damage following the ischemia-reperfusion or traumatic injury (18). In recent years, it has been shown that resveratrol exerts antidepressant effects via the modulation of hypothalamicpituitary (HPA) axis activity and oxidative-nitrosative stress and inhibition of the production of inflammatory cytokines (19-21). Based on an increasing evidence, neurotrophins, a protein family including the nerve growth factor (NGF), brain-derived neurotrophic fac­tor (BDNF), neurotrophin 3 (NT-3), NT-4/5, and NT-6 act as the potential mediators of antidepressant responses (22, 23). Neurotrophins mediate their effects by the ligation of two major types of receptors: a high affinity tyrosine kinase (Trk) receptor and a low affinity pan-neurotrophin receptor (p75N­TR), thus trigger a number of cel­lular responses including the long-term trophic effects as well as the rapid chemo­tropic and morphogenetic actions on the developing neurons, synaptic transmission, and neuronal excitability (24-27). Neurotrophins are also implicated in the neuronal plasticity and exhibit neuroprotective effects that might be of therapeutic significance in the neuropsychiatric disorders (28). Indeed, dysfunction of the neurotrophin-related signalling mechanisms is involved in the pathogenesis of a number of psychiatric disorders (23-26). The prototypical neurotrophin, NGF, which regulates the stress and cognitive function (29,30) and mediates the action of a wide variety of psychotropic agents (31-35), along with the eCBs which regulates the neuroprotective processes (36), are implicated in the mechanism of action of resveratrol (37) that will be discussed herein. RESEVERATROL AND NGF Following the discovery of the first neurotrophin, NGF, by the Nobel Laureate Rita Levi-Montalcini (38), further

Resveratrol, NGF, and endocannabinoids studies showed that NGF as well as other neurotrophins (e.g. BDNF) affect the neuronal survival and differentiation, exerts a regulatory role in the memory and attention tasks, in neuroendocrine-immune interaction, and neurological disorders (39, 41). Be­cause of its antioxidant, angiogenic and metabotrophic (including insulinotropic) prop­erties, NGF is implicated in the molecular mechanisms of cardiometabolic diseases (41, 42, 48 and references therein), accelerates corneal and skin wound healing (43, 44) and plays a fundamental role in tissue engineering (45). Over the last decade, it has been suggested that electroconvulsive treatment or antidepressant drugs may act by increasing neurotrophin contents in the cen­tral nervous system (CNS). Based on the ability of NGF to induce the release of hypothalamic vasopressin which plays a crucial role in the formation of social bonding (46), it was suggested that NGF by modulating the neuroendocrine functions is implicated in the molecular mechanisms of emotions. In this respect, the involvement of NGF and BDNF in the pathogenesis of neuropsychiatric disorders (39, 41, 47-49) and in the mechanisms of action of a wide range of psychotropic agents (31-35, 41, 50) were reported. These findings might be of great significance in optimal management of neuropsychiatric disorders. Recently, the involvement of NGF in the mech­anism of action of resveratrol has also been reported (37). The elevation of brain NGF levels following chronic treatment with resveratrol indicates that the neurotrophic effect is a slow-developing process. The sustained enhancement of NGF protein contents in the brain regions which regulate the emotional activity may be considered as a mechanism by which resveratrol exerts its therapeutic effects in the neuropsychiatric disorders. Since NGF exerts the stimulatory effect on the cell proliferation (51), therefore, the elevation of frontal cortex NGF following the chronic administration of resveratrol might be of therapeutic significance against the stress-induced reduction in cell proliferation in the frontal cortex (52). Resveratrol also elevates NGF level in the hippocampus (37). It is noteworthy that NGF plays a pivotal role in learning, hippocampal plasticity, and neurogenesis (29, 30), and induces acetylcholine release in the hippocampus (49) leading to the improved cognitive performance. Therefore, resveratrol by enhancement of the hippocampal NGF may stimulate the hippocampal neurogenesis and improve the cognitive function. Moreover, this nutraceutical increases NGF contents in the olfactory bulb and amygdala (37). The critical role of NGF in the development, regeneration, and mainte­nance of the Biomed Rev 26, 2015

15 olfactory system of mammals has been well-documented (29, 30, 53). NGF also facilitates the cholinergic neurotransmis­sion between the amygdala and nucleus basalis (54) leading to the improvement of cognitive function. Altogether, it appears that elevation of brain NGF, also BDNF, contents constitutes a major part of the biochemical altera­tions induced by certain psychotropic agents (31-35, 41, 50) including resveratrol (37). RESVERATROL AND THE ENDOCANNABINOID SIGNALING Over the last decade, the eCBs, a group of neuromodu­ latory lipids and their receptors which regulates the neuronal proliferation and maturation (55), synaptic plasticity (56), emotional reactivity (57), neurotensin neurotransmission (58-59) and neurotrophin signalling (60, 61), has emerged as a topic of great interest in neuroscience and pharmacology. The endocan­nabinoids, anandamide and 2-ara­chidonoylglycerol (2-AG), are produced on-demand by the lipid precursors in the neuronal cell membrane and activate two types of G protein-coupled receptors, cannabinoid CB1 and CB2, leading to a wide variety of pathological and physiological processes. The eCBs exerts neuroremodulatory action in various types of diseases (62). In this context, development of anandamide uptake blockers, cannabinoid receptor agonists, and selective inhibitors of endocannabinoid degradation has been the focus of intense research (63, 64). In transsynaptic neuronal changes due to the neurodegenerative processes (65), microglia-induced neurotoxicity, and cytotoxicity induced by the excitatory amino acids (66), the eCBs exerts protective effects (67). In general, the endocannabinoids exert their neuroprotective effects through a variety of mechanisms including the prevention of calcium influx, inhibition of NO and/or glutamate release, and activation of antioxidative mechanisms. Furthermore, activation of the CB1 receptors leads to the stimulation of phosphoinositide 3-kinase (PI3-K)/ AKT signalling pathway and promotion of the cell survival (68). According to the protective effects of the eCBs against the excitotoxic damage and neuronal insult (58, 67), this ubiquitous signalling system might be an emerging target for the therapeutic interventions against the neurological disorders. In Parkinson’s disease (PD), the most prevalent neurodegenerative disorder affecting the basal ganglia, dopamine depletion results in a cascade of neurochemical events within the basal ganglia. Endocannabinoids and their synthesizing and degrading enzymes are abundantly found in the basal ganglia (69). Indeed, the striatal eCBs undergoes a profound neurophysiological alteration in order

16 to restore the homeostasis within the basal ganglia (70). In Huntington’s disease, the CB1 receptor agonists and inhibitors of eCB transport reduce the hyperkinesia associated with the disease (71). Stimulation of the CB1 or CB2 receptors results in the anti-inflammatory effects in multiple sclerosis (72). Furthermore, CB1 agonists promote the oligodendro­ cyte survival and mRNA expression of myelin protein (73) suggesting that the eCBs not only attenuates the symptoms of the disease, but also improves the function of oligodendrocytes that may be due to its antiinflammatory and neuroprotective effects. In the spinal cord injury, a cascade of cellular and molecular events which occur following the initial damage, is a main target for therapeutic interventions including the application of cannabinoids. In an animal model of the spinal cord injury, a significant increase of the eCB contents has been reported (74) suggesting that activation of the eCBs is a part of the neuroprotective response which is triggered following the injury. In epilepsy, the imbalance between the inhibitory and excitatory neural circuits leads to the excitotoxicity and neuronal death (75-77). Based on the regulatory effects of the eCBs on the inhibitory and excitatory transmissions and the enhancement of the eCB contents in epilepsy (78), this ubiquitous signalling system may be a promising target for antiepileptic therapies. In ischemic stroke, recombinant tissue plasminogen activator improves the functional outcome in patients, however, it should be administered within a short period of time following the onset of symptoms. The nonpsychoactive component of cannabis, cannabidiol, has been shown to inhibit the voltage-sensitive Ca2+ channels leading to the reduction of excitotoxicity. Administration of cannabidiol 6 h after the cerebral ischemia has led to the neuroprotective effects. Furthermore, cannabidiol has preserved the regional cerebral blood flow and improved the motor coordination even at 3 days after the cerebral ischemia (79) indicating its longlasting preventive effects on the post-ischemic cerebrovascular events. In Alzheimer’s disease which is associated with neuroinflammation, neurodegeneration, and cognitive impairment (80), the eCBs is of therapeutic significance because of its anti-neuroinflammatory and neuroprotective effects (62, 81). In β-amyloid-induced neurotoxicity, the eCBs may exert protective effects through the inhibition of NO release and activation of mitogen-activated protein kinase pathway (82). In amyotrophic lateral sclerosis, one of most the debilitating Biomed Rev 26, 2015

Hassanzadeh, Atyabi and Dinarvand neurodegenerative disorder which is characterised by the degeneration of motor neurons, the eCBs has shown antiinflammatory and neuroprotective effects by the prevention of excitotoxic damage and preservation of glutamate homeostasis (83). Following the identification of the endocannabinoid binding sites in substan­tia gelatinosa, analgesic effects of the eCBs in both acute and chronic pain were identified (84, 85). The interaction between the opioid signalling and eCBs is also well-documented (86). Based on these multitarget bioactivities of the eCBs, its potential implication in the mechanism through which resveratrol regulates brain NGF contents has been investigated. It has been found that resveratrol affects brain NGF levels under the regulatory drive of CB1 receptors (37). Furthermore, the brain eCB contents are increased following the chronic treatment with resveratrol (37). It appears that the eCBs activation is required, at least in part, for the neuroprotective actions of resveratrol against the various types of neurological disorders. Regarding the mood disorders, compounds which affect the eCBs function have been shown to regulate the monoaminemediated neurotransmission and activity of HPA axis (87). In the limbic brain regions where the neuronal activity is altered in depression (88), endocannabinoids are found at moderate to high levels (87). Indeed, the regulatory role of the eCB enhancers on mood (57), has opened a new line of research in antide­pressant drug discovery and development of novel antidepressants. Cannabinoids may be beneficial against the anxiety-related disorders as the CB1 receptor antagonist, SR141716A, has been shown to induce anxiety-like responses (89). Chronic exposure to a wide range of psychotropic agents has resulted in a significant enhancement of eCB contents in the brain regions which regulate the synaptic plasticity and emotional behaviour (32-35, 37). In fact, the brain regional distribu­tion of endocannabinoids following the administration of psychotropic agents represents the important role of the eCBs in the development of effective coping strategies to the emotional responses. As recently reported, four-week oncedaily injections of resveratrol results in a sustained elevation of annadamide and 2-AG contents in the brain regions implicated in the modulation of emotional behaviour and synaptic plasticity (37). This finding indicate the existence of an intrinsic eCB tone which is involved in the mechanism of action of resveratrol. Following chronic administration of resveratrol, NGF and endocannabinoids have been increased in the same brain regions suggesting that the regulatory effect of resveratrol

Resveratrol, NGF, and endocannabinoids on brain NGF contents depends on the coordinated release of endocannabinoids. It appears that a balance between NGF and eCB signalling mediates the pharmacological effects of this natural polypheonlic compound. In addition to its intrinsic radical scavenging activity, resveratrol reduces the production of tumour necrosis factor-α, IL-1β, NF-κB, NO, and the expression of iNOS gene. This natural polyphenol upregulates the activities of protein kinase C and heme oxygenase leading to the neuroprotective effects. In fact, multiple intracellular signalling pathways and molecular targets are involved in the mechanism of action of resveratrol (90). Involvement of the extracellular signal-regulated kinase cascade, noradrenaline and serotonin system, and HPA axis in the antidepressantlike effects of resveratrol has been previously demonstrated (91,92). Based on the recent findings, the brain NGF and eCBs are also implicated in the mechanism of action of resveratrol. In this context, resveratrol by affecting NGF and eCBs might be of therapeutic significance in a wide variety of diseases which are associated with abnormal NGF or eCBs. Resveratrol enhances the brain NGF contents under the regulatory drive of CB1 receptors indicating the importance of the endogenous cannabinoid activity in the neurotrphic action of this nutraceutical. In recent years, smart delivery of drugs, neurotrophins, and nutrients has been the focus of intense research. In nanotechnology-based approaches, nanoencapsulation of nutraceuticals, application of nanosensors and computational modelling have been the emerging topics (93-97). In this respect, designing the nanoformulations of resveratrol has led to the increased cellular uptake and improved bioavailability (98, 99). This may result in a better efficacy, reduced dose and potential side effects of this nutraceutical which has shown multi-spectrum therapeutic applications. CONCLUSION For over a decade, natural products with antioxidant, antiinflammatory, metabotrophic and neuroptotective effects have been the focus of intense research. Resveratrol, a naturally occurring polyphenolic compound, has shown multi-spectrum therapeutic applications. Besides its therapeutic potential in ischemia, cancer, diabetes, and cardiometabolic disorders, resveratrol has proved to be a promising therapeutic agent against the neurodegenerative disorders. In recent years, identification of the biological functions of the eCBs has resulted in a better understanding of the pathological processes which occur in the CNS. Biomed Rev 26, 2015

17 Compounds which regulate the activity of the eCBs, have shown beneficial effects against various disorders in which the currently available drugs have shown limited efficacy. The eCBs as the modulator of the neurodegenerative and neuroinflammatory processes is indeed a promising target for the pharmacotherapy of chronic CNS disorders. Resveratrol by affecting the eCBs and NGF with well-documented regulatory actions in the neuroprotective processes and emotional activity might be a valuable therapeutic agent against a wide variety of disorders associated with abnormal eCB or NGF signalling. Conflict of interest: the authors declare no conflict of interest. REFERENCES 1. Tapsell LC,  Hemphill I,  Cobiac L,  Patch CS,  Sullivan DR, Fenech M, et al. Health benefits of herbs and spices: the past, the present, the future. Med J Aust 2006; 185: S4–S24. PMD: 17022438. 2. Lastra CA, Villegas I. Resveratrol as an anti-inflammatory and anti-aging agent: mechanism and clinical implications. Mol Nutr Food Res 2005; 49: 405-430. PMD: 17956300. 3. Cal C, Garban H, Jazirehi A, Yeh C, Mizutani Y, Bonavida B. Resveratrol and cancer: chemoprevention, apoptosis, and chemoimmunosensitizing activities. Curr Med ChemAnti-Cancer Agents 2003; 3: 77-93. PMD: 12678904. 4. Hassanzadeh P. Colorectal cancer and NF-κB signaling pathway. Gastroenterol Hepatol Bed Bench 2011; 4: 127132. PMD: 24834170. 5. Hung LM, Chen JK, Huang SS, Lee RS, Su MJ. Cardioprotective effect of resveratrol, a natural antioxidant derived from grapes. Cardiovasc Res 2000; 47:549-555. PMD: 10963727. 6. Gulcin I. Antioxidant properties of resveratrol: a structureactivity insight. Innov Food Sci Emerg 2010; 11: 210-218. DOI: 10.1016/j.ifset.2009.07.002. 7. Hassanzadeh P. A quick look at obesity; the enemy within. Gastroenterol Hepatol Bed Bench 2011; 4: 186-191. PMD: 4017438. 8. Patel KR, Scott E, Brown VA, Gescher AJ, Steward WP, Brown K. Clinical trials of resveratrol. Ann NY Acad Sci 2011; 1215: 161–169. DOI: 10.1111/j.17496632.2010.05853.x. 9. Sharma S, Misra CS, Arumugam S, Roy S, Shah V, Davis JA, et al. Antidiabetic activity of resveratrol, a known SIRT1 activator in a genetic model for type-2 diabetes. Phytother Res 2011; 25: 67-73. DOI: 10.1002/ptr.3221.

18 10. Okawara M, Katsuki H, Kurimoto E, Shibata H, Kume T, Akaike A. Resveratrol protects dopaminergic neurons in midbrain slice culture from multiple insults. Biochem Pharmacol 2007; 73: 550-560. PMD: 17147953. 11. Sahu SS, Madhyastha S, Rao GM. Neuroprotective effect of resveratrol against prenatal stress induced cognitive impairment and possible involvement of Na+, K+-ATPase activity. Pharmacol Biochem Behav 2013; 103: 520-525. DOI: 10.1016/j.pbb.2012.09.012. 12. Jung JC, Lim E, Lee Y, Kang JM, Kim H, Jang S, et al. Synthesis of novel trans-stilbene derivatives and evaluation of their potent antioxidant and neuroprotective effects. Eur J Med Chem 2009; 44: 3166-3174. DOI: 10.1016/j.ejmech.2009.03.011. 13. Pangeni R, Sahni JK, Sharma JAS, Baboota S. Resveratrol: review on therapeutic potential and recent advances in drug delivery. Exp Opin Drug Deliv 2014; 11: 1285–1298. DOI: 10.1517/17425247.2014.919253. 14. Chachay VS, Kirkpatrick CMJ, Hickman IJ, Ferguson M, Prins JB, Martin JH. Resveratrol-Pills to replace a healthy diet? Br J Clin Pharmacol 2011; 72: 27–38. DOI: 10.1111/j.1365-2125.2011.03966.x. 15. Han YS, Zheng WH, Bastianetto S, Chabot JG, Quirion R. Neuroprotective effects of resveratrol against β-amyloidinduced neurotoxicity in rat hippocampal neurons: Involvement of protein kinase C. Br J Pharmacol 2004; 141: 997–1005. PMD: 15028639. 16. Ferretta A, Gaballo A, Tanzarella P, Capitanio N, Nico B, Annese T, et al. Effect of resveratrol on mitochondrial function: Implications in parkin-associated familiar Parkinson’s disease. Biochim Biophys Acta 2014; 1842: 902–915. DOI: 10.1016/j.bbadis. 17. Wang J, Zhang Y, Tang L, Zhang N, Fan D. Protective effects of resveratrol through the up-regulation of SIRT1 expression in the mutant hSOD1-G93A-bearing motor neuronlike cell culture model of amyotrophic lateral sclerosis. Neurosci Lett 2011; 503: 250–255. DOI: 10.1016/j.neulet.2011.08.047. 18. Virmani A, Pinto L, Binienda Z, Ali S. Food, nutrigenomics, and neurodegeneration: neuroprotection by what you eat. Mol Neurobiol 2013; 48: 353-362. DOI: 10.1007/s12035013-8498-3. 19. Ahmeda RF, Abdel-Rahmana RF, Abdallaha HMI, Saleha DO, Farid OAHA, Hessin AF. Antidepressant-like effect of resveratrol in a subchronic model of depression. J Arab Soc Med Res 2014; 9: 48–53. DOI: 10.4103/16874293.145619. Biomed Rev 26, 2015

Hassanzadeh, Atyabi and Dinarvand 20. Ge JF, Peng L, Cheng JQ, Pan CX, Tang J, Chen FH, et al. Antidepressant-like effect of resveratrol: involvement of antioxidant effect and peripheral regulation on HPA axis. Pharmacol Biochem Behav 2013; 114-115: 64–69. DOI: 10.1016/j.pbb.2013.10.028. 21. Hurley LL, Akinfiresoye L, Kalejaiye O, Tizabi Y. Antidepressant effects of resveratrol in an animal model of depression. Behav Brain Res 2014; 268: 1–7. DOI: 10.1016/j.bbr.2014.03.052. 22. Berton O, Nestler EJ. New approaches to antidepressant drug discovery: Beyond monoamines. Nat Rev Neurosci 2006; 7: 137-151. PMD: 16429123. 23. Castren E, Voikar V, Rantamaki T. Role of neurotrophic factors in depression. Curr Opin Pharmacol 2007; 7: 18–21. PMD: 17049922. 24. Gallo G, Letourneau PC. Localized sources of neuro­ trophins initiate axon collateral sprouting. J Neurosci 1998; 18: 5403–5414. PMD: 9651222. 25. Lohof AM, Ip NY, Poo MM. Potentiation of developing neuromuscular synapses by the neurotrophins NT-3 and BDNF. Nature 1993; 363: 350–353. PMD: 8497318. 26. Lesser SS, Sherwood NT, Lo DC. Neurotrophins differ­ entially regulate voltage-gated ion channels. Mol Cell Neurosci 1997; 10: 173–183. PMD: 9532579. 27. Hassanzadeh P, Arbabi E. The effects of progesterone on glial cell line-derived neurotrophic factor secretion from C6 glioma cells. Iran J Basic Med Sci 2012, 15: 10461052. PMD: 23493386. 28. Schulte-Herbrüggen O, Jockers-Scherübl MC, Hellweg R. Neurotrophins: from pathophysiology to treatment in Alzheimer’s disease. Curr Alzheimer Res 2008; 5: 38-44. PMD: 18288930. 29. Lad SP, Neet KE, Mufson EJ. Nerve growth factor: structure, function and therapeutic implications for Alzheimer’s disease. Curr Drug Targets CNS Neurol Disord 2003; 2: 315–334. PMD: 14529363. 30. Aloe L, Rocco ML, Balzamino BO, Micera A. Nerve growth factor: a focus on neuroscience and therapy. Curr Neuropharmacol 2015; 13: 294-303. PMD: 26411962. 31. Hassanzadeh P, Hassanzadeh A. Effects of different psychotropic agents on the central nerve growth factor protein. Iran J Basic Med Sci 2010; 13: 202–209. 32. Hassanzadeh P, Rahimpour S. The cannabinergic system is implicated in the upregulation of central NGF protein by psychotropic drugs. Psychopharmacology 2011; 215: 129–141. DOI: 10.1007/s00213-010-2120-4.

Resveratrol, NGF, and endocannabinoids 33. Hassanzadeh P, Hassanzadeh A. Involvement of the neurotrophin and cannabinoid systems in the mechanisms of action of neurokinin receptor antagonists. Eur Neuropsychopharmacol 2011; 21: 905–917. DOI: 10.1016/j.euroneuro.2011.01.002. 34. Hassanzadeh P, Hassanzadeh A. The CB1 receptormediated endocannabinoid signaling and NGF: The novel targets of curcumin. Neurochem Res 2012; 37: 1112-1120. DOI: 10.1007/s11064-012-0716-2. 35. Hassanzadeh P, Hassanzadeh A. Implication of NGF and endocannabinoid signalling in the mechanism of action of sesamol: a multi-target natural compound with therapeutic potential. Psychopharmacology 2013; 229: 571–578. DOI: 10.1007/s00213-013-3111-z. 36. Hampson AJ, Grimaldi M, Axelrod J, Wink D. Cannabidiol and (-)Delta9-tetrahydrocannabinol are neuroprotective antioxidants. Proc Natl Acad Sci USA 1998; 95: 8268– 8273. PMD: 9653176. 37. Hassanzadeh P, Arbabi E, Atyabi F, Dinarvand R. The endocannabinoid system and NGF are involved in the mechanism of action of resveratrol: A multi-target nutraceutical with therapeutic potential in neuropsychiatric disorders. Psychopharmacology 2015; (in press). 38. Levi-Montalcini R. The nerve growth factor and the neuroscience chess board. Prog Brain Res 2004; 146: 525–527. PMD: 14699984. 39. Triaca V. Homage to Rita Levi-Montalcini. Molecular mechanisms of Alzheimer’s disease: NGF modulation of APP processing. Adipobiology 2013; 5: 7-18 40. Fiore M, Chaldakov GN, Aloe L. Nerve growth factor as a signalling molecule for nerve cells and also for the neuroendocrine-immune systems. Rev Neurosci 2009; 20: 133-145. PMD: 19774790. 41. Yanev S, Aloe L, Fiore F, Chaldakov GN. Neurotrophic and metabotrophic potential of nerve growth factor and brainderived neurotrophic factor: Linking cardiometabolic and neuropsychiatric diseases. World J Pharmacol 2013; 2: 92-99. DOI:10.5497/wjp.v2.i4.92 42. Chaldakov GN, Fiore M, Stankulov IS, Manni L, Hris­tova MG, Antonelli A, et al. Neurotrophin presence in human coronary atherosclerosis and metabolic syn­drome: a role for NGF and BDNF in cardiovascular dis­ease? Prog Brain Res 2004; 146: 279–289. PMD: 14699970. 43. Kawamoto K, Matsuda H. Nerve growth factor and wound healing. Prog Brain Res 2004; 146: 369-384. . PMD: 14699974. Biomed Rev 26, 2015

19 44. Tuveri MA, Triaca V, Aloe L. The nerve growth fac­ tor induces cutaneous ulcer healing in non-responder transplanted skin. Ann Ist Super Sanita 2006; 42: 94-96. PMD: 16801731. 45. Hassanzadeh P. Tissue engineering and growth factors. Updated evidence. Biomed Rev 2012; 23: 19-35. DOI: http://dx.doi.org/10.14748/bmr.v23.26. 46. Kendrick KM. The neurobiology of social bonds. J Neu­ roendocrinol 2004; 16: 1007–1008. PMD: 15667456. 47. Angelucci F, Aloe L, Vasquez PJ, Mathe AA. Mapping the differences in the brain concentration of brain-de­rived neurotrophic factor (BDNF) and nerve growth factor (NGF) in an animal model of depression. Neu­roreport 2000; 11: 1369-1373. PMD: 10817624. 48. Chaldakov GN, Tonchev A, Aloe L. NGF and BDNF: from nerves to adipose tissue, from neurokines to me­tabokines. Relevance to neuropsychiatric and cardiometabolic diseases. Riv Psichiatr 2009; 44: 79-87. PMD: 20066808. 49. Alleva E, Petruzzi S, Cirulli F, Aloe L. NGF regulatory role in stress and coping of rodents and humans. Phar­ macol Biochem Behav 1996; 54: 65-72. PMD: 8728540. 50. Hellweg R, Lang UE, Nagel M, Baumgartner A. Subchronic treatment with lithium increases nerve growth factor content in distinct brain regions of adult rats. Mol Psychiatry 2002; 7: 604-608. PMD: 12140783. 51. Cheng S, Ma M, Ma Y, Wang Z, Xu G, Liu X. Combination therapy with intranasal NGF and electroacupuncture enhanced cell proliferation and survival in rats after stroke. Neurol Res 2009; 31: 753–758. doi: 10.1179/174313209X382557. 52. Banasr M, Valentine GW, Li XY, Gourley SL, Taylor JR, Duman RS. Chronic unpredictable stress decreases cell proliferation in the cerebral cortex of the adult rat. Biol Psychiat 2007; 62: 496-504. PMD: 17585885. 53. Miwa T, Moriizumi T, Horikawa I, Uramoto N, Ishima­ru T, Nishimura T, et al. Role of nerve growth factor in the olfactory system. Microsc Res Tech 2002; 58: 197-203. PMD: 12203698. 54. Moises HC, Womble MD, Washburn MS, Williams LR. Nerve growth factor facilitates cholinergic neurotrans­ mission between nucleus basalis and the amygdala in rat: an electrophysiological analysis. J Neurosci 1995; 15: 8131-8142. PMD: 8613748. 55. Fernandez-Ruiz JJ, Berrendero F, Herna´ndez ML, Ra­ mos JA. The endogenous cannabinoid system and brain development. Trends Neurosci 2000; 23: 14-20. PMD: 10631784.

20 56. Hassanzadeh P, Hassanzadeh A. Cannabinoid CB1 receptors mediate neurokinin A-induced synaptic plasticity in the spinal locomotor network. J Neurol Neurophysiol 2013; 4: 3. DOI: http://dx.doi.org/10.4172/2155-9562.S1.015. 57. Bambico FR, Duranti A, Tontini A, Tarzia G, Gobbi G. Endocannabinoids in the treatment of mood disorders: evidence from animal models. Curr Pharm Des 2009; 15: 1623–1646. PMD: 19442178. 58. Hassanzadeh P, Rostami F. Cannabinoid CB1 receptors are involved in neuroleptic-induced enhancement of brain neurotensin. Iran J Basic Med Sci 2014; 17: 181-188. PMD: 24847420. 59. Hassanzadeh P, Arbabi E. Cannabinoid CB1 receptors mediate the gastroprotective effect of neurotensin. Iran J Basic Med Sci 2012; 25: 803-810. PMD: 23492756. 60. Williams EJ, Walsh FS, Doherty P. The FGF receptor uses the endocannabinoid signaling system to couple to an axonal growth response. J Cell Biol 2003; 160: 481- 486. PMD: 12578907. 61. Hassanzadeh P. The endocannabinoid system: critical for the neurotrophic action of psychotropic drugs. Biomed Rev 2010; 21: 31-46. 62. Di Marzo V, Melck D, Bisogno T, De Petrocellis L. En­ docannabinoids: endogenous cannabinoid receptor lig­ands with neuroremodulatory action. Trends Neurosci 1998; 21: 521–528. PMD: 9881850. 63. Boger DL, Sato H, Lerner AE, Hedrick MP, Fecik RA, Miyauchi H, et al. Exceptionally potent inhibitors of fatty acid amide hydrolase: the enzyme responsible for degradation of endogenous oleamide and anandamide. Proc Natl Acad Sci USA 2000; 97: 5044–5049. PMD: 10805767. 64. Casanova ML, Blazquez C, Martinez-Palacio J, Vil­ lanueva C, Fernandez-Acenero MJ, Huffman JW, et al. Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors. J Clin In­vest 2003; 111: 43–50. PMD: 1251587. 65. Hassanzadeh P, Ahmadiani A. Nitric oxide and c-Jun N-Terminal Kinase are involved in the development of dark neurons induced by inflammatory pain. Synapse 2006; 59: 101–106. doi: 10.1002/syn.20219. 66. Hassanzadeh P. Effect of celecoxib on the peripheral NO production. Iran J Basic Med Sci 2009; 12: 43-50. 67. van der Stelt M, Di Marzo V. Cannabinoid receptors and their role in neuroprotection. Neuromol Med 2005; 7: 37–50. PMD: 16052037. Biomed Rev 26, 2015

Hassanzadeh, Atyabi and Dinarvand 68. Hassanzadeh P. Discovery of the endocannabinoid system: A breakthrough in neuroscience. Arch Neurosci 2014; 2: e15030. DOI: 10.5812/archneurosci.15030. 69. Di Marzo V, Hill MP, Bisogno T, Crossman AR, Brotchie JM. Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson’s disease. FASEB J 2000; 14: 1432–1438. PMD: 10877836. 70. Gubellini P, Picconi B, Bari M, Battista N, Calabresi P, Centonze D, et al. Experimental parkinsonism alters endocannabinoid degradation: implications for striatal glutamatergic transmission. J Neurosci 2002; 22: 6900– 6907. PMD: 12177188. 71. Lastres-Becker I, De Miguel R, and Fernandez-Ruiz JJ. The endocannabinoid system and Huntigton’s disease. Curr Drug Targets CNS Neurol Disord 2003; 2: 335–347. PMD: 18781982. 72. Arevalo-Martin A, Vela JM, Molina-Holgado E, Bor­ rell J, Guaza C. Therapeutic action of cannabinoids in a murine model of multiple sclerosis. J Neurosci. 2003; 23: 2511–2516. PMD: 12684434. 73. Molina-Holgado E, Vela JM, Arévalo-Martín A, Al­ mazán G, Molina-Holgado F, Borrell J, et al. Cannabi­ noids promote oligodendrocyte progenitor survival: involvement of cannabinoid receptors and phosphati­ dylinositol-3 kinase/Akt signaling. J Neurosci 2002; 22: 9742-9753. PMD: 12427829. 74. Garcia-Ovejero D, Arevalo-Martin A, Petrosino S, Docagne F, Hagen C, et al. The endocannabinoid system is modulated in response to spinal cord injury in rats. Neurobiol Dis 2009; 33: 57–71. doi: 10.1016/j. nbd.2008.09.015. 75. McCormick DA, Contreras D. On the cellular and network bases of epileptic seizures. Annu Rev Physiol 2001; 63: 815–846. PMD: 11181977. 76. Hassanzadeh P, Arbabi E, Rostami F. The ameliorative effects of sesamol against seizures, cognitive impairment and oxidative stress in the experimental model of epilepsy. Iran J Basic Med Sci 2014; 17: 100-107. PMD: 2471892. 77. Hassanzadeh P, Arbabi E, Atyabi F, Dinarvand R, Rostami F. Ferulic acid exhibits antiepileptogenic effect and prevents oxidative stress and cognitive impairment in the pentylenetetrazole kindling model of epilepsy in rats. Iran J Basic Med Sci 2015 (in press).

Resveratrol, NGF, and endocannabinoids 78. Wallace MJ, Martin BR, De Lorenzo RJ. Evidence for a physiological role of endocannabinoids in the modulation of seizure threshold and severity. Eur J Pharmacol. 2002; 452: 295–301. PMD: 12359270. 79. Hayakawa K, Mishima K, Irie K, Hazekawa M, Mishima S, Fujioka M, et al. Cannabidiol prevents a post-ischemic injury progressively induced by cerebral ischemia via a high-mobility group box1-inhibiting mechanism. Neuropharmacology 2008; 55: 1280–1286. DOI: 10.1016/j.neuropharm.2008.06.040. 80. Weksler ME, Gouras G, Relkin NR, Szabo P. The immune system, amyloid-β peptide, and Alzheimer’s disease. Immunol Rev 2005; 205: 244–256. PMD: 15882358. 81. Iuvone T, Esposito G, Esposito R, Santamaria R, Di Rosa M, Izzo AA. Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on beta-amyloid-induced toxicity in PC12 cells. J Neurochem 2004; 89:134–141. PMD: 15030397. 82. Van der Stelt M, Mazzola C, Esposito G, Matial I, Petrosino S, De Filippis D, et al. Endocannabinoids and β-amyloid-induced neurotoxicity in vivo: effect of pharmacological elevation of endocannabinoid levels. Cell Mol Life Sci 2006; 63:1410–1424. PMD: 16732431. 83. Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A, et al. CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science 2003; 302: 84–88. PMD: 14526074. 84. Hohmann AG, Walker JM. Cannabinoid suppression of noxious heat evoked activity in wide dynamic range neurons in the lumbar dorsal horn of the rat. J Neurophysiol 1999; 81:575–583. PMD: 10036261. 85. GÜhring H, Hamza M, Sergejeva M, Ates M, Kotalla CE, Ledent C. A role for endocannabinoids in indometh­acin induced spinal antinociception. Eur J Pharmacol 2002; 454:153–163. PMD:12421642. 86. Welch SP, Eads M. Synergistic interactions of endog­enous opioids and cannabinoid systems. Brain Res 1999; 848: 183–190. PMD: 1061271. 87. Hassanzadeh P, Hassanzadeh A. The role of the endo­ cannabinoids in suppression of the HPA axis activity by doxepin. Iran J Basic Med Sci 2011; 14: 414-421. PMD: 23493814. 88. Manji HK, Drevets WC, Charney DS. The cellular neu­

Biomed Rev 26, 2015

21 robiology of depression. Nat Med 2001; 7: 541–547. PMD: 11329053. 89. Navarro M, Hernandez E, Munoz RM, del Acro I, Vil­ lanua MA, Carrera MRA. Acute administration of the CB1 cannabinoid receptor antagonist SR141716A in­duces anxiety-like responses in the rat. Neuroreport 1997; 8: 491-496. PMD: 9080435. 90. Pirola L, Frojdo S. Resveratrol: one molecule, many targets. IUBMB Life 2008; 60: 323–332. PMD: 18421779. 91. Wang Z, Gu J, Wang X, Xie K, Luan Q, Wan N, et al. Antidepressant-like activity of resveratrol treatment in the forced swim test and tail suspension test in mice: the HPA axis, BDNF expression and phosphorylation of ERK. Pharmacol Biochem Behav 2013; 112: 104–110. DOI: 10.1016/j.pbb.2013.10.007. 92. Xu Y, Wang Z, You W, Zhang X, Li S, Barish PA, et al. Antidepressant-like effect of trans-resveratrol: Involvement of serotonin and noradrenaline system. Eur Neuropsychopharmacol 2010; 20: 405-413. DOI: 10.1016/j.euroneuro.2010.02.013. 93. Hassanzadeh P, Fullwood I, Sothi S, Aldulaimi D. Cancer nanotechnology. Gastroenterol Hepatol Bed Bench 2011; 4: 63-69. PMD: 24834159. 94. Hassanzadeh P. Nanopharmaceurticals: Innovative theranostics for the neurological disorders. Biomed Rev 2014; 25: 25-34. 95. Hassanzadeh P. New perspectives in biosensor technology. Gastroenterol Hepatol Bed Bench 2010; 3: 105-107. 96. Hassanzadeh P, Arbabi E, Rostami F, Atyabi F, Dinarvand R. Carbon nanotubes prolong the regulatory action of nerve growth factor on the endocannabinoid signaling. Physiol Pharmacol 2015; (in press). 97. Hassanzadeh P. Computational modelling: Moonlighting on the neuroscience and medicine. Biomed Rev 2013; 24: 25-31. 98. Bharali DJ, Siddiqui IA, Adhami VM, Chamcheu JC, Aldahmash AM, Mukhtar H, et al. Nanoparticle delivery of natural products in the prevention and treatment of cancers: current status and future prospects. Cancers 2011; 3: 4024-4045. DOI: 10.3390/cancers3044024. 99. Teskac K, Kristl J. The evidence for solid lipid nanoparticles mediated cell uptake of resveratrol. Int J Pharm 2010; 390: 61-69. DOI: 10.1016/j.ijpharm.2009.10.011.

Biomedical Reviews 2015; 26: 23-36

© Bul­garian Society for Cell Biology ISSN 1314-1929 (online)

GOLD NANOPARTICLES: A PROMISING THERAPEUTIC APPROACH

Harsharan Pal Singh1, Ashmeet Kaur2, Ishpreet Kaur2, Harpal Singh Buttar3, and Sukhwinder Kaur Bhullar4,5 1 Department of Quality Assurance, AIMIL Pharmaceuticals (I) Limited, New Delhi, India 2 Department of Quality Assurance, Delhi Institute of Pharmaceutical Sciences & Research, New Delhi, India 3 Adjunct Professor, Department of Pathology & Laboratory Medicine, Faculty of Medicine, University of Ottawa, Ontario, Canada 4 Department of Mechanical Engineering, Bursa Technical University, Bursa, Turkey 5 Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada

Nanotechnology is rapidly advancing and will leave no field untouched by its ground breaking innovations. Nanoparticles are molecules with a diameter ranging from 10-100 nm. Nanotechnology has promising biomedical applications and most noteworthy amongst them are noble metal particles. For instance, gold nanoparticles (AuNPs) provide a unique blend of physical and optical properties, chemical inertness, and high surface to volume ratio. They can be synthesized as well as functionalised to support various ligands on their surface. Their surface functionalization and diverse properties render the gold nanoparticles highly useful for drug delivery and gene carrier for therapeutic purposes and as molecular probes for disease diagnosis. The foundation for the usage of AuNPs in therapeutics and diagnosis was laid by the ancient studies done with ruby gold for curing diseases in middle ages. Presently, AuNPs have become available in different types such as spheres, rods, shells, cages and SERS particles which vary in shape, size and physical properties. The biomedical applications of these particles include drug and gene delivery, cancer diagnosis and therapy, determination of biological molecules and microorganisms, detection of disease etiology, immunoassay, enzyme immobilization, etc. Overall, the focus of this review is to highlight that AuNPs provide an excellent platform for the discovery of new therapies, cure for certain cancers, molecular probe for diagnostic purposes, as well as gene carriers and drug delivery vehicles. Biomed Rev 2015; 26: 23-36. Key words: gold nanoparticles, cancer treatment, drug delivery system, gold nanocarrier therapy

Received 2 December 2015, revised 12 December 2015, accepted 14 December 2015. Correspondence to Mr. Harsharan Pal Singh, Department of Quality Assurance, AIMIL Pharmaceuticals (I) Limited, New Delhi, India Tel.: +91-9999054899, E-mail: [email protected]

24 INTRODUCTION Nanotechnology is a multifaceted research field that deals with the nanometer sized entities ranging from 10-100 nm (1), and can be defined as the characterization, design, production and application of devices, structures and systems by controlling shape and size at a nanometer scale. It embraces diverse fields like chemistry, physics, material sciences, molecular biology, and medicine. Recently, potential uses of nanomaterials have attracted ample amount of attention of basic and applied scientists (2, 5, 7, 8). Although the nanoparticles size ranges from one to several hundred of nanometers, they are comparatively smaller than large biomolecules such as antibodies, enzymes and receptors as well as neurons and red blood cells (1, 2-8). The nano-sized particles have unique optical, magnetic, electronic and structural properties (9). This property makes the nanoparticles highly promising for a wide range of biomedical applications such as molecular diagnosis, cellular imaging and targeted drug delivery depending upon the composite shape or structure of the nanoparticles (10). The well-studied nanoparticles include carbon nanotubes, liposomes, quantum dots, magnetic nanoparticles, polymeric particles and metallic nanoparticles (7, 11). Namely, the unique and distinctive characteristics of nanoparticles are: (a) small size which ranges from 1-100 nm, (b) large surface-to-volume ratio, (c) shape, size and composition which determines the physical and chemical properties, (d) qualitative and quantitative target-binding properties, and (e) some nanostructures show high robustness (3). The applications of metal nanoparticles in medicine and biomedical research are rather a new phenomenon and an overwhelming number of publications have appeared in the world literature during the past 10 years. A few unique properties of nanoparticles are their high reactivity towards the living cells, stability at high temperatures, and translocation into the cell organelles. These particles also demonstrate unique optical properties which make them capable of producing quantum effects suitable for imaging. These characteristics of nanoparticles make them highly useful in diagnostics and therapeutics. Among all the metal nanoparticles, the most commonly studied are gold, silver, iron, and titanium oxide nanoparticles (9). Gold nanoparticles were used by the Romans for the purpose of decorating and staining of glass. It is nearly 150 years ago when Michael Faraday noticed that the properties of colloidal gold solution were different from that of bulk metalic gold. Over the last half-century, different high yielding Biomed Rev 26, 2015

Singh, Kaur, Kaur, Buttar and Bhullar techniques for the synthesis of gold nanoparticles (AuNPs, nanogold), including spherical and non-spherical shapes have been developed (12). The AuNPs are biocompatible and non-toxic which makes them more advantageous over other metal nanoparticles. They have physico-chemical inertness, and are easy to synthesize and fabricate. The AuNPs exhibits optical properties related to plasmon resonance as well as functionalisation with molecular probes. These unique physico-chemical properties make them compatible for broad range of biomedical applications. Presently, the biomedical applications of AuNPs have expanded to divergent fields like genomics, microorganism detection and control, targeted drug delivery, optical imaging, biosensors, immunoassays, monitoring of biological activity of cells and tissues, exploiting resonance scattering, photo-thermolysis of cancer cells, and in vivo photo-acoustic techniques (9, 12, 13). HISTORICAL PERSPECTIVE OF GOLD NANOPARTICLES Metallic gold has a melting point of 1064°C and a boiling point of 280°C. This precious metal does not react with water or oxygen and is an excellent conductor of electricity (14). In 1857, Michael Faraday explored the ruby gold nanoparticles and was astonished by the ruby color of the colloidal particles. His studies on the Au particles led to the birth of the modern day colloidal chemistry and the foundation of nanotechnology. Faraday observed that metallic Au was dispersed uniformly in both ruby glass and ruby fluid which was based upon its physico-chemical characteristics. Some 100 years later these ruby-colored colloids were found to be stable and their size was determined with the aid of electron microscope to be in ranges of 2-6 nm (15, 16). Zsigmondy who started to investigate upon the opacity and color of the ruby glass, tried to reproduce Au colloids through different methods. He combined Faraday’s work and introduced a new procedure called, ‘seed mediated method’. This method is still being used for the synthesis of nanoparticles. He also came up with an ultra-microscope for characterization of size, shape and structure of nanoparticles. Another scientist, Svedberg introduced ultracentrifuge and through it demonstrated the dependence of the motion of macromolecules (colloids) on their shape and size (15). In the middle ages, colloidal gold was used for curing wide variety of diseases such as arthritis, heart disorders, epilepsy, dysentery, venereal diseases and tumors and also for the diagnosis of syphilis, a method which was used until the 20th century. Towards the end of 16th century, colloidal gold was

Biomedical applications of gold nanoparticles routinely used to make ruby glass and for coloring ceramics by techniques which are still used for such purpose (14). SYNTHESIS OF GOLD COLLOIDS Gold nanospheres were the first ones to be discovered and studied, followed by the rest of the subtypes. Based upon shape, size and physical properties the AuNPs are categorised into different subtypes: nanospheres, nanorods, nanoshells and nanocages Also, AuNPs which have great surface-enhanced Raman-scattering properties are called “SERS nanoparticles” (1, 13, 15). (Fig. 1) GOLD NANOSPHERES It was proposed a method for the synthesis of gold nanospheres of 2 nm to 100 nm diameter which was based on the reduction of aqueous tetrachloroauric acid (HAuCl4) with sodium citrate in water at 90-100°C. The reducing agent and the experimental condition can be varied as desired. As this procedure is reasonably simple and environmentally friendly, it is the most commonly used method for the synthesis of nanospheres. By varying the molar citrate:gold ratio, the size of the nanospheres can be restrained (1, 14, 16, 17). Usually, small amounts of citrate will generate larger nanospheres (1, 14). The nanospheres obtained through this process generally have a broad distribution of size and shape. Mostly, the nanoparticles produced fall in the size range of 12- 60 nm with a relative size distribution of 10-15% and show a non-uniform and irregular shape such as ellipsoids, quasi-spheres, and triangles (16). However, this method has limitation of restricted use of water as the solvent and low yield (1, 14). A second method known as ‘Burst method’ is capable of producing thermally and air stable nanospheres which are more mono-dispersed and their size can be controlled up to < 10 nm of diameter (1, 14, 17). In this method, AuCl4- is transferred

25 using a phase transfer reagent tetraoctylammonium bromide from the aqueous solution to toluene and then reduced in the presence of dodecanethiol (C12­­­­H25SH) with aqueous sodium borohydride. The ratio of thiol: gold controls the size outcome of the nanospheres (16). A larger thiol:gold ratio and the faster addition of the reducing agent under cold conditions will produce smaller and more mono-dispersed nanospheres (1, 14). GOLD NANORODS There is a wide variety of strategies proposed for the synthesis of Au-nanorods. The most commonly used method for production of Au-nanorods is the ‘Seed-mediated synthesis’. The chemical reduction of gold salt takes place in the presence of a strong reducing agent like sodium borohydride (NaBH4) forming gold seeds. Then, these seeds are added to a gold salt solution containing a weak reducing agent such as hexadecyltrimethylammonium bromide and ascorbic acid for continuation of growth. The Au seeds formed in the solution with strong reducing agent acts as site for nucleation for formation of nanorods (1, 14, 17-19). The ratio of gold seeds:gold precursor is important for controlling the length to width ratio of the nanorods (20, 21). Furthermore, with the addition of AgNO3 to the solution, the yield could be greatly increased (22). The electrochemical deposition of gold into pores of the nanoporous polycarbamate or alumina template membranes is an emblematic method of synthesis of nanorods. This method is called ‘template method’. The pore diameter of the template membrane is the pre-determinant for the nanorod diameter, while the amount of gold deposited within the membrane pores is the pre-determinant for the nanorod length. A low yield due to formation of only a single monolayer of the Au-nanorods is a fundamental disadvantage of this method. Apart from the above described methods, several other procedures: namely growth of nanorods on mica surface,

Figure 1. Schematic illustrations of different types of gold nanoparticles. Biomed Rev 26, 2015

26 bio-reduction, and photochemical synthesis as well as electrochemical synthesis for the growth and fabrication of nanorods have been explored (1, 14). GOLD NANOSHELLS Gold nanoshells are a type of spherical nanoparticle which consists of a dielectric core of materials such as silica, polystyrene or sodium sulphide coated by a thin layer of metal (usually Au) (8, 23). Silica cores are grown by the reduction of tetraethyl orthosilicate in ethanol through the process developed by Stöber. The silica nanoparticles are coated with gold by using a seeded growth technique. An amine-terminated silane is used as a liner molecule for the attachment of small gold nanospheres of diameter 2-4 nm to the silica core. This allows the additional gold molecules to be reduced until the seed particles are consolidated into a complete shell. The diameter of the silica core and the amount if gold deposited on the surface of the core determines the diameter of the gold nanoshell and the shell thickness, respectively. Other methods of production of gold nanoshell are being continuously investigated. The thermo-sensitive core-shell particles can be used as the template for in situ synthesis. For controlling the thickness of the Au nanoshell along with significant reduction of particle aggregation, the micro-gel can be used as core material (1, 14, 19, 23). GOLD NANOCAGES Gold nanocages containing controlled pore size on their surface were synthesized in 2006 by the galavanic replacement reaction between aqueous HAuCl4 and truncated- silver nanocubes. The silver nanostructures can be developed by polyol reduction, in which AgNO3 is reduced by ethylene glycol to develop silver atoms and then nanocrystals or seeds. The morphology of nanocrystals can be controlled by experimental conditions. Through the addition of more silver atoms the desired seeds can be produced. This can be done by controlling the silver seed crystalline structures in the presence of poly-(vinylpyrrolidone). Poly-(vinylpyyrolidone) is a polymer capable of selectively binding to the surface (14, 19, 24). The galvanic replacement process is used to remodel the silver nanostructures into internal hollow space within Au nanostructures. Thus, it acts as a sacrificial template for the synthesis of Au nanocages (1, 9). The dimension and wall thickness of the resultant Au nanocages can be precisely controlled by adjusting the molar ratio of silver : HAuCl4 (1). Biomed Rev 26, 2015

Singh, Kaur, Kaur, Buttar and Bhullar Au nanocages may provide some major advantages such as: (i) their surface plasmon resonance peaks could be adjusted by changing the ratio of Ag nanocubes : HAuCl 4. Thus, covering the entire spectral region from 500 to 1200 nm, (ii) their absorption coefficients could be varied by controlling the number of truncated corners and void sizes, (iii) the Au nanocages could still show resonance peaks in the near-IR region with remarkably small size of about 50 nm, and (iv) surface modifications of Au nanocages could be achieved and employed in various biomedical applications (14). SURFACE ENHANCED RAMAN SCATTERING NANOPARTICLES Surface enhanced Raman scattering (SERS) is a technique in which the nanoparticles are labeled with groups having activity in the Raman spectral region. In 2002, a new method was reported in which gold nanospheres of diameter ~13 nm were modified with a group cyanine-3. To monitor the presence of specific target DNA strands, alkythiol-capped oligonucleotide strands were used as probes. Also, in one study conducted in 2008, gold nanospheres of diameter ~60 nm were encased with a Raman reporter and then stabilized with a layer of thiolated polyethylene glycol. This technique appears to have more merits than that of other conventional ones, such as chemiluminescence and fluorescence. It provides better sensitivity, robustness, superior performance in blood, high levels of multiplexing and other biological matrices (13-15).

Cellular uptake and fate of gold nanoparticles Cellular uptake of nanoparticles is mainly through endocytosis. For nanostructures, it is considered to be via receptor-mediated endocytosis involving an interaction between cell membrane receptors and ligands on the surface of the NPs (30). The cellular uptake and the intracellular fate of the nanoparticles largely depend on size, surface functionality, shape, charge and hydrophobicity (25, 30). The optimal sized nanoparticles have smallest internalization time. The particles of diameter 20-50 nm have most efficient uptake while apoptosis is enhanced in 40-50 nm range. The variation in curvature of the AuNPs is the reason for shape dependent uptake, rod-shaped AuNPs have lower uptake than their spherical counterparts. The positively charged AuNPs have greater uptake efficiency than that for neutral and negatively charged AuNPs (26-29). In depth understanding regarding the uptake and accumulation of nanoparticles in living systems is vital for the successful usage in pharmaceuticals. The cellular uptake is often influenced by the surface charge of the carrier and hydrodynamic radius of

Biomedical applications of gold nanoparticles nanoparticles. The route of administration also determines the biodistribution of nanoparticles. Generally, the nanoparticles have a more prolonged retention in the lymph nodes, in comparison to delivery via intramuscular, subcutaneous or topical routes (31). BIOMEDICAL APPLICATIONS OF GOLD NANOPARTICLES

As a Drug Delivery System Drug delivery systems (DDSs) are used to increase the efficacy of a wide variety of pharmaceutical payloads, including small drug molecules or large biomolecules such as proteins and genetic materials. DDSs provide the positive characteristics to the ‘free’ payload through enhancing solubility, biodistribution, in vivo stability and pharmacokinetic properties. DDSs can be functionalized with ligands to target a specific tissue or cell type, providing a targeted drug delivery system (32-37). Furthermore, they can be loaded with huge amounts of drugs to act as reservoirs, rendering controlled and sustained release of drugs to maintain the levels within the therapeutic window (32, 33). The AuNPs have been effectively utilised as a drug delivery system. The transport and release factors play a critical role in the effectiveness of a drug delivery system. The drug can be loaded onto the nanosystem in two ways, either by covalent conjugation or non-covalent conjugation. Covalent conjugation usually requires intracellular processing of a pro-drug, whereas non-covalent conjugation employs active drug itself (9,25). Out of the several approaches used for fabrication of the AuNPs, the most common ones that are employed for targeted delivery applications includes surface modification of AuNPs with cationic polymers or reactive functional groups (e.g. amine, carboxyl and thiol groups). Another approach is drug encapsulation on AuNPs using layerby-layer (LbL) conjugation. These complexes provide better protection of the encapsulated drug from enzymatic metabolic degradation, allowing prolonged half-life (t1/2) and improved drug efficacy. Through the LbL approach, the amount of drug loaded can be efficiently controlled depending on the number of layers. Further, the delivery to a target cell can be achieved by immobilization of cell-specific targeting molecule onto the AuNP surface (38). For therapy, the release of drug molecule from nanoparticle is a prerequisite and can be triggered by internal (e.g. pH or glutathione, GSH) or external (light) stimuli. Internal stimulus controls the release in a biological manner whereas external stimulus has a spatio-temporal control over the release (16, 39). The release in case of covalent conjugation is well Biomed Rev 26, 2015

27 controlled, while drug molecules that are non-covalently conjugated suffer non-specific release or other types of interactions. Endogenously triggered release of AuNPs is based on glutathione (GSH) concentration in the plasma or across the cell membrane (9, 16). There exists a substantially higher intracellular GSH concentration (1-10 mM) compared to the extracellular thiol concentration (GSH 2µM, cysteine 8µM) and this higher intercellular concentration is exploited by the GSH-mediated DDS. The pro-drug bound to the AuNPs can be either released via place-exchange reactions or via disulphide exchange. Moreover, surface cysteine of proteins in the bloodstream can participate in thiol-disulfide exchange, resulting in protein carrier conjugates which can alter bioavailability and pharmacokinetic properties (3, 16, 39-41). The monolayer of the nanoparticles can provide steric shielding against this interaction, hence enabling their use in vivo. Externally controlled release of payload is a corresponding tool for site and time dependent delivery (40, 41). The IR radiation triggers local heating of the metal nanoparticles which can result in the controlled release of the therapeutic agents or dyes bound to the particles. When there is adsorption of the molecule onto the surface of the nanoparticle, the temperature increase would often result in desorption of the less stably conjugated molecule. This property of metal nanoparticles can be utilized in development of light regulated photothermal release system. These systems can be of two types: (i) direct desorption of the payload, and (ii) use of a thermally responsible polymer for triggering the release (16, 17).

Application of AuNPs for gene delivery Gene therapy is envisioned an excellent approach for the treatment of acquired and genetic diseases. Induction of target gene expression and protein synthesis can be achieved by use of DNA or RNA vectors (25). Viral and non-viral systems have been most frequently used for gene delivery. However, the viral and non-viral systems have limitation for causing immune responses and low transfection efficiency, respectively. Gold nanoparticles are considered suitable candidates for gene delivery, since they not only provide a high surface-to-volume ratio but also have a small size that maximises payload to carrier ratio (38, 39). With PEGylated AuNPs, gene expression is enhanced by about 100-folds as compared to naked DNA (9, 42-47). This is so because the PEGylation under the normal cell culture conditions stabilizes the AuNP/DNA complex by providing it

28 with a protective layer for a longer time period. This procedure helps to increase transgene expression and reduces cytotoxicity (38). These functionalized nanoparticles are also used for the delivery of amino acids and small interfering RNA (siRNA) (9, 42-49). It has been reported that mixed monolayer protected gold clusters (MMPCs) functionalized to amine containing alkyl chain exhibit effective intracellular plasmid DNA delivery and induction of target protein synthesis. The in vivo gene transfection was determined by charge density of MMPCs and alkyl chain length used in fabrication, whereas the protein induction was ascertained by MMPCs to pDNA ratio. The monolayer coverage of MMPCs and MPCs allow regulation of hydrophobicity and charge to maximize transfection efficiency and minimize toxicity (38, 41). A considerably superior transfection vector is provided by the amphiphilic particles which demonstrate that the hydrophobicity of nanoparticles improves the efficiency of cellular uptake and/ or the consequent release of the DNA from endosomal vesicles (39). AuNPs functionalized with polyethyleneimine (PEI) and chitosan provides a higher transfection efficiency for gene delivery, so these are used as a delivery vector with reduced toxicity in rabbit cornea (9, 48, 49). The siRNA delivery extensively exploits the use of AuNPs as a nanocarrier. Terminal end of the siRNA was modified to contain a functional thiol group for extensive immobilization of siRNA on the AuNPs. Thiol-modified siRNA through Authiol interactions exhibited a strong adsorption on the surface of AuNPs. When this nanocomplex is transported into the cell, free siRNA is released by the reductive cytoplasmic environment and finally it triggers the RNA interference. In addition, the end-functionalized siRNA can also be coupled to the polymer coated AuNPs through chemical linkages. Acidliable or reducible bonds can be used for selective delivery of conjugated siRNAs under acid-pH or reductive environment of the cells (38). The photo-cleavable linkers are also used for the delivery of adsorbed genetic material. The property of AuNPs heating on absorption of IR radiation is utilized for cleavage of the sensitive linkers. A photoliable nanoparticle was designed to convert the nature of surface from cationic to anionic upon irradiation. The particles were designed to have a photo-cleavable o-nitrobenzyl ester moiety and a quaternary ammonium salt as the end group. On irradiation, the linker cleaves to create an anionic carboxylate group and releases the DNA (39).

Biomed Rev 26, 2015

Singh, Kaur, Kaur, Buttar and Bhullar Application of AuNPs for chemical drug delivery AuNPs are functionalized with various chemical drug entities and biomolecules for specific destruction of cancer and bacterial cells. Several of the anticancer drugs such as methotrexate, adriamycin, paclitaxel and doxorubicin have been studied for delivery through AuNP conjugation (3, 28). Tumor necrosis factor-alpha (TNF-α), has been investigated for anticancer therapy through the usage of nanoparticles. In the nanoparticle delivery system containing a PEG coated AuNP onto which TNF-α is loaded. This approach leads to maximal tumor damage and minimal cytotoxicity. Also, the nanoparticles based delivery coupled with local heating results in enhanced therapeutic efficacy (1, 14). AuNPs having high surface to volume ratio, inherent low toxicity, and sustainable stability provide them with qualities suitable for the design of new drug delivery strategies. The optimization of characteristics such as non-immunogenicity, and enhanced cellular bioavailability are the key issues which need to be kept in mind during the engineering of particle surface. Such novel approach would serve as a useful alternative tool for the traditional delivery systems. Thus, the AuNPs seem to be an emerging framework for drug and gene delivery systems (38).

Applications of nanotechnology for cancer diagnosis and therapy Anti-cancer nanotechnology is a versatile area which has potential applications in battling different types of cancers, including molecular diagnosis, molecular imaging, bioinformatics and targeted drug delivery. The growth of anticancer nanotechnology carries the potential for personalized oncology. Thus, it may someday be helpful in such a way that protein and genetic biomarkers could be used for the diagnosis and treatment of cancer on the basis of molecular profiles of each individual patient (1). Gold nanoparticles are being extensively researched for their applications in cancer diagnosis and treatment. In order to optimize the structures of nanoparticle so as to become more efficient diagnostic and therapeutic tools, it is extremely crucial to have understanding of their behavior in the tumor microenvironment. Penetration and accumulation in the hypoxic centers of cancer cells for therapeutics is often difficult and attributed to the large intercapillary distances and variable blood flow typically in solid tumors. Whereas, the outer shell of solid tumor is often the site for angiogenesis and improper lymphatic flow. Angiogenic blood vessels have gaps of nearly 600 nm. The

Biomedical applications of gold nanoparticles vascularized regions are liable to be ‘leaky’ and resulting in enhanced permeability and retention effects. Owing to their hyperpermeability, tumor cells passively absorb the nanoparticles leading in high concentration (nearly 10-fold more) of the carrier in the tumor tissue. Also, the photothermal properties of nanoparticles help in the active targeting of the molecules and therapeutic agents into the tumor cells (1, 9, 31, 38, 42-57).

Cancer diagnosis The optical properties of AuNPs along with their ability to form conjugates have been utilized widely in the diagnosis of cancer. Functionalised AuNPs with oligonucleotides, peptides, flourophores, aptamers, antibodies, organic dyes or other biomolecules have been studied for holding a promising role in imaging of cancer cells (42-49). Oligonucleotide conjugated AuNPs have been reported for the detection of polynucleotides and proteins (like p53, a tumor suppressor gene) with the help of various detection methods, for example, gel electrophoresis, chronocolormetry, atomic force microscopy, amplified voltametric detection, scanometric assay, SPR imaging and Raman spectroscopy (1). Gold nanoparticles having a glutathione cap (GSH) with COOH grouping and fluorescein isothiocynate (FITC) tag along with folic acid were used to target carcinoma cells. Due to the expression of folic acid receptors on the HeLa cells, the AuNPs interact only with them, thus, providing a sensitive and easy method for detection of cancer cells, and differentiating them from non-cancerous cells (9, 42, 46-49). The various neuroendocrine carcinomas overexpress somatostatin receptors. This trait has been exploited for the detection of these carcinomas by use of functionalized AuNPs with octreotide peptide, a synthetic analogue of somatostatin. The development of bioimaging agents based on the use of such receptors can help in tumor diagnosis at an early stage. Owing to the increased capacity of recognized as the protein receptor and enhanced fluorescence properties, octreotide functionalized AuNPs show more interaction than the AuNPs alone towards the tumor cells (42-49, 58). Likewise, coumarin dye and PEG AuNPs show effective internalization into the human breast cancer cells. The dual functionalization of fluorescent dye along with biomolecule can be used for bioimaging and subsequent drug delivery (9, 42-49). Monoclonal antibodies (mAb) conjugation has also been helpful in the imaging of carcinomas. A PEG modified nanoparticle with covalently conjugated mAb Herceptin has Biomed Rev 26, 2015

29 enabled detection of breast carcinoma. Similarly, other mAb can also be used for diagnosis of various cancers (3, 9). There are other techniques for visualization of carcinomas, including Multiphoton Plasmon resonance microscopy, photoacoustic tomography (PAT), third-harmonic microscopy and optical coherence microscopy. These are new and promising techniques in the field of cancer diagnosis (9). PAT is a hybrid imaging technique that utilises light for rapid heating of elements within the tissue, leading to the production of photoacoustic wave (because of thermoelastic expansion) generation that can be detected with an ultrasonic transducer. The NIR-absorbing gold nanoparticles can significantly enhance the image contrast when used in this technique, due to the more considerable differences in optical absorption (hence generating a stronger photoacoustic wave) than the endogenous tissue chromophores. Raman spectroscopy is another but most promising imaging technique with respect to gold nanoparticlebased contrast agent. The SERS nanoparticles consisting of a gold core, a Raman-active molecular layer, and an outermost silica coating were used for Raman imaging in vivo. Multiple SERS nanoparticles with different NIR absorption wavelengths can allow concurrent imaging of many tumor markers, and may have considerable potential in clinical applications (1).

Cancer therapy Conventional treatment methods for cancer therapy include radiation therapy, chemotherapy and surgery. AuNPs due to their unique properties have received most attention for cancer therapy (1). The AuNP conjugates have been studied for enhancement in outcomes of radiotherapy as well as in a light-based therapy against cancer. Use of conjugated AuNPs ensures selective destruction of the targeted tumor cells. Radiotherapy has been widely used tool for cancer treatment in the past with the main objective for destruction of cancer cell DNA strands. The goal was achieved through irradiation of cancer cells by focussing highly energetic and penetrating gamma-rays, X-rays, photon and electron radiations. AuNPs have a higher probability of absorption of radiation and production of photoelectrons, X-ray fluorescence and Auger electrons as they contain heavy atoms with a large amount of electrons. Photoelectrons have better penetration through cells to produce their cytostatic effects. Thus, it provides an enhancement to the dose effectiveness of radiation in comparison to radiotherapy used solely (59). Unfortunately, the normal cells are also adversely affected by radiotherapy. The local surface plasmon resonance property of AuNPs

30 was explored for therapeutic purposes due to photothermal therapy (PTT) of tumor cells and design of light-triggered drug delivery (LTDD) system (59). In photothermal therapy, upon exposure to NIR light, the AuNPs convert absorbed light to heat via electron-electron collision leading to hot electrons. This heating effect with the use of AuNPs can cause cell death due to the alteration of normal cellular mechanism via apoptotic mechanism. A further increase in heating could result in cell cycle disruption (10, 11, 16, 59). There are in vitro studies that illustrate the use of various antibodies which are capable of producing selective destruction of the targeted cell. A comparison amongst the various types of nanoparticles, gold nanorods shows optimal light absorption and heat transduction. Also, they are the most efficient photothermal contrast agents, with the highest efficiency of light absorption and heat transduction. Another application of optical properties of AuNPs is LTDD. In this system, AuNPs should be capped with agents such as biomolecules, polymers, molecular species, etc., which can act as reservoirs for the heat-generated release of chemotherapeutic drug on irradiation. These cytotoxic drugs can be loaded onto the nanoparticles by use of several strategies such as surface modification of the AuNPs to allow interaction with the drug that either supramolecular or electrostatic, hollow nanostructures designed to store drug in them like nanoshells and nanocages and insertion of nanoparticles into a highly thermosensitive structure like liposomes and temperature responsive hydrogels. The best designed photoactive Aubased nanocarriers is gold nanocage-thermosensitive gel and porous-gold-nanoshell-PEG. They are capable of storing large amounts of almost any drug molecule and are biologically inert. Both of these designs are NIR photoactive, with sizes (40 - 60 nm) that allow using the EPR effect to reach the tumor cells (59).

Detection of biological molecules Based on the divergent and unique properties as well as using various strategies, AuNPs may be used for the detection of biological molecules like enzymes, DNA, proteins, antigens and antibodies (13, 29). The AuNP’s use in detection of oligonucleotide was first found by Mirkin and colleagues. They found that single stranded oligonucleotide targets could be detected by use of two dissimilar Au-nanoprobes (thiol-linked functionalized oligonucleotide). Each of the Au-nanoprobes was functionalized with oligonucleotides that were complementary to one of the target oligonucleotide (38). Biomed Rev 26, 2015

Singh, Kaur, Kaur, Buttar and Bhullar Upon introduction of the target into the Au-nanoprobe solution resulted in a colour change from red-to-blue. The change was due to formation of polymeric network (cross linking mechanism), which led to the nanoparticles close enough to produce colour change. This method has been expanded to a real time screening assay for endonuclease activity. Another easy-to-use and inexpensive assay was discovered which utilizes an increase in the salt concentration to induce aggregation to Au-nanoprobes. Here, the aggregation of Aunanoprobes does not result in the presence of complementary strands, rather occurs when non-complementary or mismatched targets are present. This method was also used in the detection of eukaryotic gene expression without the use of PCR amplification or retro-transcription steps (18, 38). Another domain for the use of AuNPs is for detection of proteins on the basis of their characteristic surface plasmons. For this purpose a bifunctional molecule is needed which is conjugated to AuNP on one side via thiol conjugation and on the other side to electron rich aromatic side of protein via diazonium moiety. The nanoparticle acting as a Raman marker enhances the vibration of the diazo-bond between the protein and the bifunctional molecule. Finally, protein could be detected by using surface enhanced Raman spectroscopy. Thrombin was used as a protein to test this method (9, 42-49). A recent involvement of the Au-nanoprobe cross-linking method is a strategy called bio-barcode assay. For the detection of proteins it shows attomolar sensitivity, while for DNA detection it has zeptomolar sensitivity (18). In this method, the analyte either protein or DNA is attached to a magnetic microparticle displaying recognition elements. Next, it attachs to a functionalised AuNP having a second recognition agent and ‘barcode’ DNA strands which are the markers, forming a sandwich complex (72). Separation of the complex is done by means of magnetic methods. In the end, the DNA barcodes are released and the analyte which is either a protein or a DNA is detected as well as quantified by the use of Au-nanoprobe sandwich assay and scanometric detection (using silver enhancement). The conventional bio-barcode assays are expensive and laborious, and depending upon sophisticated instruments, modifications have been developed that utilizes colorimetric, electrochemical, chemiluminescent and fluorimetric detection techniques (18). A successful use of this method has been studied in the detection and measurement of concentrations of amyloid-β-derived diffusible ligand, a potential marker present at cerebrospinal fluid  (CSF) of Alzheimer’s disease affected patients (72).

Biomedical applications of gold nanoparticles

31

Table 1. Biomedical applications of polymeric nanomaterial loaded with AuNPs Polymeric nanomaterial loaded with AuNPs

Applications

Reference

5-Aminolevulinic acid incorporated poly (vinyl alcohol) nanofibre

Colon cancer

(60)

Indomethacin-eluting stent

Tracheal regeneration

(61)

PLGA/collagen with PLGA/drug as nano-fibre core

Repairing/healing wounds

(62)

Drug-eluting nanofibres

Contraception

(63)

Biodegradable PLGA nanofibrous scaffolds with Vancomycin

Brain tissue drug delivery

(64, 65)

Biodegradable PLGA nanofibrous scaffolds with lidocaine

Epidural space drug delivery applications

(66)

Paclitaxel-eluting PCL nano-fibrous structure

Benign cardiac stricture

(67)

AuNPs loaded BSA/PVA nano-fibrous scaffolds

Cardiac tissue regeneration

(68)

AuNPs incorporated in polymethylglutarimide (PMGI) nano-fibers

Enhancing attachment and differentiation of mammalian cells, enhancing HeLa cell attachment and potentiating cardiomyocyte differentiation of human pluripotent stem cells

(69)

Chitin nano-fibers (CNFs) with metallic gold nanoparticles (AuNPs)

Cosmetic, pharmaceuticals, catalysts, electrical, electronic and optical devices.

(70)

Stretchable poly ( -caprolactone) (PCL) gold nano particles coated force sensors

Hand rehabilitation

(70, 71)

Another usefulness of AuNPs has been for the detection of biologically active materials such as vitamin E and aflatoxin A. Vitamin E analogue functionalised with AuNPs via selfassembly of thiol ligand was used to evaluate the free radical scavenging activity. For detection of aflatoxins, antibodies against AFB1 functionalised on AuNPs using electrodeposition on cysteamine functionalized AuNPs were used. Detection of aflatoxin AFB1 was highly efficient and required less response time (9, 46-49).

Detection of microorganisms AuNPs have been used for the detection bacteria and Biomed Rev 26, 2015

viruses by using various biological, molecular and microbiological methods (18, 42-49). An application for the use of Au-nanoprobes was for the detection of Microbacterium tuberculosis (MTB) and its complex (MTBC). This consists of a AuNP-based colorimetric assay which involves detection of nested PCR-amplified DNA targets of MTB and MTBC by using two probes generated with thiol-modified oligonucleotides conjugated AuNPs. On hybridisation of the two probes with their target caused aggregation of AuNPs and consequently resulted in change of colour from red to blue/purple. A similar colorimetric assay was developed for detection

32 of unamplified HCV (define HCV) RNA from clinical specimens. After extraction of HCV RNA from the patient serum, it was mixed with a specific oligonucleotide sequence in a salt containing hybridization buffer. Further, the mixture was denatured, annealed, and cooled to room temperature, then unmodified 15 nm AuNPs were added to it. The solution changed its colour to blue if there was aggregation of AuNPs in HCV positive specimens, while remained red in the negative specimens. There are various other examples for detection of microorganisms with the use of AuNPs (18, 47-49).

AuNP biosensors for detection of disease etiology An array of biosensors has been developed for basic research and application of AuNPs for detecting specific biomolecules involved in disease etiology. A nanosensor was used for the detection of cholesterol by immobilizing cholesterol oxidase on the basis of amperometric changes (9, 42-49, 73). The nanosensors used were made from gold-platinum alloy nanoparticles and showed to have high sensitivity, selectivity, fast response and good reproducibility. The principle of the detection was based upon hydrogen peroxide activity. Further, AuNP can aid detection of uric acid. Uric acid is a crucial metabolite of purines and its abnormal levels can lead to various metabolic disorders like cardiovascular diseases, gout, kidney damage, pneumonia, hyperuriceamia, and Lesch-Nyhan syndrome. The detection can be achieved by using amperometric methods, which are superior to several other methods, including enzymatic, colorimetric and electrochemical methods in a way that it can detect uric acid in whole blood, serum, and urine with detection limit as low as 50 nM (9, 74). Also, AuNPs have been developed for the determination of choline in various human samples as well as in colorimetric biosensors for the detection of proteinase activity assay (9, 43-48). Several ‘universal’ colorimetric biosensor methods have been devised for the detection of a wide range of targets including proteins, nucleic acids, DNA, small molecules, and ions. Unmodified AuNPs, ssDNA probe, and a conjugated water soluble cationic polyelectrolyte are used in this biosensor. The colourimetric technique employed in the detection is the sequestration of the conjugated polyelectrolyte to ssDNA probe that causes aggregation of the AuNPs to produce a colour change from red to blue. This happens because the polyelectrolyte is not present to stabilise the nanoparticle. When a complementary target is present, a weak bond is formed between the probe target dsDNA and conjugated Biomed Rev 26, 2015

Singh, Kaur, Kaur, Buttar and Bhullar polyelectrolyte. Now, the polyelectrolyte remains free to stabilise the AuNP against aggregation. Target concentration can be determined by the use of absorbance ratio. This method has also been used to detect the aptamers which bind to specific molecular targets. Thrombin aptamer, anti-cocaine aptamer, and a mercury-responsive sequence that folds in the presence of Hg (II) were used in this assay for the detection of their respective targets (18).

Single nucleotide polymorphisms detection Single nucleotide polymorphisms (SNPs) detection is also an extension of the AuNP’s applications. This method is appropriate for the detection of point mutation and polymorphisms in various genes associated with diseases and metabolic disorders. The technique is used to assist in finding the explanation behind individual genetic variability, which is correlated with individual susceptibility to various diseases like β-thalessemia, diabetes mellitus, some cancers and individual responses to therapeutic agents. One of the most successful uses of nanoprobes method was in the clinical diagnosis of Mycobaterial tuberculosis. Another system utilised functionalised AuNP for the detection and characterisation of human p53 gene. This technique depicts potential for cancer diagnosis (9, 18, 72).

Immunoassay AuNPs have been applied in the designing of various immunoassays. They have shown potential in the enhancement of standard enzyme-linked immunosorbent assays (ELISA) signals via conjugation with antibodies or coupling with sliver enhancement. Besides ELISA, other techniques such as electrochemical methods, conductometery, colorimetry etc. have also been used for the detection purposes. AuNPs have been studied for the novel enhancement of immunochromatographic strips which increase the detection limit of hCG (72). Here, both the primary and the secondary antibodies were conjugated to AuNPs. A biochip detection of protein was designed using AuNPs and silver enhancement. In this procedure antigen is sandwiched in between immobilised antibody (antibody immobilised onto the surface of a chip with an electrode array using standard micro-electro mechanical systems-MEMS technology) and another AuNP labelled antibody. On addition of silver enhancement solution, reduction of silver ions to metallic silver with hydroquinone catalysed by AuNP occurs, followed by deposition of metallic silver onto the surface of AuNPs increasing the size of the particles. The final product results in the easy detection of

Biomedical applications of gold nanoparticles change in electric conduction and determination of protein via conductometric methods (18). The new sensing platforms were designed to replace the standard ELISA method as they do not demand secondary antibody as well as allows increase in sensitivity. The use of mono- and poly-clonal antibodies has been replaced by the immunosensors utilising single chain fragment variable recombinant antibodies (scFv). These scFvs are small heterodimers consisting of antibodies with both variable heavy (VH) and light (LH) chains linked by a stabilising peptide linker. A colorimetric assay was developed with the use of scFv functionalised AuNPs having a cysteine or histidine in its linker region. The application of AuNPs in another immunoassay is under development and is considered to have a great potential for future use (9, 75).

Enzyme immobilisation Enzyme immobilisation matrices have been made using AuNPs. The ability of the AuNPs is utilised to attach and functionalise the enzymes, and this process makes the enzymes more thermally stable in comparison to the free enzyme. Additionally, hollow nanospheres containing active enzymes have been synthesised. By this method, enzymes remain active for a long period of time and it helps in the detection of small molecules which can enter in the nanoshells (9, 43-50). CONCLUSION The novel development of AuNPs for drug or gene carriers and as molecular nanoprobes provides a useful bridging tool over the traditional delivery vehicles. AuNPs represent an immense scope in biomedical applications due their unique properties of high surface/volume ratio, low inherent toxicity and surface engineering. The availability of different synthetic methods for creating AuNPs of different sizes and shapes bestows a versatile toolbox for the fabrication of conjugates with enhanced affinity for targeted cell receptors, coherent internalisation, high biocompatibility, enhanced permeation into tumor cells, and long circulation half-life (t1/2) of conjugates or medicaments. Furthermore, the optical and electronic properties of AuNPs are extremely useful for monitoring and detecting the biological molecules microbes of interest. Recent research on the successful utilisation of AuNPs in diagnosis of some diseases and targeted drug delivery system has set a platform for the future development of new applications in biomedical sciences. Overall, the AuNPs offer a bright scope for the diagnosis and treatment of diseases. Biomed Rev 26, 2015

33 However, there are important issues that need attention such as immune response, unwanted cytotoxicity, and long-term adverse consequences. Conflict of interest: the authors declare no conflict of interest. REFERENCES 1. Cai W, Gao T, Hong H, Sun J. Application of Au nanoparticles in cancer nanotechnology. Nanotechnol Sci Appl 2008; 1:17-32. DOI: 10.2147/NSA.S3788 2. Pankhurst QA, Connolly J, Jones SK, Dobson J. Applications of magnetic nanoparticles in biomedicine. J Phys Appl Phys 2003; 36:R167-R181. DOI: 10.1088/0022-3727/36/13/201 3. Mazzola L. Commercializing nanotechnology. Nat Biotechnol 2003; 21:1137-1143. DOI:10.1038/NBT10031137 4. Parak WJ, Gerion D, Pellegrino T, Zanchet D, Micheel C, Williams CS, et al. Biological applications of colloidal nanocrystals. Nanotechnology 2003; 14:R15-R27. DOI:10.1088/0957-4484/14/7/201 5. Nam JM, Thaxton CC, Mirkin CA. Nanoparticlesbased bio-bar codes for the ultrasensitive detection of proteins. Science 2003; 301:1884-1886. DOI:  10.1126/ science.1088755 6. Reich DH, Tanase M, Hultgren A, Bauer LA, Chen CS, Meyer GJ. Biological applications of multifunctional magnetic nanowires. J Appl Phys 2003; 93:7275-7280. DOI: 10.1063/1.155867 7. El Fray M, Strzalkowska D, Mandoli C, Pagliari F, Di Nardo P, Traversa E. Influence of ceria nanoparticles on chemical structure and properties of segmented polyesters. Mater Sci Eng C Mater Biol Appl 2015; 53:15-22. DOI: 10.1016/j.msec.2015.04.010 8. Roy I, Ohulchanskyy TY, Pudavar HE, Bergey EJ, Oseroff AR, Morgan J, et al. Ceramic-based nanoparticles entrapping water-insoluble photosensitizing anticancer drugs: a novel drug-carrier system for photodynamic therapy. J Am Chem Soc 2003; 125:7860-7865. DOI: 10.1021/ja0343095 9. Raghavendra R, Arunachalam KD, Annamalai SK, Arunachalam AM. Diagnostics and therapeutic application of gold nanoparticles. Int J Pharm Pharm Sci 2014; 6:74-87. 10. Huang X, El-Sayed MA. Gold nanoparticles: Optical properties and implementations in cancer diagnosis and photothermal therapy. J Adv Res 2010; 1:13-28. DOI:10.1016/j.jare.2010.02.002

34 11. Huang X, Jain PK, El-Sayed IH, El-Sayed MA. Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy. Nanomedicine 2007; 2:681-693. DOI:10.2217/17435889.2.5.681 12. Giljohann DA, Seferos DS, Daniel WL, Massich MD, Patel PC, Mirkin CA. Gold nanoparticles for biology and medicine. Angew Chem Int Ed 2010; 49:3280-3294.  DOI: 10.1002/anie.200904359 13. Parida UK , Nayak PL. Biomedical applications of gold nanoparticles: Opportunity and challenges. World J Nano Sci Technol 2012; 1:10-25. DOI: 10.5829/idosi. wjnst.2012.1.2.202 14. Das M, Shim KH, An SSA, Yi DK. Review on gold nanoparticles and their applications. Toxicol Envir Health Sci 2011; 3:193-205. DOI: 10.1007/s13530-011-0109-y 15. Faraday M. The Bakerian Lecture: Experimental Relations of Au (and Other Metals) to Light. Phil Trans R Soc Lond 1857; 147:145-181. 16. Voliani V, Signore G, Nifosí R, Ricci F, Luin S, Beltram F. Smart Delivery and Controlled Drug Release with Gold Nanoparticles. New Front Nanomed Recent Patents Nanomed 2012; 2:34-44. DOI: 10.2174/1877912311202010034 17. Zhou J, Ralston J, Sedev R, Beattie DA. Functionalized gold nanoparticles: Synthesis, structure and colloid stability. J Colloid Interface Sci 2009; 331:251-262. DOI: 10.1016/j.jcis.2008.12.002. 18. Azzazy HME, Mansour MMH, Samir TM, Ricardo F. Gold nanoparticles in the clinical laboratory: principles of preparation and applications. Clin Chem Lab Med 2012; 50:193-209. DOI: 10.1515/CCLM.2011.732. 19. de la Fuente JM, Grazu V. Inorganic nanoparticles vs organic nanoparticles. Nanobiotechnology 2012; 4:3-30. 20. Jana NR, Gearheart L, Murphy CJ. Seed-mediated growth approach for shape-controlled synthesis of spheroidal and rod-like gold nanoparticles using a surfactant template. Adv Mater 2001;13:1389-1393. DOI:  10.1002/15214095(200109)13:18