Nanoparticulate Contrast Agents for Multimodality Molecular Imaging

Review Journal of Biomedical Nanotechnology

Copyright © 2016 American Scientific Publishers All rights reserved Printed in the United States of America

Vol. 12, 1553–1584, 2016 www.aspbs.com/jbn

Nanoparticulate Contrast Agents for Multimodality Molecular Imaging Yang Xia1 , Murukeshan Vadakke Matham1 ∗ , Haibin Su2 , Parasuraman Padmanabhan3 ∗ , and Balázs Gulyás3 1

School of Mechanical and Aerospace Engineering, Center for Optical and Laser Engineering (COLE), Nanyang Technological University (NTU), Singapore, 639798 2 School of Material Science and Engineering, Nanyang Technological University (NTU), Singapore, 639673 3 Lee Kong Chian School of Medicine, Nanyang Technological University (NTU), Singapore, 636921

Molecular imaging is rapidly developing as a powerful tool in research and medical diagnostic. By integrating complementary signal reporters into a single nanoparticulate contrast agent, multimodal molecular imaging can be performed as scalable images with high sensitivity, resolution and specificity. In this review, multifunctional nanoparticles (MFNPs) are classified into four types: conjugation, encapsulation, core/shell, and co-doping. Further, new constructs of MFNPs were reported recently which have used nanoparticulate contrast agent such as quantum dots (QDs), iron oxide nanoparticles (IONPs), Upconversion nanoparticles (UCNPs), carbon based nanoparticles, gold nanoparticles (Au-NPs), Metal-Organic Frameworks (MOFs), dendrimers and porphyrins based nanoparticles. Different surface modification strategies were also Delivered by Ingenta to: Nanyang Technological University developed as well as ligands are attached to those NPs to render theAug biocompatibility and enable specific targeting. These IP: 155.69.24.171 On: Tue, 02 2016 01:40:53 new development in MFNPs are expected to introduce a paradigm shiftPublishers in multi-modal molecular imaging and thereby Copyright: American Scientific opening up an era of personalized medicine and new diagnostic medical imaging tools.

KEYWORDS: Multimodal Imaging, Molecular Imaging, Multifunctional Nanoparticles, Contrast Agents, Surface Functionalization, Theranostic.

CONTENTS Introduction . . . . . . . . . . . . . . . Nanoparticle Design . . . . . . . . . Modalities to be Integrated . . . Configuration of MFNP . . . . . Size . . . . . . . . . . . . . . . . . . Surface Treatment . . . . . . . . . Quantum Dots . . . . . . . . . . . . . Iron Oxide Nanoparticles . . . . . . Lanthanide Upconversion Particles Carbon-Based Nanoprobes . . . . . Carbon Nanotubes . . . . . . . . . Graphene/Graphene Oxide . . . . Carbon Dots . . . . . . . . . . . . Gold Nanoprobes . . . . . . . . . . . Metal-Organic Frameworks . . . . . Porphyrins-Based Nanoprobes . . . Dendrimer-Based Nanoprobes . . . Nanoprobes for Pet Imaging . . . . ∗

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Authors to whom correspondence should be addressed. Emails: [email protected], [email protected] Received: 9 July 2015 Revised/Accepted: 7 September 2015

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Targeting Ligands . . . . . Conclusion . . . . . . . . . Acknowledgement . . . References and Notes .

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INTRODUCTION Over the past decade, molecular imaging has emerged as a powerful tool to investigate the biological activities in the cellular and subcellular level.1–3 Usually, molecular imaging utilizes a specially designed biomarker, which can target to specific cells or molecules, and generate detectable signals. Compared to the clinically available medical imaging techniques, most of them mainly focuses on macroscopic physical, physiological or functional alterations; the multimodal molecular imaging is capable of revealing the underlying molecular and cellular pathway, which is responsible for certain biological phenomena.2 Novel molecular imaging applications are expected to enhance our understanding of

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Yang Xia is currently a Ph.D. student in School of Mechanical and Aerospace Engineering (MAE), Nanyang Technological University (NTU). He received his bachelor’s degree in Material Science and Engineering with first class honor from NTU in 2014 and started his Ph.D. at the same year under the supervision of A/P Vadakke Matham Murukeshan and Professor BalázsGulyás. His main research interests focus on diagnostic molecular imaging and multimodal nanoprobe development.

Murukeshan Vadakke Matham is an Associate Professor with the School of MAE and deputy director of COLE, NTU. His main research interests are biomedical optics, nanoscale optics, and applied optics for metrology. He has published over 250 research articles in leading journals and conference proceedings and has 6 patents and 8 innovations disclosures. He is a Fellow of the Institute of Physics and a member of SPIE.

Haibin Su is an Associate Professor at NTU. He graduated from Stony Brook University, while performing his thesis projects in Center for Data Intensive Computing at Brookhaven National Laboratory. Then he went to California Institute of Technology for taking a 3-year PostDoc position in Professor William A. Goddard’s research center Delivered Ingenta to: His Nanyang Technological University prior to by joining NTU. research interest includes development and application of IP: 155.69.24.171 On: Tue, 02 Aug 2016 01:40:53 theoretical and computational physical science: i.e., quantum-mechanical and classical Copyright: American Scientific Publishers simulations and modeling of the structural, kinetic, and dynamic properties of complex functional systems, in particular emergent collective properties at multiple spatial and temporal scales.

Parasuraman Padmanabhan is a Deputy Director, Translational neuroscience in Lee Kong Chian (LKC) School of Medicine, NTU. His expertise in multimodal molecular imaging approaches in preclinical area of research. He has a very strong and solid scientific background in cell and molecular biology, mizrobial genetics, probe development and molecular imaging. He has had the opportunity in his studies and in his professional scientific career, which had taken him across many destinations to experience a wide range of scientific environmental settings. He has more than twenty five years of research and teaching experience gained from several leading universities, such as Cornell and Stanford University, USA and also from SBIC, a A∗ STAR R&D Research Institute, which is one of the premier institute in Asean region. Balázs Gulyás is a Professor in LKC School of Medicine, NTU. Prior to this appointment, Professor Gulyás spent most of his scientific career at the world-renowned Karolinska Institutet, Stockholm, Sweden. During the past years his research focused on molecular neuroimaging with PET, with special regard to neurological and psychiatric diseases and their “humanised” small animal disease models. Professor Gulyás has published numerous books, written over 35 book chapters and contributed to over 180 research papers in peer reviewed scientific journals. He is a member of Academia Europaea (The Academy of Europe), the Hungarian Academy of Sciences and the Royal Belgian Academy of Medical Science.

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pathological development, facilitate drug discovery and development.4 Currently, multiple modalities are being applied to perform molecular imaging. However, no single modality can provide all the required information due to their inherent limitation. Table I summarizes the advantages and disadvantages of commonly used molecular imaging techniques. The idea of combing two or more complementary modalities together to obtain synergic effect is widely accepted in the research community.1 2 6 7 For example, optical imaging is highly sensitive, and can achieve a temporal resolution in the second scale, but its spatial resolution and in vivo penetration depth are limited. On the other hand magnetic resonance imaging (MRI) is capable to achieve high spatial resolution Table I.

and practically infinite penetration depth.2 By combining these two modalities, High resolution and excellent sensitivity can be obtained simultaneously.6 7 While great effort has been made in designing hybrid molecular imaging instruments (e.g., PET/CT and PET/MRI system.6 8 ), the requirement of a single probe with multiple signaling capacities has arisen. Compared to individual single modality contrast agents, multimodality probe provides better image registration, consistent pharmacokinetics and reduced administration load for the patient.6 Among the few candidates for multimodality imaging probe, multifunctional nanoparticles (MFNPs) has been attracting most of the researchers’ interests due to a number of advantages: stronger and more stable contrast enhancement (e.g., quantum dots vs. fluorescent

Advantages and disadvantages for commonly used molecular imaging techniques.

Imaging technique

Contrast mechanism

Advantages

Optical imaging16–18

Photon emission

• • • •

MRI19–21

Proton density and relaxation time

• • • •

PET22 23

-ray emission

SPECT24 25

-ray emission

CT26

X-ray attenuation

Photoacoustic tomography27 28

Photon absorption

Optical coherence tomography29–31

Light reflection

Ultrasound32 33

Acoustic echo

Highly sensitive High temporal resolution Non-ionizing Low cost

Disadvantages • Low penetration • Relatively low spatial resolution • Less chances for clinics

Unlimited penetration • Low sensitivity High spatial resolution • Long image acquisition Non-ionizing time Capable to image soft • High cost Delivered by Ingenta to: Nanyang Technological University tissue IP: 155.69.24.171 On: Tue, 02 Aug 2016 01:40:53 • Fully translational


Copyright: American Scientific Publishers • Unlimited penetration • Highly sensitive • Capable for quantitative imaging • Fully translational

Contrast agent • • • • •

Luciferin Fluorescent dye Quantum Dots Carbon nanoparticles Lanthanide particles

• Gadolinium • Iron Oxide • Manganese

• Low spatial resolution • Radiation exposure • Cyclotron is needed to produce positron emission isotope • High cost • Radiation exposure • Low spatial resolution

• Non-metal Isotope:18 F,11 C • Metal Isotope:64 Cu,86 Y, 68 Ga.

• Limited molecular imaging application • Radiation exposure

• Iodine • Barium • Gold particles

• Deeper penetration compare to optical imaging • Scalable imaging • Non-ionizing

• Deeper penetration and higher resolution cannot be obtained simultaneously.

• Organic dye • Gold nanoparticles • Carbon Nanotube

• High spatial resolution • Able to extract phase information • Non-ionizing • Real-time imaging • Able to detect cyst and blood flow • Non-ionizing • Low cost

• Limited tissue penetration

• Gold nanoparticles • Microsphere • Magnetic particles (Magnetomotive OCT) • Gas-filled microbubble • Liposomes • Perfluorocarbon droplets

• Unlimited penetration • Can be combined with radiotherapy. • Less expensive than PET • Fully translational • Unlimited penetration • High spatial resolution • Anatomical imaging • Fully translational

• Imaging quality is operator-dependent • Limited contrast agent application

• Isotope: 99m Tc, 111 In, 177 Lu

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dye9 ) large surface area for modification and ligand Modalities to be Integrated attachment,10 prolonged circulation time11 (However, this The modalities that the MFNP can be used should may also lead to increased cytotoxicity12), and better be complementary to each other. The aforementioned tumor targeting through enhanced permeability and retenMRI/Optical integration is one of the most widely studtion (EPR) effect.13 Nanoparticles are normally 100–10000 ied combinations in MFNPs. Other combinations including MRI/PET, Optical/PET and Optical/CT have also been times smaller than cells and hence can be easily interextensively explored. More-over, MFNPs with triple or nalized by cells.14 More excitingly, wide loading ability more signal-reporting capabilities have been synthesized of nanoparticles has made it possible to simultaneously with carefully designed protocols.35–38 A major challenge load diagnostic and therapeutic moiety into one package, 15 in the design of MFNPs is to balance the sensitiviwhich initiate the idea of theranostics. In contrast to the ties between different modalities, which may vary up to conventional “diagnostic followed by therapy” approach, 3 orders of magnitude.39 Luckily, the large surface area theranostics helps to eliminate the discrepancy in biodisof NPs can serve to overcome this problem by controlling tribution and selectivity between separated imaging and the moiety ratio between two contrast agents. It is also therapeutic agent (Fig. 1). The ultimate goal of theranosimportant that the integration of multiple agents should not tics is to shift the current generalized medicine, which produce detrimental effects to the performance factor of applies similar therapy to all patients with the same diseach agent. Different imaging modalities rely on different ease, into an era of personalized medicine, i.e., tailorproperties to generate the contrast and hence their permade therapy for an individual patient. So far, a series formances are evaluated accordingly. For example, MRI of attempts has been reported to integrate MFNPs with contrast agents enhance the contrast by accelerating the agents for Gene therapy, chemotherapy, hyperthermia ther15 relaxation processes of neighboring water protons. There apy, photodynamic therapy, and radionuclide therapy. are two types of relaxation process: longitudinal relaxIn this review, we have summarized some of the recent ation (T1 and transverse relaxation (T2 . One particular progress on developing MFNPs. The main focus is on intecontrast agent mainly accelerates on the relaxation prograted MRI/Optical probes while some probes that can cesses and hence MRI agents can be classified into T1 and be applied in other imaging modalities are also included. T2 agents. The ability of one agent to affect the relaxThose nanoparticles are made either by de novo synthesis ation process is defined as r1 or r2 relaxivity, correspondor by integration of commercially available contrast to: agent. Delivered by Ingenta Nanyang Technological University ing to the two types of relaxations.40 For optical imaging, Most of the nanoprobes described in this review On: wereTue, 02 Aug IP: 155.69.24.171 2016 01:40:53 the quantum yield and excitation/emission wavelength are Copyright: American Scientific Publishers reported in the past 5 years (from 2009). For nanoprobes very important for the imaging resolution, signal-to-noise that have been reported before 2009, several reviews are ratio and penetration depth.9 An MRI/Optical bi-modal already available.6 7 34 probe synthesized by integrating a MRI agent and an optical agent should exhibit similar relaxivity and optical propNANOPARTICLE DESIGN erties compared to the stand-alone contrast agent. While some of the MFNPs used for molecular imaging Configuration of MFNP are produced as by product or ‘happy coincidence’ from researches targeted for non-imaging application, most of We classify the configuration of MFNPs into the followthe reported MFNPs are carefully designed to meet a cering four categories: conjugation, encapsulation, core/shell, tain requirement. The following four factors need to be co-doping (Fig. 2). MFNPs with conjugation or core/shell determined during the design of MFNPs. configuration are normally synthesized through multi-step

Figure 1.

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size control.9 For Gd3+ doped MFNPs, the size of NPs determines the number of surface Gd3+ , and consequently affects the NPs’ relaxivity.43 Surface Treatment Surface treatment is very important to control the MFNPs’ circulation time, biocompatibility and targeting efficiency. Many as synthesized NPs are coated with hydrophobic molecules. For bio-imaging application, those NPs need to be dispersed in an aqueous solution by hydrophilic capping agent. The capping can also provide surface functional groups to conjugate with other functional moieties.44 One widely used capping material is polyethylene glycol (PEG) and its derivatives. Surface PEG layer can shield the nanoparticles from reticuloendothelial system Figure 2. Four MFNPs configuration—(a) Conjugation: two (RES) by minimizing plasma protein binding, and this imaging moieties are conjugated by a bifunctional chemical kind of capped nanoparticle are often referred as stealth linker. (b) Encapsulation: a matrix structure (i.e., micelle or silparticle. PEGlytion is widely used to increase the cirica particle) is used to entrap nanoparticles. (c) Core/Shell: an culation time of NPs.45–47 PEGylation has also been outer shell that possesses different function compare to the used to enhance tumor targeting of NPs due to the core particles. (d) Co-doping: different agents are doped into one host particle, the host particle can also act as one imaging high affinity between cancer cells and PEG cappings.48 49 agent. Other capping agents that do the same thing have also been exploited, including poly acrylic acid (PAA),12 50 polyethyleneimine (PEI),51 and polyhedral oligomeric reaction that could be more time consuming and complisilsesquioxanes (POSS).52 To further enhance the specific cated. Co-doped MFNPs can be prepared by facile onetargeting and internalization of nanoprobe, biorecognition step routes and are generally smaller in size. For different ligands, such as antibodies, peptides and aptamers, are MFNPs, certain configuration may provide the best inteDelivered by Ingenta to: Nanyang Technological University conjugated to the MFNPs’ surface.53 Due to different gration. Appropriate configurationIP: can155.69.24.171 also help to reduce On: Tue,often 02 Aug 2016 01:40:53 surface treatment, MFNPs with the same core size may the interference between contrast agents. However, the Scientific Copyright: American Publishers have much different hydrodynamic diameter, which leads actual configuration of MFNPs may be a mixture of two to different circulation and clearance behavior. of more types. An encapsulation complex can be further conjugated to targeting agent, and a core/shell nanoparticle may also be doped to yield more functions. QUANTUM DOTS Size The size of MFNP plays a major role in determines its biodistribution, clearance, contrast enhancement and the cells that can be targeted. Small nanoparticles (100 nm) are quickly cleared from plasma to the spleen, liver and bone marrow. Hence those NPs should be designed to target those organs. On the other hand, smaller NPs (240 nm for MagFL-PEN) due the multiple encapsulation and linking process. Besides fluorescent probes, IONPs can also be integrated with light absorbing materials to generate MRI/Photoacoustic bimodal probes. Song et al. doped ultra-small IONPs into NIR absorbing polypyrrole (PPy) 1561


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to form IONP@PPy nanocomposites, and then further modified the nanocomposites with PEG to obtain IONP@PPy-PEG nanoparticles. To prove the bimodality of the synthesized nanoparticles, tumor-bearing mice were injected with IONP@PPy-PEG nanoparticles and then imaged in MR and photoacoustic system (Fig. 7). The excellent NIR absorbance of PPy was also utilized for photothermal therapy.77

have lower energy than the absorbed photons. Upconvertion luminescence (UCL) is a phenomenon that light with shorter wavelength is emitted after sequential absorption of longer wavelength light. Lanthanide doped nanoparticles (Ln-NPs) is so far the most widely studied upconversion molecular probe. Ln-NPs are commonly made by doping trivalent lanthanide ions to a host lattice.78 79 Due to the high X-ray attenuation of lanthanide elements, LnNPs can be readily used as Optical/CT bimodal contrast agent.80–82 In addition, Gadolinium ions are often co-doped LANTHANIDE UPCONVERSION PARTICLES with upconverting lanthanide ions to introduce the MR All the previously mentioned optical imaging is based on property, and since doped Gd3+ ions are confined in a more downconversion phenomenon, that is, the emitted photons rigid crystal matrix, it is considered much less toxic compared to the clinically available Gd3+ -chelate complexes.40 Moreover, the Gd3+ ions may enhance the PL by acting as an intermediator which harvest the excitation energy and then transfer it to the nearby luminescent Ln ions.83 84 SPION can be incorporated with Ln-NPs in a core/shell configuration.81 85 Ln-NPs have also been reported being labeled with radioactive isotopes and then used as contrast agent for PET.86 87 or SPECT.82 AREF4 (A-alkali metal, RE-rare earth elements) fluorides such as NaYF4 is considered one of the mostly efficient host materials for upconverting lanthanide due to its lattice consistency with Ln ions, chemical stability and low phonon vibration.79 In order to produce MRI/Optical bimodal probe, NaGdY4 is often used instead Delivered by Ingenta to: Nanyang Technological University of NaYF , in which the IP: 155.69.24.171 On: Tue, 02 Aug 42016 01:40:53 Yttrium ions are substituted by Copyright: American Scientific Publishers Gadolinium ions.88–91 One example of such kind of codoped nanoprobes was reported by Zhou et al.86 The NaY02 Gd06 Yb018 Er002 F4 nanoparticles were synthesized in organic solvent with hydrophobic oleic acid capping. Further ligand exchange replaced the oleic acid coating with hydrophilic citrate coating. The shape of the citrate capped NPs (cit-NPs) are slightly elliptical with average core size of 19–22 nm (determined by TEM) and hydrodynamic size of 28.2 nm (determined by DLS). The citNPs were additionally functionalized by incubation with 18 F containing solution and became a PET/MR/UCL triple functional probe. However, the as-prepared cit-NPs have a green light emission when excited by a CW 980 nm laser. The short penetration of green light has limited the in vivo optical imaging capability of the tri-modality probe. The author conducted in vivo MR and PET imaging on mice but the optical imaging was performed in vitro Figure 7. (a) T2 -weighted MR images and (c) photoacouswith KB cells tissue section. Another work reported by tic images of the tumor bearing mice. The IONP@PPy-PEG the same group88 synthesized Tm3+ /Er3+ /Yb3+ co-doped nanoparticles were injected to the mice intravenously. MR NaGdF4 NPs which demonstrated two types of UCL: NIRimages were taken at various time post injection. Photoacousto-Vis and NIR-to-NIR. The NIR emission is attributed tic images were taken before and one day after the injection. to Tm3+ transitions while the visible emission is caused (b) Quantification of MR signals from the tumor at different time post injection. (d) Quantification of photoacoustic sigby Er3+ transitions. In vivo UCL imaging on mouse and nals from the tumor at 24 h post injection. Reprinted with ex vivo UCL imaging on the harvested organs were conpermission from [77], X. Song, et al., Ultra-small iron oxide ducted. The synthesized NPs provided strong contrast doped polypyrrole nanoparticles for in vivo multimodal imagagainst the background and predominantly accumulated in ing guided photothermal therapy. Adv. Funct. Mater. 24, 1194 in the liver and spleen. One problem with this UCL NPs is (2014). © 2014, John Wiley and Sons. 1562


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the wide size distribution of 25 and 60 nm (hydrodynamic NPs and MR imaging on the respected cell pellet. Their diameter); this could lead to inconsistent biological distriworks provided a good example of combined doping and bution and clearance. The size of NPs also affect the T1 core/shell strategy. A more detailed protocol on how to relaxivity since smaller NPs carry more surface Gd3+ ions, synthesize core/shell, Ln-doped NaGdF4 NPs has been which is responsible for the relaxation enhancement.92 reported by Wang et al. recently.96 The core NaGdF4 was first synthesized by co-precipitation of lanthanide fluoride Li et al. reported Ln ions doped NaGdF4 NPs through and long-chain hydrocarbon, unsaturated fatty acids was a microwave-assisted method.93 The emission wavelength also added as the capping agent to control the particle and intensities can be tuned by manipulating the type and growth. Then the pre-synthesized core NPs was used as concentration of doped Ln ions (Yb3+ , Er3+ , Tm3+ and template according to which the outer shell grew epitaxiHo3+ . The NaGdF4 NPs were further functionalized by ally. Lanthanides doping was achieved concomitantly with encapsulation with amine terminated SiO2 shell (NaGdF4 particle growth, and different Ln ions can be doped into core-40∼65 nm, SiO2 shell–3 nm) and then conjugated different compartments to achieve multi-color emission. with folic acid. The upconversion imaging capability and Cheng et al. integrated Ln-NP with ultrasmall SPION specific targeting to cells with folate receptors was tested through a layer-by-layer self-assembly process.85 PAA on cancer cells (HeLa and SK-OV-3 cells). KGdF4 , another AREF4 type fluoride, has also been uticoated hexagonal Ln-NPs (NaYF4 :Yb3+ /Er3+ were mixed 51 83 94 lized for multimodal bio-imaging application. with dopamine modified ultrasmall SPIONs (5 nm). The Wong SPIONs were attached to the Ln-NPs by electrostatic et al. synthesized Yb3+ , Er3+ doped KGdF4 NPs with attraction. Then negatively charged Au seeds were added polyethyleneimine (PEI) or 6-aminocaproic acid (6AA) and formed an Au shell on the surface of the SPION-LnNP capping through a one-pot hydrothermal reaction.51 The complexes. The synthesized nanoparticles were further sizes of the doped MFNPs are relatively small (14 ± 2 nm functionalized with PEGylation through gold-thiol bondfor PEI capped NPs and 13 ± 2 for 6AA capped NPs, ing. Hydrodynamic diameter of the PEGylated MFNPs estimated by TEM). The crystal structure of this KGdF4 was measured to be 230 nm. Folic acids were also conjuMFNPs remained cubic at this small size, this is congated to the MFNPs in order to target cancer cell specifsidered as an advantage over the Ln doped KYF4 NPs, ically. The MFNPs have a high transverse (T2 relaxivity in which the crystal phase transforms from hexagonal to 95 cubic and dimes the upconversion luminescence. of 352.8 mM−1 s−1University In vitro (7T). Successful UCL and MR imagDelivered by Ingenta to: Nanyang Technological upconversion imaging was performed on HeLa cells incuing were performed in vitro and in vivo (Fig. 8). What’s IP: 155.69.24.171 On: Tue, 02 Aug 2016 01:40:53 Copyright: bated with the synthesized KGdF4 MFNPs. more, thePublishers gold shell, which has excellent surface plasThe American cellular Scientific mon absorption, can be utilized for photo thermal therapy uptake efficiency of PEI capped NPs was found much (PTT). Cancer cells incubated with the MFNPs were effechigher than that of 6AA capped NPs. Although no MR tively destructed after exposure to NIR laser. However, the imagining was performed, the high magnetic mass suscepgold shell also causes quenching to the upconversion lumitibility (862 × 10−5 emu g−1 Oe−1 of the PEI-MFNPs nescence. Luckily, due to the intermediate SPION layer, indicates the potential of this particle as optical/MRI the quenching effect is much weaker compared to Ln-NPs bimodal contrast agent. with directly-attached gold shell.97 Fe3 O4 can also be inteTo further enhance the photo-stability, Chen et al. syn91 thesized Ln-NPs with a core/shell configuration. The grated as core particles. In the work by Zhu et al., Fe3 O4 Tm3+ doped NaYbF4 core was synthesized first to provide NPs with were first encapsulated with SiO2 shell through the NIR-to-NIR upconversion photoluminescence. Then a an Stöber method and then further capped with a lanshell of NaGdF4 was grown on the NaYbF4 :Tm3+ core thanide carbonate layer (Lu, Yb, Er/Tm(OH)CO3 . After to protect the PL and induce MR property. Compared calcination 600 C for 6 h, the Ln carbonate layer was to bare NaYbF4 :Tm3+ NPs, the luminescence intensitransformed into Yb3+ /Er3+ /Tm3+ doped Lu2 O3 layer. To 3+ ties of core/shell NaYbF4 :Tm /NaGdF4 NPs are 3 times get the final MFNPs, HF and NaF were added to convert the lanthanide oxide to lanthanide fluoride, and also etch higher. This PL enhancement is caused by the suppresout the SiO2 layer.81 sion of quenching effect and the activation of upconvert3+ 3+ ing ions in the core surface (the Yb and Tm ions Fluorides in simpler form (i.e., CaF2 ,98 GdF3 43 99 and in the core surface are provided a similar lattice enviSrF100 2 have also been doped with various Ln ions to ronment with those inside the core matrix). In addition, produce multifunctional Ln-NPs. Passuello et al. synthe PL decay of the core/shell NPs was much slower thesized Ln-doped (Er3+ /Yb3+ and Tm3+ /Yb3+ GdF3 than that of the bare NPs. The T1 relaxivity was meaNPs by hydrothermal treatment on mixed lanthanide sured to be 2.6 mM−1 s−1 . The same group also reported chloride/NH4F solution.43 PEG was also added as the cap3+ 3+ another similar core/shell NP with a NaGdF4 :Er , Yb ping agent. Rod-shape NPs (average size: 85 × 150 nm) were produced while uncapped GdF3 NPs were sphercore and a NaGdF4 shell.89 The bimodal capability of this core/shell NPs was demonstrated by wide-field cell imagical (average size: 65–75 nm). The PEG layers reduce ing on breast cancer cells incubated with the core/shell the water molecules around Ln ions that could quench J. Biomed. Nanotechnol. 12, 1553–1584, 2016

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performed on mice to demonstrate the potential of GdF3 NPs as T2 MRI contrast agent. Gadolinium oxide NPs can be directly used as an MRI/Optical bimodal probe.92 They are also very good host material to upconverting lanthanide ions101 102 and have been more widely exploited as bimodal probe (Gd2 O3 ,103 104 Gd2 O2 S80 105 . Paik et al. reported an Er3+ /Yb3+ doped Gd2 O3 NPs as potential bimodal imaging probe.104 What is particularly interesting about Paik’s work is that through the addition LiOH as shape directing agent, the shape of the Gd2 O3 NPs can be tuned from tripodal to triangular shape. The morphology of NPs has been reported to have influence on cellular uptake,106 107 biodistribution.108 and cytotoxicity.109 As most multimodal probes are in spherical shape, Paik’s work represents an alternative perspective to design target-specific, biocompatible nanoprobes by shape controlling. Apart from doping, Gd2 O3 NPs can also be conjugated with other imaging probes. Recently, Li et al. conjugated PEGlyted Gd2 O3 NPs with aptamer functionalized silver nanoclusters (aptamer-Ag NCs).110 The conjugation was achieved by covalent bonding between the carboxylic groups on the surface of PEG-Gd2 O3 NPs and the amino groups from the aptamer. The fluorescence of aptamer-Ag NCs and the relaxivity of Gd2 O3 NPs were well retained after the conjugation. Subsequently, MR/Optical imaging was conducted on MCF-7 cells to demonstrate the dual-modal Delivered by Ingenta to: Nanyang Technological University imaging capability of PEG-Gd IP: 155.69.24.171 On: Tue, 02 Aug 2016 01:40:53 2 O3 /aptamer-Ag nanoprobe. Copyright: American Scientific EnhancedPublishers cells uptake of the nanoprobe was also observed due to the presence of aptamers as targeting ligands. Currently, the clinical MRI community is paying more Figure 8. In vivo UCL/MR bimodal imaging of KB tumor bearand more attention on high magnetic field strength ing mouse. (a) Bright field, (b) UCL and (c) merged images (>3T) MRI as it can provide image with higher of the mouse one hour after intravenous injection of PEGsignal-to-noise ratio.111 112 At this strong magnetic field, MFNPs. The arrows in (a–c) indicate the tumor position. (d) Ex vivo imaging of harvested liver, spleen, lung, bone Gd3+ based contrast agents exhibit lower relaxivity and and tumor 24 h after the injection. Clearly MFNPs accumumagnetization.111 113 Effort has been invested to explore lated in those sites since UCL signals from other organs the potential of other paramagnetic lanthanide ions, includare neglectable. (e) T2 -weighted images of KB-tumor bearing Dy3+ , Pr3+ , Sm3+ , Ho3+ , Er3+ , and Yb3+ , as MRI ing nude mice with PEG-MFNPs injection. (f) T2 -weighted contrast agents.114 115 Compare to Gd3+ ions, those paraimages of KB-tumor bearing nude mice without PEG-MFNPs injection. (g) UCL in vivo lymphangiography mapping using magnetic ions have shorter electronic relaxation time MFNPs. (h) MR lymphangiography mapping before and after and mainly affect T2 contrast. Among those lanthanides, the MFNPs injection. Reprinted with permission from [85], Dy3+ is attracting most of the research interest due to L. Cheng, et al., Facile preparation of multifunctional upconits short relaxation and high magnetic moment.114 And version nanoprobes for multimodal imaging and dual-targeted photothermal therapy. Angewandte Chemie 123, 7523 (2011). just like other lanthanide ions, the optical property of © 2011, John Wiley and Sons. Dy3+ can be manipulated through doping in order to be applied for optical imaging. One good example was reported by Das and his colleague.111 Dy2 O3 nanocrystals the emission, resulted in an enhanced emission comwith an average diameter of 3 nm were synthesized pared to the uncapped ones. The MR properties were through a stepwise thermolysis of Tb-Dy-oleate complex measured at 4.7T. The T1 relaxivity is considerably at 280 C. Tb3+ ions were doped to enhance the luminessmall (0.05 mM−1 s− 1 for Tm3+ /Yb3+ doped NPs and −1 −1 3+ 3+ cence. The transverse relaxivity (T2 was measured to be 0.02 mM s for Er /Yb doped NPs). This is caused 2.17 mM−1 s−1 at 7T magnetic field. The contrast enhanceby the decreased surface Gd3+ ions due to large partiment is demonstrated by phantom imaging (Fig. 9(a)). cle size. The T2 relaxivites were measured to be 22.6 ± A clear dose-dependent contrast can be observed, which 3.4 mM−1 s−1 and 15.8 ± 2.5 mM−1 s−1 . Considering indicates the good sensitivity of the Dy2 O3 nanocrystal. the high the T2 /T1 ratios, T2 weighted MR imaging was 1564


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photoacoustic imaging.27 SWCTs have also been utilized in Raman imaging due to its unique Raman signature and large scattering cross-section.122 The good echogenicity of multi walled CNTs (MWNTs) can be utilized for contrast enhancement in ultrasound imaging.123 Metal catalysts are often used in the fabrication CNTs and the residual metal ions enable CNTs to function as MRI contrast agents.124 Besides exploiting the intrinsic properties of CNTs, additional imaging modalities can be integrated with CNTs through conjugation. The large surface area of CNTs can be functionalized and then linked with other imaging probes, including radio-isotopes,125 iron oxide,126 127 Gd3+ chelates,128 lanthanide nanocrystals129 and QDs.130 By sealing the endings of CNT, a large internal cavity can be created, in which a great amount of imaging and therFigure 9. (a) T2 -weighted MR images of 1% agarose gel containing different concentrations of Dy2 O3 :Tb3+ NPs. (b) Nude apeutic agents can be loaded.131 132 mouse with xenografted breast tumor cells (MCF-7), the white Recently, Chao et al. demonstrated human mesarrow indicate the position of tumor. (c) In vivo T2 -weighted MR enchymal stem cells (hMSCs) labeling with protamineimage of tumor regions. The injection site (right arrow) shows conjugated SWNTs and successfully conducted in vivo clear positive contrast enhancement by setting the un-injected left tumor as reference. Reprinted with permission from [111], Raman/PAT/MRI triple modality imaging.124 The SWNTs G. K. Das, et al., Single-phase Dy2 O3 : Tb3+ nanocrystals as were synthesized by high-pressure CO disproportionadual-modal contrast agent for high field magnetic resonance tion method, facilitated by metal catalyst. To make the and optical imaging. Chem. Mater. 23, 2439 (2011). © 2011, SWNTs water dispersible, they were further PEGylated American Chemical Society. through sonication with C18PMH-PEG-NH2, a PEGgrafted amphiphilic polymer. Protamine was also conjuIn vivo MR imaging was conducted on a tumor-bearing gate to the PEG-SWNTs to enhance the cellular uptake. nude mouse (Fig. 9(b)). The contrast enhancement can A considerable amount of ferric nanoparticles was found Delivered the by Ingenta Nanyang Technological University be clearly observed by comparing injectionto:site remain in the functionalized SWNTs, which brings a IP:The 155.69.24.171 On: Tue, 02 Aug 2016 01:40:53 and un-injected site (Fig. 9(c)). optical imaging Copyright: American Scientific high T2 Publishers relaxivity of 482.6 m M−1 s−1 . In vivo stem potential of Dy2 O3 nanocrystal was demonstrated by cell mapping was performed on a nude mouse model. confocal imaging of human lung epithelial cells (BEAShMSCs with and without SWNTs labeling were injected 2B) that has been incubated the nanocrystal. A bright to a mouse subcutaneously. And then the mouse was green fluorescence was observed. However, the imagimaged by Raman spectroscopic mapping, MR imaging ing was based on down-conversion with an excitation of and photoacoustic imaging. In all three imaging modal275 nm. The same group also reported another Dy3+ based ities, the site with SWNT-labeled stem cells injection nanocrystal: NaDyF4 :Yb3+ /NaGdF4 :Yb3+ , Er3+ . The upshows notable differences compared to the control site converting poisoning effect of Dy3+ ions was utilized to (Fig. 10). generated NIR-to-VIS upconversion fluorescence. A phoInstead of working as contrast agents, Chen et al. tosensitizer (chlorin e6) was also conjugated to the NPs to utilized CNTs as a nano-platform to which other 116 make it a theranostic nanoprobe. imaging probes are attached.132 The internal cavity of CNTs was filled with Fe3 O4 nanoparticles while the CARBON-BASED NANOPROBES exterior surface was modified with dispersing agent Carbon Nanotubes poly(sodium 4-styrenesulfonate) (PSS). CdTe QDs was encapsulated with a thin SiO2 layer (HQDs) and then Ever since its discovery in 1991,117 Carbon Nanotubes capped with poly(allylamine) (PAH). By conjugating the (CNTs) have become one of the hottest materials in the HQDs and CNTs, a magnetofluorescent nano-complex research community due to its unique physical, chemical was created. Transferrin was attached to the CNTs and biological properties. In the bio-imaging field, CNT, to enhance the tumor targeting capability. Doxorubicin particularly single walled CNTs (SWNTs), based nanohydrochloride (DOX) was also loaded to the CNTs, probes have also been extensively investigated.118 119 There which make the nano-complex a theranostic ensemble are several characters that make CNTs a good bio-imaging (Fig. 11). The drug delivery and fluorescent imaging probe: With proper functionalization such as PEGylation, functionalities were demonstrated in HeLa cell. HowCNTs have been proved to have very low toxicity by varever, the Fe3 O4 nanoparticles were only utilized for ious in vitro and in vivo studies.120 SWNTs exhibit strong magnetically guided drug delivery, no MR imaging was fluorescence emission in the NIR region.121 The high NIR conducted. absorbance of CNTs makes it a good contrast agent for J. Biomed. Nanotechnol. 12, 1553–1584, 2016

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Figure 10. Raman/MR/photoacoustic triple-modal imaging of a nude mouse after injection with hMSCs. (A) T2 -weighted MR image. The left arrow indicate the injection site of unlabeled stem cells (control site) while right arrow points to the injection site of SWNTlabeled stem cells. B, C) Raman images of control site (B) and SWNT-labeled site (C). (D, E) Photoacoustc images of control site (D) and SWNT-labeled site (E). The circles indicate the location where the stem cells are injected. Reprinted with permission from [124], C. Wang, et al., Protamine functionalized single-walled carbon nanotubes for stem cell labeling and in vivo raman/magnetic resonance/photoacoustic triple-modal imaging. Adv. Funct. Mater. 22, 2363 (2012). © 2012, John Wiley and Sons.

Graphene/Graphene Oxide therapeutic/diagnostic agents can be then conjugated with the functionalized GO/RGO. Graphene is a carbon monolayer in which the atoms In the work reported by Shi et al., A MRI/CT dualare arranged in hexagonal (honeycomb) lattice,133 it can modality probe was synthesized by crosslinking graphene be described as an unzipped SWNT. Graphene shares oxide with IONPs and gold nanoparticles (AuNPs).136 many similar features with CNTs, including large surface 134 Here, graphene oxide was used as a nanoplatform to intearea for drug/probe loading and high NIR absorbance. grate IONPs and AuNPs. The IONPs and GO was first Therefore graphene and its derivative (e.g., graphene Deliveredoxide by Ingenta Nanyang Technological University integrated by a one-step hydrothermal reaction and then oxide (GO) and reduced graphene (RGO))to:have IP: 155.69.24.171 On: Tue, 02 Aug 2016 01:40:53 AUNPs are attached to the GO through seeded crystalalso been studied extensively in the bio-imaging comCopyright: American Scientific Publishers lization. The as prepared AuNPs-IONPs-GO nanocomposmunity. Due the presence of large amount of reacite was further functionalized with lipoic acid modified tive oxygen groups, GO and RGO are more commonly used for biological applications as a versatile nanoPEG (LA-PEG) and folic acid conjugated LA-PEG platform. Those reactive groups can be utilized to crosslink (LA-PEG-FA). In vivo MRI/CT dual-model imaging was with hydrophilic capping molecules to prevent aggreconducted with tumor-bearing BALB/c mice. Clear congation and to reduce cytotoxicity.135 Then additional trast enhancement can be observed in both MR and CT

Figure 11. Schematic illustration of the preparation route of Water-Dispersible DOX-Fe3 O4 @CNT-HQDs-Trf nano-complex. Reprinted with permission from [132], M. L. Chen, et al., Quantum dots conjugated with Fe3 O4 -filled carbon nanotubes for cancertargeted imaging and magnetically guided drug delivery. Langmuir 28, 16469 (2012). © American Chemical Society.

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images. In addition, the high NIR absorbance of nanocomCarbon Dots posite was utilized to perform photothermal therapy (PTT). Carbon dots (CDs) are quantum sized (