In Vivo Fluorescence Imaging and the Diagnosis of Stem Cells Using

Jan 23, 2017 - In this review, we report the QD types available for stem cells, the methods of ... (B) The fluorescence images of stem cells labeled w...
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In Vivo Fluorescence Imaging and the Diagnosis of Stem Cells Using Quantum Dots for Regenerative Medicine Hiroshi Yukawa*,†,‡ and Yoshinobu Baba*,†,‡,§,∥,#

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Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ‡ ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan § Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ∥ Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-cho, Takamatsu 761-0395, Japan # College of Pharmacy, Kaohsiung Medical University, Shin-Chuan 1 st Rd., Kaohsiung, 807, Taiwan, R.O.C.

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magnetic resonance imaging (MRI),10 positron emission computerized-tomography (PET),11 and single-photon emission computed tomography (SPECT),12 have been implemented in clinical practice. However, these modalities are meant to be used to diagnose tissues and organs, so the highly sensitive detection of transplanted stem cells using such techniques is very difficult. Fluorescence imaging is expected to contribute to the development of stem cell transplantation, as fluorescence imaging can detect transplanted stem cells at the cellular level.13−15 However, the detection and diagnosis of transplanted stem cells in vivo using conventional fluorescent probes such as fluorescent proteins and organic fluorescent probes is almost impossible due to the inhibition caused by the autofluorescence and scattering/absorbance derived from the body.16,17 Recently, quantum dots (QDs) with entirely different fluorescent properties from conventional fluorescent probes have received focus as a potential solution to these problems. QDs achieved superhigh resolution, superhigh sensitivity, superlong duration, energy savings, and low costs and were first applied to 4K/8K displays in 2013.18 Our groups have explored stem cell labeling using QDs and in vivo fluorescence imaging of transplanted stem cells, demonstrating in vivo imaging diagnostic techniques for transplanted stem cells by measuring the fluorescence of QDs. In this review, we report the QD types available for stem cells, the methods of stem cell labeling using QDs, in vivo fluorescence imaging systems, and in vivo fluorescence imaging data of transplanted stem cells labeled with QDs, focusing largely on findings from recent papers. The future prospects for the application of QDs to regenerative medicine, including stem cell therapy, will also be discussed.

CONTENTS

Quantum Dots Types CdSe/ZnS QDs Cd-Free QDs Nonmetal QDs Stem Cell Labeling by QDs Physical Methods Chemical Methods Other Methods In Vivo Fluorescence Imaging System Fluorescence Imaging Open-Air Fluorescence Imaging and 3D Fluorescence Tomography NIR-II Fluorescence/Luminescence Imaging In Vivo Fluorescence Imaging of Stem Cells Labeled with QDs Subcutaneous Transplantation Intravenous and Other Transplantation Routes NIR-II Imaging Summary and Outlook Author Information Corresponding Authors ORCID Author Contributions Notes Biographies Acknowledgments References

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tem cell therapy plays a large role in regenerative medicine for many diseases such as lung and liver diseases, as the regeneration of these organs is very difficult.1−3 Indeed, stem cell therapy using somatic stem cells, progenitor cells, and mature cells differentiated from stem cells has been applied in clinical practice.4,5 Therefore, the detection and diagnosis of the behavior, accumulation and engraftment of transplanted stem cells in vivo are essential for ensuring the safety and maximum therapeutic effect of stem cell transplantation. However, in vivo imaging modalities for detecting transplanted stem cells are not sufficient at present.6−8 A number of modalities, such as ultrasonic diagnostics, roentgen diagnosis, X-ray computed tomography (CT),9 © 2017 American Chemical Society

QUANTUM DOTS TYPES CdSe/ZnS QDs. Semiconductor QDs have recently been used in interdisciplinary fields with bioassays, such as ELISAs. These inorganic fluorescence nanocrystals mainly comprise periodic groups of semiconductor materials. The energy levels in the semiconductor nanocrystals are quantized owing to the quantum-confinement effect19,20 and can be regulated by the crystal sizes. Near-infrared (NIR) fluorescence (>700 nm) is required for in vivo fluorescence imaging to ensure high body Published: January 23, 2017 2671

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permeability, and CdSe-based QDs can produce NIR fluorescence. For the application of QDs in visualizing stem cells as well as other biomedical applications, coating the CdSe core with a ZnS layer is also indispensable.21,22 The protection of the CdSe core by the ZnS layer suppresses the toxicity of the CdSe core due to leaching out to the adjacent spaces and enhances the fluorescence quantum yield. Furthermore, the water solubility of QDs are generally realized by the addition of functional groups, aptamers,21−23 antibodies,24 and peptides25 to the surface of QDs (Figure 1 and Figure 2A).

Figure 1. Illustrations of various kinds of quantum dots (QDs) for cells labeling.

CdSe/ZnS QDs have already been used to visualize various kinds of stem cells, such as bone marrow stem cells (BMSCs),26,27 adipose tissue-derived stem cells (ASCs) (166,148,141),28−30 embryonic stem (ES) cells,31 and induced pluripotent stem (iPS) cells16 with high efficiency, and to determine the influence of QDs on the cell self-multiplication ability and multilineage potential of stem cells. The in vivo fluorescence imaging of transplanted stem cells labeled with CdSe/ZnS QDs was first achieved predominantly in mouse models, and several hundreds of subcutaneously transplanted stem cells were able to be detected. In addition, the accumulation rate of transplanted stem cells in tissues and organs were analyzed based on the fluorescence of CdSe/ZnS QDs.30,32 However, there remain some concerns regarding the influence of Cd on stem cells. Hsieh et al. reported that the osteogenesis of BMSCs was inhibited by CdSe/ZnS QDs,27 although the coating and delivery at an optimum concentration were reported to prevent any harmful effects.33 Cd-Free QDs. As mentioned above, well-developed QDs contain toxic elements including (but not limited to) Cd, Te, Pb, Hg, and Se. Thus, novel QDs with lower cytotoxicity (especially Cd-free alternatives) have been investigated for the further advancement of QD technology.34−37 There have been many reports on cadmium-free QDs, such as AgInS2/ZnS (ZnSZAIS),38,39 CuInS2/ZnS,40−42 InP/ZnS,43 and CuO QDs (Figure 1). These QDs not including Cd have been introduced into different kinds of cells using various labeling methods, resulting in a significant decrease in the cytotoxicity. In addition, there have been some reports of Cd-free QDs producing NIR fluorescence; therefore, Cd-free QDs are expected to be useful for in vivo fluorescence imaging of transplanted stem cells.44,45 Indeed, AgInS2/ZnS QDs showed extremely low cytotoxicity in ASCs compared with CdSe/ZnS QDs and exerted no

Figure 2. Fluorescence images of stem cells labeled with QDs. (A) The morphologies and fluorescence images of stem cells labeled with CdSe/ ZnS QDs. (a) The morphologies and fluorescence images of mouse ASCs labeled with CdSe/ZnS QDs655 using octa-arginine (R8) peptides (reprinted from Biomaterials, Vol. 31, Yukawa, H.; Kagami, Y.; Watanabe, M.; Oishi, K.; Miyamoto, Y.; Okamoto, Y.; Tokeshi, M.; Kaji, N.; Noguchi, H.; Ono, K.; Sawada, M.; Baba, Y.; Hamajima, N.; Hayashi, S. Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells, pp 4094−4103 (ref 30) Copyright 2010, with permission from Elsevier). (b) The fluorescence images of human MSCs labeled with four kinds of CdSe/ZnS QDs (QDs525, 565, 605, 655) using PEI molecules (reprinted from Biomaterials, Vol. 35, (92), Yang, H. N.; Park, J. S.; Jeon, S. Y.; Park, W.; Na, K.; Park, K.H. The effect of quantum dot size and poly(ethylenimine) coating on the efficiency of gene delivery into human mesenchymal stem cells, pp 8439−8449 (ref 92) Copyright 2014, with permission from Elsevier.). The scale bar: 20 μm. (c) The morphologies and fluorescence images of breast cancer stem cells labeled with CD44 antibody-CdSe/ZnS QDs525, CD24 antibodyCdSe/ZnS QDs565 and ALDH1 antibody-CdSe/ZnS QDs625 (Reproduced from Shim, Y.; Song, J. M. Chem. Commun. 2015, 51, 2118−2121 (ref 93), with permission of The Royal Society of 2672

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with NIR fluorescence have been used in the novel daunorubicin sensing and imaging of prostate cancer cells. The cytotoxicity of CuO QDs was analyzed using mouse C2C12 cells but not stem cells.47 Furthermore, the strong fluorescence intensity and high quantum yield of Cd-free QDs remains to be established. Thus, additional studies and inspection are necessary before Cd-free QDs can be applied to stem cell labeling and imaging (Figure 2B,a). Nonmetal QDs. Recent studies have reported that silicon QDs and carbon (or graphene) QDs not including heavy-metal elements are useful for bioimaging and cell labeling (Figure 1).32,48−53 These QDs have been confirmed to show very low cytotoxicity, like Cd-Free QDs, and their fluorescence color depends on the QD size. Carbon (or graphene) QDs have also been used for stem cell labeling. Carbon QDs were confirmed to be taken up into human neural stem cells (hNSCs) in a concentration- and time-dependent manner via endocytosis. Furthermore, no significant change was found in the viability, proliferation, metabolic activity, or differentiation potential of hNSCs after treatment with carbon QDs.33 A turn-on orange-red fluorescent nanosensor based on rhodamine B derivativefunctionalized graphene QDs was developed for Fe3+ detection with high sensitivity and selectivity in cancer stem cells.54 However, the application of carbon (or graphene) QDs to stem cell labeling and imaging is extremely limited. Furthermore, few studies have examined the stem cell labeling and imaging of Si QDs. Accordingly, further studies will be required for the application of nonmetal QDs to stem cell labeling and imaging. (Figure 2B,b−d).

Figure 2. continued Chemistry). (d) The nuclear labeling of human MSCs using CdSe/ZnS QDs525 with SV40-NLS-TP peptides (as used in ref 94). White arrows show the nuclear labels. The scale bar: 100 μm. (Reprinted by permission from Macmillian Publishers Ltd.: Scientif ic Reports, Narayanan, K.; Yen, S. K.; Dou, Q.; Padmanabhan, P.; Sudhaharan, T.; Ahmed, S.; Ying, J. Y.; Selvan, S. T. Sci. Rep. 2013, 3, 2184 (ref 94). Copyright 2013). (B) The fluorescence images of stem cells labeled with Cd-free QDs. (a) The fluorescence images of mouse ASCs labeled with AgInS2/ZnS QDs using using octa-arginine (R8) peptides (Reproduced from Kim, J.; Song, S. H.; Jin, Y.; Park, H. J.; Yoon, H.; Jeon, S.; Cho, S. W. Nanoscale 2016, 8, 8512−8519 (ref 48), with permission of The Royal Society of Chemistry). (b) The fluorescence images of human ASCs labeled with graphene QDs at different concentrations (Reproduced from Guo, R.; Zhou, S.; Li, Y.; Li, X.; Fan, L.; Voelcker, N. H. ACS Appl. Mater. Interfaces 2015, 7, 23958−23966 (ref 54) Copyright 2015 American Chemical Society). (c) The fluorescence and merged images of rat MSCs labeled with carbon QDs (Reproduced from Liu, J. H.; Cao, L.; LeCroy, G. E.; Wang, P.; Meziani, M. J.; Dong, Y.; Liu, Y.; Luo, P. G.; Sun, Y. P. ACS Appl. Mater. Interfaces 2015, 7, 19439− 19445 (ref 32) Copyright 2015 American Chemical Society). (d) The fluorescence images of rabbit MSCs labeled with Si nanoparticles using nanocomposite film labeling method. The scale bar: 50 μm. (Reproduced from Gao,Y.; Lim, J.; Yeo, D. C.; Liao, S.; Lans, M.; Wang, Y.; Teoh, S. H.; Goh, B. T.; Xu, C. ACS Appl. Mater. Interfaces 2016, 8, 6336−6343 (ref 72) Copyright 2016 American Chemical Society).

influence on the multipotency of stem cells.46 However, the application of these QDs to stem cells is limited. CuInS2 QDs

Figure 3. Illustrations of major cell labeling methods. (A) Physical labeling methods of QDs for cells. (a) Electroporation using microdevice (Reproduced from Sun, C.; Cao, Z.; Wu, M.; Lu, C. Anal. Chem. 2014, 86, 11403−11409 (ref 90). Copyright 2014 American Chemical Society). (b) Photoporation using pulse laser (Reproduced from Xiong, R.; Joris, F.; Liang, S.; De Rycke, R.; Lippens, S.; Demeester, J.; Skirtach, A.; Raemdonck, K.; Himmelreich, U.; De Smedt, S. C.; Braeckmans, K. Nano Lett. 2016, in press (ref 64) Copyright 2016 American Chemical Society). (B) Chemical labeling methods of QDs for cells. (a) Chemical substances such as polyamideamine (PAMAM), polyethylenimine (PEI), and stearyl-triphenyl phosphonium (STPP) (as used in refs65−68). (b) Cationic liposomes such as Lipofectamine and Lipofectin (as used in ref 11). (c) Cell penetrating peptides (CPPs) such as HIV/TAT, HSV/VP22, and oligo-arginine (as used in ref 91). (d) Antibody for stem cell targeting specificity of QDs (Reproduced with permission from Proceedings of the National Academy of Sciences USA Han, H. S.; Niemeyer, E.; Huang, Y.; Kamoun, W. S.; Martin, J. D.; Bhaumik, J.; Chen, Y.; Roberge, S.; Cui, J.; Martin, M. R.; Fukumura, D.; Jain, R. K.; Bawendi, M. G.; Duda, D. G. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 1350−1355 (ref 71). (C) Nanocomposite film labeling method of nanoparticles (Reproduced from Gao, Y.; Lim, J.; Yeo, D. C.; Liao, S.; Lans, M.; Wang, Y.; Teoh, S. H.; Goh, B. T.; Xu, C. ACS Appl. Mater. Interfaces 2016, 8, 6336−6343 (ref 72) Copyright 2016 American Chemical Society). 2673

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Figure 4. In vivo fluorescence imaging systems and comparison of the fluorescence intensity between normal and alfalfa-free feed. (A) In vivo fluorescence imaging systems. (a) Maestro Dynamic (Copyright 2016−2017 PerkinElmer, Inc. All rights reserved. Printed with permission). (b) Clairvivo OPT plus (SHIMADZU). (c) IVIS Spectrum CT (Copyright 2016−2017 PerkinElmer, Inc. All rights reserved. Printed with permission). (d) Solaris (Copyright 2016−2017 PerkinElmer, Inc. All rights reserved. Printed with permission). (e) FMT4000 (Copyright 2016−2017 PerkinElmer, Inc. All rights reserved. Printed with permission). (f) SAI-1000 (SHIMADZU). (B) Comparison of the fluorescence intensity between normal and alfalfafree feed (reprinted from Biomaterials, Vol. 33, Yukawa, H.; Watanabe, M.; Kaji, N.; Okamoto, Y.; Tokeshi, M.; Miyamoto, Y.; Noguchi, H.; Baba, Y.; Hayashi, S. Monitoring transplanted adipose tissue-derived stem cells combined with heparin in the liver by fluorescence imaging using quantum dots, pp 2177−2186 (ref 74) Copyright 2012, with permission from Elsevier). (a) Fluorescence image of normal and alfalfa-free feed. (b) In vivo fluorescence image of mouse fed on normal feed. (c) Fluorescence intensities of normal and alfalfa-free feed.

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STEM CELL LABELING BY QDS Physical Methods. Electroporation, which is the application of an external electric field to permeabilize the plasma membrane, has been used for the introduction of functional substrates such as chemical compounds, proteins, and genes into cells (Figure 2A,a).55−59 Several groups have previously studied the delivery of QDs into cells by electroporation.60−63 However, there are few published papers on the application of electroporation to stem cell labeling, due largely to the complicated operations involved and high cytotoxicity to stem cells associated with electroporation. Most recently, photoporation has been reported to be useful for the introduction of QDs into cells (Figure 2A,b).64 Photoporation induced the cytosolic delivery of QDs, resulting in 3-fold higher loading of QDs with improved stability and reduced cytotoxicity compared with classic endocytic uptake. In addition, interestingly, cytosolic delivery by photoporation prevented the asymmetric inheritance of labels by daughter cells over subsequent cell generations. However, few studies have

examined the influence of photoporation on the specificity of stem cells, so further development of this approach in the future is awaited. Chemical Methods. Some kinds of polymers, such as polyethylenimine (PEI), polyamideamine (PAMAM), and stearyl triphenylphosphonium (STPP), have been reported to induce the transduction of QDs into stem cells.65−68 These polymers have a positive electric charge and thus can interact with the negatively charged cell surface with high efficiency. In addition, these polymers interact with a variety of negatively charged molecules or materials directly, such as DNA molecules and carboxyl- or sulfo-group-coated QDs, at high efficiency. However, the strong positive charge affects the stability of the cell surface and leads to cell death; as such, charge modulation is necessary for stem cell labeling of QDs using these polymers (Figure 3B,a). Cationic liposomes such as lipofectamine and lipofectin (DOTMA) are transfection agents and have also been reported to be useful for the transduction of negatively charged QDs into 2674

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cells.29 QDs encapsulated with these cationic liposomes have been reported to be made relatively quickly and were transduced through endocytosis at high efficiency. In addition, stem cells labeled with QDs using cationic liposomes were found to maintain cells’ self-replication ability and pluripotency. However, as with cationic polymers, charge modulation is necessary for stem cell labeling of QDs. Thus, the optimum mixing ratio of QDs and cationic liposomes must be determined for stem cell labeling without cytotoxicity (Figure 3B,b). Cell-penetrating peptides (CPPs) such as HIV/TAT, HSV/ VP22, and oligo-arginine also are known to be useful for stem cell labeling. These peptides include approximately 8 to 12 cationic amino acids, such as arginine and lysine, and can contact the negatively charged cell membrane at very high efficiency. CPPs are joined with QDs mainly through covalent bonds or electrostatic interactions. Covalently joined CPPs-QDs are very stable, whereas the synthesis and purification of CPPsQDs demand a significant amount of time and effort (Figure 3B,c).46−69 Electrostatically bound CPPs-QDs were conjugated by the positively charged amino acids of CPPs and the negatively charged functional groups of the QDs. These electrostatically bound CPPs-QDs exist stably in cell culture medium and the synthesis method of CPPs-QDs is very speedy compared with that of chemical binding. The cytotoxicity of CPPs to stem cells was reported to be lower than that with chemical polymers and cationic liposomes.30 In addition, CPPs-QDs did not influence the stem cells features, such as the self-propagation and multilineage potential. Furthermore, recent studies indicated that QDs could label iPS cells using CPPs with high efficiency, and the iPS cells kept their undifferentiated state and multipotency.48 Therefore, CPPs are convenient peptides for cell labeling with QDs; however, the validation of the optimum volume of CPPs is indispensable.70 Antibodies (Abs) are expected to be available for ensuring the stem-cell-targeting specificity of QDs. It was reported that QDs and Abs were conjugated by coupling whole Abs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine-norbornene cycloaddition, and these conjugates were used for in vivo single-cell labeling in bone marrow.71 These QD-Abs diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells. Therefore, this labeling method is useful in a wide range of structural and functional stem cell imaging systems to investigate the interactions between stem cells and their environment in damaged or diseased tissues and organs. However, the synthesis and purification of QD-Abs may be more time-consuming than when using CPPs (Figure 3B,d). Other Methods. Nanocomposite film including nanoparticles (NPs) has been recently shown to induce the transduction of NPs to adherent bone marrow stem cells (BMSCs) without concerns of freely suspended NPs.72 This method uses a nanocomposite film prepared by the homogeneous dispersal of NPs within a biodegradable polymeric film, such as polycaprolactone (PCL). Once BMSCs are seeded onto the film, they adhere, spread, and filter into the film through the micropores formed during the film fabrication. The preembedded NPs are internalized by the stem cells during this infiltration process. This method appears to be useful for achieving adherent stem-cell-targeting specificity of NPs, such as BMSCs including bone marrow, thereby making it a very attractive method. However, the transduction time is relatively long (about 3 days), and few studies have examined the

transduction of QDs into stem cells. Therefore, further development of this approach in the future is expected (Figure 3C).

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IN VIVO FLUORESCENCE IMAGING SYSTEM Fluorescence Imaging. In vivo fluorescence/luminescence imaging systems, which can identify and measure the fluorescence or emission derive from the body have been developed for small and medium-sized animals,73 including Maestro Dynamic, Clairvivo OPT plus, IVIS spectrum CT, and Solaris (Figure 4A,a−f). However, with this technique, the autofluorescence of normal food including alfalfa must be paid attention.74 Indeed, autofluorescence derived from the mouse provided normal food was observed from the gastrointestinal tract by the excitation of the ultraviolet and visible regions. In order to inhibit the influence on in vivo fluorescence imaging, the animals were provided feed including no fluorescence components (alfalfa-free feed) for at least 1 week. The fluorescence intensity of the alfalfa-free feed was decreased by approximately 90% in comparison to that of normal feed (Figure 4B,a−c). The Maestro system is a multispectral imaging system specialized for fluorescence imaging. By removing autofluorescence, it is possible to detect only the target portion using fluorescent markers with high sensitivity. In addition, it is possible to separate multiple fluorescent signals easily. As key features of this system, the detection wavelength region and spatial resolution are 500−950 nm and 25 μm, respectively. The multispectral image acquisition time is 2−10 s, and the maximum exposure time is 20 min (Figure 4A,a). The Clairvivo OPT plus system enables the observation of deep tissue using an excitation light source with a high-intensity near-infrared region (semiconductor laser, LD). Using this system, we can observe the fluorescence from five directions simultaneously, indicating simultaneous measurements in a short time from five directions using excitation light and multi mirrors (Figure 4A,b). The IVIS System, including IVIS Spectrum CT, is an expandable, sensitive imaging system that is easy to use for both fluorescence and bioluminescence imaging in vivo. This system includes a highly sensitive charge-coupled device (CCD) camera, light-tight imaging chamber, and complete automation and analysis capabilities. As the leading optical imaging platform for in vivo analysis, IVIS systems include a range of practical accessories developed through experience in research laboratories worldwide. As key detailed features of this system, the detector is a cooled CCD camera (2048 × 2048 pixels), and the minimum pixel resolution is 20 μm. Optical and microcomputed tomography (CT) technology are integrated, allowing for threedimensional (3D) optical tomography of fluorescence and bioluminescence (Figure 4A,c). Open-Air Fluorescence Imaging and 3D Fluorescence Tomography. Solaris is an open-air in vivo fluorescence imaging system targeting small animals as well as relatively large animals, such as monkeys and dogs. As key features, this system detects the 470−800 nm wavelength fluorescence as multiplex imaging images using 4 kinds of fluorescence filters and is equipped with liquid tunable filter technology. This technology reduces the autofluorescence derived from animals and addresses spectral unmixing. Furthermore, this system employs the white light used in surgical operation rooms (Figure 4A,b). The MSOT systems, including FMT4000, are next-generation molecular imaging systems that combine the molecular 2675

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Figure 5. Detection and multiplex in vivo imaging capability of QDs in subcutaneous transplantation. (A) Multiplex in vivo imaging of stem cells labeled with QDs. (a) In vivo fluorescence image and fluorescence wavelength of mouse subcutaneously transplanted mouse ASCs labeled with QDs655, 705, and 800 into the back of the mice with a single excitation light source (reprinted from Biomaterials, Vol. 31, Yukawa, H.; Kagami, Y.; Watanabe, M.; Oishi, K.; Miyamoto, Y.; Okamoto, Y.; Tokeshi, M.; Kaji, N.; Noguchi, H.; Ono, K.; Sawada, M.; Baba, Y.; Hamajima, N.; Hayashi, S. Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells, pp 4094−4103 (ref 30) Copyright 2010, with permission from Elsevier). (b) In vivo fluorescence image of mouse subcutaneously transplanted mouse ES cells labeled with QDs525, 565, 605, 655, 705, and 800 into the back of the mice with a single excitation light source (Lin, S.; Xie, X.; Patel, M. R.; Yang, Y. H.; Li, Z.; Cao, F.; Gheysens, O.; Zhang, Y.; Gambhir, S. S.; Rao, J. H.; Wu, J. C. BMC Biotechnol. 2007, 7, 67 (ref 79)). (c) In vivo fluorescence images of mouse subcutaneously transplanted stem cells (0.5, 1.0, and 3.0 × 105 cells) labeled with QDs655 using R8 peptides into the backs of the mice after 1 h, 1 day, 2 days, 5 days, and 7 days (reprinted from Biomaterials, Vol. 31, Yukawa, H.; Kagami, Y.; Watanabe, M.; Oishi, K.; Miyamoto, Y.; Okamoto, Y.; Tokeshi, M.; Kaji, N.; Noguchi, H.; Ono, K.; Sawada, M.; Baba, Y.; Hamajima, N.; Hayashi, S. Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells, pp 4094−4103 (ref 30) Copyright 2010, with permission from Elsevier). (B) In vivo imaging of subcutaneously transplanted stem cells in combination with scaffolds. (a) In vivo imaging of human ASCs labeled with graphene QDs 24 h after transplantation using a methacrylated hyaluronic acid hydrogel (Reproduced from Kim, J.; Song, S. H.; Jin, Y.; Park, H. J.; Yoon, H.; Jeon, S.; Cho, S. W. Nanoscale 2016, 8, 8512−8519 (ref 48), with permission of The Royal Society of Chemistry). (b) In vivo imaging of subcutaneously transplanted collagen scaffold loaded with human MSCs (hMSCs) labeled with Ag2S QDs. Fluorescence images at day 0, 10, and 30 after transplantation of (1) blank collagen scaffold, (2) a collagen scaffold seeded with hMSCs, and (3) a collagen scaffold seeded with hMSCs labeled with Ag2S QDs (reprinted from Biomaterials, Vol. 53, Chen, G.; Tian, F.; Li, C.; Zhang, Y.; Weng, Z.; Zhang, Y.; Peng, R.; Wang, Q. In vivo real-time visualization of mesenchymal stem cells tropism for cutaneous regeneration using NIR-II fluorescence imaging, pp 265−273 (ref 44) Copyright 2015, with permission from Elsevier). (c) Long-term in vivo imaging of subcutaneously transplanted insulin producing cells labeled by photoporation (Reproduced from Xiong, R.; Joris, F.; Liang, S.; De Rycke, R.; Lippens, S.; Demeester, J.; Skirtach, A.; Raemdonck, K.; Himmelreich, U.; De Smedt, S. C.; Braeckmans, K. Nano Lett. 2016, in press (ref 64) Copyright 2016 American Chemical Society).

μm. Whole-body small animal imaging is available; as such, this imaging system is expected to be useful for drug development in regenerative medicine (Figure 4A,e). NIR-II Fluorescence/Luminescence Imaging. It was recently reported that fluorescence/luminescence imaging in the second near-infrared window (NIR-II; wavelength > 1 000

specificity of optical imaging and the depth and resolution of ultrasonic imaging. This system accumulates drugs in the living body of mice and rats and then visualizes the hypoxic conditions of target organs and tissues. This system can measure 3D tomograms in real time. As key features, the penetration depth is about 2−4 cm, and the cross-sectional in-plane resolution is 150 2676

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Figure 6. In vivo and ex vivo imaging of stem cells labeled with QDs after intravenous transplantation. (A) In vivo and ex vivo fluorescence images after tail vein injection of rat MSCs labeled with CdSe/ZnS QDs. (a) In vivo fluorescence images after tail vein injection of rat MSCs labeled with QDs (From Liu, H.; Tang, W.; Li, C.; Lv, P.; Wang, Z.; Liu, Y.; Zhang, C.; Bao, Y.; Chen, H.; Meng, X.; Song, Y.; Xia, X.; Pan, F.; Cui, D.; Shi, Y. Nanoscale Res. Lett. 2015, 10, 265 (ref 83), with kind permission from Springer Science and Business Media). The time points of 1, 3, 6, and 12 h, respectively. (b) Ex vivo fluorescence images of five important organs of rats with diabetes transplanted of rat MSCs labeled with CdSe/ZnS QDs. (c) The ratio of the fluorescence intensity (RFI) of each organ equals the fluorescence intensity of the organ divided by the total fluorescence intensity of all five organs in the normal control group and MSC treatment group. (B) In vivo and ex vivo fluorescence images of mice with acute liver failure after transplantation of mouse ASCs labeled with CdSe/ZnS QDs using R8 (reprinted from Biomaterials, Vol. 33, Yukawa, H.; Watanabe, M.; Kaji, N.; Okamoto, Y.; Tokeshi, M.; Miyamoto, Y.; Noguchi, H.; Baba, Y.; Hayashi, S. Monitoring transplanted adipose tissue-derived stem cells combined with heparin in the liver by fluorescence imaging using quantum dots, pp 2177−2186 (ref 74) Copyright 2012, with permission from Elsevier). (a,b) In vivo fluorescence imaging of mouse ASCs labeled with CdSe/ZnS QDs655 (a), 800 (b). (c) Ex vitro fluorescence imaging of mouse ASCs labeled with CdSe/ZnS QDs800 with or without heparin. (d) The ratio of the fluorescence intensity (RFI) of each organ equals the fluorescence intensity of the organ divided by the total fluorescence intensity of five organs with or without heparin. (C) In combination without heparin (a) or with heparin (b) without laparotomy. (C) Transfection of VEGF genes into endothelial progenitor cells and in vivo imaging using CdSe/ZnS QDs in as ischemia hind limb model (reprinted from Biomaterials, Vol. 33, Yang, H. N.; Park, J. S.; Woo, D. G.; Jeon, S. Y.; Park, K. H. Transfection of VEGF165 genes into endothelial progenitor cells and in vivo imaging using quantum dots in an ischemia hind limb model, pp 8670−8684 (ref 67) Copyright 2012, with permission from Elsevier). (a) Schematic diagram of in vivo imaging of transplanted EPCs. (b) In vivo imaging of CdSe/ZnS QDs taken up by EPCs (upper) and determination of QDs residence or apoptosis of transplanted EPCs by xenozene and confocal laser microscopy (as used in ref 67). 2677

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application of photoporation to stem cell labeling and in vivo imaging using QDs. Intravenous and Other Transplantation Routes. Stem cell therapy via intravenous transplantation is expected to be available in clinical applications for certain diseases of the liver, pancreas, and lungs.80−82 In vivo fluorescence imaging of intravenous transplanted stem cells labeled with QDs makes it possible to monitor the transplanted stem cells in vivo and measure their rate of engraftment in tissues and organs. In vivo fluorescence images at different time points (1, 3, 6, and 12 h) after tail vein injection of MSCs labeled with CdSe/ZnS QDs in diabetes and normal groups are shown in Figure 6A,a. At 6 h after transplantation, the MSCs labeled with CdSe/ZnS QDs accumulated in the pathological tissue of the pancreas, and the strongest fluorescence intensity was detected. The transplanted MSCs mainly accumulated in the liver in the normal group, while the dominant signals were in the pancreas in diabetic mice. In addition, five major organs (heart, lungs, pancreas, spleen, and liver) were harvested and imaged (Figure 6A,b). Ex vivo fluorescence images of these organs in diabetic rats transplanted with MSCs labeled with CdSe/ZnS QDs are shown in Figure 6A,c. The pancreas fluorescence signal of diabetic rats was much higher than that of the control animals. Of note, the strongest signal of CdSe/ZnS QDs was noted in the liver.83 In vivo fluorescence imaging using QDs shows that transplanted MSCs target the pathological pancreas and play an important role in the treatment of diabetes. In a model of acute liver failure, greater accumulation of transplanted ASCs in the liver was found when heparin was administered simultaneously than when the ASCs were transplanted alone (Figure 6B).74 In these cases, CdSe/ZnS QDs with near-infrared fluorescence were shown to be useful for the in vivo fluorescence imaging of transplanted stem cells, as the liver is a relatively deep site, and the autofluorescence is strong (Figure 6B,a,b). Indeed, intravenously transplanted ASCs labeled with CdSe/ZnS QDs800 were able to be observed in the liver and lungs even without laparotomy; however, transplanted ASCs labeled with QDs655 were unable to be detected (Figure 6B,c,d).74 The efficacy of the simultaneous administration of heparin for acute liver treatment with ASC transplantation was clarified using in vivo fluorescence imaging technology. In vivo imaging of transplanted endothelial progenitor cells (EPCs) transfected with the VEGF gene using coupling of CdSe/ZnS QDs nanoparticles with PEI is shown in Figure 6C,a.67 To evaluate the long-term duration of QDs in EPCs, in vivo imaging of QDs was performed and the residence of QDs in EPCs and survival EPCs in nude mice were assessed. The red fluorescence of the QDs in nude mice slowly disappeared in a time-dependent manner. However, the red fluorescence of the QDs was still able to be detected at 28 days after transfection (Figure 6C,b). In addition, to confirm the residence of QDs in EPCs, we sacrificed hind limb ischemia model nude mice transplanted with QDs-loaded EPCs. The transplanted tissues with green corresponding to cell or tissue death, red corresponding to QDs, and blue corresponding to the cell nucleus are shown in Figure 6C,c. These studies suggest that in vivo fluorescence imaging using QDs may become increasingly important in the development of stem cell therapy for regenerative medicine. NIR-II Imaging. Organic fluorescent dye, carbon nanotubes, and inorganic nanoparticle have already been developed as NIRII fluorescence probes. Since organic fluorescent dyes with benzobisthiazole (BBTD) structure show NIR-II fluorescence

nm) is an ideal strategy for in vivo imaging due to its deeper tissue penetration, lower autofluorescence derived from the body, and higher spatial and temporal resolution than fluorescence imaging in the first near-infrared window (NIR-I, 700−950 nm).54,75,76 The SAI-1000 system is a new in vivo NIR-II fluorescent imaging system that can acquire high-definition images using the wavelength range of ≥1000 nm. This system can perform observations in real time and save the findings as an animation. As key features, the detection wavelength region and the field of view are 900−1 700 nm and 50 mm × 40 mm, respectively. In addition, this system uses a cooled InGaAs area camera as a detector and a 980 nm laser diode as the excitation light source. It is also lightweight, compact, and easy to move. However, there are not currently enough NIR-II fluorescence/luminescence imaging systems available to enable its widespread adoption (Figure 4A,f).

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IN VIVO FLUORESCENCE IMAGING OF STEM CELLS LABELED WITH QDS Subcutaneous Transplantation. The in vivo fluorescence imaging of subcutaneously transplanted stem cells labeled with QDs in mice has been continually reported.44,48,77,78 The fluorescence intensity derived from QDs can be observed and measured quantitatively using an in vivo fluorescence imaging system after the subcutaneous transplantation of various kinds of stem cells labeled with QDs into the back of mice. Indeed, subcutaneously transplanted ASCs labeled with CdSe/ZnS QDs655, 705, and 800 on the back of the mouse were able to be clearly detected (Figure 5A,a).30 Multicolor imaging of transplanted ES cells labeled with six kinds of CdSe/ZnS QDs525, 565, 605, 655, 705, and 800 was achieved with high contrast (Figure 5A,b).79 In addition, the number of transplanted ASCs labeled with CdSe/ZnS QDs655 was able to be determined based on the fluorescence intensity of the QDs (Figure 5A,c).30 These results were obtained thanks to the fluorescence specificities of CdSe/ZnS QDs, such as a narrow fluorescence wavelength and a strong fluorescence intensity. In addition, stem cell therapy in combination with a scaffold was found to induce the accumulation and engraftment of transplanted stem cells at the location of the injection. Methacrylated hyaluronic acid (HA-ME) hydrogel containing human ASCs (hASCs) labeled with graphene QDs (GQDs) were transplanted into the dorsal region of athymic mice. The hASCs labeled with GQDs were able to be observed after 24 h transplantation at high efficiency compared to hASCs transplantation alone without HA-ME (Figure 5B,a).48 The in vivo imaging of a subcutaneously transplanted collagen scaffold loaded with human mesenchymal stem cells (hMSCs) labeled with Ag2S QDs is shown in Figure 5B,b. Because of the strong and stable NIR-II fluorescence imaging of Ag2S QDs, the fluorescence signals were able to be easily detected for up to 1 month at the wound site transplanted with a collagen scaffold seeded with labeled hMSCs, demonstrating the advantages of QDs for long-term in vivo imaging.44 In contrast, photoporation has been reported to result in 3-fold higher loading of InP QDs with improved signal stability and reduced cytotoxicity compared with endocytic transduction. This method has allowed for the marked improvement in the long term cell visibility in vivo, with cells labeled with fluorescent dextran able to be tracked for up to 2 months in Swiss nude mice compared with only 2 weeks for cells labeled by endocytosis (Figure 5C).64 Future studies should further explore the 2678

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Figure 7. In vivo NIR-II fluorescence imaging. (A) In vivo NIR-II fluorescence imaging using lanthanide nanoparticles (LNPs) (Reprinted by permission from Macmillian Publishers Ltd.: SCIENTIFIC REPORTS, Fukushima, S.; Furukawa, T.; Niioka, H.; Ichimiya, M.; Sannomiya, T.; Tanaka, N.; Onoshima, D.; Yukawa, H.; Baba, Y.; Ashida, M.; Miyake, J.; Araki, T.; Hashimoto, M., Sci. Rep. 2016, 6, 25950 (ref 86). Copyright 2016). (a) Normalized fluorescence spectra of Y2O3: Tm, Yb (solid) and Y2O3: Er, Yb (dotted) phosphors under 980 nm NIR-II light irradiation. (b) NIR-II fluorescence images of Y2O3: Er, Yb under 980 nm excitation. (c) In vivo NIR-II fluorescence imaging of a rat transplanted C2H12 cell sheet labeled with Y2O3: Er, Yb nanoparticles under 980 nm excitation (as used in ref 86). (B) In vivo NIR-II fluorescence imaging using AgInTe2 QDs. (Reproduced from Kameyama, T.; Ishigami, Y.; Yukawa, H.; Shimada, T.; Baba, Y.; Ishikawa, T.; Kuwabata, S.; Torimoto, T. Nanoscale 2016, 8, 5435−5440 (ref 45), with permission of The Royal Society of Chemistry). (a) Schematic illustration of DSPC-AgInTe2 nanocomposite. (b) Absorption and fluorescence spectra of DSPC-AgInTe2 nanocomposites under 700 nm excitation. (c) In vivo NIR-II imaging of a mouse injected of a 50 mm3 portion of DSPC-AgInTe2 dispersion. The concentration of DSPC-AgInTe2 nanocomposites were 50 (i), 25 (ii), 12.5 (iii), 6.25 (iv), and 0 nmol (particles) dm−3 (v).

(peak at about 1 070 nm), this probe has been used for in vivo NIR-II fluorescence imaging of blood vessels of the brain in mice.84 Single-walled carbon nanotubes showing NIR-II fluorescence (1 300 nm) have been noted in carbon nanotubes and similarly applied for the in vivo imaging of blood vessels of the brain in mice.85 In addition, lanthanide nanoparticles (LNPs) and QDs are focused in inorganic nanoparticles. Y2O3: Ln, Yb (Ln = Tm [810 and 1 630 nm], Ho [1 200 nm], Er [1 530 nm]) nanoparticles with NIR-II fluorescence have already been developed (Figure 7A,a). In vitro and in vivo NIR-II fluorescence imaging of calculated HeLa cells and transplanted C2C12 cells labeled with these nanoparticles, respectively, have been performed (Figure 7A,b,c).86 Several reports have described QDs with NIR-II fluorescence. Ag2S QDs with strong and stable NIR-II fluorescence have already been developed. In vivo NIR-II fluorescence imaging of transplanted labeled hMSCs with a collagen scaffold was performed, and the fluorescence signals were able to be detected up to 1 month at the wound site. AgInTe2 nanoparticles were confirmed to show strong and stable NIR-II fluorescence (1 100 nm), and water-soluble AgInTe2 nanoparticles encapsulated with 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC) liposome (DSPC-AgInTe2) have been developed (Figure 7B,a,b). In vivo NIR-II fluorescence of injected DSPC-AgInTe2 was able to be observed using a SAI1000 imaging system (Figure 7B,c).87 However, in vivo NIR-II fluorescence imaging technology, including NIR-II probes, has yet to be established; as such, further studies are needed to clarify its utility in stem cell imaging.

to stem transplantation in regenerative medicine, which demands high-resolution imaging at the cellular level. The potential for clinical applications of QDs have extended with the exploitation of extremely low cytotoxic QDs that do not include heavy metals like Cd as well as the development of NIR-II (over 1 000 nm) fluorescence QDs with strong body permeability.87,88 Of note, clinical tests using fluorescence nanoparticles given from basic studies have already started in the U.S.,89 and research and development in this field is becoming more important. Furthermore, more and more development of QDs such as the synthesis of hybrid nanoparticles of QDs and other functional molecules may enable the diagnosis and initiation of stem cell therapy in vivo at the same time.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: ++81-52-789-5654. Fax: ++81-52-789-5177. E-mail: h. [email protected]. *Phone: ++81-52-789-4664. Fax: ++81-52-789-4666. E-mail: [email protected]. ORCID

Hiroshi Yukawa: 0000-0002-9352-1520 Author Contributions

This manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript. Notes

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The authors declare no competing financial interest.

SUMMARY AND OUTLOOK In this review, we described current studies and topics regarding the in vivo imaging of transplanted stem cells using QDs. In vivo fluorescence imaging technology using QDs will likely contribute

Biographies Hiroshi Yukawa is an Designated Lecture of the Graduate School of Engineering at Nagoya University, Japan. He obtained a Master degree in Engineering from Tokyo University, Japan, in 2002. He joined the 2679

DOI: 10.1021/acs.analchem.6b04763 Anal. Chem. 2017, 89, 2671−2681

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Drug Development Group, R&D Company, KOBAYASHI Pharmaceutical Co., Ltd. In 2002. He earned his Ph.D. in Medical Science from Nagoya University in 2010 under the supervision of Professor. M. Hamaguchi and earned his Ph.D. in Engineering from Tokyo University in 2011 under the supervision of Professor K. Araki. He has been employed in the Graduate School of Engineering at Nagoya University from 2012. His current research focus is the application of nanoparticles including quantum dots to stem cell labeling and in vivo imaging diagnosis of transplanted stem cells. Yoshinobu Baba is a Professor of the Department of Applied Chemistry, Nagoya University. He is an Associate Editor of Analytical Chemistry and serves as an editorial/advisory board member for over 15 scientific journals. He has received over 160 awards for his contribution in the field of nanotechnology. He is the author or coauthor of 931 publications and is also an inventor on over 90 patents. He has delivered more than 875 plenary and invited lectures at conferences. His work has been cited on 356 occasions by newspapers and television.

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ACKNOWLEDGMENTS This paper was mainly supported by the Japan Agency for Medical Research and Development (AMED) through its “Research Center Network for Realization of Regenerative Medicine”. This work was partially supported by JSPS KAKENHI Grant Number JP26790006. We appreciate the help of Yoko Tsutsui and Tomoko Arimoto (Nagoya University) for submitting this paper.

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DOI: 10.1021/acs.analchem.6b04763 Anal. Chem. 2017, 89, 2671−2681