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Human Induced Pluripotent Stem Cells for Tumor Targeted Delivery of Gold Nanorods and Enhanced Photothermal Therapy yanlei liu, meng yang , jingpu zhang , xiao zhi , Chao Li, Chunlei Zhang, Fei Pan, kan wang , Yuming Yang, jesus martinez de la fuente , and Daxiang Cui ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b07172 • Publication Date (Web): 13 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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Human Induced Pluripotent Stem Cells for Tumor Targeted Delivery of Gold Nanorods and Enhanced Photothermal Therapy Yanlei Liua,b,1, Meng Yanga,b,1, Jingpu Zhangb, Xiao Zhib, Chao Lib, Chunlei Zhanga, Fei Pana,c, Kan Wanga, Yuming Yanga, Jesus Martinez de la Fuentea2, Daxiang Cuia,c* a

Institute of Nano Biomedicine and Engineering, Key Laboratory for Thin Film and Microfabrication of the

Ministry of Education, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering;

b

School of Biomedical Engineering, cNational Center for Translational Medicine,

Collaborative Innovational Center for System Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China 2

Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, Zaragoza, 50018, Spain

* To whom correspondence should be addressed. Tel: 0086-21-34206886; Fax: 0086-21-34206886; Email: [email protected] 1

These authors equally contributed to this article

ABSTRACT How to improve effective accumulation and intratumoral distribution of plasmonic gold nanoparticles has become a great challenge for photothermal therapy of tumor. Herein, we reported

a

nanoplatform

with

photothermal

therapeutic

effects

by

fabricating

Au

nanorods@SiO2@CXCR4 nanoparticles and loading the prepared nanoparticles into the human induced pluripotent stem cells(AuNRs-iPS). In virtue of the prominent optical properties of Au nanorods@SiO2@CXCR4 and remarkable tumor target migration ability of iPS cells, the Au nanorods delivery mediated by iPS cells via the nanoplatform AuNRs-iPS was found to have a prolonged retention time and spatially even distribution in MGC803 tumor-bearing nude mice observed by photoacoustic tomography and two-photon luminescence. Based on these improvements, the nanoplatform displayed a robust migration capacity to target the tumor site and to improve photothermal therapeutic efficacy on inhibiting the growth of tumors in xenograft mice under a low laser power density. The combination of gold nanorods with human iPS cells as a theranostic platform paves an alternative road for cancer theranostics and holds great promise for clinical translation in the near future.

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KEYWORDS:

Au

nanorods

·

CXCR4

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·

induced

pluripotent

stem

cells · photothermal therapy

Scheme 1 AuNRs@SiO2@CXCR4 loaded human iPS cells for Target Delivery and Intratumoral Homogeneous Distribution of AuNRs and Enhanced Photothermal Therapy

As representative plasmonic nanoparticles with strong and tunable surface plasmon resonance (SPR) at near-infrared light region, gold nanoparticles have been extensively explored for disease theranostic applications.1-3 Most of the gold-based nanoplatforms use the plasmon-enhanced two-photon luminescence and localized photothermal effect to improve bioimaging in parallel with localized hyperthermia for photothermal therapy (PTT) and photoenhanced chemotherapy.4-6 Thermoablative technology offers several practical advantages over the surgical resection such as lower morbidity, avoiding damages to the surrounding healthy tissue, and reduced costs by shortening hospitalization time. The ultimate goal of cancer therapy is to selectively kill tumor cells without damages to the surrounding normal cells. Plasmonic photothermal therapy is being developed in an effort to localize heating to the targeted tumor tissue with little damage to the surrounding normal tissues,7,8 but it is extremely challenging to allow the nanoparticles to be transported throughout the tumor and to reach an effective concentration at the same time. Plasmonic photothermal therapy utilizes gold nanoparticles to convert non-harmful laser light into thermal energy, and gold nanoparticle-mediated photothermal therapy involves delivery of nanoparticles to the tumor tissue followed by irradiation to produce a localized elevation in temperature. Therefore, it is critical to make the nanoparticles evenly distributed in an effective


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concentration throughout tumors.9 In recent decades, a great effort has gone into developing promising gold nanoparticles with difference shapes, such as nanosphere, nanoshells, nanostars and nanorods. Effective accumulation of gold nanoparticles in tumors is crucial for nanoparticles-mediated cancer treatment. As a common strategy, the surface of gold nanoparticles is modified with cell-specific ligands for active tumor targeting to enhance their preferable accumulation,10,11 but the strategy often decreases the blood circulation time of ligand-modified gold nanoparticles. Moreover, a long circulation time of nanoparticles in the blood is a prerequisite for targeted delivery.2 Therefore, a long circulation time and active tumor-targeted ability of nanoplatform are needed for effective accumulation of gold nanoparticles in tumors. In recent years, with the discovery that many stem cells exhibit the intrinsic characteristics of targeting tumors, tumor therapy strategy based on multiple types of stem cells is emerging, which utilize these cells as a good carrier to load the anticancer agents and nanoparticles.9,12,13 Especially, induced pluripotent stem cells (iPS) pioneered by Yamanaka in 2006 are able to be generated directly from adult cells.14 Due to their capacity of cell trafficking, iPS cells have been recognized as promising strategy for tumor therapy in the last decade.15 One of the major regulators of cell trafficking is chemokines and their receptors. More than 50 different kinds of chemokines and 20 matched chemokine receptors have been reported including stromal derived factor-1 (SDF-1) and G-protein coupled seven-span transmembrane receptor CXCR4.16 It has been proved that both stem cells and cancer cells express CXCR4 on their membrane, and SDF-1/CXCR4 play a key role not only in stem cells’ homing to tumor site12 but also in tumor metastasis.17 Moreover, the tumor tropism of iPS has been studied in mouse tumor and SDF-1/CXCR4 is proposed to be involved in this process.18 However, one concern about the application of stem cells in tumor therapy is that stem cells are capable to form tumors by themselves or even promote tumor growth. Nevertheless, the therapies based on stem cells as vehicles of nanoparticle result in the death of stem cells after photothermal therapy, thereby effectively avoiding the tumor formation concern.12 In the present work, to develop a nanoplatform with improved photothermal therapeutic efficacy, AuNRs-iPS was established by loading the nanoparticle into iPS cells. As shown in Scheme 1, in the platform, AuNRs@SiO2@CXCR4 was composed of mesoporous silica-coated gold nanorods and the conjugated antibody of CXCR4 that was utilized to improve the loading efficiency into iPS cells, and the iPS cells were used as transporters to target the tumor. The basic


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characteristics, photothermal properties and the tumor targeting capacity of the nanoplatform (AuNRs-iPS) were studied. Moreover, the improved intratumor distribution of AuNRs delivered by iPS via the nanoplatform was investigated using the photoacoustic tomography (PA) and two-photon fluorescence imaging. Based on the validation of the platform, it was finally applied to the photothermal therapy in MGC803 tumor-bearing mice. The strategy remarkably inhibited the growth of MGC803 tumors and improved the photothermal therapy in vivo.

RESULTS AND DISCUSSION CTAB-stabilized AuNRs were synthesized by the seed-mediated growth method1. Transmission electron microscope (TEM) image showed that AuNRs have a mean length of 65.0 ± 7.5 nm and width of 12.0 ± 1.5 nm (Figure 1A, Figure S2A and C). The formation of silica coating around AuNRs (AuNRs@SiO2) made it 86.0 ± 5.5 nm in length and 28.0 ± 6.5 nm in width, and the thickness of the silica layer was 10.50 ± 2.1 nm (Figure 1B, Figure S2B and D ). As shown in Figure 1C, a longitudinal SPR (LSPR) maximum for the original AuNRs was at around 805 nm, and the peak slightly red-shifted to 817 nm for AuNRs@SiO2, followed by another red-shift to 830 nm after CXCR4 antibody was conjugated to the AuNRs@SiO2. Silica is extremely stable, has quite good biocompatibility, and it can be easily used to modify surface with silane chemistry without significant affect on the properties of the original materials.19 After AuNRs are coated with silica, the zeta potential of AuNRs changed from positive charge to negative due to the OH groups on the surface of [email protected] Previous studies reported that the cellular uptake of positively charged nanoparticles is apparently higher than that of negatively charged nanoparticles.18 Thus, in order to improve the cellular uptake of AuNRs@SiO2, based on the specific antibody-antigen interaction of CXCR4, CXCR4 antibody were conjugated to AuNRs@SiO2 using APTES and succinic anhydride which avoided the concern of weak electrostatic adsorption when the nanoparticle was uptaken by the cells. Figure 1D shows the FTIR spectra of AuNRs@SiO2, AuNRs@SiO2-COOH and AuNRs@SiO2@CXCR4. All of them have the following absorptions: the absorption band of -OH at 3442 cm-1, the vibration peaks of C=C at 1634 cm-1 and the Si-OH bending vibration peaks at 963 cm-1. Comparing with the IR spectra of AuNRs@SiO2, a new absorption band appeared for AuNRs@SiO2-COOH at 1560 cm-1


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which belongs to C=O stretching vibration, in addition, the Si-OH bending vibration peaks at 953 cm-1 dramatically decreased. When APTES and CXCR4 antibody were conjugated with AuNRs@SiO2-COOH, the carbonyl vibration peaks at 1560 cm-1 increased dramatically and the Si-O-Si stretching vibration peaks at 1063cm-1 presented with a small shift to 1075 cm-1, which is ascribed to the hydroxyl stretching. These results suggested that CXCR4 antibody was successfully conjugated with AuNRs@SiO2. In this work, the iPS cells were used as a transporter loaded with AuNRs@SiO2@CXCR4, in which the primary issue is to confirm the activity of iPS cells before a sufficient amount of AuNRs are loaded to reach up to the level that achieves the therapeutic purpose in the targeted tumor site. It is difficult to use CCK8 to test the toxicity of AuNRs@SiO2 and AuNRs@SiO2@CXCR4 for the clone state growth of iPS cells (Figure S4A). Thereby, we chose HDF-1 cells as the cell source to finish the toxicity experiment. Data obtained from CCK-8 assay indicated almost no cytotoxicity of AuNRs@SiO2 and AuNRs@SiO2@CXCR4 in the concentration range of 0-80 µg/mL (Figure S3). To determine the optimum concentration of AuNRs@SiO2@CXCR4 for iPS incubation, the cytotoxicity caused by different amounts of AuNRs@SiO2@CXCR4 was assessed by apoptosis assay using flow cytometry. As shown in Figure 2, after iPS cells were exposed to a series of AuNRs@SiO2@CXCR4 in different concentrations, we observed that iPS cells exposed to lower concentration (0-40 µg/mL) suffered the minimal apoptosis and necrosis (with cells mortality < 15 %), whereas those in the presence of 80 µg/mL AuNRs@SiO2@CXCR4 induced significant apoptosis. The increased death in the nanoparticle-treated cells could be attributed to an increased cellular uptake. Finally, a dose of 40 µg/mL AuNRs was used for the further experiments. Most of the AuNRs in stem cells were found to be resident in lysosomes but a portion of these AuNRs were finally excluded to the extracellular environment.21 To assess the time course over which nanoparticles are up-taken and retained within cells, we carried out quantitative measurements on the elemental gold content in iPS cells treated with different formulations of nanoparticles by using ICP-MS. As shown in Figure 3B, the uptake of AuNRs@SiO2 and AuNRs@SiO2@CXCR4 significantly increased during the first 2-4 h, followed by a weak increase later, and finally reached a plateau at 8 h. Compared with AuNRs@SiO2 groups, the elemental gold content of the AuNRs@SiO2@CXCR4 groups was higher at each time point. As a


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result, the 8 h incubation period after which the amount of AuNRs@SiO2@CXCR4 loading into iPS reached maximum was adopted for the further in vivo experiments (28µg / 1.0×106 cells). In order to compare the difference between AuNRs@SiO2 and AuNRs@SiO2@CXCR4 in terms of the uptake by iPS cells, two-photon luminescence of the AuNRs core was conducted based on their distinguished two-photon property. As shown in Figure 3A, after iPS cells were incubated with the two nanoparticle groups separately for 24 h, we observed the bright luminescence

in

the

cytoplasm,

which

suggested

that

both

AuNRs@SiO2

and

AuNRs@SiO2@CXCR4 were able to be uptaken by iPS cells. Moreover, it is notable that the fluorescence intensity in AuNRs@SiO2@CXCR4-treated cells was higher than that in AuNRs@SiO2-treated cells. All these results confirmed that much more AuNRs@SiO2@CXCR4 were taken up by iPS cells than AuNRs@SiO2. In order to verify the expression of CXCR4 on iPS cells, qRT-PCR and western-blot were conducted. As shown in Figure S4, iPS cells with the typical clone state were successfully reprogrammed from HDF-1 cells. Moreover, CXCR4 was proved to be expressed in the iPS cells rather than HDF-1 cells in both the gene and protein levels. To evaluate the ability of AuNRs-loaded iPS cells to migrate toward tumor, transwell Boyden migration assay was conducted on these cells induced by conditioned medium prepared from MGC803 and unconditioned medium DMEM in the 24-well matrigel invasion chamber. The results showed that the migration of iPS cells was promoted by the MGC803 conditioned medium and maintained at the same level after loading AuNRs (Figure 3C, Figure S5).The iPS cells have been reported as delivery vehicles to transport nanoparticles into the tumors.22,23 Similarly, AuNRs-iPS cells in our study were capable to migrate from the injection site to the tumor site. To explore the potential influence caused by the application of iPS cells for in vivo gastric cancer cells targeting, we initially tested the effect of iPS cells conditioned medium (CM) and cultured iPS supernatant on the proliferation of MGC803 cells. In Figure S6 A, the CCK-8 results showed that 25% CM dilution medium cannot reduce the proliferation of MGC803 cells, but 50% and 75% CM dilution medium significantly reduced the proliferation of MGC803 cells. In addition, no significant effect of gastric cancer cells MGC803 conditioned medium was found on gastric cancer cells (Figure S6 B). The results confirmed that iPS cells conditioned medium inhibited the proliferation of MGC803 cells. Whether adult stem cells themselves support or


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suppress tumor growth has been widely studied, but showed contradictory results.24 In this study, we demonstrated the suppressing effect of iPS supernatant on gastric cancer cell MGC803 in vitro. We speculated that some cell factors secreted out of the iPS cells acted a key role in inhibiting MGC803 cells proliferation, but what these factors are and how they manipulate the cancer cells remain to be answered. Before the application of AuNRs-loaded iPS cells to photo-thermal therapy, it is necessary to learn the intracellular distribution of AuNRs in iPS cells. Thus, transmission electron microscopic was used intuitively to display the internalization of these nanoparticles. As shown in Figure 4 (B, C, D, denoted by red arrows), AuNRs@SiO2@CXCR4 were located in lysosomes and endosomes in aggregated status, and no nanoparticles were observed in the nucleus. Although AuNRs@SiO2@CXCR4 were internalized by iPS cells through endocytosis, a portion of AuNRs@SiO2@CXCR4 were found to be expelled by cell exocytosis (Figure 4E, F denoted by green arrows, Figure S7A, B denoted by red arrows). Subsequently, these expelled AuNRs can be re-endocytosed by the cells consistent with the previous reports.25 We speculated that the expelled AuNRs@SiO2@CXCR4 were specifically recognized and endocytosed by the surrounding cancer cells and cancer stem cells, because both of them have high CXCR4 expression, which further improved intratumoral distribution of AuNRs. Considering that a portion of internalized AuNRs are eventually expelled from iPS cells, the therapeutic effect of AuNRs retaining in iPS cells was examined regarding to in vitro photothermal ablation capacity of AuNRs@SiO2 and AuNRs@SiO2@CXCR4 for iPS cells upon NIR laser irradiation. Cells viabilities of iPS cells were evaluated by staining with dead-live calcein AM. As shown in Figure 5, almost no damage was observed in the control groups of normal iPS cells without cultured nanoparticles. However, there existed the nearly equal amount of dying cells stained in red and live cells stained in green in the AuNRs groups, while in the AuNRs-iPS groups, most of the cells were killed with almost no live cells left. These results confirmed that although a portion of internalized AuNRs@SiO2@CXCR4 was expelled from iPS cells, the remaining AuNRs@SiO2@CXCR4 meet the criteria for therapy after 8 h incubation. To confirm that iPS cells can track the gastric cancer cells in mice, in vivo fluorescence imaging was employed to longitudinally monitor the distribution using iPS-GFP cells in tumor-bearing mice. As shown in Figure S8 A (a-e), one day after tail vein injection, the


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fluorescent signals were distributed around the whole body. The fluorescent signals began to accumulate in the tumor region the second day after injection, and the fluorescent signals in the tumor regions increased to the climax on the 4th day after tail vein injection. In addition, the ex vivo imaging was also performed in internal organs and tumors from the mice the 4th day after the treatment. As shown in Figure S8 C and D, the fluorescent signals were dominatingly located in tumor site, and only a small number of iPS-GFP cells were located in liver and lung. The results suggested that iPS cells as the vehicles succeeded in targeting and homing to tumor in mice. Effective accumulation of nanoparticles and more uniform distribution in tumors are crucial for nanoparticles-mediated cancer therapy.

2, 26

In order to investigate whether the iPS-mediated

delivery of AuNRs can increase the effective accumulation and improve the pattern of distribution, the AuNRs distribution in tumor after injections of free AuNRs@SiO2 or AuNRs-iPS were detected by PA imaging and two-photon fluorescence imaging. Because of the linear relationship between the PA signal amplitude and the concentration of gold nanoparticles, PA imaging is recognized as an excellent technology to monitor the accumulation and distribution of nanoparticles in tumor27. In our study, the accumulation and distribution of AuNRs in tumors were real-time monitored using a PA scanner. It can be seen in Figure 6 that the vascular system of the tumors was clear in the first 24 h in all the nanoparticle-treated groups (horizontal plane images of tumor vascular). However, the PA signal in the group treated with AuNRs@SiO2 became faint since 24th h post-injection, and only weak signal was left at 36th h post-injection, whereas a strong signal from the intravascular system was kept clear up to 36 h in tumors treated by AuNRs-iPS. The results suggested that iPS cells prolonged the retention time of AuNRs and increased their accumulation quantity in tumor sites. Additionally, the two-photon fluorescence imaging of the sections from tumor tissues (Figure 7) told us that the majority of AuNRs were distributed in the shallow tumor tissue with little remaining in the deep tissue for the free AuNRs@SiO2 groups. By contrast, in the AuNRs-iPS groups, AuNRs was distributed more evenly throughout the tumor. As a result, the introduction of iPS cells as transporters delivered larger quantity of AuNRs to tumor tissue and improved the intratumoral distribution of AuNRs. To investigate the in vivo antitumor efficacy of AuNRs-iPS based on photothermal therapy (PTT), the photothermal effect of AuNRs was monitored using an infrared thermal imaging


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camera. Although AuNRs has excellent photothermal conversion efficiency to generate a high temperature within a short laser-exposure time in vitro, wilder temperature produced by AuNRs will induce irreversible damage to healthy tissues.

28

To ensure that the temperature reached the

threshold for thermotherapy on the premise of the safe healthy tissues, laser with a low power density (1.5W/cm2) was applied and the laser irradiation was limited to 3 min so that the tumor temperature was lower than 45 ºC.

29

Compared with the tumor injected with PBS buffer

increasing to 38.5 ºC upon irradiation, the tumor temperature in the AuNRs-iPS groups reached the highest 44.8 ºC, while the temperature for AuNRs@SiO2-treated tumors was 43.4 ºC (Figure 8A). After the laser was removed, temperatures of the tumor site quickly declined to a normal value in 1 min, suggesting that the laser irradiation-treatment is safe. A single large heat source is proposed as an inefficient means to deliver energy to a large volume of tissue. 30 The nanoplatform, AuNRs-iPS, developed by us showed an increased temperature for photothermal therapy. The spatially even distribution of the AuNRs delivered by iPS cells contributed to creating a more uniform thermal profile to an effective therapeutic level throughout the tumor, and thus was helpful to achieve a better therapeutic effect. 26 Compared with the PBS buffer and PBS buffer /irradiation groups, the tumor volumes in both the treated groups with AuNRs@SiO2 and AuNRs-iPS showed a significant decrease at the end of 4 weeks of therapy (Figure 8B). In particular, the AuNRs-iPS treated group, showed a better effect on inhibiting the tumor growth than the AuNRs@SiO2 treated group, demonstrating an effective thermal therapy using our improved nanoplatform. In addition, the survivability of mice in AuNRs-iPS treated group was markedly higher than that in the rest groups including PBS group, PBS+laser group,or AuNRs@SiO2 +laser group (Figure 8C). The mechanism underlying the thermal therapy of AuNRs-iPS was investigated in terms of the related proteins. The caspase-3 protein, a member of the cysteine-aspartic acid protease family, is activated in the apoptotic cell both by extrinsic and intrinsic pathways.

31

Hsp70 is a family

member of conserved ubiquitously expressed heat shock proteins. Hsp70 is known to be overexpressed in cells under heat stress in order to protect the cell from thermal damage.32 In our study, activated caspase-3 and Hsp70 protein were found to be overexpressed in the MGC803 tumors treated by AuNRs-iPS (Figure 8D). The results revealed that the thermal therapy effect of AuNRs-iPS worked by inducing apoptosis in tumor cells.


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At the 20th day treatments, the mice were sacrificed and the tumors were collected for H&E staining. As shown in Figure 8E, some damaged cells were observed in tumors in the AuNRs@SiO2+laser groups, while much more damaged cells were observed in the AuNRs-iPS groups. But, in the PBS and PBS+laser groups, no obvious damaged tumor cells were found. These results clearly demonstrated that based on the application of the iPS vehicles, AuNRs-iPS performed better than free AuNRs@SiO2 in inhibiting the in vivo tumor growth.

CONCLUSION In summary, we have successfully designed and fabricated a nanoplatform AuNRs-iPS by loading AuNRs@SiO2@CXCR4 into iPS cells to transport AuNRs to MGC803 tumor in mice for photothermal therapy, which showed low cytotoxicity and improved biocompatibility. By conjugating CXCR4 antibody with AuNRs@SiO2, the loading ability into iPS cells and photothermal effect of AuNRs@SiO2@CXCR4 were both improved. In addition, after loaded with the nanoparticle, AuNRs@SiO2@CXCR4, the iPS cells still have the ability to migrate toward tumor in vitro and track the gastric cancer cells in vivo. Most importantly, with the help of the photoacoustic tomography and two-photon luminescence, the AuNRs delivery mediated by iPS cells via the nanoplatform AuNRs-iPS was found to have a prolonged retention time and spatially even distribution in MGC803 tumor tissue in mice. Based on these improvements, the strategy exhibited good photothermal therapeutic efficacy by inhibiting the growth of tumors in mice at a low laser power density. The combination of nanoparticles with iPS cells in our strategy paves a novel way for cancer therapy and holds great promise in the future.

EXPERIMENTAL SECTION Synthesis of the AuNRs@SiO2. In a typical experiment, AuNRs were synthesized according to the seed-mediated template- assisted protocol. Mesoporous silica coating on AuNRs was carried out according to Gorelikov and Matsuurra method with some modifications.

33,34

To remove

excess reagents, 40 mL of as-prepared AuNRs solution was centrifuged twice at 11000 rpm for 6 min, and the precipitate redispersed in 20 mL deionized water. 4 mL of 25 mg/ mL PVP aqueous solution was added to 20 mL of the AuNRs solution, and then gently stirred for 15 h. The mixture was centrifuged once at 8500 rpm for 6 min and the precipitate was dispersed in 10 mL of deionized water. Then, 100 µL of 0.1M NaOH solution was added under stirring. Following this


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step, three injections of 15 µL 20% TEOS in methanol was added under gentle stirring at 30 minute intervals and then the mixed solution was gently stirred for 3 days at 28 ºC. Synthesis of AuNRs@SiO2@CXCR4. As-prepared AuNRs@SiO2 were collected by centrifugation at 11000 rpm for 6 min and were washed twice with DI water and twice with ethanol, then gently resuspended into 5 mL N,N-dimethyl-Formamide for further functionalization. 400µL APTS and 168.89 µg succinic anhydride were added in 20 mL N,N-dimethyl-Formamide and allowed to react under vigorous stirring at 65 ºC for 3 h. Subsequently, 5 mL AuNRs@SiO2 solution was added and vigorously stirred at 65 ºC for 5 h. Finally, the resultant was washed three times with DI water, and dried at -50 ºC for 12 h to obtain the AuNRs@SiO2-COOH. Figure S1 illustrates the synthetic pathway for making the AuNRs@SiO2-COOH. Covalent binding of CXCR4 antibody to the AuNRs@SiO2-COOH was carried out according to the standard EDC-NHS reaction method with some modifications. AuNRs@SiO2-COOH was activated by an EDC/NHS solution for 30 min. Next, 1.5 µL CXCR4 antibody were added to form a mixed solution and allowed to react under gentle stirring at room temperature overnight. The resultants were washed three times by centrifugation at 11000 rpm for 6 min with DI water in order to remove the unreacted chemical. Finally, the purified AuNRs@SiO2@CXCR4 was redispersed into 5 mL PBS for the next characterization and application. The morphology of the nanoparticles was characterized by transmission electron microscopy operated at an accelerating voltage of 200 kV. UV-vis spectra were measured with a Shimadzu UV-2450 UV-visible spectrophotometer. Cell culture. Human gastric cancer cell lines of MGC803, human dermal fibroblast (HDF), GES-1 and MEF were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The iPS cells and green fluorescent protein of iPS cells (iPS-GFP) were cultured in hiPS medium. All cell lines were maintained at 37 ºC in a humidified incubator containing 5% CO2. The cells were passaged when cells reached 80% confluence. Animals and tumor model. Female BALB/c athymic nude mice, 6-8 weeks of age and weighting 18-23 g, were purchased from Shanghai LAC Laboratory Animal Co. Ltd., and housed in a SPF grade animal center. The use of all mice in this study complied with the current ethical considerations: Approval of institutional Animal Care and Use Committee of Shanghai Jiao Tong


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University (SCXK-2012-0002). The mice were anesthetized by isoflurane and 2×106 MGC803 cells suspended in 50 µL PBS were transplanted into the mice subcutaneous. Effect of iPS cells conditioned medium on gastric cancer cells. To obtain conditioned medium (CM), iPS cells were plate on 90 mm plates with 10 mL of stem cell medium. After 48 h incubation, CM was harvested and passed through a 0.22 µm syringe filter to remove cellular debris. Effects of iPS cells conditioned medium on gastric cancer cells were evaluated by CCK-8 assay as followed. Gastric cancer cell line MGC803 were plated at a density of 1×104 cells per well on 96-well plate with DMEM medium containing 10% FBS at 37 ºC. After 48 h incubation, the medium was replaced with CM, and cells were then incubated with different compositions of CM as following: 100% DMEM medium, 75% DMEM medium + 25% CM, 50% DMEM medium + 50% CM, 25% DMEM medium + 75% CM and 100% CM, 100% iPS cells complete culture medium, for each experiment. After 24 h incubation, MGC803 cells were rinsed with PBS twice and cell viability was assessed by the CCK-8 method. Similarly, we collected the MGC803 conditioned media after 48 h culturing. The collected conditional media were used to treat MGC803 in different concentrations. After 24h treatments, CCK8 was used to assess the cell viability. In vivo imaging of iPS cells (iPS-GFP) in tumor-bearing mice. To evaluate in vivo iPS cells distribution, whole-animal imaging and ex vivo organ imaging were performed using the in vivo imaging systems (IVIS-100 imaging system, Caliper). The tumor reached a size of ≈ 150-200 mm3 before the distribution studies. The iPS cells (GFP, 1×106 cells) were intravenously injected into the tumor-bearing mice. Time-course fluorescent images (excitation: 470 nm; emission: 535 nm; integration; 15 s) were acquired on a Bruker In-Vivo F PRO imaging system. After the in vivo imaging, the mice were anesthetized before sacrifice. The tumor and major organs were collected, and then were imaged by the Bruker In-Vivo F PRO imaging system with the same parameters. Apoptosis assay and cytotoxicity assessment. To determine the cytotoxicity of AuNRs@SiO2@CXCR4, the apoptotic and necrotic cell distribution were determined by Annexic V-FITC/PI Apoptosis Detection Kit. The iPS cells were plated in triplicate at a density of 2×104 cells / well in 12-well microplates. After 48 h incubation, the medium was replaced with fresh medium and a series concentration of AuNRs@SiO2@CXCR4 (equivalent to 0-80 µg/mL Au) were added to the medium and incubated at 37 ºC for 24 h, respectively. Then, the iPS cells were


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trypsinized, harvested, washed with PBS and resuspended in 200µL of binding buffer containing 5 µL Annexin and 10µL PI. After incubation at room temperature for 15 min in the dark, 400 µL of binding buffer was added to each sample before washing and the sample were analyzed by BD FACS Calibur® (BD Biosciences, Mountain View, CA). HDF-1 cells were plated at a density of 1×104 cells per well on 96-well plate with DMEM medium containing 10% FBS at 37 ºC. After 48 h incubation, the medium was replaced, and cells were then incubated with various concentrations of AuNRs@SiO2 and AuNRs@SiO2@CXCR4 (0-80 µg/mL), respectively. After 24 h incubation, HDF cells were rinsed with PBS twice and cell viability was assessed by the CCK-8 method. Cell uptake and localization. Two-photon luminescence 35 of the AuNRs core was used to assess the cellular uptake of AuNRs@SiO2 or AuNRs@SiO2@CXCR4 with the same elemental Au concentration. Briefly, iPS cells were incubated with AuNRs@SiO2 (40 µg/mL) or equivalent dose of AuNRs@SiO2@CXCR4 for 24 h, and then were washed three times with PBS to remove unloaded nanoparticles. Finally, two-photon luminescence images of AuNRs were obtained using a two-photon fluorescent microscope with excitation at 780 nm and emission at 601-657 nm. One part of the cells treated with AuNRs@SiO2@CXCR4 were collected, embedded and made into the specimens for transmission electron microscopy (TEM). The Fourier transform infrared (FTIR) spectra of nanoparticles were measured in the range of 400-4000 cm-1 with an EQUINOX 55 spectrometer using potassium bromide disks. Quantification of AuNRs@SiO2@CXCR4-loaded iPS cells. To measure the amount of AuNRs@SiO2@CXCR4 uptake by iPS cells, iPS cells were incubated with AuNRs@SiO2 or Au@SiO2@CXCR4 with the same elemental Au concentration for 2, 4, 8, 16 and 32 h. At each time point, the iPS cells were washed three times with PBS to remove unloaded nanoparticles, then the AuNRs@SiO2 and AuNRs@SiO2@CXCR4-loaded iPS cells were diluted into 1mL PBS with 2% HNO3, the gold concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS). In vitro transwell boyden migration assay. As previously described, 500 µL of target media (either DMEM or MGC803 culture supernatant medium) per well was added in a 24-well microplate. Suspensions of iPS or AuNRs-iPS in DMEM (1×105 cells/100 µL) were placed in the upper transwell chamber and incubated at 37 ºC overnight. After incubation, the cells not migrating to the bottom side were swept off by cotton swab. The transwell chambers were placed


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in a new 24-well cell culture plate containing DAPI and incubated for 5 min and then washed twice with PBS. After that, the numbers of migrated cells were counted under fluorescence microscope. In vitro photothermal effect of AuNRs-iPS cells. iPS cells were incubated with PBS(as control groups), AuNRs@SiO2 (40 µg/mL) or equivalent dose of AuNRs@SiO2@CXCR4 for 8 h , and were carefully rinsed three times with PBS to remove the unloaded nanoparticles. The Au nanoparticles-loaded iPS cells were irradiated by NIR laser at 808 nm with a power of 0.8 W/cm2 for 3 min. After heating, laser-treated and untreated iPS cells were identified by calein AM live solution staining. 36 Photoacoustic imaging. Nude mice were implanted with MGC-803 cells in the back. When the tumors grew into about 50 mm3 in size, photoacoustic (PA) imaging of AuNRs was implemented by Endra Nexus 128 PA scanner (Ann tbor, MI). Tumor-bearing mice were intratumorally injected with 120 µL free AuNRs@SiO2 (50 µg) or AuNRs-iPS cells (the same elemental Au concentration within 1.8×106 cells) after anesthetized with isoflurane, and then were placed in the protruding tip of the bowl. Mice tail vein-injected with PBS were used as controls. The images of the tumor sites were consistently kept to facilitate the comparisons. PA imaging of the site exposed to the 800 nm irradiation by NIR laser was acquired according to different injection times (0, 4, 12, 24, 36 h), and pre-injection scans were set as controls. Reconstruction of single wave-length optoacoustic images was achieved by Osirix imaging software by displaying with transverse maximum intensity projection and an UCLA modality. Two-photo fluorescent microscope observation of tumor sections. After tail vein injections of PBS, free AuNRs@SiO2 (50 µg) or AuNRs-iPS cells (The same elemental Au concentration within 1.8×106 cells), the nude mice with MGC803 tumors were sacrificed, then the tumors were frozen in Tissue Tek OCT and sectioned on a leica CM 1510 S cryostat (Sakura Funetek USA). Sections (8 µm) were collected on positively charged slides for two-photon luminescence imaging. The images were obtained using a two-photo fluorescent microscope with excitation at 780 nm and emission at 601-657 nm. In vivo photothermal therapy of AuNRs-iPS cells. The nude mice with MGC-803 tumors were anaesthetized with isoflurance before tail vein injections of PBS, free AuNRs@SiO2 (50 µg) or AuNRs-iPS cells (The same concentration of elemental Au within 1.8×106 cells). Three days


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later, the tumor region was irradiated with an 808 nm laser. To prevent the epidermis of tumor region from burnt during the laser-treatment process, the laser treatment was carried out for 3min at a power density of 1.5 W/cm2 (30 s interval after every 1 min of the exposure). Simultaneously, the temperature variation of tumor sites was monitored by using an infrared thermal imaging camera. Tumor size was measured by using digital vernier caliper every 25 days. After the experiment, the mice were sacrificed and the tumors were excised and weighed, then the tumors were washed three times with PBS buffer for further investigation on H&E staining. Western Blot. Total proteins were harvested from iPS cells, HDF-1, MGC803, GES-1 lines, normal tumor tissue and thermal treatment tumor tissue. GAPDH, CXCR4, Caspase 3 and HSP70, were analyzed by Western Blot. Statistical analysis. Data were presented as mean ± SD unless otherwise stated. Statistical significance was determined using a two-tailed student′s test (P < 0.05) unless otherwise stated. Conflict of interest：The authors declare no competing financial interest. Acknowledgement. This work is supported by the National Key Basic Research Program (973 Project) (No. 2015CB931802), National Natural Scientific Fund (No. 81225010, 81327002, and 31100717), 863 Project of China (2014AA020700), Shanghai Science and Technology Fund (No. 13NM1401500 and 15DZ2252000). Supporting Information Available: Details of physical, chemical and biological characterization

of the AuNRs-iPS. This material is available free of charge via the internet at http://pubs.acs.org.

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Figures and caption

Figure 1. TEM images of (A) AuNRs and (B) AuNRs@SiO2. (C) UV-vis spectra of AuNRs, AuNRs@SiO2 and AuNRs@SiO2@CXCR4. (D) FTIR spectra of AuNRs@SiO2, AuNRs@SiO2-COOH and AuNRs@SiO2@CXCR4.


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Figure 2. Flow cytometric analysis of iPS cells death induced by AuNRs@SiO2@CXCR4.

Figure 3. Cellular uptake of AuNRs@SiO2 and AuNRs@SiO2@CXCR4. (A)Two-photon laser scanning confocal microscopy images of AuNRs@SiO2 and AuNRs@SiO2@CXCR4 in iPS cells. The untreated cells were used as controls. The excitation wavelength =780 nm. All scale bars are 100 µm. (B) Quantitative cellular uptake of AuNRs@SiO2 and AuNRs@SiO2@CXCR4 by iPS cells were measured using ICP-MS. (C) In vitro transwell Boyden migration assay: untreated iPS cells and AuNRs@SiO2@CXCR4 -treated iPS cells


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(28 µg/ 106 cells) were compared.

Figure 4. Representative TEM images of iPS cells. (A) Normal iPS cell. (B, C, D, E, F) Ultrastructures of iPS cells after 24 h incubation with AuNRs@SiO2@CXCR4. Red arrows and green arrow point the location of AuNRs@SiO2@CXCR4. The red arrows point the AuNRs@SiO2@CXCR4 located in the lysosome, the green arrows point AuNRs@SiO2@CXCR4 being exocytosed by the iPS cells.

Figure 5. Photothermal therapy effects on iPS cells. iPS cells were incubated with PBS (as control groups), AuNRs@SiO2 or AuNRs@SiO2@CXCR4 for 8 h at 37 ºC, and then exposed to a 808 nm laser for 3 min. Representative 10× images are shown after laser exposure


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(Live-dead staining with calcein PI and calcein-M): the first panel: bright field, the second panel: fluorescence field, live cells are stained in green, the third panel: fluorescence field, dead cells are stained in red. All scale bars are 100 µm.

Figure 6. PA imaging in vivo in the UCLA mold: Representative PA sequential images of tumor vascular acquired before injection and after injection (2, 8, 24 and 36 h) of the PBS (the first panel), AuNRs@SiO2 (the second panel) and AuNRs-iPS (the last panel).

Figure 7. Two-photon laser scanning confocal microscopy images of tumors sections 5 days after injection of (A) PBS, (B) AuNRs@SiO2 and (C) AuNRs-iPS. All scale bars are 200 µm.


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Figure 8. Antitumor activity of AuNRs-iPS in vivo . (A) Infrared microscopic imaging: the nude mouse of GC subcutaneous xenograft upon NIR laser irradiation (808 nm, 1.5 W/cm2, 3min) 3 days after PBS, AuNRs@SiO2 and AuNRs-iPS injection. (B) Tumor growth in the various groups after laser irradiation, #P

Human Induced Pluripotent Stem Cells for Tumor ... - ACS Publications

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