Cell Medicine, Vol. 6, pp. 91–97, 2013 Printed in the USA. All rights reserved. Copyright 2013 Cognizant Comm. Corp.
2155-1790/13 $90.00 + .00 DOI: http://dx.doi.org/10.3727/215517913X674261 www.cognizantcommunication.com
Adipose Tissue-Derived Stem Cell Imaging Using Cadmium-Free Quantum Dots Yoshiyuki Miyazaki,* Hiroshi Yukawa,† Hiroyasu Nishi,‡ Yukihiro Okamoto,† Noritada Kaji,*† Tsukasa Torimoto,‡ and Yoshinobu Baba*†§ *Department of Applied Chemistry, Nagoya University Graduate School of Engineering, Furo-cho, Chikusa-ku, Nagoya, Japan †FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan ‡Department of Crystalline Materials Science, Nagoya University Graduate School of Engineering, Furo-cho, Chikusa-ku, Nagoya, Japan §Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho, Takamatsu, Japan
Quantum dots (QDs) have received much attention for biomolecule and cell imaging applications because of their superior optical properties such as high quantum efficiency, size-tunable emission, and resistance to photobleaching process. However, QDs that are commercially available contain cadmium (Cd), a highly toxic element. Thus, the development of Cd-free and less toxic QDs is strongly desired. In this study, we developed Cd-free QDs (ZnS-coated ZnS-AgInS2 solid solution nanoparticles with a sulfo group: ZnS-ZAIS-SO3H) and investigated the ability of this material to label stem cells. ZnS-ZAIS-SO3H could be transduced into mouse adipose tissue-derived stem cells (mASCs) using octaarginine peptides (R8), known as cell-penetrating peptides. The optimal ratio of ZnS-ZAIS-SO3H:R8 was found to be 1:100 for labeling mASCs. More than 80% of mASCs labeled with 500 nM ZnS-ZAIS-SO3H were found to be alive, and the proliferation rates of labeled mASCs were maintained at the same rate as that of nonlabeled mASCs. In addition, no abnormalities in the morphology of mASCs labeled with ZnS-ZAIS-SO3H could be observed. These data suggest that ZnS-ZAISSO3H may be effective for the labeling of mASCs. Key words: Cadmium-free quantum dots (Cd-free QDs); Octaarginine (R8); Adipose tissue-derived stem cells (ASCs)
INTRODUCTION Quantum dots (QDs) have several distinctive photoluminescence advantages such as high quantum yields, narrow emission bandwidths, and resistance to photo bleaching in comparison with conventional organic probes (9,18). Because of these fluorescence characteristics, QDs have been widely applied for ultrasensitive sensing of small quantities of substances or cells (1,10). In fact, QD-based Western blot technology enables us to detect tracer proteins related to diseases (2). The fabrication of QDs and lectin conjugates has been reported to be useful for low cytometric identification of leukemia cells (22). In addition, QDs have recently been investigated for the fluorescence imaging and diagnosis of living cells. We have already reported that transplanted stem cells in mice could be monitored with high sensitivity by QDs containing cadmium (Cd) with near-infrared region wavelength (20,21).
However, their biological applications are limited because of the presence of Cd, a very toxic element. Most commercially available QDs contain Cd in their core. Especially in the case of medical applications of QDs, the involvement of Cd is a serious problem. Several reports have shown the cytotoxicity of free Cd2+ ions released from QDs to different types of cells (5,8). We also confirmed that QDs containing Cd are cytotoxic to mesenchymal stem cells at concentrations greater than 16 nM (20). To resolve this problem, new types of QDs such as coated and Cd-free QDs have been proposed (3,6,7,12). Kirchner et al. showed that silicacoated QDs have no cytotoxic effects on cell lines examined with concentrations as high as 30 µM (12). This article suggests that the encapsulation of QDs including Cd with silica can reduce the degree of leaching of Cd as the toxic element from the core. However, even if QDs have been encapsulated accurately, the perfect block
Received April 30, 2013; final acceptance October 23, 2013. Online prepub date: October 29, 2013. Address correspondence to Hiroshi Yukawa, FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Tel: +81-52-789-5654; Fax: 81-52-789-5117; E-mail: [email protected]
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of free Cd2+ ions released from QDs is thought to be substantially difficult. On the other hand, Cd-free QDs, which maintain the fluorescence properties of QDs containing Cd, attract considerable attention as safe and nontoxic probes for biological and clinical applications (11,15). Mandal et al. reported that QDs with a zinc sulfide (ZnS) shell and a copper indium sulfide (CuInS2) (CIS) core, which have high quantum yields and long emission lifetimes emitting from the visible to near-infrared spectral region, are available for bioimaging of highly autofluorescent human breast cancer cells (14). To date, we have developed Cd-free QDs, ZnS-coated silver indium sulfide (AgInS2) solid solution nanoparticles (ZnS-ZAIS), and demonstrated the superior optical properties and the easy regulation of fluorescence wavelength region by particle composition change (16,17). However, the biological and medical applications of ZnS-ZAIS such as the imaging of live cells have remained unexplored. In this study, we addressed the addition of a sulfo group (SO3H) to ZnS-ZAIS; the newly developed ZnS-ZAISSO3H is soluble in water. Next, we checked the optical properties and particle size and investigated whether stem cells can be labeled with ZnS-ZAIS-SO3H efficiently and safely. Herein, we used mouse adipose tissue-derived stem cells (mASCs) as stem cells to compare with our previous data on the influence of QDs containing Cd on mASCs. MATERIALS AND METHODS Animals Three 7- to 14-month-old female C57BL/6 mice were purchased from Japan SLC, Hamamatsu, Japan. The mice were housed in a controlled environment (12-h light/dark cycles at 22°C) with free access to water and a standard chow diet before being killed. All conditions and handling of animals in this study were conducted with protocols approved by the Nagoya University Committee on Animal Use and Care. Isolation and Culture of mASCs The 7- to 14-month-old female C57BL/6 mice were killed by cervical dislocation; adipose tissue specimens in the inguinal groove were isolated and washed extensively with Hank’s balanced salt solution or phosphatebuffered saline (both Gibco, Life Technologies Japan, Tokyo, Japan) to remove the blood cells. The isolated adipose tissue specimens were cut finely and digested with 1 ml of 1 mg/ml type I collagenase (274 U/mg, Koken Co., Ltd., Tokyo, Japan) at 37°C in a shaking water bath for 45 min. The cells were filtered using 250-mm nylon cell strainers (BD Biosciences, Tokyo, Japan) and suspended in Dulbecco's modified Eagle medium/F12 (DMEM/ F12; Gibco) containing 20% fetal bovine serum (FBS;
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Trace Scientific Ltd., Melbourne, Australia; Uin 53141, Lot B01249-500) and 100 U/ml penicillin/streptomycin (Invitrogen Corporation, Auckland, New Zealand) (culture medium). They were centrifuged at 780 × g for 5 min at room temperature, and mASCs were obtained from the pellet. They were washed three times by suspension and centrifugation in culture medium and then were incubated overnight in culture medium at 37°C with 5% CO2. Synthesis of Oleylamine-Modified ZnS-AgInS2 Solid Solution Nanoparticles (ZAIS) ZAIS covered with oleylamine was prepared according to the procedure paper (17) with a slight modification. Silver diethyldithiocarbamate [Ag(S2CNEt2)] (13.6 mg; Wako Pure Chemical Industries, Osaka, Japan), indium tris(diethyldithiocarbamate) [In(S2CNEt2)3] (29.7 mg; Sigma-Aldrich, St. Louis, MO, USA), and zinc bis (diethyldithiocarbamate) [Zn(S2CNEt2)2] (6.8 mg: SigmaAldrich) were dispersed in oleylamine (3.0 ml; Tokyo Chemical Industry, Tokyo, Japan), and the reaction solution was heated at 180°C for 30 min under N2 atmosphere. The resulting suspension was subjected to centrifugation for 5 min at 2,600 × g to remove large particles. ZAIS was separated from the supernatant by addition of methanol (Wako Pure Chemical Industries), followed by centrifugation for 5 min at 2,600 × g. Synthesis of ZnS-Coated ZAIS Nanoparticles (ZnS-ZAIS) ZnS-ZAIS was prepared according to the procedure paper (16). ZAIS nanoparticles prepared as above, zinc acetate dihydrate (11.8 mg; Wako Pure Chemical Industries), and thioacetamide (4.0 mg; Kishida Chemical, Osaka, Japan) were dissolved in oleylamine (2.0 ml), and the reaction solution was heat treated at 180°C for 30 min under N2 atmosphere. After filtration through a syringe filter (Advantec, Tokyo, Japan), the nanoparticles were separated from the solution by the addition of methanol, followed by centrifugation for 5 min at 2,600 × g. Synthesis of ZnS-ZAIS Modified With Sulfonic Acid (ZnS-ZAIS-SO3H) ZnS-ZAIS-SO3H was prepared by a similar procedure to that previously described (23). The ZnS-ZAIS prepared above was dissolved in chloroform (1.0 ml; Wako Pure Chemical Industry). A mixture of sodium 2-mercaptoethanesulfonate (MES) (164 mg; Tokyo Chemi cal Industry), tetramethylammonium hydride (Nacalai Tesque, Kyoto, Japan) 25% methanol solution (730 ml), and methanol (1.0 ml) was added into the ZnS-ZAIS nanoparticle solution, and the reaction solution was heated for 1.5 h at 70°C under N2 atmosphere. After the solvent was removed under reduced pressure, the crude product
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was dissolved in methanol. Chloroform was then added to precipitate ZnS-ZAIS-SO3H, followed by centrifugation for 5 min at 2,600 × g. This purification process was repeated several times to remove residual reagents. The resulting precipitate was dried under vacuum and dissolved in MilliQ water (Millipore, Billerica, MA, USA). ZnS-ZAIS-SO3H Properties The absorbance spectra, photoluminescence spectra, size distribution, and zeta potential of ZnS-ZAIS-SO3H were measured. The absorption spectra were measured using an Agilent Technology 8453A UV-visible spectrophotometer (Santa Clara, CA, USA). The photoluminescence spectra were measured using a photonic multichannel analyzer (PMA-12; Hamamatsu Photonics, Shizuoka, Japan). The size distribution and the zeta potential of ZnS-ZAIS-SO3H and R8–ZnS-ZAIS-SO3H complex were measured using a dynamic light-scattering spectrophotometer (ZETASIZER Nano-ZS, Malvern Instru ments Limited Japan, Hyogo, Japan). Preparation and Transduction Method of R8–ZnS-ZAIS-SO3H Complex To determine the optimal ZnS-ZAIS-SO3H concentration and the ratio of ZnS-ZAIS-SO3H and octaarginine peptides (R8; Sigma-Aldrich Corporation, Tokyo, Japan) to transduce into mASCs, ZnS-ZAIS-SO3H and R8 were mixed for 20 min at room temperature in various ratios (1:10, 1:100, 1:1,000), and R8–ZnS-ZAIS-SO3H complexes were obtained. In this time, the mixed solution was placed in a dark place. Next, mASCs were incubated with the R8–ZnS-ZAIS-SO3H complexes in transduction medium (DMEM/F12, 2% FBS, 100 U/ml penicillin/ streptomycin) at 37°C. After 4 h of incubation, the cells were washed using transduction medium. Then, the transduction of ZnS-ZAIS-SO3H into mASCs was confirmed by conventional fluorescence microscopy. Cytotoxicity of ZnS-ZAIS-SO3H to mASCs mASCs (2 × 104 cells) were seeded in 96-well plates (BD Falcon; BD Biosciences) with 100 ml of culture medium for 1 day, and they were replaced with 100 ml of the R8–ZnS-ZAIS-SO3H complexes in transduction medium at 37°C. ZnS-ZAIS-SO3H and R8 were mixed at the optimal ratio of 1:100. After 4 h of incubation, the cells were washed and incubated for 1 day. After that, viable cells were counted using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). CCK-8 reagent (10 ml) was added to each well, and the reaction was allowed to proceed for 1.5 h. The absorbance of samples at 450 nm was measured against a background control using a microplate reader (POLARstar OPTIMA; BMG LABTECH, Ortenberg, Germany).
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Proliferation of mASCs Labeled With ZnS-ZAIS-SO3H mASCs (4 × 103 cells) were seeded in a 96-well plate with 100 ml of culture medium for 1 day and then transduced with ZnS-ZAIS-SO3H using R8 at various concentrations for 4 h. ZnS-ZAIS-SO3H (0, 12.5, 25, 50, 100 nM) and R8 were mixed at the optimal ratio of 1:100, and then each complex was added to mASCs. Next, the cells were washed, and the medium was replaced with 100 ml of new culture medium. After 2, 4, and 5 days, viable cells were counted using the CCK-8 in the same way as the cytotoxic method. RESULTS ZnS-ZAIS-SO3H Properties Absorbance and photoluminescence spectra of ZnSZAIS-SO3H are shown (Fig. 1A). The fluorescence maximum wavelength was 673 nm. The quantum yield at 365 nm excitation in water was 0.67. The results of size distribution and zeta potential of ZnS-ZAIS-SO3H and R8–ZnS-ZAIS-SO3H complex are shown (Fig. 1B, a, b). The size peak was about 10 nm in both particles. The zeta potentials of ZnS-ZAIS-SO3H and R8–ZnS-ZAIS-SO3H were −7.33 and +10.34 mV, respectively. Transduction of ZnS-ZAIS-SO3H Into mASCs Using R8 To test whether mASCs could be labeled with ZnSZAIS-SO3H (0, 62.5, 125, 250, 500 nM) using R8, the morphologies and red fluorescence derived from mASCs labeled with R8–ZnS-ZAIS-SO3H complex (mixed at the ratio of 1:1,000) were checked using fluorescence microscopy. The red fluorescence derived from mASCs labeled with more than 250 nM ZnS-ZAIS-SO3H could be detected. On the other hand, no morphology changes could be observed in all conditions (Fig. 2). Optimal Concentration Ratio of ZnS-ZAIS-SO3H and R8 To investigate the optimal concentration ratio of ZnS-ZAIS-SO3H and R8, 500 nM ZnS-ZAIS-SO3H was mixed with various concentrations of R8 (0, 5, 50, 500 µM), respectively, and these complexes were transduced into mASCs for 4 h. The red fluorescence derived from labeled mASCs could be detected most efficiently in the condition in which mASCs were labeled with the complex consisting of 500 nM ZnS-ZAIS-SO3H and 50 µM R8 (Fig. 3). These results suggest that the optimal concentration ratio of ZnS-ZAIS-SO3H:R8 is 1:100 for labeling mASCs. Cytotoxicity and Proliferation of mASCs Labeled With ZnS-ZAIS-SO3H To examine the influence of ZnS-ZAIS-SO3H on the cytotoxicity and proliferation of mASCs, ZnS-ZAISSO3H was transduced into mASCs using R8 under various
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Figure 1. Absorbance spectra, fluorescence spectra, size distribution, and zeta potential. (A) The absorbance and fluorescence spectra of ZnS-coated ZnS-AgInS2 solid solution nanoparticles modified with sulfonic acid (ZnS-ZAIS-SO3H) in water. The peak of fluorescence spectrum is 673 nm. (B) The size distribution and zeta potential of (a) ZnS-ZAIS-SO3H (10 nM) and (b) octaarginine peptides (R8)–ZnS-ZAIS-SO3H complex (ZnS-ZAIS-SO3H: 10 nM, R8: 10 µM) in water. (a) The size peak is about 10 nm. The zeta potential average shows −7.33 mV. (b) The size peak is about 10 nm. The zeta potential average shows +10.34 mV.
concentrations for 4 h. More than 80% of mASCs labeled with 500 nM ZnS-ZAIS-SO3H were confirmed to be alive (Fig. 4A). Moreover, the influence on the proliferation rate of mASCs was checked within the noncytotoxic range of concentrations. No significant differences were observed between the proliferation rates of mASCs labeled with ZnS-ZAIS-SO3H and that of normal mASCs (Fig. 4B). DISCUSSION In this study, we developed new Cd-free QDs, watersoluble ZnS-ZAIS-SO3H after addition of a sulfo group, and examined whether ZnS-ZAIS-SO3H is available for live cell imaging. The addition of a sulfo group into ZnS-ZAIS did not affect the absorbance and fluorescence wavelength. The surface charge of ZnS-ZAISSO3H changed to retain a negative charge in water. We confirmed that ZnS-ZAIS-SO3H alone was generally unable to label living cells with a negative charge on their
membranes. Thus, we used R8, known as a cell-penetrating peptide, for cell labeling by ZnS-ZAIS-SO3H based on our previous reports (4,13). Cell-penetrating peptides are thought to be a useful tool for QDs transduction because of their low cytotoxicity and high transduction efficiency. We have already ascertained that R8 was useful for transduction of QDs into mASCs (20). R8–ZnS-ZAIS-SO3H was positively charged and could label mASCs effectively. The particle size distribution was found to shift to a bigger size, and more than 50-nm size of nanoparticles was discovered. These results can indicate that ZnS-ZAIS-SO3H interacted with R8, and R8–ZnS-ZAIS-SO3H complexes were formed in water. On the other hand, the optimal concentration ratio of R8 and ZnS-ZAIS-SO3H changed in comparison with the optimal ratio of 1:10,000 for R8 and QDs, which are commercially available and containing Cd (Cd-QDs). This change is attributed to the number and the electronic charge of the sulfo group introduced on ZnS-ZAIS.
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conditions were not completely the same, this remarkable difference is thought to be mainly explained by Cd (11,14,19). The proliferation rate of mASCs was confirmed to be at the same level as that of normal mASCs under noncytotoxic concentrations. In this study, ZnS-ZAIS-SO3H did not affect the self-renewal ability of mASCs, whereas the maintenance of differentiation ability of mASCs labeled with ZnS-ZAIS-SO3H has not yet been confirmed. Thus, further studies are needed to confirm that ZnS-ZAIS-SO3H does not influence the differentiation ability of mASCs for biological and clinical applications. In conclusion, we developed Cd-free QDs, ZnS-ZAISSO3H, and demonstrated the utility of this material and R8
Figure 2. Transduction of ZnS-ZAIS-SO3H into mASCs using R8. (A, C, E, G, I) The morphologies of mouse adipose stem cells (mASCs) transduced with ZnS-ZAIS-SO3H (0, 62.5, 125, 250, 500 nM) using R8 (ZnS-ZAIS-SO3H:R8 = 1:1000). (B, D, F, H, J) The red fluorescence derived from mASCs transduced with ZnS-ZAIS-SO3H (0, 62.5, 125, 250, 500 nM) using R8 (ZnS-ZAIS-SO3H:R8 = 1:1000).
We confirmed that ZnS-ZAIS-SO3H could improve the cytotoxicity to mASCs drastically as compared with Cd-QDs. In fact, the cytotoxicity of Cd-QDs to mASCs was ascertained to be higher than 16 nM. However, the cytotoxicity of ZnS-ZAIS-SO3H to mASCs was ascertained to be greater than 500 nM. Although the examination
Figure 3. Optimal concentration ratio of ZnS-ZAIS-SO3H and R8. (A, C, E, G) The morphology of mASCs transduced with ZnS-ZAIS-SO3H (500 nM) using R8 (0, 5, 50, 500 µM). (B, D, F, H) The red fluorescence derived from mASCs transduced with ZnS-ZAIS-SO3H (500 nM) using R8 (0, 5, 50, 500 µM). The optimal concentration ratio of ZnS-ZAIS-SO3H and R8 was proved to be 1:100.
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Figure 4. Cytotoxicity and proliferation of mASCs labeled with QDs. (A) The cytotoxicity of mASCs labeled with various concentrations of ZnS-ZAIS-SO3H (0, 62.5, 125, 500 nM) using R8 at the optimal ratio. No significant cytotoxicity could be found in all concentrations. (B) The proliferation rate of mASCs labeled with nontoxic concentrations of ZnS-ZAIS-SO3H (0, 12.5, 25, 50, 100 nM) using R8 at the optimal ratio. No significant differences could be confirmed in all concentrations. QDs, quantum dots.
complex for fluorescence labeling of mASCs using R8. The optimal concentration ratio of ZnS-ZAIS-SO3H:R8 was 1:100 for delivery into mASCs. No significant cytotoxicity could be confirmed in mASCs labeled with less than or equal to 500 nM ZnS-ZAIS-SO3H. In addition, mASCs labeled with 100 nM ZnS-ZAIS-SO3H using R8 had no adverse effect on cell proliferation. This study provides the potential of Cd-free QDs, ZnS-ZAIS-SO3H, for the development of safe and effective mASC imaging. ACKNOWLEDGMENT: This research is partially supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)” and partially supported by the Japan Science and Technology Agency (JST) through its “Research Center Network for Realization of Regenerative Medicine.” The authors declare no conflict of interest.
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CuInS2/ZnS quantum dots for sentinel lymph node imaging with reduced toxicity. ACS Nano 4(5):2531–2538; 2010. 16. Torimoto, T.; Adachi, T.; Okazaki, K.; Sakuraoka, M.; Shibayama, T.; Ohtani, B.; Kudo, A.; Kuwabata, S. Facile synthesis of ZnS-AgInS2 solid solution nanoparticles for a color-adjustable luminophore. J. Am. Chem. Soc. 129(41):12388–12389; 2007. 17. Torimoto, T.; Ogawa, S.; Adachi, T.; Kameyama, T.; Okazaki, K.; Shibayama, T.; Kudo, A.; Kuwabata, S. Remarkable photoluminescence enhancement of ZnS-AgInS2 solid solution nanoparticles by post-synthesis treatment. Chem. Commun. 46(12):2082–2084; 2010. 18. Xu, X.; Liu, X.; Nie, Z.; Pan, Y.; Guo, M.; Yao, S. Labelfree fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots. Anal. Chem. 83(1):52–59; 2011. 19. Ye, L.; Yong, K. T.; Liu, L.; Roy, I.; Hu, R.; Zhu, J.; Cai, H.; Law, W. C.; Liu, J.; Wang, K.; Liu, J.; Liu, Y.; Hu, Y.; Zhang, X.; Swihart, M. T.; Prasad, P. N. A pilot study in non-human primates shows no adverse response to intravenous injection of quantum dots. Nat. Nanotechnol. 7(7):453–458; 2012.
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2155-1790/13 $90.00 + .00 DOI: http://dx.doi.org/10.3727/215517913X674261 www.cognizantcommunication.com
Adipose Tissue-Derived Stem Cell Imaging Using Cadmium-Free Quantum Dots Yoshiyuki Miyazaki,* Hiroshi Yukawa,† Hiroyasu Nishi,‡ Yukihiro Okamoto,† Noritada Kaji,*† Tsukasa Torimoto,‡ and Yoshinobu Baba*†§ *Department of Applied Chemistry, Nagoya University Graduate School of Engineering, Furo-cho, Chikusa-ku, Nagoya, Japan †FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan ‡Department of Crystalline Materials Science, Nagoya University Graduate School of Engineering, Furo-cho, Chikusa-ku, Nagoya, Japan §Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho, Takamatsu, Japan
Quantum dots (QDs) have received much attention for biomolecule and cell imaging applications because of their superior optical properties such as high quantum efficiency, size-tunable emission, and resistance to photobleaching process. However, QDs that are commercially available contain cadmium (Cd), a highly toxic element. Thus, the development of Cd-free and less toxic QDs is strongly desired. In this study, we developed Cd-free QDs (ZnS-coated ZnS-AgInS2 solid solution nanoparticles with a sulfo group: ZnS-ZAIS-SO3H) and investigated the ability of this material to label stem cells. ZnS-ZAIS-SO3H could be transduced into mouse adipose tissue-derived stem cells (mASCs) using octaarginine peptides (R8), known as cell-penetrating peptides. The optimal ratio of ZnS-ZAIS-SO3H:R8 was found to be 1:100 for labeling mASCs. More than 80% of mASCs labeled with 500 nM ZnS-ZAIS-SO3H were found to be alive, and the proliferation rates of labeled mASCs were maintained at the same rate as that of nonlabeled mASCs. In addition, no abnormalities in the morphology of mASCs labeled with ZnS-ZAIS-SO3H could be observed. These data suggest that ZnS-ZAISSO3H may be effective for the labeling of mASCs. Key words: Cadmium-free quantum dots (Cd-free QDs); Octaarginine (R8); Adipose tissue-derived stem cells (ASCs)
INTRODUCTION Quantum dots (QDs) have several distinctive photoluminescence advantages such as high quantum yields, narrow emission bandwidths, and resistance to photo bleaching in comparison with conventional organic probes (9,18). Because of these fluorescence characteristics, QDs have been widely applied for ultrasensitive sensing of small quantities of substances or cells (1,10). In fact, QD-based Western blot technology enables us to detect tracer proteins related to diseases (2). The fabrication of QDs and lectin conjugates has been reported to be useful for low cytometric identification of leukemia cells (22). In addition, QDs have recently been investigated for the fluorescence imaging and diagnosis of living cells. We have already reported that transplanted stem cells in mice could be monitored with high sensitivity by QDs containing cadmium (Cd) with near-infrared region wavelength (20,21).
However, their biological applications are limited because of the presence of Cd, a very toxic element. Most commercially available QDs contain Cd in their core. Especially in the case of medical applications of QDs, the involvement of Cd is a serious problem. Several reports have shown the cytotoxicity of free Cd2+ ions released from QDs to different types of cells (5,8). We also confirmed that QDs containing Cd are cytotoxic to mesenchymal stem cells at concentrations greater than 16 nM (20). To resolve this problem, new types of QDs such as coated and Cd-free QDs have been proposed (3,6,7,12). Kirchner et al. showed that silicacoated QDs have no cytotoxic effects on cell lines examined with concentrations as high as 30 µM (12). This article suggests that the encapsulation of QDs including Cd with silica can reduce the degree of leaching of Cd as the toxic element from the core. However, even if QDs have been encapsulated accurately, the perfect block
Received April 30, 2013; final acceptance October 23, 2013. Online prepub date: October 29, 2013. Address correspondence to Hiroshi Yukawa, FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Tel: +81-52-789-5654; Fax: 81-52-789-5117; E-mail: [email protected]
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of free Cd2+ ions released from QDs is thought to be substantially difficult. On the other hand, Cd-free QDs, which maintain the fluorescence properties of QDs containing Cd, attract considerable attention as safe and nontoxic probes for biological and clinical applications (11,15). Mandal et al. reported that QDs with a zinc sulfide (ZnS) shell and a copper indium sulfide (CuInS2) (CIS) core, which have high quantum yields and long emission lifetimes emitting from the visible to near-infrared spectral region, are available for bioimaging of highly autofluorescent human breast cancer cells (14). To date, we have developed Cd-free QDs, ZnS-coated silver indium sulfide (AgInS2) solid solution nanoparticles (ZnS-ZAIS), and demonstrated the superior optical properties and the easy regulation of fluorescence wavelength region by particle composition change (16,17). However, the biological and medical applications of ZnS-ZAIS such as the imaging of live cells have remained unexplored. In this study, we addressed the addition of a sulfo group (SO3H) to ZnS-ZAIS; the newly developed ZnS-ZAISSO3H is soluble in water. Next, we checked the optical properties and particle size and investigated whether stem cells can be labeled with ZnS-ZAIS-SO3H efficiently and safely. Herein, we used mouse adipose tissue-derived stem cells (mASCs) as stem cells to compare with our previous data on the influence of QDs containing Cd on mASCs. MATERIALS AND METHODS Animals Three 7- to 14-month-old female C57BL/6 mice were purchased from Japan SLC, Hamamatsu, Japan. The mice were housed in a controlled environment (12-h light/dark cycles at 22°C) with free access to water and a standard chow diet before being killed. All conditions and handling of animals in this study were conducted with protocols approved by the Nagoya University Committee on Animal Use and Care. Isolation and Culture of mASCs The 7- to 14-month-old female C57BL/6 mice were killed by cervical dislocation; adipose tissue specimens in the inguinal groove were isolated and washed extensively with Hank’s balanced salt solution or phosphatebuffered saline (both Gibco, Life Technologies Japan, Tokyo, Japan) to remove the blood cells. The isolated adipose tissue specimens were cut finely and digested with 1 ml of 1 mg/ml type I collagenase (274 U/mg, Koken Co., Ltd., Tokyo, Japan) at 37°C in a shaking water bath for 45 min. The cells were filtered using 250-mm nylon cell strainers (BD Biosciences, Tokyo, Japan) and suspended in Dulbecco's modified Eagle medium/F12 (DMEM/ F12; Gibco) containing 20% fetal bovine serum (FBS;
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Trace Scientific Ltd., Melbourne, Australia; Uin 53141, Lot B01249-500) and 100 U/ml penicillin/streptomycin (Invitrogen Corporation, Auckland, New Zealand) (culture medium). They were centrifuged at 780 × g for 5 min at room temperature, and mASCs were obtained from the pellet. They were washed three times by suspension and centrifugation in culture medium and then were incubated overnight in culture medium at 37°C with 5% CO2. Synthesis of Oleylamine-Modified ZnS-AgInS2 Solid Solution Nanoparticles (ZAIS) ZAIS covered with oleylamine was prepared according to the procedure paper (17) with a slight modification. Silver diethyldithiocarbamate [Ag(S2CNEt2)] (13.6 mg; Wako Pure Chemical Industries, Osaka, Japan), indium tris(diethyldithiocarbamate) [In(S2CNEt2)3] (29.7 mg; Sigma-Aldrich, St. Louis, MO, USA), and zinc bis (diethyldithiocarbamate) [Zn(S2CNEt2)2] (6.8 mg: SigmaAldrich) were dispersed in oleylamine (3.0 ml; Tokyo Chemical Industry, Tokyo, Japan), and the reaction solution was heated at 180°C for 30 min under N2 atmosphere. The resulting suspension was subjected to centrifugation for 5 min at 2,600 × g to remove large particles. ZAIS was separated from the supernatant by addition of methanol (Wako Pure Chemical Industries), followed by centrifugation for 5 min at 2,600 × g. Synthesis of ZnS-Coated ZAIS Nanoparticles (ZnS-ZAIS) ZnS-ZAIS was prepared according to the procedure paper (16). ZAIS nanoparticles prepared as above, zinc acetate dihydrate (11.8 mg; Wako Pure Chemical Industries), and thioacetamide (4.0 mg; Kishida Chemical, Osaka, Japan) were dissolved in oleylamine (2.0 ml), and the reaction solution was heat treated at 180°C for 30 min under N2 atmosphere. After filtration through a syringe filter (Advantec, Tokyo, Japan), the nanoparticles were separated from the solution by the addition of methanol, followed by centrifugation for 5 min at 2,600 × g. Synthesis of ZnS-ZAIS Modified With Sulfonic Acid (ZnS-ZAIS-SO3H) ZnS-ZAIS-SO3H was prepared by a similar procedure to that previously described (23). The ZnS-ZAIS prepared above was dissolved in chloroform (1.0 ml; Wako Pure Chemical Industry). A mixture of sodium 2-mercaptoethanesulfonate (MES) (164 mg; Tokyo Chemi cal Industry), tetramethylammonium hydride (Nacalai Tesque, Kyoto, Japan) 25% methanol solution (730 ml), and methanol (1.0 ml) was added into the ZnS-ZAIS nanoparticle solution, and the reaction solution was heated for 1.5 h at 70°C under N2 atmosphere. After the solvent was removed under reduced pressure, the crude product
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was dissolved in methanol. Chloroform was then added to precipitate ZnS-ZAIS-SO3H, followed by centrifugation for 5 min at 2,600 × g. This purification process was repeated several times to remove residual reagents. The resulting precipitate was dried under vacuum and dissolved in MilliQ water (Millipore, Billerica, MA, USA). ZnS-ZAIS-SO3H Properties The absorbance spectra, photoluminescence spectra, size distribution, and zeta potential of ZnS-ZAIS-SO3H were measured. The absorption spectra were measured using an Agilent Technology 8453A UV-visible spectrophotometer (Santa Clara, CA, USA). The photoluminescence spectra were measured using a photonic multichannel analyzer (PMA-12; Hamamatsu Photonics, Shizuoka, Japan). The size distribution and the zeta potential of ZnS-ZAIS-SO3H and R8–ZnS-ZAIS-SO3H complex were measured using a dynamic light-scattering spectrophotometer (ZETASIZER Nano-ZS, Malvern Instru ments Limited Japan, Hyogo, Japan). Preparation and Transduction Method of R8–ZnS-ZAIS-SO3H Complex To determine the optimal ZnS-ZAIS-SO3H concentration and the ratio of ZnS-ZAIS-SO3H and octaarginine peptides (R8; Sigma-Aldrich Corporation, Tokyo, Japan) to transduce into mASCs, ZnS-ZAIS-SO3H and R8 were mixed for 20 min at room temperature in various ratios (1:10, 1:100, 1:1,000), and R8–ZnS-ZAIS-SO3H complexes were obtained. In this time, the mixed solution was placed in a dark place. Next, mASCs were incubated with the R8–ZnS-ZAIS-SO3H complexes in transduction medium (DMEM/F12, 2% FBS, 100 U/ml penicillin/ streptomycin) at 37°C. After 4 h of incubation, the cells were washed using transduction medium. Then, the transduction of ZnS-ZAIS-SO3H into mASCs was confirmed by conventional fluorescence microscopy. Cytotoxicity of ZnS-ZAIS-SO3H to mASCs mASCs (2 × 104 cells) were seeded in 96-well plates (BD Falcon; BD Biosciences) with 100 ml of culture medium for 1 day, and they were replaced with 100 ml of the R8–ZnS-ZAIS-SO3H complexes in transduction medium at 37°C. ZnS-ZAIS-SO3H and R8 were mixed at the optimal ratio of 1:100. After 4 h of incubation, the cells were washed and incubated for 1 day. After that, viable cells were counted using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). CCK-8 reagent (10 ml) was added to each well, and the reaction was allowed to proceed for 1.5 h. The absorbance of samples at 450 nm was measured against a background control using a microplate reader (POLARstar OPTIMA; BMG LABTECH, Ortenberg, Germany).
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Proliferation of mASCs Labeled With ZnS-ZAIS-SO3H mASCs (4 × 103 cells) were seeded in a 96-well plate with 100 ml of culture medium for 1 day and then transduced with ZnS-ZAIS-SO3H using R8 at various concentrations for 4 h. ZnS-ZAIS-SO3H (0, 12.5, 25, 50, 100 nM) and R8 were mixed at the optimal ratio of 1:100, and then each complex was added to mASCs. Next, the cells were washed, and the medium was replaced with 100 ml of new culture medium. After 2, 4, and 5 days, viable cells were counted using the CCK-8 in the same way as the cytotoxic method. RESULTS ZnS-ZAIS-SO3H Properties Absorbance and photoluminescence spectra of ZnSZAIS-SO3H are shown (Fig. 1A). The fluorescence maximum wavelength was 673 nm. The quantum yield at 365 nm excitation in water was 0.67. The results of size distribution and zeta potential of ZnS-ZAIS-SO3H and R8–ZnS-ZAIS-SO3H complex are shown (Fig. 1B, a, b). The size peak was about 10 nm in both particles. The zeta potentials of ZnS-ZAIS-SO3H and R8–ZnS-ZAIS-SO3H were −7.33 and +10.34 mV, respectively. Transduction of ZnS-ZAIS-SO3H Into mASCs Using R8 To test whether mASCs could be labeled with ZnSZAIS-SO3H (0, 62.5, 125, 250, 500 nM) using R8, the morphologies and red fluorescence derived from mASCs labeled with R8–ZnS-ZAIS-SO3H complex (mixed at the ratio of 1:1,000) were checked using fluorescence microscopy. The red fluorescence derived from mASCs labeled with more than 250 nM ZnS-ZAIS-SO3H could be detected. On the other hand, no morphology changes could be observed in all conditions (Fig. 2). Optimal Concentration Ratio of ZnS-ZAIS-SO3H and R8 To investigate the optimal concentration ratio of ZnS-ZAIS-SO3H and R8, 500 nM ZnS-ZAIS-SO3H was mixed with various concentrations of R8 (0, 5, 50, 500 µM), respectively, and these complexes were transduced into mASCs for 4 h. The red fluorescence derived from labeled mASCs could be detected most efficiently in the condition in which mASCs were labeled with the complex consisting of 500 nM ZnS-ZAIS-SO3H and 50 µM R8 (Fig. 3). These results suggest that the optimal concentration ratio of ZnS-ZAIS-SO3H:R8 is 1:100 for labeling mASCs. Cytotoxicity and Proliferation of mASCs Labeled With ZnS-ZAIS-SO3H To examine the influence of ZnS-ZAIS-SO3H on the cytotoxicity and proliferation of mASCs, ZnS-ZAISSO3H was transduced into mASCs using R8 under various
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Figure 1. Absorbance spectra, fluorescence spectra, size distribution, and zeta potential. (A) The absorbance and fluorescence spectra of ZnS-coated ZnS-AgInS2 solid solution nanoparticles modified with sulfonic acid (ZnS-ZAIS-SO3H) in water. The peak of fluorescence spectrum is 673 nm. (B) The size distribution and zeta potential of (a) ZnS-ZAIS-SO3H (10 nM) and (b) octaarginine peptides (R8)–ZnS-ZAIS-SO3H complex (ZnS-ZAIS-SO3H: 10 nM, R8: 10 µM) in water. (a) The size peak is about 10 nm. The zeta potential average shows −7.33 mV. (b) The size peak is about 10 nm. The zeta potential average shows +10.34 mV.
concentrations for 4 h. More than 80% of mASCs labeled with 500 nM ZnS-ZAIS-SO3H were confirmed to be alive (Fig. 4A). Moreover, the influence on the proliferation rate of mASCs was checked within the noncytotoxic range of concentrations. No significant differences were observed between the proliferation rates of mASCs labeled with ZnS-ZAIS-SO3H and that of normal mASCs (Fig. 4B). DISCUSSION In this study, we developed new Cd-free QDs, watersoluble ZnS-ZAIS-SO3H after addition of a sulfo group, and examined whether ZnS-ZAIS-SO3H is available for live cell imaging. The addition of a sulfo group into ZnS-ZAIS did not affect the absorbance and fluorescence wavelength. The surface charge of ZnS-ZAISSO3H changed to retain a negative charge in water. We confirmed that ZnS-ZAIS-SO3H alone was generally unable to label living cells with a negative charge on their
membranes. Thus, we used R8, known as a cell-penetrating peptide, for cell labeling by ZnS-ZAIS-SO3H based on our previous reports (4,13). Cell-penetrating peptides are thought to be a useful tool for QDs transduction because of their low cytotoxicity and high transduction efficiency. We have already ascertained that R8 was useful for transduction of QDs into mASCs (20). R8–ZnS-ZAIS-SO3H was positively charged and could label mASCs effectively. The particle size distribution was found to shift to a bigger size, and more than 50-nm size of nanoparticles was discovered. These results can indicate that ZnS-ZAIS-SO3H interacted with R8, and R8–ZnS-ZAIS-SO3H complexes were formed in water. On the other hand, the optimal concentration ratio of R8 and ZnS-ZAIS-SO3H changed in comparison with the optimal ratio of 1:10,000 for R8 and QDs, which are commercially available and containing Cd (Cd-QDs). This change is attributed to the number and the electronic charge of the sulfo group introduced on ZnS-ZAIS.
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conditions were not completely the same, this remarkable difference is thought to be mainly explained by Cd (11,14,19). The proliferation rate of mASCs was confirmed to be at the same level as that of normal mASCs under noncytotoxic concentrations. In this study, ZnS-ZAIS-SO3H did not affect the self-renewal ability of mASCs, whereas the maintenance of differentiation ability of mASCs labeled with ZnS-ZAIS-SO3H has not yet been confirmed. Thus, further studies are needed to confirm that ZnS-ZAIS-SO3H does not influence the differentiation ability of mASCs for biological and clinical applications. In conclusion, we developed Cd-free QDs, ZnS-ZAISSO3H, and demonstrated the utility of this material and R8
Figure 2. Transduction of ZnS-ZAIS-SO3H into mASCs using R8. (A, C, E, G, I) The morphologies of mouse adipose stem cells (mASCs) transduced with ZnS-ZAIS-SO3H (0, 62.5, 125, 250, 500 nM) using R8 (ZnS-ZAIS-SO3H:R8 = 1:1000). (B, D, F, H, J) The red fluorescence derived from mASCs transduced with ZnS-ZAIS-SO3H (0, 62.5, 125, 250, 500 nM) using R8 (ZnS-ZAIS-SO3H:R8 = 1:1000).
We confirmed that ZnS-ZAIS-SO3H could improve the cytotoxicity to mASCs drastically as compared with Cd-QDs. In fact, the cytotoxicity of Cd-QDs to mASCs was ascertained to be higher than 16 nM. However, the cytotoxicity of ZnS-ZAIS-SO3H to mASCs was ascertained to be greater than 500 nM. Although the examination
Figure 3. Optimal concentration ratio of ZnS-ZAIS-SO3H and R8. (A, C, E, G) The morphology of mASCs transduced with ZnS-ZAIS-SO3H (500 nM) using R8 (0, 5, 50, 500 µM). (B, D, F, H) The red fluorescence derived from mASCs transduced with ZnS-ZAIS-SO3H (500 nM) using R8 (0, 5, 50, 500 µM). The optimal concentration ratio of ZnS-ZAIS-SO3H and R8 was proved to be 1:100.
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Figure 4. Cytotoxicity and proliferation of mASCs labeled with QDs. (A) The cytotoxicity of mASCs labeled with various concentrations of ZnS-ZAIS-SO3H (0, 62.5, 125, 500 nM) using R8 at the optimal ratio. No significant cytotoxicity could be found in all concentrations. (B) The proliferation rate of mASCs labeled with nontoxic concentrations of ZnS-ZAIS-SO3H (0, 12.5, 25, 50, 100 nM) using R8 at the optimal ratio. No significant differences could be confirmed in all concentrations. QDs, quantum dots.
complex for fluorescence labeling of mASCs using R8. The optimal concentration ratio of ZnS-ZAIS-SO3H:R8 was 1:100 for delivery into mASCs. No significant cytotoxicity could be confirmed in mASCs labeled with less than or equal to 500 nM ZnS-ZAIS-SO3H. In addition, mASCs labeled with 100 nM ZnS-ZAIS-SO3H using R8 had no adverse effect on cell proliferation. This study provides the potential of Cd-free QDs, ZnS-ZAIS-SO3H, for the development of safe and effective mASC imaging. ACKNOWLEDGMENT: This research is partially supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)” and partially supported by the Japan Science and Technology Agency (JST) through its “Research Center Network for Realization of Regenerative Medicine.” The authors declare no conflict of interest.
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