Mesoporous Silica Nanoparticles as a Delivery System for Hydrophobic Anticancer Drugs

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Cellular imaging DOI: 10.1002/smll.200700493

Mesoporous Silica Nanoparticles Improve Magnetic Labeling Efficiency in Human Stem Cells Hon-Man Liu,y Si-Han Wu,y Chen-Wen Lu, Ming Yao, Jong-Kai Hsiao, Yann Hung, Yu-Shen Lin, Chung-Yuan Mou, Chung-Shi Yang, Dong-Ming Huang,* and Yao-Chang Chen

Tumblerlike magnetic/fluorescein isothiocyanate (FITC)-labeled mesoporous silica nanoparticles, [email protected], have been developed, which are composed of silica-coated core–shell superparamagnetic iron oxide ([email protected]) nanoparticles co-condensed with FITC-incorporated mesoporous silica. [email protected] can label human mesenchymal stem cells (hMSCs) through endocytosis efficiently for magnetic resonance imaging (MRI) in vitro and in vivo, as manifested by using a clinical 1.5-T MRI system with requirements of simultaneous low incubation dosage of iron, low detection cell numbers, and short incubation time. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes and osteocytes, which can still be readily detected by MRI. Moreover, a higher MRI signal intensity decrease is observed in [email protected] cells than in [email protected] cells. This is the first report that MCM-41-type MSNs are advantageous to cellular uptake, as manifested by a higher labeling efficiency of [email protected] than [email protected]

Keywords:     

biocompatible materials magnetic resonance imaging mesoporous materials nanoparticles stem cells

[] C.-W. Lu, Dr. C.-S. Yang, Dr. D.-M. Huang Center for Nanomedicine Research National Health Research Institutes 35 Keyan Rd., Zhunan Town, Miaoli County 350 (Taiwan) E-mail: [email protected] Dr. H.-M. Liu,y Dr. J.-K. Hsiao Department of Medical Imaging National Taiwan University Hospital and College of Medicine National Taiwan University 7 Chung-Shan South Rd. Taipei 100 (Taiwan) S.-H. Wu,y Dr. Y. Hung, Y.-S. Lin, Dr. C.-Y. Mou Department of Chemistry National Taiwan University 1 Sec. 4, Roosevelt Rd. Taipei 106 (Taiwan) Dr. M. Yao, Dr. Y.-C. Chen Department of Laboratory Medicine National Taiwan University Hospital and College of Medicine National Taiwan University 7 Chung-Shan South Rd. Taipei 100 (Taiwan) [y] These authors contributed equally to this work. : Supporting Information is available on the WWW under http:// www.small-journal.com or from the author. small 2008, 4, No. 5, 619–626

1. Introduction Tracking the distribution of stem cells in vivo for distinguishing whether cellular regeneration originated from an exogenous cell source is crucial to their therapeutic use. Traditionally, the techniques for examining stem-cell transplantation in animal models are performed by postmortem histological analysis and consequently cannot be applied in clinical studies. Thus, the development of techniques to noninvasively monitor the fate and distribution of transplanted stem cells is definitely important and has attracted great research efforts. Among these, magnetic resonance imaging (MRI) is an ideal imaging modality for the biodistribution of magnetically labeled cells.[1–3] To track stem cells by MRI, cells must be labeled magnetically by endocytic internalization.[3–5] However, cellular tracking by these nanoparticles suffers from low intracellular labeling efficiency, as manifested by the requirement of a long-term incubation or a high concentration

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of particles with cells.[1,2,6–8] Furthermore, in most studies MRI systems with higher field strength than the standard clinical magnetic resonance (MR) imagers of 1.5 T were usually employed to compensate for the limited MRI sensitivity due to the low intracellular labeling efficiency,[2,6,7,9] while a few reports used large numbers of cells for 1.5-T imagers.[8,10,11] Hence, efficient cellular-internalizing methods are highly desirable. A promising strategy to improve internalizing efficiency is to link the particles to an HIV tat peptide or monoclonal antibody;[9] however, the key issue regarding the biosafety of a xenogeneic protein should be seriously considered. For stem-cell tracking in particular, the biological effects of the internalized particles should be safeguarded. Recently, silica (SiO2) was demonstrated to serve as a good nanomaterial for bifunctional or multifunctional cellular labeling because it can be easily surface functionalized for bioconjugation, and is quite biocompatible and resistant to biodegradation in cellular environments.[12–14] In particular, mesoporous silica nanoparticles (MSNs), in the form of stable aqueous dispersions, are emerging as an ideal agent for biomedical imaging, drug delivery, and gene therapy.[13,15–19] In our previous study, we developed novel fluorescein isothiocyanate (FITC)-incorporated MSNs (FITC-MSNs) that can be efficiently internalized into human mesenchymal stem cells (hMSCs) without affecting the viability, growth, and differentiation of these cells.[13] More recently, we have developed FITC-incorporated silica-coated core–shell superparamagnetic iron oxide (SPIO) nanoparticles, [email protected] (FITC), to magnetically and efficiently label hMSCs in vitro and in vivo.[14] Furthermore, we have synthesized a dualfunctional material by conjugating nonfluorescent silica core–shell SPIO ([email protected]) with FITC-MSNs (termed [email protected]) and have demonstrated that [email protected] can be internalized into NIH 3T3 cells.[18] In the present study, we investigated whether hMSCs could be efficiently and harmlessly labeled with [email protected] and then imaged with a clinical MRI analyzer at low cell number. Furthermore, we compared the MRI signal intensities of hMSCs labeled with [email protected] and [email protected] to determine the effects of MSNs on magnetic labeling efficiency.

2. Results and Discussion 2.1. Cellular Uptake Behavior of [email protected] in hMSCs Using Flow Cytometry and Confocal Microscopy [email protected] were synthesized as described in our previous study[18] and illustrated in Figure 1. In the cell-uptake experiments of [email protected], hMSCs were isolated,

Figure 1. Synthetic procedure of tumblerlike magnetic/FITC-labeled mesoporous silica nanoparticles ([email protected]). The mesoporous structure is not shown.

Table 1. Cellular uptake of [email protected] in hMSCs using flow cytometry. [email protected]

0

0.3

1

3

10

30

1.0[a] 27.4[b] 1.2 25.1 1.2 23.1

1.3 27.4 1.9 29.9 2.9 30.3

3.2 35.5 6.9 29.0 16.3 31.3

11.6 33.8 46.1 35.9 66.6 44.0

63.9 53.4 88.9 96.6 96.9 205.6

98.3 288.9 99.3 685.3 99.7 1192.2

S1

[mg mL

]

0.5 h 1h 2h

[a]The numbers of positively labeled cells (defined as a fluorescence value >10) are represented as the percentage of total cells counted. [b]The mean fluorescence intensity of [email protected] cells was noted.

expanded, and then incubated with different concentrations of [email protected] suspensions in serum-free low-glucose Dulbecco’s modified Eagle’s medium (DMEM) for various incubation times. After the indicated time, the cells were harvested by trypsinization; cellular uptake of [email protected] was determined semiquantitatively by the incorporated fluorescence intensity through flow cytometry (Becton Dickenson, Mississauga, CA). The data showed that the uptake of [email protected] was dependent on time (30 min, 1 h, and 2 h) and dose (3, 10, and 30 mg mL1; Table 1). The uptake of [email protected] began significantly as early as 30 min after incubation with 10 mg mL1 of nanoparticles or as low as 3 mg mL1 of nanoparticles for 1 h incubation. Since endocytosis is known as the main mechanism of cellular internalization for the magnetic nanoparticle vectors, we used various pharmacological inhibitors of endocytosis to explore the underlying uptake mechanism. Flow cytometry data (Table 2) showed that the uptake of [email protected] was inhibited by phenylarsine oxide (PAO; a clathrin inhibitor)

Table 2. Effects of various inhibitors on the uptake of [email protected]

Control [email protected] (30 mg mLS1) for 1 h

DMSO

PAO (3 mM)

CytD (3 mM)

Filipin (3 mg mLS1)

Wort (100 nM)

Noco (10 mM)

2.6[a] 85.4

2.0 65.5

2.1 60.5

2.2 88.2

2.7 88.3

2.0 85.8

[a]The numbers of positively labeled cells (defined as a fluorescence value >10) are represented as the percentage of total cells counted.

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Figure 2. hMSCs were treated with [email protected] (30 mg mL1) for 2 h and then incubated with LysoTracker Red for another 30 min. LysoTracker Red-labeled organelles (late endosomes/lysosomes) (a) and the cellular distribution of green fluorescent [email protected] (b) were analyzed by a Zeiss Axiovert 100 M confocal unit. (c) Co-localization of green fluorescent [email protected] with late endosomes/lysosomes (overlap of a and b).

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Figure 3. a) Representative MR images of centrifuged hMSC pellets in Eppendorf tubes placed in a water bath. Each cell pellet contained 1.2  105 cells after treatment with various concentrations of [email protected] for 1 or 2 h before scanning by the 1.5-T MRI system. The change in signal intensity was more significant in the group treated with 30 mg mL1 [email protected] compared to that treated with 10 mg mL1 [email protected] at both incubation times. The signal intensity change is more obvious after incubation of [email protected] for 2 h compared to only 1 h. b) Sensitivity of in vitro MRI of [email protected] hMSCs. Cell numbers ranging from 3  103 to 1.2  105 were scanned after treatment with 30 mg mL1 of [email protected] for 1 h. Top: unlabeled cells with identical cell numbers (control). Bottom: labeled cells detected as darkened spots at the bottom of the test tubes. c) The internalization mechanism of [email protected] was also evidenced by MRI. [email protected] cells treated or untreated with various inhibitors were scanned at an identical cell number of 2.4  105.

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and cytochalasin D (CytD; an actin inhibitor) but not by filipin (a caveola inhibitor), wortmannin (Wort; an inhibitor of macropinocytosis), or nocodazole (Noco; a microtubule inhibitor), which suggests that clathrin-coated pit endocytosis and actin microfilaments might be involved in the uptake of [email protected] Typical histograms of the fluorescence intensity of hMSCs that were incubated with various concentrations of [email protected] for different times are shown in the Supporting Information (Figure S1). In the endocytic pathway, ingested molecules are delivered to early endosomes and then (through late endosomes) to lysosomes. Therefore, we studied the co-localization of LysoTracker Red, a marker for late endosomes and lysosomes with red fluorescence, with [email protected] (green fluorescence). Meanwhile, to avoid the artifact of cytochemical fixing, live cells after incubation with [email protected] and LysoTracker Red were visualized with a Zeiss Axiovert 100 M confocal unit. Indeed, [email protected] could be internalized into hMSCs by endocytosis, which was evidenced by co-localization of LysoTracker Red (Figure 2a) and [email protected] (Figure 2b) in late endosomes and lysosomes that exhibited orange to yellow fluorescence (Figure 2c).

were processed for comparison by T2WI. As shown in Figure 3c, cells labeled with [email protected] and treated with PAO or CytD exhibited a lower MRI intensity decrease, which indicated inhibition of particle uptake by these inhibitors.

2.3. The Biocompatibility of [email protected] To evaluate the possible cytotoxic effect of [email protected], cell viability was examined by MTT reduction assay (see Experimental Section). No evidence of cytotoxicity was observed 2 or 4 h after treatment with [email protected], nor was the cell proliferation affected after [email protected] treatment in growth medium for 24 h (Figure 4a). In addition, we investigated the differentiation potential of labeled hMSCs into adipocytes and osteocytes to examine possible adverse effects of [email protected] on the functions of stem cells. Adipogenic differentiation with the formation and accumulation of lipid vacuoles was demonstrated by Oil Red O stain. Osteogenic differentiation was observed by Fast Blue stain of alkaline phosphatase (Sigma–Aldrich). As shown in Figure 4b, adipogenic differentiation of hMSCs was observed only in cells grown in adipogenic medium and not in regular growth medium. hMSCs treated with [email protected] did differentiate into

2.2. Cellular Uptake Behavior of [email protected] in hMSCs by MRI The highly efficient intracellular uptake and the underlying mechanisms were confirmed by a clinical 1.5-T MRI system (Signa Excite, GE Healthcare, USA). The MR images of centrifuged cell pellets in test tubes placed in a water bath were easily detected. In the T2-weighted image mode (T2WI), cells exposed to 30 mg mL1 of [email protected] for 1 h could be readily detected. The T2WI of labeled hMSCs showed that the MRI signal also displayed a dose- and time-dependent behavior (Figure 3a) as in flow cytometry studies. Hence, for studying the biological effects and the mechanism of uptake of [email protected], an incubation of hMSCs with 30 mg mL1 of [email protected] for 1 h was chosen. The fact that a small number of hMSCs with a short-term incubation were easily imaged with a clinical 1.5-T MRI analyzer implied a high cellular labeling efficiency of [email protected] To investigate the limit of labeling, a series of diluted labeled hMSCs was processed for MRI. Figure 3b shows that the minimum detectable number of hMSCs is around 1.2  104 to 3  104. No signal intensity difference could be detected in all of the samples in the unlabeled control group. Similar inhibitory effects on the uptake of [email protected] for the inhibitors used in flow cytometry were also verified by MRI. To improve the MRI sensitivity, 2.4  105 cells

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Figure 4. a) hMSCs were incubated in the absence (control) or presence of 30 mg mL1 of [email protected] for 2 and 4 h. The cytotoxicity of [email protected] was determined by a MTT assay immediately after incubation. The effect of [email protected] on cell proliferation was examined after hMSCs were allowed to grow in regular growth medium for 24 h after a 1-h incubation with [email protected] Data are expressed as mean  stand error of three determinations (each in triplicate). b,c) The effects of [email protected] on adipogenic (b) and osteogenic (c) differentiation of hMSCs. Cells were first treated without (top panels) or with 30 mg mL1 of [email protected] (bottom panels) for 1 h and then incubated with regular growth medium as negative control (all left panels), adipogenic medium (right panels in b), or osteogenic medium (right panels in c) for differentiation. Original magnification, 200. The images represent three independent experiments. d) hMSCs were first incubated in the absence (control) or presence of 30 mg mL1 of [email protected] for 1 h, and then cells were incubated with regular growth medium (Reg.), adipogenic medium (Adi.), and osteogenic medium (Ost.) for 7 days. After incubation, cells were harvested, centrifuged, and scanned with a 1.5-T MRI system. T2WIs showed there were significant differences of signal intensities between the treated cells and the control. Close-up images of (b) and (c) are shown in the Supporting Information (Figures S2a and S2b, respectively).

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adipocytes, and there was no obvious difference in Oil Red O stain assay between [email protected] control cells and untreated control cells. Figure 4c shows that [email protected] did not affect the osteogenic differentiation induced by incubating hMSCs with osteogenic medium. These data indicated that [email protected] were biocompatible. Moreover, after incubation with different media for 7 days, the cells prelabeled with [email protected] for 1 h could be detected by T2WI in a clinical 1.5-T MR imager (Figure 4d).

2.4. Effects of MSNs on Improving Cellular Uptake of Magnetic Nanoparticles As mentioned above, one of the major problems inherent to stem-cell tracking is the low labeling efficiency of magnetic nanoparticles. Significant efforts have been devoted to improving the labeling efficiency, and several reports have explored the factors (e.g., particle material, particle size, particle shape, and the surface properties of nanoparticles) influencing the cellular uptake of nanoparticles.[20–24] In previous studies, we found that FITC-incorporated MSNs (FITC-MSNs) could be efficiently internalized into hMSCs,[13] and we also demonstrated that SPIO nanoparticles coated with FITC-incorporated silica, [email protected](FITC), could efficiently label the same cells.[14] In the present study, our data showed that [email protected], a novel kind of magnetic nanoparticle composed of two types of nanoparticles described in previous studies, exerted a highly magnetic labeling efficiency. In view of the magnetic properties of [email protected], which were acquired from SPIO nanoparticles, we compared the MR images of hMSCs labeled with [email protected] or [email protected] to evaluate the difference in labeling efficiency. To avoid the variations in the nanoparticle uptake efficiency, hMSCs from three independent donors were incubated with either 30 mg mL1 of [email protected] or different concentrations (10, 15, 20, 30, 45, or 60 mg mL1) of [email protected] for 1 h and then processed for MRI at the same time. Although the labeling efficiency varied in hMSCs from different donors, Figure 5a shows that the MRI signal intensity loss of hMSCs labeled with 30 mg mL1 of [email protected] was greater than that of those treated with 30 mg mL1 of [email protected] Quantitative analysis by measuring the MRI intensity contrast induced by labeled hMSCs revealed superior MRI signal change in cells labeled with 30 mg mL1 of [email protected] over those labeled with 30 mg mL1 of [email protected] by Student’s t-test (P < 0.02; Figure 5b), but there is no statistically significant diffrence between hMSCs labeled with 45 mg mL1 of [email protected] and 30 mg mL1 of [email protected] (P ¼ 0.19). The data show that the labeling efficiency of [email protected] was somewhat higher than that of [email protected] Moreover, given the consideration that the iron contents of [email protected] and [email protected] determined by inductively coupled plasma atomic-emission spectrometry are about 0.9 and 1.7 wt%, respectively, [email protected] displayed much greater labeling efficiency than [email protected] as manifested by the need for a much lower concentration of iron to express similar MRI intensities in [email protected] hMSCs. Since [email protected] small 2008, 4, No. 5, 619–626

Figure 5. Effects of MSNs on improving cellular uptake of magnetic nanoparticles. a) 1.2  105 hMSCs from three independent donors were incubated in the absence (control) or presence of various concentrations (10, 15, 20, 30, 45, and 60 mg mL1) of [email protected] or 30 mg mL1 of [email protected] for 1 h and then scanned by a 1.5-T MRI system. b) Quantification of the labeling effect based on MR images. The volume of signal intensity loss produced by labeled hMSCs was measured. hMSCs treated with 30 mg mL1 of [email protected] exhibited a higher degree of signal intensity change than an identical number of [email protected] hMSCs (P ¼ 0.016). The signal intensity changes between hMSCs treated with 45 mg mL1 of [email protected] and 30 mg mL1 of [email protected] were statistically identical (P ¼ 0.19).

differ from [email protected] only in the attachment of MSNs, we suggest that the higher labeling efficiency of [email protected] was derived from the MSNs.

2.5. In Vivo MRI of hMSCs Labeled with [email protected] We conducted the MRI of hMSCs labeled with [email protected] in a nude mice model. Nude mice were implanted with hMSCs at the olfactory cortex of the brain. Clinical 1.5-T MR images of the mouse brain 8 h after implantation revealed a dark signal at the frontal cortex, which represents the labeling efficiency and imaging ability of [email protected] (Figure 6a). The animal was imaged by MRI 9 days after hMSC implantation and these [email protected] hMSCs could still be visualized (Figure 6b). The finding proved that this labeling method is capable of long-term observation of stem cells in living animals. In addition, compared to more than ten million cells needed for stem-cell therapy,[25] the capability of imaging such low cell numbers (1  105) delivered into mouse brain also indicate the feasibility of monitoring stem-cell migration to different areas in the organism. The high efficiency of stem-cell labeling and

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Figure 6. Clinical 1.5-T MR images of nude mouse 8 h (a) and 9 days (b) after implantation of [email protected] hMSCs at the frontal cortex. a) 1.0  105 hMSCs were injected into the frontal cortex of a nude mouse. Under clinical 1.5-T MR scanning, the hMSCs revealed a dark dot at the frontal cortex (arrow). b) Repeated MR scanning was carried out 9 days after hMSC implantation. The stem cells could still be visualized as a black dot at the frontal cortex. No migration of these cells is found (arrow head).

imaging also provides a good strategy for tracking stem-cell migration. Although not shown here, the migration of stem cells could be imaged if an injury animal model was used and a more sensitive MR pulse sequence, such as a gradient echo sequence, was added for animal scanning. The pulse sequences used here are spin echo sequences, which are ideal for anatomical demonstration. Thereafter, this method of cell labeling fulfils the needs of stem-cell therapy.

3. Conclusions We have demonstrated that as-synthesized [email protected] can efficiently label hMSCs as manifested by the following: 1) the application procedure used to label hMSCs is fast (only 30 min to 1 h incubation) and simple; 2) the concentration of iron oxide for efficient labeling is low (0.27 mg mL1 of culture medium); and 3) the labeled cells can be visualized in a clinical 1.5-T MRI system with a minimal detectable cell number of about 104 in vitro and with an in vivo detection quantity of 1.2  105 cells. Moreover, [email protected] are nontoxic without affecting the cell viability, growth, and differentiation of hMSCs. The labeled cells remain detectable by MRI after long-term growth or differentiation, which is further evidence for the biocompatibility and durability of [email protected] Above all, this is the first report to demonstrate that MCM-41-type MSNs are advantageous to the cellular uptake of nanoparticles. With biocompatibility and efficient labeling, we suggest that MSNs can serve as a new multifunctional stem-cell tracking agent in addition to other biomedical applications of nanotechnology.

4. Experimental Section Culture of hMSCs from bone marrow: hMSCs were isolated from bone marrow of normal donors as described previously,[16]

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and were cultured in regular growth medium consisting of low-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 U mLS1), and streptomycin (100 mg mLS1). All cultures were kept in an atmosphere of 5% CO2 and 95% air at 37-C. Flow cytometric detection of uptake of [email protected]: hMSCs were seeded at 1.2 T 105 cells per well in six-well plates and allowed to attach for 24 h. To determine the [email protected] uptake and loading contents, the cells were incubated with different concentrations of [email protected] suspension in serumfree medium for various incubation times. Treated cells were then washed three times with phosphate-buffered saline (PBS: 137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4), and then harvested by trypsinization. After centrifugation, the cell pellet was washed once and resuspended with PBS containing 2% FBS. The fluorescent dye incorporated in [email protected] served as a marker to semiquantitatively determine their cellular uptake, which was analyzed by FACSCalibur flow cytometry and CellQuest Pro software (Becton Dickenson, Mississauga, CA). Cellular internalization of [email protected]: hMSCs were plated 24 h before the experiment in chamber slides or poly-D-lysine-coated glass cover slips at 5 T 103 cells per cm2. To study the intracellular distribution of [email protected], the cells were incubated first with a suspension of [email protected] for 2 h, and then with 1 mM LysoTracker Red (Molecular Probes) in serum-free medium for another 30 min. After incubation, the cells were washed three times with PBS and visualized with serum-free medium. Images were acquired with a Zeiss Axiovert 100 M confocal unit. MRI in vitro and statistics: MRI was performed using a clinical 1.5-T MR system (Signa Excite, GE Healthcare, USA). The cell samples were centrifuged and placed in a water tank. The tank was then placed in an eight-channel head coil. Two-dimensional T2-weighted gradient echo pulse (GRE) sequences were used (TR/ TE ¼ 550/15 ms, FA ¼ 15). The slice thickness was 1.4 mm with a 0.03-mm gap and the field of view (FOV) was 14 T 10.5 cm. The total scan time was 4 min and 43 s with a number of excitations (NEX) of 3. The images were then analyzed at the workstation provided by GE Healthcare. For quantitative analysis of the signal intensity of these cell pellets, the areas of signal intensity loss produced by labeled hMSCs in each MRI scanning slice were calculated and all the areas in the slice were summed. For comparison of each group, Student’s t-test was used and statistical significance was obtained if the probability was less than 0.05. Cell viability and proliferation assay: In vitro cell viability experiments were performed as follows. After incubation with 30 mg mLS1 of [email protected] in serum-free medium for 2 and 4 h, cells were incubated with fresh serum-free medium containing 0.5 mg mLS1 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 1 h at 37 -C for cytotoxicity assay. For proliferation assay, the cells after treatment with 30 mg mLS1 of [email protected] for 1 h were allowed to grow in regular growth medium for 24 h followed by incubation with MTT reagent. The amount of dark blue formazan dye generated by the live cells was proportional to the number of live cells and the absorbance at 570 nm was measured using a microplate reader. Cell numbers were determined from a standard plot of known cell numbers versus the corresponding absorbance density.

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In vitro differentiation studies and cytochemical analysis: To induce adipogenic differentiation, hMSCs were first incubated with 30 mg mLS1 of [email protected] for 1 h followed by washing with PBS, and then cultured in adipogenic medium or regular growth medium as control for 7 days. Medium changes were carried out twice weekly. Adipogenic medium consisted of high-glucose DMEM supplemented with isobutyl-1-methylxanthine (0.5 mM, Sigma–Aldrich), dexamethasone (1 mM, Sigma–Aldrich), insulin (10 ng mLS1, Sigma–Aldrich), indomethacin (50 mM, Sigma–Aldrich), and 10% FBS. To induce osteogenic differentiation, hMSCs were incubated first with 30 mg mLS1 of [email protected] for 1 h followed by PBS washing, and then treated with osteogenic medium for 1 week, changing the medium twice weekly. Osteogenic medium consisted of a-minimal essential medium (Gibco) supplemented with dexamethasone (1 mM), b-glycerol phosphate (50 mM, Sigma–Aldrich), and 50 mg mLS1 ascorbic acid (Sigma–Aldrich). Adipogenic differentiation was assessed by the cellular accumulation of neutral lipid vacuoles after cells were fixed with 4% formaldehyde and stained with Oil Red O (Sigma–Aldrich). For evaluation of osteogenic differentiation, cells were fixed with citrate-buffered acetone and stained with Fast Blue RR Salt (Sigma–Aldrich) to detect the alkaline phosphatase activity. Mechanism of uptake of [email protected]: There are multiple pathways for particle internalization including clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis, and non-clathrin–non-caveolae-dependent endocytosis. To study the mechanism of uptake of [email protected], hMSCs were incubated with various inhibitors (Sigma–Aldrich) as described below and with a suspension of [email protected] (30 mg mLS1) for 1 h. PAO, a trivalent arsenical, inhibits clathrin-mediated endocytosis by reacting with vicinal sulfhydryls to form stable ring structures. Filipin interacts with 3-b-hydroxysterols in the plasma membrane to form filipin–sterol complexes and subsequently to cause the filamentous caveolin-1-coat, a rapid disassembly that leads to inhibition of caveolae-mediated endocytosis. CytD, a potent inhibitor of actin polymerization, was used to inhibit actin-related phagocytosis and non-clathrin–non-caveolae-dependent endocytosis. Noco disrupts microtubules by binding to b-tubulin and preventing formation of one of the two interchain disulfide linkages, thus inhibiting microtubule dynamics and blocking endocytotic vesicle trafficking. Wort was used to inhibit macropinocytosis by blocking phosphatidylinositol 3-kinase. The effects of inhibitors on cellular [email protected] uptake were examined by flow cytometry and MRI. MRI of magnetically labeled hMSCs in nude mice: Male nude mice (6 weeks of age) were obtained from the National Laboratory Animal Center, Taiwan, and maintained in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. hMSCs were treated with [email protected] (1 mg mLS1) for 1 h, and then harvested by trypsinization and suspended at 1 T 107 cells mLS1 in PBS. An adult female nude mouse was anesthetized with ketamine (100 mg kgS1) mixed with xylazine (5 mg kgS1) into the intraperitoneal space. Stereotaxic injection of hMSCs was performed as described previously.[26] Briefly, a Kopf apparatus and a Hamilton syringe with a bevel-tipped 30-gauge needle were used. hMSCs labeled with [email protected] at 1 T 105 cells were infused in 10-mL aliquots at 0.1-mm intervals small 2008, 4, No. 5, 619–626

with approximately 4000 cells at each interval. The injection track began ventrally, ending in the frontal cortex. Injections were performed at the rate of 0.3 mL minS1 and the needle was left in place for 3 min before withdrawal. After 8 h and after 9 days of hMSC implantation, animal MRI studies were carried out on mice under Isoflurane gas anesthesia (Forane, Abbott). The animal was then scanned under the same clinical 1.5-T MRI system as in the in vitro study. A custom-made radio-frequency (RF) coil for RF excitation and signal reception was used. The RF coil was made as a cylindrical volume resonator with an inner diameter of 38 mm and length of 82 mm. Fast spin echo pulse sequences were used (TR/TE ¼ 3000/90 ms, echo chain ¼ 1/1, bandwidth ¼ 31.2 kHz). The slice thickness was 0.8 mm with a 0.2-mm gap and the FOV was 5 T 2 cm. The scan time was 6.5 min at the NEX of 16. The images were then analyzed at the workstation provided by the vendor.

Acknowledgements This work is supported by grants from the National Health Research Institutes (Grants NM-095-PP-02 and NM-096-PP-02), Taiwan, and from the National Science Council (NSC 95-2120-M-002-009 and NSC 95-2320-B-400-013) of Taiwan. We thank Dr. Dar-Ming Lai at the Department of Surgery, National Taiwan University Hospital and College of Medicine, National Taiwan University, for cell implantation.

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