Effect of size and morphology of functionalized upconversion nanoparticle labeled probe on stem cell absorption

Synthesis of functional upconversion nanoparticles

All chemicals used in the synthesis were purchased from Sigma-Aldrich and used as received without further purification. The synthesis of NaYF₄: Gd³⁺/Er³⁺ nanocrystals (Ball shape) were done following a reported procedure with some modifications.^28,29 In this synthesis, A precursor solution of the lanthanide chlorides (0.78 mmol YCl₃·6H₂O, 0.20 mmol GdCl₃·6H₂O and 0.02 mmol ErCl₃·6H₂O) were taken in a 100 mL 3-necked flask and 30 mL sodium oleate and 10 mL of oleic acid were added. The flask was then stirred at room temperature for 30 min to get a homogenous solution. Subsequently, 12 mL ethanol solution containing 5 mmol NaF and 4 mL H₂O was added dropwise into the flask. The resulting solution was stirred again at room temperature for 10 min to get a homogenous solution. The reaction vessel was then heated up to 160°C under a gentle flow of argon and held at that temperature for 12 h. The flask was cooled to room temperature and added an ethanol solution with CaCl₂ (3 mmol). And then, another ethanol solution with Na₃PO₄ (2 mmol) was injected. The reaction was continued for 1 h under stirring. The (NaYF₄: Gd³⁺/Er³⁺)/Ca₃(PO₄)₂ nanoparticles were precipitated by adding ethanol, separated by centrifugation, washed with ethanol several times, and re-dispersed in cyclohexane for future use. The synthesis of other size (NaYF₄: Gd³⁺/Er³⁺)/Ca₃(PO₄)₂ UCNPs is shown in the Table 1.

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Table 1.

The proportion of synthetic materials.

Sample	Ethanol/mL	NaF/mM	H2O/mL	T/°c	t/h	Shape	Size
B-FUCs	12	5	4	160	12	Ball	83 nm
S-FUCs	12	6	4	180	24	Square	105 nm
H-FUCs	18	10	6	200	24	Hexagon	235 nm
R-FUCs	24	16	6	240	48	Rod	1.2 μm

UCNPs coated with a functional polymer were prepared by a commonly used the LBL assembly technique for nanoparticles. Branched NH₂-PEG-NH₂ (Mw ∼2000) and sodium hyaluronate (Mw ∼10,000) were dissolved in DI water to prepare a stock solution (10 mg/mL), respectively. Diluted hydrochloric acid was added to adjust the pH value of stock solution to 7.4. The stock solution of UCNPs was mixed vigorously with the same volume of NH₂-PEG-NH₂ solution. After 24 h of reaction, the NH₂-PEG-coated UCNPs were purified by three Centrifugal washings. The resultant solution was filtered through a 0.22 mm syringe filter to remove large aggregates. Excess polymers were removed by centrifugation of FUC-NPs and repeated water washing.

Isolation and expansion of human umbilical cord mesenchymal stem cells

HUCMSCs were obtained from Human umbilical cords which were harvested after normal deliveries. After removed the vessels, the umbilical cords were minced and then incubated with 0.2% collagenase type II (Sigma, MO, USA) for 2 h at 37°C, finally passed through a 100 μm filter to collected cell suspensions. The centrifuged cell pellets were re-suspended in Human Umbilical Cord Mesenchymal Stem Cell Growth Medium (Cyagen Biosciences Inc., Catalog No. HUXUC-90011) and then plated in T-75 cell culture flasks. After 3 days of culture, the growth medium was replaced and the remnants of the cord fragments were removed. The HUCMSCs were cultured until they reached to 80% confluence. All cell cultures were maintained under the 5% CO₂, atmosphere at 37°C.

Live/dead cells viability assay

HUCMSCs were cultured on glass coverslips at a density of 1 × 10⁵ cells well-6 at 37°C for 24 h. After treatment with FUC-NPs for 24 h, the cells was harvested and washing with PBS for three times. The fluorescent dyes were prepared by mixing 10 mg of acridine orange (AO) and 10 mg of ethidium bromide (EB) in 10 mL of phosphate buffer solution (PBS, 0.01 mol/L, pH 7.4). Then, the cells were stained with 100 mL of fluorescent dyes for 15 min, and observed under an inverted fluorescence microscope.

Apoptosis analysis

Apoptosis was detected with an annexin V-FITC kit purchased from TOYOBO (Japan) according to the manufacturer’s instructions. 1 × 10⁶ HUCMSCs were grown in 6-well culture dishes for 24 h at 37 °C/5% CO₂. Then the cells incubated with FUC-NPs for 24 h, respectively. After trypsination, cells were centrifuged at 1000 min/r for 5 min, re-suspended in 200 μL of binding buffer. Subsequently, 5 μL of FITClabeled annexin V and 5 μL of the PI stock solution were added to the cell suspension for 15 min in the dark at room temperature. Stained cells were analyzed with a flow cytometry (BD FACSAria).

Lactate dehydrogenase analysis

The HUCMSCs membrane integrity was measured by lactate dehydrogenase (LDH) leakage assay, which using a cytotoxicity detection kit (Promega Cat. 7891) following the vendor’s protocol. In this experiment, the HUCMSCs -free medium was used as the negative control, while HUCMSCs lysed by 1% Triton-X-100 were used as the positive control. The assay was performed using a microplate reader.

Reactive oxygen species assay

ROS production was evaluated using a hydroethidine (DHE) probe known be oxidized by various oxidative agents. In brief, cells were treated with FUC-NPs for 1 day. Then collected the cells and re-suspended in cell culture medium containing 1 μM HE, and analyzed using a flow cytometry and Leica laser-scanning microscope.

Human umbilical cord mesenchymal stem cells labeling with functional upconversion nanoparticles

FUC-NPs at various concentrations were incubated with HUCMSCs in 12-well culture plates for 4 h, followed by repeated washing with PBS to remove free FUC-NPs. Confocal luminescence imaging of cells was performed with a modified flow cytometry and a modified Leica laser-scanning microscope using 980 nm external laser as the excitation source.

Transmission electron microscopy

For TEM, 5 × 10⁶ HUCMSCs were trypsinized and further processed as described previously. After fixation and prior embedding, fixed cells were centrifuged for pellet formation. TEM imaging was conducted on transmission electron microscope (TEM) (JEM-2010, JEOL, Japan) and measured by Image J Software. ³⁰

Induced differentiations of functional upconversion nanoparticles labeled human umbilical cord mesenchymal stem cells

Differentiation capacity of FUC-NPs labeled HUCMSCs was assessed by osteogenic and adipogenic differentiation induction experiments. For osteogenic differentiation, HUCMSCs with or without FUC-NPs labeling were seeded at a density of 1 × 10⁴ cells/cm² in a 12-well culture plate. When the cells reached confluent, osteogenic differentiation was L-DMEM supplemented with 10% FBS, dexamethasone (10 mM), ascorbate (0.1 mM), and β-glycerolphosphate (10 mM). The labeled HUCMSCs were cultured for 21 days, and the culture medium was replaced every 3 days. The cells were then fixed and stained by Alizarin red S.

The adipogenic induction culture was initiated by adding an induction culture containing L-DMEM cell medium supplemented with 10% FBS, dexamethasone (10 μM), indomethacin (0.2 mM), 3-isobuty-1 methylxanthine (IBMX, 0.5 mM) and insulin (4 μg/mL). The HUCMSCs were cultured for 18 days and the induction medium was changed every 2 days. Then HUCMSCs were fixed and stained with Oil-red-O.

HUCMSCs were allowed to grow into full confluence in 6-well plates precoated with 0.1% gelatin and then incubated with 5 μg/mL Mitomycin C at 37°C, 5% CO₂ for 2 h to inactivate HUCMSCs. Monolayer inactivated HUCMSCs were wounded by scratching with 1 mL pipette tip. Fresh Stem cell growth medium was added with FUC-NPs. Images were taken by Nikon digital camera after 24 h of incubation at 37°C, 5% CO₂.

Characterization of functional upconversion nanoparticles nanoparticles

TEM images of the FUC-NPs nanoparticles were obtained from a TEM (JEM-2010, JEOL, Japan). The TEM grids were coated with the nanoparticles by drop casting a dilute dispersion of the nanoparticles (hexane dispersions of hydrophobic nanoparticles and ethanol dispersions of the hydrophilic nanoparticles) and air dried before imaging. TEM imaging showed that the FUC-NPs are monodispersed in size (Figure 1).The size distribution was calculated for ∼83 nm (Ball shape, B-FUCs), 105 nm (Square shape, S-FUCs), ∼235 nm (Hexagon shape, H-FUCs) and ∼1.2 μm (Rod shape, R-FUCs), respectively. This result was met with the hydrodynamic nanoparticle size of FUC-NPs measured by DLS (Figure S1A). The X-ray diffraction (XRD) patterns of the nanoparticles were acquired using a Bruker D4 X-ray diffractometer with a Cu-K_α source (γ = 0.15418 nm) operating. The XRD patterns were collected at a sampling width of 0.1° (2θ) and scan speed of 8° min⁻¹. The XRD pattern (Figure S1B) shows that the FUC-NPs nanoparticles retain their phase after modification, and the peaks match well with the standard spectral lines of β-NaYF₄ (JCPDS #16–0334).^28,29 FT-IR spectra were obtained from an EQUINOX55 FT-IR spectrometer with a resolution of 2 cm⁻¹ and averaged over three scans. The samples were taken as dry powder and pelletized with KBr and placed on the sample holder. Comparison of the FT-IR spectra of the (NaYF₄: Gd³⁺/Er³⁺)/Ca₃(PO₄)₂ UCNPs with that of the NH₂-PEG-coated UCNPs indicated that the oleates were modified with NH₂-PEG-NH₂. The FT-IR spectrum of the (NaYF₄: Gd³⁺/Er³⁺)/Ca₃(PO₄)₂ UCNPs (Figure S1C) shows the carboxylate stretching at 1637 and 1564 cm⁻¹, the appearance of oleate -CH₂ symmetric peaks at 2926 and 2855 cm⁻¹ and the characteristic peaks of Ca₃(PO₄)₂ at 601 and 561 cm⁻¹. On the other hand, the FT-IR spectrum of the NH₂-PEG-coated UCNPs shows a strong N-H bending peak at 2926 cm⁻¹ suggested the successful PEG conjugation. The broad peak at 3442 cm⁻¹ can be due to the O-H stretching of oleate or aspartate and the adsorbed water molecules. The upconversion luminescence (UCL) spectra of FUC-NPs were measured using a 980 nm laser as the excitation light, showing UCL peaks around ∼530 nm and ∼590 nm (Figure S1D). The UCL peaks were not sharp due to the low power of excitation light instrument.

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Figure 1.

The TEM image of functional upconversion nanoparticles, including B-FUCs (ball shape, 83 nm), S-FUCs (square shape, 105 nm), H-FUCs (hexagon shape, 235 nm) and R-FUCs (rod shape, 1.2 μm).

Functional upconversion nanoparticles cellular cytotoxicity

Many reports have shown the toxicology of UCNPs in vitro and in vivo. It generally accepted that the cellular toxicity of UCNPs is closely linked to their size and the surface chemistry.^22,24 Before using FUC-NPs for stem cell labeling, we must understand how FUC-NPs with different sizes and shapes interact with HUCMSCs. Numbers of methods from various aspects were used to evaluate the potential cellular cytotoxicity of FUC-NPs to HUCMSCs.

In order to determine the optimal time and nanoparticle concentration for stem cell labeling, HUCMSCs were treated with different concentrations of four shapes FUC-NPs (0.25, 5, 10, 20 or 40 μg/mL) for 48 h, and different time for 1 day, 3 days or 7 days with 5 μg/mL. After incubation, we examined the cytotoxicity of the different shapes nanoparticles at the aforementioned concentrations and time points. The MTT assay showed that nearly 95% of HUCMSCs were viable following nanoparticle concentrations up to 40 μg/mL and exposure 48 h. There was no significant difference in cell viability between the FUC-NPs labeled groups and unlabeled control (p > 0.05). When the treatment concentrations were 0.25 and 5 μg/mL, the cell viability slightly increased compared to the untreated control at each shapes nanoparticles examined (Figure 2(a)). There was no significant toxicity between 10 μg/mL and 20 μg/mL doses at this exposure time. To investigate the impact of different shapes FUC-NPs labeling on the proliferation capability of HUCMSCs, HUCMSCs were incubated with FUC-NPs at 5 μg/mL for 24 h. After removal of un-internalized UCNPs, the cells were cultured for another 1 day, 3 days or 7 days before being subjected to MTT assay. The short-term proliferation of HUCMSCs was minimally increased by the FUC-NPs labeling. All groups enhanced a little increases in the number of viable cells, with about 14% increase in viable cells for HUCMSCs treated with 5 μg/mL of B-FUCs and S-FUCs after 7 days culture as compared to the unlabeled HUCMSCs (Figure 2(b)).

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Figure 2.

(a) Cytotoxicity in HUCMSCs exposed to B-FUCs, S-FUCs, H-FUCs and R-FUCs for 48 h. (b) Cytotoxicity in HUCMSCs exposed to B-FUCs, S-FUCs, H-FUCs and R-FUCs e for 1, 3, 7 days, respectively. The cytotoxicity was determined by MTT reduction method. Values are the mean ± SD of three independent experiments.

The Live/Dead cells viability assay was performed with HUCMSCs stained using AO&EB fluorescent dyes to further confirm the cellular cytotoxicity of four shapes nanoparticles. In this test, the HUCMSCs cells that appeared green/red when visualized under fluorescence microscopy were considered live/dead with intact/damaged membranes. Fluorescence micrographs of HUCMSCs cells before and after treatment with B-FUCs, S-FUCs, H-FUCs and R-FUCs for 24 h are shown in Figure 3(a). The HUCMSCs of the control group fluoresced green (Figure 3(a)), indicating that the cells were alive. After treatment with different nanoparticles for 24 h, the red fluorescence was hardly observed, suggesting that the HUCMSCs were also alive (Figure 3(a)). Therefore, the result confirmed that the FUC-NPs with a low toxicity can’t damage the cell membranes.

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Figure 3.

(a) The live/dead cells viability assay was performed with HUCMSCs stained using AO&EB fluorescent dyes to further confirm the cellular of nanoparticles. (b) Flow cytometry of apoptotic HUCMSCs as assessed by annexin V-FITC/PI fluorescent intensity. HUCMSCs treated with B-FUCs, S-FUCs, H-FUCs and R-FUCs, respectively. All the concentration is 5 μg/mL.

To determine whether nanoparticles shape or size-induced apoptosis, HUCMSC cells were cultured in the presence and absence of the B-FUCs, S-FUCs, H-FUCs and R-FUCs and analyzed by annexin V–FITC/PI double staining. Figure 3(b) indicates the results of bivariate FITC-Annexin V/PI flow cytometry of HUCMSCs after incubation with four nanoparticles for 24 h. The Q3 quadrant of the histograms represents the viable cells, which exclude PI and are negative for FITC-Annexin V binding. Our data indicate that over 95% HUCMSC cells were alive, which also confirms the low cellular cytotoxicity of the FUC-NPs.

So as to further look for any potential cell damage caused by FUC-NPs, the release of lactate dehydrogenase (LDH) from FUC-NPs labeled HUCMSCs was examined. Released LDH can lead to cell necrosis, which is an indicator of cell membrane damage. Data show that the levels of LDH release from FUC-NPs labeled HUCMSCs were normal compared to that of unlabeled control, suggesting no obvious cell membrane damage induced by FUC-NPs (Figure 4(a)). In this assay, 1% Triton-X-100 lysed HUCMSCs was used as the positive control. Intracellular peroxide and superoxide such as O²⁻ or H₂O₂ can damage biomacromolecules such as lipids, DNA and proteins, resulting in a high degree of cellular toxicity. In this study, the reactive oxygen species (ROS) levels in HUCMSCs were evaluated using a dihydroethidine (DHE) probe. No significant increase in the percentage of DHE positive cells after treatment with four shapes of FUC-NPs was observed, indicating minimal oxidative stress induced to stem cells after FUC-NPs -labeling (Figures 4(b) and (c)).

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Figure 4.

(a) The release of lactate dehydrogenase (LDH) from FUC-NPs labeled HUCMSCs was examined. (b) and (c) The reactive oxygen species (ROS) levels in HUCMSCs were evaluated using a dihydroethidine (DHE) probe. HUCMSCs treated with 5 μg/mL B-FUCs, S-FUCs, H-FUCs and R-FUCs, respectively. Values are the mean ± SD of three independent experiments.

Test of functional upconversion nanoparticles cellular-uptake under different conditions

Next, we used FUC-NPs for stem cell labeling. HUCMSCs were obtained from human umbilical cords which were harvested from Guangzhou Overseas Chinese Hospital after normal deliveries by the standard protocol and characterized by their specific surface antigen expressions using flow cytometry (Figure S2).^1,31,32 To further evaluate whether cell phenotype changes after FUC-NPs labeling, MSCs-related markers such as CD45, CD73, CD105, were measured when HUCMSCs were treated with FUC-NPs for 48 h. As shown in Figure S2, four FUC-NPs will not change the HUCMSCs specific surface antigen expressions, indicating that FUC-NPs were without toxic effects on HUCMSCs. For cell labeling, HUCMSCs were incubated with FUC-NPs for 12 h (Figure 5(a)). After washing with PBS to remove free nanoparticles, stem cells labeled with FUC-NPs have been analyzed using flow cytometry. The results for nanoparticles B-FUCs, S-FUCs, H-FUCs and R-FUCs are shown in Figure 5(a). Upon excitation, all the cell samples incubated with the nanoparticles displayed higher emission intensities, compared with the autofluorescence of untreated HUCMSCs, indicating efficient cellular internalization of the nanoparticles. The emission intensity of HUCMSCs treated with S-FUCs is higher than those treated with nanoparticles B-FUCs, H-FUCs and R-FUCs (Figure 5(a)). The possible reason is in accordance with S-FUCs is more effectively interiorized by the cells.

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Figure 5.

(a) The cell take-up of nanoparticles B-FUCs, S-FUCs, H-FUCs and R-FUCs, respectively. (b) The cell take-up of nanoparticles B-FUCs at different expose time. (c) The cell take-up of nanoparticles B-FUCs at different concentration. (d) The cell take-up of nanoparticles B-FUCs at different temperature.

The cellular-uptake properties of S-FUCs have been studied in more detail using flow cytometry. The results of HUCMSCs treated with nanoparticles S-FUCs at a concentration of 5 μg/mL for 1, 12, 24 and 48 h are illustrated in Figure 5(b). The data suggests that the cellular uptake of the nanoparticles S-FUCs was a time-dependent process. Additionally, after treatment with nanoparticles S-FUCs (0.25, 5, 10 or 20 μg/mL) for 24 h, led to a clear increase of cellular-uptake efficiency, as revealed by flow cytometry results (Figure 5(c)) and confocal microscopy (Supporting information Figure S3). Interestingly, incubation of the HUCMSCs with nanoparticles S-FUCs at 4°C resulted in much reduced cellular-uptake efficiency (Figure 5(d)). This suggests that the interiorization of the nanoparticles were an energyrequiring process such as endocytosis.

Evaluation of the cellular internalization of functional upconversion nanoparticles into human umbilical cord mesenchymal stem cells

In order to determine the cellular internalization of FUC-NPs into stem cell, HUMSCs were treated with four nanoparticles (5 μg/mL) for 24 h. After incubation, the cells were co-stain for cytoskeletal actin and nuclei, then washed and imaged using a confocal microscope. The upconversion luminescence was observed the localization of FUC-NPs signal within the cytoplasm of HUCMSCs, demonstrating that the stem cells were successfully labeled with the FUC-NPs (Figure 6). The level of FUC-NPs uptake in individual cells was proportional to the nanoparticle treatment concentration (Supporting information Figure S3). Moreover, S-FUCs have shown a better cell-uptake property for stem cell labeling. Nest, the S-FUCs that entered the cells were clearly present in the cytosol of HUCMSCs, as detected by TEM at 24 h after sample treatment (Figure 7).

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Figure 6.

Co-localization of FUC-NPs signal (green) with HUCMSCs actin (red) and nuclei (blue) following incubation with 5 μg/mL FUC-NPs for 24 h.

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Figure 7.

TEM analysis of cellular uptake of S-FUCs into HUCMSCs.

Stem cell labeling with functional upconversion nanoparticles

One potential challenge for in vivo tracking of FUC-NPs-labeled stem cells is the possibility that the uptaken nanoparticles could be leaked out from stem cells over time by exocytosis, leading to the unexpected labeling of other surrounding cells and introducing false positive signals. ³³ From this, we investigated this issue by a transwell culture system. ²⁰ HUCMSCs labeled with FUC-NPs (5 μg/mL, 4 h) were grown on the porous membrane in the upper compartment, while unlabeled HUCMSCs were cultured in the lower compartment of the transwell culture system (Figure 8). The porous membrane, which separated the upper and lower compartments, allowed the passing of free nanoparticles but not cells. The labeled and unlabeled cells were co-cultured for 7 days and no obvious FUC-NPs signal in unlabeled cells cultured in the lower compartment throughout this culture period, the data supporting that FUC-NPs could be used in stem cell tracking. And we also tested how the UCL intensity from S-FUCs-labeled HUCMSCs cultured in the upper compartment changed over time. As shown in Figure S4, the cells in the bottom compartment remained free of any S-FUCs signal throughout the culture period, suggesting that there were no particles excytosed from the labeled cells in the upper compartment.

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Figure 8.

Long-term imaging of MFNP-labeled HUCMSCs in vitro. HUCMSCs with and without FUC-NPs-labeling were cultured in upper and lower compartments, respectively, which were separated by a layer of porous membrane (pore size = 1 μm). Confocal UCL images of HUCMSCs grown in upper and lower compartments were taken after 3 days of incubation. Green and blue colors represent UCL signals and blue fluorescence from FUC-NPs and DAPI, respectively.

To further look for the way that those nanoparticles can be transported into cells, the HUCMSCs was incubated with S-FUCs at 4°C, 25°C, 37°C. Interestingly, no significant UCL signals were observed from incubation of the cells with S-FUCs under 4°C, as revealed by confocal microscope (Figure S5). It suggests that the cellular uptake of those nanoparticles could likely via energy requiring process such as endocytosis. ³⁴

Further carefully study is needed to determine the detailed cell entry pathways of those relatively nanoparticles. Caveolae-mediated pinocytosis, clathrin receptor-mediated endocytosis, and macropinocytosis-mediated phagocytosis are the main endocytic pathways for active cell transport mechanism of internalizing macromolecules, particles, and small molecules. Caveolae-mediated pinocytosis occurs via a cholesterol sensitive channel wherein caveolar vesicles are formed and fused with caveosomes. This mechanism is not significantly contributed to constitutive endocytosis. ³⁵ Clathrin receptor-mediated endocytosis is started through the formation of clathrin-coated vesicles and leads to the formation of endosomes, which is the most outstanding one for cellular entry. Macropinocytosis-mediated phagocytosis is an efficient pathway for the nonselective endocytosis of large-sized particles, which is a process involving the formation of large vesicles by the closing of ruffling membrane domains. ³⁶ In this study, we investigated the internalization pathways of FUC-NPs nanoparticles using three different types of endocytosis inhibitor. We used 5 mM β-cyclodextrin to inhibit caveolae-mediated pinocytosis, 0.45 M hypertonic sucrose to inhibit clathrin receptor-mediated endocytosis, and 5 μM cytochalasin D to inhibit macropinocytosis-mediated phagocytosis.

The upconversion luminescence of cells which incubated with FUC-NPs nanoparticles after interaction with endocytosis inhibitors for 4 h (Figure 9). As shown in Figure 9, both the B-FUCs and S-FUCs upconversion luminescence of HUCMSCs treated with inhibitors was weaker than that of cells incubated without inhibitor, especially clearly for cells the cultured with sucrose. Meanwhile, the upconversion luminescence intensity of cells in the presence of β-cyclodextrin and cytochalasin D were similar to that of HUCMSCs in the absence of inhibitor, illustrating that both B-FUCs and S-FUCs entered the cells by clathrin receptor-mediated endocytosis. Interestingly, the entry of H-FUCs was blocked in the presence of sucrose and cytochalasin D, but was unaffected by β-cyclodextrin. However, the cellular uptake of R-FUCs significantly decreased only treatment with cytochalasin D. The inhibition study showed that FUC-NPs nanoparticles entered HUCMSCs cells by clathrin receptor-mediated endocytosis, or macropinocytosis-mediated phagocytosis. And the most prominent pathway of FUC-NPs was depended on the size of nanoparticles.

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Figure 9.

Confocal laser scanning microscopy images of HUCMSCs cells after incubation with FUC-NPs containing different endocytosis inhibitor, (a) none, (b) β- Cyclodextrin, (c) sucrose, and (d) Cytochalasin D. FUC-NPs-labeling dosage was 5 μg/mL.

Differentiation of functional upconversion nanoparticles labeled human umbilical cord mesenchymal stem cells

We next assessed the effect of FUC-NPs-labeling to the on the osteogenic and adipogenic differentiations capability of HUCMSCs. Osteogenic differentiation (Figure 10(a)) and adipogenic differentiation (Figure 10(b)), Alizarin-red-S (ARS) and Oil-red-O (ORO) staining were carried out to visualize calcium deposits and lipid vacuoles, respectively. HUCMSCs labeled with FUC-NPs (5 μg/mL, 4 h) were cultured in osteogenic or adipogenic media for 21 days or 18 days and their differentiation potency was compared with normal unlabeled HUCMSCs. Light microscopy reveals that HUCMSCs labeled with FUC-NPs were able to undergo osteogeneic and adipogenic differentiation. And our data suggest that B-FUCs and S-FUCs labeling slightly promoted the osteogeneic differentiation of HUCMSCs. We speculated that it can be thank to their little size and no exocytosis. Besides, a modified wound scratch assay was carried out to analyze the migration of FUC-NPs labeled HUCMSCs. Migration was analyzed in a modified wound scratch assay. Compared to the unlabeled control, B-FUCs labeled HUCMSCs exhibit an increase in migration rate while R-FUCs labeled HUCMSCs show a little decrease (Figure S6).

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Figure 10.

Osteogenic and adipogenic differentiation of FUC-NPs labeled HUCMSCs. (a) Alizarin red S staining of unlabeled rMSCs after 21 days culture in adipogenic (negative staining control) and osteogenic media (positive control), and labeled HUCMSCs after 21 days culture in osteogenic media. (b) Oil red O staining of unlabeled HUCMSCs after 18 days culture in osteogenic (negative staining control) and adipogenic media (positive control), and labeled HUCMSCs after 18 days culture in adipogenic media.