Labeling mesenchymal cells with DMSA-coated gold and iron oxide nanoparticles: assessment of biocompatibility and potential applications

Abstract

Background: Nanoparticles' unique features have been highly explored in cellular therapies. However, nanoparticles can be cytotoxic. The cytotoxicity can be overcome by coating the nanoparticles with an appropriated surface modification. Nanoparticle coating influences biocompatibility between nanoparticles and cells and may affect some cell properties. Here, we evaluated the biocompatibility of gold and maghemite nanoparticles functionalized with 2,3-dimercaptosuccinic acid (DMSA), Au-DMSA and γ-Fe2O3-DMSA respectively, with human mesenchymal stem cells. Also, we tested these nanoparticles as tracers for mesenchymal stem cells in vivo tracking by computed tomography and as agents for mesenchymal stem cells magnetic targeting.

Results: Significant cell death was not observed in MTT, Trypan Blue and light microscopy analyses. However, ultra-structural alterations as swollen and degenerated mitochondria, high amounts of myelin figures and structures similar to apoptotic bodies were detected in some mesenchymal stem cells. Au-DMSA and γ-Fe2O3-DMSA labeling did not affect mesenchymal stem cells adipogenesis and osteogenesis differentiation, proliferation rates or lymphocyte suppression capability. The uptake measurements indicated that both inorganic nanoparticles were well uptaken by mesenchymal stem cells. However, Au-DMSA could not be detected in microtomograph after being incorporated by mesenchymal stem cells. γ-Fe2O3-DMSA labeled cells were magnetically responsive in vitro and after infused in vivo in an experimental model of lung silicosis.

Conclusion: In terms of biocompatibility, the use of γ-Fe2O3-DMSA and Au-DMSA as tracers for mesenchymal stem cells was assured. However, Au-DMSA shown to be not suitable for visualization and tracking of these cells in vivo by standard computed microtomography. Otherwise, γ-Fe2O3-DMSA shows to be a promising agent for mesenchymal stem cells magnetic targeting.

Keywords: Biocompatibility; Computed microtomography; DMSA-nanoparticles; Gold nanoparticles; Iron oxide nanoparticle; Magnetic targeting; Mesenchymal stem cells.

Fig. 1

Transmission electron microscopy analysis of γ-Fe₂O₃-DMSA and Au-DMSA. a TEM micrographs of Fe-DMSA nanoparticles, bars 50 nm. b TEM micrographs of Au-DMSA nanoparticles, bars 50 nm

Fig. 2

Cell viability assessment by MTT and Trypan-blue staining. a MTT assay of γ-Fe₂O₃-DMSA labeled MSC. The data express average percentage and standard deviation of MSC that have remained viable after exposure to γ-Fe₂O₃-DMSA in four different concentrations at three different exposure times. b MTT assay of Au-DMSA labeled MSC. The average percentage and standard deviation of viable MSC are represented after 24 h of exposure to three different concentrations of Au-DMSA. (*) Significant reduction between the cells of the three experimental groups compared to the control group (p < 0.01). c MTT assay of Au-DMSA labeled MSC 4, 24, 48 and 72 h after incubation with the nanoparticles (90 µg/mL) during 24 h. There were significant differences between control cells and labeled cells only 4 and 24 h after exposure (p < 0.05). d Cell viability test by trypan-blue staining. The data express the mean percentage and standard deviation of MSC that remained alive after 24 h of exposure to 52 and 90 µg/mL of Au-DMSA and 60 and 80 µg/mL of γ-Fe₂O₃-DMSA

Fig. 3

Analysis of MSC morphology by Instant Prov staining kit. a Negative control group. b Positive control group, with pyknotic nuclei (blue arrows) and normal nuclei (black arrows). c MSC incubated with Au-DMSA (90 μg/mL) and d with γ-Fe₂O₃-DMSA (80 μg/mL) for 24 h. Bars 50 micrometers (μm)

Fig. 4

Transmission electron microscopy micrographs of labeled MSC. a Unlabeled MSC (control). b γ-Fe₂O₃-DMSA labeled MSC (80 µg/mL); the white arrows point some of the uptaken nanoparticles. c MSC labeled with Au-DMSA (90 µg/mL); the white arrowheads point some of the uptaken nanoparticles. d Cells exposed to Au-DMSA for 24 h presented myelin figures (black arrows) and e more electron-lucent structures (white arrows) compared to unlabeled cells. f In turn, after 24 h of exposure toγ-Fe₂O₃-DMSA, MSC presented signs of mitochondrial toxicity: mitochondria full of nanoparticles are swollen and degenerated (black arrows), as compared to organelles without them (white arrows). g These cells also presented some myelin figures (black arrows). h Lastly, γ-Fe₂O₃-DMSA nanoparticles were stored in vesicles (white arrows). Nu nucleus, M mitochondria, RER rough endoplasmic reticulum, L lipid

Fig. 5

MSC osteogenic differentiation assay. a–c Cytochemical analysis of differentiated MSC monolayers in light microscopy, with Alizarin Red, in order to evidence the formation of mineralized nodules. These nodules were present in control cells (a), in γ-Fe₂O₃-DMSA labeled cells, (b) and in Au-DMSA labeled cells (c). d Quantification of alizarin red incorporated in monolayers of differentiated and non-differentiated MSC; (*) Significant reduction in the group “MSC + Au-DMSA” compared to the groups “Unmarked MSC” and “MSC + γ-Fe₂O₃-DMSA” (p < 0.05). e Measurement of alkaline phosphatase (ALP) activity: data are represented as the average ratio of ALP activity and total protein content, with the respective standard deviations. There was no significant difference between control and experimental groups (p > 0.05). Bars 100 µm

Fig. 6

MSC adipogenic differentiation assay. a–c Oil Red O cytochemical analysis of differentiated MSC monolayers in light microscopy, with in order to evidence the formation of intracellular lipid vesicles. These vesicles were seen in control cells (a), in γ-Fe₂O₃-DMSA labeled cells, (b) and in Au-DMSA labeled cells (c). d Quantification of Oil Red O incorporated in monolayers of differentiated and non-differentiated MSC, with no significant difference between control and experimental groups (p > 0.05). Bars 50 µm

Fig. 7

a, b MSC proliferation curves. a Cells were incubated for 24 h with DMEM-LG (filled circle), or with DMEM-LG with diluted γ-Fe₂O₃-DMSA (filled square) (80 μg/mL), then were plated and counted after different times. There was no significant difference between the experimental groups (p > 0.05) in any count times. b Cells were incubated for 24 h with DMEM-LG (filled circle), or with DMEM-LG with diluted Au-DMSA (filled square) (90 μg/mL), then were plated and counted after different times. (*) Significant increase in Au-DMSA labeled MSC amount compared to the control group only on the second day (p < 0.05). c Analysis by flow cytometry of CFSE-marked lymphocytes, co-cultured with labeled and unlabeled MSC. The spectra shown are representative of assays performed in triplicate. Gray Line Lymphocytes not marked with CFSE; Black line not activated marked lymphocytes; Red Line activated marked lymphocytes; Blue line activated lymphocytes co-cultured with MSC; Yellow line activated lymphocytes co-cultured with γ-Fe₂O₃-DMSA labeled MSC; Green line activated lymphocytes co-cultured with Au-DMSA labeled MSC

Fig. 8

Au-DMSA potential as a tracer for MSC tracking in computed microtomography. a–c Analysis of MSC precipitates in Sky-Scan 1640 microtomograph in which cross-sections of samples in Eppendorf tubes are represented: a water, b unlabeled MSC, c MSC labeled with Au-DMSA. d–g Sky-Scan 1640 images of longitudinal sections of mice. The animals were immediately analyzed after intranasal instillation with unlabeled cells (d) or with Au-DMSA labeled cells (e). Five days later, animals instilled with unlabeled cells (f) and Au-DMSA labeled cells (g) were analyzed again. Aw airways; H heart; L liver

Fig. 9

γ-Fe₂O₃-DMSA as a potential agent for MSC magnetic targeting. a–d Magnetic responsiveness test in vitro. a, b γ-Fe₂O₃-DMSA labeled cells were seeded in culture plates with a fixed plastic piece (a) or a circular magnet (b). The region in orange corresponds to the site where the materials were fixed. c, d The culture plates were examined by light microscopy in order to demonstrate the difference in amount of cells present in regions highlighted in red. e Iron measurement by colorimetric dosage of Prussian blue. Data refer to the mean ± SD from the mass of iron present in lungs, divided by lung’s weight. f–h Histological analysis of MSC retention in silicotic mice lungs. The slides were stained with Prussian Blue technique and contrasted with neutral red, evidencing iron from the γ-Fe₂O₃-DMSA in blue. f Healthy animal treated with saline g Animals treated with γ-Fe₂O₃-DMSA labeled MSC without external magnets h Animal treated with labeled MSC and with external magnets