Biological Potential of Polyethylene Glycol (PEG)-Functionalized Graphene Quantum Dots in In Vitro Neural Stem/Progenitor Cells

Abstract

Stem cell therapy is one of the novel and prospective fields. The ability of stem cells to differentiate into different lineages makes them attractive candidates for several therapies. It is essential to understand the cell fate, distribution, and function of transplanted cells in the local microenvironment before their applications. Therefore, it is necessary to develop an accurate and reliable labeling method of stem cells for imaging techniques to track their translocation after transplantation. The graphitic quantum dots (GQDs) are selected among various stem cell labeling and tracking strategies which have high photoluminescence ability, photostability, relatively low cytotoxicity, tunable surface functional groups, and delivering capacity. Since GQDs interact easily with the cell and interfere with cell behavior through surface functional groups, an appropriate surface modification needs to be considered to get close to the ideal labeling nanoprobes. In this study, polyethylene glycol (PEG) is used to improve biocompatibility while simultaneously maintaining the photoluminescent potentials of GQDs. The biochemically inert PEG successfully covered the surface of GQDs. The PEG-GQDs composites show adequate bioimaging capabilities when internalized into neural stem/progenitor cells (NSPCs). Furthermore, the bio-inertness of the PEG-GQDs is confirmed. Herein, we introduce the PEG-GQDs as a valuable tool for stem cell labeling and tracking for biomedical therapies in the field of neural regeneration.

Keywords: biocompatibility; cytotoxicity; neural stem/progenitor cells (NSPCs); polyethylene glycol functionalized-graphene quantum dots (PEG-GQDs); the visible bio labeling system.

Figure 1

(A) Schematic illustration of the GQDs and PEG-GQDs fabrication. (B) Particle size distribution taken from transmission electron microscope (TEM) images (bar) and Dynamic light scattering (DLS, line) of the GQDs and PEG-GQDs. (C) Photograph of cyclohexane/water hydrophilicity/miscibility test. (D) Zeta potential of PEG-GQDs. (E) X-ray photoelectron spectroscopy (XPS) C 1s peaks. (F) Fourier-transform infrared spectroscopy (FTIR) of the PEG, GQDs, and PEG-GQDs.

Figure 2

Optical fluorescence of PEG-GQDs prepared in DW at various concentrations and visualized at three different wavelengths (405 nm, 488 nm, and 555 nm). (A) Optical fluorescence images of the PEG-GQDs at different exposure times (405 nm: 1000 ms; 488 nm: 1500 ms; 555 nm: 700 ms). (B) The quantification figure of the fluorescence intensity for the PEG-GQDs at different exposure time.

Figure 3

Cytotoxicity test of PEG-GQDs in rat neural stem/progenitor cells (rNPSCs). The amounts of 10,000 rNPSCs were seeded on the Matrigel pre-coated 96-well plate. After the cells grew for 24 h, the gradient concentrations of PEG-GQDs were added to the cells. The series of cell viability tests were then checked by a CCK assay (A) and live-dead staining (B) after 24 h of incubation. (A) In the Cell counting Kit-8 (CCK-8) assay, the rNSPCs were treated with CCK-8 reagent diluted with prewarmed media and incubated for 2 h at 37 °C. Then, the absorbance values were measured with a microplate reader at 450 nm. (B) For the live/dead assay, we used the live/dead viability cytotoxicity kit. The cells were incubated with Calcein AM and Ethidium homodimer-1 for 30 min. Live cells were distinguished by the green fluorescence of Calcein AM and dead cells were distinguished by the red fluorescence of Ethidium homodimer-1. Then, images were obtained with a fluorescence microscope at 40×. Scale bar: 500 µm. The error bars represent the standard deviation of the mean (n = 3). “*” indicates a significant difference between the control and experimental groups (p < 0.05). “***” indicates a significant difference between the control and experimental groups (p < 0.001).

Figure 4

The absorption and intracellular location of PEG-GQDs in rat neural stem/progenitor cells (rNPSCs). (A) Confocal microscopy imaging of PEG-GQDs (320 µg/mL) in rNSPC cells. (B) Fewer PEG-GQDs were absorbed in the cell. (C) The aggregated PEG-GQDs were observed in the cell. The yellow square represents the region of interest. Green fluorescence was cell body stained with Phalloidin and blue fluorescence was from PEG-GQDs. (D) The rNSPCs were incubated with or without 320 µg/mL PEG-GQDs for 0 h, 2 h, 4 h, 16 h, 24 h, and 48 h at 37 °C, 5% CO₂, and 100% humidity incubator. As the incubation time increased, more cells were detected with the PEG-GQDs (320 µg/mL) by the Fluorescence-activated cell sorting (FACS) method. The Y coordinate axes represent the count of cell number. (E) The percentage of cells detected with PB450-A fluorescence signals of PEG-GQDs (320 µg/mL) in rNSPC cells were summarized. The uptake of PEG-GQDs increased over the time and reached saturation after 24 h. Scale bar: 50 µm. The error bars represent the standard deviation of the mean (n = 3). “a” indicates a significant difference between the 0 h and experimental groups (p < 0.0001). “b” indicates a significant difference between the 24 h and experimental groups (p < 0.005). “c” indicates a significant difference between the 24 h and experimental groups (p < 0.0001).

Figure 5

PEG-GQDs did not affect the differentiation potential of rat neural stem/progenitor cells (rNPSCs). (A) The rNSPCs were incubated for 24 h without or with 320 µg/mL PEG-GQDs. Subsequently, the growth media was replaced with Neurobasal plus media to induce differentiation. After 7 days of differentiation, immunocytochemistry assays were performed to estimate the differentiation potential of rNSPCs. Representative microphotographs demonstrated β tubulin III (Tuj1)- and Glial fibrillary acidic protein (GFAP)-positive cells. The nuclei were counterstained with Hoechst (blue). Scale bar: 50 µm. (B) The percentage of Tuj1- and GFAP-positive cells were normalized with Hoechst- positive total cells numbers. There was no significant difference compared with the non-treated control group. “N.S.” indicates no difference between the control and experimental groups (n = 15, p > 0.05).