Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

Abstract

Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a 'green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

Figure 1

UV–vis absorption spectra of SLE, GO, and S-rGO suspensions in water.

Figure 2

XRD patterns of GO (A) and S-rGO (B).

Figure 3

Hydrodynamic size distribution of GO (A) and S-rGO (B).

Figure 4

FTIR spectra of GO and S-rGO.

Figure 5

SEM images of GO (A) and S-rGO (B).

Figure 6

Raman spectra of GO (A) and S-rGO (B).

Figure 7

AFM images of GO (A) and S-rGO (B).

Figure 8

Effect of GO and S-rGO on cell viability of PMEF cells. Cell viability of PMEF cells was determined using WST-8 assay after a 24-h exposure to different concentrations of GO or S-rGO. The results represent the means of three separate experiments, and error bars represent the standard error of the mean. GO-treated groups showed statistically significant differences from the control group by Student’s t test (p < 0.05).

Figure 9

Effect of GO and S-rGO on LDH leakage in PMEF cells. LDH leakage was measured by changes in optical densities due to NAD⁺ reduction which were monitored at 490 nm, as described in the ‘Methods’ section, using cytotoxicity detection lactate dehydrogenase kit. The results represent the means of three separate experiments, and error bars represent the standard error of the mean. GO-treated groups showed statistically significant differences from the control group by Student’s t test (p < 0.05).

Figure 10

The effect of GO and S-rGO on alkaline phosphatase activity. PMEF cells were treated with various concentrations of GO and S-rGO for 4 days. ALP activity was measured as described in the ‘Methods’ section. The results represent the means of three separate experiments, and error bars represent the standard error of the mean. GO- and S-rGO-treated groups showed statistically significant differences from the control group by Student’s t test (p < 0.05).