Green synthesis of graphene and its cytotoxic effects in human breast cancer cells

Abstract

Background: This paper describes an environmentally friendly ("green") approach for the synthesis of soluble graphene using Bacillus marisflavi biomass as a reducing and stabilizing agent under mild conditions in aqueous solution. In addition, the study reported here investigated the cytotoxicity effects of graphene oxide (GO) and bacterially reduced graphene oxide (B-rGO) on the inhibition of cell viability, reactive oxygen species (ROS) generation, and membrane integrity in human breast cancer cells.

Methods: The reduction of GO was characterized by ultraviolet-visible spectroscopy. Size distribution was analyzed by dynamic light scattering. Further, X-ray diffraction and high-resolution scanning electron microscopy were used to investigate the crystallinity of graphene and the morphologies of prepared graphene, respectively. The formation of defects further supports the bio-functionalization of graphene, as indicated in the Raman spectrum of B-rGO. Surface morphology and the thickness of the GO and B-rGO were analyzed using atomic force microscopy, while the biocompatibility of GO and B-rGO were investigated using WST-8 assays on MCF-7 cells. Finally, cellular toxicity was evaluated by ROS generation and membrane integrity assays.

Results: In this study, we demonstrated an environmentally friendly, cost-effective, and simple method for the preparation of water-soluble graphene using bacterial biomass. This reduction method avoids the use of toxic reagents such as hydrazine and hydrazine hydrate. The synthesized soluble graphene was confirmed using various analytical techniques. Our results suggest that both GO and B-rGO exhibit toxicity to MCF-7 cells in a dose-dependent manner, with a dose > 60 μg/mL exhibiting obvious cytotoxicity effects, such as decreasing cell viability, increasing ROS generation, and releasing of lactate dehydrogenase.

Conclusion: We developed a green and a simple approach to produce graphene using bacterial biomass as a reducing and stabilizing agent. The proposed approach confers B-rGO with great potential for various biological and biomedical applications.

Keywords: Bacillus marisflavi; Raman spectroscopy; graphene oxide; reduced graphene oxide; ultraviolet–visible spectroscopy.

Figure 1

Photograph of graphene oxide (left) and bacterially reduced graphene oxide (right) at a concentration of 500 μg/mL.

Figure 2

Ultraviolet–visible absorption spectra of graphene oxide (GO) and the reduced graphene oxide (rGO) suspension reduced by bacterial biomass (50 μg/mL). Abbreviation: Abs, absorption spectrum.

Figure 3

X-ray diffraction patterns of (A) graphene oxide and (B) bacterially reduced graphene oxide. Note: The arrow indicates the position of crystalline peak of reduced graphene oxide. The dot represent the intensity value of peak.

Figure 4

Hydrodynamic size distribution of (A) graphene oxide and (B) bacterially reduced graphene oxide (500 μg/mL) measured by dynamic light scattering at room temperature in deionized water.

Figure 5

Scanning electron microscopy images of (A) graphene oxide and (B) bacterially reduced graphene oxide.

Figure 6

Raman spectra of (A) graphene oxide and (B) bacterially reduced graphene oxide.

Figure 7

Atomic force microscopy images of (A) graphene oxide and (B) bacterially reduced graphene oxide.

Figure 8

The effect of graphene oxide (GO) and bacterially reduced graphene oxide (B-rGO) on cell viability of MCF-7 cells. Notes: The cell viability of MCF-7 cells was determined by WST-8 assay after 24 hours of exposure to different concentrations of GO or B-rGO. The results represent the means of three separate experiments and error bars represent the standard error of the mean. Treated groups showed statistically significant differences from the control group, as determined using Student’s t-test (P < 0.05).

Figure 9

Generation of reactive oxygen species (ROS) in graphene oxide (GO)-and bacterially reduced graphene oxide (B-rGO)-treated MCF-7 cells. Notes: The relative fluorescence of 2′,7′-dichlorofluorescein was measured using a spectrofluorometer with excitation at 485 nm and emission at 530 nm. The results represent the means of three separate experiments and the error bars represent the standard error of the mean. Treated groups showed statistically significant differences from the control group, as determined by Student’s t-test (P < 0.05).

Figure 10

The effect of graphene oxide (GO) and bacterially reduced graphene oxide (B-rGO) on lactate dehydrogenase (LDH) activity in MCF-7 cells. Notes: LDH activity was measured by changes in optical densities due to nicotinamide adenine dinucleotide reduction, monitored at 490 nm, as described in “Materials and methods,” using a cytotoxicity detection lactate dehydrogenase kit. The results represent the means of three separate experiments and the error bars represent the standard error of the mean. Treated groups showed statistically significant differences from the control group, as determined by Student’s t-test (P < 0.05).

Figure 11

The effect of graphene oxide (GO) and bacterially reduced graphene oxide (B-rGO) on the mortality of MCF-7 cells. Notes: The mortality of MCF-7 cells was determined using trypan blue assay after 24 hours of exposure to different concentrations of GO or B-rGO. The results represent the means of three separate experiments and the error bars represent the standard error of the mean. Treated groups showed statistically significant differences from the control group, as determined by Student’s t-test (P < 0.05).