Ginkgo biloba: a natural reducing agent for the synthesis of cytocompatible graphene

Abstract

Background: Graphene is a novel two-dimensional planar nanocomposite material consisting of rings of carbon atoms with a hexagonal lattice structure. Graphene exhibits unique physical, chemical, mechanical, electrical, elasticity, and cytocompatible properties that lead to many potential biomedical applications. Nevertheless, the water-insoluble property of graphene restricts its application in various aspects of biomedical fields. Therefore, the objective of this work was to find a novel biological approach for an efficient method to synthesize water-soluble and cytocompatible graphene using Ginkgo biloba extract (GbE) as a reducing and stabilizing agent. In addition, we investigated the biocompatibility effects of graphene in MDA-MB-231 human breast cancer cells.

Materials and methods: Synthesized graphene oxide (GO) and GbE-reduced GO (Gb-rGO) were characterized using various sequences of techniques: ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and Raman spectroscopy. Biocompatibility of GO and Gb-rGO was assessed in human breast cancer cells using a series of assays, including cell viability, apoptosis, and alkaline phosphatase (ALP) activity.

Results: The successful synthesis of graphene was confirmed by UV-vis spectroscopy and FTIR. DLS analysis was performed to determine the average size of GO and Gb-rGO. X-ray diffraction studies confirmed the crystalline nature of graphene. SEM was used to investigate the surface morphologies of GO and Gb-rGO. AFM was employed to investigate the morphologies of prepared graphene and the height profile of GO and Gb-rGO. The formation of defects in Gb-rGO was confirmed by Raman spectroscopy. The biocompatibility of the prepared GO and Gb-rGO was investigated using a water-soluble tetrazolium 8 assay on human breast cancer cells. GO exhibited a dose-dependent toxicity, whereas Gb-rGO-treated cells showed significant biocompatibility and increased ALP activity compared to GO.

Conclusion: In this work, a nontoxic natural reducing agent of GbE was used to prepare soluble graphene. The as-prepared Gb-rGO showed significant biocompatibility with human cancer cells. This simple, cost-effective, and green procedure offers an alternative route for large-scale production of rGO, and could be used for various biomedical applications, such as tissue engineering, drug delivery, biosensing, and molecular imaging.

Keywords: Fourier-transform infrared spectroscopy; Raman spectroscopy; UV-visible spectroscopy; alkaline phosphatase activity; atomic force microscopy; biocompatibility; cell viability; graphene; scanning electron microscopy.

Figure 1

Digital photographs of aqueous dispersions of Ginkgo biloba (Gb) extracts (0.5 mg/mL) (A) of graphene oxide (GO) before (B) and after 12 hours (C) and 24 hours (D) reduction of GO using Gb extract.

Figure 2

Ultraviolet-visible absorption spectra of graphene oxide (GO) and Ginkgo biloba extract-reduced GO (Gb-rGO) suspension in water. Abbreviations: AU, arbitrary unit; h, hours.

Figure 3

X-ray diffraction pattern of graphene oxide (A) and Ginkgo biloba extract-reduced graphene oxide (B). Abbreviation: AU, arbitrary unit.

Figure 4

Fourier-transform infrared spectra of graphene oxide (GO) and Ginkgo biloba extract-reduced GO (Gb-rGO). Abbreviation: AU, arbitrary unit.

Figure 5

Hydrodynamic size distribution of graphene oxide (A) and Ginkgo biloba extract-reduced graphene oxide (B). Abbreviations: PDI, polydispersity index; Z, size average.

Figure 6

Raman spectra of graphene oxide (A) and Ginkgo biloba extract-reduced graphene oxide (B). Abbreviations: AU, arbitrary unit; 2D, two-dimensional; D, D band; G, G band.

Figure 7

Scanning electron microscopy images of graphene oxide (A) and Ginkgo biloba extract-reduced graphene oxide (B).

Figure 8

Atomic force microscopy images of graphene oxide (A) and Ginkgo biloba extract-reduced graphene oxide (B).

Figure 9

Effect of graphene oxide (GO) and Ginkgo biloba extract-reduced GO (Gb-rGO) on cell viability of MDA-MB-231 human cancer cells. Cell viability of MDA-MB-231 cells was determined by water-soluble tetrazolium 8 assay after 24-hour exposure to different concentrations of GO or Gb-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). Abbreviation: AU, arbitrary unit.

Figure 10

(A and B) Detection of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Fluorescent staining of MDA-MB-231 cells after 24 hours’ treatment with graphene oxide (GO) and Ginkgo biloba extract-reduced GO (Gb-rGO) (100 μg/mL) using TUNEL assay. Representative images are shown for apoptotic deoxyribonucleic acid fragmentation (red staining) and corresponding nuclei (blue staining). The arrows indicate the detachment of cells from the dish (A). MDA-MB-231 cells were treated with GO and Gb-rGO (100 μg/mL) for 24 hours. The percentage of death cells was quantified using the cell-death detection kit. The experiments were performed in triplicate; data shown represent means ± standard deviation of three independent experiments. *P<0.05 compared with untreated cells (B). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; CON, control.

Figure 11

The effect of graphene oxide (GO) and Ginkgo biloba extract-reduced GO (Gb-rGO) on alkaline phosphatase (ALP) activity. MDA-MB-231 cells were treated with various concentrations of GO and Gb-rGO for 5 days. 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).