title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
authors.forename: Sangiliyandi
authors.initials: S
authors.lastname: Gurunathan
authors.forename: Jae Woong
authors.initials: JW
authors.lastname: Han
authors.forename: Vasuki
authors.initials: V
authors.lastname: Eppakayala
authors.forename: Jin-Hoi
authors.initials: JH
authors.lastname: Kim
mesh_terms: D016923:Cell Death / Q000187:drug effects*; D002462:Cell Membrane / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects*; D006108:Graphite / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity; D055772:Green Chemistry Technology; D006801:Humans; D061986:MCF-7 Cells; D008855:Microscopy, Electron, Scanning; D010087:Oxides / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity; D010316:Particle Size; D017382:Reactive Oxygen Species / Q000378:metabolism; D013057:Spectrum Analysis
chemical_list: D010087:Oxides; D017382:Reactive Oxygen Species; D006108:Graphite
keywords: Bacillus marisflavi; Raman spectroscopy; graphene oxide; reduced graphene oxide; ultraviolet–visible spectroscopy