The Study Of Nanotoxic Effects On Graphene And Its Derivatives

Graphene is a two-dimensional carbon nanomaterial with only a single atomic layer thickness.For the unique physical and chemical properties,graphene and its derivatives have been widely studied by researchers,exhibiting great potential in energy storage,optics and biomedical field.Therefore,in order to promote its application in biomedical practice,it is imperative to figure out the toxic effects of graphene based nanomaterials.Nowadays,it is controversial on whether graphene possess toxic or side effects on human health and the underlying molecular mechanism is poorly understood,which urged us to have a deep understanding of their potential toxicity hazards and their mechanisms.In this paper,both experimental and theoretical approaches are employed to explore the toxicity and potential mechanism of graphene and its derivatives from many aspects.Our study provides a theoretical guidance for biomedical applications of graphene and its derivatives.Chapter 1: Toxicitic effects of varying degree reduced graphene oxide(rGO)produced by different doses of ?-rayObjective: Current study is focus on exploring a clean and simple method for producing varying degree reduced graphene oxide and investigating their toxicitic effects.Methods: ?-ray was used to reduce graphene oxide(GO).UV spectroscopy X-ray photoelectron spectroscopy(XPS)and atomic force microscopy(AFM)were employed to characterize rGO.CCK-8,morphology,apoptosis autophage and reactive oxygen production(ROS)were evaluated to investigate the cytotoxicity of rGO with different oxygen levels.Colony formation and electron microscopy were performed to detect rGO-induced bacterial toxicity.Results:?-ray could dose-dependently reduce GO to rGO resulted by higher?-ray dose could induce stronger cytotoxic effects to A549 cells,relating to ROS generation,apoptosis and autophagy occurrence.On the other hand,rGO resulted by middle?-ray dose was found most toxic to bacteria.Chapter 2: Study on the interaction between GO nanosheets and actin filamentsObjective: To explore the new nanotoxic mechanism induced by GO nanosheets in A549 cells.Methods: The morphology of A549 cells was observed by optical microscopy after GO treatment.Electron microscopy imaging and immunofluorescence were employed to investigate the relationship between GO nanosheets and actin filaments.GO induced the invasive ability changes were detected by scratch and single cell tracking experiments.Results: GO treatment could produce a lot of light spots in A549 cells.Those light spots were holes which generated from the damage of actin filaments MD simulation showed that GO nanosheets could insert into the inter-strand gap of actin tetramer(helical repeating unit of actin filament),which eventually leaded to the disruption of actin filaments.GO treatment could significantly affect migration of cells.Chapter 3: Study on protein coating mitigates the cytotoxicity of graphene oxideObjective: To investigate the molecular mechanism by which protein coating mitigates the cytotoxicity caused by GO.Methods: Protein adsorption of GO was analyzed by AFM and protein quantitative.CCK-8 and Live/dead kit were used to evaluate the cytotoxicity of GO.Electron microscopy imagings were applicated to observe the damage of the cell membrane.The effect of protein coating(also called protein corona)on the interaction between GO and membrane was analyzed by MD simulation and molecular interaction detection.Side scattered light(SSC)signal was detected to investigated the cellular uptake of GO.The effects of protein corona on the biological damage induced by GO were analyzed by detecting ROS generation,apoptosis and cell cycle.Results: GO nanosheets can quickly adsorb protein to form a structure called corona.Reduced cytotoxicity and the interaction between the phospholipids and graphene surface of bare GO nanosheets were found by protein corona due to an unfavorable steric effect.The underlying mechanism was related to decreased cellular uptake of GO,reduced generation of ROS and apoptosis,and finally alleviated cell cycle arrest.Conclusions:(1)?-ray could dose-dependently reduce GO to rGO.rGO reduced by higher?-ray was more toxic to mammal cells.However,with the increasing of reduced degree to an extent,bactericidal ability of rGO decreased significantly.(2)GO treatment induced light spots in cells,which have been recognized as holes,resulting from the damage of actin filaments induced by GO nanosheets.GO nanosheets could damage actin filaments by direct interaction,which further weaken the migration ability of cells.(3)GO nanosheets could adsorb biological macromolecules(especially proteins)rapidly after they enter the biological micro-environment.Adsorbed protein weakened the interaction between the phospholipids and graphene surface,thereby reducing the physical damage induced by GO.Protein corona mitigated the biological damage caused by bared GO via reducing cellular uptake of GO.In this paper,we focus on the nanotoxic effects of graphene and its derivatives.We have investigated the toxicity of rGO with different oxygen levels,the interaction between GO nanosheets and actin filaments and the effect of protein corona on cytotoxicity caused by GO.Our studies complement the nanotoxicology of graphene materials and provide new insights into the mechanism of the cytotoxicity induced by graphene and its derivatives,providing reference and theoretical guidance for its future biomedical applications.