The Study On Interaction And Mechanism Of PEGylated Graphene Oxide With Cells

The unique two-dimensional structure endows graphene with excellent performance,including efficient loading,photothermal effect and photoluminescence,which has attracted booming interests in biomedical applications.The existing findings have paid more attention on its functions on treatment effect.but ignored special biological effects that induced by two-dimensional structure of graphene.In this thesis,the biological effects of PEGylated graphene oxide(GO-PEG)on two kinds of interacted cells(macrophage and vascular endothelial cell)after in vivo injection were evaluated.Furthermore,in vivo immune activation effect and special stealth-but-activated mechanism induced by GO-PEG were discovered,and the biosecurity of GO-PEG was evaluated.This thesis mainly included the following issues:1.GO-PEG with superior biocompatibility was prepared,and its in vivo effects on macrophage activation were investigated.On the basis of graphene oxide(GO),GO-PEG was synthesized by PEGylated chemical decoration,which had the lateral dimensional size of 200-300 nm,thickness of 5-10 nm and negatively charged on the surface.In vivo study indicated that the level of cytokine secretion after single intraperitoneal injection was dose-dependent while the level of cytokine secretion after multiple intraperitoneal injection was frequency-independent.Based on the above results,we further investigated the mechanism of immune activation stimulated by GO-PEG,and found that GO-PEG not only induced aggregation of phospholipid clusters by interaction with cell membrane,but also initiated integrin αvβ8 mediated mechanotransduction to gather talin in inner membrane,thus leading to the promotion of inflammatory cytokines secreted by macrophages.2.Cellular behaviors of vascular endothelial cell were studied after GO-PEG stimulation,and the special stealth-but-activated effect of GO-PEG was discovered.PEGylation improved biocompatibility of GO-PEG to vascular endothelial cell and endowed it with stealth effect.Without internalization,GO-PEG still activated vascular endothelial cell and promoted secretion of inflammatory,chemotactic and adhesive cytokines.The stealth-but-activated effect of GO-PEG included cell morphology change(including the increase of cell area,length and width,the decrease of nucleus roundness and cell roundness)and accelerated membrane fluidity while maintaining cell membrane integrity.3.The initial activation site on vascular endothelial cell membrane after stealth-but-activated effect induced by GO-PEG was revealed,and the corresponding microscopic mechanism was presented.Cav1.3 L-typed calcium channel was determined as an initial activation site by gene sequencing analysis and antagonist inhibition experiments,which played an important role in vascular endothelial cell activation.Due to GO-PEG absorption onto/insertion into membrane,the up-regulation of Cav1.3 L-typed calcium channel increased Ca2+ influx and transformed extracellular mechanical signals into internal chemical signals,which further promoted cytokine secretion and finally activated vascular endothelial cell.4.Biosecurity of GO-PEG after intravenous injection was investigated,and the impact of GO-PEG on atherosclerosis was evaluated on the basis of atherosclerotic animal model.In vivo toxicity experiments showed that single intravenous injection of low dose was nontoxic,while single intravenous injection of high dose induced inflammation and inflammatory infiltrates in liver.In addition,multiple intravenous injections of low dose induced slight inflammation and dysfunction in liver and kidney,which could recover after administration.In the atherosclerotic animal model,chronic intravenous injection of GO-PEG induced vascular inflammation and accelerated the development of atherosclerosis,suggesting the potential pathological effects of chronic intravenous injection.The aforementioned experiments evaluated the biosecurity of GO-PEG in vivo,and laid the foundation for safe and reasonable application of two-dimensional materials.