Preparation Of Carbon-based Nanomaterials And Their Biological Effects

In recent years,carbon-based nanomaterials related investigations is very active in nanotechnology.And a variety of carbon nanomaterials were prepared,such as fullerenes and their derivatives,carbon dots and graphene quantum dots(GQDs).Carbon nanomaterials possess fascinating biological activity,such as antioxidant,antibacterial,antiviral,anti-tumor,anti-radiation,chemotherapy sensitization,autophagy induction and drug loading,which show great potential applications in biomedicine.Due to the hydrophilic property and free radical scavenging ability,some carbon nanomaterials can be used to treat ischemia-reperfusion injury,neurodegenerative diseases,ionizing radiation-induced damage,and the prevention of chemotherapeutic drug-mediated toxicity.However,some literatures have reported the toxicity of such materials.Although the research objects in biological system are various,studies about biological effects of nanomaterials usually focus on a certain level,thus bring limitation about the elucidation of biosafety evaluation.Therefore,it is essential to explore their biological effects at different levels before the application of carbon nanomaterials in biological systems.In this thesis,water-soluble fullerene(Nano-C60)and fullerenol were prepared.Subsequently,varieties of methods were conducted to study the biological effects of carbon nanomaterials(fullerene,fullerenol and graphene quantum dots)on serum proteins,cell and isolated mitochondria.The results will provide a comprehensive understanding of bio-behavior of carbon nanomaterials,thus promoting their safety application in biological fields.This thesis consists of six chapters.Chapter 1:This chapter introduced a comprehensive overview of basic properties and biological applications of carbon-based materials including fullerene,fullerenol and graphene quantum dots(GQDs),as well as interactions of such materials with serum proteins,cells and isolated mitochondria.Chapter 2:Soluble Nano-C60 nanoparticles were prepared according to solvent exchange method and characterized by UV-Vis absorption spectroscopy,MALDI-TOF-MS,TEM and DLS.After that,a variety of spectroscopic measurements as well as isothermal titration calorimetry(ITC)were applied to study the interaction between Nano-C60 with human transferrin(Tf).The results of fluorescence spectra indicated that the intrinsic fluorescence quenching of Tf was a static process due to the formation of Nano-C60/Tf complex.Electrostatic interaction played a major role in the binding process.In addition,the results of synchronous fluorescence and CD spectra demonstrated that the secondary conformation of Tf has been changed by Nano-C60.Moreover,the results of ITC indicated that the binding process is spontaneous and driven by enthalpy.Chapter 3:Hydroxyl groups were introduced on the surface of fullerene to obtained polyhydrolated fullerene derivateive(fullerenol,C60(OH)44).The structure of fullerenol was determined by IR spectra,Thermogravimetric analysis(TGA)and elemental analysis.And the size distribution in aqueous solutions was measured by DLS.As a step toward the elucidation of basic behavior in biological systems,a variety of spectroscopic measurements as well as isothermal titration calorimetry(ITC)were applied to study the interaction between fullerenol with bovine serum albumin(BSA)and y-globulins.The results indicated that intrinsic fluorescence of proteins could be effectively quenched by the dynamic mechanism.The affinity values of both proteins bound to fullerenol were of the same order of magnitude.Moreover,fullerenol had little influence on the secondary structure of proteins.Meanwhile,ITC results showed that the interaction between fullerenol and BSA is exothermic process,while the interaction with y-globulins is endothermic.In both cases,enthalpy and entropy changes are always balanced to get a favorable free energy change(?G<0),ensuring that the interaction process is spontaneous.Chapter 4:The effects of fullerenol on cell viability,autophagy and lysosomal function of human gastric tumor cell line SGC-7901 were investigated by MTT method,flow cytometer,laser confocal microscopy,TEM and western blot.Fullerenol had no influence on cell viability under nutrient-rich condition.When serum was deprived,cytotoxicity occurred in a concentration-and time-dependent manner,along with massive vacuoles in cytoplasm and large amount of ROS generation.Fullerenol treatment led to the accumulation of autophagosomes,as evidenced by the increased fluorescent intensity of Monodansylcadaverine(MDC)staining cells,elevated level of LC3 protein,and observation of auotphagosomes in cytoplasm.Subsequent results of p62 level demonstrated that the accumulation of autophagosomes resulted from the blockade of autophagic flux.Moreover,Fullerenol disrupted autophagic flux by impairing lysosomal function,including lysosome membrane permeabilization(LMP),alkaline of lysosomes,and reduced activity of capthesin B.Suppression of essential autophagy may contribute to cell death under starvation condition.Furthermore,fullerenol-induced cell death could be alleviated by 3-MA.Therefore,the ability of fullerenol to kill tumor cells under starvation may provide a new strategy for the treatment of gastric cancer.Chapter 5:The effects of fullerenol on isolated mitochondria were investigated using a variety of methods including UV-Vis absorbance spectra,fluorescence spectra,flow cytometer,a Clark-type oxygen electrode and TEM.Our studies demonstrated that fullerenol induced mitochondrial permeability transition(MPT),swelling of rat liver mitochondria,collapse of membrane potential and enhancement of mitochondrial membrane permeability to H+ and K+.In addition,fullerenol had a significant impact on mitochondrial inner membrane in polar protein regions,influencing the mitochondrial membrane fluidity.Effects of fullerenol on the respiration of isolated mitochondria demonstrated that fullerenol not only affected the respiration chain but also damaged the inner membrane.All results of this chapter can provide comprehensive information to elucidate the possible toxic mechanism of fullerenol at subcellular level.Chapter 6:The effects of GQDs on human gastric cancer cell line SGC-7901 were investigated by MTT method,laser confocal microscopy,western blot and TEM.GQDs can be internalized into cells and GQDs-labeled cells exhibited bright yellow fluorescence in a form of aggregated dots.And GQDs had little influence on the cell viability of SGC-7901 cells.Meanwhile,GQDs elevated the level of autophagy related protein LC3-II/LC3-I and induced autophagic structure that observed by TEM.Subsequent results of LC3 turn over and p62 degradation demonstrated the activation of autophagy.Besides,GQDs effectively mitigated H2O2-induced cytotoxicity in SGC-7901 cells,making it a potential antioxidant.