Biological Interactions Of Graphene Based Nanomaterials And Its Biomedical Applications

The pending wide use of graphene based materials urges a biosafety assessment and safe design of nanomaterial that demonstrate applicability to human medicine. In biological microenvironment, biomolecules will interact with graphene forming corona and endow graphene a new biological identity. Moreover, the reaction between graphene and normal cellular homeostasis may lead to oxidative stress, lipid peroxidation, protein denaturation, and subsequent cell death.In this doctoral dissertation, we used experimental and simulation–based techniques to characterize the adsorption of proteins onto graphene oxide(GO). By a deep understanding of the basic interaction mechanism, the graphene-induced clearance of mature Aβ amyloid fibrils has been investigated. Afterwards, we found both direct and indirect generation of reactive oxygen species leading to oxidative stress in bacterial is the leading mechanism proposed for the toxicity of GO under simulated sunlight exposure. Finally, the turnover of anti- and pro- oxidant properites of graphene quantum dots(GQD) in presence or absence of light was systemically investigated.The main methods, results and conclusions of this dissertation are summarized as follows:1. The adsorption of four high-abundance blood proteins onto the carbon-based nanomaterial GO and reduced GO(rGO) were investigated via experimental(Atomic force microscopy, fluorescence spectroscopy, surface plasmon resonance and so on) and simulation-based(molecular dynamics, MD) approaches. Among the proteins in question, we observe competitive binding to the GO surface that features a mélange of distinct packing modes. MD simulations reveal that the protein adsorption is mainly enthalpically driven through strong π-π stacking interactions between GO and aromatic protein residues, in addition to hydrophobic interactions. Overall, these results were in line with previous findings related to adsorption of serum proteins onto single-walled carbon nanotubes(SWCNTs), but GO exhibits a dramatic enhancement of adsorption capacity compared to this one-dimensional carbon form. Encouragingly, protein-coated GO resulted in a markedly less cytotoxicity than pristine and protein-coated SWCNTs, suggesting a useful role for this planar nanomaterial in biomedical applications.2. Based on the interaction of graphene and proteins, we continue to investigate the graphene-induced clearance of mature amyloid fibrils in Alzheimer’s disease(AD). Current therapies for AD can provide a moderate symptomatic reduction or delay progression at various stages of the disease, but such treatments ultimately do not arrest the advancement of AD. We here provide both experimental and computational evidence that pristine graphene and GO nanosheets can inhibit Aβ peptide monomer fibrillation and disassemble and clear mature amyloid fibrils, thus impacting the central molecular superstructures correlated with AD pathogenesis. MD simulations reveal that graphene nanosheets can penetrate and extract a large number of peptides from preformed amyloid fibrils; these effects seem to be related to exceptionally strong dispersion interactions between peptides and graphene that are further enhanced by strong π-π stacking between the aromatic residues of extracted Aβ peptides and the graphene surface. Atomic force microscopy images confirm these predictions by demonstrating that mature amyloid fibrils can be cut into pieces and cleared by GO. Thioflavin fluorescence assays further illustrate the detailed dynamic processes by which GO induces inhibition and clearance of monomer aggregation and mature amyloid fibrils, respectively. Cell viability and ROS assays indicate that GO can indeed mitigate cytotoxicity of Aβ peptide amyloids. Our findings provide new insights into the underlying molecular mechanisms that define graphene-amyloid interaction and suggest that further research on nanotherapies for Alzheimer’s and other protein aggregationrelated diseases is warranted.3. Bacterial inactivation by light-induced oxidative stress of GO and its chemical transformation was studied. It has been reported that GO is a promising material for the development of antimicrobial agent due to contact-based or oxidative stress-mediated antimicrobial activity. Here we demonstrated the enhanced antibacterial efficiency of GO under simulated sunlight exposure. Using electron spin resonance spectroscopy with spin trapping and spin labeling techniques, we observed that irradiated GO had a strong oxidizing property proved by the increased depletion of different kinds of antioxidant, which is ascribed to photogenerated charge carriers. Moreover, the results from UV-visible spectroscopy, X-ray photoelectron spectroscopy and electron spin resonance spectroscopy revealed the GO themselves were reduced by remaining irradiated electron. These findings unveiled enhanced antibacterial mechanism of GO under sunlight exposure and rapid phototransforms that are likely to have great potential for use in understanding the graphene based phototoxicity.4. Crossover between anti- and pro- oxidant activities of GQD in the absence or presence of light was studied. GQD is a class of zero dimensional graphene-based materials with excellent luminescence properties, showing promises for a variety of medical, biological, and optoelectrical applications. We found that GQD can protect cells against oxidative damage, due to the efficient manner in which GQD quench reactive oxygen species(ROS) and nitrogen-centered free radicals. These free radicals react with a wide range of biological targets and play an important role in cellular physiology and pathophysiology. However, when exposed to blue light, GQD exhibit significant phototoxicity, increasing intracellular ROS levels and reducing cell viability. For the first time, ESR was employed to systemically reveal the mechanism for generation of a series of free radicals during photoexcitation of GQD. We propose that singlet oxygen is generated by photoexcited GQD via energy transfer and electron transfer pathways. Hydroxyl radicals and superoxide radical anions result from band-toband transition and creation of electron-hole pair, respectively reacting with surrounding molecular oxygen or H2 O. Additionally, we found upon photoexcitation, GQD accelerated the oxidation of nonenzymic antioxidants including ascorbate and glutathione, and promoted lipid peroxidation, reactions possibly related to the phototoxicity of GQD. The results indicated that GQD could be potentially exploited for cytoprotective or cytotoxic anticancer/ antibacterial applications.In conclusion, we studied the interaction between graphene based material and biological microenvironment(such as proteins, cellular redox system), including its applicable potentials and mechanism. And we believe that if we understood the fundamental interaction of this new carbon nanosheet with biological molecules, the biomedical application and safty of graphene will be intelligently exploited.