Biosafety Study Of Surface Engineered Graphene

It’s of crucial importance for graphene oxide (GO) complexes bioapplication by investigating the effect of surface engineering to biological outcomes. In this thesis, different surface-engineered GO complexes were synthesized to investigate their in vitro behaviors through cellular internalization, viability and stress response of macrophage. During these processes the distinct property of nGO-PEG in cytokine induction was unveiled, and further related mechanism in molecular level as well as in vivo results were adequately studied.This thesis mainly included the following issues:1. We engineered different GO complexes and investigated their effects on the viability of peritoneal macrophages. On the basis of pristine nGO, we decorated its surface by polyethylene glycol (PEG), bovine serum albumin (BSA), and polyetherimide (PEI). In contrast to pristine nGO, decoration of PEG and BSA hindered endocytosis and improved their benignancy to macrophages. On the contrary, nGO-PEI commenced with a favorable endocytosis but suffered stagnation afterwards because of the compromised macrophage viability. After nGO, nGO-BSA and nGO-PEI were internalized by cells, they tended to interact with mitochondria. Such interactions disrupted the normal potential and integrity of mitochondria and then elicited an alteration in reactive oxygen species and cytochrome c. These responses further initiated the activation of caspase family and finally dictated cells into apoptosis. The results above established that surface attributes of GO could shape their interaction with macrophages and result in disparate biological outcomes.2. We further dissected the impact of nGO complexes to macrophage stress response, and uncovered the unique property of nGO-PEG in macrophage activation. Compared to internalized nGO complexes, nGO-PEG triggered high grade of cytokines on the extracellular milieu in spite of negligible internalization. This peculiar attribute of nGO-PEG could not be found on PEG per se or simple mixture of nGO & PEG. Moreover, we also noted the phenomenon of cytokine downturn by second stimulus, which indicated nGO-PEG induced macrophage activation might be one-off behavior. Via the comparison of cytokine level among different materials, we’re amazed to find that 2 D materials were better at cytokine induction than any other dimensioned ones. By observing the mutual interaction between nGO and cell membrane, we also discovered that more cell protrusions were generated with a membrane integrity, and membrane motility was accelerated, which further encouraged cell migration.3. By virtue of computational simulation, genechip and quantigene analysis, we presented the inner signaling pathway of macrophage activation. Computational simulation work indicated that nGO-PEG could interact with cells by partial insertion into the lipid membranes or face-on configurations onto membranes, which facilitated its faster diffusion across cell exterior and activated cytokine-related receptors. The antibody blocking and siRNA interference tests further unsealed the key role of integrin β8 in priming cytokine secretion. The upregulation of integrin β8 resulted in the recruitment and autophosphorylation of FAK and subsequently activated MAPK/PI3k-related intracellular signaling pathways, which enhanced the expression of nuclear transcription factor. These events transformed the external stimulus carried by nGO-PEG into chemical signals and ultimately gave rise to macrophage activation.4. On the foundation of the in vitro results, we systematically evaluated the in vivo biological safety of nGO-PEG by intraperitoneal injection. The short-term toxicity test demonstrated that the cytokine expression level in peritoneal exudate correlated with the dosage of nGO-PEG and decreased over time as well as inflammatory cells. The histotomy and hematological analysis discovered that nGO-PEG didn’t cause obvious toxicity even at high dose. Further long-term toxicity assay proved that the cytokine secretion profile was independent with treating times, and the capability of calling cytokines was reduced by the process of metabolism and cell tolerance to nGO-PEG. The continual administration of nGO-PEG caused temporary lesions to the liver and peritoneum. Advances above reminded us the peculiar property of pegylated 2D materials in cytokine induction and also paved the way to rationally design graphene-based complexes with safety and high performance.