Study On The Cell Biological Effect Of Dual-Porymer- Functionalized Graphene Oxide

Development of gene transfection vectors with low toxicity and high transfection efficiency remains a key challenge in gene therapy. In recent years, functionalized graphene materials, due to their unique physiochemical properties, have attracted widespread attention and have shown promising potentials for biomedical applications. Previous studies have demonstrated that dual-polymer-functionalized nanosized GO outperforms commercial reagent lipofectamine 2000 in terms of siRNA or DNA transfection. However, notable cytotoxicity of the materials was still noted at high concentrations. To understand the underlying mechanism and thereby provide useful information for the development of ideal gene transfection reagents, we herein systematically studied the mechanism of nGO-PEG-PEI-mediated toxicity.We first studied the cytotoxicity of nGO-PEG-PEI. MTT assay showed that nGO-PEG-PEI led to appreciable reduction in the cell viability in different cell lines at about 10μg/mL. Annexin V-FITC/PI dual staining assay revealed that the cytotoxicity was mainly attributed to necrosis rather than apoptosis. The difference in MTT and Annexin V-FITC/PI dual staining assays indicated that nGO-PEG-PEI may also affect the cell cycle. Such hypothesis was further supported by flow cytometry results which demonstrated that nGO-PEG-PEI induced S phase arrest in HeLa cells. Additionally, majority of the dead cells were in S phase, indicating that the cells failed to transit from S phase to G2 phase and ultimately died via necrosis. For cells treated with nGO-PEG-PEI and pre-synchronized in G1 phase, the entire cell cycle became longer and there was a prominent delay of transition into S phase.Next, we probed the mechanism underlying the S phase arrest induced by nGO-PEG-PEI. The results indicated that nGO-PEG-PEI induced serious DNA damage. Western blotting results showed that the protein phosphorylation levels that are critical to active intra-S-phase checkpoint were highly increased, including ATM/ATR and their substrates Chk2/Chkl and Cdc25A. Besides, nGO-PEG-PEI-induced S phase arrest as well as alteration of cell morphology could be inhibited by AZD7762, an inhibitor of Cdc25A. All of these results collectively substantiated that after entering the cells, nGO-PEG-PEI induced DNA damage via some unknown signaling pathways and activated Cdc25A and accordingly the intra-S-phase checkpoint through both the ATM-Chk2 and ATR-Chkl signaling pathways, ultimately leading to S phase defect and extended cell cycle length or even cell necrosis.This study therefore unravels the mechanism of nGO-PEG-PEI-induced cell necrosis and provides important theoretical basis for understanding the biological effect of GO and GO derivatives, which helps the rational design of effective yet safe graphene-based gene delivery vectors. It also affords useful information for the study on cell biology effect and biomedical applications of nanomaterials.