Deciphering the underlying mechanisms of oxidation-state dependent cytotoxicity of graphene oxide on mammalian cells.

The promising broad applications of graphene oxide (GO) derivatives in biomedicine have raised concerns about their safety on biological organisms. However, correlations between the physicochemical properties, especially oxidation degree of GOs and their toxicity, and the underlying mechanisms are not well understood. Herein, we evaluated the cytotoxicity of three GO samples with various oxidation degrees on mouse embryo fibroblasts (MEFs). Three samples can be internalized by MEFs observed via transmission electron microscopy (TEM), and were well tolerant by MEFs at lower doses (below 25μg/ml) but significantly toxic at 50 and 100μg/ml via Cytell Imaging System. More importantly, as the oxidation degree decreased, GO derivatives led to a higher degree of cytotoxicity and apoptosis. Meanwhile, three GOs stimulated dramatic enhancement in reactive oxygen species (ROS) production in MEFs, where the less oxidized GO produced a higher level of ROS, suggesting the major role of oxidative stress in the oxidation-degree dependent toxicity of GOs. Results from electron spin resonance (ESR) spectrometry showed a strong association of the lower oxidation degree of GOs with their stronger indirect oxidative damage through facilitating H2O2 decomposition into OH and higher direct oxidative abilities on cells. The theoretical simulation revealed the key contributions of carboxyl groups and aromatic domain size of nanosheets to varying the energy barrier of H2O2 decomposition reaction. These systematic explorations in the chemical mechanisms unravel the key physicochemical properties that would lead to the diverse toxic profiles of the GO nanosheets with different oxygenation levels, and offer us new clues in the molecular design of carbon nanomaterials for their safe applications in biomedicine.