Toxicity of carbon nanomaterials: A model to predict ROS production from easily measurable surface characteristics

Toxicity pathways of engineered carbon nanomaterials, which have many and diverse industrial applications, include oxidative stress primarily due to the generation of reactive oxygen species (ROS). This study on 13 carbon black, 6 Graphene nanoplatelets (GNP), and 3 nanodiamond materials identified physicochemical properties driving their ROS production. ROS production was cross-validated using different assays: electron paramagnetic resonance (EPR) with and without (bulk) CPH spin probe, and cellular and acellular fluorescence measurements using the DCFH₂-DA probe. ROS production was highly correlated between assays, however, the EPR spin-count without CPH (i.e., intrinsic ROS of bulk materials) was not correlated with the surface-related ROS production captured by the other assays. ROS production was also closely linked to cell membrane damage through hemolytic potential (r = 0.96). Among physicochemical properties, ROS production was primarily related to BET specific surface area (SSA). The variation in ROS production not explained by SSA was 4-fold for sp²-hybridized carbon black and GNP materials and 100-fold when also considering sp³-hybridized carbon nanodiamond materials. This variation in ROS production was related to surface properties and could be accurately modelled (R² = 0.99) based on surface-sensitive analysis using x-ray photoelectron spectroscopy (XPS). The XPS-based model showed that ROS production increased with carbon sp²-hybridization and sulfur oxide surface groups while it was inhibited by the total non-oxidized sulfur and oxygen surface content. Surface functionalization thus appears to regulate ROS production of common carbon nanomaterials by at least a factor of 4, and simple surface-specific analyses may be useful in the design of safer carbon nanomaterials.