In vitro toxicological evaluation of graphene oxide and its heparin derivatives in African green monkey kidney (Vero) and human keratinocyte (HaCaT) cells.

Graphene oxide (GO) has emerged as a versatile nanomaterial (NM) for several biomedical applications attributed to its large surface area, abundance of oxygenated groups, and ability to interface with biomolecules. However, concerns regarding cytotoxicity, often linked to oxidative stress, membrane disruption, and excessive cellular uptake, limit therapeutic potential. Surface functionalization with biocompatible polymers, such as heparin, was explored as a strategy to improve performance. The aim of this study was to examine the biocompatibility of GO and heparin with GO (GO@Heparin 2.5 or 5%) utilizing Vero (epithelial) and HaCaT (keratinocyte) cells by MTT assay, nitric oxide (NO) quantification, and DCFH-DA measurements of reactive oxygen species (ROS). In Vero cells, GO@Heparin maintained or increased metabolic activity and markedly reduced ROS production at low to intermediate concentrations, whereas HaCaT cells exhibited more limited response. Heparin alone increased cell viability and decreased oxidative stress, supporting intrinsic cytoprotective activity. At higher nominal concentrations (≥125 µg/ml), both GO and GO@Heparin induced concentration‑ and time‑dependent cytotoxic effects. Overall, heparin functionalization was associated with improved cell viability and favorable modulation of ROS generation compared to pristine GO. Considering the previously reported antiviral activity of both GO and heparin, GO@Heparin represents a promising hybrid NM for biomedical and antiviral applications.