Intracellular localization and toxicity of graphene oxide and reduced graphene oxide nanoplatelets to mussel hemocytes in vitro.

Recently, graphene materials have attracted tremendous research interest due to their unique physicochemical properties that hold great promise in electronics, energy, materials and biomedical areas. Graphene oxide (GO) is one of the most extensively studied graphene derivatives. In order to improve GO electrical properties, nanoplatelets are chemically reduced, thus increasing nanoplatelet conductivity. This reduced GO (rGO) shows different properties and behavior compared to GO. Graphene-based wastes are expected to end up in the marine environment. Here we aimed to assess the potential toxic effects of GO and rGO to marine organisms by using in vitro assays with mussel (Mytilus galloprovincialis) hemocytes. Cells were exposed to a wide range of concentrations (up to 100mg/L) of GO (with and without polyvinylpyrrolidone-PVP as stabilizing agent: GO and GO-PVP) and rGO with PVP (rGO-PVP) to assess cytotoxicity and cell membrane integrity. Then, cells were exposed to sublethal concentrations of GO and rGO-PVP to assess their subcellular distribution through transmission electron microscopy (TEM) and to evaluate their effects on ROS production. GO, GO-PVP and rGO-PVP showed low and concentration-dependent cytotoxicity. rGO-PVP (LC50=29.902 and 33.94mg/L depending on the origin) was more toxic than GO (LC50=49.84 and 54.51mg/L depending on the origin) and GO-PVP (LC50=43.72mg/L). PVP was not toxic to hemocytes but increased bioavailability and toxicity of nanoplatelets. At TEM, GO and rGO-PVP nanoplatelets caused invaginations and perforations of the plasma membrane, which agrees with the observed decrease in cell membrane integrity. Nanoplatelets were internalized, at a higher extent for rGO-PVP than for GO, and found in the cytosol and in endolysosomal vesicles of hemocytes. Both GO and rGO-PVP increased ROS production at the highest sublethal concentration tested. In conclusion, GO, GO-PVP and rGO-PVP are not highly toxic to mussel cells but they cause membrane damage and their toxicity is ROS-mediated. Finally, in vitro assays with mussel hemocytes are sensitive tools to detect toxic effects of graphene-based nanomaterials.