Oxidized graphene nanoparticles as a delivery system for the pro-apoptotic sphingolipid C₆ ceramide

Sphingolipids such as ceramide have attracted much attention as possible anticancer agents due to their potent pro-apoptotic effects. However, due to their extreme hydrophobicity, there is currently no clinically approved delivery method for in vivo use as a therapeutic agent. To this end, we have developed a novel method for loading the short-chain C₆ ceramide onto oxidized graphene nanoribbons (O-GNRs) and graphene nanoplatelets (GNPs). Mass spectrometry revealed loading efficiencies of 57% and 51.5% for C₆ ceramide onto O-GNRs and GNPs, respectively. The PrestoBlue viability assay revealed that 100 µg/mL of C₆ ceramide-loaded O-GNRs and C₆ ceramide-loaded GNPs reduced HeLa cell viability by approximately 93% and approximately 76%, respectively, compared to untreated HeLa cells, while equal concentrations of these nanoparticles without C₆ ceramide did not significantly reduce HeLa cell viability. We confirmed that this cytotoxicity was apoptotic in nature via capase-3 activity and Hoechst staining. Using live-cell confocal imaging with the fluorescent NBD-ceramide loaded on O-GNRs, we observed robust uptake into HeLa cells within 30 min while NBD-ceramide on its own was uptaken much more rapidly. Transmission electron microscopy confirmed that C₆ ceramide-loaded O-GNRs were actually entering cells. Taken together, these data show that O-GNRs are a promising delivery agent for ceramide. To our knowledge, this study is the first to use such a loading method. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 25-37, 2019.