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

Abstract 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.