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Vitamin C, which promotes oxidative stress, has emerged as an alternative to conventional chemotherapy for treating cancers. However, owing to its cytotoxicity, vitamin C should be delivered to cells in its oxidized form, dehydroascorbate (DHA). Glutathione (GSH)-responsive nanoparticles (G-NPs) have been prepared as potential carriers of DHA by incorporating diselenide-based amphiphilic lipids into the liposomal membrane. Bare liposomes without diselenide-based lipids were prepared as control NPs; these exhibited particle sizes and surface charges comparable to those of G-NPs. DHA was physically encapsulated in NPs using the dehydration-rehydration method. DHA-loaded G-NPs induced much higher GSH depletion in HT29 colon cancer cells than DHA-loaded controls. Interestingly, DHA-loaded G-NPs significantly increased the intracellular levels of reactive oxygen species, implying enhanced oxidative stress by stimuli-responsive DHA release. Consequently, DHA-loaded G-NPs exhibited significant cytotoxicity against HT29 colon cancer cells. Overall, G-NPs might serve as potential carriers for intracellular delivery of DHA during cancer treatment.