An improved superoxide-generating nanodevice for oxidative stress studies in cultured cells

The effects of reactive oxygen species on cells have attracted great attention from both physiological and pathological aspects. Superoxide (O₂^-) is the primary reactive oxygen species formed in animals. We previously developed an O₂^--generating nanodevice consisting of NADPH oxidase 2 (Nox2) and modulated activating factors. However, the device was subsequently found to be unstable in a standard culture medium. Here we improved the device in stability by cross-linking. This new nanodevice, Device II, had a half-life of 3 h at 37 °C in the medium. Device II induced cell death in 80% of HEK293 cells after 24 h of incubation. Superoxide dismutase alone did not diminish the effect of the device, but eliminated the effect when used together with catalase, confirming that the cell death was caused by H₂O₂ derived from O₂^-. Flow cytometric analyses revealed that Device II induced caspase-3 activation in HEK293 cells, suggesting that the cell death proceeded largely through apoptosis.