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

• Our previously developed O2−-generating tool was unstable in culture medium.
• A new nanodevice, Device II, was prepared by cross-linking of the original device.
• The new nanodevice was much more stable in culture medium than the original device.
• Device II efficiently induced cell death mainly through apoptosis.

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.