Ratiometric fluorophores to ONOO– in living cells and monitoring endothelial dysfunction

Peroxynitrite (ONOO^–), generated from nitric oxide and superoxide, is implicated in cellular dysfunction and death. To detect ONOO^– in live cells, we developed two hemicyanine-based ratiometric fluorophores, P-mito and P-boc. These ratiometric fluorophores were successfully used to monitor ONOO^– fluctuations in cancer cells and endothelial cells. P-mito predominantly accumulated in the mitochondria of live cells and, upon selective reaction with ONOO^–, increased the ratio of the fluorescence intensity from blue channel to that from red channel. Treatment with an ONOO^– scavenger, N-acetyl cysteine, suppressed this ratio increase. Notably, in a model of endothelial dysfunction induced by treating pulmonary arterial endothelial cells with TGFβ2 and IL-1β, our system successful demonstrated increased ONOO^– levels, effectively highlighting the utility of the hemicyanine-based fluorescence response to ONOO^–. Therefore, our hemicyanine-based ONOO^– detection system provides an effective platform for spatiotemporal monitoring and quantitative analysis of ONOO^– within live cells, enabling molecular-level research into various ONOO^–-related pathological conditions.