A ratiometric fluorescent probe for assessing mitochondrial phospholipid peroxidation within living cells

Mitochondrial oxidative damage contributes to a wide range of pathologies, and lipid peroxidation of the mitochondrial inner membrane is a major component of this disruption. However, despite its importance, there are no methods to assess mitochondrial lipid peroxidation within cells specifically. To address this unmet need we have developed a ratiometric, fluorescent, mitochondria-targeted lipid peroxidation probe, MitoPerOx. This compound is derived from the C11-BODIPY(581/591) probe, which contains a boron dipyromethane difluoride (BODIPY) fluorophore conjugated via a dienyl link to a phenyl group. In response to lipid peroxidation the fluorescence emission maximum shifts from ∼590 to ∼520nm. To target this probe to the matrix-facing surface of the mitochondrial inner membrane we attached a triphenylphosphonium lipophilic cation, which leads to its selective uptake into mitochondria in cells, driven by the mitochondrial membrane potential. Here we report on the development and characterization of MitoPerOx. We found that MitoPerOx was taken up very rapidly into mitochondria within cells, where it responded to changes in mitochondrial lipid peroxidation that could be measured by fluorimetry, confocal microscopy, and epifluorescence live cell imaging. Importantly, the peroxidation-sensitive change in fluorescence at 520nm relative to that at 590nm enabled the use of the probe as a ratiometric fluorescent probe, greatly facilitating assessment of mitochondrial lipid peroxidation in cells.