Singlet Oxygen Flux, Associated Lipid Photooxidation, and Membrane Expansion Dynamics Visualized on Giant Unilamellar...

The physical properties of lipid membranes depend on their lipid composition. Photosensitized singlet oxygen (¹O₂) provides a handle to spatiotemporally control the generation of lipid hydroperoxides via the ene reaction, enabling fundamental studies on membrane dynamics in response to chemical composition changes. Critical to relating the physical properties of the lipid membrane to hydroperoxide formation is the availability of a sensitive reporter to quantify the arrival of ¹O₂. Here, we show that a fluorogenic α-tocopherol analogue, H₄BPMHC, undergoes a >360-fold emission intensity enhancement in liposomes following a reaction with ¹O₂. Rapid quenching of ¹O₂ by the probe (k_q = 4.9 × 10⁸ M^-1 s^-1) ensures zero-order kinetics of probe consumption. The remarkable intensity enhancement of H₄BPMHC upon ¹O₂ trapping, its linear temporal behavior, and its protective role in outcompeting membrane damage provide a sensitive and reliable method to quantify the ¹O₂ flux on lipid membranes. Armed with this probe, fluorescence microscopy studies were devised to enable (i) monitoring the flux of photosensitized ¹O₂ into giant unilamellar vesicles (GUVs), (ii) establishing the onset of the ene reaction with the double bonds of monounsaturated lipids, and (iii) visualizing the ensuing collective membrane expansion dynamics associated with molecular changes in the lipid structure upon hydroperoxide formation. A correlation was observed between the time for antioxidant H₄BPMHC consumption by ¹O₂ and the onset of membrane fluctuations and surface expansion. Together, our imaging studies with H₄BPMHC in GUVs provide a methodology to explore the intimate relationship between photosensitizer activity, chemical insult, membrane morphology, and its collective dynamics.