Integrity of Membrane Structures in Giant Unilamellar Vesicles as Assay for Antioxidants and Prooxidants

We have attempted to evaluate, on the basis of optical microscopy for a single giant unilamellar vesicle (GUV), the potency of antioxidants in protecting GUV membranes from oxidative destruction. Photosensitized membrane budding of GUVs prepared from soybean phosphatidylcholine with chlorophyll a (Chl a) and β-carotene (β-Car) as photosensitizer and protector, respectively, were followed by microscopic imaging. A dimensionless entropy parameter, ΔE, as derived from the time-resolved microscopic images, was employed to describe the evolution of morphological variation of GUVs. As an indication of membrane instability, the budding process showed three successive temporal regimes as a common feature: a lag phase prior to the initiation of budding characterized by LP (in s), a budding phase when ΔE increased with a rate of k_ΔE (in s^-1), and an ending phase with morphology stabilized at a constant ΔE_end (dimensionless). We show that the phase-associated parameters can be objectively obtained by fitting the ΔE-t kinetics curves to a Boltzmann function and that all of the parameters are rather sensitive to β-Car concentration. As for the efficacy of these parameters in quantifying the protection potency of β-Car, k_ΔE is shown to be most sensitive for β-Car in a concentration regime of biological significance of <1 × 10^-7 M, whereas LP and ΔE_end are more sensitive for β-Car concentrations exceeding 1 × 10^-7 M. Furthermore, based on the results of GUV imaging and fluorescence and Raman spectroscopies, we have revealed for different phases the mechanistic interplay among ¹O₂* diffusion, PC-OOH accumulation, Chl a and/or β-Car consumption, and the morphological variation. The developed assay should be valuable for characterizing the potency of antioxidants or prooxidants in the protection or destruction of the membrane integrity of GUVs.