Kinetic Study of Photo-Induced Lipid Oxidation in Giant Unilamellar Vesicles

Abstract

Despite association of lipid oxidation to many key clinical concerns, the specific molecular mechanisms and dynamics of its roles in etiology and pathogenesis are not well understood. We have studied the kinetics of photo-induced phospholipid bilayer oxidation using giant unilamellar vesicles (GUVs) as model membranes. Incorporating rhodamine-labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanoamine (Rh-DPPE) as photosensitizer to generate highly reactive singlet oxygen at the surface of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) GUVs, we observed several morphological changes during oxidation of the monounsaturated lipid bilayer. Within minutes of irradiation, the initial and brief growth of membrane area was accompanied by vesicle flattening and high-amplitude membrane fluctuations. After reaching a maximum area, the membrane began contracting, causing the GUVs to become tumid with a surface tension sufficient to exceed the critical limit for pore formation. The periodic micron-scale openings of pores could be quantitatively observed as step-decreases in vesicle size beyond pre-oxidation dimensions. The growth and shrinkage behaviors were analyzed as consecutive first-order rate-limiting steps within the overall oxidative process to estimate molecular size changes and the rate constants of the underlying chemical events. Dependence of these constants on irradiation intensity and on concentrations of Rh-DPPE and DOPC were considered. Furthermore, experiments varying membrane DOPC content were repeated at ten-fold increased aqueous viscosity to slow down fluid leakage and allow for more accurate measurement of pore closure rates. The calculated decreased line tension with higher DOPC concentration suggested pore edge-stabilizing geometry of oxidation products. In agreement with existing simulation results, we observed that photo-induced lipid oxidation consecutively expanded and reduced membrane area of DOPC GUVs, and our analysis suggested that these actions resulted from two key reaction steps occurring at different rates.