Membrane heterogeneities and fusogenicity in phosphatidylcholine-phosphatidic acid rigid vesicles as a function of pH and lipid chain mismatch.

The role of pH-dependent lipid heterogeneities on the fusogenicity of membranes was evaluated on model lipid bilayers in the form of unilamellar vesicles composed of lipid pairs at a fixed equimolar ratio of phosphatidylcholine (PC) and phosphatidic acid (PA) headgroups. The pH and the hydrophobic composition (lipid acyl tails) of membranes were systematically altered, and their effect on vesicle aggregation, membrane fusogenicity, content release, and content mixing was evaluated. Lowering pH increases the extent of protonated PA headgroups forming phase-separated PA-rich heterogeneities and giving rise to molecular packing defects originating at the phase boundaries. Phase boundaries within the membrane's hydrophobic portion are affected by the lipid acyl-tail dynamics (fluidity) and the matching or nonmatching lengths of the acyl tails of lipid pairs comprising the membrane. The aggregates' size increases with lowering pH and is independent of the membrane's hydrophobic composition. Contrary to aggregation, the initial rates of lipid mixing are proportional to pH and also depend on membrane's hydrophobic composition. The apparent lipid-mixing rate constants are greater for membranes containing lipid pairs with mismatched acyl-tail lengths, followed by pairs with matching acyl tails in the gel state and by pairs with matching tails where one lipid is close to its transition temperature. Addition of cholesterol conserves the independence of vesicle aggregation from the membrane's hydrophobic composition. However, it decreases the aggregation rates and inverts the tendency for fusion, among the three types of hydrophobic compositions, suggesting a role of cholesterol's preferential partition in different regions of membrane's heterogeneities. We propose a phenomenological model where the membrane phase boundaries containing molecular packing defects act as "sticking points" on the vesicle exterior via which vesicles aggregate upon contact followed by defect merging via intervesicle lipid exchange and mixing. Such heterogeneous bilayers in the form of drug encapsulating liposomes may potentially improve the therapeutic efficacy by fusing with endosomal membranes, thus increasing drug bioavailability.