Vesicle lamellarity has a significant influence on lipid packing, fluidity, and permeability of the membranes and thus affects bilayer-molecule interactions. Although it is commonly assumed that freeze/thaw cycles and extrusion through submicron filters produce unilamellar vesicles, some multilamellar vesicles (MLV) remain. The stacked bilayer arrangement of MLVs can affect observations from vesicle-based assays (e.g. dye leakage or fluidity assays). Also, MLVs are not representative of the single-bilayer architecture characteristic of the cell’s plasma membrane, thus adding to the challenges of translating insights from vesicle-based assays to cellular systems.
In this study, we investigate the effect of lipid composition on lamellarity and bilayer structure in vesicles with simple and complex lipid compositions, specifically, anionic and zwitterionic phospholipids and sterols. Our results show that vesicles containing zwitterionic phosphatidylcholine (POPC) lipids only, have a low fraction of externally accessible lipid (facc), indicating a high degree multilamellarity. Adding ergosterol, the facc further decreased indicating that sterols significantly increase the presence of MLVs. This was accompanied by bilayer structural changes including reduced area per lipid, increased the bilayer and hydrophobic thickness, signifying an altered membrane fluidity compared to PC vesicles. The addition of phosphatidylethanolamine (POPE) caused yet further reduction of facc was further amplifying the structural changes caused by ergosterol. The addition of negatively charged lipid phosphatidylserine (POPS) caused a significant increase in facc compared to PC or PC-ergo vesicles, without significant changes in the structural parameters.
Our results demonstrate the ergosterol and POPE significantly increase the degree of multilamellarity in vesicles, while causing bilayer structural modification, and that at least 10% of POPS is required to form predominately unilamellar vesicles.