Correlation between composition of the outer layer and phase asymmetry for vesicles ruptured by phospholipase D

Spherical phospholipid bilayers, vesicles, were prepared by using the layer-by-layer double emulsion technique, which allows individual layers to be formed asymmetrically. Phases of the layers were adjusted by selecting the lipid tail group. The head group composition of the vesicle outer layer varied 0-100 % of phosphatidylcholine (PC) by 10 % under the condition that the diameter of the vesicle was kept constant. On the outer layer of the vesicle, the phospholipase D (PLD) reacted to convert PC to phosphatidic acid. The reaction induced a curvature change of the vesicles, which eventually led them to rupture. Response time from the PLD injection to the rupture was measured against the different compositions of the outer layer at each phase (solid and liquid) using the fluorescence intensity change of pH-sensitive dye encapsulated in the vesicles. From this measurement, the rupture caused by the PLD reaction was analyzed with respect to the phase asymmetry of the layers and the composition of the outer layer. These results were interpreted with the lipid density and stability of the layers. It was observed that the solid phase of the outer layer had a variance in response time according to the phase of the inner layer, whereas the liquid phase did not. Additionally, the response of the solid phase of the outer layer at the liquid phase of the inner layer was faster than at the solid phase of the inner layer as a result of its stability.