Effect of formulation method and lipid composition on lipid distribution across membrane leaflets in asymmetric lipid vesicles

Lipid-based delivery systems such as liposomes face challenges such as low encapsulation efficiency, immunogenicity and limited endosomal escape hinder the widespread adoption of liposomes as delivery vehicles. To improve their efficacy, attempts have been made to adapt them to mimic extracellular vesicles (EVs), which are produced by cells for communication and have desirable characteristics including innate biocompatibility. We hypothesise that EV membranes, like the cell membranes they are derived from, have different lipid distributions in their inner and outer leaflets (i.e. are asymmetric). This asymmetry affects EV biophysical and biochemical properties - however, methods to formulated asymmetric vesicles on large length scales have limited control over lipid composition and size, selectivity towards certain phospholipid headgroups, and low throughput. In this proposal, we address these limitations by comparing the chemical and physical properties of vesicles formulated with a range of lipid compositions. The data we acquire will help facilitate the design of asymmetric vesicles that mimic the lipid compositions found in EVs, and shed light on the suitability of various formulation methods for different membrane compositions. This will help bridge the gap between synthetic and biological vesicles, allowing us to evaluate the role lipid composition plays in vesicle structure, fusion mechanisms and membrane stiffness.