Effect of PEG, Lipid Composition and Formulation on Vesicle Lamellarity: A Small Angle Neutron Scattering Study

Abstract

Liposomes are vesicles comprising a phospholipid bilayer and inner aqueous cavity, and are well-established systems for drug delivery applications. They are able to encapsulate both hydrophobic and hydrophilic compounds. Among the parameters to account for when designing a liposomal drug carrier lamellarity (number of consecutive lipid bilayers within one vesicle) is very important: it affects both encapsulation efficiency and efflux rate through the membrane of the encapsulated drug. Despite the relevance of liposomes as drug carriers, a comprehensive study on how lipid composition and formulation method affect vesicle lamellarity is still lacking. Here, we combined small-angle neutron scattering (SANS) and cryo-TEM to elucidate the membrane structure of vesicles prepared using three different formulation methods (film hydration followed by freeze-thawing (FT) or agitation on a shaker (AS), and reverse phase evaporation (REV)) and extruded through a 100 nm membrane. We examined two phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), both widely used in the liposome field. Our results show that POPC vesicles prepared via the FT or AS methods retain a substantial amount of multilamellarity after extrusion through 100 nm membranes. This is also true for DPPC vesicles, although the extent of residual multilamellarity is much lower. The introduction of a poly(ethylene)glycol (PEG)-modified lipid in the vesicle composition results in the formation of unilamellar vesicles after extrusion when the PEGylated lipid content is at least 0.5 mol% for POPC vesicles and 0.1 mol% for DPPC vesicles. As for the REV method, unextruded DPPC vesicles are already unilamellar, while unextruded POPC vesicles still exhibit some bilamellarity. Extruded, REV-prepared POPC vesicles are unilamellar instead. In conclusion, our results provide important insights into the effect of formulation method and lipid composition on producing liposomes with a defined membrane structure.