Insertion stability of poly(ethylene glycol)-cholesteryl-based lipid anchors in liposome membranes

Liposomes consist of a hydrophilic core surrounded by a phospholipid (PL) bilayer. In human blood, the half-life of such artificial vesicles is limited. To prolong their stability in the circulation, liposomal bilayers can be modified by inserting poly(ethylene glycol) (PEG) molecules using either PL or sterols as membrane anchors. This establishes a hydrophilic steric barrier, reducing the adsorption of serum proteins, recognition and elimination by cells of the immune system. In addition, targeting ligands (such as antibodies) are frequently coupled to the distal end of the PEG chains to direct the vesicles (then called 'immuno-liposomes') to specific cell types, such as tumor cells. To our knowledge, experiments on the stability of ligand anchoring have so far only been conducted with PL-based PEGs and not with sterol-based PEGs after insertion via the sterol-based post-insertion technique (SPIT). Therefore, our study examines the insertion stability of PEG-cholesteryl ester (Chol-PEG) molecules with PEG chains of 1000, 1500 and 2000Da molecular mass which have been inserted into the membranes of liposomes using SPIT. For this study we used different acceptor media and multiple analytical techniques, including pulsed-field-gradient nuclear magnetic resonance (PFG-NMR), free-flow electrophoresis, size exclusion chromatography and ultracentrifugation. The obtained data consistently showed that a higher molar mass of PEG chains positively correlates with higher release from the liposome membranes. Furthermore, we could detect and quantify the migration of Chol-PEG molecules from radioactively double-labeled surface-modified liposomes to negatively charged acceptor liposomes via free-flow electrophoresis. Insertion of Chol-PEG molecules into the membrane of preformed liposomes using SPIT is an essential step for the functionalization of liposomes with the aim of specific targeting. For the first time, we present a kinetic analysis of this insertion process using PFG-NMR, showing that insertion into the liposomal membranes takes place within 90s for Chol-PEG1000 molecules.