Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

Abstract

In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG 2000-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl- sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl- sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine (DMPE-PEG 2000), 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine (DPPE-PEG 2000), or 1,2-distearoyl- sn-glycero-3-phosphoethanolamine (DSPE-PEG 2000). Transfer of DSPE-PEG 2000 to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG 2000 was transferred efficiently even at 4°C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG 2000 to become evenly distributed between the two liposome populations ( T EQ) at 4°C and 37°C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.