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 degrees 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 degrees C and 37 degrees 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.

Developing Lipid Nanoparticle-Based siRNA Therapeutics for Hepatocellular Carcinoma Using an Integrated Approach

“…PEG-lipids with longer acyl chain confer stronger interaction with membrane lipids thus less likely to be extracted/exchanged, and consequently, PEGs with longer lipid anchor are often associated with a slower rate of deshielding (15,(19)(20)(21). Our results suggest that PEG-DMPE may be shed rapidly to expose the "naked" lipid particle consisting of fusogenic lipid D-Lin-DMA, which allows it to fuse with the cell membrane to achieve efficient siRNA transfection in vitro.…”

Prevention of Antibody-Mediated Elimination of Ligand-Targeted Liposomes by Using Poly(Ethylene glycol)-Modified Lipids

“…This is consistent with data indicating that DSPE-PEG 2000 and DPPE-PEG 2000 are retained well in the membrane after i.v. administration (Li et al, 2001b). When the concentration of PEG-lipid (DPPE-PEG 2000 ) was lowered to 2%, the protective effect of this lipid was no longer evident.…”

“…Exchange rates in the order of minutes have been measured both in vitro and in vivo when this lipid is incorporated in neutral liposomes (Holland et al, 1996;Li et al, 2001b). The protective effect of this PEG-lipid on Bxliposomes was also investigated in this study.…”

“…However, this level of liposomal lipid represents only 12% of the injected dose and is significantly less than that observed after injection of the nonhaptenized formulation (DSPC/Chol). The limited effectiveness of DMPE-PEG 2000 in protecting Bx-liposomes from rapid elimination in vivo is probably due to the rapid loss of this PEG-lipid out of the liposomal bilayer (Holland et al, 1996;Li et al, 2001b).…”

“…DSPE-PEG is known for its retention in the liposomal bilayer, and is thus probably not an ideal PEG-lipid for re-exposing liposomal ligands. It remains to be tested whether DPPE-PEG, with an intermediate exchange rate (Li et al, 2001b), can be released from liposomes in the time frame that is suitable for maximal target tissue localization as well as optimal dissociation to reveal the surfaceassociated targeting ligand.…”