No abstract

A novel acid-labile poly(ethylene glycol) (PEG)-conjugated lipid, (R)-1,2-di-O-(1′Z,9′Z-octadecadienyl)glyceryl-3-(ω-methoxy-poly(ethylene glycolate, MW5000) (BVEP), a neutral PEG-derivatized analogue of diplasmenylcholine, has been used at low molar ratios to disperse the nonlamellar, fusogenic lipid 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as unilamellar liposomes. It was anticipated that acidcatalyzed hydrolysis of the vinyl ether linkages would destabilize BVEP/DOPE liposomes by removal of the water-soluble, sterically stabilizing PEG layer, thereby promoting contents release and membranemembrane fusion. This paper describes the hydrolysis rates, contents release rates, and fusion kinetics of BVEP-stabilized DOPE liposomes at 1:99, 3:97, and 5:95 molar ratios of BVEP/DOPE. Calcein leakage kinetics indicate that 3:97 BVEP/DOPE liposomes offer the best stability at pH 7.4 while retaining favorable leakage properties at pH 4.5 (t50%release ≈ 4 h). N-Rhodamine phosphatidylethanolamine/N-nitrobenzoxadiazole phosphatidylethanolamine lipid mixing assays show that membrane fusion occurs on a much slower time scale than leakage in these systems, with ∼12% lipid mixing occurring over a 24 h time period at pH 2.0. No appreciable membrane fusion occurred in these liposomes at either pH 7.4 or 4.5 when monitored for up to 3 days. 31 P NMR spectra at pH 7.4 contain a single isotropic line shape, consistent with the presence of large liposomes. The 31 P NMR line shape did not change significantly even after long exposure times at pH 4.0; however, Mn 2+ addition experiments with acid-treated samples produced linebroadened spectra, indicating that all the phosphorus sites were continuous with the bulk water phase. Time-dependent cryogenic transmission electron microscopy experiments indicate that extensive liposome collapse to give small dense aggregates occurs over a 1-4 h period when 3:97 BVEP/DOPE liposomes are acidified to pH 4.5. Taken together, these results suggest that acid-catalyzed hydrolysis of BVEP/DOPE liposomes does result in dePEGylative triggering; however, the primary outcome of this cleavage process is contents leakage and liposome collapse to give <100 nm particles that are presumed to be inverted hexagonal phase structures, with membrane lipid mixing occurring on a kinetically slower time scale. † Dedicated to the memory of David F. O'Brien, deceased July 2002.

An acid-cleavable PEG lipid, 1′-(4′-cholesteryloxy-3′-butenyl)-ω-methoxy-polyethylene [112] glycolate (CVEP), has been developed that produces stable liposomes when dispersed as a minor component (0.5-5 mol%) in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cleavage of CVEP at mildly acidic pH's results in dePEGylation of the latently fusogenic DOPE liposomes, thereby triggering the onset of contents release. This paper describes the synthesis of CVEP via a six step sequence starting from the readily available precursors 1,4-butanediol, cholesterol, and mPEG acid. The hydrolysis rates and release kinetics from CVEP:DOPE liposome dispersions as a function of CVEP loading, as well as the cryogenic transmission electron microscopy and pH-dependent monolayer properties of 9:91 CVEP:DOPE mixtures, also are reported. When folate-receptor positive KB cells were exposed to calcein-loaded 5:95 CVEP:DOPE liposomes containing 0.1 mol% folatemodified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene[76] glycolamide (folate-PEG-DSPE), efficient delivery of the calcein cargo to the cytoplasm of the cells was observed as determined by fluorescence microscopy and flow cytometry. Fluorescence resonance energy transfer analysis of lipid mixing in these cells was consistent with membrane-membrane fusion between the liposome and endosomal membranes.