1-2-dielaidoylphosphatidylethanolamine and cholesteryl-succinate

Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

Topics: Animals; Anions; Cattle; Cell Line; Cholesterol Esters; Detergents; DNA; Drug Carriers; Drug Stability; Gene Transfer Techniques; Glycosaminoglycans; Haplorhini; Hydrogen-Ion Concentration; Imines; Lipids; Liposomes; Particle Size; Phosphatidylethanolamines; Plasmids; Polyethylenes; Transfection

Cholesteryl hemisuccinate (CHEMS) is an acidic cholesterol ester that self-assembles into bilayers in alkaline and neutral aqueous media and is commonly employed in mixtures with dioleoylphosphatidylethanolamine (DOPE) to form 'pH sensitive' fusogenic vesicles. We show here that CHEMS itself exhibits pH sensitive polymorphism. This is evident from the fusogenic properties of large unilamellar vesicles (LUV) composed of CHEMS and direct visualization employing freeze-fracture electron microscopy. Below pH 4.3, LUV composed of CHEMS undergo fusion as monitored by lipid mixing assays and freeze-fracture electron micrographs reveal the characteristic striated signature of H( parallel) phase lipid. It is suggested that the pH dependent phase preferences of CHEMS contribute to the pH sensitivity of LUV composed of mixtures of CHEMS and DOPE.

Topics: Cholesterol Esters; Freeze Fracturing; Hydrogen-Ion Concentration; In Vitro Techniques; Liposomes; Membrane Fusion; Microscopy, Electron; Phosphatidylethanolamines; Water

The objective of this study is to present a rational strategy to target macromolecules to the nucleus via the endocytic pathway. The two major barriers in this route to the nucleus are known as endosomal escape and nuclear transport. pH-sensitive liposomes were used in order to achieve endosomal escape under the conditions of low pH in endosomes. Bovine serum albumin (alb) served as a model compound to be delivered to nucleus and was encapsulated into the pH-sensitive liposomes. The liposomes are composed of dioleoyl phosphatidyl ethanolamine: cholesterylhemisuccinate. They were taken up by rat peritoneal macrophages via endocytosis and subsequently underwent degradation, principally by lysosomal enzymes. By using pH-sensitive liposomes, intracellular degradation was reduced by a significant extent, as expected, via endosomal escape. Cytosolic delivery of FITC-labelled alb was also detected by confocal microscopy. Selective targeting to the nucleus was performed by adding the nuclear localization signal (NLS) of the SV-40 large T antigen to the FITC-alb, which were then encapsulated into the pH-sensitive liposomes. Confocal microscopy revealed that FITC-alb, in the presence of NLS was successfully delivered into nucleus, while no transport was observed in the absence of NLS. These results provide a useful strategy for the nuclear targeting of macromolecules using pH-sensitive liposomes in conjunction with NLS.

Topics: Animals; Cattle; Cell Nucleus; Cholesterol Esters; Drug Carriers; Endocytosis; Endosomes; Fluorescein-5-isothiocyanate; Hydrogen-Ion Concentration; Liposomes; Macrophages, Peritoneal; Male; Phosphatidylethanolamines; Rats; Rats, Wistar; Serum Albumin, Bovine