Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin

Abstract

Cationic liposomes are used to deliver genes into cells in vitro and in vivo. The present study is aimed to characterize the electrostatic parameters of cationic, large unilamellar vesicles, 110±20 nm in size, composed of DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, DMRIE/DOPE 1/1, or DC–CHOL/DOPE (mole ratio 1/1). {Abbreviations: DOTAP, N-(1-(2,3-dioleoyloxy)propyl)- N, N, N-trimethylammonium chloride; DOPE, 1,2-dioleoyl- sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; DC–CHOL, 3β[ N-( N′, N′-dimethylaminoethane)carbamoyl]cholesterol}. The cationic liposomes had a large positive surface potential and a high pH at the liposomal surface in 20 mM Hepes buffer (pH 7.4) as monitored by the pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin. In contrast to DOTAP and DMRIE which were 100% charged, DC–CHOL in DC–CHOL/DOPE (1/1) liposomes was only about 50% charged in 20 mM Hepes buffer (pH 7.4). This might result in an easier dissociation of bilayers containing DC–CHOL from the plasmid DNA (which is necessary to enable transcription), in a decrease of the charge on the external surfaces of the liposomes or DNA–lipid complexes, and in an increase in release of the DNA–lipid complex into the cytosol from the endosomes. Other electrostatic characteristics found were that the primary amine group of DOPE in cationic liposomes dissociated at high (>7.9) pH bulk and that a salt bridge was likely between the quaternary amine of DOTAP or DMRIE and the phosphate group of DOPE or DOPC, but not between the tertiary amine of DC–CHOL and the phosphate group of DOPE. The liposomes containing DOTAP were unstable upon dilution, probably due to the high critical aggregation concentration of DOTAP, 7×10 −5 M. This might also be a mechanism of the dissociation of bilayers containing DOTAP from the plasmid DNA.