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


BACKGROUND
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, 3beta[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) pHbulk 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 X 10(-5) M. This might also be a mechanism of the dissociation of bilayers containing DOTAP from the plasmid DNA.
Zuidam
N J
NJ
Department of Biochemistry, The Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Barenholz
Y
Y
D002784:Cholesterol; D002851:Chromatography, High Pressure Liquid; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D008961:Models, Structural; D008968:Molecular Conformation; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013499:Surface Properties; D014468:Umbelliferones
D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D014468:Umbelliferones; C087276:(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium; C031827:4-heptadecylumbelliferone; C020888:1,2-dielaidoylphosphatidylethanolamine; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane