4-heptadecylumbelliferone and 1-2-oleoylphosphatidylcholine

Microphase separation behaviors of cationic liposomes have been investigated using a pH-sensitive fluorescent probe with 4-heptadecyl-7-hydroxycoumarin (HHC), 1,6-diphenyl-1,3,5-hexatriene, and 6-lauroyl-2-dimethylaminonaphthalene, and to estimate localized electrostatic potentials. Shifts of the apparent pKa values of HHC were observed in cationic liposomes in proportion to the amount of cationic lipids. Two pKa values were obtained with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/3β-[N(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Ch) liposomes, while only one pKa value was generated with either DOPC/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or DOPC/dimethyldioctadecylammonium-bromide (DODAB) liposomes. The physicochemical membrane property analyses, focusing on membrane fluidity and membrane polarity, revealed heterogeneity among DOPC/DC-Ch liposomes. By analyzing the pH titration curves using sigmoidal fitting, the localized electrostatic potentials were estimated. For DOPC/DOTAP = (7/3), the membrane was in the liquid-disordered phase and the density of cationic molecules was 0.41 cation/nm(2). For DOPC/DC-Ch = (7/3), the membrane was heterogeneous and the densities of cationic molecules in liquid-disordered and liquid-ordered phases were 0.25 and 1.24 cation/nm(2), respectively. We thereby conclude that the DC-Ch molecules can form nanodomains when these molecules are concentrated to 59%.

Topics: 2-Naphthylamine; Cholesterol; Diphenylhexatriene; Fatty Acids, Monounsaturated; Fluorescent Dyes; Hydrogen-Ion Concentration; Laurates; Liposomes; Membrane Fluidity; Phosphatidylcholines; Quaternary Ammonium Compounds; Spectrometry, Fluorescence; Umbelliferones

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). {. 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.

Topics: Cholesterol; Chromatography, High Pressure Liquid; Fatty Acids, Monounsaturated; Fluorescent Dyes; Gene Transfer Techniques; Hydrogen-Ion Concentration; Kinetics; Lipids; Liposomes; Models, Structural; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Static Electricity; Structure-Activity Relationship; Surface Properties; Umbelliferones