4-heptadecylumbelliferone and laurdan

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

Lipoplexes, which are spontaneously formed complexes between oligonucleotide (ODN) and cationic lipid, can be used to deliver ODNs into cells, both in vitro and in vivo. The present study was aimed at characterizing the interactions associated with the formation of lipoplexes, specifically in terms of electrostatics, hydration and particle size. Large unilamellar vesicles (approximately 100 nm diameter), composed of either DOTAP, DOTAP/cholesterol (mole ratio 1:1) or DOTAP/DOPE (mole ratio 1:1) were employed as a model of cationic liposomes. Neutral vesicles ( approximately 100 nm diameter), composed of DOPC/DOPE (mole ratio 1:1), were employed as control liposomes. After ODN addition to vesicles, at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin. In separate 'mirror image' experiments, liposomes were added at different mole ratios to fluorescein isothiocyanate-labeled ODNs, thus yielding data about changes in the pH near the ODN molecules induced by the complexation with the cationic lipid. Particle size distribution and turbidity fluctuations were analyzed by the use of photon correlation spectroscopy and static light-scattering, respectively. In additional fluorescent probe studies, TMADPH was used to quantify membrane defects while laurdan was used to measure the level of hydration at the water-lipid interface. The results indicate that mutual neutralization of cationic lipids by ODNs and vice versa is a spontaneous reaction and that this neutralization is the main driving force for lipoplex generation. When lipid neutralization is partial, induced membrane defects cause the lipoplexes to exhibit increased size instability.

Topics: 2-Naphthylamine; Cations; Cholesterol; Diphenylhexatriene; Fatty Acids, Monounsaturated; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Laurates; Lipids; Liposomes; Oligodeoxyribonucleotides, Antisense; Oligonucleotides; Particle Size; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Static Electricity; Thionucleotides; Umbelliferones