1-2-oleoylphosphatidylcholine and pyranine

We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-xylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by 31P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH <6.5, (iii) an increase in the mean diameter of liposomes for pH <6.0, and (iv) a mixing of liposome membranes for pH <5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5-7.0 to 6.5-6.0 and (ii) a release of approximately 15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellularly after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.

Topics: Animals; Arylsulfonates; Biophysics; Cells, Cultured; Cholesterol Esters; Hydrogen-Ion Concentration; Liposomes; Macrophages; Magnetic Resonance Spectroscopy; Mice; Particle Size; Permeability; Phosphatidylcholines; Phosphatidylethanolamines

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.

Topics: Animals; Arylsulfonates; Binding Sites; Cations; Cell Line; Endocytosis; Fatty Acids, Monounsaturated; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Liposomes; Macrophages; Mice; Microscopy, Fluorescence; Oleic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Quaternary Ammonium Compounds; Rhodamines; Spectrometry, Fluorescence; Surface Properties

A dioleoylphosphatidylcholine unilamellar vesicle model system was used to determine proton permeability. The fluorescence of the pH reporter group, pyranine, trapped within vesicles with a difference in pH across the bilayer, was digitized and analyzed with numerical integration. When H+ flux was initiated by the acidification of the external buffer (acid jump), the apparent H+ permeability was found to be a linear function of the reciprocal of the internal H+ concentration with the slope inversely proportional to the initial size of the H+ gradient. When flux was initiated by the alkalinization of the external buffer (base jump), the apparent permeability coefficient was constant for each external H+ concentration. However, the value of the apparent permeability was linearly dependent on the reciprocal of the external H+. The possibility that carbonates (carbon dioxide, carbonic acid, bicarbonate and carbonate) could be acting as proton carriers was tested by adding millimolar concentrations of bicarbonate to solutions greatly reduced in carbonates. The slopes of the graphs of apparent permeability coefficient vs. reciprocal H+ were linear functions of added bicarbonate concentration for both acid and base jump conditions. These observations were interpreted in terms of a model suggesting that carbonic acid or carbon dioxide together with bicarbonate was an efficient proton carrier across phospholipid bilayers.

Topics: Arylsulfonates; Bicarbonates; Buffers; Cell Membrane Permeability; Fluorescent Dyes; Hydrogen-Ion Concentration; Lipid Bilayers; Liposomes; Phosphatidylcholines; Protons