Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient (original) (raw)
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Span 80 vesicles have a more fluid, flexible and “wet” surface than phospholipid liposomes
Colloids and Surfaces B: Biointerfaces, 2011
The surface properties of Span 80 vesicles at various cholesterol contents, together with those of various liposomes, were characterized by using fluorescence probes. The membrane fluidity of the Span 80 vesicles was measured by 1,6-diphenyl-1.3.5-hexatriene (DPH) and trimethlyammonium-DPH (TMA-DPH), and the results suggested that the surface of the Span 80 vesicles was fluid due to the lateral diffusion of Span 80 molecules. The depolarization measured by TMA-DPH and the headgroup mobility measured by dielectric dispersion analysis indicated the high mobility of the head group of Span 80 vesicles. This suggested that the surface of Span 80 vesicles was flexible due to the head group structure of Span 80, sorbitol. In addition, spectrophotometric analysis with 6-dodecanoyl-N, N-dimethyl-2-naphthylamine and 8-anilino-1-naphthalenesulfonic acid indicated that the water molecules could easily invade into the interior of the Span 80 vesicle membrane, suggesting that the membrane surface was more wet than the liposome surface. These surface properties indicated that the protein could interact with the interior of vesicle membranes, which was similar to the case of cholesterol. Thus the present results confirmed that the Span 80 vesicle surfaces showed the unique characteristics of fluidity, flexibility, and "wetness", whereas the liposome surfaces did not.
Chemistry and Physics of Lipids, 2000
Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristicsin terms of lamellarity and morphology -of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicle's preparation method.
Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into a mixture of phospholipid and organic solvent and the organic solvent is subsequently removed by evaporation under reduced pressure. These vesicles can be made from various lipids or mixtures of lipids and have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles. Most importantly, a substantial fraction of the aqueous phase (up to 65% at low salt concentrations) is entrapped within the vesicles, encapsulating even large mac-
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
Previous observations on serum-induced leakage of liposome contents from egg phosphatidylcholine liposomes (Allen, T.M. and Cleland, L.G. (1980) Biochim. Biophys. Acta 597, 418--426) have been extended in order to examine the role of the phase transition and phospholipid backbone in leakage. The high-density lipoprotein (HDL) fraction has been purified from human serum and the rate of transfer of radioactively labelled phospholipids from sonicated liposomes to high~lensity lipoproteins has been examined. Results obtained from the caicein dequenching method for serum-induced leakage of liposome contents showed that as the proportion of solid phospholipid (distearoyl phosphatidylcholine, Tc = 56°C) increased, relative to the proportion of egg phosphatidylcholine, the half-time for retention of liposome contents at 37°C in the presence of serum also increased. Including increasing amounts of bovine brain sphingomyelin (Tc = 30 ° C) in egg phosphatidylcholine liposomes also substantially decreased leakage from liposomes in the presence of serum at 37°C. 14C-labeiled egg phosphatidylcholine was found to transfer readily from liposomes to purified HDL, as did 14C-labelled dioleoyl phosphatidylcholine. Including cholesterol in egg phosphatidylcholine liposomes decreased the rate of transfer of phospholipid to HDL. ~4C-labelled distearoyl phosphatidylcholine did not exchange readily with HDL. These .results are consistent with the interpretation that tightening bilayer packing prevents the apolipoprotein-mediated transfer of phospholipid to HDL and slows the leakage of liposome contents associated with this transfer.
Structural Components of Liposomes and Characterization Tools
Liposomes are the artificially formulated spherical vesicles which are composed of lipid bilayer having encapsulation ability of both hydrophilic and lipophilic drugs in order to prevent them from degradation. The controlled and targeted release ability of liposomes makes them a unique drug delivery system. Liposomes are combination of phospholipids in an aqueous media resulting in bilayered structures. Liposomes can be characterized by various methods. Liposomes can be used in controlling and targeting drug delivery system. Liposomes have various therapeutic application including cancer, ocular, infectious diseases and also various applications in diagnostics, industrial and gene therapy. With the advancement of science and technology, liposomes will have a remarkable future in the pharmaceutical market.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
Small unilamellar phosphatidylseriae/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca ~÷ concentration was increased to a threshold of 3--5 mM in 100 mM NaC1, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 tim diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2÷-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca :÷ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca 2÷ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca 2÷ concentration. Abbreviation: Hepes, N-2-hydroxyethylpiperazine-N~-2-ethanesulfonic acid.