Lipid suspensions/solutions

Prepare a liposome suspension, containing PE, at a total-lipid concentration of 5mg/ml in 0.1M sodium phosphate, 0.15M NaCl, pH 6.8. Maintain all lipid-containing solutions under an inert gas atmosphere. Degas all buffers and bubble them with nitrogen or argon prior to use. 

The most common methods for preparing liposomes32, which are not discussed here in detail, are the ultrasonication of lipid suspensions in water 6), the injection of alcoholic or etheral solutions of lipid into water, the dialysis of lipid-surfactant mixtures, and the removal of lipid films on glass surfaces by simple hand-shaking in water. 

Preparing LUVs from lipid suspension. The solution is subjected to several (typically four) freeze-thaw cycles in order to decrease the lamellarity. 

It is convenient to use a DHPC stock solution for bicelle preparation. As DHPC is extremely hygroscopic, it is otherwise difficult to estimate the true amount of DHPC that is actually added to the lipid suspension. Furthermore, unless a stock solution is prepared, DHPC should be handled in a dry atmosphere. It is important for the estimation of bicelle size (related to the g-value) that the relative concentrations of lipids and DHPC can be controlled. 

Lipid suspensions are also known to enhance the bioavailability of hydrophobic drugs. Unlike lipid solutions, suspended drug needs to undergo additional dissolution before the absorption. Therefore, factors such as drug particle size and amount suspended may also influence the bioavailability. 

Phospholipids. For the removal of ionic contaminants from raw zwitterionic phospholipids, most lipids were purified twice by mixed-bed ionic exchange (Amberlite AB-2) of methanolic solutions. (About Ig of lipid in lOmL of MeOH). With both runs the first ImL of the eluate was discarded. The main fraction of the solution was evaporated at 40°C under dry N2 and recryst three times from n-pentane. The resulting white powder was dried for about 4h at 50° under reduced pressure and stored at 3°. Some samples were purified by mixed-bed ion exchange of aqueous suspensions of the crystal/liquid crystal phase. [Kaatze et al. J Phys Chem 89 2565 7955.]... 

Seiler [250] proposed a way of estimating the extent of hydrogen bonding in solute partitioning between water and a lipid phase by measuring the so-called A log P parameter. The latter parameter is usually defined as the difference between the partition coefficient of a solute measured in the octanol-water system and that measured in an inert alkane-water suspension AlogP = log Kp oet — log Kp aik. 

Reverse-phase evaporation in a nitrogen atmosphere was used to prepare lipids. A lipid film previously formed was redissolved in diethyl ether and an aqueous phase containing the dyebath components added to the phospholipid solution. The resulting two-phase system was sonicated at 70 W and 5 °C for 3 minutes to obtain an emulsion. The solvent was removed at 20 °C by rotary evaporation under vacuum, the material forming a viscous gel and then an aqueous solution. The vesicle suspension was extruded through a polycarbonate membrane to obtain a uniform size distribution (400 nm). 

Fig. 9 Surface modification of cells with ssDNA-PEG-lipid. (a) Real-time monitoring of PEG-lipid incorporation into a supported lipid membrane by SPR. (r) A suspension of small unilamellar vesicles (SUV) of egg yolk lecithin (70 pg/mL) was applied to a CH3-SAM surface. A PEG-lipid solution (100 pg/mL) was then applied, (ii) Three types of PEG-lipids were compared PEG-DMPE (C14), PEG-DPPE (C16), and PEG-DSPE (C18) with acyl chains of 14, 16, and 18 carbons, respectively, (b) Confocal laser scanning microscopic image of an CCRF-CEM cell displays immobilized FITC-oligo(dA)2o hybridized to membrane-incorporated oligo(dT)20-PEG-lipid. (c) SPR sensorigrams of interaction between oligo(dA)2o-urokinase and the oligo (dT)2o-PEG-lipid incorporated into the cell surface, (i) BSA solution was applied to block nonspecific sites on the oligo(dT)20-incorporated substrate, (ii) Oligo(dA)20-urokinase (solid line) or oligo(dT)20-urokinase (dotted line) was applied...

Dissolve the protein or peptide to be conjugated at a concentration of lOmg/ml in 0.5M sodium carbonate, pH 9.5. Mix the activated liposome suspension with the polypeptide solution at the desired molar ratio to effect the conjugation. Mixing the equivalent of 4 mg of protein per mg of total lipid usually results in acceptable conjugates. 

Liposomes containing PE lipid components may be activated with these crosslinkers to contain iodoacetyl derivatives on their surface (Figure 22.29). The reaction conditions described in Chapter 5, Section 1.5 may be used, substituting a liposome suspension for the initial protein being modified in that protocol. The derivatives are stable enough in aqueous solution to allow purification of the modified vesicles from excess reagent (by dialysis or gel filtration) without... 

In the present study we have investigated the process of photodynamic inactivation of influenza vims in the allantoic fluid of chicken embryos. This inactivation has been realized by C60 water suspension used as a photosensitizer. Similar studies have been carried out previously by Kaserman and Kempf (1997, 1998). Unlike the latter studies, in which viruses were inactivated in salt solutions (buffer), our experiments were performed in a natural biological fluid that contains all typical components (proteins, lipids, salts, etc.). Comparing the viral inactivation over time in our experiments with previous results we conclude that the process described by Kaserman and Kempf (1997, 1998) was more time-consuming, a fact that may significantly restrict its practical use. 

The procedure chosen for the preparation of lipid complexes of AmB was nanoprecipitation. This procedure has been developed in our laboratory for a number of years and can be applied to the formulation of a number of different colloidal systems liposomes, microemulsions, polymeric nanoparticles (nanospheres and nanocapsules), complexes, and pure drug particles (14-16). Briefly, the substances of interest are dissolved in a solvent A and this solution is poured into a nonsolvent B of the substance that is miscible with the solvent A. As the solvent diffuses, the dissolved material is stranded as small particles, typically 100 to 400 nm in diameter. The solvent is usually an alcohol, acetone, or tetrahydrofuran and the nonsolvent A is usually water or aqueous buffer, with or without a hydrophilic surfactant to improve colloid stability after formation. Solvent A can be removed by evaporation under vacuum, which can also be used to concentrate the suspension. The concentration of the substance of interest in the organic solvent and the proportions of the two solvents are the main parameters influencing the final size of the particles. For liposomes, this method is similar to the ethanol injection technique proposed by Batzii and Korn in 1973 (17), which is however limited to 40 mM of lipids in ethanol and 10% of ethanol in final aqueous suspension. 

The chloroform solution of lipids (Solution A) is placed in a 50-mL round-bottomed spherical Quick-fit flask. Following evaporation of the solvent in a rotary evaporator at about 37°C, a thin lipid film is formed on the walls of the flask. The film is flushed for about 60 seconds with oxygen-free nitrogen (N2) to ensure complete solvent removal and to replace air. Two milliliters of distilled water and a few glass beads are added into the flask, the stopper is replaced, and the flask shaken vigorously by hand or mechanically until the lipid film has been transformed into a milky suspension. This process is carried out above the liquid-crystalline transition temperature (7/) of the phospholipid component of liposomes (> 7/) by prewarming the water... 

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