Multilamellar vesicle preparation

Liposomes are members of a family of vesicular structures which can vary widely in their physicochemical properties. Basically, a liposome is built of one or more lipid bilayers surrounding an aqueous core. The backbone of the bilayer consists of phospholipids the major phospholipid is usually phosphatidylcholine (PC), a neutral lipid. Size, number of bilayers, bilayer charge, and bilayer rigidity are critical parameters controlling the fate of liposomes in vitro and in vivo. Dependent on the preparation procedure unilamellar or multilamellar vesicles can be produced. The diameter of these vesicles can range from 25 nm up to 50 ym—a 2000-fold size difference. 

Vesicles [10, 11] these aggregates of insoluble natural or artificial amphiphiles in water can have various shapes (spherical, cylindrical). Depending on the preparation conditions, small unilamellar or large multilamellar vesicles can be produced. The structures meet the self-organization criterion, because they are, albeit on a long time scale, dynamic and not in thermodynamic equilibrium, which would in many cases be a macroscopically phase separated lamellar phase. 

Liposomes are formed due to the amphiphilic character of lipids which assemble into bilayers by the force of hydrophobic interaction. Similar assemblies of lipids form microspheres when neutral lipids, such as triglycerides, are dispersed with phospholipids. Liposomes are conventionally classified into three groups by their morphology, i.e., multilamellar vesicle (MLV), small unilamellar vesicle (SUV), and large unilamellar vesicle (LUV). This classification of liposomes is useful when liposomes are used as models for biomembranes. However, when liposomes are used as capsules for drugs, size and homogeneity of the liposomes are more important than the number of lamellars in a liposome. Therefore, "sized" liposomes are preferred. These are prepared by extrusion through a polycarbonate... 

Since soy lecithin ( 20% extract from Avanti) was selected as a basis for absorption modeling, and since 37 % of its content is unspecified, it is important to at least establish that there are no titratable substituents near physiological pH. Asymmetric triglycerides, the suspected unspecified components, are not expected to ionize. Suspensions of multilamellar vesicles of soy lecithin were prepared and titrated across the physiological pH range, in both directions. The versatile Bjerrum plots (Chapter 3) were used to display the titration data in Fig. 7.33. (Please note the extremely expanded scale for %.) It is clear that there are no ionizable groups... 

Entrapment of plasmid DNA and/or protein into liposomes entails the preparation of a lipid film from which multilamellar vesicles and, eventually, small unilamellar vesicles (SUVs) are produced. SUVs are then mixed with the plasmid DNA and/or protein destined for entrapment and dehydrated. The dry cake is subsequently broken up and rehydrated to generate multilamellar dehydration-rehydration vesicles (DRV) containing the plasmid DNA and/or protein. On centrifugation, liposome-entrapped vaccines are separated from nonentrapped materials. When required, the DRV are reduced in size by microfluidization in the presence or absence of nonentrapped materials or by employing an alternative method (7) of DRV production, which utilizes sucrose (see below). 

Figure 5 shows small multilamellar vesicles under the electron microscope, visible as concentric spheres. Note the fascinating texture of the spheres - the actually fluid bilayer appears structured, frozen in time at the moment of preparation. 

There are several other ways to entrap solutes inside the liposomes, and the entrapping efficiency depends on the structure of liposomes (small unilamellar, large unilamellar, multilamellar, vesicles, etc.) and from the technique for liposome preparation (Roseman etal., 1978 Cullis etal., 1987 Walde and Ishikawa, 2001). 

Materials and Methods. PS was purified from bovine brain in this laboratory as previously described (45). Dispersions of multilamellar vesicles and sonicated unilamellar vesicles were prepared as described earlier (15, 45) in a standard buffer containing 100mm NaCl, 7mm L-histi-dine N-tris-(hydroxymethyl) methyl 2-amino ethane-sulfonic acid (TES), and approximately 0.1mm EDTA, adjusted to pH 7.4. CF containing vesicles were prepared by hydration and sonication in a solution of 100mm CF, 0.1mm EDTA, and 1/10 (v/v) of the standard buffer, adjusted to pH 7.4, separated by passage through a Sephadex G-75 (1.0 X 20 cm) column equilibrated with the standard (0.1M NaCl) buffer and stored on ice. 

Liposomes used for transfection are either large unilamellar vesicles (LUVs) of 100 to 200 nm in diameter or small unilamellar vesicles (SUVs) of 20 to 100 nm. Liu et al.124 have reported that for a given liposome composition, multilamellar vesicles (MLVs) of 300 to 700 nm in diameter exhibit higher transfection efficiency than SUVs. However, more recent studies on the nature of the liposome-DNA complex (or lipoplex) revealed that lipoplexes from SUVs or MLVs do not differ significantly in size. On the other hand, the composition of the medium, not the type of the liposome used in the preparation of the lipoplex, plays a key role in determining the final size of the complex. And the transfection efficiency is also shown to depend on the final size of the complexes but not the type of the liposome.125... 

In conventional film-shaking liposome preparation, lipids (see Note 2) are dissolved in chloroform and the solvent removed by evaporation to leave a lipid film. Insulin-containing citrate buffer (pH 4.0) is added to hydrate the lipid film. Mechanical shaking of the mixture results in the formation of multilamellar vesicles. 

A block copolymer effective as a controlled release agents of biologically active materials have been prepared. This agent consisted of ethylene oxide-propylene sulfide-ethylene oxide teipolymer that had been end-capped with a selected cysteine-containing peptide. These materials resist degradation prior to reaching their intended targets because they behave as multilamellar vesicles. 

Multilamellar vesicles are the most commonly used model membrane systems. It is important to note that in order to simplify the parameters of the study, in most cases the model membranes are prepared exclusively ftom phospholipids and they do not contain other molecules, usually present in biological membranes that have an important role in their fiinctionality. The complexity of real membranes is not close to the artificial model membranes and these systems, i.e. liposomes, are not an absolute analog of the biological membranes. 

Numerous techniques for the preparation of liposomes have been described. Typical procedures involve the hydration of lipid mixtures in buffer, resulting in the formation of large multilamellar vesicles (MLV). These are of limited use in... 

The exchangeable pool of lipids in the two membranes can be easily determined. When the substrates were prepared by the procedure of DiCorleto and Zilversmit (1977), 70% of the unilamellar vesicle phosphatidylcholine was available for exchange and 7% of the phosphatidylcholine of the phosphatidylcholine phosphatidylethanolamine cardio-lipin (70 25 5, mol%) multilamellar vesicles. Knowledge of the size of the exchangeable pool of lipid is important for predicting the linear range of the assay and determining kinetic parameters. 

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