Liposome formation method

Figure 1 Influence of liposome formation method on TRP2 content of liposomes.

The sensors discussed so far are based on ligands covalently bound to the polymer backbone. Other methods of detection - often referred to as mix and detect methods - work by simple noncovalent incorporation of the polymer with the ligand of interest. Reichert et al. generated liposomes of polydiacetylene with sialic acid for the same purpose of detection as Charych s surface-bound polymers, but realized that covalent functionalization of the polymer was not necessary [17]. Through simple mixing of the lipid-bound sialic acid with the polymer before sonication and liposome formation, they were able to form a functional colorimetric recognition system (Fig. 8). 

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. 

Liposome Preparation Techniques In most cases, liposomes are named by the preparation method used for their formation, Such as sonicated, dehydrated-rehy-drated vesicle (DRV), reverse-phase evaporation (REV), one step, and extruded. Several reviews have summarized available liposome preparation methods [91,124, 125], Liposome formation happens spontaneously when phospholipids are dispersed in water. However, the preparation of drug-encapsulating liposomes with high drug encapsulation and specific size and lamellarity is not always an easy task. The most important methods are highlighted below. 

Talsma, H., Van Steenbergen, M. J., Borchert, J. C. H., Crommelin, D. J. A. (1994), A novel technique for the one-step preparation of liposomes and nonionic surfactant vesicles without the use of organic solvents. Liposome formation in a continuous gas stream The bubble method,/. Pharm. Sci., 83, 276-280. 

Based on the modes of lipid dispersion, the methods of liposomes formation can be classified into three categories mechanical dispersion, solvent dispersion, and detergent solubilization (175). These generally involve the following stages as evident in Figure 8.23. 

Liposome-mediated gene delivery is dependent on numerous factors, such as, the formulation of the liposomes including the cationic lipid/neutral lipid ratio, how the liposomes are prepared, the cationic liposome/DNA charge ratio of the complex of cationic liposome and DNA (lipoplex), and the method used to produce the lipoplex. Recently, it was reported that the way in which a liposome was prepared affected transfection efficiency (1), and formation method of lipoplex affected size of lipoplex in which large ones increased the efficiency of transfection (2-7). 

Notably, liposomes composed of 3 -[N- N N -dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) together withdioleoylphosphatidylethanolamine (DOPE) (DC-Chol/ DOPE liposome) have been classified as one of the most efficient vectors for the transfection of DNA into cells (8-10) and in clinical trials (11, 12). It has been demonstrated that a 3 2 or 1 1 molar ratio of DC-Chol/DOPE liposome results in high transfection efficiency (10). In these cases, liposomes are mostly prepared by the dry-film method. To further improve the transfection efficiency, it is necessary to evaluate DC-Chol/DOPE liposome from formulation and preparation method of liposome to formation method of their lipoplex. 

FIGURE 58.4 Schematic representation of the mechanisms of liposome formation (a) film-forming method and (b) organic solvent injection method. (From Zhang, K. et al., Powder TechnoL, 190(3), 39355, 2009.)... 

A method resulting in improved encapsulation of aqueous phase by MLV is the so-called dehydration-rehydration procedure (Kirby and Gregoriadis, 1984 Shew and Deamer, 1985). The lipid (usually preformed liposomes) is dried (by either lyophilization or evaporation) in the presence of the aqueous solute to be entrapped, thus forming a mixed film with solute trapped between layers. Subsequent gradual rehydration with a minimum of aqueous phase leads to the formation of MLV with a high entrapment of the aqueous solutes added. 

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