Liposome procedure

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. 

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. 

As illustrated above there exist a large variety of techniques for preparing liposomes. From a pharmaceutical point of view, optimum liposome preparation techniques would avoid the use of organic solvent and detergents (which are difficult to remove), would exhibit a high trapping efficiency, would yield well-defined vesicles which can be produced in a reproducible way, and would be rapid and amenable to scale-up procedures (see Sec. VIII). 

Liposome size can range from around 20 nm to around 50 pm. To a certain extent, the mean diameter and distribution of the diameters can be controlled by sizing procedures after the formation of the initial liposome dispersion or by a careful selection of the preparation conditions (cf. Sec. II). Several techniques can be used to determine mean particle size and particle size distribution (Groves, 1984). 

Relatively few articles have been published on the industrial manufacturing of liposomes (Fildes, 1981 Rao, 1984 Ostro, 1988 Van Hoogevest and Fankhauser, 1989 Martin, 1989). A large number of patents describing procedures for large-scale production of... 

Kirby, C. J., and Gregoriadis, G. (1984). A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions, in Liposome Technology, Vol. 1 (G. Gregoriadis, ed.), CRC Press, Boca Raton, pp. 19-27. 

Szoka, F., and Papahadjopoulos, D. (1978). Procedure for preparation of liposomes with large aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Acad. Sci. USA. 75, 4194-4198. 

Virosomes are virus-mimicking systems that contain liposomal bilayer and pH-dependent protein impregnated in the liposomal wall. Virosomes are produced by a detergent dialysis procedure. Many researchers have demonstrated that the virosomes facilitate the leakage of the encapsulated drugs from the endosomes into the cytoplasm. This is, however, complicated technology and, so far, no virosome products are used in the clinical practice. 

The determination of partition coefficients using liposomes as a lipid phase require that the sample be equilibrated with a suspension of liposomes, followed by a separation procedure, before the sample is quantitated in the fraction free of the lipid component. 

Native chemical ligation also can be extended to the conjugation of peptides or proteins to other molecules or surfaces. For instance, Reulen et al. (2007) prepared liposomes that contained cysteine-PEG-phospholipid derivatives and then coupled thioester-modified peptides or proteins to form a protein-liposome conjugate. Using this procedure, approximately 100 molecules of a collagen binding protein could be coupled to the cysteine-containing liposomes. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

The biotinylated liposomes prepared by this procedure may be stored under an inert-gas atmosphere at 4°C for long periods without degradation. 

Bz s-imidoesters like DMS may be used to couple proteins to PE-containing liposomes by crosslinking with the amines on both molecules (Figure 22.24). However, single-step crosslinking procedures using homobifunctional reagents are particularly subject to uncontrollable polymerization of protein in solution. Polymerization is possible because the procedure is done with the liposomes, protein, and crosslinker all in solution at the same time. 

The following generalized method is based on the procedure described by Heath et al. (1981) for the coupling of immunoglobulins to liposomes containing glycosphingolipids. 

A variety of lipid adjuvants and protein mediators have also been shown to influence the immune response to antigens encapsulated in liposomes. The most widely used examples of such adjuvants for practical immunization procedures are endotoxin (including lipid A and lipopolysaccharide) and numerous types of lipophilic derivatives of muramyl dipeptide. 

Liposome Formation. The pioneering investigations of Bang-ham (5) have shown that thin films of natural phospholipids form bilayer assemblies if they are lyophilized in excess water by simple handshaking above the phase transition temperature. While this procedure results in the formation of large, multibilayered spherical structures, by ultrasonication of such lipid dispersions small unilamellar liposomes are formed (16), which are schematically shown in Figure 10. Additional metTiods for liposome preparation are described in a number of reviews (17,44,45,46). 

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