Liposomes preparation

Adjuvants are substances which can modify the immune response of an antigen (139,140). With better understanding of the functions of different arms of the immune system, it is possible to explore the effects of an adjuvant, such that the protective efficacy of a vaccine can be improved. At present, aluminum salt is the only adjuvant approved for use in human vaccines. New adjuvants such as QS-21, 3D-MPL, MF-59, and other liposome preparations are being evaluated. Several of these adjuvants have been in clinical trial, but none have been approved for human use. IL-12 has been proposed as an adjuvant which can specifically promote T-helper 1 ceU response, and can be a very promising adjuvant for future vaccine development. 

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). 

Peroxidation of lipids is another factor which must be considered in the safety evaluation of liposome administration. Smith and coworkers (1983) demonstrated that lipid peroxides can play an important role in liver toxicity. Allen et al. (1984) showed that liposomes protected by an antioxidant caused less MPS impairment than liposomes subjected to mild oxidizing conditions. From the study of Kunimoto et al. (1981) it can be concluded that the level of peroxidation in freshly prepared liposome preparations and those on storage strongly depends both on the phospholipid fatty acid composition and on the head group of the phospholipid. Addition of appropriate antioxidants to liposomes composed of lipids which are liable to peroxidation and designed for use in human studies is therefore necessary. 

For a number of liposome preparations—both injectables and locally administered products—the therapeutic advantages over existing formulations have been proven in animal models clinical trials with liposome preparations are now under way. So far, clinical studies showed no significant toxic effects which could be ascribed to the lipid components of the liposomes used. 

Batzri, S., and Korn, E. D. (1973). Single bilayer liposomes prepared without sonication, Biochim. Biophys. Acta, 298, 1015-1019. 

Crommelin, D. J. A., and Storm, G. (1987). Pharmaceutical aspects of liposomes Preparation, characterization, and stability, in Controlled Drug Delivery (B. W. Muller, ed.), Wissenschaftliche Verlagsgescellschaft mbH, Stuttgart, pp. 80-91,... 

Klein, R. A. (1970). The detection of oxidation in liposome preparations, Biochim. Biophys. Acta, 210, 486-489. 

Lichtenberg, D., and Barenholz, Y. (1988). Liposomes Preparation, characterization and preservation, in Methods of Biological Analysis. Vol. 33 (D Glick, ed.), John Wiley and Sons, New York, pp. 337-461. 

Ohsawa, T., Matsukawa, Y., Takakura, Y., Hashida, M., and Sezaki, H. (1985). Fate of lipid and encapsulated drug after intramuscular administration of liposomes prepared by the freezethawing method in rats, Chem. Pharm. Bull., 5013-5022. 

Storm, G., Van Bloois, L., Brouwer, M., and Crommelin, D. J. A. (1985). The interaction of cytostatics with adsorbents in aqueous media. The potential implications for liposome preparation, Biochim. Biophys. Acta. 818, 343-351. 

Szoka, F., and Papahadjopoulos, D. (1981). Liposomes Preparation and characterization, in Liposomes From Physical Structure to Therapeutic Applications (C. G. Knight, ed.), Elsevier, Amsterdam, pp. 51-82. 

Osterherg, T. Svensson, M. Lundahl, P, Chromatographic retention of drug molecules on immohilized liposomes prepared from egg phospholipids and from chemically pure phospholipids, Eur. J. Pharm. Sci. 12, 427 139 (2001). 

Fig. 13. Mannosylated prophyrin clusters prepared by Ballut et al. for liposome preparation.

An example of a lipid mixture preparation based on mass would be to dissolve 100 mg of PC, 40 mg of cholesterol, and 10 mg of PG in 5 ml of chloroform/methanol solution. When using activated PE components, inclusion of 10 mg of the PE derivative to this recipe will result in a stable liposome preparation. 

Regardless of their method of fabrication, most liposome preparations need to be further classified and purified before use. To remove excess aqueous components that were not encapsulated during the vesicle formation process, gel filtration using a column of Sephadex G-50 or dialysis can be employed. To fractionate the liposome population according to size, gel filtration using a column of Sepharose 2B or 4B should be done. 

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

Characterizing the resultant complex for the amount of protein per liposome is somewhat more difficult than in other protein conjugation applications. The protein-liposome composition is highly dependent on the size of each liposomal particle, the amount of protein charged to the reaction, and the mole quantity of reactive lipid present in the bilayer construction. An approach to solving this problem is presented by Hutchinson et al. (1989). In analyzing at least 17 different protein-liposome preparations, the ratio of proteindipid content (pg protein/pg lipid) in most of the complexes ranged from a low of about 4 to as much as 675. In some instances, however, up to 6,000 molecules of a particular protein could be incorporated into each liposome. 

Mayhew, E., Lazo, R., Vail, W.J., King, J., and Green, A.M. (1984) Characterization of liposomes prepared using a microemulsifier. Biochim. Biophys. Acta 775, 169-174. 

Figure 5.30 Temperature dependence of molar ellipticity at 218 nm for liposomes prepared from L-DMPC, L-DPPC (39), and L-POPC (40). Reprinted with permission from Ref. 134. Copyright 1997 by the American Chemical Society.

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). 

Several cell lines were used to inveshgate the role of PS oxidahon in apoptosis. Preferenhal oxidahon of PS was observed in human leukemia HL-60 cells (Fabisiak et al, 1998, 2000 Kawai et al, 2000), and normal human keratinocytes (Shvedova et al, 2001). Similarly, in pheochromocytoma PC 12 cells exposed to a radical-generating anhneoplashc dmg, neocarzinostatin, extemalizahon of PS was potentiated by its selechve oxidation in whole cells (Schor et al, 1999). In contrast, this selechve PS oxidahon did not occur in liposomes prepared from mixtures of PnA-labeled phospholipids extracted from the ceUs and exposed to oxidants imder the same conditions (Fabisiak et al, 1998 Kagan et al, 2000 Shvedova et al,... 

Many types of liposomes of different lipid composition and different sizes having a transmembrane AS gradient were prepared (10). These liposomes varied (i) in their liposome-forming phosphatidylcholine (PC), being with and without cholesterol and/or lipopolymer (ii) in their size and (iii) in their method of preparation. The approaches for preparing these different liposome formulations varies in their lipid hydration and downsizing. Table 1 in Haran et al. (10) gives a partial list of such liposome preparations. In all cases the scheme of liposome preparation can be summarized as described in Table 4. 

DOX concentration was determined spectrophotometrically based on the molar extinction coefficient of 125000 OD M (38) in a dual-beam spectrophotometer (either Perkin-Elmer Lambda 3B or Kontron Uvikon 860). The DOX quantification was confirmed by HPLC (49-51). Purity of DOX and its degree of degradation during the processes of liposome preparation and liposome storage were determined by a combination of HPLC and TLC, as described by Barenholz leave et al. (38,49,50). 

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