Preparation of Liposomes

Mixtures of phospholipids in aqueous solution will spontaneously associate to form liposomal structures. To prepare liposomes having morphologies useful for bioconjugate or delivery techniques, it is necessary to control this assemblage to create vesicles of the proper size and shape. Many methods are available to accomplish this goal, however all of them have at least several steps in common (1) dissolving the lipid mixture in organic solvent, (2) dispersion in an aqueous phase, and (3) fractionation to isolate the correct liposomal population. 

The correct ratio of lipid constituents is important to form stable liposomes. For instance, a reliable liposomal composition for encapsulating aqueous substances may contain molar ratios of lecithin cholesterol negatively charged phospholipid (e.g., phosphatidyl glycerol (PG)) of 0.9 1 0.1. A composition that is typical when an activated phosphatidylethanolamine (PE) derivative is included may contain molar ratios of phosphatidylcholine (PC) cholesterol PG derivatized PE of 8 10 1 1. Another typical composition using a maleimide derivative of PE without PG is PC male-imide-PE cholesterol of 85 15 50 (Friede et al., 1993). In general, to maintain membrane stability, the PE derivative should not exceed a concentration ratio of about l-10mol PE per lOOmol of total lipid. 

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. 

Once the desired mixture of lipid components is dissolved and homogenized in organic solvent, one of several techniques may be used to disperse the liposomes in aqueous solution. These methods may be broadly classified as (1) mechanical dispersion, (2) detergent-assisted solubilization, and (3) solvent-mediated dispersion. 

Solvent-mediated dispersion techniques used to create liposomes first involve dissolving the lipid mixture in an organic solvent to create a homogeneous solution, and then introducing this solution into an aqueous phase. The solvent may or may not be soluble in the aqueous phase to effect this process. There also may be components dissolved in the aqueous phase to be encapsulated in the developing liposomes. 

Probably the most popular option is mechanical dispersion, simply because the greatest number of methods that utilize it have been developed. When using mechanical means to form vesicles, the lipid solution first is dried to remove all traces of organic solvent prior to dispersion in an aqueous media. The dispersion process is the key to 

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Upon the spontaneous rearrangement of anhydrous phospholipids in the presence of water into a hydrated bilayer structure, a portion of the aqueous phase is entrapped within a continuous, closed bilayer structure. By this process water-soluble compounds are passively entrapped in liposomes. The efficiency of encapsulation varies and depends, for example, on the method of preparation of liposomes and the phospholipid concentration during preparation. Different parameters can be used to describe the encapsulation efficiency ... 

A. (1986a). Preparation of liposomes via detergent removal from mixed micelles by dilution. The effect of bilayer composition and process parameters on liposome characteristics, Pharm. Weekbl. 

Olson, F., Hunt, C. A., Szoka, F., Vail, W. J., and Papahadjopou-los, D. (1979). Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes, Biochim. Biophys. Acta. 557, 9-23. 

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. 

Lasch, J., Weissig, V., and Brandi, M., Preparation of liposomes, in Liposomes A Practical Approach, Torchihn 1. and Weissig, V., Eds., Oxford University Press, New York, 2003, 3. 

Cortesi, R., Esposito, E., Gambarin, S., Telloli, P., Menegatti, E., and Nastruzzi, C., Preparation of liposomes by reverse-phase evaporation using alternative organic solvents, Journal of Microencapsulation, 1999, 16, 251-256. 

Principles of Preparation of Liposomes Having Transmembrane Ammonium Ion Gradient... 

Table 4 Steps in Preparation of Liposomes Having a Transmembrane Ammonium Sulfate Gradient...

Schelte P, et al. Differential reactivity of maleimide and bromoacetyl functions with thiols application to the preparation of liposomal diepitope constructs. Bioconjugate Chem 2000 11 118. 

Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages mechanism of action, preparation of liposomes and applications. J Immunol Methods 1994 174 83-93. 

Lasch J, Weissig V, Brandi M. Preparation of liposomes. In Torchilin VP, Weissig V, eds. Liposomes-A Practical Approach. 2nd ed. Oxford Oxford University Press, 2003 3-30. 

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