Fig. 1. Chemical structures of DOTAP and DOPE, two commonly used lipids in liposomal formulations.

include neutral components such as DOPE or cholesterol that can introduce endosomal disruption properties (27). It was originally believed that fusion with cell membranes was the primary mechanism to gain cytoplasmic access. It is now widely accepted that this process occurs through an endocytic or phagocytic event, and access to the cytoplasm is mediated through a destabilization of the endosomal membrane (28).

Alternatively, it has been shown that plasmid DNA can be encapsulated within a lipid vesicle called a dehydrated-rehydrated vesicle (DRV) (29). DRVs are formed by freeze drying lipoplexes to increase the association of plasmid DNA with the flattened lipo-somal vesicles. Subsequent rehydration of these dried complexes results in the apparent entrapment of plasmid inside the lipid bilayer. Although early studies demonstrated that cationic liposomes seem to inhibit expression of plasmid DNA when delivered instrmus-cularly, DRVs have shown the ability to generate improved cellular immunity and secretion of 100x greater IgG1 levels than that obtainable by cationic lipoplexes or naked DNA (29). Various lipids can be included in these formulations to increased immune response (30) and enhance oral delivery of DRVs (31). Finally, the incorporation of viral fusogenic peptides from hemaglutinating virus Japan (HVJ) or influenza into liposomes can enhance responses against tumor-associated antigens (Table 1).

Cationic lipid formulations are relatively easy to prepare, and protect the plasmid DNA from nucleases in the extracellular environment. However, the polycationic nature of the lipoplex/liposomal formulations impart a degree of cellular promiscuity in the transfection of cells, along with an inherent ability to bind to serum proteins. This can severely limit the stability and targetability of plasmid/lipid formulations. Creating targeted liposome vectors with improved serum stability could significantly enhance the potency of liposomal delivery vectors.

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