Biol. Pharm. Bull. 29(6) 1223—1228 (2006)

infectious disease. Most of the vaccines available today are injected parenterally, and they are not effective for inducing immunity at mucosal surfaces. In contrast, oral vaccines elicit both mucosal and systemic immune responses and have the advantages of self-administration. However, the oral administration of vaccines has the problem of antigen degradation by gastric acid and proteolytic enzymes in the digestive system, and extremely large doses are required to achieve an adequate immune response. Therefore, the use of particles such as liposomes as a carrier to prevent the degradation of antigens has been investigated. Liposomes are attractive as an antigen delivery carrier since their particle size, surface charge and membrane fluidity can be easily controlled. Besides, the natural tendency of liposomes to interact with macrophages has served as the primary rationale for utilizing liposomes as a carrier of antigens. However, usual liposomes can be rapidly degraded by bile salts and other components of the gastrointestinal intraluminal environment. Therefore, stabilized liposomes such as polysaccharidecoated liposomes and polymerized liposomes have been investigated. In previous studies, double liposomes (DL), in which several small liposomes (SL) were encapsulated in a large liposome, were prepared by a glass-filter method and a glassbeads (GB) method. Salmon calcitoninand insulinloaded DL exerted good hypocalcemic and hypoglycemic effects via oral administration, respectively. We suggest that the use of DL as a vaccine career will be advantageous for improving the protection of the antigen. That is, it is hypothesized regarding the concept of vaccine delivery using DL that the outer liposomes are degraded by lipase, bile salts etc. after protecting the inner liposomes until they reach the lower small intestine, and then SL are taken up by the M cells in the intestinal lumen. The strength of the bilayer can be adjusted by changing the lipid compositions of SL and the outer lipid. In the present study, we prepared SL containing ovalbumin (OVA) as a model antigen by the GB method, and then obtained DL by encapsulation of SL in a large liposome by the GB method or the reverse-phase evaporation (REV) method. SL were sized to a desired particle diameter to be encapsulated in a large liposome by sequential sonication and extrusion. The morphological structure of DL was examined using confocal laser scanning microscopy and scanning electron microscopy by the freeze-fracture method. In vitro characteristics of SL and DL were determined. Furthermore, systemic and mucosal immune responses following oral administration of the liposomes to mice were examined.