The feasibility of creating a s.c. depot for sustained protein delivery with the goal of enhancing antigen immunogenicity was investigated. The depot was designed as antigen-laden liposomes of hydrogenated egg phosphatidylcholine and cholesterol (1:1 molar ratio) encapsulated in alginatepoly(L-lysine) microcapsules and evaluated using iodinated bovine serum albumin (BSA) as a model antigen. The in vivo release behavior of the liposomes and microencapsulated liposomes (MELs) was evaluated from the BSA serum concentration proffles after s.c. injection into rats and the pharmacokinetic parameters of '5I-labeled BSA appearance after s.c. or i.v.injections of BSA in saline. Maximal BSA concentrations were detected 11 h after s.c. injection in all rats. The BSA serum concentrations decreased rapidly in rats injected with BSA in saline or Freund's adjuvant and less rapidly in rats injected with BSA in liposomes or MELs. Four to 5 weeks after injection, BSA-associated radioactivity was detected only in sera of rats injected with BSA in liposomes or MELs. Fifty days after injection, 50% of the originally injected BSA was recovered from the s.c. sites of rats injected with BSA in MELs; no radioactivity was recovered from the other three groups of rats. The antigen-reactive antibody levels induced in rats immunized with BSA in MELs were 2-to 3-fold higher than those obtained in rats immunized with BSA in liposomes, saline, or Freund's adjuvant. More significantly, high antibody levels were maintained for more than 150 days after a single injection of BSA in MELs, suggesting that MELs can serve as a long-term singledose immunization vehicle.Adjuvants are often used to increase antigen-induced immune responses. Two commonly used adjuvants are Freund's adjuvant (FA) and alum. However, chronic inflammation at the injection site makes FA unacceptable for human use (1). Although these reactions are less severe when alum is used, inflammation is still observed at injection sites, and not all proteins adsorb well to alum (2).Liposomes have been widely investigated as antigen carriers, due to their greater biocompatibility and their potential as a generic system for antigen encapsulation. They have been used to induce humoral immunity for a variety of antigens (3,4). Although the antibody titers were usually higher than that observed when antigen was injected in saline, they were lower than the levels induced when animals were immunized with FA (5, 6). Moreover, at least two injections of antigen in liposomes were generally needed to elicit an immune response (4, 7). This might be due to liposome instability in the host. Macrophages and high density lipoproteins destroy liposomes at injection sites (8-10). Although the rate of liposome clearance in vivo can be regulated by many factors such as the type of phospholipids, the preparation procedure, and vesicle size (11), most liposomal preparations sustain peptide delivery for, at best, up to 2 weeks when injected s.c. or i.m. (12, 13).One approach to extend antigen delivery ...

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