Vaccination is the most effective way to prevent infection and avoid the influenza pandemic of H5N1 subtype influenza.H5N 1 split vaccine is of interest for safety profiles, but its immunogenicity is relatively low.Thus, adjuvants are needed to increase the efficacy of split vaccines.Here, aimed to enhance the immunogenicity of H5N 1 split vaccine, we developed a novel antigen delivery system based on quatemized chitosan hydrogel microparticles (Gel MPs) with multiple mechanisms of immunity enhancement.Gel MPs were prepared by premix membrane emulsification combining with self solidification in a simple and mild way.Gel MPs, ionic cross-linking with glycerophosphate (GP), were high water content, pH sensitive and positive charged.By measuring their interactions with BMDCs in vitro and antigen retention ability at local injection site, Gel MPs were proved to be superior as a vaccine delivery system due to their ability to: (1) improve antigen uptake by BMDCs and enhance the endosomal escape of antigen into cytoplasm due to positive surface charges, particulate morphology and pH sensitivity of Gel MPs; (2) dramatically activate BMDCs, especially up-regulation the expression of MHC I and IL-12 production; (3) form an antigen depot and recruit the inflammatory cells at the injection site thus further enhancing antigen capture by APCs.Furthermore, intramuscular vaccination in vivo indicated that Gel MPs/antigen elicited highest antigen-specific IgG in serum along with pronounced T cell mediated immune responses in comparison to aluminium salts (Alum), lipopolysaccharide (LPS) and covalent cross-linking quatemized chitosan MPs (GC MPs).Detailed characterization of both IgG1 and IgG2a antibodies as well as the secretion of IFN-γ and IL-4 by splenocytes revealed that Gel MPs could enhance mixed Thl/Th2 immune responses.In conclusion, these results demonstrate that Gel MPs are efficient antigen delivery vehicles based on multiple mechanisms to enhance both humoral and cellular immune responses with a mixed Thl/Th2 immunity against H5N1 split antigen.Beyond that, considering the multiple immune enhancement mechanisms of Gel MPs, it will be possible to deliver DNA vaccines and most likely, other split and/or subunit vaccines against bacterial and virus infections.Moreover, these findings should offer positive contributions to the rational design and development of efficient and safe antigen delivery systems.