Preparation and immunopotentiating activity of a novel oil-in-water nanoemulsion adjuvant based on trimethyl chitosan

BACKGROUND: Subunit vaccines offer excellent safety profiles but often suffer from limited immunogenicity, requiring potent adjuvants to elicit robust protection. We hypothesized that combining a positively charged biopolymer, trimethyl chitosan (TMC), with a nanoemulsion (NE) would create a synergistic delivery system to enhance immune responses. This study aimed to formulate a TMC-loaded nanoemulsion (TMC-NE) and evaluate its potential as a safe, effective adjuvant for subunit vaccines. METHODS: TMC was synthesized via the quaternization of chitosan. Its modification and cyto-compatibility were verified by FT-IR, Zeta potential analysis, and cytotoxicity assays. Based on the pseudo-ternary phase diagram, a stable oil-in-water blank nanoemulsion (NE) was prepared. Subsequently, TMC-loaded nanoemulsion (TMC-NE) was prepared with TMC aqueous solution as the aqueous phase. The formulation was characterized for its droplet size, polydispersity index (PDI), and viscosity, and its safety in vivo was evaluated in mice. Using ovalbumin (OVA) as the model antigen, the immune adjuvant activity of TMC-NE was evaluated in mice by intramuscular injection, and compared with the NE and a commercial similar adjuvant M902. Humoral and cellular immune responses were analyzed by measuring serum IgG antibody levels and splenic lymphocyte proliferation, as well as the expression of cytokines IL-4 and IFN-γ. RESULTS: Chitosan was successfully quaternized, yielding TMC with low cytotoxicity. The formulated TMC-NE was clear and transparent, with a mean droplet size of 25.41 ± 2.13 nm, a PDI of 0.231, and suitable viscosity. In vivo, TMC-NE showed no significant tissue toxicity. Immunization studies revealed that TMC-NE stimulated IgG antibody levels comparable to the M902 adjuvant and significantly higher than the blank NE. Furthermore, TMC-NE significantly increased the expression of IL-4 and IFN-γ and promoted lymphocyte proliferation and T lymphocyte levels, indicating the induction of both humoral and cellular immune responses. CONCLUSIONS: The TMC-loaded nanoemulsion (TMC-NE) is characterized as a safe and effective antigen delivery system with potent immune adjuvant properties. These findings strongly support its prospective application as a novel vaccine adjuvant.