Development of Squalene-Based Oil-in-Water Emulsion Adjuvants Using a Self-Emulsifying Drug Delivery System for Enhanced Antigen-Specific Antibody Titers

A recombinant protein cannot induce sufficient immune response by itself. Various substances, including cytokine and mineral, have been used as adjuvants to enhance the immunogenicity and efficacy of vaccines; however, most of them induce excessive immune responses or exhibit cytotoxicity. In this study, a self-emulsifying drug delivery system (SEDDS), an isotropic mixture of oil, surfactant, and solvent, was designed for oil-in-water emulsions as a non-toxic adjuvant to increase immune response to antigens.Squalene-based oil-in-water emulsions were prepared by SEDDS to assess its value as an adjuvant. Fifteen emulsions (F1-F15) were prepared by stirring two types of surfactants (Span® 85 and Kolliphor® RH40), and squalene and carboxymethyl cellulose (CMC) were added at different ratios. The physical properties and viscosity of the 15 emulsions were evaluated by measuring droplet size, zeta potential, and polydispersity index. The toxic effect of emulsions was assessed by acute toxicity test in mice. Mice were immunized twice with 1:1 mixtures of antigen and adjuvant (15 emulsions, phosphate-buffered saline, and commercial alum-based adjuvant). Antigen-specific antibody titers from immunized mice serum were measured by an indirect enzyme-linked immunosorbent assay.All emulsions exhibited droplet sizes ranging from 322 to 812 nm and maintained zeta potential values between -30 mV to -10 mV for 4 weeks, indicating good physical stability as a vaccine adjuvant. Additionally, all emulsions were non-toxic, and they induced humoral immunity at a similar level compared to commercial alum-based adjuvant in the first immunization. However, 12% squalene-based oil-in-water emulsion containing 0.5% of ultra-high viscosity CMC (F15) showed significantly higher immune response than a commercial adjuvant in the second immunization.Squalene-based oil-in-water emulsions could be conveniently prepared using SEDDS technique and are non-toxic and stable at room temperature storage. Moreover, squalene-based oil-in-water emulsions show enhanced immune induction with antigen; hence, they can possibly be used as effective adjuvants.