Development and in vitro cellular evaluation of optimised multiple emulsion and liposomes-in-emulsion adjuvant...

The global need for effective vaccines has gained more significance after the COVID-19 pandemic. Subunit vaccines based on emulsions and liposomes offer several advantages, including biocompatibility, biodegradability, and compositional flexibility. This study aimed to develop and optimize a dual delivery system incorporating ovalbumin (OVA) liposomes in a multiple W/O/W emulsion system for vaccine delivery. A 4 × 3 × 3 factorial design was employed to optimize the emulsion, investigating the effects of phase ratios and surfactant concentrations (Span 80 and Tween 80) on emulsion key properties, including droplet size, viscosity, zeta potential, pH, and stability. The optimised emulsion was selected based on droplet size, viscosity, and stability. Optimised HTAB cationic liposomes were also prepared, and subsequently, three adjuvant systems were developed: liposomes (FL-O), multiple emulsion (FE-O), and liposomes-in-multiple emulsion (FLE-O). In vitro release studies showed that liposomal formulations (FL-O and FLE-O) provided sustained antigen release compared to the emulsion-only system (FE-O). Incorporation of liposomes also enhanced the stability of the multiple emulsion system. Cellular studies revealed that all systems supported intracellular antigen processing, with emulsion-based formulations showing the highest cellular uptake and association, though some cytotoxicity was observed. Overall, this study demonstrates the potential of combining liposomes with multiple emulsions to develop stable, customizable vaccine adjuvant systems with adjustable release and cellular interaction profiles, based on formulation design.