Development of pH responsive novel emulsion adjuvant for oral immunization and in vivo evaluation

• pH responsive multiple emulsion was developed for effective oral vaccine delivery.
• Quil A was added to the internal aqueous phase and squalene was used as oil phase.
• Addition of Quil A has resulted in increased stability and strong immune response.
• Pectin in the external aqueous phase provided additional protection from acidic pH.
• Developed formulation induced balanced Th1 and Th2 immune response.

The present work has been envisaged to develop a pH responsive multiple emulsion (W/O/W) adjuvant for successful antigen delivery via oral route. The formulation was prepared by double emulsion technique using squalene as oil phase and a combination of antigens and Quil A as internal aqueous phase. Squalene emulsion (O/W) adjuvants are well accepted for parenteral administration. Instead of using conventional surfactants, Quil A was incorporated in the internal aqueous compartment for the first time. The microscopic analysis confirmed the formation of the W/O/W multiple emulsions with globule diameter ranging from 7 to 12 μm. Considerably, addition of Quil A resulted in marked improvement in both in vitro stability and immune response. Furthermore, addition of pectin to the external aqueous phase resulted in improved in vitro stability. A formulation with gel like structure was formed when pectin was used as stabilizer and also accounted with a significant increase in viscosity (from 275.3 cP to 330.4 cP) in the simulated gastric fluid, which reverts back to 290.2 cP after exposure to simulated intestinal fluid. Induction of strong mucosal and systemic immune response suggested that the developed formulation could be useful for the delivery of multiple biomolecules via oral route.

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