Dual role of ascorbyl Palmitate as coagel and adjuvant in experimental antivenom development

An ideal vaccine adjuvant should be biocompatible, enable antigen storage and controlled release, and effectively stimulate the immune system. In this study, we evaluated the dual functionality of ascorbyl palmitate (ASC16): as an adjuvant additive in its dispersed state (120 µM), and as a coagel formed at low temperatures and high ASC16 concentrations (Coa-ASC16_PEG), for use in the antivenom development. Coa-ASC16_PEG was prepared by mixing ASC16 with polyethylene glycol 400 (PEG400) and solubilising the mixture at 64 °C for 2 min; subsequently, the formulation was allowed to cool to 40 °C and whole Crotalus durissus terrificus venom, which naturally contains a complex mixture of proteins, was incorporated for 10 s, including major toxins such as crotoxin, serine proteinases, L-amino acid oxidase, and phosphodiesterases. To produce experimental antivenom, mice were immunized with Coa-ASC16_PEG or Freund's adjuvant, with or without the addition of dispersed ASC16. The physicochemical analyses showed that the Coa-ASC16_PEG exhibited a semi-crystalline, viscoelastic structure capable of effectively encapsulating and releasing proteins. The addition of dispersed ASC16 enhanced the immune response induced by both adjuvants (Freund's and Coa-ASC16_PEG), yielding up to a 1.2-fold increase in venom-specific IgG titres and an approximately 2.7-fold increase in IgG antibody avidity. ASC16-based formulations were associated with reduced local adverse reactions compared with Freund's adjuvant-based formulations. In conclusion, we propose an alternative ASC16-based formulation obtained at low temperatures that preserves key physicochemical properties and shows minimal impact at the inoculation site. Moreover, the incorporation of dispersed ASC16 as an additive enhances the humoral immune response. Taken together, our results obtained in a murine model position ASC16 as a promising adjuvant, demonstrating reduced local reactogenicity compared with Freund's adjuvant.