Optimization, characterization, and immunological evaluation of cubosomes loaded with Arnebia euchroma (Royle) Johnst.-derived polysaccharide as an adjuvant.

Arnebia euchroma (Royle) Johnst.-derived polysaccharide (ARP) exhibits immunomodulatory and immunoenhancing activities while lipid-based cubosomes, known for their high drug-loading capacity and stability, have demonstrated great potential as vaccine adjuvant delivery systems. Herein, systematic process optimization yielded ARP-loaded cubosomes (ARPC) with ARP-to-phytantriol and stabilizer F127-to-phytantriol mass ratios of 2.8:10 and 2:10, respectively, in an aqueous-phase volume of 19 mL. ARPC exhibited favorable characteristics, including an encapsulation efficiency of 70.35% ± 1.15%, a particle size of 383.6 ± 3.6 nm, a polydispersity index (PDI) of 0.183 ± 0.016, a zeta potential of -24.4 ± 0.4 mV, and good storage stability at 4 °C. ARPC significantly promoted lymphocyte proliferation, activated macrophages, and enhanced the secretion of nitric oxide and proinflammatory cytokine (TNF-α, IL-1β, and IL-6) in a dose-dependent manner in vitro. In addition, the in vivo immunoadjuvant activity of ARPC was evaluated through subcutaneous injection in mice. Using ovalbumin as a model antigen, ARPC demonstrated good biosafety, eliciting robust and balanced humoral and cellular immune responses through the secretion of high levels of ovalbumin-specific antibodies and key cytokines (TNF-α, IFN-γ, IL-4, and IL-17). Further, proportions of CD4+ and CD8+ T cells increased upon restimulation. Results confirmed that cubosomal encapsulation enhanced the stability and immunomodulatory efficacy of ARP, positioning ARPC as a potential vaccine delivery system and immunoadjuvant candidate.