Adjuvant potency of Astragaloside VII embedded cholesterol nanoparticles for H3N2 influenza vaccine

Adjuvants are substances that enhance the immune response to a specific antigen. Among the various candidates for novel vaccine adjuvants, saponins stand out due to their unique immunomodulatory properties. In this study, we aimed to develop PEG (polyethylene glycol)/cholesterol-based lipid nanoparticles (LNPs) for delivering Astragaloside VII (AST-VII) and boosting its adjuvant potential for the influenza vaccine. To form PEG/cholesterol/AST-VII-based LNPs (PEG300: Chol-AST-VII LNPs), we tested three primary solvents (acetone, ethanol, and chloroform) to assess their impact on hydrodynamic particle size, distribution, surface chemistry, and colloidal stability. The resulting nanoparticles were mixed with inactivated H3N2 influenza antigen and tested on PMA (phorbol 12-myristate 13-acetate)-ionomycin treated human whole blood to evaluate cytokine release. Using chloroform as the desolvation agent, PEG300: Chol-AST-VII LNPs (80.2 ± 7.7 nm) were successfully produced. Co-treatment with PMA-ionomycin, AST-VII, and PEG300: Chol-AST-VII LNPs significantly increased the levels of IL-2 and IFN-γ compared to PMA-ionomycin treatment alone. In the presence of H3N2, AST-VII enhanced IL-17A production, while PEG300: Chol-AST-VII LNPs promoted IFN-γ production. Hemolysis was observed only at the highest concentration (250 μg/mL) of PEG300: Chol-AST-VII LNPs. Overall, AST-VII and AST-VII-loaded LNPs demonstrated potential as effective adjuvants for inactivated H3N2 vaccines in vitro, stimulating cytokine responses via the Th1/Th17 pathway.