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

Abstract

Adjuvants are substances that increase the immune response to a given antigen. In the development of novel vaccine adjuvants/systems, saponins are one of the most attractive molecules due to their altered immunomodulatory activities. In this study, we tried to develop PEG (polyethylene glycol)/cholesterol-based lipid nanoparticles (LNPs) to deliver the Astragaloside VII (AST-VII) and potentiate adjuvant properties of AST-VII for the influenza vaccine. In the formation of PEG/cholesterol/AST-VII-based LNPs (PEG300: Chol-AST-VII LNPs), 3 different primary solvents (acetone, ethanol, and chloroform) were evaluated, employing their effects on hydrodynamic particle size, distribution, surface chemistry, and colloidal stability. Prepared nanoparticles were simply admixtured with inactivated influenza antigen (H3N2) and applied to PMA (phorbol 12-myristate 13-acetate)-ionomycin treated human whole blood to evaluate their cytokine release profile. PEG300: Chol-AST-VII LNPs (80.2 ± 7.7 nm) were obtained using chloroform as a desolvation agent. Co-treatment of PMA-ionomycin with AST-VII and PEG300: Chol-AST-VII LNPs significantly increased the levels of IL-2 and IFN-g, compared to PMA-ionomycin alone. In the presence of H3N2, AST-VII was able to augment IL-17A, while PEG300: Chol-AST-VII LNPs stimulated the production of IFN-g. Hemolysis was only observed in PEG300: Chol-AST-VII LNPs (250 μg/mL) treatment. AST-VII and AST-VII-integrated LNPs could be used as efficacious adjuvants for an inactivated H3N2 vaccine in vitro, and cytokine response through Th1/Th17 route was reported.

Keywords: Astragaloside VII; Immunomodulatory activity; cholesterol; influenza; nanocarriers; triterpenoid saponins.

Figure 1

Schematic illustration of the synthesis route applied for the formation of PEG300: Chol-AST-VII LNPs through the nanoprecipitation method in the presence of solvent/water interphase. TEM image of an as-synthesized nanovesicle and magnified image of the nanovesicle membrane.

Figure 2

DLS results of PEG300: Chol-AST-VII LNPs. Hydrodynamic particle size distribution by intensity graph of PEG300: Chol-AST-VII LNPs made using (a) acetone; (b) ethanol; and (c) chloroform as a primary solvent for the nanoprecipitation method. Dependence of (d) ζ-Potential and (e) hydrodynamic size of nanocarriers on the pH and primary solvent type.

Figure 3

TEM and FTIR analysis of LNPs. (a) images of PEG300: Chol-AST-VII nanocarriers prepared using different primary solvents before and after storage at room temperature over 15 min. No precipitation was observed over time; (b) transmission electron microscope image of PEG300: Chol-AST-VII LNPs made up using chloroform as a primary solvent; (c) FT-IR spectra of AST-VII integrated nanocarriers prepared using chloroform (red line), acetone (blue line), and ethanol (green line), spectra corresponding to (d) PEG300: Chol-AST-VII LNPs prepared in chloroform/water (i) and each component of LNPs: cholesterol (ii), Polyethylene glycol (iii), and AST-VII (iv). The characteristic peaks of each component are illustrated by asterisks (*); the arrow points to the interassociated hydrogen bonds formed by carrier formation.

Figure 4

Chemical representation of each component of LNPs. a) AST-VII, cholesterol and polyethylene glycol; b) schematic representation of LNP formation in chloroform/water interphase.

Figure 5

Cytokine profile of adjuvants and adjuvanted H3N2. Cytokine levels of PMA-ionomycin stimulated hWB treated with ASTVII, QS-21, and PEG300: Chol-AST-VII LNPs mixed w/without inactivate H3N2. The cell culture supernatants were analyzed for a) IL-2; b) IFN-γ; and c) IL-17A by ELISA. The significant difference is defined between treated groups and PMA-ionomycin: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Schematic representation of the possible mechanism of action for PEG300: Chol-AST-VII LNPs. Inactivated H3N2 vaccine combined with AST-VII-carrying LNPs, and this combination was taken by APC (i). After internalization of the adjuvant-antigen combination, APC (immature state) was processed and presented the viral antigens on the MHC molecules (mature state-activated) (ii) to naïve CD8⁺ and CD4⁺ T-cells (iii). Activated T-cells secreted Th1 type cytokines (i.e. IL-2, IFN-g).