Immunoenhancement effects of chitosan-modified ginseng stem-leaf saponins-encapsulated cubosomes as an adjuvant

Purpose: Conventional adjuvants such as aluminum and Freund’s adjuvants have limitations, including poor cell-mediated immunity and local toxicity. Ginseng stem-leaf saponins (GSLS) have immunomodulatory/adjuvant activity, but improved delivery systems are needed. Cubosomes are stable lipid cubic liquid crystalline nanoparticles with high membrane surface area, and chitosan modification can add positive charge and improve antigen delivery.

Hypothesis: If GSLS is encapsulated in chitosan-modified cubosomes and co-delivered with a model antigen such as ovalbumin (OVA), then the formulation will enhance macrophage antigen uptake, cytokine/NO/iNOS responses, and induce stronger humoral and cellular immune responses with low toxicity.

Aims: Prepare and characterize chitosan-modified GSLS-encapsulated cubosomes (Cub-GSLS^CS). - Evaluate storage stability and sustained release of GSLS and OVA. - Assess macrophage cytotoxicity, antigen uptake, cytokine secretion, NO production, and iNOS activity in vitro. - Evaluate adjuvant activity in OVA-immunized mice by measuring T cell subsets, antigen-specific antibody responses, and histopathology.

Delivery system: Platform: Cubosomes — lipid cubic liquid crystalline nanoparticles with an Im3m nanostructure. - Payload: Ginseng stem-leaf saponins (GSLS); OVA as model antigen; OVA-FITC for uptake studies. - Surface modification: Chitosan (CS) to confer positive charge and modify the nanoparticle surface. - Formulations: Cub, Cub-GSLS, Cub-GSLS^CS; OVA-loaded versions: Cub-OVA, Cub-GSLS-OVA, Cub-GSLS^CS-OVA. - Physicochemical properties: Average diameter ~190–205 nm; PDI < 0.3; zeta potential: Cub and Cub-GSLS ~ −22 to −21 mV; Cub-GSLS^CS ~ +29.9 mV. - Encapsulation efficiency: GSLS ~65% in Cub-GSLS and ~60% in Cub-GSLS^CS; OVA encapsulation efficiency ~63% for CS-modified cubosomes, significantly higher than unmodified.

Approach: In vitro: Peritoneal macrophages from ICR mice; cytotoxicity by MTT; cellular uptake by confocal microscopy and flow cytometry; cytokine levels by ELISA; NO by Griess reagent; iNOS activity by assay kit. - In vivo: Six-week-old ICR mice; subcutaneous immunization twice at 2-week intervals (days 0 and 14); groups: PBS control, free OVA, CFA-OVA, GSLS-OVA, Cub-OVA, Cub-GSLS-OVA, and Cub-GSLS^CS-OVA; OVA concentration 250 µg/mL; mice sacrificed on days 21, 28, 35, and 42. - No infectious or tumor challenge model was used.

Key methods: Dynamic light scattering for size, PDI, and zeta potential. - TEM for morphology. - SAXS for liquid crystalline phase identification. - Encapsulation efficiency and in vitro release (dialysis). - MTT cytotoxicity assay. - CLSM and flow cytometry for antigen uptake. - ELISA for IL-6, IL-12, TNF-α, and OVA-specific IgG/IgG1/IgG2a. - Griess assay for NO; iNOS activity assay. - Flow cytometry for CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. - H&E histopathology of major organs and spleen.

Key results: Nanoparticle characterization: Cub-GSLS^CS showed Im3m nanostructure, ~205 nm size, positive zeta potential (+29.9 mV), GSLS encapsulation efficiency ~60–65%, and OVA encapsulation efficiency ~63%. Nanoparticles were stable for 28 days at 4°C and showed sustained release. - In vitro macrophage effects: Cub-GSLS^CS significantly enhanced OVA-FITC uptake and increased secretion of IL-6, IL-12, and TNF-α compared with other groups. It also increased NO production and iNOS activity. - In vivo immune response: Cub-GSLS^CS-OVA increased the CD4⁺/CD8⁺ T cell ratio and induced high levels of OVA-specific IgG, IgG1, and IgG2a, generally comparable to CFA-OVA and higher than free OVA or unmodified cubosomes. IgG1 and IgG2a patterns suggested a mixed Th1/Th2 response. - Toxicity: No significant macrophage cytotoxicity at safe concentrations; histopathology of heart, liver, spleen, lung, and kidney showed normal tissue structure. Spleen showed enlarged lymphatic nodules and germinal centers, indicating immune activation.

Interpretation: The authors conclude that Cub-GSLS^CS is a promising vaccine delivery and adjuvant system. It enhances macrophage phagocytic function, promotes macrophage activation and cytokine production, and induces strong antigen-specific humoral and cellular immune responses with low toxicity. The system may provide a foundation for clinical application of cubosome-based adjuvants.

Limitations: Only OVA was used as a model antigen; no infectious disease or tumor challenge was tested. - Only ICR mice were used; no human or large-animal validation. - Follow-up was relatively short (up to 42 days), with no survival or long-term memory analysis. - Detailed biodistribution, clearance, and mechanistic receptor studies were not reported. - Some units/concentrations in the provided text appear inconsistent, and full citation details were not included in the supplied file content.