Purpose: Mucosal vaccination can stimulate both mucosal and systemic immunity, but free protein antigens generally induce poor systemic immune responses and require adjuvants. There is a need for a safe, effective nasal vaccine carrier that improves antigen stability, uptake, and immunogenicity, especially for poorly immunogenic protein antigens.
Hypothesis: If bovine serum albumin (BSA) is encapsulated in hybrid poly(β-amino ester)–poly(D,L-lactide-co-glycolide) (PbAE–PLGA) nanoparticles, then nasal vaccination with these cationic, mucoadhesive particles will induce stronger humoral, cellular, and mucosal immune responses than free antigen, without additional adjuvant.
Aims: Synthesize and characterize PbAE–PLGA hybrid nanoparticles loaded with BSA. - Optimize particle size, zeta potential, encapsulation efficiency, and physical stability. - Evaluate antigen integrity and in vitro cytotoxicity in respiratory epithelial cell lines. - Assess in vivo immunoadjuvanticity after intranasal (i.n.) and subcutaneous (s.c.) administration in mice, including antibody titers, mucosal IgA, and cytokine profiles.
Delivery system:
Component: Polymer; Details: Poly(β-amino ester) (PbAE) synthesized by Michael addition of bisphenol-A-ethoxylate diacrylate and ethylenediamine; blended with PLGA
Component: PLGA types; Details: RG502 (7–17 kDa) or RG503 (24–38 kDa)
Component: Polymer ratio; Details: PbAE/PLGA = 1:4 (m/m)
Component: Nanoparticle type; Details: Hybrid polymeric nanoparticles
Component: Payload; Details: Bovine serum albumin (BSA), model protein antigen (~66 kDa)
Component: Preparation; Details: Water-in-oil-in-water (W/O/W) double emulsion solvent evaporation
Component: Size / charge; Details: ~235 nm (RG503) or ~244 nm (RG502); zeta potential ~ +13.8 to +20.1 mV
Component: Encapsulation efficiency; Details: ~91% (RG503); ~84% (RG502)
Component: Targeting ligand; Details: None
Component: Key feature; Details: Cationic surface for mucoadhesion; sustained antigen release; no additional adjuvant required
Approach: In vitro: Calu-3 and A549 respiratory epithelial cell lines for MTT cytotoxicity; SDS-PAGE for BSA integrity. - In vivo: Female BALB/c mice (6–8 weeks), 7 groups of 5 mice. - Administration: Intranasal (20 µL) or subcutaneous (100 µL), equivalent to 20 µg BSA; boosted on day 14. - Groups: i.n. PbAE–PLGA–BSA, free BSA, CpG ODN–BSA; s.c. PbAE–PLGA–BSA, free BSA, alum–BSA; untreated control. - Disease context: Vaccine delivery, not a disease challenge model. - No pathogen challenge or protection study.
Key methods: Physicochemical characterization: Photon correlation spectroscopy (size, PDI), zeta potential, SEM. - Encapsulation efficiency: Bicinchoninic acid assay. - Antigen integrity: SDS-PAGE. - Cytotoxicity: MTT assay in Calu-3 and A549 cells. - Antibody responses: ELISA for serum IgG, IgG1, IgG2a; vaginal wash secretory IgA (sIgA). - Cytokine responses: ELISA for IL-2, IL-4, IL-6, IL-10, IFN-γ after splenocyte re-stimulation with BSA. - Statistics: One-way ANOVA with Newman–Keuls multiple comparison test; p < 0.05.
Key results: Particle properties: RG503 formulation: 234.77 ± 4.49 nm, PDI 0.15, zeta +13.8 ± 4.09 mV, EE 91.16 ± 2.92%. RG502 formulation: EE 84.60 ± 0.67%, zeta +20.1 ± 4.8 mV. Stable for 24 weeks at +4 °C. - Cytotoxicity: Dose-dependent; hybrid nanoparticles less toxic than PbAE polymer alone in both Calu-3 and A549 cells. BSA integrity retained by SDS-PAGE. - Intranasal immunization: PbAE–PLGA–BSA induced higher IgG titers than free BSA (p < 0.05) and slightly higher than CpG ODN–BSA (not significant). At day 21, IgG titers were 4.5-fold higher than CpG ODN–BSA and 102-fold higher than free BSA. At day 253, 60-fold higher than free BSA. - Subcutaneous immunization: At day 13, 75-fold higher serum IgG than free BSA; at day 253, 10-fold higher IgG than alum–BSA. - Mucosal immunity: BSA-specific sIgA in vaginal washes was increased after i.n. PbAE–PLGA–BSA. - Cytokines: High IL-2 and IFN-γ (Th1), plus elevated IL-4, IL-6, IL-10 (Th2), indicating balanced Th1/Th2 activation. IgG1/IgG2a ratios for i.n. nanoparticles were 2.55 and 2.74 at days 13 and 21.
Interpretation: The authors claim that hybrid PbAE–PLGA nanoparticles are promising nasal vaccine carriers for poorly immunogenic protein antigens. They induce strong systemic humoral and cellular immune responses, stimulate mucosal immunity, and provide long-lasting antibody production without additional adjuvant. This platform may improve nasal vaccination against protein/peptide antigens.
Limitations: Model antigen only: BSA was used; no clinically relevant pathogen antigen or challenge model. - No protection/efficacy data: No pathogen challenge, survival, or functional protection. - No in vivo toxicity: Only in vitro cytotoxicity in two cell lines. - Mechanism not fully resolved: Immune enhancement attributed to mucoadhesion and sustained release, but direct uptake/trafficking not deeply studied. - Single animal species: Mice only; no large-animal or human data. - No targeting ligand: Delivery relies on passive/mucosal interactions. - Stability: Zeta potential decreased over 24 weeks, likely due to PbAE degradation. - No direct comparison with all clinical adjuvants or with viral vectors.
