Study On Uniform PLGA Nanoparticles As Vaccine Delivery And Adjuvant Systems Utilizing Various Antigen Loading Modes

Biodegradable polymer-based nanoparticles (NPs) could function as effective antigen delivery and adjuvant systems. Antigen loading by NPs could be mainly through surface conjugation and encapsulation, with some particular issues currently hampering their development. The surface inherent chemical inactivity of polymeric NPs such as poly (D,L-lactic-co-glycolic acid) (PLGA), raised a big challenge for safe and efficient antigen loading by surface conjugation. And for encapsulating antigens, the NPs could not realize the adjustable antigen intracellular release resulting in weak immune response. Therefore, this dissertation proposed pathogen-mimicking polymeric NPs based on dopamine polymerization and pH-responsive polymeric NPs with rapid antigen release behavior focusing on the primary restrictions in these two areas. In detail, this thesis mainly included the following issues:1) NPs conjugated antigen on the surface as antigen delivery and adjuvant system. Uniform-sized PLGA NPs of ～900 nm were fabricated using a premixed membrane emulsification technique followed by O/W emulsion-diffusion-evaporation method. NP surfaces were coated and functionalized utilizing the mussel-inspired biomimetic polydopamine (pD). The incorporation of hepatitis B surface antigen (HBsAg) and TLR9 agonist unmethylated cytosine-guanine (CpG) motif was achieved with the pD surface. The NPs possessed pathogen-mimicking manners owing to their size, shape, structure and surface molecular immune-activating properties given by CpG. The biocompatibility and biosafety of these pathogen-mimicking NPs were confirmed. Pathogen-mimicking NPs might hold great potential as vaccine delivery and adjuvant system due to their ability to:1) enhance cytokine secretion including inflammatory cytokines and chemokines and immune cell recruitment at the injection site; 2) significantly activate and maturate dendritic cells (DCs); 3) induce stronger humoral and cellular immune responses in vivo.2) With the respect of antigen encapsulated in NPs, pH-responsive PLGA NPs with rapid antigen (OVA) intracellular release behavior in DCs were fabricated. Using the W/O/W emulsion-diffusion-extraction method combined with the premixed membrane emulsification technique, uniform-sized pH-responsive PLGA NPs with rapid antigen release behavior by adjusting the structure to thin shells and large inner space and adding NH4HCO3 in the inner water phase acting as an antigen release promoter were successfully prepared. And antigens loaded in the NPs retained activity from the analysis results of fluorescent and CD spectra. Under different pH values (7.4,6.5, and 5.0) to evaluate antigen intracellular release behavior in the endosome/lysosome and cytoplasm environment of DC, the NPs possessed pH-responsive and rapid antigen release behaviors.3) The adjuvanticity of pH-responsive PLGA NPs with rapid antigen intracellular release behavior were further investigated including antigen presentation, DC activation and maturation, and the subsequent in vivo immune response. After uptake by DCs, antigens released rapidly from the NPs could be crosspresented (230% of that crosspresented by normal NP-antigen). Moreover, the NPs induced up-regulation of costimulatory molecules and stimulated cytokine production of DCs. Mouse immunization with pH-responsive PLGA NPs induced greater lymphocyte activation, more antigen-specific CD8+ T cells, stronger cytotoxic capacity (IFN-γ and granzyme B, increased by about 105% and 79% than normal NPs, respectively), enhanced antigen-specific IgG antibodies compared to alum. The NPs also improved generation of memory T cells to protect against reinfection.4) Based on the aforementioned investigations, the dual-immunopotentiator-loaded pH-responsive PLGA NPs were constructed and their adjuvanticity was further evaluated. By incorporating imiquimod (IMQ) and conjugating CpG through dopamine polymerization onto the surface of pH-responsive PLGA NPs, the efficacy of NPs exhibited great enhancement in activating antigen-presenting cells, enhancing crosspresentation and eliciting PRRs recognition. Dual-immunopotentiator-loaded NPs showed superior adjuvanticity including potent humoral and cellular immune responses. Considering adjuvanticity and safety profiles, the as-constructed NPs were promising robust vaccine adjuvant.In conclusion, these results indicated that PLGA NPs-based antigen delivery and adjuvant systems with superior activity could be achieved by manipulating antigen loading modes and combining molecular immunopotentiators with NPs. These might have significant implications for rational vaccine design, and break new ground for the development of vaccine adjuvants in the future.