Effects Of Particle Introduction On Emulsion Adjuvanticity

Developing efficient and safe vaccines is an effective means of preventing outbreaks of infectious diseases.Due to the weak immunogenicity of the new antigens,the development of adjuvants has become one of the focuses of vaccine research and development.Compound adjuvants are promising adjuvants because they can simultaneously improve immune strength and change the type of immunity.Emulsion adjuvants for clinical applications have received widespread attention because they are able to induce considerable humoral immunity,but they cannot deliver antigens intracellularly or induce cellular immunity.Meanwhile,particle adjuvants can be effectively endocytosed by cells and induce favorable cellular immunity,but they don't have the ability to simulate the deformability of natural pathogens.Therefore,this dissertation proposes to introduce particles into emulsions to form compound adjuvants.After reasonable design,they can realize the study of cell uptake and intracellular behavior,so as to achieve synergistic immune effects.After that,the influence of different distributions of particles in emulsion on the mechanism of action also can be investigated.Detailed work done in this dissertation are listed below:1.Multiple preparation techniques of chitosan particles were developed,providing diversified chitosan particles for investigating particle-emulsion compound adjuvants.We prepared micron and submicron particles employing the novel premix membrane emulsification technology and chitosan thermosensitive hydrogel.And nanoparticles were prepared by self-solidification,ionic-crosslinking,or nanoprecipitated method utilizing ionic interaction between molecules.Size,homogeneity,surface charge and hydrophilicity of chitosan particles were characterized.These particle candidates were then introduced into emulsion to construct stable particle-emulsion compound adjuvants.2.Constructing a novel particles-in-oil-in-water emulsion(CSSE),which was capable of simulating the dynamic endocytosis process of pathogens to achieve high uptake efficiency and eventually enhanced the immune response.Particles and antigens were embedded in the interior of emulsion.With the assistance of particles,CSSE exhibited amplified deformability and the vaccine-cell contact zone was increased.Additionally,its configurational transitions,which offered sustained exposure of sheltered uptake signals including antigens and stimulator chitosan during endocytosis,resulted in efficient antigen delivery to APCs and immunocyte activation.As a result,CSSE formulation robustly induced both humoral and cellular immunity against foot-and-mouth disease virus(FMDV),with an adjuvant-sparing effect,compared to the commercial adjuvant ISA206.These advantages prove CSSE to be a promising veterinary vaccine adjuvant with superior safety and effectiveness.3.Constructing special Pickering emulsion(CSPE)with the characteristic of changing from particles-stabilized emulsion to polymer-stabilized emulsion,which facilitated efficient and controllable lysosomal escape,thus inducing excellent cellular immunity.CSPE underwent mechanical deformation on the cell membrane,mimicking the deformability of natural pathogens,which was favorable for efficient uptake of vaccines and cells activation.Chitosan nanoparticles could convert to molecular chains and amino groups on them were protonated.CSPE imposed the spatial restriction effect of interface on the particles,which facilitated its conversion to polymer-stabilized emulsion and achieves proton accumulation effect simultaneously.By adjusting the number of protons,the regulation of lysosomal escape efficiency was realized.Eventually,by utilizing the efficient lysosomal escape and polymer-stabilized emulsion,the antigen cross-presentation was conspicuously ameliorated.In comparison with commercial adjuvants,the Pickering emulsion significantly promoted cellular immunity,especially the CTL immune response,with Thl-bias.As a result,Pickering emulsion showed excellent performance in EG7-OVA lymphoma and B16-MUC1 melanoma,resulting in a remarkable reduction in tumor growth and extension in survival time.4.Studying the effects of particle distributions in emulsion(outside/surface/inside)on the mechanism of action,and adjuvanticity.Three compound adjuvant formulations composed of chitosan particles and squalene emulsion were successfully constructed.The particles were distributed inside the emulsion droplets,on the surface,and over the water phase,respectively.It was concluded that distributing particles into the emulsion or on its surface induced significant antigen-depot effect,strong cells recruitment,and simultaneous delivery to lymph node.Robust Th2 or Thl cytokines were secreted at the injection site induced by emulsions with particles distributed inside or on the surface,respectively.The results showed that particles in the emulsion droplets induced Th2 immune bias,and particles on the surface of emulsion resulted in Th1 bias.Meanwhile,emulsions with particles distributed outside induced weaker humoral and cellular immunity than the above two formulations.