One-pot Synthesis Of Antigen-Conjugated Gold Nanoparticles And The Application As Vaccine Adjuvants

Adjuvants play the key roles in the field of research and development of vaccines.In recent years,various nanomaterials,such as gold nanoparticles(GNPs),have shown numerous promising biomedical applications.Some studies have shown that GNPs can enhance humoral or even cellular immune responses,which considered to be novel adjuvant candidates.It has already been revealed that the adjuvanticity of GNPs is associated with their particle sizes,shapes,and surface modifications.However,the effects of binding manner between antigens and GNPs on immune response have not been unveiled.In this thesis,the model antigen ovalbumin(OVA)modified GNPs(OVA@GNPs)from one-pot synthesis was compared with the physical mixture of OVA and GNPs(OVA+GNPs)to explore the effect of antigenGNPs binding mode on animal immune response.Our goal is to develop a simple and easy way to develop vaccine adjuvants with low toxicity and high stability GNPs which can cause efficient immune responses.The physical mixture(OVA+GNPs)and covalent complex(OVA@GNPs)of OVA and GNPs were prepared,respectively.The concentration of gold ions for binding most of OVA in solution was optimized during the formation of OVA@GNPs.The physical and chemical properties and structure information were characterized using SDS-PAGE,UV-vis absorption spectrum,transmission electron microscope,atomic force microscope,infrared spectrum and circular dichroism spectrum.The differences between two antigen-GNPs complexes in their stability,cytotoxicity and cellular uptake in vitro were investigated.Titer of specific IgG antibody in the serum of subcutaneously immunized mouse was determined through ELISA.The main organs of immunized mice were sliced and stained with hematoxylin-eosin.Furthermore,the skin sections of cutaneous immunized animals were stained,and specific antibodies in serum were also determined.After optimization,the concentration of gold ions was 1.2 mM when there was 1 mg/mL OVA in reaction solutions.Through polyacrylamide gel electrophoresis and other characterization methods,OVA was shown to be mainly combined with GNPs though physical interactions and there was no structural changes of OVA in the mixture of OVA and GNPs;while in the case of OVA@GNPs,OVA was covalently conjugated with GNPs and the secondary structure of OVA was changed.But binding off OVA with specific antibody was not affected in vitro.In vitro stability study showed that both kinds of antigen-GNPs complexes had good stability during their storage,and stability of OVA@GNPs was better than OVA+GNPs.Within the testing concentration range,no obvious cytotoxicity was observed for either kind of complexes.After subcutaneous immunization,the titer of specific antibodies against OVA in OVA@GNPs group were not only significantly higher than that in OVA group and OVA+GNPs group but also almost equivalent to Aluminum adjuvant group.We also showed the dosage of OVA@GNPs was only 1/5 of aluminum adjuvant to reach equivalent immune responses.The results of IgG antibody subtyping demonstrate that IgG1 was the main antibody in each group,suggesting that T cell-dependent humoral immunity was the main immune response.No obvious histopathological changes were found in the main organs of immunized mice after hematoxylin-eosin staining.Both antigen-GNPs complexes are safe to use in vivo.The results of skin penetration and transdermal immunization showed that the ability of small particle GNPs as protein transdermal carrier is very limited.Chitosan and carbomer gel may have very limited positive effects on transdermal immunity.Conclusively,through comparing two kinds of antigen-GNPs complexes,OVA@GNPs and OVA+GNPs,it was shown that the binding mode of antigen with GNPs affects their stability in vitro and immune responses in vivo.The covalent complexes of OVA and GNPs prepared by one-step synthesis can effectively induce a stronger humoral immune response in mice than OVA+GNPs and be comparable to Aluminum adjuvant.These results demonstrate the potential applications of one-pot synthesis approach for producing antigen protein-conjugated gold nanoparticles for vaccine delivery.