Fabrication Of CaCO₃ Microspheres And Its Application As Vaccine Adjuvants For Hepatitis B Vaccine

Calcium carbonate(CaCO3)microspheres(MPs)are pH-sensitive and easy to achieve lysosomal escape to enhance cellular immunity.Furthermore,calcium ions can promote the activation of immune cells,which thus makes the CaCO3 MPs possess great potential as vaccine adjuvants.The simplest preparation method of CaCO3 MPs is the double decomposition reaction of calcium salts and carbonates(metathesis method).However,there are still some problems for this method,including broad particle size distribution,poor reproducibility and so on.In particular,CaCO3 MPs with small particle size are not available in aqueous solutions without additives.Therefore,in this paper,the reaction parameters of CaCO3 MPs prepared by metathesis method were optimized and the smaller MPs were also prepared by using different dispersion technique.The effects of physical and chemical properties(surface charge,particle size,preparation forms,etc.)on the adjuvanticity and the underlying mechanisms were investigated,which would provide new ideas and theoretical basis for the rational design of inorganic nano-/microparticle adjuvant.In detail,this thesis mainly included the following five issues:1)The CaCO3 MPs were prepared by utilizing the reaction of calcium chloride and sodium carbonate.The morphologies and size distribution were systematically optimized with various reaction parameters.Under the optimal conditions,the CaCO3 MPs with uniform size(around 4 ?m)and loading Hepatitis B surface antigen(HBsAg)were obtained.Protamine and polyglutamic acid(PGA)were coated on the surface of particles with layer-by-layer(LBL)self-assembly technique to obtain HBsAg-loaded MPs with different surface charges.The immune cell activation,cytokines and antibody secretion levels were assessed at the level of animals.The in vivo results showed that the MPs with positive charges could induce higher levels of humoral and cellular immune responses,which revealed the excellent adjuvant effect.2)In order to further prepare smaller CaCO3 MPs,based on the above optimal preparation conditions,various uniform-sized and spherical CaC03 MPs with average diameters range from 1 ?m to 4 ?m were facilely and rapidly fabricated in the absence of additives via different dispersion techniques including mechanical stirring,homogenization,and ultrasonication.The antigen loading efficiency was high and the integrity of the antigen structure ensured the immunogenicity.Compared with pure antigen and 4 ?m MPs,1 ?m CaCO3 MPs were more easily uptaken by dendritic cells(DCs),and could promote the maturation and activation of DCs with the expression of more CD40 and CD83 as well as secretion of more cytokines.Furthermore,1?m MPs could improve both humoral and cellular immune response in vivo,which were superior to aluminum adjuvant and wouldprovide better immune protection.3)Combined with the advantages of particle size and surface charge,1 ?m CaCO3 MPs with positive surface charges were fabricated by ultrasonication dispersion technique and LBL self-assembly technique.Compared with pure antigen and uncoated MPs,the residence time of the positively charged MPs was moderate at injection site,and it was easy to be engulfed up by DCs to enhance the level of antigen cross-presentation.In addition,they could promote the activation of B,CD4~+ and CD8~+ T lymphocytes,so as to stimulate the secretion of numerous cytokines,generate higher antibody levels,activate cytotoxic T lymphocytes(CTL)and enhance specific immune responses.4)In order to further enhance the adjuvanticity,Poly I:C,a kind of immunostimulant,was introduced on the surface of HBsAg-loaded CaCO3 MPs by utilizing its negatively charged property.The complex formulation including HBsAg and Poly I:C was obtained and evaluated with the immune responses.In vitro studies showed that,compared with the pure antigen and the CaCO3 MPs without Poly I:C,the complex formulation promoted the maturation and activation of DCs with secreting abundant inflammatory cytokines IL-1?.Moreover,it also promoted the splenocyte proliferation and the cytokines levels of INF-y and IL-6.The activation levels of CD8~+ T cells of the complex formulation were significantly higher than those of the pure antigen(3 times)and the CaCO3 MPs without Poly I:C(2 times),inducing higher level of CTL activation which exhibited the potential of removing intracellular infection efficiently.5)In order to investigate the relationship between the formulation type and adjuvant activity,HBsAg-loaded CaCO3 MPs were firstly coated with polydopamine,then modified with the DC-targeted molecule Anti-DEC205.The DC-targeted microcapsules(MCs)were finally obtained through removing the CaCO3 core by EDTA.Compared with pure antigen and CaCO3 MPs,the introduction of DC target molecules could significantly enhance antigen uptake of DCs and promote the expression of surface costimulatory molecules and MHC ?/? molecules in vitro.They could facilitate the secretion of cytokines,produce a robust immune memory.Compared with DC-targeted CaCO3 MPs,the DC-targeted MCs could generate higher antibody levels,providing more humoral immune protection.In conclusion,the reaction conditions of CaCO3 MPs prepared by metathesis method were optimized in the absence of additives.The effect of particle size,surface charge,immunostimulant and targeting molecule on the adjuvanticity were investigated.These results indicated that by virtue of reasonable selection and rational design,safe and efficient particulate adjuvant vaccine would be obtained with broad prospects and promising clinical applications.