Preparation Of Natural Polysaccharides Nano-vaccine And Study Of Its Application On Tumor Immunotherapy

Cancer immunotherapy as a revolutionary cancer treatment aims to activate or boost the inherent immunological systems to recognize and kill cancer cells.Drug delivery systems have greatly promoted the delivery of antigen in the development of cancer immunotherapy,and the selection of safe and effective materials is crucial for the construction of delivery system.Natural polysaccharide is a kind of natural polymer material with degradability,good biocompatibility and immunomodulation.It has a good application prospect as a delivery carrier for vaccine.Therefore,this study designed and constructed two kinds of nano-vaccines with natural polysaccharides as the carrier,with the purpose of enhancing the body’s immune response and improving the antitumor effect.(1)Chitosan/calcium phosphate nanosheet vaccine(OVA@CSC/CaP):First,chitosan was modified with catechol group,and then the modified chitosan aqueous solution was stirred with PBS solution for 10 min,then CaCl2 solution was added to stir for another 10 min to obtain a CSC/CaP antigen delivery carrier with a sheet structure.The modification of catechol groups has improved the solubility of chitosan in neutral aqueous solutions.The antigen was loaded into the nanosheets by a co-precipitation method during its preparation.X-ray powder diffraction(XRD)results show that the nanosheets are composed of CaHPO4·2H2O crystals,and the nanosheets can be efficiently internalized by dendritic cells(DCs)via macropinocytosis.Lysotracker Red and Lamp-1(CD 107a)were used to label the lysosomes in the cells,and it was observed that the nanosheets could promote the escape of antigens from lysosomes.Moreover,DCs activation was triggered by the nanosheet along with up-regulation of co-stimulation marker expression and secretion of Th1-type cytokines.More importantly,compared with free antigen,the nanosheets significantly increase the ability of antigen cross-presentation.Therefore,the chitosan/calcium phosphate nanosheets as vaccine carriers for cross-presentation of exogenous antigens is worthy of further study.(2)Antigen and adjuvant co-delivery nano-vaccine(APPC):Alginate(ALG)and polyethyleneimine(PEI)are used as carrier materials,and liver cancer specific polypeptide antigen phosphatidylinositol 3 peptide(Glypican-3127-136,AMFKNNYPSL)is used as antigen,and CpG oligodeoxynucleotide(CpG)is used as an adjuvant to form APPC through electrostatic interaction.First,the particle size and morphology of APPC were characterized by dynamic light scattering and transmission electron microscopy,and the uptake of APPC by DCs was assessed by flow cytometry and confocal laser scanning microscopy(CLSM).The immunoregulatory capacity of APPC on DCs was examined in vitro and in vivo.The experimental results show that nano-vaccine has a spherical structure with uniform particle size,and can simultaneously increase the uptake of antigens and adjuvants by DCs,up-regulate the expression of costimulatory molecules on the surface of DCs and the secretion of cytokines,and can increase the proportion of CD3+CD4+T and CD3+CD8+T cells in spleen and have specific immune killing effect on liver cancer cells in vivo.The nano-vaccine prepared by this method can enhance the T cellular immune response of the body,and it is worth further studying on its application in liver cancer immunotherapy.