Phosphorylated Chitosan As Vaccine Delivery System For Intramuscular Immunization

In the filed of immunotherapy,immune vaccinations have received more and more attention for disease prevention and treatment.In immune vaccination,efficient vaccine adjuvants are necessary due to the weak immunogenicity of vaccines.Some traditional vaccine adjuvants have been widely used but have shown obvious limitations such as poor bio-safety.Therefore,researchers make a great effort to develop more functional novel immune adjuvants such as chitosan-based immune adjuvants.However,chitosan is poorly water-soluble,which greatly limits its application as immune adjuvants,regardless of its good biocompatibility,biodegradability and other biological activities.In this work,we prepared a water-soluble chitosan derivative phosphorylated chitosan(PCS)and evaluated its potential as a novel immune adjuvant.PCS was found to retain the good biocompatibility and biodegradability of chitosan and found to be pH-sensitive: specifically,it was water-soluble at pH<7.0 but began to gel at pH>7.0.By virtue of this,neutral PCS aqueous solutions containing ovalbumin(OVA)antigen was intramuscularly injected into test mice,which would transform to an OVA-containing gel network for OVA immunization.So far,the use of PCS as an adjuvant for immunotherapy has not been studied.Therefore,with good physical and chemical properties,PCS can be designed as a novel immune adjuvant or vaccine delivery system for immunotherapy,which have better application prospects than chitosan.The main contents of our study are as follows: 1.The synthesis,characterization and properties research of PCSIn this paper,chitosan was modified with homogeneous method,and PCS was synthesized with good water solubility.Then the PCS was characterized by FTIR,1H-NMR and XRD.The content of phosphorus was determined by ICP.The solubility and pH sensitivity of PCS were studied by titration.The CCK-8 method was used to determine the in vitro cytotoxicity of PCS and the biosafety was evaluated.The rheological property of PCS was measured by rheometer.The encapsulation efficiency and in vitro release behavior of PCS on OVA antigen were determined by UV spectroscopy.The effect of PCS on the secondary structure of OVA was studied by CD method.2.Study on the immune efficiency of PCS as vaccine delivery systemMice were immunized with different vaccine formulations by intramuscular injection.At 10 days after the third immunization,the blood samples were collected.Then,the serum was separated and stored.The spleen was taken to prepare different concentrations of splenocytes suspension.The levels of IFN-?,IL-12,IL-4,IL-10 and the titers of IgG,IgG1 and IgG2 a were determined by ELISA.The proliferation of splenocytes was detected by CCK-8 method.The numbers of IFN-?-and IL-4-secreted cells were further measured by ELISpot method.The memory T cell immune response and the activation of CD11c+ dendritic cells were detected by flow cytometry.Fluorescence in vivo imaging method can visually monitor the residence time and slow release of OVA antigen at the injection site.The antigen was taken by APC and presented to the spleen by drainage lymph nodes by immunohistochemistry.Finally,the effect of different vaccine formulations on immunological function and histological structure of spleen was evaluated by H&E staining.The results showed that the use of 30 mg/mL PCS as vaccine delivery system contributed to significantly higher level of antigen-specific immune responses,including higher level of antigen-specific IgG,IgG1 and Ig G2 a antibodies,IFN-?,IL-12 and IL-4 cytokines secretion by splenocytes,as well as memory CD4+ and CD8+ T cells.In vivo imaging and immunohistochemistry assays suggests that the improved immunization efficacy may be attributed to the controlled-release of antigen from injection site by PCS gel network,and then prolonged antigen stimuli to the immune system.From the results,PCS could be developed as a promising vaccine delivery system for immunotherapy.