Study On Immunoenhancement Activity Of Mannose Glycosylated Chitosan PLGA Microspheres

Vaccination is the most effective way to control animal infectious diseases.However,although many farms have been vaccinated strictly according to the immunization procedures,the animals are still difficult to produce an effective immune response due to a variety of reasons,thus some infectious diseases still epidemic frequently.It is reported that the use of immune adjuvant or antigen presentation system can improve the immune effect of the vaccine.In this study,PLGA was used as an antigen carrier to encapsulate OVA,which was approved by FDA and EMA.PLGA microspheres were prepared by double emulsion solvent evaporation method,and the Mannose Glycosylated Chitosan was adsorpted on the surface of microspheres.The physicochemical properties,drug loading of microspheres,and immunomodulation activity was evaluated by series of experiment in vitro and in vivo.The current study provides the research basis for the using of mannose chitosan PLGA microsphere act as an antigen presentation system targeting macrophages and dendritic cells.The main research progress and achievements are as follows:1.Synthesis and identification of PLGA microspheres with mannose and chitosan:mannose modified chitosan was prepared by reductive ammoniation,Element analysis results show that the synthesis is successful and stable product properties.L9（3³）orthogonal experiment was used to optimize the dosage of PLGA,OVA and PVA.The results showed that the optimum conditions for the synthesis of PLGA microspheres were:PLGA,40mg,OVA,50mg,PVA concentration,2%,8000r/min shear,emulsification time,60s,oil/water ratio,1:10.According to the optimal formulation,the size of microspheres is about 6.45μm,the drug loading is about 7.9%,and the reproducibility well.The physical and chemical properties of FITC-MPs are similar to MPs.2.The modulation effect of MAN-CS-PLGA MPs on the mouse macrophage（RAW264.7）in vitro was investigated by series of experiment.In this study,RAW264.7 cells was treated with FITC-MAN-CS-PLGA MPs for 8 hours in vitro,and the macrophage viability and safty concentration was determined by CCK-8 method.fluorescence intensity of FITC-CS-PLGA MPs treated macrophages was observed by laser confocal microscopy.RAW264.7 was treated with 20μL FITC MPS（containing 40μg·m L-1 FITC-OVA）for 12h,and the macrophages were lysed and the fluorescence intensity was detected by fluorescence intensity reader.The results showed that when the concentration was 10μg.m L^-1-60μg.m L^-1,The viability of macrophages treated with FITC-MAN-CS-PLGA MPs was more than 100%（P<0.01）,and FITC-CS-PLGA MPs could significantly improve the proliferation of macrophages.Furthermore,The results of laser confocal microscopy showed that the fluorescence intensity of FITC-CS-PLGA MPs treated macrophages was higher than that of other groups.In addition,the fluorescence intensity in FITC-CS-PLGA MPs treated macrophages significantly higher than other group.The present results indicated that PLGA microspheres with chitosan and mannose chitosan could significantly improved the proliferation of macrophage,increased phagocytic efficiency of macrophages to FITC-CS-PLGA MPs,and also significantly promoted the antigen（OVA）presentation efficiency of macrophage.3.The effect of mannose chitosan PLGA microspheres on immune response in mice.ICR mice（n=110）were randomly divided into five groups（n=22）.Each group subcutaneously injected with different formulation（saline,OVA,PLGA MPs,CS-PLGA MPs and MAN-CS-PLGA MPs）.After that,the animals were subcutaneously immunized once at one weeks intervals.The percentage of CD3⁺CD4⁺and CD3⁺CD8⁺T cells,frequency of surface molecules（CD80、CD86,and MHC-II）in CD11c⁺DCs,OVA-specific Ig G,and Ig G（Ig G1,Ig G2a,and Ig G2b）antibody titer,concentration of serum cytokine（IL-2,IFN-γ,IL-4,and IL-6）were determined by flow cytometry or ELISA.The result showed that MAN-CS-PLGA MPs C significantly increased the ratio of CD3⁺CD4⁺/CD3⁺CD8⁺T cells.（P<0.01）.MAN-CS-PLGA MPs C also significantly enhanced CD80,CD86 and MHC II expression in DCs（P<0.05）,and improved OVA-specific Ig G,Ig G1,Ig G2a,and Ig G2b antibody.Moreover,MAN-CS-PLGA MPs promoted the concentration of IL-2,IFN-γ,IL-4,and IL-6in serum of mice.The current data showed that MAN-CS-PLGA MPs can significantly improve the balance Th1/Th2 type immune responses of immunized mice,thus enhancing the cellular and humoral immune responses of mice,The mechanism of immune enhancement may be to activate the initial T cells to initiate immune response by promoting the maturation of dendritic cells and improving the efficiency of antigen presentation of dendritic cells.In conclusion,MAN-CS-PLGA MPs can target mannose receptor on the surface of macrophage in vitro and improve the efficiency of macrophage internalizing antigen.MAN-CS-PLGA MPs can induce the balanced Th1/Th2 humoral and cellular immune responses.The mechanism of its immunoenhancement may be to enhance the phagocytosis of dendritic cells and macrophages to microsphere by targeting mannose receptor so as to improve the efficiency of antigen presentation and promote the maturation of dendritic cells to activate the initial T cells to initiate immune response.