Study On Immune Activity Of DEC-205-Targeting Astragalus Polysaccharide Long-circulating Liposomes

Astragalus is the dried root of Astragalus monogholicus Bunge or Astragalus membranaceus（Fisch）Bunge.It has the functions of enhancing the body’s immune function,protecting the liver,diuresis,anti-aging,anti-stress,antihypertensive,and wider antibacterial effects.It has been widely used in clinical treatment.Polysaccharides have the advantages of many biological activities,such as anti-tumor,lowering blood sugar,and improving immunity,also obtain the advantages of lower toxicity,as an important active substance in Chinese medicinal materials.In the present study,synthetic phospholipids DSPC and cholesterol are used as membrane materials,and small unilamellar liposomes are prepared by thin-film dispersion method,the immunomodulator astragalus polysaccharide and model antigen ovalbumin was encapsulated by long-circling liposomes,and the surface of liposomes were conjugated by DEC-205 antibody,a DEC-205-targeting long-circulation nanoliposome vaccine was obtained and the immunomodulation effects on ICR mice were investigated by series of experiment.The current study may provide a basis for develop an antigen and immunomodulator loaded DCs targeting nanoliposome delivery system.The main research methods are as follows:Firstly,the process of liposome preparation was studied,and the preparation process of long-circulation nanoliposomes was optimized through a single factor test and orthogonal test.Taking the encapsulation rate of ovalbumin in liposomes as the index,the membrane-material ratio,lipid-drug ratio,ultrasonic time,and the amount of polyethylene glycol as influencing factors,the preparation process was optimized to obtain the best encapsulation rate.Secondly,the morphological characteristics of nanoliposomes were observed through transmission electron microscopy,and the particle size and Zeta potential of nanoliposomes were detected by laser particle size analyzer.The in vitro drug release effect was observed by detecting the leakage of astragalus polysaccharides in liposomes.Finally,the immunemodulation effect targeting nanoliposome was explored by animal experiment.224 4-week-old female ICR mice were randomly divided into 8 groups,28 in each group,Naive group,ovalbumin group,astragalus polysaccharide group,ovalbumin+astragalus polysaccharide group,ovalbumin+aluminum adjuvant Group,blank nanoliposome group,ovalbumin+astragalus polysaccharide long-circulation nanoliposome group,and DEC-205 targeting ovalbumin+astragalus polysaccharide long-circulation nanoliposome group.All the eight groups were immunized synchronously,subcutaneously injected into the neck,and two immunizations were separated by one week.The vaccine antigen content was the same in each group,except for the Naive group,the astragalus polysaccharide group,and the blank liposome group,which has no antigen.On days 7,14,21,28,and 35 after the first immunization,3 mice were taken from each group for blood collection.Detect the concentration changes of antibodies Ig G,Ig G1,Ig G2a,Ig G2b in the serum of mice and the IL-2,IL-4,IL-6 and IFN-γin the serum,and the bodyweight,Spleen weight and thymus weight of the mouse were recorded after each blood collection,to calculate the immune organ index of the mice.On the second day after the first immunization,3 mice were taken from each group,and the spleen was collected aseptically.The expression levels of costimulatory molecules CD80,CD86 on the surface of dendritic cells and the main histocompatibility MHCⅡwere detected by flow cytometry,to judge the maturation of dendritic cells.On the 7th day after the second immunization,6 mice were taken from each group and the spleen was taken aseptically.3 mice were tested for the proliferation ability of splenic lymphocytes.3 mice were tested for T lymphocyte differentiation by flow cytometry.The results showed that the optimized process of preparation of long-circulation nanoliposomes were:lipid/drug ratio,4:1;phospholipid/cholesterol ratio,4:1;the ultrasonic time,20min;and the concentration of DSPE-PEG₂₀₀₀,5%.Under the optimal conditions,the encapsulation rate of OVA in liposomes is 34.71%,and the encapsulation rate of Astragalus polysaccharides is 20.91%.Observation of nanoliposomes through transmission electron microscopy showed that the liposomes had a single-layered membrane structure,uniform and round,and the membrane structure was complete,all of which were spherical or near-spherical.The drugs and lipids wrapped inside the liposomes could be seen.The results of particle size,polydispersity coefficient,and Zeta potential showed that the liposomes were negatively charged,the particle size distribution range was narrow,and the distribution was normal unimodal.The liposome homogenization effect was nice,and those results is consistent with the TEM observation.The in vitro release test of APS showed that the cumulative release rate of APS was less than 20%in 3d and less than 40%in 7d,and the release was stable.Serum test results show that the concentration of antibodies and antibody subsets in the serum can maintain a high level for a period,and the expression of IL-2,IL-4,IL-6,and IFN-γin the serum is up-regulated,indicating the long circulation liposomes can continuously release antigens,stimulate the body’s immune system,and improve immune efficiency.The spleen index and thymus index of mice increased,and the proliferation capacity of T lymphocytes and B lymphocytes increased.The expression of CD80,CD86,and MHCⅡmolecules on the surface of DC cells was up-regulated,and the number of CD3⁺T cells,CD4⁺T cells and CD8⁺T cells increased,the ratio of CD4⁺/CD8⁺increased,indicating that liposomes can enhance the body’s immunity by stimulating the maturation of dendritic cells and the proliferation and differentiation of T lymphocytes and B lymphocytes while improving the immune effect of the vaccine.In summary,the optimal process for preparing long-circulating liposomes are:lipid/drug ratio,4:1;phospholipid/cholesterol ratio,4:1;the ultrasonic time,20min;and the concentration of DSPE-PEG₂₀₀₀,5%.Under optimal conditions,the encapsulation rate of OVA in liposomes is 34.71%,and the encapsulation rate of astragalus polysaccharides is 20.91%.The liposomes have a single-layer membrane structure,which is uniform and round,and the surface is smooth.The DEC-205mediated liposome can improve the OVA-specific antibody titer and the concentration of IL-2,IL-4,IL-6,and IFN-γin serum,stimulate the maturation of dendritic cells,and up-regulate the proliferation of T lymphocytes and B lymphocytes.Taken together,the DEC-205 mediated liposome can act as an DCs targeting antigen and APS delivering system to enhance the balance Th1/Th2 immune response of mice.