Study On Preparation And Immunological Enhancement Activity Of Chinese Yam Polysaccharide PLGA Nanoparticles

Rhizoma discorea,also named as known as the Chinese yam,hemp yam,Bai Shao,potato and so on.Modern pharmacological studies have shown that the main active ingredient in Chinese yam is Chinese yam polysaccharide(CYP),with immune enhancement,anti-oxidation,anti-aging,hypoglycemic,hypolipidemic,anti-mutation,anti-tumor,regulating intestinal microecology,liver protection and so on.However,CYP is a macromolecule polysaccharide,if the CYP is used directly in clinical practice,there is a limitation of the development of clinical application that the polysaccharide has a rapid metabolism in the body,a short time of action,a low concentration of action,a large clinical use and a low bioavailability.The development of new formulations is the application of CYP in clinical research.Poly(lactide-co-glycolide)(PLGA)is a synthetic polymer copolymer with good biocompatibility,good biodegradability and non-toxic and non-specific immunogenicity.PLGA as the nano-materials for preparaing the nanoparticles on the drug have a sustained release and targeting,can protect the biological activity of drugs and greatly improve the bioavailability of drugs,reduce the side effects of drugsThe preparation of nanoparticles on the drug have a sustained release and targeting,can protect the biological activity of drugs and greatly improve the bioavailability of drugs,and reduce the side effects of drugs,which triggering the researchers’ extensive attention and inquiry.The encapsulation of CYP into the PLGA nanoparticles as an injection can avoid the shortcomings of CYP in the body with short half-life and reduce the amount of CYP.At the same time,it can improve the absorption of CYP,prolong the time of drug action,increase the bioavailability of CYP,and enhance the immmostimulatory activity of CYP.In this paper,the CYP-PLGA nanoparticles(CYPP)were prepared by double emulsion solvent evaporation method.The CYPP was optimized by response surface methodology.The immunoenhancement of the nanoparticles was studied by in vivo and in vitro experiments.The test consists of the following four parts:Experiment 1 Preparation and optimization of CYP-PLGA nanoparticles The CYP-PLGA nanoparticles were prepared by double emulsion solvent evaporation method and the optimal preparation conditions of PLGA nanoparticles were optimized by response surface methodology.The CYPP and free CYP was separated by the microcolumn centrifugation assay.The concentration of CYP was determined by phenol concentrated sulfuric acid method,and then the encapsulation efficiency of nanoparticles was calculated.Based on the encapsulation efficiency and drug loading of CYPP,the volume ratio of the internal water phase to the oil phase,the volume ratio of the primary emulsion to the external water phase,the concentration of F68 and the concentration of PLGA were investigated.The results showed that the volume ratio of the internal water phase to the oil phase,the volume ratio of the primary emulsion to the external water phase and the concentration of F68 had a greater effect on the preparation of CYPP.Based on the results of single factor test,the response surface method was used to explore the two effects of these three factors in the preparation of nanoparticles.The optimal conditions for the preparation of CYPP were as follows:the volume ratio of the internal water phase to the oil phase was 1:9,the volume ratio of the primary emulsion to the external water phase was 1:10,and concentration of F68 was 0.7%.The validation test was carried out according to the optimum preparation conditions.The entrapment efficiency of the nanoparticles was 65.56±0.23%,and which was less than that of the predicted values of 66.82%.After the optimized conditions,CYPP was prepared.The morphology of the nanoparticles were observed under transmission electron microscopy and scanning electron microscopy.It can be seen that the nanoparticles are similar to sphere,and the shape is uniform and the surface is smooth and compact.The particle size was 212.3±3.3 nm,PDI value of 0.121±0.013,less than 0.3,indicating uniform distribution of nanoparticles,uniform size,stable shape,good dispersion.The potential of the nanoparticles is negative,indicating that the nanoparticles with negative charge.In vitro release results showed that the cumulative release rate of CYPP was significantly lower than that of free CYP at 48 h,indicating that CYPP release CYP was significantly slower than free CYP.Experiment 2 Effect of CYPP nanoparticles on spleen lymphocyte proliferation on mice in vitro Lymphocyte proliferation was measured by MTT assay.Flow cytometry was used to classify and compare the lymphocytes.First,the maximum safe concentration of CYP and CYPP was measured.CYP and CYPP were diluted from 0.8 mg mL’1 to 11 concentrations,and co-cultured with spleen cells.After 44h of in vitro culture,the absorbance was measured by MTT method at 570 nm,and the maximum safe concentration was selected.Five concentrations below the maximum safe concentration,which was alone or co-cultured with LPS for 44 h in vitro.The effects of CYP and CYPP on the proliferation of B lymphocytes were observed by MTT assay.The effects of CYP and CYPP on the proliferation of T lymphocytes were observed by adding CYP,CYPP and BP alone or synergistic PHA to the spleen lymphocytes of mice at the maximum concentration of five concentrations.The results showed that the maximum safe concentration of CYPP(400 μg·mL-1)was higher than that of CYP(200 μg ·mL-1).In concentration of 200 μg·mL-1,the effect of CYPP in single or synergistic with LPS or PHA had the best effect on spleen lymphocyte proliferation at the concentration of 200 μg·mL-1,which was significantly higher than that of CYP,BP and cell control group.When the concentration range was 25-200 μg·mL-1,the free CYP,CYPP and BP can effectively stimulate the proliferation of lymphocyte,and CYPP is better than CYP group and BP group.The ratio of CD4+ to CD8+ was significantly higher than the free CYP and BP,indicating that CYPP group are more effective in immunophenotyping T cells than free CYP and BP.Experiment 3 Effects of the CYPP nanoparticles on the immune function of dendritic cells in vitro In order to investigate the effect of CYPP nanoparticles on the immune function of dendritic cells derived from bone marrow in mice.First,the uptake ability of dendritic cells was explored by confocal laser scanning microscopy.In order to verify that the green fluorescence around the cells was a labeled drug from the cells,which the ultrastructure of the cells was observed by transmission electron microscopy.The results of these two parts showed that the effect of CYPP on the uptake of dendritic cells was greater than that of CYP and BP alone.Meanwhile,it was verified that dendritic cells had taken the nanoparticles into the cells.Flow cytometry was used to detect the expression of MHC II and co-stimulatory molecules(CD80 and CD86)in the dendritic cells under the stimulation of three drugs.The results showed that the expressions of MHC class II molecules and costimulatory molecules of CD80 and CD86 on the surface of dendritic cells were stimulated by CYPP,which were significantly higher than those of the CYP group,the BP group and the cell control group.The results showed that the stimulation,uptake and maturation of the dendritic cells were triggered by encapsulated with PLGA nanoparticles,are stronger than the individual CYP and blank nanoparticles.Indicated that CYPP can exert its function effectively for both specific and non-specific immunity.Experiment 4 Immune adjuvant activity of CYPP on OVA antigen In order to investigate the adjuvant activity of the OVA mode antigens encapsulated to CYPP nanoparticles to further verify the immunoregulatory effect of the new adjuvant of CYPP nanoparticles.144 Balb/c mice aged 6 weeks were randomly divided into 6 groups(n=24).The mice were immunized with subcutaneous injection of OVA antigen vaccine with different components.After 24 hours of first immunization,the draing lymph nodes such as mandibular lymph nodes,shoulder lymph nodes and inguinal lymph nodes were taken to detect the activation of dendritic cells.Blood samples were collected from the Day 14,Day 21,Day 28,Day 35 and Day 42 after the first immunization.Serum samples were used to detect the antibody titers of OVA-specific IgG,total IgG,IgG2a and IgGl in serum by ELISA assay.The spleen was isolated from the mouse and the lymphocytes were isolated and cultured.After 66-68 h,the lymphocyte proliferation was measured by MTT assay.The culture supernatant of spleen lymphocyte was isolated to determine the levels of IL-4,IL-6,IFN-y and IL-2 by ELISA assay.On the Day 35 after the first immunization,the spleen of mouse was taken to make tissue sections and the spleen histological changes were observed.The results showed that CYPP as the adjuvant to encapsulate OVA model antigen immunized with mice,which the degree of activation of dendritic cells in drainage lymph nodes was higher than that in other groups.The antibody level and proliferation of T and B lymphocytes of CYPP/OVA group were significantly higher than the CYP/OVA and BP/OVA.The increase of splenic nodules,thickening of the lymphatic sheath around the white pulp artery and other histological changes that indicated the enhancement of immune function.The results showed that PLGA nanoparticles could significantly improve the efficacy of CYP on specific immune enhancement and prolong the time of drug action to protect and enhance the immune effect and sustained release of antigen.