Study On Preparation And Immunological Enhancement Activity Of PHY Encapsulated PLA Nanospheres

Poria cocos,also known as Fu Tu,Fu Ling,Song Fu Ling,are as the same value as fungi with active ingredients:polysaccharides,alkaloids,saponins,terpenoids,polyphenols and other substances.Pachyman(PHY)is a kind of fungal polysaccharide,which mainly composed of ?-(1?3)-D-glucan and distributed in the fruiting body,mycelium and its fermentation broth.PHY has a variety of biological activities,such as:anti-tumor,immune regulation,liver protection and so on.However,the molecular weight of PHY is too large,and the drug release rate is too fast,which may lead to excessive doses demand,increased side effects,and inappropriate for clinical.Therefore,it is necessary to solve the problem.Poly lactic acid is a kind of new biodegradable polymer material with good biocompatibility,non-toxic and non-irritating.It can produce lactic acid monomer by hydrolyzed in the human body,oxidized by lactate dehydrogenase to generate pyruvic acid,participate in the tricarboxylic acid cycle,and finally generate water and carbon dioxide exclude from the human body.As peptides,proteins,enzymes,vaccines and other drugs are susceptible to decomposed by gastrointestinal acid/alkali substances and a variety of digestive enzymes,so it can only employ parenteral routes of administration during clinical application.It can protect the drug from damage by encapsulated in PLA nanoparticles and effectively control the degradation of nanoparticles in vivo.In this study,PHY encapsulated PLA nanospheres(PHYP)was prepared by double emulsion method.The optimum preparation conditions of PHYP nanospheres were optimized by response surface methodology.A series of characterization and the immunestimulatory activity of PHYP nanospheres were exploded in vitro and in vivo.The test is divided into the following four parts as follows:Experiment 1 Preparation and optimization of the preparation condition of Pachyman encapsulated PLA nanospheres Pachyman encapsulated PLA nanospheres were prepared by double emulsion solvent method.Consider the encapsulation efficiency and drug loading as the evaluation indicator,and explore the impact of the four factors(concentration of F68,concentration of PL A,ratio of drug to PL A,and ratio of primary emulsion to EAP)by the one-variable-at-a-time experiments.It was demonstrated that the effects of F68 concentration(w/v),PLA concentration(mg·mL-1)and PHY to PLA ratio(w1/o)on encapsulation efficiency and drug loading were significant.Based on the results of one-variable-at-a-time experiments,the effects of F68 concentration(w/v),PLA concentration(mg·mL-1)and PHY to PLA ratio(w1/o)on encapsulation efficiency were assessed by response surface methodology.The optimal preparation conditions were as follows:F68 concentration was 0.33%,PLA concentration was 30 mg·mL-1,PHY and PLA ratio was 10.25:1.According to the optimal preparation conditions,the acceptability test was carried out,and the encapsulation efficiency of the obtained PHYP nanospheres was 58.21±0.56%.The particle size of PHYP were 258.1±3.76 nm tested by DLS and the morphology was spherical,smooth with uniform in size observed under transmission electron microscope.The drug release assay was explored by dialysis method.It was found that under the same conditions,most of the PHY was released in the first few hours,and PHYP was slowly released in the next few days after a sudden release,and finally reached its peak.The stability of the PHYP nanospheres in different media was investigated.It was found that PHYP were less stable in the medium containing serum than in the serum-free medium.Experiment 2 Effect of PHYP on lymphocyte proliferation in mice In order to observe the effect of PHYP on the proliferation of lymphocytes in mice,the maximum safe concentration of PHYP and PHY was measured and the maximum safe concentration was selected.Mice spleen lymphocytes were was co-cultured with PHA or LPS for 48 h in vitro,and the proliferation of lymphocytes was investigated by MTT method.Finally,PHYP and PHY with the three concentrations below the maximum safe concentration were co-cultured with the isolated lymphocyte cell suspension.After 48h incubation with CD3,CD4 and CD8 at 4 ?,the cells were tested by flow cytometry.It was found that the maximum safe concentration of PHYP was lower than PHY.The effect of PHYP on the proliferation of lymphocytes was evident when the concentration was 31.25 ?g·mL-1(P<0.05).PHYP stimulated the strongest proliferation of T lymphocytes in concert with PHA with the concentration of 62.5 ?g·mL-1,when it stimulated B lymphocytes in concert with LPS with the concentration of 7.813 ?g·mL-1.PHYP,PHY and PLA could stimulate lymphocyte proliferation in the concentration range of 62.5 to 3.906 ?g·mL-1,and the effect of PHYP was superior to PHY and PLA.The middle concentration group of PHYP abtained the highest proportion of CD4/CD8,and was significantly higher than that of PHY group.Experiment 3 Effect of maturation on the Bone Marrow Dendritic Cells by PHYP In order to study the effect of PHYP nanospheres on bone marrow dendritic cells in mice,the effect of PHYP on the expression of cell surfaces and the phagocytosis of dendritic cells on antigen and PHYP were investigated.In the study of the expression of co-stimulatory molecules on BMDC,the bone marrow mononuclear cells of mice were collectted,induced,and the medium was changed overnight.After incubation for 7 days in vitro,PHYP,PHY and PLA nanospheres were added and co-cultured with cell suspension for 24 h,and then incubated with CD86 and CD80 antibodies.The effect was investigated by flow cytometry.The effect of BMDC on antigen uptake with fluorescent dyes was observed under laser confocal microscopy after staining of the nuclei.The phagocytosis of PHYP by BMDCs was demonstrated by ultrathin treatment.The results showed that PHYP reached a significant expression of CD86 and CD80 on the dendritic cell surface compared with other control groups.At the same time,the results of laser confocalization showed that the antigen uptake of BMDCs was significantly increased under the stimulation of PHYP.Finally,the images of TEM showed the phagocytosis of PHYP nanopsheres by BMDCs,indicating that the differentiation and maturation of BMDCs by PHYP nanospheres were achieved through uptaken by dendritic cells.Experiment 4 Effects of PHYP nanospheres on OVA antigen-specific immune response In order to explore the immune response of PHYP nanospheres on OVA-immunized mice,90 BALB/c mice aged 4-6 weeks were randomly divided into 6 groups(n= 15),subcutaneously inoculated on day 0 for the first time,and on day 14 for the second time.The serum of the mice was collected by the method of eyeballing on day 28,day 35,day 42 and day 49 after the first inoculation,respectively.The concentration of OVA specific IgG antibody and the content of IgG1 and IgG2a from serum were measured.Lymphocytes were cultured on day 7,14 and 35 after the second immunization.The proliferation of lymphocytes was measured by MTT assay at 48 h,and the activation of T lymphocytes was detected by flow cytometry.The levels of cytokines IFN-y,IL-2,IL-4 and IL-6 in the supernatant were detected by ELISA.The effects of PHYP on the expression of dendritic cells on CD86,CD80 and MHC ? in lymph nodes were investigated in the hippocampal and inguinal lymph nodes of mice after 24 hours of inoculation.It was concluded that OVA-PHYP significantly promoted the production of OVA-specific antibodies in serum,stimulated the secretion of cytokines in mice spleen cells,increased the proliferation rate of spleen lymphocytes in mice,caused more CD3+CD4+,CD3+CD8+lymphocytes,and promoted the expression of CD86,CD80 and MHC ? on the dendritic cells in lymph nodes.