Study On Immunological Enhancement Activity And Mechanism Of Alhagi Honey Polysaccharide Encapsulated PLGA Nanoparticles And Pickering Emulsion

Alhagi honey is a kind of traditional Chinese nourishing medicine,and its main active ingredient,Alhagi honey polysaccharides(AHP),can effectively enhance the immunity of animal bodies.However,the efficacy of AHP is limited in vivo,specific performance of fast metabolism,short action time,and poor targeting.Poly D,L-lacticco-glycolic acid(PLGA)is a kind of biodegradable polymer material and the nanoparticles prepared by PLGA have characteristics like good biosafety,drug loading capacity,sustained release,and accurate targeting.Encapsulating AHP in PLGA not only can help the effective delivery of AHP,but also can take advantage of the synergy between them,which might induce more effective immunoenhancement activity.In this study,AHP encapsulated PLGA nanoparticles and cationic modified nanoparticles were prepared.In addition,their quality and immunoenhancement activity were evaluated to select the optimal preparation for technical parameters.Then,based on the selected nanoparticles,a new Pickering emulsion adjuvant that could load a large amount of antigen by simple mixing and further induce effective immune responses was developed.After that,the immune enhancement activity of the nanoparticles and Picking emulsion was compared and screened,and the immune enhancement mechanism of the best adjuvant was studied.All experiments are divided into the following eight parts:Experiment 1 Preparation and quality evaluation of cationic polymer modified Alhagi honey polysaccharide encapsulated PLGA nanoparticles.The aims of this experiment were to prepare the AHP encapsulated PLGA nanoparticles(AHPP)and the cationic polymer modified AHPP with good stability.The AHP was isolated by water extraction and alcohol precipitation method and purified by column chromatography method,then the structure of the purified polysaccharides was detected and verified.Based on that,the AHPP and three kinds of cationic polymer modified nanoparticles were prepared by double emulsion solvent evaporation method.Finally,the encapsulation efficiency,characterization,and stability of the nanoparticles were tested.The results demonstrated that the AHP were homogeneous polysaccharides with a molecular weight of 46.8 kDa.AHP could be successfully encapsulated into PLGA nanoparticles.The nanoparticles were spherical in shape and the particle size was about 200 nm.The charge of AHPP became positive after being modified with CS,PEI or ?PL.The PEI and ?PL modification had no impact to the structure,particle size or encapsulation efficiency of AHPP.Although the particle size of AHPP became larger and the encapsulation efficiency of AHP decreased with CS modification,the integrity of AHPP was not damaged too much.Besides,the stability of AHPP was improved after PEI and ?PL modification.The above results indicated that AHP was successfully encapsulated into PLGA nanoparticles and three kinds of cationic polymer materials could be successfully modified on the surface of AHPP.The AHPP and its three derived cationic polymer modified nanoparticles were all spherical in shape and had good stability.Experiment 2 The effect of AHP encapsulated PLGA nanoparticles on the function of mice peritoneal macrophages in vitro.The aims of this experiment were to observe the effects of AHP,AHPP,CS-AHPP,PEI-AHPP,and ?PL-AHPP on macrophages(M?),including phagocytosis,surface molecule expression and cytokine secretion.In this experiment,the safe concentration of different nanoparticles on M? was firstly measured.Then the phagocytosis,surface molecule expression,and cytokine secretion of Mcp were determined by laser confocal,flow cytometry,and ELISA methods under the safe drug concentration.The results demonstrated that the cationic polymer modification could significantly enhance the phagocytic activity of M? to AHPP.Compared with AHP,AHPP could significantly stimulated the expression of MHC ?+,CD86+,and CD80+,the secretion of iNOS,NO,and cytokines(IFN ?,IL-1? and IL-12)on macrophages.In additional,the phagocytic activity and surface co-stimulatory molecules expression of AHPP on macrophages were further improved after three kinds of cationic polymer modification,and PEIAHPP exhibited the strongest activity among all the treatment groups.Experiment 3 Quality evaluation of Alhagi honey polysaccharide encapsulated PLGA nanoparticles as vaccine adjuvant.The aims of this experiment were to evaluate the morphological characteristics,particle size,stability,and drug release characteristics of AHPP,CS-AHPP,PEI-AHPP,and PL-AHPP as vaccine adjuvants.In this experiment,Ovalbumin(OVA)was used as the model antigen.OVA was loaded on AHPP and other three kinds of cationic polymer modified nanoparticles,then the antigen loading rate,characterization changes,in vitro antigen release and stability of different nanoparticles were detected.The results showed that the antigen loading efficacy of OVA loaded AHPP(AHPP/OVA)was 60.37±1.79%,that was to say,the antigen loading efficacy of AHPP/OVA was increased due to electrostatic adsorption after cationic polymer modification.The results of scanning electron microscope and transmission electron microscope showed that all the nanoparticles had the same structure and morphology,and the structure would not be damaged greatly after freeze-drying.All nanoparticles were able to release antigen slowly within 35 days,showed good sustained release behaviors.The results of the stability test illustrated that the AHPP began to show double peaks at days 14 and the nanoparticles aggregated at days 21,while PEI-AHPP and ?PL-AHPP showed double peaks at days 28 and 21,respectively,and no nanoparticle aggregated within 28 days,which confirmed the nanoparticles had good stability.The experimental results showed that the AHPP/OVA and three derived cationic polymer modified nanoparticles had good antigen sustained release effects in vitro.The loading efficacy and stability of AHPP/OVA were improved after cationic polymer modified in which PEI-AHPP/OVA showed the best stability.Experiment 4 Immunoenhancement activity of alhagi honey polysaccharide encapsulated PLGA nanoparticles on mice immunized with OVA vaccine.The aims of this experiment were to observe the immunoenhancement activity of AHPP,CS-AHPP,PEI-AHPP,and ?PL-AHPP.In this experiment,OVA was used as a model antigen and the OVA loaded nanoparticles were intramuscularly injected into the mice.Then the spleen lymphocyte proliferation,spleen T lymphocyte differentiation,secretion of OVA-specific 1gG antibodies,subtype antibodies,serum cytokines,and mouse spleen histological changes were detected.The results showed that all AHPP/OVA and the three cationic polymer modified nanoparticles could improve the proliferation of T cells and B cells.The ratio of CD4+/CD8+T cells in PEIAHPP/OVA group was significantly higher than all the other groups(P<0.05).The PEI-AHPP/OVA and ?PL-AHPP/OVA groups induced high levels of OVA-lgG antibodies and antibody subtypes,and the ratio of IgG2a/IgG1 in PEI-AHPP/OVA group was significantly higher than that in other groups(P<0.05),which showed the type of immune response was inclined to Th1 type.The PEI-AHPP/OVA group induced high levels of both Th1 type cytokines(TNF-?,IFN-?)and Th2 type cytokines(IL-4,IL-6).Among all the experimental groups,PEI-AHPP/OVA and ?PL-AHPP/OVA group showed the most significant increase in splenic corpuscle and lymphocytes.The experimental results showed that the immunoenhancement activity of AHPP/OVA was further enhanced after being modified by PEI and ?PL,and the PEI-AHPP/OVA nanoadjuvant could induce strong and long-term cellular and humoral immune responses in mice.Experiment 5 Preparation and quality evaluation of Pickering emulsion of alhagi honey polysaccharide encapsulated PLGA nanoparticles.In order to improve the antigen loading efficacy of PEI-AHPP and AHPP,and prepared a new vaccine adjuvant which can load more antigens and induce effective immune response through simple mixing with antigen,two novel Pickering emulsion adjuvants were developed.In this experiment,PEI-PPAS or PPAS were prepared by assembling PEI-AHPP or AHPP with oil-phase squalene via phacoemulsification.The physical forms,stabilities,loading efficacies,morphological structures and in vitro antigen release behaviors of the two kinds of Pickering emulsion adjuvants were measured.The results showed that the PEI-PPAS and PPAS were both about 1?m in size and the PDI were less than 0.3,the charge of PEI-PPAS and PPAS was 11.43±1.16 and-23.00±1.87,respectively.Both of them had good stability,and no aggregation or decomposition within 35 days at 37?.The antigen loading efficacies of PEI-PPAS and PPAS were 98.58±1.05%and 63.09±2.48%,respectively.Both Pickering emulsion adjuvants had rough strawberry-like surfaces,antigens could hence be absorbed on their surfaces.Furthermore,these Pickering emulsion adjuvants showed excellent sustained antigen release behaviors in vitro.The experimental results showed that the new Pickering emulsion adjuvants PEI-PPAS and PPAS had similar shapes,could carry numerous antigens by adsorption effects,had good stabilities and antigen sustained release behaviors in vitro.By contrast,the PEI-PPAS had higher antigen loading efficacy in the two adjuvants.Experiment 6 Immunoenhancement activity of Pickering emulsion of alhagi honey polysaccharide encapsulated PLGA nanoparticles on mice immunized with OVA vaccine.In order to detect the best antigen loading mode and immunoenhancement activity of PPAS and PEI-PPAS as immune adj uvants,mice were immunized with these Picking emulsions.In this experiment,OVA was loaded on or into two different of Pickering emulsion adjuvants by adsorption or encapsulation,and the mice were immunized by intramuscular injection.Then the OVA-specific 1gG antibodies,subtype antibodies,serum cytokines,spleen T lymphocyte differentiation,lymph nodes DCs surface molecules expression,serum biochemical index,and histological changes of heart,liver,spleen,lung and kidney in mice were detected.The results showed that in all the experimental groups,both PEI-AHPP/OVA and PEI-PPAS(Ads)/OVA which was prepared by OVA adsorption could stimulate the strong and long-time secretion of OVA-IgG,OVA-IgG1,OVA-IgG2a and OVA-IgG2b in the body.PEIPPAS(Ads)/OVA and PPAS(Ads)/OVA could stimulate the expression of Th2 type cytokines,PEI-PPAS(Ads)/OVA and PEI-AHPP/OVA increased the expression of Th1 type cytokines,PEI-PPAS(Ads)/OVA activated the differentiation of splenic T lymphocytes CD3e+CD4+and CD3e+CD8a+,promoted the expression of the molecules CD11c+MHC I+,CD11c+MHC II+and CD11c+ CD86+of dendritic cells(DC)in lymph node in the meantime.The serum biochemical indexes and organ histology results showed that all the experimental adjuvants had good safety,the antigen adsorbed PEIPPAS adjuvant could induce strong and long-term cellular and humoral immune responses.Experiment 7 Immunoenhancement activity of alhagi honey polysaccharide encapsulated PLGA nanoparticles and Pickering emulsion on chicken immunized with H9N2 vaccine.The aims of this experiment were to investigate the immunoenhancement activity of AHPP,PEI-AHPP,PPAS,and PEI-PPAS for H9N2 vaccine.In this experiment,119 chicks were divided into 7 groups and immunized with four different H9N2 antigen adsorbed vaccine adjuvants.After the second immunization,chicken hemagglutination inhibitory titer,spleen T lymphocyte differentiation,spleen cytokine mRNA expression level,and histological changes of each immune organ were measured.The results showed that in all the experimental groups,PEI-AHPP/H9N2,PPAS/H9N2,and PEIPPAS/H9N2 all could produce high hemagglutination inhibition(HI)titers,induce high levels of INF-?,IL-6,and IL-12p40 expression,high levels of CD3e+CD4+and CD3e+CD8+expression in spleen T lymphocytes.Among all the experimental groups,PEI-PPAS/H9N2 group had the strongest induction effect on CD3+CD4+and showed the highest immune organ index to chicken thymus and bursa of Fabricius.HE stained sections of immune organs showed that all the adjuvant groups had no toxic effect on chicken immune organs,and the PPAS/H9N2 and PEI-PPAS/H9N2 groups significantly enhanced the immune responses of chicken spleen.The experimental results indicated that all the PEI-AHPP/H9N2,PPAS/H9N2 and PEI-PPAS/H9N2 had good immune adjuvant activities,among which PEI-PPAS/H9N2 exhibited strongest immunoenhancement activity,and could induced more effective and long-times cellular and humoral immune responses in chicks.Experiment 8 Immunoenhancement mechanism of Pickering emulsion of alhagi honey polysaccharide encapsulated PLGA nanoparticlesThe objective of this experiment was to explore the mechanism of the PEI-PPAS adjuvants to exert long-lasting and strong cellular and humoral immune responses.In this experiment the Fluorescent marked PEI-PPAS was injected into mice,and the migration of PEI-PPAS in mice was detected by in vivo fluorescence test.The main signal pathway and the mechanism of PEI-PPAS/OVA on DCs were determinded by high-throughput sequencing,flow cytometry and laser confocal detection methods.The results showed that the fluorescence intensity of PEI-PPAS/OVA group was significantly higher than that of OVA group(P<0.05),which showed the good sustained release effect of PEI-PPAS.The total fluorescence intensity of lymph nodes of PEI-PPAS/OVA group was significantly higher than that in OVA and Alum/OVA groups(P<0.05),which showed the good lymph node targeting of PEI-PPAS.Highthroughput sequencing results showed that PEI-PPAS/OVA stimulated DCs in a variety of ways,most of which are phagocytosis.High-throughput sequencing results combined with flow cytometry and confocal detection showed that after PEIPPAS/OVA was phagocytosed by DCs,it was presented by MHC II molecules through the lysosomal pathway,causing a strong humoral immune response;escape through lysosomes,and passed MHC I Molecular presentation causes a strong cellular immune response.The results of high-throughput sequencing combined with flow cytometry and confocal detection showed that after PEI-PPAS/OVA was phagocytosed by DCs,parts of PEI-PPAS/OVA is treated by lysosomes and passed through the MHC II molecular presentation pathway,inducing a strong humoral immune response;part of PEI-PPAS/OVA escapes through the lysosome and passed through the MHC I molecular presentation pathway,inducing a strong cellular immune response.It showed that PEI-PPAS produced a long-term and effective immune response through good slow-release effect and lymph node targeting,and induced a strong humoral and cellular immune response through the presentation of MHC II and MHC I molecules presentation pathway.In summary,PEI-PPAS prepared by assembly based on PEI-AHPP as a vaccine adjuvant for OVA and H9N2 antigens can stimulate the body to produce long-term and strong cellular and humoral immune responses through various pathways.PEI-PPAS adjuvant has higher antigen loading rate and immune enhancement activity than the other nano-adjuvant groups.Therefore,PEI-PPAS is a promising immune immune enhancer for vaccines.