Study On The Adjuvant Activity And Mechanism Of Polyethylenimine-coated PLGA Nanoparticles-encapsulated Angelica Sinensis Polysaccharide

Angelica sinensi polysaccharide(ASP),one of the main active components of the Chinese herbal medicine Angelica sinensis,has the immune-enhancing effect.Modern pharmacological studies have found that ASP can be used as an immunopotentiator to promote the development of the immune organs,promote the proliferation and activation of T and B lymphocytes,improve the functions of immune cells such as macrophages and dendritic cells,enhance the expressions of antibodies and cytokines,and improve the immune responses.However,as a water-soluble polysaccharide,ASP has some disadvantages,such as easy degradation,short half-life,poor targeting,low bioavailability,and high dosage,which limits the clinical application of ASP in livestock and poultry.Based on this problem,we selected Poly(lactic-co-glycolic acid)(PLGA)nanoparticles as the carrier of ASP.The cationic PLGA nanoparticle-based adjuvant delivery systems have good biodegradability and biodegradability,could load antigens and immunopotentiators,effectively protect antigens and immunopotentiators from degradation,provide a controlled and sustained release of antigens and immunopotentiators,target antigen-presenting cells and lymph nodes,enhance the effects of immunopotentiators and improve the immunogenicity of antigens,thereby enhancing the immune responses.In this study,the cationic polymer polyethylenimine(PEI)was used to modify the surface of PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide to prepare a new cationic Angelica polysaccharide nano-delivery system(ASP-PLGA-PEI),and explore the adjuvant activity and action mechanisms.First,the preparation conditions of the ASP-PLGA-PEI were optimized,and the stability of the ASP-PLGA-PEI was determined.Secondly,the effects of the ASP-PLGA-PEI on the function of macrophages in vitro were investigated.Next,the ASP-PLGA-PEI was utilized as the adjuvants of different antigens and vaccines(PCV2 vaccines,chicken H9N2 inactivated vaccines and OVA antigen)to induce the immune responses in vivo experiments,and the safety of ASP-PLGA-PEI-based vaccines were evaluated.In addition,the effects of different antigen loading modes and immunization administration routes of ASP-PLGA-PEI on immune responses were investigated.The best antigen loading mode and administration route were determined,and the mechanism of its adjuvant activity were clarified.Finally,the signal pathway of ASP-PLGA-PEI to promote the activation of dendritic cells(DCs),and the endocytic pathway taken up by DCs were investigated to illustrate its potential mechanism.The experimental study is divided into the following seven parts:Experiment 1 Preparation and quality evaluation of polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharideThe polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide(ASP-PLGA-PEI)were prepared by the double emulsion solvent evaporation method.The ASP was encapsulated into PLGA nanoparticles,and the cationic polymer polyethyleneimine(PEI)was coated on the surface of nanoparticles to modify the charges to be positive.In this experiment,the content of PEI in the nanoparticles system was optimized.The particle size,zeta potential,polydispersity index(PDI),and the encapsulation efficiency of ASP with different mass ratio of PLGA to PEI(w/w)were determined to evaluate the best preparation conditions.Secondly,the morphology and distribution of ASP-PLGA-PEI under the best conditions were observed by transmission electron microscope(TEM).Finally,the ASP-PLGA-PEI solution was stored at 4℃,and the stability was determined.The results demonstrated that the optimal ratio of PLGA to PEI(w/w)at 10:1 was chosen to fabricate the ASP-PLGA-PEI nanoparticles.The particle size was 257.5±13.45 nm,the PDI was 0.211±0.017,the zeta potential was 41.8±0.23m V,and the encapsulation efficiency of ASP was 64.53±2.019%.The morphology of the ASP-PLGA-PEI was smooth spherical nanoparticles with uniform size distribution.The ASP-PLGA-PEI is relatively stable for 28 days at 4°C.The particle size,zeta potential and polydispersity index did not change with large amplitude over 28 days,and the encapsulation efficiency of ASP was slowly decreased over 28 days.The results indicated that the ASP-PLGA-PEI were successfully prepared with high ASP-encapsulation efficiency and good stability.Experiment2Effectsofpolyethylenimine-coatedPLGA nanoparticles-encapsulated Angelica sinensis polysaccharide on mouse peritoneal macrophages in vitroThe goal of this experiment was to investigate the effects of ASP-PLGA-PEI on the functions of macrophages in vitro.Firstly,the ASP-PLGA-PEI was cultured with macrophages,and the cell viability was determined by MTT.Secondly,to evaluate the effects of ASP-PLGA-PEI on the activation of macrophages,the expressions of surface molecules on macrophages were determined by flow cytometry,and the the expressions of cytokines in the cell supernatant were determined by ELISA kits.Finally,to investigate the antigen uptake by macrophages,the ASP-PLGA-PEI and OVA-FITC antigen were mixed,and the antigen uptake efficiency was determined after incubation with macrophages by using flow cytometry.The images of antigen uptake by macrophages were observed by confocal laser scanning microscopy(CLSM).The results demonstrated that the ASP-PLGA-PEI significantly promoted the expressions of molecules MHCII and CD86,and promoted the secretions of cytokines IL-1βand IL-12p70,which was significantly higher than that of ASP and ASP-PLGA.In addition,the ASP-PLGA-PEI effectively adsorbed antigens,and promoted the antigen uptake by macrophages.While,the ASP and ASP-PLGA failed to promote the antigen uptake by macrophages.The results suggested that the ASP-PLGA-PEI could effectively activate macrophages and enhance the antigen uptake by macrophages,thereby promoting immune response.Experiment 3 Adjuvant activity of polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide for PCV2 vaccineThis experiment aimed to evaluate the adjuvant activity of ASP-PLGA-PEI for porcine circovirus type 2(PCV2)vaccine in vivo.Firstly,the ASP-PLGA-PEI and PCV2 antigen solution were mixed,then the characterization and stability of nanoparticles vaccines were determined.Secondly,the mice were subcutaneously immunized with the nanoparticles vaccines,and the ISA-206 adjuvant was used as the positive group.To evaluate the immune resposnes induced by ASP-PLGA-PEI-PCV2,the levels of PCV2-specific antibody Ig G and subtypes Ig G1,Ig G2a and Ig G2b,and the expression levels of cytokines IFN-γ,TNF-α,IL-4 and IL-6 in the serum were determined.In addition,the histological analysis of the spleens was carried out.The results showed that after ASP-PLGA-PEI adsorbed PCV2antigen,the particle size obviously increased,and the PCV2 antigen adsorption rate was30.47±0.790%.The ASP-PLGA-PEI nanoparticles vaccine could remain stable at 4℃over 14 days.In vivo experiment demonstrated that the ASP-PLGA-PEI as the adjuvant for PCV2 antigen promoted the strong and long-lasting specific antibody expression,increased the expressions of Th1-type related antibodies Ig G2a and Ig G2b and Th2-type related antibody Ig G1.The levels of Th1 type antibody were stronger than that of ISA-206adjuvant.In addition,the ASP-PLGA-PEI-PCV2 vaccine promoted the expressions of Th1and Th2 cytokines,promoted the enlargement of spleen nodules,and improved the activation of splenic lymphocytes.The results suggested that ASP-PLGA-PEI as an adjuvant for the PCV2 vaccine could effectively adsorb antigen,induce a strong and long-lasting immune response,and elict a mixed Th1 and Th2 immune responses.Experiment 4 Comparison of Polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide with Alum and oil-based adjuvants as adjuvants for H9N2 vaccineIn this experiment,we further explored the adjuvant activity of ASP-PLGA-PEI for inactivated H9N2 vaccine on the basis of the previous experimnets,and compared to the common aluminum adjuvant(Alum)and oil adjuvant(ISA-206).Firstly,the ASP-PLGA-PEI and the inactivated H9N2 vaccine were mixed to develop the nanoparticles vaccines,and the characterizations of nanoparticle vaccines were then determined.Secondly,those differernt H9N2 vaccines were subcutaneously injected into chickens,and the HI antibody titers and antigen-specifc Ig G in serum were determined at different time points.The proportion of CD3~+CD4~+and CD3~+CD8~+T cells in peripheral blood lymphocytes were determined by flow cytometry.And the m RNA expression levels of IFN-γand IL-10 in peripheral blood lymphocyte were measured,and the expression levels of IFN-γand IL-10 in serum were determined.Finally,the organ indexs of immune organs were measured,the histological analysis was performed,the safety of nanoparticles vaccines was evaluated,and the expression of CD4~+and CD8~+T cells in the spleen and thymus was measured by immunofluorescence.The results showed that ASP-PLGA-PEI,as an adjuvant for the inactivated H9N2 vaccine,induced high levels of HI titers and anti-specific Ig G antibody,promoted the activation of CD4~+and CD8~+T cells,increased the expression of IFN-γ,and promoted humoral and cellular immune responses.The Alum and ISA-206 adjuvants intelligently induced high levels of antibody responses,but failed to promote the activation of CD4~+and CD8~+T cells.The results suggested that ASP-PLGA-PEI,as an adjuvant for inactivated H9N2 vaccine,could simultaneously induce strong humoral immune response and cellular immune response in chickens,and better than aluminum adjuvant and ISA-206 adjuvant.Experiment 5 Antigen loading mode of polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide affect immune responsesThe purpose of this experiment was to expore the adjuvant activity of ASP-PLGA-PEI for subunit vaccines,investigate the effect of the antigen loading mode of cationic PLGA nanoparticle-based vaccine delivery system on the immune responses,and clarify the mechanism of nanoparticle adjuvants.Firstly,three different OVA-loaded and ASP-encapsulated PLGA nanoparticles with different surface charges and antigen loading modes were developed.The OVA was used as the model antigen,and the PEI was used modify the surface charges of nanoparticles.The three nanoparticles were designed as antigen-encapsulated with negatively charged(ASP-PLGA/OVA),antigen-encapsulated with positive charged(ASP-PLGA/OVA-PEI)and antigen-adsorbed with positive charged(ASP-PLGA-PEI-OVA).The characterization of the three nanoparticles vaccines and the release of ASP and OVA in vitro were determined.Next,the antigen uptake of the three nanoparticles and the co-localization of the antigen and lysosomes were determined by flow cytometry and CLSM.Finally,the three nanoparticles vaccines were injected subcutaneously into mice.To evaluate the immnune responses induced by three nanoparticles vaccines,the levels of OVA-specific antibody Ig G and its subtype Ig G2a and Ig G1 in serum,the activation of CD4~+and CD8~+T cells in the spleen lymphocytes,and the response of memory T cells were measured.The results showed that all three nanoparticles provided a contolled and sustained release of ASP in vitro.The antigen-encapsulated nanoparticles provided a contolled and sustained release of OVA,while the antigen-adsorbed nanoparticles failed to slowly release OVA.The cationic modified PLGA nanoparticles(ASP-PLGA/OVA-PEI and ASP-PLGA-PEI-OVA)promoted the antigen escape from lysosomes and improved the cytoplasmic antigen presentation.In vivo experiment results demonstrated that cationic modified PLGA nanoparticles promoted the activation of CD4~+and CD8~+T cells,and improved the response of memory T cells.In addition,antigen-encapsulated nanoparticles(ASP-PLGA/OVA and ASP-PLGA/OVA-PEI)induced strong and long-lasting expressions of antigen-specific antibody,compared to antigen-adsorbed nanoparticles.In summary,the results sugested that ASP-PLGA/OVA-PEI nanoparticles,which were cationic nanoparticles with antigen-encapsulated,could promote the activation of CD4~+and CD8~+T cells and memory T cells,effectively induce the expression of antigen-specific antibodies and antibody subtypes,and induce a stronger immune response.Experiment 6 Administration routes of polyethylenimine-coated PLGA nanoparticles-encapsulatd Angelica Sinensis polysaccharide vaccine delivery system affect immune responsesThe goal of this experiment was to further investigate how the administration routes(intramuscular injection(i.m.)and subcutaneous injection(s.c.))affect the immune responses and clarify the mechanism of cationic PLGA nanoparticle-encapsulatd Angelica sinensis polysaccharide vaccine delivery system.The ASP-PLGA/OVA-PEI,which was proved the optimal nanoparticles vaccines in the previous experiments,was chosed as the nanoparticle vaccines(NPs)in this study.The NPs vaccine was intramuscularly and subcutaneously injected(i.m.and s.c.)into mice,and the immune responses induced by these two delivery routes were compared.Firstly,the activation of DCs in the lymph nodesand available antigen in draining lymph nodes were determined.Secondly,the levels of anti-specific antibody Ig G and subtypes Ig G2a and Ig G1 in the serum were measured at different time points.And the expressions of cytokines IFN-γ,TNF-αand IL-6 in the serum were determined.Finally,the changes in the subset of T lymphocytes and the activation of cytotoxic T lymphocytes were measured.The results showed that both i.m.and s.c.administration of NPs vaccines elicited strong antigen-specific Ig G,Ig G1,and Ig G2a antibody responses,with no significant difference.However,compared with i.m.,NPs with s.c.promoted the activation of lymph node DCs,improved the antigen transport into draining lymph nodes,enhanced the secretion of cytokines IFN-γ,TNF-αand IL-6,increased the ratio of CD4~+/CD8~+of T cells,promoted the activation of cytotoxic T lymphocytes response,and induced a stronger cellular immune response.The results indicated that compared with i.m.,cationic PLGA nanoparticle-encapsulatd Angelica sinensis polysaccharide vaccine delivery system with subcutaneous injection was more suitable.Experiment 7 Polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide promote DCs activation and associated molecular mechanismsIn this experiment,we investigated the effects of ASP-PLGA-PEI on activation and maturation of the bone marrow-derived dendritic cells,and further explored the underlying mechanism.Firstly,the dendritic cells derived from mouse bone marrow were cultured in vitro.After incubation with ASP-PLGA-PEI,the cell vaiabilty was determined by using MTT.Secondly,to evaluate the activation and maturation of DCs with stimulation of ASP-PLGA-PEI,the expressions of co-stimulatory molecules CD40,CD80 and CD86 on DCs were measured,and the expression levels of cytokines TNF-αand IL-12p70 were determined.Next,after incubation with ASP-PLGA-PEI,the transcriptome sequencing(RNA-seq)of DCs was performed to analyze the differentially expressed genes(DEGs)and differentially expressed genes KEGG enrichment pathway.And the RT-q PCR method was used to verify the transcriptome sequencing results.The Western blot method was used to determine and verify the associated signal pathway.Finally,the potential endocytosis pathway of cationic PLGA-based nanoparticles by DCs was determined by using different endocytosis pathway inhibitors.The results showed that ASP-PLGA-PEI significantly up-regulated the expression levels of molecules CD40,CD80 and CD86,and promoted the expression levels of cytokines TNF-αand IL-12p70.Differentially expressed genes analysis showed 2812 genes were differentially expressed in a significant manner,in which 1323genes were up-regulated and 1489 genes were down-regulated.Furthermore,ASP-PLGA-PEI promoted the expression of p JAK2 protein in the JAK2/STAT3 pathway,and the expression of co-stimulatory molecules and cytokines induced by ASP-PLGA-PEI nanoparticles were decreased with the presence of the inhibitor of JAK2/STAT3 signaling pathway.In addition,the nanoparticles were internalized by DCs mainly through the clathrin-mediated endocytosis and micropinocytosis.These results demonstrated that the ASP-PLGA-PEI could promote the DCs activation and maturation via a complex interaction network,in which the JAK2/STAT3 signaling pathway plays a crucial role in regulating the expression of co-stimulatory molecules and cytokines.