| 1 ObjecticesInhalation of foreign pathogenic microorgamisms such as influenza virus and Mycobacterium tuberculosis into body through the respiratory tract can cause a variety of diseases and seriously affect human health.Compared with the subcutaneous injection of vaccine,nasal mucosal immunization can cause local mucosal immune responses and directly resist the invasion of microorganisms.The physiological environment of nasal cavity can prevent the pathogen from entering the body.However,it also limits the effectiveness of direct intranasal immunization of inactivated or attenuated viral antigens.Therefore,the discovery of novel nasal mucosal vaccine adjuvants is essential in the application of nasal immunization.Curdlan is a linear β-1,3-glucan produced by Alcaligenes faecalis,which is water-insoluble.Curdlan sulfate(CS)obtained by sulfation of 2’-OH and 6’-OH is not only soluble in water,but also has the activities of anti-oxidation,anti-HIV infection and immune regulation.Our previous study found that CS could interact with hepatitis B virus(HBV),inhibit the process of virus entry,and thus have anti-HBV activity.CS could specifically bind to dectin-1 receptor and then activate antigen-presenting cells(APCs)through MAPKs pathway to induce the production of cytokines.As a hepatitis B vaccine adjuvant,CS could improve the immunogenicity of HBsAg and provoke the humoral and cellular immunity.Therefore,we intend to use CS as an immunostimulant to improve the immunogenicity of antigens.To enhance the efficient delivery of antigens,we chose to prepare nanocarriers suitable for protein permeable absorption.6-O-2’-hydroxylpropyltrimethyl ammonium chloride chitosan(O-HTCC)is a water-soluble 6-O-quaternized chitoan.O-HTCC can act as a good protein carrier because the quaternary amination modification of 6’-OH increases the positive charge density.Using the charge properties of CS and O-HTCC,we prepared positively charged CS/O-HTCC nanoparticles.The application of CS/O-HTCC nanoparticles as a nasal mucosal vaccine adjuvant was studied by evaluating the antigen carrier activity and effectiveness of intranasal immunization.Afterwards,the effects of CS/O-HTCC nanoparticles on immune cells and mucosal layers were evaluated to investigate the mechanism of action of CS/O-HTCC nanoparticles as nasal mucosal vaccine adjuvants.2 Method2.1 The preparation and evaluation of physicochemical properties of CS/O-HTCCnanoparticlesCurdlan was modified by sulfur trioxide-pyridine system and chitosan was modified by benzaldehyde-glycidyltrimethylammonium chloride system to form CS and O-HTCC.The CS/O-HTCC nanoparticles were obtained by ionic gelation method,and the particle size,zeta potential and polydispersity of the nanoparticles were detected by laser particle size analyzer.The carrier efficiency and in vitro release were detected by SDS-PAGE.2.2 In vivo study of CS/O-HTCC nanoparticles acting as nasal mucosal vaccine adjuvantOvalbumin(OVA)as a model antigen was adsorbed to CS/O-HTCC nanoparticles.Balb/c mice were immunized intranasally on day 1,15 and 29.The OVA-specific antibodies in serum and mucosal lavage fluid of the immunized mice were detected by ELISA.The proliferation and transformation activity of spleen lymphocytes were detected by MTT assay.The neutral erythrocyte reagent was used to detect the phagocytic ability of macrophages.The surface markers of immune cells were detected by flow cytometry.2.3 In vitro immunostimulatory activity and mechanism study of CS/O-HTCC nanoparticlesMice peritoneal macrophages were isolated.The phagocytic ability of macrophages was detected by flow cytometry.The cellular distribution of nanoparticles in macrophages was observed by confocal microscopy and transmission electron microscopy.The expression of iNOS and Cox-2 was detected by Western blot.The transcription levels of iNOS,Cox-2,IL-1β,IL-6 and TNF-α were detected by RT-PCR.The content of NO was detected by Griess reagent method,and the content of IL-1β,IL-6 and TNF-α were detected by ELISA.Mice bone marrow-derived dendritic cells(BMDCs)were isolated.The expressions of surface markers were detected by flow cytometry.The transcription levels of CXCL-10,IFNAR1,IFNAR2 and IFN-β were detected by RT-PCR.The productions of IL-6 and IL-12 were detected by ELISA kit.The amount of IFN+lymphocytes in BMDCs/lymphocytes co-cultere system was detected by flow cytometry to evaluate the functional activation of BMDCs.Mice spleen lymphocytes were isolated.The proliferation of lymphocytes was detected by CCK-8 assay.The expression levels of p-AKT,p-ERK,p-P38,p-JNK,p-NF-κB,Bax,Bcl-2 and Caspase-3 were detected by Western blot.The transcription levels of CTLA-4,IL-2 and TGF-β were detected by RT-PCR.2.4 Transmembrane transport activity and mechanism study of CS/O-HTCC nanoparticlesCalu-3 cell line was used to construct in vitro nasal mucosa model.The cytotoxicity of nanoparticles was detected by CCK-8 method.The ability of nanoparticles to be taken up by epithelial cells was detected by fluorescence microscopy and flow cytometry.The transmucosal ability of CS/O-HTCC nanoparticles was detected by fluorescence microplate reader.Endocytic mechanism studies:Using endocytic inhibitors(chlorpromazine hydrochloride,genistein and cytochalasin D)stimulate epithelial cells.Flow cytometry was used to detect the effect of CS/O-HTCC nanoparticles on antigen endocytosis.Study of the mechanism of paracellular transportion pathway:Western blot was used to detect the expression of tight junction-associated proteins and adherent junction-associated proteins.3 Results3.1 Preparation and evaluation of CS/O-HTCC nanoparticlesCurdlan sulfate(a degree of sulfation of 13.74%)and 6-O-quaternized chitosan were prepared.According to the concentration ratio of 1:5,CS/O-HTCC nanoparticles with a particle size of 167.03±5.88 nm,a potential of 50.10±0.85 mV and a uniform dispersion(PDI=0.12±0.01)were prepared by dropwise addition.OVA could be adsorbed onto CS/O-HTCC nanoparticles at a ratio of 4:1:5.The nanoparticles had highly loading efficiency(72.60%±2.21%)and sustained release under physiological conditions of pH 7.4.3.2 Nasal immunization with OVA/CS/O-HTCC nanoparticles can enhance systemic and local mucosal immune responses in miceCompared with antigen alone,CS/O-HTCC nanoparticles as nasal mucosal vaccine adjuvants could significantly increase the proliferation(###p<0.001)and phagocytic activity(###p<0.001)of macrophages and promote recruitment and phenotypic activation of APCs in spleen after nasal immunization.Compared with the OVA treatment group,the proliferation(###p<0.001)and transformation(###p<0.001)activity of spleen lymphocytes were significantly improved after intranasal immunization of mice with OVA/CS/O-HTCC nanoparticles.CS and O-HTCC acting as vaccine adjuvants could promote the aggregation of CD3+CD4+T cells(*p<0.05,**p<0.01)and CD3+CD8+T cells(*p<0.05,*p<0.05)in spleen,but had no significant effect on helper T cells activation(p>0.05).After intranasal immunization with OVA/CS/O-HTCC nanoparticles,the amount of CD4+T cells(***p<0.001)and CD8+T cells(**p<0.01)in spleen increased more significantly,as well as the activated CD4+T cells(**p<0.01).After intranasal immunization with OVA/CS/O-HTCC nanoparticles,the antibody titers of OVA-specific IgG,IgG1,IgG2a and IgA in the serum were significantly increased,and the deviation of IgG1/IgG2a caused by OVA was significantly corrected(##p<0.01).By measuring the secretion levels of Thl and Th2 cytokines,it was also confirmed that compared with OVA,OVA/CS/O-HTCC nanoparticles could induce IFN-γ secretion after nasal immunization(#p<0.05),transforming the immune type to Th1 type.Through the detection of sIgA in mucosal lavage fluid,it was confirmed thatCS/O-HTCC nanoparticles as nasal mucosal vaccine adjuvants could significantly stimulate proximal mucosal immunity(###p<0.001)and distal mucosal immune response(#p<0.001).3.3 CS/O-HTCC nanoparticles induce pleiotropic activation of antigen-presentingcells For macrophages:①CS/O-HTCC nanoparticles could not only significantlyincrease the phagocytic capacity of cells(***p<0.001),but also help the antigens to be efficiently taken up.This activity is mainly dependent on the activity of O-HTCC.②By observing the subcellular localization of the antigen after ingestion,it was found that the antigens carried by the CS/O-HTCC nanoparticles were phagocytized by macrophages and lysosomal escape occurred.This effect may be related to the acid sensitivity of CS/O-HTCC nanoparticles.③CS/O-HTCC nanoparticles could significantly enhance the transcription,expression and secretion of various inflammatory factors,which are mainly dependent on the activity of CS.For dendritic cells:①CS/O-HTCC nanoparticles stimulated BMDCs tosignificantly increase the positive expression of CD40,CD80 and CD86 molecules,indicating that nanoparticles can cause phenotypic activation of dendritic cells.②After incubation with CS/O-HTCC nanoparticles,activated DCs were co-incubated with lymphocytes,and it was found that the DCs could significantly increase the positive expression of IFN-y in lymphocytes(***p<0.001).It is indicated that nanoparticles can induce functional activation of dendritic cells.③CS/OHTCC nanoparticles significantly stimulated the expression of MHC I and MHC II molecules on the surface of BMDCs simultaneously,suggesting that CS/O-HTCC nanoparticles can stimulate the cross-presentation of dendritic cells,which is mainly dependent on the activity of O-HTCC.④CS/O-HTCC nanoparticles could significantly increase the transcription of type I interferon-related genes,which depended on the activity of O-HTCC.⑤CS/O-HTCC nanoparticles induced high secretion levels of IL-6(***p<0.001)and IL-12(***p<0.001),which was mainly dependent on the activity of CS3.4 CS/O-HTCC nanoparticles directly stimulate lymphocytic proliferation activity through multiple pathwaysCS/O-HTCC nanoparticles treating lymphocytes directly can stimulate IL-2 transcription,down-regulate the transcription levels of CTLA-4 and TGF-β,upregulate the expression of p-AKT,p-ERK,and p-JNK and the phosphorylation of NF-κB.Furthermore,CS/O-HTCC nanoparticles downregulated of the expression of Bax,increased the expression of Bcl-2,thereby inhibited the activation of Caspase-3.Ultimately CS/O-HTCC nanoparticles significantly increased the growth rate of lymphocytes(*p<0.05).3.5 CS/O-HTCC nanoparticles could help the antigen penetrate the nasal mucosa,be taken up by submucosal APCs,and transport into cervical lymph nodeThe CS/O-HTCC nanoparticles did not affect the growth of epithelial cells at different concentrations,indicating that the nanoparticles had no obvious toxicity to the nasal mucosa.CS/O-HTCC nanoparticles could not only be taken up by epithelial cells(*p<0.05),but also effectively penetrate the dense nasal mucosa(***p<0.001).The study of permeation mechanism:①CS/O-HTCC nanoparticles could significantly inhibit the expression of TJP 1,OCLN,p-Claudin 4 and CDH 1(*p<0.05,**p<0.01,***p<0.001,**p<0.001),thereby opening tight junctions and adhesive junctions between epithelial cells.②Chlorpromazine hydrochloride,genistein and cytochalasin D could significantly inhibit FITC-OVA/CS/O-HTCC nanoparticles endocytosis(***p<0.001,**p<0.001,**p<0.001),confirming that CS/O-HTCC nanoparticles assist antigens taken up by epithelial cells via clathrin-mediated endocytosis,caveolin-mediated endocytosis and macrocytosis.CS/O-HTCC nanoparticles could stimulate phenotypic activation of submucosal APCs.By measuring the content of OVA in cervical lymph nodes at different time after nasal drops,we found that CS/O-HTCC nanoparticles could significantly increase the content of antigen into lymph nodes and prolong the duration of action.At 2h,the antigen content in the lymph nodes peaked.Through immunofluorescence experiments,we found that in FITC-OVA treatment group,the fluorescence signal in the cervical lymph nodes was weak;while after FITC-OVA/CS/O-HTCC nanoparticles dropped intranasally,macrophages and dendritic cells carried the ingested antigen entering the depth of the cervical lymph nodes and the fluorescence intensity was significantly higher.4 Novelties and Conclusions:(1)Uniform and positively charged CS/O-HTCC nanoparticles were prepared for the first time by ionic gelation method and the protein carrier effect of CS/O-HTCC nanoparticles was firstly confirmed.(2)The nasal mucosal vaccine adjuvant activity of CS/O-HTCC nanoparticles was firstly evaluated for the first time.After intranasal immunization,CS/O-HTCC nanoparticles could increase the immunogenicity of the loaded antigen,change the immunological type of the antigen,effectively activate the immune cells,promote the production of antigen-specific antibodies in serum and mucosal lavage,and enhance the systemic immune and mucosal immune responses.(3)The immunomodulatory activity of CS/O-HTCC nanoparticles was fistly evaluated.It was confirmed that CS/O-HTCC nanoparticles could pleiotropically stimulate the activation and maturation of macrophages and dendritic cells and directly stimulate the proliferation of lymphocytes.(4)The transmembrane transport capacity of CS/O-HTCC nanoparticles was firstly studied.It was confirmed that CS/O-HTCC nanoparticles could promote antigens taken up by epithelial cells via clathrin-mediated endocytosis,caveolin-mediated endocytosis and macrocytosis.When it entered the nasal mucosal epithelial cells,it crossed the dense nasal mucosal barrier by opening tight junctions and adhesive junctions between mucosal epithelial cells.CS/O-HTCC nanoparticles could assist the antigen taken by submucosal APCs and transported to the cervical draining lymph nodes to elicit a further immune response. |
PDF Full Text Request