| Lycium barbarum is a traditional Chinese nourishing medicine that be commonly used."Compendium of Materia Medica" records:"Goji nourish liver and kidney,nourish lung and produce body fluid,enhance immunity,delay aging,whitening back to black,teeth fall and grow again." Lycium barbarum polysaccharide(LBP)is the main active ingredient extracted from goji berries and has a wide range of pharmacological activities such as anti-aging,anti-tumor,anti-oxidation,immunomodulation and so on.However,LBP molecular weight between 24-24 1kd,molecular weight is large,there are many defects,such as fast metabolism,difficult to maintain a high concentration of plasma,targeting is poor,action range is not concentrated,large clinical dosage and so on.Liposomes,a kind of bilayered vesicles formed by self-assembly of phospholipid molecules in an aqueous medium,are widely used as a vehicle for delivering various therapeutic agents due to their high biocompatibility,diverse and high-loading capacity.And liposomes are easy for preparation and surface decoration to engender multifunctional features.Liposomes are safe and effective drug-loading systems and can be used as adjuvant.And LBP have the function of regulating the body’s immunity.In this study,liposomes were used as a delivery carrier to encapsulate LBP,which were used to improve the immune protection effect of the vaccine and enhance cellular immunity and humoral immunity of the body.Firstly,reverse-phase evaporation was used to maximize the loading of LBP into liposomes to achieve efficient loading and targeted delivery of polysaccharides.Secondly,the characterizations and quality of Lycium barbarum polysaccharide liposome(LBPL)were evaluated,and then the experiments on immune cells in vitro were carried out.Furthermore,LBPL was encapsulated with two different types of antigens and tested in vivo to detect the immune enhancement effect on animals and its immune adjuvant activity.Finally,the effect of LBPL on antigen-presenting cells macrophages and dendritic cells(DCs)and the mechanism by which Toll-like receptor 4(TLR4)pathway activates immunopotentiation were investigated.The experiments were divided into the following eight parts:Experiment 1 Preparation of Lycium barbarum polysaccharide liposome and optimization of its preparation conditionsThe purpose of this experiment was to screen out the optimal preparation methods and conditions for Lycium barbarum polysaccharide liposome(LBPL).LBPL were prepared by reverse-phase evaporation method and optimized by response surface methodology.The liposomes and unencapsulated drugs were separated by microcolumn centrifugation,the concentration of LBP was determined by phenol-concentrated sulfuric acid method,and the encapsulation rate of liposomes was calculated.Taking the encapsulation efficiency and drug loading of LBPL as indicators,the effects of four single factors on the preparation of LBPL,such as ratio of lipid to drug,ultrasonic time,water bath temperature and ratio of soybean phospholipids to cholesterol,were investigated.The results showed that three single factors(ratio of lipid to drug,ultrasonic time and ratio of soybean phospholipids to cholesterol)had a greater impact on the preparation of LBPL.According to the results of single-factor test,response surface analysis was used to study the influence of three imoportant factors on the preparation of LBPL by taking the encapsulation rate as the response value,totally 17 tests were conducted.The experimental results showed that the optimal preparation conditions for LBPL:the ratio of lipid to drug ratio was 25:1,the ultrasonic time was 14mins,and the ratio of membrane to material ratio was 2.4:1.According to the verification test of the optimal preparation conditions,the encapsulation rate of liposomes obtained was 86.37±0.63%,with a relatively small error compared with the predicted value of 87.671%.It indicated that the encapsulation efficiency of LBPL prepared by reverse phase evaporation method was high.The response surface method can optimize the preparation conditions of LBPL very well,and the encapsulation efficiency reached more than 85%.Experiment 2 The quality evaluation of Lycium barbarum polysaccharide liposomeThis study was designed to evaluate the morphological characteristics,particle size,stability and drug release of LBPL.LBPL prepared under the optimal conditions obtained in the previous chapter,and the ultrastructure of LBPL was observed under transmission electron microscope.The particle size and potential of LBPL were measured by laser particle size analyzer.LBPL and blank liposomes stored at 4℃,and the changes in liposome size and polydispersity index(PDI)were monitored at four time points of 7,15,30,and 45 days,respectively.LBPL and LBP were added into dialysis bags to simulate the humoral environment,and the polysaccharide release curve was measured within 96 h.The results showed that the LBPL particles were nearly spherical with uniform shape and size.The average particle size was 120.7 ± 0.84 nm,which is electrically neutral.Within 45 days,the particle size and PDI of LBPL changed little.The polysaccharides in LBPL were slowly released under the conditions of pH 7.4.It indicated that the morphology of LBPL was well characterized,the particle size is uniform and the stability of LBPL was good.The liposome as a drug carrier had a good sustained release effect on the encapsulated LBP at pH 7.4.Experiment 3 Effects of Lycium barbarum polysaccharide liposome on proliferation and differentiation of mice spleen lymphocytesThe purpose of this experiment was to investigate the maximum safe concentration of LBPL and the effects of LBPL on mice spleen T and B lymphocyte proliferation and T lymphocyte phenotype.The maximum safe concentration of LBPL and LBP on mouse spleen lymphocytes were firstly determined by MTT assay.After 48h of treatment with 11 concentrations of LBPL and LBP,the maximum concentration that was not significantly lower than that of the cell of control group was selected as the maximum safe concentration.Then,five concentrations below the safe concentration of LBPL and LBP were selected to stimulate T lymphocytes proliferation either alone or in combination with PHA,and B lymphocytes proliferation was stimulated by alone or in combination with LPS.Finally,the three concentrations after the maximum safe concentration of LBPL and LBP were added to lymphocytes for 48 hours.CD3,CD4 and CD8 antibodies were added to cells and incubated for 0.5 h at 4℃ in the dark,and the cells were detected by flow cytometry.The results showed that LBPL had a more significant proliferative effect than LBP,and combined with PHA to promote the proliferation of T lymphocytes,and promoted the proliferation of B lymphocytes synergistically with LPS.LBPL was stronger than the effect of LBP on lymphocytes proliferation,and the effective range is large.The proportion of lymphocyte CD3+ phenotype in the group of high LBPL concentration was the highest,and the proportion of lymphocyte CD4/CD8 phenotype in the medium concentration group was the highest,and the groups of high and medium LBPL concentration were significantly higher than the other control groups.It indicated that the safe concentration of LBP after liposome encapsulation increased and the effects of LBP on spleen lymphocyte proliferation and phenotypic differentiation were significantly improved by liposome encapsulation.Experiment 4 Effects of Lycium barbarum polysaccharide liposome on immune function of macrophagesThis study was designed to investigate the effects of LBPL in activating mouse peritoneal macrophage immune function.Mouse peritoneal macrophages were isolated and cultured in vitro.In the experiment of phagocytosis of FITC-labeled Escherichia coli,the fluorescence intensity was detected by flow cytometry,and the percentage of phagocytosis was calculated.LBPL and LBP were respectively treated mouse peritoneal macrophages,and the levels of pro-inflammatory cytokines,NO and INOS secreted from mouse peritoneal macrophages were detected by ELISA.The results showed that LBPL activated macrophages and enhanced their phagocytic function.The mean fluorescence intensity of LBPL low concentration group was the highest,which was significantly higher than other groups including LPS positive control group(P<0.05).The levels of IL-1β,TNF-α,IFN-y and inflammatory factors NO and iNOS secreted by macrophages increased to varying degrees with the stimulation of LBPL.It indicated that LBPL could promote the phagocytic function of macrophages significantly.The low concentration of LBPL had the highest rate of action to macrophages phagocytized fluorescently labeled E.coli and the fluorescence intensity of per cell was the highest.And LBPL activated the immune function of macrophages.Experiment 5 Effects of Lycium barbarum polysaccharide liposome on maturation and antigen presentation of bone marrow dendritic cellsThe purpose of this experiment was to investigate the effects of LBPL on the maturation of antigen presenting cells DCs,cytokine secretion and antigen presentation.In vitro,tibia and femur of mice were isolated to prepare bone marrow mononuclear cell suspension.LBPL,LBP and BL were added to cells after 15 h.In addition,a cell control group(BC)was set and LPS was used as the positive control group.After cultured for 48 h,the effect of LBPL on proliferation of DCs precursor cells was detected by MTT method.Induced by stimulating factor,differentiated into immature DCs.On the seventh day,125μg·mL-1 LBPL,LBP and BL were treated the cells for 48 h.MHC-Ⅱ,CD86 and CD80 antibodies were added to cells in the dark for 0.5 h.Flow cytometry was used to detect the expression of the surface co-stimulatory molecules of bone marrow derived dendritic cells.The effects of LBPL on the secretion of cytokines IL-12p40 and TNF-α of dendritic cells were detected by ELISA.The effect of LBPL on DCs uptaking FITC-labeled antigen was observed by laser confocal microscopy.The results showed that LBPL significantly promoted the proliferation of dendritic precursor cells compared with other control groups.The expression of co-stimulatory molecules MHC-Π,CD80 and CD86 on the surface of dendritic cells increased significantly after stimulation with LBPL.LBPL significantly increased the levels of cytokines IL-12p40 and TNF-α secreted by dendritic cells.The antigen uptake of DCs was significantly increased after the stimulation of LBPL.It indicated that encapsulation of LBP in liposomes had a more significant effect on promoting maturation of DCs and the ability on stimulating DCs to uptake antigens was improved.Experiment 6 Adjuvant activity of Lycium barbarum polysaccharide liposome on mice immunized with OVA vaccineThe purpose of this study was to investigate the immunoadjuvant effect of LBPL on mice immunized with encapsulated antigen OVA vaccine.OVA(250 μg·mL-1)was wrapped in LBPL and the rate of encapsulation was increased by repeated freeze-thaw cycles.The particle size and PDI of LBPL-OVA stored at 4℃ for 45 d were measured.The stability of encapsulation efficiency of LBPL-OVA at 4℃ and 37℃ was observed.LBPL-OVA was used to detect the antigen release profile at specific time points within 120 h in neutral(pH 7.4)and acidic(pH 5.0)phosphate buffers.120 BALB/c mice aged 5 weeks were randomly divided into 6 groups:LBPL-OVA,BL-OVA,LBP-OVA,OVA,CFA-OVA,BC,and each mouse was subcutaneously immunized with 0.2 mL.Immunize once a week for a total of three immunizations.At 24h and 48h after the first immunization,the lymph nodes of the mice in each experimental group were used to detect the dendritic cell phenotype by flow cytometry.7 days after the first immunization,the lymph nodes of the mice in each experimental group were taken for immunohistochemical sections.On the 14th and 28th day after the third immunization,4 mice were randomly selected from each group,and the spleens were sterilely obtained.The MTT method was used to determine the stimulation indexes of antigen re-stimulation,T and B lymphocytes by PHA and LPS,respectively.The phenotypic changes of T lymphocytes were detected by flow cytometry.Serum was collected from blood samples on days 7,14,21 and 28,and concentrations of OVA-specific IgG,IgG1 and IgG2a and cytokines IFN-γ,TNF-α,IL-4 and IL-6 were determined by ELISA.The results showed that the encapsulation efficiency of LBPL-OVA was higher and more stable at 4℃ and the antigen was slowly and slowly released at pH 7.4.After LBPL-OVA immunized mice,the expression levels of three surface molecules on DCs were significantly increased in lymph nodes;the amount of antigen in lymph nodes increased;the lymphocyte stimulation index increased;the ratio of CD3+subpopulation in lymphocytes and proportion of CD4+/CD8+ T cells increased;the amount of antibodies and cytokines in serum increased.The ratio of IgG2a/IgG1 in the LBPL-OVA group was significantly higher than the other groups.It indicated that LBPL-OVA activated dendritic cells in draining lymph nodes and antigenic dose of DCs in lymph nodes were increased,effectively stimulating CD4+CD8+T cell proliferation.LBPL-OVA significantly improved the body’s humoral and cellular immune responses and had a tendency to Th1 type immune response.Experiment 7 Adjuvant activity of Lycium barbarum polysaccharide liposome on mice immuned with PCV2 vaccineThe purpose of this experiment was to study the immunoadjuvant activity of LBPL against PCV2 as an antigen.The LBPL and PCV2 antigen were mixed and stirred at a volume ratio of 3:1,and the encapsulation efficiency was increased by repeated freeze-thaw cycles.Then the particle size and PDI size of LBPL-PCV2 were detected.125 BALB/c mice aged 5 weeks were randomly divided into 5 groups with 25 mice in each group.Subcutaneous immunization was performed twice,with an interval of 2 weeks.The vaccine groups were LBPL-PCV2(the volume ratio of LBPL to PCV2 was 3:1,the same below),BL-PCV2,LBP-PCV2,Positive control group W/O-PCV2,PBS was used as blank control group(BC).Mice blood samples were collected after the second immunization,and antibodies,Th1 type cytokines(IFN-y and TNF-α)and Th2 type cytokines(IL-4)in serum were measured.Two weeks after the second immunization,the spleens of mice in each group were collected,subjected to H&E staining,and the histomorphological changes of the spleens were observed under a microscope.The results showed that the average particle size of LBPL-PCV2 was 122.1 ± 0.78 nm,and the polydispersity index(PDI)was 0.248.LBPL as an immunoadjuvant significantly increased the production of PCV2-specific antibody IgG and antibody IgG1,and promoted the secretion of cytokines IFN-y,TNF-αand IL-4.The most obvious changes occurred in the spleen of the LBPL-PCV2 group.The peripheral lymphatic sheath in the middle of the white pulp was thickened,the number and volume of the splenic corpuscle were increased,and full of lymphocytes were found,and the spleen germinal center was obvious.It indicated that LBPL-PCV2 stimulated the body to produce effective Thl and Th2 type immune responses.As a vaccine adjuvant,LBPL has a good improvement and stimulating effect on the spleen.Experiment 8 Effect of Lycium barbarum polysaccharide liposome on activating the TLR4/MyD88/NF-KB pathway of dendritic cellsThis study was designed to explore the mechanism by which LBPL exerts immunopotentiating effect.Mouse bone marrow-derived dendritic cells(DCs)were isolated and cultured,and induced by granulocyte-macrophage colony-stimulating factor to differentiate into immature dendritic cells.Cells were treated with three different concentrations of LBPL,PLH(125 μg·mL-1 LBPL),PLM(62.5 μg·mL-1 LBPL),and PLL(31.25 μg·mL-1 LBPL).After 24 hours of drug treatment,the mRNA expression levels of TLR4 signaling pathway-related molecules in dendritic cells were analyzed by qPCR.The protein expression levels of TLR4,TRAF6,MyD88 and NF-κB in the nucleus of DCs treated by different drug groups were analyzed by Western Blot.The experimental data showed that the relative transcription of NF-κB mRNA in dendritic cells was significantly increased with the treatment of three concentrations of LBPL.At the concentration 125μg·mL-1 of LBPL group,the mRNA relative transcription of the four related molecules in the TLR4 signaling pathway were significantly increased,and the expression was the highest in three concentrations.The protein expression levels of TLR4,TRAF6,MyD88 and NF-κB in DCs nucleus of the PLH group were higher than those of the other groups except the LPS positive control.These results indicated that LBPL played a positive role in the mRNA and protein expression of related molecules in the TLR4/MyD88/NF-κB pathway of DCs.LBPL had effect on DCs by activating the TLR4/MyD88/NF-κB pathway,thereby exerting an immunopotentiating effect. |
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