Study On Immunological Enhancement Activity Of Ganoderma Lucidum Polysaccharide Liposome And Cubosome

Ganoderma lucidum is a fungus belonging to the genus Basidiomycetes and a tonic drug of traditional Chinese medicine.Modern pharmacological studies have shown that Ganoderma lucidum polysaccharide was the main active ingredient in Ganoderma lucidum,which has the effects of regulating immunity,anti-oxidation,anti-aging,anti-tumor,liver protection,and strengthen heart.However,Ganoderma lucidum polysaccharides(GLP)belong to macromolecular polysaccharides.If they are directly administered,there are defects such as rapid metabolism of polysaccharides in the body,short action time,inconsistency of the scope of action,large clinical dosage,and low bioavailability,which limited the development of clinical applications of Ganoderma lucidum polysaccharides.Therefore,to expand the clinical application of Ganoderma lucidum polysaccharides,it is necessary to develop new delivery system.Liposomes are microscopic vesicles that have a phospholipid bilayer similar to a biofilm structure.Liposomes can provide effective delivery of drugs and serve as vaccine delivery systems and adjuvants.Cubosomes has a honeycomb structure,formed by amphiphilic lipids dispersing in aqueous solution and self-assemble into closed lipid bilayers containing bicontinuous water and lipid regions.Cubosomes can encapsulate drugs with various polarities and doses,and have diverse drug encapsulation,which offer many application functions,such as the protection of drug activity,control of drug release,targeted drug delivery,and prevention of aggregation between drug molecules.Cubosomes can effectively deliver drugs or antigens due to their unique internal structure.Encapsulating GLP in liposomes and Cubosomes not only can help the effective delivery of GLP,but also can take advantage of both and make up for the deficiency of both,which induce more effective immunopotentiation.In this experiment,Ganoderma lucidum polysaccharide liposomes and cubic liquid crystals were prepared,their characterization characteristics and stability were analyzed,the effect of their adjuvant activity were tested in vitro and vivo and were divided into the following eight parts:Experiment 1 Preparation and optimization of the preparation condition of Ganoderma lucidum polysaccharide liposomes Ganoderma lucidum polysaccharide liposomes were prepared by reverse phase evaporation and optimized by response surface methodology.Taking the encapsulation efficiency and drug loading of GLPL as indicators,the effects of four single factors on the preparation of GLPL were investigated.The results showed that three single factors(ratio of soybean phospholipid to cholesterol,ratio of soybean phospholipid to tween-80 and ultrasonic time)had a greater impact on the preparation of GLPL.Based on the results of single-factor experiments,response surface methodology was used to study the effect of three factors on the GLPL preparation.The optimal preparation conditions for the GLPL were as follows,soybean phosphatide to cholesterol ratio of 11:1,soybean phosphatide to tween 80 ratios of 10.5:1 and ultrasonic time(min)of 11.Under these conditions,the GLPL were small homogenous vesicles and the experimental EE was 71.43 ± 0.49%,relative error is 1.6%.The GLPL prepared by the optimal preparation conditions were observed by TEM.The GLPL were nearly spherical and uniform in size.The particle size of the GLPLs was 164.3±9.2nm.Experiment 2 Effect of GLPL on proliferation and differentiation of mouse spleen lymphocytes To study the effect of GLPL on the proliferation and differentiation of mouse spleen lymphocytes.In this experiment,different concentrations of GLPL were added to mouse spleen lymphocytes in single or synergistic with LPS or PHA.After 44 hours of culture,the effect of GLPL on the proliferation and differentiation of mouse splenic lymphocytes was measured by MTT assay and flow cytometry.The results showed that GLPL could significantly promote the proliferation of mouse spleen lymphocytes in single or synergistic with LPS or PHA.At the concentration of 62.5?g/mL,GLPL induced most powerful proliferation of lymphocytes,and significantly better than GLP,BL and blank control group.Compared with GLP,GLPL can more effectively promote the differentiation of T lymphocytes to CD4.The experiments showed that the effect of GLP encapsulated by liposomes on the proliferation of spleen lymphocytes and the differentiation of splenic lymphocytes were significantly enhanced.Experiment 3 The effect of GLPL on the function of mouse peritoneal macrophages To study the effects of GLPL on the phagocytosis and cytokine secretion of mouse peritoneal macrophages,different concentrations of GLPL,GLP and BL were added into macrophages suspension.Flow cytometry was used to determine the effect of drugs on the phagocytosis of fluorescently labeled E.coli by peritoneal macrophages and the expression of MHC II.ELISA was used to determine the effect of the drug on cytokines and inflammatory factors NO and iNOS.The results showed that GLPL can significantly enhance the phagocytosis of mouse peritoneal macrophages and the expression of MHC II,and can significantly promote the secretion of IL-1?,TNF-?,IFN-? and NO by macrophages,and promote the activity of iNOS.The experiments showed that the effect of GLP encapsulated by liposomes on macrophage activation were significantly enhanced.Experiment 4 Stability evaluation and immunoenhancement of GLPL encapsulating OVA To study the stability and adjuvant activity of GLPL,the OVA was wrapped in GLPL by repeated freezing and thawing.To study the stability of nanoparticles,the BL/OVA and GLPL/OVA dispersion was put into a tube and stored at two different temperatures(4? and 37?).The encapsulation efficiency of OVA was determined.At different time points,an aliquot of the sample in 4? was taken out,and the particle size and polydispersity index(PDI)was determined as measures of physical stability of the GLPL dispersion.120 BALB/c mice,age 5 weeks,were immunized and divided into 6 groups.All injections were given subcutaneously and mice were inoculated 3 times at one-week intervals.DCs activation was determined by removing sciatic and popliteal lymph nodes 24h and 48h after vaccinated with different vaccine formulations.The expression of surface molecules was detected by flow cytometry.At 14 days after the final immunization,splenic lymphocytes were isolated from the immunized mice and were assessed for spleen lymphocyte proliferation index and the differentiation by MTT assay and fluorescence activated cell sorting(FACS).The levels of IgG,IgGl,IgG2a,IL-4,IL-6,TNF-?,and IFN-y were measured in the supernatant and serum.The results showed that when GLPL/OVA was stored at 4?,the rate of decrease of encapsulation rate was lower than that of 37? particle size and PDIhave less change in 28 days.GLPL can significantly promote the maturation of DCs in lymph nodes of mice,promote the proliferation and differentiation of splenic lymphocytes,and promote the levels of OVA-specific antibodies,IFN-y,TNF-? and IL-4,and the ratio of IgG2a/IgG1 in mice,and significantly better than GLP.Thus,the GLPL/OVA formulation could not onlyhave a stronger ability to promote the maturation of dendritic cells in lymph nodes of mice,but only stimulated splenic lymphocyte proliferation and can significantly enhance humoral and cellular immunity in mice.Experiment 5 Immunoenhancement of GLPL on PCV-2-immunized mice To study the adjuvant activity of GLPL as adjuvant for PCV-2 vaccine,100 mice were divided into 5 groups and immunized with PCV-2 vaccine with different adjuvants,one week later,these mice were immunized again.The levels of IgG5 antibody subtypes(IgGl,IgG2a,IgG2b,IgG3)and cytokines(IL-4,IFN-?,IL-17,IL-12p70,TNF-?)were measured using ELISA 7,14,21 days after the first immunization in serum.The spleens of mice were collected,and tissue sections were made 21 days after the first immunization.HE staining was used to observe the histological changes of the spleens in each group.The results showed that GLPL was more effective in promoting the levels of PCV-2 specific IgG,antibody subtypes(IgGI,IgG2a,IgG2b,IgG3)and cytokines(IL-4,IFN-?,IL-17,TNF-?)compared with the GLP group and the BL group.Spleen histological results show that GLPL can significantly stimulate the enlargement of splenic corpuscle in mice.The experiment showed that GLPL,as an immunopotentiators of PCV-2 seedlings,can significantly increase the specific humoral and cellular immune responses of mice to PCV-2,and the effect is better than that of GLP.Experiment 6 The preparation and characterization of GLPC To prepare Ganoderma lucidum polysaccharide cubosomes with good stability and high encapsulation efficiency,the preparation method and dispersion medium of cubic liquid crystal was optimized,and its basic characterization was analyzed and tested.It was found that the encapsulation rate of cubic liquid crystal prepared by ethanol dispersion method was higher.In addition,the effects of different dispersion media were compared.It was found that the cubic liquid crystal with double distilled water as the dispersion medium was more stable.The characterization showed that the average entrapment efficiency of GLPC was 89.5±3.51%.Particle size analysis revealed that the particle size of GLPC slightly increased after encapsulating GLP.SAXS results showed that the crystal form of GLPC was Pn3m.The experiments show that the preparation of GLPC with the ethanol dispersion method is better.Experiment 7 Immunoenhancement of GLPC on PCV-2-immunized mice To study the adjuvant activity and mechanism of action of GLPC for PCV-2 vaccine.GLPC,GLP and Cub were used as adjuvant of PCV-2 respectively.The levels of IgG,antibody subtypes(IgG1,IgG2a,IgG2b,IgG3)and cytokines(IL-4,IFN-?,IL-17,IL-12p70,TNF-?)were measured by ELISA method 7,14,21 days after the first immunization in serum.The lymph nodes were collected after 24h and 48h of immunization,and the status of DCs in lymph nodes were measured.The antigen content and the level of central memory cells and effector memory cells were measured by immunohistochemistry and flow cytometry.The results showed that GLPC can effectively promote the level of PCV-2 specific IgG.In addition,GLPC can significantly promote the levels of PCV-2 specific IgG,antibody subtypes(IgG1,IgG2a,IgG2b,IgG3)and cytokines(IL-4,IFN-y,IL-17,TNF-?)in serum.Then,GLPC can stimulate the maturation of DCs in lymph nodes of mice more strongly,promote the transport of antigens from subcutaneous to lymph nodes and promote the level of central memory cells and effector memory cells in lymph nodes,and significantly better than GLP.The experiments have shown that GLPC can significantly promote the maturation of DCs in the lymph nodes and the generation of memory T cells,thereby generating the stronger humoral and cellular immune responses to PCV-2.Experiment 8 Immunoenhancement of modified GLPC on OVA-immunized mice To load OVA onto the surface of GLPC and study its adjuvant mechanism,CTAB and PDDAC were respectively modified to the surface of GLPC and cationic GLPC was prepared.The cationic GLPC were used as OVA adjuvant to immunize mice and GLPC were used as controls.At 24h and 48h after the first immunization,the draining lymph nodes were collected and the surface molecules of dendritic cell were detected by flow cytometry.14days after the third immunization,the spleens were aseptically extracted and lymphocyte proliferation and differentiation were measured by MTT assay and flow cytometry.Mice sera were collected at 14 and 28days after the third immunization,the levels of OVA-specific IgG,IgG1,IgG2a and cytokines IFN-?,TNF-?,IL-4,IL-6 were measured by ELISA.At 28 days after the third immunization,the gene expression changes of the spleen of mice were detected d by transcriptomic sequencing.Compared with GLPC,PDDAC-GLPC,as an OVA immunopotentiator,not only effectively promotes the production of OVA-specific IgG,increases the secretion of cytokines,stimulates the proliferation of splenic CD4+and CD8+T cells,but also significantly promotes the maturation of mouse lymph nodes and is better than GLPC.As OVA immunopotentiator,the adjuvant activity of CTAB-GLPC were better than GLPC group except that the effect of increasing specific IgG was not significantly different from that of GLPC.The immune-enhancing effect of DDAC-GLPC was attributed to activation of the spleen mainly through the NOD-like receptor pathway,PI3K-ATK signaling pathway,cytokine interaction,and leukocyte transendothelial migration.The experiments have shown PDDAC-GLPC-OVA could significantly promoteactivation of dendritic cells powerfully and4 signaling pathway,producedstronger humoral and cellular immune response.