Study On Immunomodulatory Effects Of Cationic-modified Polygonatum Sibiricum Polysaccharide Cubosomes

Polygonatum sibiricum polysaccharide(PSP)is one of the effective components of Polygonatum sibiricum,the dried rhizome of Polygonatum.It has effects of spleen nourishing and lung moistening.PSP has been proved that it has the biological functions of anti-tumor and anti-virus,and has the function of immune enhancement as most Chinese traditional medicine polysaccharides.In recent years,more and more nanomaterials have been used in drug delivery system.The applications of effective ingredients of traditional Chinese medicine in combination with nanomaterials in the treatment or diagnosis of diseases have attracted extensive attention.Cubic liquid crystalline nanoparticle is a kind of micro drug delivery system with good biocompatibility,slow-release ability and other advantages.The dispersion of it in water referred to cubosomes.In general,cubosomes are the nanostructured entities which have the same structure as the parent phase but much lower viscosity.Cubosomes encapsulated with polysaccharides can improve the targeting of the function of polysaccharides and increase the bioavailability of polysaccharides.Cubosomes were negatively charged and most protein antigens also exhibit negative electricity.Therefore,in order to adsorb more protein antigens,Cetyltrimethylammonium bromide(CTAB)was selected as a kind of cationic surfactant modifier.CTAB-modified Polygonatum sibiricum polysaccharide cubosomes(CTAB-modified PSP-Cubs)were prepared firstly and the response surface method was used to design an optimal preparation condition.The in vivo and in vitro experiments were used to prove the immune enhancement and immune adjuvant activity of CTAB-modified PSP-Cubs.The paper is divided into the following four parts:Experiment 1 Optimization of preparation conditions for CTAB-modified Polygonatum sibiricum polysaccharide cubosomes Preparation of CTAB-modified Polygonatum sibiricum polysaccharide cubosomes(CTAB-modified PSP-Cubs)was based on the principle of large to small.Briefly,a larger particle size cubic liquid crystal system was first obtained.It is then dispersed into a cubic liquid crystal system having a small particle size through energy input.Three factors that had significant impact on the encapsulation efficiency(EE)of CTAB-modified PSP-Cubs were selected based on previous experimental:the mass percentage of PSP to GMO,the mass percentage of F127 to GMO and the mass percentage of water to GMO.Response surface software was used to design and optimize the experiment,and the most suitable preparation conditions were selected according to 17 combinations.The optimal conditions were as follow:the mass percentage of PSP to GMO,F127 to GMO,and the aqueous phase to GMO was 1.4%,9%,and 50%,respectively.The EE of CTAB-modified PSP-Cubs was 62.4±2.6%.Experiment 2 Characterizations of CTAB-modified Polygonatum sibiricum polysaccharide cubosomes In order to conduct quality assessment of the CTAB-modified Polygonatum sibiricum polysaccharide cubosomes,transmission electron microscope(TEM)was used to observe the surface morphology of cubosomes.The particle size,polydispersity index(PDI)and zeta potential of cubosomes were measured by Malvern potentiometer based on dynamic light scattering(DLS).To identify the nanostructure of CTAB-modified PSP-Cubs,Small-angle X-ray scattering(SAXS)was used.The particle size of CTAB-modified PSP-Cubs were 427.7±8.0 nm,with a good dispersion.CTAB-modified PSP-Cubs were square-like nano-level particles with a Pn3m structure,with positive charges on the surface.A well-characterized CTAB-modified PSP-Cubs formulation as potential immune adjuvant has been proposed.Experiment 3 Effects of CTAB-modified Polygonatum sibiricum polysaccharide cubosomes in vitro The purpose of this part is to observe the effects of CTAB-modified PSP-Cubs on lymphocytes and macrophages in vitro.MTT assay was used to measure the safety concentration of cubosomes for lymphocytes and their effects on spleen lymphocytes proliferation.The effect of CTAB-modified PSP-Cubs on the ratio of CD4+ to CD8+ was evaluated by flow cytometry.The influences of CTAB-modified PSP-Cubs on the expression of surface activation molecules of macrophages and the secretion of TNF-α and IFN-y cytokines by macrophages in vitro were detected by confocal laser scanning microscopy,flow cytometry and ELISA,respectively.Results showed that the safe concentration of CTAB-modified PSP-Cubs for lymphocytes was 39.06 μg/mL and the nanoparticles were able to stimulate lymphocytes proliferation.CTAB-modified PSP-Cubs could cause an increase in CD4+/CD8+ ratio.The effect of CTAB-modified PSP-Cubs on the uptake of macrophages was better than that of PSP and Cubs alone.In addition,the results also showed that CTAB-modified PSP-Cubs could promote the expression of CD86 and CD80 molecules as well as cytokines in macrophages compared with PSP and control groups.These results indicated that CTAB-modified PSP-Cubs could strengthen the immune response in vitro.Experiment 4 Effects of CTAB-modified Polygonatum sibiricum polysaccharide cubosomes in vivo This part was to further study the immunomodulation of CTAB-modified PSP-Cubs with OVA(CTAB-modified PSP-Cubs/OVA)in immunized mice.Firstly,the safety evaluation of CTAB-modified PSP-Cubs was carried out.For in vivo experiments,six-week-old ICR mice were divided into 7 groups randomly.The mice were immunized by dorsal subcutaneous injection of CTAB-modified PSP-Cubs/OVA on day 0 and day 14.The activation of dendritic cells in lymph nodes was analyzed 96 h after the first immunization.On the 7th and 35th days after the second immunization,lymphocyte proliferation was analyzed by MTT assay.The ratios of CD4+to CD8+were analyzed on the 21st day,while histological analysis of the spleen was observed on the 28th day after the second immunization.On the 7th,14th and 21st days after the second immunization,blood were collected and the concentrations of OVA-specific IgG in serum were determined.The cytokines concentrations on the 7th day after the second immunization were determined by ELISA.As the results showed,CTAB-modified PSP-Cubs/OVA could promote the production of OVA-specific IgG in serum and the secretion of both TH1 and TH2 cytokines compared with other groups.The ratio of CD4+ to CD8+ in CTAB-modified PSP-Cubs/OVA group was significantly increased compared with other groups.CTAB-modified PSP-Cubs/OVA could significantly activate dendritic cells and promote lymphocyte proliferation.The results indicated that CTAB-modified PSP-Cubs/OVA could promote the secretion of related cytokines and the proliferation of lymphocytes,stimulate the cellular immune response and increase the level of humoral immunity.Above all,CTAB-modified PSP-Cubs had good adjuvant activity.