Research On The Nanometerization Of Ganoderma Lucidum Polysaccharide And Its Immune Regulation And Anti-tumor Effects On Dendritic Cells

Objective:To investigate the immunomodμlatory activity of Ganoderma Lucidum polysaccharide-Gold nanocomposites(GLP-Au)on dendritic cells(DC)and the anti-tumor effect when combinated with chemotherapeutic drug doxorubicin(DOX)compared to the free Ganoderma Lucidum polysaccharide(GLP).Methods:1.After the treatment of removing proteins and pigments,GLP was further purified with DEAE-32 cellulose anion exchange column and Sephacryl S-300 gel filtration chromatography.The molecular weight and the monosaccharide composition of GLP were also examined.2.GLP was reacted with cysteamin and stabilized by sodium borohydride to form GLP-SH,which was further reacted with chloroauric acid to form GLP-Au nanocomposite.3.A series of characterizations were conducted to verify whether GLP is combined with gold nanoparticles.4.To compare the immunomodμlatory activities of GLP-Au and free GLP on DC,the expression of surface antigen molecules(CD80/CD86/CD40/MHCII),endocytosis ability,activity of acid phosphatase(ACP),and the expression of cytokines secreted by DC were evaluated.5.The immunostimulatory effects of GLP-Au and GLP were further confirmed by DC-mediated T cell proliferation assay.6.The immunostimulatory effects of GLP-Au in vivo were initially explored and compared to free GLP.7.The anti-tumor effects of GLP-Au were performed in 4T1 tumor-bearing mice compared with GLP combined with DOX.Result:GLP was extracted,isolated,and detected for molecular weight by HPGPC.The molecular weight was 15.1 KDa.The monosaccharide component of GLP was analyzed by GC-MS.GLP mainly contained arabinose,glucose,galactose,and a small amount of rhamnose,xylose and mannose with their mole ratio of 18.6:38.8:25.6:4.72:4.49:7.75.GLP-Au nanocomposite was demonstrated to be spherical observed in transmission electron microscope(TEM).The UV-Vis absorption spectrum and X-ray diffraction pattern showed that the presence of GLP and Au in GLP-Au nanocomposites.After treatment with GLP-Au and GLP at 10,20,40μg/ml concentration,the expression of surface antigen molecules significantly increased.GLP-Au showed stronger stimulating effects than GLP at the same polysaccharide concentration.Both GLP and GLP-Au reduced DC endocytosis,while GLP-Au further reduced phagocytosis of DC after treatment at all concentrations compared to free GLP.ACP activity was decreased in DCs after the treatment of 10,20,40μg/ml of GLP or GLP-Au.ACP activity in GLP-Au-treated DC was further reduced than in GLP-treated DC.GLP-Au significantly increased the m RNA expression of IL-6,IL-12,IL-1β,TNF-αand IFN-γ.Cytokine expression stimulated by GLP-Au was much stronger than GLP alone.GLP-Au activated DC through TLR4,and the cellular uptake of GLP-Au also partially contributed to DC maturation.Compared to GLP,GLP-Au has a stronger immunostimulatory effects on DC-mediated T cell proliferation.GLP-Au exhibited strong inhibitory effects on 4T1 tumor growth and pulmonary metastasis when combined with doxorubicin.GLP-Au recovered body weight loss by doxorubicin and increased the percentage of CD4~+/CD44~+memory T cells.Conclusion:Our results showed that GLP-Au in vitro was able to induce stronger DC activation(phenotypic markers and functions)as well as robust T cell responses(CD4/CD8+T cell proliferation)than GLP.Secondly,combination therapy showed that GLP-Au exhibited stronger effects on tumor growth and metastasis than free GLP when DOX was co-treated.We hypothesized that chemotherapeutic drugs such as DOX may destruct a small amount of tumor cells,promoting the release of tumor antigen that can be captured by DC.During the tumor antigen presentation,GLP-Au facilitated the expression of co-stimulatory factors such as CD40/80/86 and MHCII for potent stimulation of T cell response(cell proliferation and functional changes).Lastly,we found that chemotherapeutic drugs may damage the immune system by lowering the percentage of CD4/CD8 T lymphocytes,while GLP-Au reversed the decline of CD4and CD8 T cell population.In addition,GLP-Au significantly induced memory T cells,which may be the important fact for effective inhibition of tumor metastasis.As the result,this study provided the groundwork for design of polysaccharide nano-formulation,which not only promoted stability and prolonged activation of immunoactive polysaccharides,but also proposed a strategy for reducing the doses of chemotherapeutic drug and polysaccharides due to their combined therapeutic effects.Other classical polysaccharide products that have been used clinically(such as lentinan or astragalus polysaccharides)can be also formulated into nanocomposites,aiming to apply nanotechnology into the modernization of traditional medicine.