Study On Immuno-Enhancement Of Lentinan Grafted Graphene Oxide

Lentinus edodes is a kind of medicinal and edible fungus,belonging to basidiomycetes of Umbelliferaeand.Lentinus edodes was originated in China,and has significant medicinal value recorded in "Daily Use Materia Medica" and "Compendium of Materia Medica".Lentinan(LNT)is the main effective component of the fruiting bodies of lentinus edodes,which has significant anti-tumor,antibacterial,antiviral and immunomodulatory effects.However,lentinan is limited in clinical application because of some disadvantages such as susceptibility to degradation,rapid metabolism and poor targeting.Graphene oxide(GO)is an oxidized derivative of graphene,has a large surface area,excellent hydrophilicity and dispersibility,multifunctional modification due to the abundant oxygen-containing functional groups on its surface,and good targeting.In this study,GO were used as a carrier,and lentinan was grafted on the surface of GO by covalent bonds to improve the bioavailability,reduce the dosage of lentinan,and promote its targeting of immune cells;GO-LNT also was used as a carrier for antigens,which would effectively deliver antigens to antigen presenting cells and form a reservoir of antigen at the injection site,keeping the body in a high-level immune response state for a long time.In this experiment,stable and safe GO-LNT was prepared,and the effect on the immune function of macrophages and the immune enhancement effect as a vaccine adjuvant in the body were studied.The test is divided into the following three parts:Experoment Ⅰ The preparation and characterization of GO-LNT The LNT was grafted onto the surface of GO by covalent bonds,and then the characteristics and stability of GO-LNT were determined by using various methods.Firstly,the microscopic morphology and structure of GO-LNT were observed by atomic force microscope,scanning electron microscope and transmission electron microscope;secondly,the grafting method and material structure of GO-LNT were detected by using Fourier-infrared spectroscopy and Raman spectroscopy;moreover,the thermal stability was detected by using a thermogravimetric analyzer;then,the particle size,potential and stability of GO-LNT were investigated by using a laser particle size analyzer;finally,the ungrafted LNT solution was collected from the washing solution in the process of preparing GO-LNT,and grafting efficiency of the LNT was calculated by the phenol-sulfuric acid method.The results showed that the GO and GO-LNT had a flaky structure with wrinkles,ultra-thin features with curling,and a smooth surface,and GO-LNT had a thickness of approximately 20-30 nm.In the infrared spectrum,the absorption peak of the primary alcohol hydroxyl group in GO-LNT partly disappeared and the new peak was changed,which has been proved that LNT is grafted on the surface of GO by esterification reaction.The thermogravimetric analysis showed that the thermal stability of GO was enhanced after LNT grafting.The particle size of GO-LNT was approximately 900-1300 nm,and the GO sheets were 550-800 nm;the measured zeta potentials of GO(-28.6 ± 0.68 m V)and GO-LNT(-29.3 ± 0.69 m V)were negatively charged.And they are stable after repeated freeze-drying and dissolution.And result showed that 71.42%of LNT was successfully grafted onto GO.Experiment Ⅱ The effect of GO-LNT on the function of macrophages.The mouse peritoneal macrophages were obtained from mouse and cultivated to detected the effects of GO-LNT on the phagocytic function and expression of surface molecule.First of all,in order to investigate the maximum safe concentration of GO-LNT and GO,the macrophages were stimulated with drugs at different concentrations,and the cell viability were determined by CCK-8 method.The effect of GO-LNT on phagocytic function and expression of surface molecule on macrophages were determined respectively by using confocal laser scanning microscopy and flow cytometer.The results showed that GO-LNT significantly promoted the expression of the surface molecules CD86 and MHC-Ⅱ on macrophages,and also boosted the phagocytosis of the antigen OVA by macrophages.However,GO-LNT couldn’t help the antigen escape from the lysosome,indicating that GO-LNT/OVA is presented by MHC-Ⅱmolecules and mainly induced humoral immune response.The result of transcriptomic analysis revealed that GO-LNT enhanced immune response of macrophages through regulating the NF-k B signaling pathway,Toll-like receptor signaling pathway and protein digestion and absorption.Experiment Ⅲ The effect of GO-LNT on the immune response of animalimmunized by OVA In order to explore the immune-enhancement of GO-LNT in body,6-week-old ICR mice were randomly divided into 6 groups.The mice were injected intramuscularly and boosted with equivalent doses 14 days later.On the 5th day after the first immunization,the draining lymph nodes were taken to detect the activation of dendritic cells.The spleen was taken to detect the maturation of splenic lymphocytes on the day 21 after the second immunization.Blood was collected on days 7,14 and 21,and ELISA was used to detect the levels of Ig G,Ig G1,Ig G2 a and Ig G2 b in the serum.The serum and main organs(heart,lung,liver,spleen and kidney)were collected on the day 35 after the second immunization for serum biochemistry assay and H&E staining to determined safety in vivo of GO and GO-LNT.Mice were injected intramuscularly with Cy5.5-labeled OVA combined with different vaccine formulations,the antigen depot effect of GO-LNT/OVA was explored by using an IVIS Spectrum visible light imager.The results showed that GO-LNT as the adjuvant to load OVA model antigen could promote the activation of dendritic cells in the lymph nodes and the maturation of splenic lymphocytes,and also increase the level of Ig G and its subtypes in the serum.The results of biochemistry assay and H&E staining showed that GO-LNT was safe in vivo.Moreover,GO-LNT could promote lymphatic targeting and prolong the retention of OVA at lymph nodes after intramuscular injection,offer opportunities to deliver drugs or antigen to enhance lymphatic drug targeting and immunity.The above results indicate that GO-LNT as the adjuvant to load OVA model antigen,could effectively promote the body’s humoral and cellular immune responses for a long time.