Study On Mucosal Immune Adjuvant Activity Of Ultra-Large Pore Mesoporous Silica Nanoparticles Loading Lycium Barbarum Polysaccharides

Mucosa is the frontline of body immune defense.Protective mucosal immune response can be effectively induced by oral administration,nose drop,ocular routes and other mucosal routes.However,the vast majority of mucosal vaccines have not yet been developed,and lack safe and effective immune adjuvants.Lycium barbarum polysaccharides(LBP),the main active component of the Lycium barbarum,which has various pharmacological activities,including antioxidant,antiviral,cell protection,and immune regulation.Compared with traditional mesoporous silica nanoparticles,ultra-large mesoporous silica nanoparticles(UCMS)have more advantages as delivery carriers for drugs or subunit vaccines.This study combines the advantages of LBP and UCMS to develop a new traditional Chinese medicine polysaccharide nano-mucosal immune adjuvant.This adjuvant can protect the antigen and better exert the immune effects of traditional Chinese medicine polysaccharides.1.The preparation and characterization evaluation of LBP-UCMSThe UCMS nanoparticles were prepared using the sol-gel method,and LBP was loaded onto the UCMS nanoparticles by adsorption to obtain ultra-large pore mesoporous silica nanoparticles loading lycium barbarum polysaccharides(LBP-UCMS).LBP-UCMS was successfully evaluated by various techniques,and its oral toxicity was evaluated by continuous administration to mice for 14 days.The results showed that LBP-UCMS was spherical with a uniform size,and the average particle size was 493.4±14.17 nm with negative charge.LBP was successfully loaded onto UCMS,and the polysaccharide loading rate was 21.92%.The release rate of LBP-UCMS was low in gastric simulated fluid,while it was as high as 88.4%in intestinal simulated fluid,indicating that UCMS can prevent LBP from degradation in gastric fluid and enable it to enter the intestine for effective action.The results of the subacute oral toxicity test showed that LBP-UCMS had good safety with no obvious toxicity to mice.2.Effects of LBP-UCMS on immune cellsThis study investigated the effects of LBP-UCMS on the immune function of mouse macrophages RAW264.7 and bone marrow-derived dendritic cells(DCs).The expression levels of surface co-stimulatory molecules on macrophages and DCs were detected by flow cytometry after treated with LBP-UCMS.The effect of LBP-UCMS on the phagocytosis of FITC-dextran by macrophages was observed under laser confocal microscopy,The impact of LBP-UCMS on the mRNA expression levels of cytokines TNF-α,IL-1β,and IL-6 secreted by macrophages was detected by RT-qPCR.The results showed that LBP-UCMS significantly increased the expression levels of surface co-stimulatory molecules MHC-II,CD86,and CD80 on macrophages and DCs.LBP-UCMS promoted the phagocytic function of macrophages,enhanced the ability of macrophages to secrete cytokines such as IL-1β,TNF-α,and IL-6,and exhibited better immune effects than LBP.3.Effects of LBP-UCMS on mucosal immunity activity of oral H9N2 inactivated vaccine in miceThis study investigated the effect and mechanism of LBP-UCMS as a mucosal adjuvant for the H9N2 subtype avian influenza inactivated vaccine via oral immunization.150 5-weekold BALB/c mice were randomly divided into six groups of 25 mice in each group,including Control,WIV,WIV+LBP,WIV+UCMS,WIV+LBP-UCMS and WIV+CT.The first oral immunization with adjuvant and antigen was administered on day 0,and followed by booster immunization 14 days later.The classical mucosal adjuvant cholera toxin(CT)as a positive control.The level of the systemic immune response induced by LBP-UCMS as a mucosal adjuvant was evaluated by serum antibody titers and changes in T cell phenotype(CD3+,CD4+/CD8+)of spleen.The level of mucosal immune response induced by LBP-UCMS as a mucosal adjuvant was evaluated by measuring the villus length,crypt depth and V/C ratio of the duodenum and jejunum,the content of slgA in jejunal washings,the mRNA expression of IgA a-chain,J-chain,and pIgR of jejunum,and the protein expression levels of TLR4,MyD88,and pIgR.The results showed that after oral immunization of mice with LBP-UCMS as a mucosal adjuvant,the antibody titers increased,the ratio of CD3+subset and CD4+/CD8+T cells in lymphocytes increased.LBP-UCMS also promoted the absorption of nutrients in the duodenum and jejunum of mice,induced the secretion of sIgA antibodies,and increased the protein expression levels of TLR4,MyD88,and pIgR.In summary,LBP-UCMS increased the level of systemic and mucosal immune responses,demonstrating its potential as a safe and effective mucosal adjuvant.