Preparation Of β-glucan-CpG Oligodeoxynucleotide Nanoparticles And Their Immunoenhancement

Objective:β-glucan is a rigid polysaccharide with a triple helix conformation.It can change the single-chain and triple helix conformations of β-glucan by changing the solution conditions.Based on the denaturation-renaturation characteristics of the triple helix structure,unique functional materials can be constructed,such as chemical sensors,one-dimensional nanocomposites and gene carriers.In the previous work,we have prepared a single-chain conformation of β-glucan and used yeast β-glucan as the raw material to conduct formylation.The formylated yeast β-glucan is an electrically neutral single-chain polysaccharide.In addition,the in vitro uptake rate,immunoregulatory activity and the mechanism of immunoregulatory activity of the polysaccharide nucleic acid complex(FG-CpG dA)were also explored.Besides,the model antigen ovalbumin(OVA)was used to immunize mice to explore the humoral and cellular immune effects of CpG oligodeoxynucleotide-loaded polysaccharide nucleic acid complex(FG-CpG dA)and its in vivo safety,so as to provide a selection of gene vectors,and lay the foundation for further research on the functionalized single-stranded β-glucan gene vector.Methods and Results:1.Polysaccharides nucleic acid complex(FG-CpG dA)was prepared using modified single-chain β-glucan and synthetic CpG dA,a nucleic acid fragment carrying poly(dA)and CpG ODN.The FG-CpG dA was verified and characterized by circular dichroism,agarose gel electrophoresis experiment,nano-particle size potential meter and atomic force microscope.The results showed that the modified single-chain β-glucan could be used as a gene carrier to carry CpG dA,and there was an interaction between the two.2.The polysaccharide nucleic acid complex(FG-CpG dA)was co-cultured with RAW264.7macrophages to demonstrate its in vitro safety through the CCK8 experiment;The investigations by fluorescence microscope and flow cytometry showed that polysaccharide nucleic acid complex(FG-CpG dA)could improve the uptake rate of nucleic acid CpG dA by RAW264.7 macrophages,and it increased with the time.The RT-qPCR,ELISA,and Western blot experiments were performed to detect the immunoregulatory effect of the polysaccharide nucleic acid complex.The polysaccharide nucleic acid complex up-regulated the expression of inflammatory factors at the mRNA and protein levels,and it was found that the immunoregulatory effect of the polysaccharide nucleic acid complex was regulated through the MAPK and NF-κB signaling pathways.3.The mice were immunized with the polysaccharide nucleic acid complex(FG-CpG dA)and the mode antigen OVA.The safety of the polysaccharide nucleic acid complex(FG-CpG dA)and the immunoenhancement were detected by observing the body weight growth trend of the mice,the expression of OVA antibody in serum,the effects of CD4+ and CD8+T lymphocytes in spleen,organ index and histopathological section.From the results of mouse body weight growth trend,organ index,and pathological sections,polysaccharide nucleic acid complex(FG-CpG dA)was found to be safe.Detection of antibody titer and T lymphocytes showed that polysaccharide nucleic acid complex(FG-CpG dA)was able to improve the humoral and cellular immune responses of mice,with immune enhancing effect,and could be used as an adjuvant.Conclusion:This experiment proved that the modified single-stranded β-glucan could be used as a gene carrier.CpG oligodeoxynucleotides could be loaded under the assistance of poly(dA)by conformational transformation to form a triple helix polysaccharide nucleic acid complex.It was confirmed at the cell level that polysaccharide nucleic acid complex(FG-CpG dA)had the immunoregulatory effect.Moreover,the immunoregulatory effect was mediated by MAPK and NF-κB signaling pathways,which could improve the humoral and cellular immune responses of OVA antigen in vivo,with good safety.The theoretical basis of the beta-glucan is enriched,and novel choices are provided for gene carriers and vaccine adjuvants.