Construction Of Immune Cells-targeting Glyco-nanocarriers For CpG ODNs Delivery

Oligodeoxyoligonucleotides（CpG ODNs）containing unmethylated cytosine-guanosine dinucleotide motifs can activate innate and adaptive immune responses.They play an important role in the immunotherapy of cancer,allergy,and infectious diseases.However,the phosphodiester skeleton structure of CpG ODNs is easily degraded by serum nucleases,so CpG ODNs have a short half-life and poor stability.Due to electrostatic repulsion,it is difficult for CpG ODNs to pass the negatively charged cell membrane,which blocks the uptake by immunity cells.These defects severely limit the application of CpG ODNs.Polysaccharides have good biocompatibility and degradability.Some polysaccharides have immune activity and can be specifically recognized by immune cells,which can mediate cellular endocytosis.Therefore,polysaccharides are expected to be used as drug delivery vehicles targeting immune cells.Based on these advantages,this paper usedβ-glucan and glycogen to construct CpG ODNs delivery vehicles.The two vehicles are aminatedβ-glucans（NH₂-Glu）and dendritic cell-targeting peptide functionalized glycogens（PG NPs）,which can load CpG ODNs and are specifically recognized by macrophage and dendritic cells,respectively.These schemes significantly enhanced the uptake of CpG ODNs,stimulated immune cells to secrete more cytokines and improved the level of immune response.（1）Aminatedβ-glucan（NH₂-Gly）as carrier to delivery CpG ODNs and synergistically enhance anti-tumor immune response.β-glucan has immunostimulatory activity and can be specifically recognized by C-type lectin-1（Dectin-1）receptor and complement receptor 3（CR3）on the surface of macrophages.β-glucan was motified with amino-groups to obtain positively charged NH₂-Glu vehicles.The NH₂-Glu/CpG NPs were finally formed through strong electrostatic interaction between cationic NH₂-Glu and negatively charged CpG ODNs.NH₂-Glu had a strong CpG ODNs condensation ability and the maximum loading capacity was about 127μg/mg.The size of NH₂-Glu/CpG NPs is about 100 nm and it can protect CpG ODNs from nuclease degradation.NH₂-Glu/CpG NPs were specifically recognized by receptors of macrophages（RAW264.7）,which effectively enhanced the uptake of CpG ODNs by RAW264.7 cells.β-glucan and CpG ODNs in NH₂-Glu/CpG NPs synergistically enhanced the secretion of interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in vitro and in vivo.It also activated the immune system and effectively inhibited the growth of tumors in mice,which had a good immunotherapy effect on tumors.In conclusion,as a simple and efficient targeted delivery carrier for CpG ODNs,NH₂-Glu has broad application prospects in the field of immunotherapy.（2）Research on dendritic cells targeting peptide-functionalized glycogen to precisely delivery CpG ODNs and enhance Th1 immune response.First,aminoated glycogen（NH₂-Gly）was synthesized,and then the carboxylated dendritic cell targeting peptide was grafted on NH₂-Gly through the amide bond to construct PG NPs carrier.The PG NPs are positively charged,with a particle size of about 150 nm,and have good cytocompatibility and blood compatibility.PG NPs had a strong CpG ODNs condensation ability and could resist the degradation of CpG ODNs by nucleases.PG-CpG NPs specifically binded to bone marrow-derived dendritic cells（BMDCs）and significantly increased the uptake of CpG ODNs.It also activated BMDCs cells,increased the expression of CD80 and CD86molecules on the cell surface,and stimulated the secretion of Th1-type cytokines.Therefore,PG-CpG NPs have great potential in modulating the imbalance of Th1/Th2 immune response.