Immunostimulatory DNA Nanohydrogel Inhibited The Proliferation Of U251 Glioma Cells

Recent years,tumor immunotherapy has become a new method of cancer treatment beyond surgery,chemotherapy and radiotherapy,which mainly depend on activating the selfimmune activity of patients to kill tumor cells.Non-methylated CpG oligonucleotide(CpG ODN)is an immunoadjuvant that can activate many immune cells,such as macrophages,dendritic cells,etc.It has been widely used because of its low toxicity.By binding to tolllike receptor 9(TLR9)in the endosome of immune cells,CpG ODN can activate both congenital and acquired immune responses,and induce Th1 antigen specific T cell responses.However,since CpG ODN intolerance to nucleases in blood and cytoplasm and the low uptake ratio by immune cells because of it negatively charged,the application of CpG ODN has been greatly limited.Recent years,researchers have found that nucleic acids show different characteristics from simple nucleic acid materials after having certain structures.They can easily penetrate cell membranes,and benefit from the fact that DNA molecules are part of the human body,so they are biocompatibility and non-toxicity.Therefore,in our study,a new immuno-stimulate DNA nanohydrogel called CpG-MCA nanohydrogel was constructed by means of rolling cycle amplification(RCA)reaction combined multi-primed chain amplification(MCA)reaction.Results showed that CpG-MCA nanohydrogel was made up of nanoflowers which diameter were range from 400 nm to 1500 nm.CpG-MCA nanohydrogel could maintain the gel properties in a certain period of time in the medium containing fetal bovine serum,and could be degraded slowly.Compared with CpG ODN,CpG-MCA nanohydrogel could be well uptake by RAW264.7 cells and stimulate the secretion of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)from RAW264.7 cells in a concentration dependent manner.In addition,the mRNA of TNF-α,IL-6 and TLR9 and were also increased with the stimulation of CpG-MCA nanohydrogel.We found that CpG-MCA nanohydrogel was nontoxicity and CpG-MCA nanohydrogel were significantly stronger in stimulating the proliferation,differentiation and migration of RAW264.7 cells compared with CpG ODN(P < 0.01).Finally,we found that CpG-MCA nanohydrogels could inhibit the proliferation of U251 cells by inducing the secretion of TNF-α from RAW264.7 cells.Our study shows that CpG-MCA nanohydrogel was much stronger than CpG ODN on promoting the proliferation,differentiation and migration of RAW264.7 cells and secreting M1-type inflammation cytokines to inhibit the proliferation of glioma cells.Our research provided an experimental basis for the future application of CpG-MCA nanohydrogel in cancer immunotherapy.