The Study Of DNA Nanoflower Structure For Tumor Immunotherapy

Unmethylated cytosine-guanine dinucleotide oligodeoxynucleotide is an immunostimulatory adjuvant and widely used in immunetherapy as an adjuvant for immunotherapy.Unmethylated cytosine-guanine dinucleotide oligo-deoxynucleotide is widely reported to have immune stimulating function,and can directly activate antigen presenting cells(APCs)through membrane receptor TLR9,such as dendritic cells(DC),As an effective immune adjuvant,CpG is now widely used in the treatment of immune tumor.Because the chemical nature of CpG is oligonucleotide,there are a lot of nuclease in serum,which makes CpG easy to be degraded by nuclease during circulation,which limits its application in vivo.Therefore,the design and development of nanocarrier to load CpG has become a research hotspot in recent years,a variety of carriers was developed to protect CpG effectively,reduce nuclease degradation,prolong the circulation time in vivo,increase internalization on CpG cells and enhance the immune stimulating effect.DNA nanomaterials have received extensive attention because of their good biocompatibility,wide size distribution and shape design.In this work,we use DNA self-assembly technique into DNA nanoflower structure,while i-Motif structure DNA complementary sequence can be designed and formed in the DNA self-assembly template and the end of CpG design under acidic conditions,so that the release amount of CpG increased,the cellular internalization and immune stimulating effect significantly improved.By designing the DNA self-assembled template chain,a sequence of i-Motif structures can be formed on the DNA template chain in acid conditions.At the same time,We add the complementary sequence of i-Motif to the end of the CpG hairpin structure.This design makes the DNA carrier in the process of rolling circle replication to form the product containing a large number of i-Motif fragments,and it can be combined with the CpG terminal sequence through the base complement pair,forming a nanoscale structure containing a large number of CpG fragments.The nanomaterials were absorbed by cells.Under lysosomal acidic condition,CpG was released from the DNA carrier and activated the TLR9 receptor on lysosomal membrane,which stimulated the cells to produce strong immune response due to the acid response of i-Motif sequence.The structure of DNA nanomaterials was obtained by self-assembly of PCR technology.Under the transmission electron microscope,the morphologies and sizes of the nanostructures were observed,and the reaction conditions such as adjusting the replication time were adjusted appropriately,and the structure was adjusted to the right size.In the PBS detection of different p H,it was found that under the acid pH value,the amount of CpG release was significantly higher than that under the pH=7 condition.This is due to the production of a DNA sequence capable of forming i-Motif structures in the process of DNA replication,which allows a high level of release of the complementary CpG under acidic conditions.Laser confocal microscopy and flow cytometry were used to detect the cell internalization of the nanoflower structure of the loaded CpG and the single strand of free CpG.The results showed that the use of DNA nanoscale vector could significantly increase the effect of cell internalization on CpG.The results of enzyme-linked immunosorbent assay(ELISA)showed that CpG loaded with DNA nanoflower vector could stimulate macrophages to secrete higher levels of immune factors.