DNA Network For Immune Cell Capture And Localized Immunotherapy

DNA as a functional polymer with sequence editability,structure designability and good biocompatibility,has attracted extensive attention in recent years.At present,DNA materials have shown superior potential in cell capture,immunotherapy and cell delivery,etc.Based on the technology of double rolling circle amplification(RCA),this thesis has designed and explored the highly specific and efficient capture ability of double RCA-based DNA network for T-cells,and the application of this material in immunotherapy.The main research contents are summarized as follows:1.The sequence design,synthesis and characterizations of DNA network were carried out.Circular templates were synthesized and rolling circle amplification reaction occurred under the catalysis of phi29 DNA polymerase to prepare DNA-chain-1 with the ability to specifically capture T-cells and blockade immune checkpoint,and DNA-chain-2 containing immunostimulatory Cp G oligonucleotides(ODN).The two DNA chains were designed with sites that can complementarily hybridize to form a DNA network,and the complementary sites were the cutting site of restriction nuclease Hha I.In addition,Hha I@TGMS nanoparticles with inflammation responsiveness were synthesized and characterized.2.The above-mentioned DNA network was used to quickly and efficiently capture T-cells from artificial mixed cell populations and digested cell populations from tumor tissue.The specific binding of nucleic acid aptamers to T-cells membranes was proved by fluorescent co-localization experiment.The high efficiency of fishing T-cells from artificial mixed cell populations was confirmed by fluorescence microscopy imaging and cell counting,and the efficient fishing of tumor-infiltrating T-cells(TIT)was proved by flow cytometry.3.The application of this DNA network for fishing TIT in immunotherapy was explored.Cell experiments were performed to verify the activation of the digested DNA network on macrophages,and label-free real-time cell analysis technology and fluorescence imaging were used to verify the killing effect of the fished TIT on melanoma cells.The orthotopic melanoma murine model validated the immune efficacy of the material fishing with TIT and loading with Hha I@TGMS nanoparticles.In this study,the technology of efficiently fishing and isolating T-cells using DNA networks promoted the further development in the field of cell separation using DNA materials,and provided a new strategy for the efficient isolation of tumor-infiltrating Tcells for immunotherapy.