Construction Of RNA-Targeted Intelligent Response Probes For Tumor Theranostic Application

Background:Recently,cancer is one of the most major diseases threatening human life and health.The development of highly effective tumor theranostic agents is an important approach against to cancer.Stimulus-responsive tumor theranostic materials base on tumor microenvironment or external physical stimuli their"intelligent" characteristics have received frequent attention in recent years.RNA as the "central pipeline" of life information transmission that mainly responsible for gene transcription and protein translation.Design RNA-targeted drugs will be able to more accurately regulate the process of life information transmission.Therefore,integrating stimulus-response and RNA-targeted concepts into probe design,then constructing RNA-targeted intelligent response tumor theranostic probes that expected to accelerate the process of "precision medicine" and achieve a new breakthrough in tumor theranostic.Objective:We focuses on the key scientific problem of precise tumor theranostic.Two types of RNA-targeted intelligent responsive tumor probes are designed and synthesized.Due to the responsiveness of the probes to the red light and the tumor microenvironment,which realized the control from the cytoplasmic RNA targeting to specific RNA regulating in the cell.The probes have sensitive tumor imaging capabilities and excellent treatment effects,providing a new method for precise tumor theranostic.Methods:In the first part,a new type of near-infrared fluorescent probe f-CR that can covalently cyclize with RNA molecules in the cytoplasm mediated by red light was designed and synthesized.The probe consists of a NIR dye(Cy 7)as a signal unit,a αvβ3 integrin targeting group cRGD and a 1O2-sensitive furan for cross-linking RNA.The probe f-CR has been proved by in vivo and in vitro experiments that a covalent cyclization and cross-linking reaction between the furan group and some bases on the RNA molecule under the initiating of 1O2,thereby anchoring the RNA molecule.RNA after modified probe.The uptake of the probe in tumor cell has significantly improved and the retention time of the tumor is prolonged,which is conducive to long-term and efficient tumor imaging.In addition,the crosslinking of the probe and RNA cause tumor cell apoptosis,realizing tumor theranostic.In the second part,a new type of small molecule probe PRDG that Furin recognizes and specifically cleaves is designed and synthesized,which is mainly composed of three parts:furin response polypeptide arginine-valine-arginine-arginine(RVRR),photoacoustic/photosensitizer drug-purpurin 18 and nuclear magnetic resonance contrast agent Gd-DOTA.The probe PRDG shows good Hif-1α siRNA carrying capacity.Under the mediation of furin,which can effectively realize the tumor-specific delivery of Hif-1α siRNA.Compared with a single treatment method,synergistic gene therapy and photodynamic therapy significantly improves the ability of the probe to kill tumor cells.Combined with photoacoustic and MRI dual-modality imaging,the precise tumor theranostic of living tumors is realized.Results:In the first part of the work,the probe f-CR can react with commercial RNA under the initiating of 1O2 in the solution that shows great RNA-specific crosslinking ability.In cell experiments,through cell imaging and gel electrophoresis experiments,which can be observed that f-CR is efficiently labeled on RNA molecules in the cytoplasm and significantly prolong the retention time of the probe in the cell.We also found that the normal biological functions of the RNA molecules are destroyed by modification that make serious tumor cell apoptosis.In the in vivo experiment,under the stimulation of 660 nm red light,the photosensitizer methylene blue(MB)generates a large amount of singlet oxygen,which triggers the crosslinking of the probe with the RNA in the tumor tissue.The uptake of the probe in the tumor tissue is increased and realizing the long-term tumor imaging.The crosslinking of the probe with RNA induces tumor cell apoptosis,which effectively inhibits tumor growth.In the second part,the probe PRDG self-assemble with Hif-1α siRNA into nanoparticles through hydrogen bonding and charge interaction in aqueous solution.Then,the PRDG@Hif-1α siRNA has disassemble under the shearing of furin to release Hif-1α siRNA.In colon cancer cell Experiments show that under the irradiation of 660 nm laser,the 1O2 also caused the lysosomal membrane to rupture while killing the cells,then release Hif-1α siRNA to the cytoplasm and Maximize treatment efficiency.In this study,photodynamic synergistic gene therapy significantly enhanced the toxicity of tumor cells,and at the same time realized the tumor theranostic by combined photoacoustic and magnetic resonance dual modal imaging.Conclusion:Based on the stimulus-responsive tumor theranostic strategy,this thesis rationally designed and synthesized two novel tumor small molecule probes f-CR and PRDG focus on RNA molecules as the target,and f-CR and cytoplasmic RNA under From the non-specific crosslinking off-CR with cytoplasmic RNA under the control of light to the precise delivery of Hif-1α siRNA to tumors mediated by furin,and the non-specific targeting of intracellular RNA to specific targets has been realized.A new type of RNA-targeted tumor theranostic strategy has been established.