Synthesis Of Methylene Blue Fluorescent Probe Targeting Esophageal Cancer And Its Application In Diagnosis And Treatment

With the increasing global aging population,the number of diagnosed cancer patients is still increasing year by year.Due to the atypical early symptoms of esophageal cancer,more than 80% of patients have developed into advanced cancer with distal metastasis when diagnosed,resulting in unsatisfactory treatment outcomes and difficult to break through 40% of the five-year survival rate.Therefore,there is an urgent need for the simple and effective diagnostic methods for screening early esophageal cancer and to achieve early intervention treatment to improve the survival rate of patients.Fluorescent molecular diagnostic probe has no radiation damage and high temporal and spatial resolution.It can play a great role in early detection,intraoperative navigation,targeted therapy and prognosis analysis of esophageal cancer,and has strong clinical transformation potential.In this thesis,a series of fluorescent molecular probes based on methylene blue(MB)dye molecules were designed and synthesized to target the microenvironment of esophageal cancer.The imaging and therapeutic functions of the synthetic diagnostic probe were systematically evaluated in vitro and on esophageal tumor bearing mice.The specific research contents are as follows:(1)Using MB,a clinical dye molecule,as the fluorescent signal group of diagnosis and treatment probe,MB-SS-CPT with glutathione(GSH)responsive function was successfully synthesized by connecting MB with camptothecin(CPT)through a straight chain compound containing disulfide bond.Once triggered by GSH,the probe MB-SSCPT can achieve stimulate release of MB and enhance the fluorescence signal.According to the linear relationship between the concentration and fluorescence intensity of MB-SS-CPT upon GSH,the detection limit concentration of GSH is calculated to be 820 n M.The experiments show that the MB-SS-CPT probe can resist the interference of a variety of amino acids and inorganic salt ions and has a strong fluorescence selectivity for in vitro GSH detection.By using fluorescence confocal imaging and flow cytometry,MB-SS-CPT probe was proved to have good stimulating response fluorescence imaging effect on esophageal cancer cells.MB-SS-CPT probe was applied to fluorescence imaging of esophageal tumor bearing mice in vivo.The fluorescence intensity of tumor site of mice received MB-SS-CPT injection was improved 2 times within 1 hours,and the total imaging time could be maintained for more than 4 hours.In addition,the antitumor therapeutic effect of the probe was also evaluated.Compared with the control group,the tumor inhibition rate of mice injected with MB-SS-CPT probe and underwent laser exposure could reach 60%,indicating that the probe has good antitumor effect.In conclusion,through the design of clinical drug of MB,a new responsive diagnosis and treatment integrated fluorescent probe is obtained,so that MB can further achieve expanded functional range and can have the targeted responsive ability toward tumor microenvironment on the basis of the original staining imaging and photodynamic.(2)Though MB-SS-CPT enabled MB with the responsive function toward tumor microenvironment,the probe structure of MB-SS-CPT and its reaction between GSH and disulfide bond could relatively prolong its response time toward GSH.Therefore,based on the structure of MB,a diagnostic probe,MB-GP,targeting dipeptidyl peptidase(DPP IV)that highly expressed on the surface of esophageal cancer cells was designed.The glycine proline dipeptide derivative(GP)on MB-GP can be recognized and hydrolyzed by DPP IV in the tumor microenvironment of esophageal cancer.When MB-GP is cut off by enzyme catalysis,MB will be released to achieve the fluorescence imaging performance and photodynamic therapy ability.MB-GP can be used to detect DPP IV protease and esophageal cancer cells in vitro.Within a certain concentration range,the fluorescence intensity of MB-GP has a linear relationship with the concentration of protease and cells.Using the linear relationship,the detection limit of DDP IV enzyme was calculated to be 1.1 ng/m L and that of esophageal cancer cells was 1300 cells/m L.MB-GP Probe was further applied to fluorescence response imaging of esophageal cancer cells at a concentration of 5 μM.The imaging effect can be achieved under the incubation condition of 1 h.Through confocal imaging and flow cytometry fluorescence intensity analysis,it was verified that the probe MB-GP had good DPP IV targeting ability.After MB-GP probe was injected into the esophageal tumor bearing mice,the MB-GP showed rapid and responsive fluorescence signals,which was enhanced more than 5 times in 1 h,and the imaging time window was more than 6 h.On the basis of the photodynamic therapy ability of the probe,the volume and mass of tumors in the mice received MB-GP treatment and underwent laser exposure were reduced 66.67% when compared to that of mice in the control group,indicating that MB-GP can effectively inhibit the growth of esophageal cancer.(3)Though the MB-GP probe could target DPP IV and has excellent performance in imaging and treatment of esophageal cancer,its performance towards esophageal cancer is based on the hydrolysis of amide bonds upon recognition of DPP IV,which may be disturbed by DPP IV isozymes and therefore could reduce the response specificity of the MB-GP probe.Therefore,in order to further improve the targeting specificity of the probe to DPP IV,the phenoxazine dyes connecting Alogliptin,a DPP IV inhibitor,was designed and synthesized to specifically target DPP IV,named probe655.The probe 655 has excellent fluorescence performance,and the Stokes shift could reach 100 nm.The binding simulation between DPP IV and probe 655,and the confocal imaging experiments showed that probe 655 processed a high affinity towards DPP IV,which could improve its targeting specificity for esophageal cancer cells.The probe655 also has considerable photodynamic characteristics,which could produce a significant number of reactive oxygen species in vitro upon laser exposure.Under the condition of low probe concentration and laser irradiation power,the probe 655 showed effective killing of esophageal cancer cells and excellent therapeutic outcomes on esophageal tumor bearing mice.