Development Of Tumor Microenvironment-responsive Probes For Tumor Theranostics Application

Background:As a growing biomedical discipline,molecular imaging provides an effective means to enable the visualization of the cellular function and uncover the mechanism of physiological and pathological processes such as cancer,cerebral disease,cardiovascular disease,etc.With the rapid development of molecular imaging technology and the in-depth study of tumor microenvironment,stimulus-responsive intelligent small molecule probes designed based on tumor microenvironment have attracted more and more attention in the field of tumor imaging and treatment.When the smart molecular probe reaches the tumor tissue or tumor cells,the structure of the probe changes under the stimulation of tumor microenvironment,so as to significantly enhance the imaging signal or therapeutic effect,thus achieving accurate detection and treatment of tumor.Common environmental stimuli include:internal environment(including highly expressed enzymes,reducing substances,low pH,reactive oxygen species,etc)and external environment(including light,temperature,ultrasound,etc).Objective:In this thesis,we developed some new smart acidic pH/glutathione(GSH)dual-stimuli responsive nanoprobes,based on tumor microenvironment response model design and application of small molecules,which can self-assemble into nanoparticles through the CBT condensation reaction in the tumor cells or under tumor microenvironment.It has demonstrated that the aggregation of probes can effectively enhance their cell uptake and prolong the retention time in tumors,ultimately achieving accurate diagnosis and eficient treatment of tumors.Method:In the first part of this research,we have successfully designed and synthesized a new smart acidic pH/glutathione(GSH)dual-stimuli responsive intelligent small molecule probe(1),which can intermolecularly undergo CBT condensation and form nanoparticle(I-NPs)in the tumor microenvironment,with the citraconic amide bond as the pH-responsive group,the disulfide bond as the reductive responsive group,and the triiodobenzoic acid as the CT imaging group.When the probe 1 entered the tumor tissue,under the stimulation of the weak acidic environment of tumor cells,the intramolecular carboxyl group of the citron amide group catalyzed the hydrolysis of amide bonds and left in the form of citric acid,thus exposing the original amino group in the cysteine structure.On the other hand,overexpression of glutathione in tumor tissue reduces disulfide bonds and exposes sulfydryl.Then 2-cyano-benzothiazole and the 1,2-amino-mercaptan formed after the response occurred a quick and efficient click condensation reaction to generate amphiphilic dimer,and through ?-? stack in situ self-assembly into nanoparticles.This tumor microenvironment responsive strategy can effectively enhance the accumulation of probes in tumors and prolong their retention time,so as to achieve the purpose of tumor-specific and long-term imaging.In the second part of the research,on the basis of the study in the first part,we further synthesized a small-molecule theranostic probe(Cy-1)which can be activated by tumor microenvironment to form aggregates in tumors resulting in enhanced tumor imaging and therapy.Based on the characteristics of low pH and overexpressed glutathione conditions,we realized the integration of tumor diagnosis and treatment in vivo by virtue of the photoacoustic,fluorescence,photothermal and other properties of hematocyanine dyes.Results:In vitro experiments,a series of studies,including HPLC,electron microscopy and ultraviolet,have proved that the probe 1 has a good response to aggregation.In addition,fluorescence experiments on cells also proved that probe 1 also had a very good effect on aggregation and retention at the cell level.The results of in vivo experiments on mice showed that probe 1 could effectively enhance the enrichment of probe in tumor and prolong its retention time after it was injected into mice via tail vein.In the second part of the work,we investigated the changes of photoacoustic,fluorescence,ultraviolet absorption and photothermal properties of the probe Cy-1 before and after aggregation in vitro,and found that its ultraviolet absorption,fluorescence,photoacoustic signal and photothermal conversion efficiency had significant change after aggregation.Subsequently,the in vivo results indicated that we have successfully achieved improved tumor accumulation and long-term imaging by monitoring the enhanced photoacoustic signals induced by the aggregation of cyanine dyes.On the other hand,the results of animal experiments showed that the probe Cy-1 had great photothermal therapeutic effect.In general,we have successfully designed and synthesized a pH/GSH dual-responsive small molecular probe which can be used for in vivo dual-modal PA/NIR imaging of tumors with good specificity and sensitivity.Meanwhile,under the guidance of photoacoustic-fluorescence imaging,more accurate photothermal treatment can be achieved.