Design And Synthesis Of Novel H₂O₂-Mediated Cross-Linkable Near-Infrared Molecular Probes For Tumor Diagnosis Application

Background:With rapid development of fluorescent labeling and optical imaging technology,it is convenient to monitor all kinds of life activities in vivo without damage in real time,and it has a great application prospect in macromolecule detection,drug screening,tumor detection and gene expression.Near-infrared imaging technology is widely used in the field of tumor imaging due to its low interference of spontaneous fluorescence of organisms and tissues detected in the spectral range and deep penetrability of tissues,which effectively improves the accuracy and sensitivity of imaging to a certain extent.Compared with traditional organic dyes,near-infrared dyes have the advantages of higher fluorescence quantum conversion,tandem biotoxicity and plastic molecular structure,etc.However,in most tumors near infrared small molecular probes are easy to be rapidly excreted metabolism,living organisms in retention time is too short,leading to tumor imaging sensitivity is not high,seriously affected the tumor dynamic real-time imaging effect for a long time.Therefore,it is of great significance to design and develop a new near-infrared small-molecule probe that can stay in the tumor focus area for a long time and conduct dynamic real-time sensitivity monitoring on the tumor,so as to realize the sensitivity diagnosis and dynamic tracing of the tumor.Research purposes:The main purpose of this paper is to design and synthesize a new hydrogen peroxy-mediated cross-linked tumor near-infrared small molecule probe.The strategic coupling of 3,5-dioxocyclohexane group that can specifically crosslink with sulfenic acid,mitochondrial targeting group triphenylphosphine(TPP)and near infrared fluorescent dye Cy5 are strategically coupled into one.To construct a cross-linking near-infrared organic small molecule probe(DATC)mediated by hydrogen peroxide,aiming to improve the accumulation and retention time of the probe in the tumor lesions,thereby enhancing the fluorescent imaging effect of living tumors.Methods:In the first part of the study,3,5-dioxycyclohexane carboxylic acid was selected as the sulfenic acid specific cross-linking reaction group,and covalently coupled with the near-infrared fluorescent dye Cy5 and the mitochondrial targeting group TPP.A hydrogen peroxide-mediated cross-linked near-infrared organic small molecule probe(DATC)was obtained.Meanwhile,we synthesized a probe(TC)without 3,5-dioxycyclohexane group as a control probe.Due to the high expression of hydrogen peroxide in the tumor site,the thiol groups of many endogenous proteins are easily oxidized into substances such as protein sulfenic acid,protein sulfinic acid and protein sulfonic acid.When the probe DATC arrived at the tumor site,3,5-dioxycyclohexane carboxylic acid is converted into oxygen generation α-β unsaturated ketene,quickly and protein sulfonic acid covalent cross-linking reaction,causing internal probe covalent bond anchor due to tumor lesions,thus enhance the probe in the amount of enrichment of tumor and prolong the residence time,and eventually achieve the goal of improving tumor in vivo fluorescence imaging sensitivity.In the second part studies,in order to further improve the tumor targeting and crosslinking efficiency of probe,on the basis of the former part of the research,choosing norbornadiene olefin carboxylic acid sulfenic acid cross-linked group for the new times,through strategic and near-infrared fluorescent Cypate coupled and tumor targeting polypeptide cRGD with active tumor targeting united into hydrogen peroxide mediated crosslinking type small molecule probe(NR-Cypate)After the probe was actively targeted to the tumor site,it was anchored to the tumor by covalent cross-linking reaction with protein sulfenic acid.The cross-linking efficiency of the probe in in vitro tumor cells and in vivo tumor tissues was studied by using fluorescence confocal microscopy technique,and its ability to improve the sensitivity of tumor imaging was systematically evaluated.The near infrared luminescence,photoacoustic and photothermal characteristics of the dye Cypate were used in combination with the multi-mode imaging technology to further study the application of multi-mode imaging and photothermal therapy in vivo tumors.Results:The results of gel electrophoresis experiments show that the probe DATC can effectively label the hydrogen peroxide pretreated bovine serum albumin.In vitro cytotoxicity studies have found that DATC has little toxicity to cells and has good biocompatibility.Secondly,the imaging results of tumor cells showed that the probe DATC had good targeting ability to the mitochondria of the cells.After incubating the probe with hydrogen peroxide-pretreated mouse breast cancer cells 4T1 for a certain period of time,the cell imaging results showed that DATC can stay in tumor cells longer than the control probe TC.Cell-level immunofluorescence experiments confirmed that DATC had a covalent association with intracellular protein sulfenic acid and had a good correlation.Finally,DATC was injected intratumorally into mice,and the small animal live optical imaging system was used to detect and track the fluorescence signal of the tumor site.It was found that DATC could stay in the tumor for up to 36 hours,while the control probe TC was in After 6 hours,the fluorescence signal in the tumor was weak.In short,we designed and synthesized a hydrogen peroxide-mediated cross-linking near-infrared organic small molecule probe.High concentrations of hydrogen peroxide in the tumor microenvironment can mediate its selective anchoring in tumor lesions,thereby improving the tumor accumulation and retention time of the probe is conducive to sensitive imaging and long-term tracking of living tumors.Conclusion:A large number of experimental studies in vivo and in vitro have proved that by strategically introducing a 3,5-dioxocyclohexane cross-linking group into a traditional organic small molecule fluorescent probe,the high concentration of hydrogen peroxide-mediated sulfenic acid cross-linking in the tumor microenvironment was used.This method enabled the probe to be selectively anchored to the tumor lesion area,which can effectively increase the tumor accumulation and retention time of the probe,which is conducive to enhancing the imaging and treatment effect of the tumor in vivo,and for the study of the sensitivity of tumor diagnosis and effective treatment provides a new way of thinking.