Engineering Reactive Sulfur Species Fluorescent Probes With High Selectivity For Detecting And Pathology Research Of Fatty Liver

Among the many active sulfur species,hydrogen sulfide（H₂S）and hydrogen polysulfide（H₂S_n）have attracted much attention because of their involvement in signal regulation of various physiological and pathological processes.Studies have shown that H₂S and H₂S_nplay important roles in the pathogenesis and repair of liver injury diseases.Therefore,the accurate detection of the content of active sulfur species（RSS）and the expression level of their metabolic regulation enzymes（sulfatase）in the organism will help people deeply analyze the occurrence and development mechanism of liver injury,realizing its early diagnosis and treatment.In recent years,due to its advantages of high sensitivity,non-invasiveness,high temporal and spatial resolution,fluorescent probe analysis technology has been widely used by researchers in the fluorescence imaging detection of RSS and their regulatory enzymes in cells and in vivo.However,the existing fluorescent probes for accurate imaging detection of RSS and related metabolic regulatory enzymes in liver injury diseases still have some problems and challenges.On the one hand,most of H₂S_nfluorescent probes based on the construction of ester bonds have poor stability,which are susceptible to the influence of hydrolytic enzymes and p H fluctuations in cells and in vivo.On the other hand,most H₂S and H₂S_nfluorescent probes are prone to interference from other high concentrations of active sulfur species（such as millimolar GSH,Cys）in bioimaging applications,resulting in non-specific false positive signals.In addition,due to their environmentally sensitive characteristics,the fluorescent signals of most fluorescent probes are easily interfered by lipid droplets when used in the detection of liver diseases such as non-alcoholic fatty liver disease（NAFLD）,which severely hinders the accurate imaging analysis of related RSS and their regulatory enzymes.For these problems,we have combined rational design and screening strategies,and developed a series of high-performance fluorescent probes for RSS and their regulating enzymes through multi-step optimization methods,such as the construction of probe molecular library and recognition site screening,the design and synthesis of ratiometric probe,and probe nano-modification.We have applied these probes to achieve precise fluorescence imaging analysis of H₂S_n,H₂S and sulfatase in living cells and in vivo,and revealed the signal pathways produced by H₂S_nand H₂S in NAFLD and the mechanism of anti-oxidative damage.Based on this,this paper mainly carries out the following work:1)In view of the problems of limited stability for the current H₂S_nrecognition site（ester bond）,we have introduced an additional electrophilic center at the 2’-position of the phenyl ring in rhodamine,and constructed a library of probe molecules with dual electrophilic centers based on the dual nucleophilic characteristics of H₂S_n.By testing the response of these compounds to various RSS,we have screened out the compound Rh Ph CO6,which had a fast response to H₂S_n（within 1 min）and good selectivity.Furthermore,fluoroboron pyrrole dye（Br-BODIPY）was selected as the energy donor,and a ratiometric fluorescent probe PPG-BOD-Rh Ph CO based on the principle of FRET was constructed based on the amphiphilic polymer self-assembly strategy.This probe had high sensitivity to H₂S_n（detection limit is 17 n M）,good selectivity,good water solubility and biocompatibility.Finally,it had been successfully used for ratiometric imaging and detection of endogenous H₂S_nin inflammatory cells.2)NAFLD,a type of chronic fatty liver disease with a high incidence,faces many problems in its pathogenesis,therapeutic targets and diagnosis.It is urgent to develop accurate detection methods for signal molecules and biomarkers in NAFLD disease.In addition,we have found that the probe PPG-BOD-Rh Ph CO could cause the leakage of donor dye in fatty liver cells during the experiment,resulting in false positive signals.In order to solve these problems,we have selected Rh Ph CO6 as an energy acceptor that has a good response to H₂S_nin the previous chapter,and a naphthalene derivative（Np）with two-photon properties as the energy donor to construct a two-photon TBET-based ratiometric probe（Np-Rh Ph CO）with high energy transfer efficiency.Then,a TBET-based ratiometric nanoprobe PPG-Np-Rh Ph CO with good photostability and biocompatibility was obtained through self-assembly of nanoparticles.This nanoprobe can detect H₂S_nwith high selectivity in the presence of biothiols（GSH and Cys）and their persulfides（GSSH and Cys SSH）.Compared with the probe PPG-BOD-Rh Ph CO,PPG-Np-Rh Ph CO has higher stability and accuracy,and successfully achieved accurate imaging analysis of intracellular endogenous H₂S_nin high-fat environments such as NAFLD.And combined with biochemical analysis such as western blot and oil red staining,the signal pathway of H₂S_nproduction in NAFLD cells has been revealed,which to our knowledge is reported for the first time.The results have shown that H₂S_nin drug-treated NAFLD was mainly regulated by 3-mercaptopyruvate sulfotransferase（MPST）and cystathionine-γ-lyase（CSE）,and the ROS/H₂S/H₂S_ncrosstalk signal pathway has been revealed,which will help the detection of NAFLD disease and the analysis of active sulfur signaling molecular pathways.3)On the basis of the previous chapter,we have further developed a near-infrared（NIR）H₂S fluorescent probe that can be used to study NAFLD in vivo.In view of the limited selectivity of traditional H₂S recognition sites,we first constructed a H₂S near-infrared probe molecular library based on the three mechanisms of H₂S thiolysis,azide reduction,and dual nucleophilic addition.By testing the response of these compounds to various reactive sulfur（RSS）,a fluorescent probe 1-CSN with high sensitivity and selectivity towards H₂S has been screened.With the unique advantages of mesoporous silica nanomaterials（MSN）,a highly stable NIR nanoprobe MSN@CSN@PEG has been constructed for H₂S imaging detection with good biocompatibility,high selectivity and sensitivity.This nanoprobe was successfully realized the visual analysis of endogenous H₂S of NAFLD in vivo,and found that H₂S levels in the liver showed an increasing trend in the later stages of NAFLD disease,which was also verified by western blot analysis.4)The expression level of sulfatase（STS）,an enzyme related to active sulfur metabolism in organisms,plays an important role in the development of hormone-dependent cancers,such as breast cancer and cervical cancer.However,so far there is no suitable tool that can be used for in-situ analysis of sulfatase（STS）in biological samples.Thus,we have developed an STS-responsive two-photon ratiometric fluorescent probe ERNath S with cell membrane penetration ability,and explored the specific recognition mechanism between ERNath S and sulfatase through molecular docking experiments.Compared with the commercial probe 4-MUS,the probe ERNath S has good cell membrane penetration ability,and successfully achieved the imaging detection of STS at the living cell and subcellular level,so that it can clearly distinguish estrogen-dependent tumor cells from normal cells.In addition,ERNath S has been successfully applied to three-dimensional imaging and two-photon ratiometric imaging detection of STS in deep tumor tissues（up to 120μm in depth）for the first time.