Construction Of Highly Selective Fluorescent Probe And Its Application In Detection Of Inflammation And Drug-induced Liver Injury

Fluorescence analysis is favored by many scientists because of its high sensitivity,good selectivity and simple operation,and fluorescence imaging has the advantages of real-time,in-situ and visualization detection,small damage to the detection sample,good reproducibility,and so on.In recent years,inflammation and drug-induced liver injury are considered to be a serious public health problem.Therefore,it is important to accurately detect these biomarkers of inflammation and drug-induced liver injury with high sensitivity and good selectivity,which will help people to timely prevent and targetedly treat these diseases.Peroxynitrite(ONOO~-)and leucine aminopeptidase(LAP)are known biomarkers of drug-induced liver injury.However,due to the strong oxidative and nucleophile properties of ONOO~-,some dyes emitted in the visible or near infrared wavelengths are easily destroyed by ONOO~-,on one hand,which can lead to a lot of fluorescent ONOO~-probes have poor selectivity,and on the other hand,will cause false signals in the detection process for ONOO~-and LAP probes and lead to inaccurate experiment results.Therefore,to solve this problem,we aim to improve the selectivity,sensitivity and accuracy of fluorescent probes in this paper,and we construct a series of two-photon and near-infrared fluorescent probes through the combination of reasonable design and dye screening strategy.These ONOO~-and LAP fluorescent probes were designed and synthesized based on coumarin,naphthalimide,rhodamine and Changsha fluorophores,respectively.The concrete contents are as follows:(1)We have developed a two-photon ratiometric fluorescent probe,MITO-CC by combining the strategies of reasonable design and dye-screening,for selectively detecting ONOO~-in test tube and biological contexts.The ratiometric probe based on FRET mechanism exhibits not only outstanding sensitivity(11.30 nM),fast response(within 20 s)toward ONOO~-,but also high selectivity upon other various biological ROS and RNS in a physiological pH aqueous solution.The probe was successfully applied to ratiometric detection of endogenous ONOO~-produced by cells and tissues with minimal cytotoxicity either by one-photon or two-photon fluorescence confocal microscopy.Moreover,probe MITO-CC is capable of monitoring ONOO~-produced by LPS stimulation in the inflamed mouse model.This probe is highly promising to be useful for biological imaging to disclose the roles of ONOO~-in various physiological and pathological conditions.(2)In order to develop universal fluorescent probes,we further designed and synthesized two ONOO~-fluorescent probes Naph-ONOO~-and Rhod-ONOO~-based on the ONOO~-mediatedα-ketoamide deprotection reaction mechanism.These probes can not only detect ONOO~-in vitro with high specificity without interference from other biological species,but also can be used for in-situ imaging and real-time monitoring of ONOO~-generation in bioorganism.Among them,the two-photon fluorescent probe Naph-ONOO~-based on naphthalene amide fluorophore can monitor ONOO~-by one/two photon fluorescence imaging.The other probe Rhod-ONOO~-,accumulated mainly in mitochondria,showed that ONOO~-was mainly produced in mitochondria during drug-induced oxidative stress.The production of ONOO~-was first tracked by CYP 450 family oxidase-mediated xenobiotic metabolism,and the molecular mechanism of drug-induced hepatotoxicity was explored.(3)In order to explore the process of drug-induced liver injury in a deeper way,we construct two small molecules NIR-ONOO~-and NIR-LAP based on the combination of reasonable design and near infrared fluorescent dyes screening strategy,which can detect the change of ONOO~-and LAP level in biological system with high selectivity and high sensitivity.In addition,by using the unique optical properties of the near infrared fluorescence imaging,probes NIR-ONOO~-and NIR-LAP were also used to accurately detect the small fluctuations of ONOO~-and LAP in APAP-induced hepatotoxicity of living cells and mice,and the therapeutic effects of several hepatoprotective drugs on liver injury in mice were successfully evaluated.The development of these two near infrared fluorescence probes provides an effective tool for the study of the complex relationship between ONOO~-/LAP and drug-induced liver injury,which is helpful for the diagnosis of drug-induced liver injury and the development of its therapeutic drugs.(4)In this chapter,we designed and synthesized a mitochondrial-targetable two photon probe,MitoTP-ONOO~-,a reaction-based fluorescent probe.The probe was capable of detecting ONOO~-level with high selectivity and sensitivity under physiological conditions and in cells,respectively.By applying probe NIR-LAP developed by the above work,we intend to use probes MitoTP-ONOO~-and NIR-LAP,based on the excellent two-photon and near-infrared properties of these probes,for real time imaging of the generation of ONOO~-and LAP in the process of APAP-induced hepatotoxicity,and reveal the different changes of ONOO~-and LAP in this process,which is expected to provide a new idea for the diagnosis and treatment of drug-induced hepatotoxicity.