| Cell,as the essential unit of morphological structure and life activity,is comprised of numerous organelles,including mitochondria,lysosomes,endoplasmic reticulum,Golgi apparatus,ribosome,nucleus and so on.A series of biologically active small molecules in cells and organelles,such as reactive oxygen species,reactive nitrogen species,reactive sulfur species,and metal ions,are vital for maintaining the normal physiological functions of cells and organelles,as well as regulating the related pathophysiological processes.These active small molecules have vivacious chemical properties,and can reversibly or irreversibly modify the structure of biological macromolecules(proteins,nucleic acids,etc.),thereby regulating the functions of related biological macromolecules.In addition,active small molecules can cross the biomembrane in many ways and serves as the signaling molecules in cellular communication.Therefore,studying the subcellular distribution and concentration changes of active small molecules is of great significance for further investigation of the pathophysiological functions of active small molecules as well as the diagnosis and treatment of related diseases.For further study of the subcellular distribution and concentration changes of active small molecules,it needs to develop in-situ imaging technology with high selectivity,high sensitivity,and high temporal and spatial resolution at the subcellular level.Since active small molecules usually present the characteristics of short lifetime,high activity,and low concentration,the fluorescence imaging technology utilizing fluorescent probes becomes an ideal tool because of its high sensitivity,high temporal and spatial resolution,and non-invasiveness.This dissertation focuses on the design and synthesis of novel molecular fluorescent probes with subcellular localization capabilities.These probes are used to fluorescence imaging of active small molecules in organelles,tissues,and living organisms.Then the probes are applied to cell and animal model research to reveal the pathological effects and functions of active small molecules.This thesis carried out the following two aspects of research work:(1)Fluorescence imaging of peroxynitrite during drug-induced cardiac toxicity by a mitochondria-targetable probeMitochondria are the major sites for biological oxidation and energy conversion.The defects or dysfunction of mitochondria can induce a variety of diseases,such as neurodegenerative diseases,metabolic diseases and cancer.Reactive oxygen species(ROS)are produced accompanying mitochondrial oxidative phosphorylation.Excess ROS can cause mitochondrial damage,release pro-apoptotic factors,and induce apoptosis.Peroxynitrite(ONOO-)is one of the most active ROS,which can lead to the oxidation or nitrification of biological macromolecules,thus inducing mitochondrial apoptosis and even cell death.Mitochondria are the primary sites for ONOO-production and biological functions.Based on the current literatures,we speculate that mitochondrial ONOO-plays an important role during the process of cardiotoxicity induced by anthracycline antibiotics.Based on the above issue,a two-photon excited and near-infrared emitting fluorescent probe with specific response to ONOO-was designed and synthesized by utilizing Nile Red derivatives as fluorophore and ?-ketoamide as recognition group.The probe can selectively,sensitively,and rapidly respond to ONOO-,and is capable of targeting mitochondria,which can achieve two-photon fluorescence imaging of ONOO-in mitochondria and tissue.By utilizing this probe,it was found that the level of mitochondrial ONOO-will rise at the early apoptotic stage after anthracycline stimulation.The elevated ONOO-plays a key role in initiating and promoting apoptosis of cardiomyocytes and acts as important factors for myocardial cells apoptosis and tissues damage.Thus,the ONOO-level can be used as an early biomarker to predict drug-induced subclinical cardiac toxicity.The study provided important clues for the diagnosis and treatment of drug-induced heart damage.(2)Two-photon imaging of formaldehyde in live cells and in vivo with a lysosome-targetable and acid p H-activated fluorescent probeLysosomes are membranous organelles containing various acidic hydrolases and have an internal acidic p H(p H 4.5-5.5).As an important acidic organelle for eukaryotic cells,lysosomes play important role in many cellular biological processes such as endocytosis,cell growth and apoptosis,autophagy,iron homeostasis,and oxidative stress.Abnormal changes in lysosomes and their internal parameters can cause lysosomal storage diseases,Alzheimer's disease,and cancer.Formaldehyde is a highly active endogenous metabolite with intracellular physiological concentrations ranging from approximately 0.1-0.4 m M.Endogenous formaldehyde can be regulated by demethylase and oxidase.Abnormally elevated formaldehyde can induce various diseases such as cancer,neurodegenerative diseases,diabetes,chronic liver and heart diseases.Lysosomes are important organelles for formaldehyde production and biological functions.The abnormity of lysosomes will affect the concentration,subcellular distribution and biological effects of formaldehyde.Therefore,monitoring the concentration changes of formaldehyde in lysosomes has a very important role in understanding the production,distribution and metabolism of formaldehyde.Furthermore,it's benefical for the prevention,diagnosis and treatment of formaldehyde-related diseases.Based on the above issue,a lysosome-targetable two-photon fluorescent probe was designed and synthesized for specifical detection of formaldehyde.The probe can solely respond to formaldehyde in the acidic environment of lysosomes and hardly respond to formaldehyde in the neutral environment of cytoplasm and other organelles.Therefore,the probe can specifically and accurately detect the formaldehyde concentration in lysosomes.In addition,by using this probe,we achieve the in situ two-photon fluorescence imaging of formaldehyde in lysosomes and mouse abdomen tissues.With the aid of this probe,it was found that N-acetylcysteine can scavenge intracellular formaldehyde,indicating potential application prospect in the prevention and treatment of formaldehyde-related diseases.In summary,two fluorescent probes respectively targeting mitochondria and lysosomes were designed and synthesized for two-photon fluorescence imaging of ONOO-and formaldehyde in cells,tissues,and living animals.Both of the probes displayed good optical properties and response performance.By applying these probes to live cells and animal models,the pathological effects and functions of ONOO-and formaldehyde were preliminarily elucidated.These results provide highly effective fluorescence imaging tools for related biological research and supply valuable clues for prevention,diagnosis,and treatment of related diseases. |
PDF Full Text Request