Lipid Droplets Localization Fluorescent Probes Based On Triphenylamine And Its Application In Imaging Analysis

Lipid droplets are a class of organelles originating from the endoplasmic reticulum and encapsulated by a single layer of phospholipid molecules,and are the main storage sites for intracellular neutral lipids（triglycerides,cholesterol esters,etc.）.The size and composition of lipid droplets in different cells and tissues vary greatly.Lipid droplets are not only the energy stores of cells,but also complex and dynamic multifunctional organelles.Lipid droplets can move along the cytoskeleton and interact with other organelles（endoplasmic reticulum,mitochondria,autophagosomes,lysosomes,etc.）and play a key role in lipid metabolism and storage,protein degradation and signal transduction Therefore,it plays a role in maintaining the stability of lipid metabolism,the energy balance of the body and the stability of the intracellular microenvironment.Studies have shown that many diseases such as obesity,fatty liver,diabetes and cardiovascular disease are often accompanied by changes in lipid droplets in the cellular microenvironment.Therefore,it is of great significance to study the dynamic changes of lipid droplets in the cellular microenvironment.As signaling molecules,reactive oxygen species participate in and regulate many physiological and pathological processes.As an important reactive oxygen species,superoxide anion plays a role in information transmission,immune regulation and blood pressure regulation.Excessive superoxide anion(O₂^·-)will cause damage to biological macromolecules such as lipids,proteins and nucleic acids in cells,thereby affecting their normal physiological functions and inducing apoptosis and the occurrence of various diseases.Therefore,it is of great significance to study the changes of O₂^·-in lipid droplets,and to explore the changes of O₂^·-in the process of lipid droplet formation and migration.In this article,four kinds of fluorescent probes targeting lipid droplets were designed and synthesized based on the structure of triphenylamine,and the synthesized probes were found to have specific responses to O₂^·-.The probe has the characteristics of high sensitivity,good selectivity and low cytotoxicity,and can be used for the imaging detection of O₂^·-in lipid droplets.With the synthesized probes TB-1,TB-2,TB-3,and TB-4,two aspects of research were carried out:1.Four small molecule fluorescent probes were synthesized based on triphenylamine.The four probes connect electron-donating groups（triphenylamine）and electron-withdrawing groups（aldehyde groups,malononitrile）through a conjugated system,with D-π-A structure,resulting in its intramolecular charge transfer properties,which is manifested as:with the increase of solvent polarity,the greater the interaction between the probe and the solvent molecule,the greater the loss of non-radiative energy,and the fluorescence of the probe.The intensity decreases,and the emission spectrum is red-shifted.The synthesized probes have the following advantages:simple preparation,stable optical properties,good biocompatibility,and low cytotoxicity.Through co-localization experiments（artificial lipid droplets,intracellular lipid droplets）with commercial localization dyes（Nile Red,BODIBY493/503,ER-Tracker Red and Hoechst 33342）,it was confirmed that the synthesized probes have good lipid droplet localization ability.Dynamic tracing of lipid droplets in cells provides efficient probe compounds.2.The synthesized lipid droplet localization probe was used to test the response to intracellular species such as reactive oxygen species.The results show that the probe TB-1,TB-2,TB-3,TB-4can react specifically with O₂^·-,and new fluorescence emission appears around 510 nm,520 nm,525 nm,and 550 nm,respectively.The study of the reaction mechanism preliminarily showed that the S atom of the benzothiadiazole part of the probe was oxidized by O₂^·-to form a sulphone,which changed the electronic push-pull structure inside the probe molecule,resulting in the change of the fluorescence emission spectrum.The probe showed good selectivity and sensitivity to O₂^·-.The probe was used for imaging detection of O₂^·-in lipid droplets in cells with good results.This study provides a new probe material and method for in situ imaging and tracing of O₂^·-in lipid droplets.