Design,Synthesis And Cell Imaging Applications Of Solvatochromic Fluorescent Dyes

Most organelles in eukaryotic cells are enclosed and isolated by biofilms,with unique components and microenvironment,which can fulfill their unique biological functions.At the same time,close cooperation and contact between organelles play a key role in cell physiological processes,apoptosis,autophagy and other pathological processes.Therefore,it is great significance to study the dynamic changes and interaction networks of organelles.Fluorescence imaging technology has the advantages of non-invasive,high sensitivity,in situ and real-time dynamic monitoring of living biological samples.The resolution(～200 nm)is equivalent to the size of organelles,so it is an important technology to study subcellular dynamic processes,and the corresponding fluorescence probe is a key tool to achieve target observation.Previously,several fluorescence probes have been reported to investigate organelle interactions and important processes such as apoptosis.For example,single molecule dichroism imaging endoplasmic reticulum and lipid droplet fluorescence probes.However,as previously reported,there is little difference in fluorescence wavelength between the endoplasmic reticulum and lipid droplets,and the fluorescence signals interfere with each other,hindering the in-depth study of their interaction.The fluorescence probes used to detect the autophagy process of mitochondria are mostly based on the pH response principle,and the pH fluctuations in mitochondria and lysozyme will cause interference.In addition,there are few reports on single fluorescent probes that distinguish different stages of apoptosis.Therefore,in view of the shortcomings of previously reported probes,this paper mainly used solvent-chromic fluorescent dyes with different fluorescence emission wavelengths in different solvent environments to develop fluorescence probes for monitoring double-organelle interaction,two-color fluorescence probes for imaging mitochondrial autophagy process without pH response,and single fluorescence probes for differentiating and observing different stages of apoptosis.Details are as follows:(Ⅰ)Based on the difference in water content of lipid droplet membrane and endoplasmic reticulum,a solvent chromotropic fluorescence probe was designed and synthesized by reversible open/closed loop reaction of Rhodamine dye,which realized the two-color observation of lipid droplet and endoplasmic reticulum.Moreover,the fluorescence wavelength of the probe in the two organelles is very different,which can eliminate the cross-color interference to the maximum extent.The probe DCTX which was sensitive to the changes of water content in the organism,displayed strong green fluorescence in the hydrophobic LDs from its ring-closed form,while existed in a ring-opened form in ER to illuminate a strong near-infrared(NIR)emission.Importantly,the spectral difference was up to 320 nm,and thus the crosstalk between two channels was negligible.With the unique probe,the lipid accumulation in cells treated with different concentrations of oleic acid,cholesterol and stearic acid have been successfully observed.The changes of LDs and ER in living cells stimulated by temperature changes and hypoxia stimulation also have been revealed.Meanwhile,the different size and distribution of LDs and ER in various tissues was also studied using the robust probe.(Ⅱ)A mitochondrial fixed fluorescence probe based on aggregation/monomer principle was constructed for the first time for two-color observation of mitochondrial autophagy process.The probe has cationic salt structure,and the monomer emits green fluorescence,which can be concentrated in mitochondria with negative membrane potential,form aggregates,and emit near infrared fluorescence.Meanwhile,the probe has benzyl chloride group,which immobilized in mitochondria via the reaction with thiol group of mitochondrial proteins,and the changes of mitochondrial membrane potential did not affect localization of it;During mitophagy,the probe molecules were delivered into autophagolysosomes together with mitochondria,which switched to monomers to give green emission.In this manner,the mitophagy procedure was visualized in dual-emissive mode.With the robust probe,the mitophagy induced by starvation,oxidative stress,and hypoxia has been successfully visualized.(Ⅲ)Based on the principle of polarity response,a fluorescence probe was designed to distinguish healthy,early apoptotic,and late apoptotic cells in three different emission colors.The probe consisted of a polar responsive fluorophore and a weakly basic secondary amine group which targeted lysosomes in living cells and localized in the high-polarity region of lysosomal membranes because of the secondary amine group protonating into quaternary ammonium salt.With the increase of lysosome pH in early apoptotic cells,the presence of the probe in the form of secondary amine leaded to increased lipid solubility which was located in the inner part of the lysosome membrane and the polarity of the environment was lower than that of the outer environment.In late apoptotic cells,due to the destruction of the lysosome,the probe immigrated from lysosomes to the lipid droplets with ultralow polarity.Therefore,the probe was in three different polar environments in living cells,early apoptotic cells and late apoptotic cells;Because of the different emission wavelength of the probe in different polar environments,the probe could realized the trichromatic differentiation observation of the cells in three different states.Using this probe,we successfully investigated the different apoptotic stages of cells treated with ultraviolet irradiation,paclitaxel and N-acetylmaleimide.(Ⅳ)A series of hydrophilic fluorescent probes with excellent membrane permeability were designed by using the mechanism of intramolecular reversible cyclization.The probes have two forms of related ring and open ring,which were closed ring in aprotic solvent and open ring in protic solvent.This kind of probes existed in the form of open loop in water environment and have strong hydration,which could avoid large particle aggregation caused by insoluble in water.At the same time,they changed from a hydrophilic open ring form to a hydrophobic closed ring form when they approach the hydrophobic layer of the phospholipid bimolecular layer,allowing them to effectively pass through highly hydrophobic phospholipid bimolecules.Therefore,this kind of probes could achieve a perfect combination of hydrophilicity and good film penetration performance.In addition,we synthesized control molecules that were unable to generate switching rings,confirming that the difference in the probe’s membrane penetration ability was indeed due to the unique intramolecular switching ring reaction,rather than the simple difference between hydroxyethyl and methyl.Using this principle,we constructed a fluorescent probe with excellent membrane penetration performance that realized the ability to stain intracellular lysosomes at an ultra-low concentration of 1 n M.