Construction Of Polarity-sensitive Fluorescence Probes For Lipid Droplets Targeting And Their Bioimaging Applications

Polarity,as an important parameter in the environment,is closely related to the activities of living organisms.In the process of cell metabolism,the polarity of the microenvironment always changes.The polarity of cells can affect the processes of cell division,growth,movement,and other processes,leading to the differentiation of cell morphology and function.Once the intracellular polarity changes abnormally,the intracellular environment will be disturbed,causing various physiological or pathological problems.Therefore,detecting polarity changes in the intracellular environment may provide insights into various physiological and pathological processes,which is crucial for monitoring the different states of cell life activities.Lipid droplets（LDs）,as the energy storage of neutral lipids and proteins,play an important role in energy storage,membrane lipid synthesis,protein degradation and signal transduction.Although some progress has been made in the detection of microenvironment polarity of organelle LDs,there is still extensive space for development.For example,most of the current measurements for biological polarity are qualitative rather than quantitative.In addition,the polarity of LDs has not been revealed in various diseases.Therefore,in this paper,coumarin and naphthylamide fluorophore were selected as fluorophores respectively to construct three types of fluorescence probes with excellent detection for polarity.The fluorescence intensity,fluorescence lifetime,and other characterization methods were used to monitor the polarity changes in LDs.The specific contents are as follows:We designed two new fluorescent probes LDs-CA and LDs-BCA based on the triphenylamine and coumarin fluorophores to monitor LDs polarity and numbers variation.Coumarin was sensitive to polarity and the diethylamino group was introduced into the 7-position of coumarin to increase the liposolubility,so that the probe could enter the LDs smoothly.On the basis of the probe LDs-CA,the addition of the BF₂ group to obtain LDs-BCA could significantly enhance the intramolecular charge transfer（ICT）effect and showed a more sensitive response to polarity.Spectral experiments showed that in polar solvents,the ICT effect of the probe is enhanced,resulting in the redshift of fluorescence emission wavelength and the significant reduction of fluorescence intensity,showing the characteristics of obvious solvation effect.Biological experiments showed that the two probes have good selectivity for LDs,and could monitor the changes in LDs’size and quantity in different growth stages,and detected the polarity differences of cells in different states.More gratifying,the probe LDs-BCA was used to image the gastric fat of living mice,and the polarity difference between normal mice and obese mice was successfully monitored,which provided a new idea for the detection of obesity.To further achieve quantitative detection of polarity,naphthylamide with long lifetime and good photostability was used to design a polar probe LYC.The probe LYC was sensitive to polarity,and the fluorescence intensity increased by more than 10 times and the fluorescence lifetime increased by 3 times from the polar solution PBS to the nonpolar solvent dioxane.The excellent linear relationship between the fluorescence lifetime and polarity（R²=0.99）indicated that the probe LYC could achieve quantitative detection of polarity through the fluorescence lifetime.LYC could target LDs in cells with a colocalization coefficient of 0.91.In addition,cell imaging experiment results showed that LYC can achieve quantitative detection of the polarity of dead and living cells and monitor the cell apoptosis process by fluorescence lifetime imaging microscopy（FLIM）.Inspired by the probes LDs-CA and LDs-BCA are sensitive to polarity,and the probe LYC shows excellent fluorescence lifetime,we designed and synthesized a new fluorescent probe CCB by aldol condensation reaction with coumarin derivative as electron acceptor group and carbazole derivative as donor group.We hoped that the change of LDs polarity caused by the NAFLD can be monitored by fluorescence lifetime change of probe CCB.The probe CCB showed good biocompatibility,long fluorescence lifetime,lipid solubility and could realize specific imaging of LDs.The fluorescence lifetime of the probe CCB had a good linear fitting relationship with the polarity and increased significantly（about 15-fold）with decreasing solvent polarity.Furthermore,CCB was successfully used to monitor NAFLD-induced polarity changes in cells and animal tissue slices by FLIM.To our knowledge,this is the first time that the LDs polarity changes are used to quantitatively diagnose the steatosis process of NAFLD by FLIM,which may enlighten to the quantitative diagnosis of NAFLD.