Microenvironment-sensitive Small-molecular Fluorescent Probes And Their Applications In Imaging Cells And Tissuses

Various organelles and microenvironments consisting in living cells are crucial for maintaining the normal life activities of cells.Different organelles display diverse microenvironments,and the relative stability of microenvironments provides a guarantee for the normal life activities of organelles.Since each organelle has a specific microenvironment,microenvironment-sensitive fluorescent probes were designed to visualize a specific organelle,and monitor the changes in the microenvironment of this organelle.Due to the discrimination of different organelles in the microenvironments,a single probe was designed to simultaneously image two organelles in dual-color,so as to study the interaction between these two organelles in the cell life activities.Therefore,this paper focuses on the following aspects:(1)A pH-sensitive monomolecular dual-target fluorescent probe for simultaneous visualizing nuclei and mitochondria in dual color;(2)Viscosity-sensitive endoplasmic reticulum fluorescent probes with a long alkyl chain;(3)Viscosity-sensitive fluorescent probes for visualizing plasma membrane and nucleolus and their applications in tissue imaging.1.In living cells,organelles do not exist in isolation.They interact with each other to form an interaction network of organelles,and jointly complete a series of life activities.Therefore,visualizing two organelles simultaneously is very significant to study interaction networks of organelles.Nuclei and mitochondria are two essential organelles in eukaryotic cells,and they play vital roles in many physiological processes such as cell proliferation and survival,cell differentiation,mitochondrial autophagy,apoptosis and signal transduction.So,visualizing nuclei and mitochondria in dual color is beneficial to study physiological processes,such as cell apoptosis.Generally,nuclei exhibit a basophilic property due to the presence of nucleic acids,whereas mitochondria maintain a basic pH(pH=?8)environment at a normal condition.So,a pH-sensitive fluorescent probe(HMBI)was designed and synthesized to simultaneously visualize nuclei and mitochondria in dual colors by taking advantage of the basophilic property of nuclei and the alkaline environment in mitochondria.In acidic environment,HMBI showed green fluorescence,while in alkaline environment,it presented red fluorescence.So,HMBI is able to discriminate nuclei and mitochondria in green and red fluorescence,respectively.In addition,HMBI can also distinguish normal cells and damaged cells.HMBI can visualize green nuclei and red mitochondria in normal cells.However,the fluorescence intensity of mitochondria is significantly reduced in damaged cells,which may ascribe to the reductions of mitochondrial membrane potential and pH in the damaged cells.In this paper,HMBI was used to observe the process of cell apoptosis:in normal cells,the probe could simultaneously image the nuclei and mitochondria;in apoptotic cells,the fluorescence intensity of mitochondria was significantly reduced,and typical apoptotic characteristics,such as shrinking and pyknotic nuclei,could be observed in the nuclei.This work provides guidances and references for the exploitation of monomolecular dual-target fluorescent probes to visualize two organelles simultaneously in dual color.2.Similar to apoptosis,endoplasmic reticulum autophagy(ER-Phagy)is a vital process involving multiple organelles,and it is critical in the quality control of endoplasmic reticulum proteins.The accumulation of unfolded or misfolded proteins in ER can lead to endoplasmic reticulum stress,causing a variety of pathological processes and serious diseases,such as tumors and neurodegenerative diseases.Therefore,the in-depth study of ER-Phagy is of great significance.Based on the strong hydrophobic interaction between the long alkyl chain and the phospholipid bimolecular layer in the ER membrane structure,a viscosity-sensitive fluorescent probe(HTMP-16)with a long alkyl chain was designed and synthesized to monitor ER-Phagy.The probe can visualize endoplasmic reticulum in normal and damaged cells,and it enables to firmly target endoplasmic reticulum,which is conducive to long-term track endoplasmic reticulum.In view of this,HTMP-16 was applied to trace and visualize the process of endoplasmic reticulum autophagy in real time.In addition,due to the viscosity-sensitive characteristic of the probe,HTMP-16 was also able to observe the changes of endoplasmic reticulum viscosity.Currently,most ER fluorescent probes(ER-tracker green and ER-tracker red)contain methyl sulfonamide groups that target the ER by binding to sulfonamides receptors located in the potassium channels of ER.However,this kind of probes blocks the KATP channel,resulting in depolarization of the cell membrane.Inspired by the easy internalization of cell membrane probes with long alkyl chain,a new type of long alkyl chain endoplasmic reticulum probe was developed considering the endoplasmic reticulum has a membrane structure similar to that of the cell membrane.Compared with the previously reported endoplasmic reticulum probe with long alkyl chain assisted by?-cyclodextrin,this paper provides a new idea for the development of endoplasmic reticulum probe.3.As a barrier of cells,plasma membrane maintains the integrity of the cell by selectively controlling the substances entering cells.This process is closely related to a series of life activities such as signal transduction,cell differentiation,and cell fusion.Meanwhile,compared with the cells cultured in culture medium,cells in tissues are surrounded by an extracellular matrix containing diverse amounts and types of collagen,proteoglycans,adhesion molecules,and growth factors,which is a major obstacle to the development of probes to visualize organelles in tissues.So it is still rare and remains a great challenge to visualize plasma membrane in tissues,because they have low signal-to-noise ratio.Due to the strong binding force between the long alkyl chain and the phospholipids of the plasma membrane and the excellent tissue permeability of the small-molecule structure probes,two viscosity-sensitive small-molecule structure probes(DSP-16 and DSP-18)with a long alkyl chain were designed and synthesized to visualize the plasma membrane in tissues.These two probes have good viscosity-sensitive properties,which are beneficial for high-fidelity visualizing plasma membranes in tissues.DSP-16 and DSP-18 can image the plasma membranes in living cells,fixed cells and rat blood red cells,indicting good targeting abilities to plasma membrane.Compared with the commercially available plasma membrane probe Dil,the two probes were able to image plasma membranes in liver tissues and T-tubule of rat skeletal muscle tissue.4.As an important organelle,nucleoli are the sites for synthesis,processing and assembly of ribosomal RNA(rRNA),which is closely related to cell proliferation and differentiation.The number,size,and activity of nucleoli are closely associated with cell proliferation,and these properties can predict cancer and other diseases.Some reports suggest that nucleolar morphology is closely related to several neurodegenerative diseases,such as Parkinson's disease.In addition,nucleoli have become diagnostic biomarkers for pathological detection of malignant tumors.Therefore,visualizing the nucleoli in tissues is of great significance.However,there are few reports on probes that can visualize nucleoli in tissues.Fluorescent probes(IVMOI and HTMP)that can visualize nucleoli in tissues were synthesized taking advantage of the excellent permeability of small-molecular probes.The two probes displayed weak fluorescence at low viscosity and strong fluorescence at high viscosity,which is beneficial for high-fidelity visualizing nucleoli in cells and tissues.In summary,a series of environment-sensitive fluorescent probes were designed and synthesized focusing on the microenvironment in cells and tissues.Based on the basophilic property of DNA in nuclei and the alkaline environment of mitochondria,a pH-sensitive probe was designed and synthesized to simultaneously visualize nuclei and mitochondria in dual color,and monitored cell apoptosis.Taking advantage of the hydrophobic interaction between hydrophobic alkyl chain and phospholipids,a endoplasmic reticulum probe with long alkyl chain was designed and synthesized,and the process of endoplasmic reticulum autophagy in cells was also visualized with the help of this probe.Utilizing the excellent permeability of small-molecular probes in tissues,a serious of viscosity-sensitive fluorescent probes with small-molecular structure were designed and synthesized to visualize plasma membrane and nucleolus in tissues.From the perspective of basic research,this paper plays a guiding role in the exploitation of new probes,such as monomolecular dual-target fluorescent probes.From the perspective of applied research,these probes are helpful to the study of cell physiological process and will promote the development of histopathology.