Design And Synthesis Of Target-Switchable Probes And Self-Reporting Photosensitizers For In Situ Monitoring Cell Viability

Cell viability is closely related to apoptosis,autophagy,necrosis and other cellular processes.During the process of cell death,the cell viability decreases significantly,and the morphology and biochemical properties of the organelles would also change obviously,such as the decrease of mitochondrial membrane potential（ΔΨ_m）,the loss of membrane integrity,the fluctuation of lysosomal pH,and so on.The regulation of specific cell viability in organism is an important guarantee to maintain cell homeostasis under physiological conditions.The abnormal regulation of cell viability can lead to many serious diseases,such as cancer.In addition,the monitoring of cell viability is essential for evaluating the efficacy of drug and toxicity of biological reagents,and screening anticancer drugs.Therefore,real-time monitoring of cell viability is of great significance to biology,pathology and medicine.At present,various techniques and methods have been used to detect cell viability,such as flow cytometry,MTT assay,transmission electron microscopy（TEM）,bioimaging and so on.Among them,bioimaging technique based on fluorescent probes is widely used due to its advantages of simple operation,minor injury to biological samples,detecting live samples,realtime and in-situ monitoring.However,the previous probes for imaging cell viability exist some shortcomings:①Fluorescence lifetime imaging technique possesses higher accuracy and precision,but there are few reported probes for detecting ΔΨ_m via fluorescence lifetime imaging,and the sensitivities of the probes are insufficient;②There have been few reports of fluorescent probes specifically detecting early or late apoptosis,and these probes have disadvantages of high toxicities,expensive costs and cumbersome operations.③ Apoptosis and autophagy are two important and closely related cellular processes.However,fluorescence probes that can distinguish apoptosis and autophagy have not been reported.④ Lysosome is an important organelle for regulating apoptosis,and lysosomal injury can easily induce apoptosis of cancer cells.However,lysosomes-targeted self-reporting photosensitizers have been reported rarely.Based on the change characteristics of organelles during cell death,in this paper,a series of target-switchable fluorescent probes were designed and synthesized,and realized the visualization of ΔΨ_m,the different stages of apoptosis,apoptosis and autophagy processes;In addition,lysosomes-targeted self-reporting photosensitizers were constructed,and realized real-time and in situ visualization of the PDT process.The main contents are as follows:1.Decreased mitochondrial membrane potential is a significant feature of reduced cell activity,in order to successfully correlate ΔΨ_m and the fluorescence lifetime of fluorescence dyes,a new strategy for obtaining the fluorescence lifetime difference with targets switching was proposed,a group of mitochondrial membrane potential dependent viscosity probes（PQO and PQP）were constructed.When ΔΨ_m was normal,the two probes accumulated in mitochondria by electrophoresis due to the large negative transmembrane potential of mitochondrion,and they targeted mitochondrial inner membrane due to their water-oil amphipathicity.When ΔΨ_m was decreased,both of them escaped from mitochondria and targeted cytoplasm and nucleoli because of their affinities to RNA,and the fluorescence lifetime of them increased significantly due to RNA had more viscous environment than that of mitochondrial inner membrane.The decrease of ΔΨ_m in cell injury induced by hydrogen peroxide was monitored successfully by PQO and PQP.This strategy can provide theoretical guidance for the design of relevant probes,the two probes can be used as powerful tools to monitor ΔΨ_m,and promote the development of the biomedical field.2.Based on the characteristics of decreased mitochondrial membrane potential in early apoptosis and loss of membrane integrity in late apoptosis,we further proposed a chainengineering strategy to regulate the membrane permeability of the probes,and successfully constructed a group of target-switchable probes to distinguish the different stages of apoptosis.The 2,7-substituted carbazole divinylpyridine salt with strong affinity to RNA was selected as the parent,and modified with different chain lengths,we obtained two probes with very similar structure（CPI-2 and CPI-3）,and the two probes was successfully used to detect the late and early apoptosis,respectively.CPI-2,which possessed short chain,could not enter living cells due to its poor membrane permeability,but it could stain the nucleoli of late apoptotic cells due to the destruction of cell membrane integrity.CPI-3 could penetrate the plasma membranes and stain the mitochondria of living cells,it could migrate to nucleoli in the early apoptosis due to the significant decrease of mitochondrial membrane potential.In addition,CPI-2 and CPI-3 were successfully used to monitor different apoptotic stages induced by different concentrations of H₂O₂,paclitaxel and rotenone.It is worth mentioning that CPI-2 and CPI-3 were used to study the apoptosis induced by heavy metal and the inhibition of cysteine to heavy metal induced apoptosis.This study provides a feasible strategy for constructing probes to detect the different stages of apoptosis,and it can promote the development of related fields.3.Using the change characteristics of lysosomal pH during apoptosis and autophagy,we constructed a target-switchable fluorescence probe（NKLR）based on aggregation/monomer principle for ratiometric and discriminative visualization of autophagy and apoptosis.NKLR equipped with alkalescent amino and showed certain affinity to RNA.In addition,RNA could induce NKLR to form aggregates with different fluorescent colors.In living cells,NKLR could simultaneously two-color image RNA（deep red channel）and lysosomes（yellow channel）in aggregation and monomer states,respectively.During autophagy,NKLR migrated from RNA to lysosomes with increased yellow fluorescence and sharply decreased deep red fluorescence.And this migration process was reversible when autophagy recovery.In contrast,during apoptosis,NKLR immigrated from lysosomes to RNA,and the yellow emission decreased and even disappeared,while the fluorescence of deep red channel was slightly increased.Overall,autophagy and apoptosis could be distinguished by the fluorescence intensity ratios of the two channels.Meanwhile,healthy cells,autophagic cells,and apoptotic cells could be distinguished by three point-to-point fluorescence images via the intracellular location and emission color of NKLR.Therefore,NKLR can be used as an ideal molecular tool to deeply reveal the relationship between autophagy and apoptosis,and provide the foundation for the research of physiology and pathology.4.Lysosome damage can lead to lysosome membrane permeability increase,which allows the release of cathepsin B/D to trigger apoptosis,so lysosome is the ideal target for photodynamic therapy（PDT）.We developed a lysosomes-targeted self-reporting photosensitizer NSLN,which could real-time and in situ monitor the PDT process.NSLN had both basic unit and cationic group that could target lysosomes in living cells and possess affinity to nucleic acids.When lysosome damage or cell death,NSLN migrated from lysosomes to nuclei.Therefore,the localization of the probe in the cell could reflect the cell viability.NSLN successfully monitored apoptosis induced by H₂O₂,rotenone and paclitaxel.NSLN could produce large amounts of reactive oxygen species under light irradiation,leading to lysosome damage and apoptosis,and it could monitor cell status via subcellular migration during PDT process.This work provides a new method for monitoring PDT processes and makes it possible to control phototoxicity.NSLN can serve as a powerful tool to promote the development of PDT.Overall,the proposed strategies can guide the design of relevant probes,and the developed fluorescent probes can be used as ideal tools to detect cell viability and promote the progress of biology,pathology and medicine and other related fields.