Designand Synthesis Of Fluorescent Probes Bearing Long Alkyl Chain For Imaging/Tracking Mitochondria And Imaging Ultrastructures In Plasma Membrane In Situ

Fluorescent probe is an important category of functional materials and plays an important role in fundamental and application research of life science and medicine.Mitochondria and plasma membrane are both crucial components in cells,which play vital roles in numerous cellular physiological processes.This dissertation focuses on the following aspects.C①fluorescent probes which can in-situ and directly image mitochondria in living tissues;②fluorescent probes that real-time track mitochondria in living cells;③fluorescent probes which can in-situ image plasma membrane;④fluorescent probes that simultaneously,in-situ,and discriminately image lipid raft/non-raft domains by dual colors in plasma membrane.The cells cultured in vitro were quite different from those in tissues.Therefore,in-situ and directly imaging mitochondria in tissues can offer more native and accurate information.Particularly,in the clinical diagnose of mitochondrial diseases such as mitochondrial myopathy,it is a routine examination item to directly observe mitochondrial morphology and number in muscle tissues from patients.Because the composition in tissues is more complex than the cultured cells,the selectivity of mitochondrial probes should be higher.However,the selectivity of available probes is inadequate for exclusively tissue imaging.In order to solve the problem,we propose a method that modifying dual targeting groups to a probe can increase its selectivity.Then we modify a C18-alkyl chain to a common cationic mitochondrial probe and obtain a new probe(Mito-MOI)with ultrahigh selectivity.The cationic moiety has electrostatic interaction with the inner membrane negative charge.Simultaneously,the C18-alkyl chain,as the second targeting group,is deeply embedded into the hydrophobic region of inner membrane through hydrophobic interaction.Therefore,the dual targeting groups(cation and C18-alkyl chain)actually endow Mito-MOI with ultrahigh selectivity.As expected,high-resolution microscopic photos showed that Mito-MOI indeed stained mitochondrial inner membrane.Moreover,in-situ and high-fidelity tissue imaging has been achieved,and particularly,four kinds of mitochondria and their crystal-like structure in muscle tissues were visualized clearly.Finally,the dynamic process of mitochondrial fission in living cells has been shown and Mito-MOI has good photostablity as well as low toxicity.The strategy employing dual targeting groups should have guidance significance for designing fluorescent probes with ultrahigh selectivity to various intracellular membranous components.Compared with in-situ and statically imaging mitochondria,long-range,real-time,in-situ,and dynamically tracking mitochondrial morphology and number during the whole cell cycle is more significant.Current mitochondrial probes are highly dependent on the large negative mitochondrial membrane potential(MMP).Once MMP decreases or vanishes,these ordinary cationic probes will move away from mitochondria,which limits their use in tracking mitochondria with fluctuant MMP in living organism.Furthermore,available mitochondrial trackers,which can react with thiols or amino acids of mitochondrial proteins and form covalent bind,will influence some properties of proteins in mitochondria and thus may damage mitochondria after staining.In order to solve the problem,we have weak interaction instead of covalent bind.And we utilize the strong hydrophobic interaction between long alkyl chain and lipid bilayer,add C 12-alkyl chain to carbazole-pyridine and indole-pyridine cationic salt,respectively,and obtain two non-reactive mitochondrial trackers(ECPI-12 and IVPI-12).When ECPI-12/IVPI-12 stain mitochondria,the cationic "head" locates at the matrix-facing surface of the inner membrane through electrostatic interaction with the negative charge of the inner membrane.Simultaneously,the long lipophilic aliphatic chain inserts into the hydrophobic region of the inner membrane through hydrophobic interaction.When mitochondria are depolarized,although the electrostatic interaction decreases,the hydrophobic interaction still remains and immobilizes the probe within mitochondria.Different from covalent bond,this hydrophobic interaction belongs to noncovalent interaction,which would not destroy mitochondrial protein.As expected,ECPI-12/IVPI-12 were able to illuminate functional mitochondria in living cells.Particularly,when MMP decreased or vanished,ECPI-12/IVPI-12 could still be retained in mitochondria,indicating that they had the ability to track mitochondrial dynamics.Through contrast experiments with ECPI-2 and IVPI-2,we proved that it was the long alkyl chain that made ECPI-12/IVPI-12 retained in mitochondria when MMP decreased.Moreover,cytotoxicity tests showed that ECPI-12/IVPI-12 had lower toxicity than MTDR,demonstrating that hydrophobic interaction was more facile than covalent bond,and brought lower injury to mitochondria.In addition,ECPI-12 and IVPI-12 had the ability to track mitophagy process in real time.And they had excellent TPEF properties and could be used in deep tissue imaging.These results demonstrate that ECPI-12 and IVPI-12 could serve as powerful tools to track the mitochondrial dynamic changes in cells and tissues during physiological and pathological processes.Two-photon excited fluorescence(TPEF)imaging has great advantages including high penetration depth to samples,low background noise,low photo-damage and photobleaching and so on.Therefore,it is significant to develop TPEF fluorescent probes for bioimaging applications.Plasma membrane is the communication medium between cell and extracellular environment,which is closely related to signal transduction,endocytosis,exocytosis,ion exchange,and so on.Hence,imaging plasma membrane in living cells allows people to get a better understanding of the relevant biological processes.Although there are many reports about plasma membrane probes,TPEF ones are rare.In addition,the reported TPEF plasma membrane probes have low two-photon active absorption cross section(δ×Φ)and the imaging results in cells are not clear enough.In order to solve the problem,we synthesized a plasma membrane probe ECPI-18 with good two-photon properties using carbazole-pyridine salt system as the platform.When excited by 860 nm,the value of two-photon absorption cross section(δ)and δ×Φ was 991 GM and 150 GM,respectively,indicating that ECPI-18 had good two-photon properties.Moreover,ECPI-18 was able to be used in TPEF imaging and plasma membrane was clearly imaged in living cells.MTT resluts showed that ECPI-18 had low cytotoxicity.And multi-staining experiments indicated ECPI-18 had good co-staining compatibility with nuclear probe and mitochondrial probe.Plasma membrane contains two kinds of ultrastructures,i.e.lipid raft and non-raft domains,collaboration of which plays essential roles in various bioactivities.Lipid raft domains participate in many physiological such as signaling,formation of protein clusters and so on.And non-raft domains are associated with the nanoclusters of the resting T cell antigen receptor.Discriminating and simultaneously imaging Lipid raft and non-raft domains is important in relevant biological and physiological research.Current fluorescent probes for imaging lipid rafts and non-raft domains in plasma membrane are rare.They have low signal to noise ratio(SNR)and the imaging results in cells are not clear enough.In order to solve the problem,we use molecular rotor and intramolecular charge transfer(ICT)mechanisms to design a probe(DSPI-18)with high SNR for imaging lipid-rafts and non-raft domains in plasma membrane.Molecular rotor effect can increase the fidelity of bioimaging and ICT enables DSPI-18 to emit different fluorescent signals in lipid rafts and non-raft domains.According to the real-color imaging results in giant unilamellar vesicles,DSPI-18 can stain raft lipid and non-raft domains and emit green and red fluorescence,respectively.The emission spectra difference between lipid-raft and non-raft domains is 50 nm.During bioimaging,DSPI-18 can stay in plasma membrane for more than 30 min,and high-fidelity fluorescent images can be obtained.In real-color imaging,clear orangered and green fluorescence are observed in the plasma membrane of SiHa cells.In addition,the orangered region in plasma membrane represents non-raft domain and the green region is lipid-raft domain.These results indicate that DSPI-18 has the potential for the investigation of lipid raft and non-raft domains.Furthermore,using molecular rotor and ICT to design a probe with high SNR is reasonable.In conclusion,we have designed a mitochondrial probe with dual targeting groups to increase the selectivity and obtained high-fidelity fluorescent images of mitochondria in intact living tissues.We also design two non-reactive mitochondrial trackers by using the hydrophobic interaction between long alkyl chain and lipid bilayer.The two trackers have no injury to mitochondrial proteins.Then we synthesize a plasma membrane probe with good two-photon properties using carbazole-pyridine salt system as the platform.Finally,using molecular rotor and ICT,we design a probe with high SNR for discriminately imaging lipid raft and non-raft domains in plasma membrane.From the viewpoint of fundamental research,the work in this dissertation reveals new strategies for fluorescent probe design,which should have guidance significance and reference value for designing other fluorescent probes.From the point of applications,these probes can serve as powerful tools for in-depth study on organelles and intracellular ultrastructures in biology and medicine.