Development Of Interface-Targeting Lipid Droplets And Two-Color Lipid Droplets And Endoplasmic Reticulum Fluorescent Probes As Well As Research Of Side-Chain Engineering Strategy For Controling The Memebrane Permeability Of Fluorescent Dyes

Cells are the basic structural and functional unit of living organisms.The ultimate goal of life science research is to reveal the dynamics and functions of all cells in living organisms.Compared with the commonly used cell biology research methods,such as radioactive tracer with radioactive hazard,as well as electron microscopy and mass spectrometry,etc.that can only be used to observe and analyze dead cells,fluorescence imaging technology supplemented by important bioanalytical materials(fluorescent probes)is equivalent to installing an endoscope on intact cells,minimizing damage to living cells,enabling the real-time and in situ observation of organelles,biomolecules and their dynamic changes in living cells.Therefore,the research content of this thesis mainly focuses on the following four aspects:(1)Development of lipid droplets and related organelle fluorescent probes,including interface-targeting fluorescent probes for high-fidelity,noise-free imaging lipid droplet,and single-molecule dual-targeting fluorescent probes that two-color image lipid droplets and endoplasmic reticulum;(2)Side chain engineering strategy and the derived fluorescent probes,including side chain engineering strategy for controlling the membrane-permeability of dyes and developing a series of novel fluorescent probes based on this strategyOrganelles are important components of eukaryotic cells.Lipid droplets are new and highly dynamic intracellular organelles,composed of a neutral core containing triglycerides and cholesterol esters and a phospholipid monolayer with a variety of proteins distributed on its surface.They play important roles in regulating cellular energy homeostasis and protecting cells from lipotoxicity and lipid apoptosis Moreover,their abnormality is closely related to various diseases such as abnormal fatty liver,type ? diabetes,atherosclerosis and obesity.Therefore,it is necessary to develop specific lipid droplets fluorescent probes for revealing lipid droplet biology and the occurrence and development of related physiology and pathology.However,existing lipid droplets probes are designed based on the hydrophobic core inside the lipid droplets.These lipophilic probes show low seltivity and cannot distinguish lipid droplets from other intracellular lipophilic environments.In order to solve this problem,based on its special hemi-membrane interface structure,we first propose an interface targeting strategy to design lipid droplet probes.As a result,amphiphilic fluorescents probe were designed and synthesized by rational introduction of a cationic salt on a lipophilic fluorophore(NBD).In this way,the NBD portion can be completely embedded in the anhydrous core of the lipid droplet by a strong hydrophobic interaction,and the cationic portion can accurately locate on the polar shell of the phospholipid monolayer by electrostatic interaction.Thus,this interface-targeting approach enables amphiphilic probes with ultrahigh selectivity to lipid droplets and achieves the high fidelity imaging.Using interface-targeting probes,we successfully monitor the changes of lipid droplets in size and morphology in living cells,and further observe abnormalities in liver tissue under a two-photon microscope Therefore,interface targeting probes are expected to be used for early detection of fatty liverBased on the above-mentioned interface-targeting lipid droplets probe research,this paper further develope single-molecule dual-targeting fluorescent probes for simultaneously imaging lipid droplets and endoplasmic reticulum in dual-color mode As an emerging highly dynamic organelle in cells,lipid droplets not only have their independent functions,but also interact with many intracellular organelles,which is an important part of the network of organelle interactions.In particular,lipid droplets have close relationship with endoplasmic reticulum.At present,a mainstream view is that lipid droplets originate from the endoplasmic reticulum.Therefore,the study of the interaction between lipid droplets and endoplasmic reticulum is useful for understanding lipid droplet biology and resolving organelles.However,current electron microscopy or lipid drop proteome methods cannot reveal the interaction between lipid droplets and endoplasmic reticulum in living cells.The combination of two fluorescent probes that separately stain the two organelles can achieve simultaneous observation of two organelles,but it brings a series of problems,including asynchronous photoresponse,complex staining process,and increased cytotoxicity.In order to solve these problems,it is imperative to develop a single-molecule dual-target two-color fluorescent probe capable of simultaneously and two-color discrimination of intracellular lipid droplets and endoplasmic reticulum in living cells,but such probes have not been reported yet.In this paper,we have made full use of the intrinsic difference of water content between the lipid droplets and the endoplasmic reticulum,and developed 3-hydroxyflavone derivatives with excitatory intramolecular proton transfer(ESIPT)properties.These probes are highly sensitive to the small change in water content.needle.On the endoplasmic reticulum with more water content,the ESIPT process is inhibited and these probes emits short-wavelength fluorescence.While in the anhydrous lipid droplets,the ESIPT effect causes the probes to emit long-wavelength fluorescence.Therefore,for the first time,simultaneous and two-color distinguishing imaging of lipid droplets and endoplasmic reticulum by a single fluorescent probe have been achieved.Using these probes,we also conducted a preliminary investigation of the interaction between lipid droplets and endoplasmic reticulum,and found that the new lipid droplets have similar fluorescent colors witrh endoplasmic reticulum,which may provide a new proof for the scientific hypothesis that lipid droplets originate from the endoplasmic reticulum..When designing and developing fluorescent dyes,we first need to consider the dye deliverability.However,due to the strict selectivity of the plasma membrane,the delivery of foreign fluorescent dyes to cells has been a challenging task.In order to efficiently deliver fluorescent dyes to living cells,various methods such as microinjection,transmembrane,and liposome have been developed.However,these methods require compulsive and direct treatment of living cells,causing irreversible damage.Compared with the above methoda,the AM ester loading technique has obvious advantages,no longer increases the membrane permeability by destroying the cells,but esterifies dye molecules for passing through the membrane.However,this method has its own limitations,which can only be used to modify fluorescent dyes containing hydroxyl or carboxyl groups.And because the AM ester is easily cleaved by the aliphatic amine,the cell culture medium is required to contain no amino acid or primary or secondary amine.In this paper,we first propose to use the side chain engineering strategy to coordinate the membrane adsorption/dissociation processes of molecules for improving dye self-delivery.Based on the same molecular backbone,a series of dyes containing different length alkyl chains were synthesized.Through systematic experimental and theoretical studies,we found that as the length of the alkyl chain increases,the membrane permeability of the molecule increases first and then decreases.At a suitable length,the membrane permeability of the molecule can be significantly enhanced,enabling the cell delivery in 30 s.Using this strategy,a class of impermeable molecule has also been successfully engineered to smoothly enter the living cells by modifying the alkyl chain of the appropriate length.The strategy is simple and easy,and does not cause damage to cells and has a certain universality,so it can be used as the preferred method to significantly improve the membrane permeability of fluorescent dyesBased on the above-mentioned side chain engineering strategy research,we found that the styrylpyridinium salt framework has excellent fluorescence properties and can be used as a fluorescent probe platform similar to fluorescein and rhodamine Therefore,based on this molecular skeleton,we aim to develop as many fluorescent probes as possible.By adjusting the side chain length of the styrylpyridinium framework,we further found that the side chain length not only affects the membrane permeability of the dye molecule,but also affects the insertion depth into the phospholipid bilayer.When the length of the alkyl side chain is between 6-10,the probe molecules have ultrafast membrane permeability and can stain the mitochondria at an ultra-low concentration of 1 nM,which can effectively avoid the problem of aggregation-induced quenching;moreover,their fluorescence intensity depends on the magnitude of the mitochondrial membrane potential,and thus they can be used to accurately monitor changes in mitochondrial membrane potential.When the alkyl chain length is 12-14,the probe molecules can not only target mitochondria,but also the strong hydrophobic interaction with the mitochondrial inner membrane lipid bilayer makes these probes unaffected by the mitochondrial membrane potential,so they can be used for long-term mitochondrial tracker.When the alkyl chain length is 18,the probe molecule can stably stain the plasma membrane,and meanwhile,its advantages of small molecular weight and excellent characteristics of two-photon red light emission make it clearly show the subtle structure of plasma membrane in rat skeletal muscle tissue.Therefore,based on the styrylpyridinium framework,we have developed ultralow concentrations mitochondrial membrane potential(MMP)probes,non-reactive mitochondrial tracers,and cytoplasmic membrane probes for imaging tissues.From the perspective of basic research,this paper proposes a new strategy for designing fluorescent probes by using the unique structural features of the interest target as well as the intrinsic difference between different targets,which is instructive for the design of future new target probes;we also propose to use the chain engineering strategy to regulate the membrane permeability of fluorescent dyes,which solves the basic problems of the delivery of fluorescent dyes.From the perspective of applied research,the fluorescent probes in this paper will help biologists and medical scientists to conduct in-depth research on lipid droplets and their interaction networks,as well as mitochondria and cell membranes.