Real-time Fluorescence Imaging Studies of Living Cells and Lipid Droplets Based on ESIPT and AIE Probes

Author:Li Yan Jie

Supervisor:hu chang hua


Degree Year:2019





Since the concept of aggregation-induced emission(AIE)was coined in 2001,it has produced significant and profound impacts to the organic luminescent materials and biomedicine.AIE Luminogens(AIEgens),as a kind of fluorescence probe with excellent stability and visibility,has displayed broad application prospects in many fields,such as the imaging with precise positioning,measurements of intracellular analysts,dynamic process monitoring,and visualization of the drug delivery.Among these applications,the imaging capability with precise positioning at subcellular structure level is an important basic research for some other applications.The regular summary of the specific staining and lit up of organelles,such as the mitochondria,lysosomes,cell membranes and lipid droplets,by some AIEgens,provides important reference to the researchers in the design of functional molecular probes and enrichment of the choice of molecular carrier materials.More and more AIEgens are explored,discovered,and widely applied in the fields of bio-imaging and analysis.So the systematic investigation of the imaging and positioning capability of the new AIEgens is important and necessary.It is valuable to explore the luminescent property and applications of the fluorescent probes,such as the AIEgens with excited-state intramolecular proton transfer(ESIPT)property,the natural medicine with AIE property,and the stimulating responsive AIEgens with excellent luminescence performance and positioning ability,but these works are still in the early stages.And there are still some problems which are needed to be solved urgently.Firstly,for the ESIPT fluorescent probes,the key structure of the proton transfer should be retained,and the modification and regulation could only be achieved from the substituent site.So the regulation degree of performance of the key illuminating site may be relatively limited,and the multi-faceted regulation are hard to be obtained.Besides,to achieve the mechanism investigation of the drugs with AIE property by optical imaging,the emission condition of the drugs and the the physiological state related to pharmacodynamics should be considered together,and the establishment of research model needs to follow the objective properties of the drugs.In addition,the species of the natural drugs with AIE property are limited and their attributes are always fixed.Therefore,it is still urgently needed to specifically design and screen imaging dyes with excellent precise positioning and luminescence properties.Simultaneously introducing a smart interface that can effectively regulate the luminescence into the probes is more appreciated,but it relies on reasonable structural design.The above limited conditions will obviously put forward higher requirements to the molecular design and imaging applications.We carried out researches related to the screening and imaging applications of ESIPT AIEgens with high-efficiency transmembrane capability,natural drug berberine hydrochloride with AIE property for real-time in situ fluorescence imaging of lipid droplets and imaging of lipid-lowering process,as well as the design and applications of AIEgen with stimuli response interface and precise positioning ability to the lipid droplits.And the main points of our work in this thesis are summarized as follows:1.The preparation and cellular imaging applications of ESIPT AIEgen with high transmembrane capability.A simple schiff-base prepared from salicylaldehyde and p-dimethylaminoaniline by the aldimine condensation was selected as the basis structure of the ESIPT probes.The amino-group contained molecules are fixed to p-dimethylaminoaniline.The ESIPT 4-N,N-dimethylaminoaniline salicylaldehyde Schiff-bases(DSS)with different substituents was obtained by choosing salicylaldehyde with different substituents.The diethylamino functionalized ESIPT Schiff-base(DSS-4-DEA)was found to exhibit superior cell penetration capability than other four molecules from the imaging results of four cell lines and it can enter and light up the cells.In the DSSs,there is a hydroxyl and a nitrogen atoms in the imine,which is the effective binding site duo to the suitable spatial distribution,so the DSSs can coordinate with many metal ions.The results showed that the fluorescence of DSSs could selectively quenched by the Cu2+,so the DSS-4-DEA was successfully used to analyze the Cu2+and fluorescence image of intracellular Cu2+.Besides,the DSS-4-DEA was used as highly efficient transmembrane carrier for the fluorescent dyes,DSS-5-Br,and intracellular delivery of drug molecules,Curcumin,with low penetration capability,and thus it confirmes the multifunctional application of DSS-4-DEA.2.Real-time AIE imaging study of BBR-Cl in lipid metabolism intervention.The AIE properties of the Berberine(BBR)molecules with different anions was investigated,and the difference in the solubility of BBR caused by anions modulates the luminescence intensity.AIE curves displayed that the anions played important roles in the AIE property of the BBRs,and the results showed that the poorly water-soluble Berberine sulfate(BBR-HSO4)had stronger fluorescence than BBR and Berberine hydrochloride(BBR-Cl).However,the luminescence intensity of these three drugs can meet the requirements of fluorescence imaging.Therefore,the most commonly used BBR-Cl was selected for the subsequent imaging study of the lipid-lowering regulation.A 3T3 adipocyte model was constructed by drug-induced differentiation and a HeLa cell model was constructed by oleic acid feeding,and they were used to study the BBR-Cl induced regulation of fat metabolism.BBR-Cl can be enriched into the lipid droplets,and the green fluorescence can be emitted from the aggregate state of the BBR-Cl.Then this signal was used to systematically study the intracellular BBR-Cl distribution,its metabolic rate,and the dynamic changes in lipid content during lipid metabolism intervention.The results of real-time imaging analysis are basically consistent with the results of Oil Red O staining.In this research,the dual-function applications of BBR-Cl for lipid-lowering therapy and fluorescent probes is fully demonstrated.3.The preparation of AIEgen with lipid droplet localization and highly sensitive imaging study.According to the structural rule and design principle of lipid droplet localization AIEgens,the imine bond which can respond to the external response was used as the connecting node to construct AIEgen with both stimuli-responsive and lipid droplet localization performance.Herein,the triphenylamine and benzonitrile structures were selected as the electron donor acceptor pair,and the prepared TPA-CN moleculehasared-shiftedemissionwavelengthcomparedtothe triphenylamine-aldehyde structure.The Schiff base structure has certain stability and is responsive to acidity below pH 4.35,showing a blue-shifted emission under acidic conditions from 530 nm to 480 nm,which is attributed to the reduction of the conjugated structure caused by the acid triggered bond breakage.Co-localization imaging study is achieved by using Nile Red,a commercially available red fluorescent lipid dye,and the results showed that TPA-CN had high-efficient lipid droplet localization ability.And for the lipid droplets with small size,TPA-CN showed higher visibility.The high stability and signal-to-background ratio allowed TPA-CN to be used for highly sensitive imaging observations of intracellular lipid droplets and for accurate acquisition of the dynamic position information of lipid droplets at submicron scale.In summary,the cell penetration and imaging capability of the ESIPT molecules with DSS structures was investigated.The DSS-4-DEA could be used for imaging analysis of intracellular Cu2+and as transmembrane carrier for efficient delivery of fluorescent dyes and drugs.Besides,the simultaneous use of BBR-Cl as a drug molecule and imaging agent was achieved by rational use of its green fluorescence.Without the addition of fluorescent indicators,the regulation of BBR-Cl on the metabolism of fat was visualized and imaged.In addition,the stimuli-responsive AIEgens TPA-CN were prepared,showing an acidic pH triggered blue shift of the fluorescence wavelength,and it has been successfully used for the precise positioning and dynamic imaging of the lipid droplets.