Design,Synthesis And Biological Imaging Of Tetraphenylethylene Based Polymer Fluorescent Probes

With the continuous improvement of science and technology,fluorescence has been studied by more and more people.The fluorescent probe,as one of the emerging fluorescent materials,which has the unique advantages of high selectivity,easy to use and high sensitivity,applied in environmental science,materials science,medicine,biology and other fields.At present,fluorescent probes fall into the following categories:fluorescent proteins,quantum dots,and organic fluorescent probes.Fluorescent proteins have high selectivity and biocompatibility,but they offer lower resistance to photobleaching and are easily decomposed by enzymes.Although quantum dots show strong fluorescence,they are unfavorable for application on the biological cells due to their cytotoxicity problems.Traditional organic fluorescent probes have good molecular regulation,good biocompatibility,low cytotoxicity,as well as the ability to biodegrade,but their light emissions are offen quenched at high concentrations or in aggregate state?aggregation-caused quenching;ACQ?,which has severely limited the use of these probes in the biological field.In this paper,we design and synthesize a series of polymer fluorescent probes based on tetraphenylethylene and its derivatives with aggregation-induced emission?AIE?effect,exploring its application in the field of biomolecule detection and cells imaging.1.The neutral polymer P1(M_w=4.834×10⁴ g mol^-1,M_w/M_n=2.28)was directly copolymerized by AIE fluorescent molecules based on tetraphenylethylene?TPE?with alkyl chains and thiophene monomers containing hydrophilic ether bonds.Treatment of P1 with trimenthlamine provide a linear polyelectrolyte P1⁺.The structures of intermediates and polymers were characterized.The photophysical properties of the macromolecules and the cells stained with P1⁺were imaged and analyzed.It was found that the fluorescent probe had a typical AIE effect and exhibited green fluorescence emission after aggregation.It was also confirmed that P1⁺formed aggregates in the DMSO-THF mixed system?1:99 v/v?with an average particle diameter of 110.1 nm by dynamic light scattering and transmission electron microscopy.P1⁺has lower cytotoxicity and good biocompatibility.The labeling characteristics of P1⁺on mouse neuroblastoma neuro-2A cell were further observed through Confocal Laser Scanning Microscopy?CLSM?and Fluorescence Lifetime Imaging Microscopy?FLIM?based on Time-Correlated Single Photon Counting?TCSPC?.As a result,it was found that P1⁺remaining in the cytomembrane shows shorter fluorescence lifetime while accumulating in the central region of the cell exhibites longer lifetime.2.An AIE fluorescent molecule based on tetraphenylethylene with ether chains was synthesized,and then a homopolymer P2(M_n=5.929×10³ g mol^-1,M_w/M_n=2.04)was obtained by Suzuki coupling reaction.Then P2 was respectively reacted with three functional groups?morpholine,triphenylphosphine and pyridine?to generate the fluorescent probe P2⁺series?P2⁺-Mor,P2⁺-PPh₃,P2⁺-Pyr?.The structures of intermediates and macromolecules were characterized,and the photophysical properties of polymers were further tested.It was found that P2⁺-Mor,P2⁺-PPh₃,and P2⁺-Pyr all exhibited AIE characteristics with green fluorescence emission,which displayed good resistance to photobleaching.After binding to macromolecule heparin,the fluorescence intensity was significantly enhanced.The minimum detection limits of heparin by the three fluorescent probes P2⁺-Mor,P2⁺-PPh₃,and P2⁺-Pyr were 0.23?M,0.17?M,and 0.28?M,respectively.The cytotoxicity of P2⁺series were evaluated by MTT assay and the results shows that they have low cytotoxicity and good biocompatibility.The co-infection experiments with Lysotracker red?commercial dyes?show that all three fluorescent probes entered the HeLa cell after 48 h incubation,and the targeting efficiency of P2⁺-PPh₃ and P2⁺-Pyr to lysosomes in HeLa cells were better than that of P2⁺-Mor.TCSPC based FLIM observations revealed that most of the P2⁺-PPh₃ accumulating mainly on the cyto membrane shows shorter fluorescence lifetime after 0.5 h staining with HeLa cells.This suggests that the entering of these fluorescent probes into cells is relatively slow.3.A series of comonomers containing thiophene with hydrophilic ether chains and tetraphenylethylene were synthesized,and then copolymerization generated P3.The polymer-based fluorescent probe P3⁺series?P3⁺-Mor,P3⁺-PPh₃,P3⁺-Pyr?with green fluorescence emission were obtained through nucleophilic substitution reaction,and show AIE characteristics.Owing to the long-chain ether linkages on thiophene monomer,the polymerization environment had been changed and the resulting molecular weight of P3 increased(M_n=3.482×10⁴ g mol^-1,M_w/M_n=1.47).The light stability test show that the P3⁺-PPh₃ and P3⁺-Pyr have better anti-photobleaching performance compared with P3⁺-Mor.In the study of targeting biomacromolecules,the lowest detection limits for heparin of P3⁺-Mor,P3⁺-PPh₃,and P3⁺-Pyr are 1.50?M,0.17?M,and 0.28?M,respectively.The three probes have low cytotoxicity and could target the lysosome of HeLa cells slowly after endocytosis.The targeting efficiency of P3⁺-PPh₃ and P3⁺-Pyr were better than that of P3⁺-Mor.Comprehensive comparison of P3⁺and P2⁺series probes revealed that the macromolecule fluorescent probes with triphenylphosphine have better targeting performance to lysosomes and obtained a promising application prospect in the field of targeting lysosomes in cancer cells and anticancer drug carriers.