Highly Emissive AIEgens with Multiple Functions: Facile Synthesis, Chromism…ias.ust.hk/ias/files/pdf/1568711643_b2.pdf · 2019. 9. 17. · emission (AIE). Moreover, TPAP-BB not

Highly Emissive AIEgens with Multiple Functions: Facile Synthesis,Chromism, Specific Lipid Droplet Imaging, Apoptosis Monitoring,and In Vivo ImagingDongfeng Dang,†,‡,# Haixiang Liu,†,# Jianguo Wang,†,§ Ming Chen,†,§ Yong Liu,†,§ Herman H.-Y. Sung,†

Ian D. Williams,† Ryan T. K. Kwok,† Jacky W. Y. Lam,† and Ben Zhong Tang*,†,§,∥

†Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration andReconstruction, Division of Life Science, Institute for Advanced Study, Institute of Molecular Functional Materials, and Departmentof Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, HongKong, China‡School of Science, MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, Xi’an JiaotongUniversity, Xi’an 710049, China§HKUST-Shenzhen Research Institute, No. 9 YuCexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen 518057, China∥NSCF Center for Luminescence from Molecular Aggregates, SCUT-HKUST Joint Research Laboratory, State Key Laboratory ofLuminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China

*S Supporting Information

ABSTRACT: The development of new luminescent materials has allowedus to gain new knowledge and opened a new opportunity for scientificachievement and social development. In this work, luminogens, namely,(4-pyridinyl)-phenyldiphenylamine (TPAP) and 3-diphenylamino-6-(2-pyridinyl)phenyldiphenylboron (TPAP-BB), were synthesized in satisfac-tory yields through convenient synthetic routes and their properties weresystematically investigated. These luminogens exhibited a moderatefluorescence quantum yield in tetrahydrofuran (14.6% for TPAP and49.0% for TPAP-BB), whereas their values in thin films were much higher(69.4% and 88.4%), demonstrating a phenomenon of aggregation-inducedemission (AIE). Moreover, TPAP-BB not only exhibited piezo-chromism(its emission color could be tuned repeatedly by grinding−heating cyclebecause of the transformation between crystalline and amorphous phases) but also showed on−off light emission process byrepeatedly fuming with acid and ammonia vapor. Furthermore, TPAP-BB exhibited impressive high photostability and lowtoxicity to living cells. It could specifically stain lipid droplets (LDs) in live HeLa cells with higher signal-to-noise ratio than NileRed, a commercial LDs agent. This dye could also be applied for real-time monitoring of cell apoptosis and in vivo imaging inMedaka fish. Such results were expected to create enthusiasm to generate new AIE luminogens for further technologicalapplications.

■ INTRODUCTIONMonitoring and localizing organelles, such as the nucleus,1,2

mitochondria,3 lysosomes,4 and cytoplasm membrane5 ineukaryotic cells, have attracted a tremendous amount ofattention in the past few decades for their critical roles incellular functions and health care. However, lipid droplets(LDs) are ignored and have been regarded just as lipid-richorganelles in cells for a long time. Interestingly, recent studiesshow that LDs are closely related to many physiologicalprocesses.6−8 For instance, cellular stress was found to triggerapoptosis to induce extensive formation of LDs in cells.9,10

Meanwhile, it should be mentioned that the accumulation ofabnormal LDs can also lead to various diseases, such asAlzheimer’s disease.11,12 Therefore, similar to other well-studied organelles, it is important to localize and track LDs in

situ and in real time not only for the understanding of theirbiological functions but also for the early diagnosis of relateddiseases.13−15 To achieve this goal, tremendous works havebeen devoted to developing various imaging techniques inrecent years, including Raman microscopy and transmissionelectron microscopy.16,17 However, although these techniqueshave been widely known and well developed, their abilities inreal-time detection and in situ monitoring are still low.Moreover, the high cost and time-consuming measurement ofthese techniques also limited their further applications inbiomedical studies. Hence, new approaches for real-time

Received: August 16, 2018Revised: October 1, 2018Published: October 2, 2018

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pubs.acs.org/cmCite This: Chem. Mater. 2018, 30, 7892−7901

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detection of LDs and monitoring their dynamic biologicalprocesses, such as apoptosis, are yet to be developed.Currently, fluorescence imaging technique using highly

emissive visualizing agents is emerging as one of the mostpowerful approaches in biomedical study for its advantages offast response, high sensitivity, as well as easy and noninvasivemanner.18−27 Among these visualizing agents, organicluminescent materials (OLMs) possess the unique features ofgood chemical stability and excellent biocompatibility. Thestructural tailorability of OLMs also enables researchers totune their properties easily to meet different needs.28−38

However, it is worth noting that although many OLMs havebeen developed and commercialized for fluorescence imaging,their drawbacks, such as inferior emission features in aggregatestate,39 high background noise, poor photostability, smallStokes shifts, and complicated synthetic process, haveprevented further realization of their potential for applications.In 2001, Tang and co-workers discovered an unusual

photophysical phenomenon of ‘‘aggregation-induced emission’’(AIE) in some propeller-like molecules and found that theseAIE luminogens (AIEgens) were generally nonemissive insolution but emit intensively in the aggregate state.40−42

Compared to commercially available OLMs, AIEgens usuallyexhibit larger Stokes shifts to minimize emission self-quenchingto impart high sensitivity.43,44 Moreover, AIEgens also exhibithigh photobleaching resistance and photostability, makingthem suitable for long-term process tracking and real-timemonitoring. Thus, AIEgens are potential materials forbiomedical applications,45,46 such as lipid droplets imaging.Recently, several easily prepared AIEgens were developed andreported13,14,19 with either photoactivatable characteristics13 ortwo-photon and near-infrared (NIR) absorption features14 forprecise spatial and temporal imaging. For instance, Tang andco-workers prepared TPA-BI to achieve the precise two-photon imaging of LDs with large Stokes shift and also largetwo-photon absorption cross section.14 However, it should bementioned that although significant progress has been achievedin this field currently, AIEgens with super brightness for theinvestigation of real-time monitoring for lipid droplets andtheir related process, such as apoptosis monitoring, is seriouslylimited. Therefore, on the basis of this consideration, in thiswork, new AIEgens abbreviated as TPAP and TPAP-BB withelectron-rich building blocks (D) and electron-deficient units(A) were facilely synthesized through a simple synthetic route(Figure 1). Because of their D−A architectures and molecularrotors introduced in molecular backbones, both TPAP andTPAP-BB exhibited intramolecular charge transfer (ICT) andAIE characteristics (Figure 1). Interestingly, in contrast to themoderate photoluminescence quantum yields (PLQYs) ofTPAP in THF solution (14.6%) and in thin film (49.0%),TPAP-BB exhibited more efficient light emission with higherPLQYs (69.4% in THF and 88.4% in thin film, respectively).In addition to its superior fluorescent performance, TPAP-BBalso showed piezo- and acido-chromisms: its emission colorand process could be turned by grinding−heating and acid−base vapor fuming cycles. Moreover, low toxicity to living cellsand also good photostability were observed for TPAP-BB toserve as a visualizing agent for LDs specific imaging with muchhigher signal-to-noise ratio than the commercial LDs dyes ofNile Red. Finally, TPAP-BB was successfully applied in thereal-time monitoring of apoptosis in HeLa cells induced byhydrogen peroxide (H2O2) and in vivo imaging of Medaka fish.Such highly emissive, multifunctional AIEgens with low

cytotoxicity is thus expected to find wide applications, notonly in biological areas but also in other fields such as opticsand sensors.

■ EXPERIMENTAL SECTIONInstruments. 1H NMR and 13C NMR spectra were measured on a

Bruker ARX 400 NMR using CDCl3 as solvent. Mass spectra wereobtained on a GCT premier CAB048 mass spectrometer operating inMALDI-TOF mode. UV−vis spectra were measured on a Milton RaySpectronic 3000 array spectrophotometer. The PL spectra werecollected on a PerkinElmer LS 55 spectrophotometer. The theoreticalstudy was carried out on a 6-31G** basis set in Gaussian09 using thedensity functional theory (DFT) approximated by B3LYP. Datacollection from single crystals was conducted using a Bruker SmartAPEXII CCD diffractometer equipped with graphite-monochromatedCu Kα radiation (λ = 1.54178 Å). The absolute quantum yield wasmeasured using an Edinburgh Instrument FLS980 Integrating sphere.The fluorescence lifetime was measured using a Hamamatsu CompactFluorescence Lifetime Spectrometer C11367. Fluorescent imageswere acquired using an Olympus BX 41 fluorescence microscope.Laser confocal scanning microscope images were collected using aZeiss laser scanning confocal microscope (LSM7 DUO) and analyzedusing ZEN 2009 software (Carl Zeiss).

X-ray Crystallography. Crystal data for TPAP include thefollowing: C23H18N2, MW = 322.39, monoclinic, P21/n, T(K) =100.01(10), a (Å) = 13.98506(19), b (Å) = 10.65635(14), c (Å) =11.37257(17), α (deg) = 90, β (deg) = 99.9799(14), γ (deg) = 90, V(Å3) = 1669.20(4), Z = 4, ρcalc (g/cm

3) = 1.283, μ (mm−1) = 0.581,F(000)= 680.0, 4806 measured reflections, 2935 independentreflections (Rint = 0.0118, Rsigma = 0.0174), GOF on F2 = 1.005, R1= 0.0352, wR2 = 0.0804 (all data). These data can be obtained free ofcharge from The Cambridge Crystallographic Data Centre (CCDC:1842802) via www.ccdc.cam.ac.uk/data_request/cif, or by [email protected], or by contacting The CambridgeCrystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK. Fax: +44 1223 336033. Crystal data for TPAP-BB includethe following: C35H27BN2, MW = 486.39, monoclinic, P21/n, T (K) =220.00(10), a (Å) = 14.85425(18), b (Å) = 10.61484(10), c (Å) =16.83686(18), α (deg) = 90, β (deg) = 94.4742(10), γ (deg) = 90, V(Å3) = 2646.67(5), Z = 4, ρcalc (g/cm

3) = 1.221, μ (mm−1) = 0.537,F(000) = 1024.0, 8124 measured reflections, 4619 independentreflections (Rint = 0.0138, Rsigma = 0.0188), GOF on F2 = 1.002, R1 =

Figure 1. Schematic diagram for molecular structures and emissionmechanism of TPAP and TPAP-BB (a, color code: gray = C; yellow =N; and purple = B). Synthetic route to TPAP and TPAP-BB (b).

Chemistry of Materials Article

DOI: 10.1021/acs.chemmater.8b03495Chem. Mater. 2018, 30, 7892−7901

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0.0389, wR2 = 0.0920 (all data). These data can be obtained free ofcharge from The Cambridge Crystallographic Data Centre (CCDC:1842801) via www.ccdc.cam.ac.uk/data_request/cif, or by [email protected], or by contacting The CambridgeCrystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK. Fax: +44 1223 336033.Synthesis of TPAP. To a solution of 4-(diphenylamino)

phenylboronic acid (compound 1, 2.0 g, 6.9 mmol) and 2-bromopyridine (compound 2, 1.1 g, 6.9 mmol) in a mixture oftoluene (80 mL) and ethanol (10 mL) were added tetrakis-(triphenylphosphine)palladium [Pd(PPh3)4] (150 mg) and potassiumcarbonate (K2CO3, 2 M, 34 mL) under a nitrogen atmosphere. Afterreflux for 20 h, the mixture was cooled and then extracted withdichloromethane (DCM) three times (3 × 50 mL). The obtainedorganic solution was washed with water (3 × 100 mL), and aftersolvent removal, the crude product was purified by columnchromatography to afford the pure TPAP as a white solid (1.85 g,83%). 1H NMR (400 MHz, CDCl3), δH = 8.68 (d, J = 4.8 Hz, 1H),7.89 (d, J = 8.6 Hz, 2H), 7.76−7.68 (m, 2H), 7.36−7.24 (m, 4H),7.20−7.16 (m, 7H), 7.10−7.06 (t, J = 7.8 Hz, 2H).Synthesis of Compound 3. In a two-neck flask, compound

TPAP (1.0 g, 3.1 mmol) and N,N-diisopropyl-ethylamine (20 mg)were dissolved in DCM and then boron tribromide solution in DCM(1.0 M, 10 mL) was added dropwise at 0−5 °C. Afterward, themixture was stirred overnight at room temperature. Then saturatedK2CO3 solution was added to quench the reaction and the resultingmixture was extracted with chloroform (3 × 80 mL). The collectedorganic layer was washed with water three times (3 × 100 mL) andthen dried over anhydrous magnesium sulfate (MgSO4). Finally, thesolvent was removed under reduced pressure to get compound 3 as anorange solid (1.12 g, 74%). 1H NMR (400 MHz, CDCl3), δH = 8.81(d, J = 5.9 Hz, 1H), 8.06−8.02 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 8.2 Hz,1H), 7.54 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H), 7.44−7.30(m, 5H), 7.24−7.10 (m, 6H), 6.98−6.96 (m, 1H).

Synthesis of TPAP-BB. To a solution of compound 3 (0.5 g, 1.0mmol) in toluene (30 mL) was added diphenylzinc (0.45 g, 2.0mmol) under a nitrogen atmosphere. Then the mixture was stirred at70 °C for 12 h. After that, 20 mL of water was added, and theobtained mixture was extracted with ethyl acetate (3 × 80 mL). Thecombined organic layer was washed with brine (3 × 100 mL), andafter solvent removal, the crude product was purified by columnchromatography to get the pure TPAP-BB as a yellow solid (0.33 g,67%). 1H NMR (400 MHz, CDCl3), δH = 8.44 (d, J = 5.3 Hz, 1H),7.98−7.95 (t, J = 7.5 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.70 (d, J =8.3 Hz, 1H), 7.49 (s, 1H), 7.30−7.17 (m, 19H), 7.09−7.06 (t, J = 7.0Hz, 2H), 6.96 (d, J = 7.6 Hz, 1H). 13C NMR (100 MHz, CDCl3), δC= 158.14, 150.60, 147.47, 143.98, 140.21, 133.12, 129.71, 129.24,127.32, 125.59, 125.30, 123.73, 123.47, 122.59, 120.35, 120.22,117.44. MALDI-MS calculated for C35H27BN2 [M]

+ 486.23; found486.2288.

Cell Culture and Cytotoxicity Study. HeLa cells were culturedin minimal essential medium supplemented with 10% fetal bovineserum and antibiotics (100 units/mL penicillin and 100 mg/mLstreptomycin) at 37 °C and 5% CO2 humidity. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was usedto evaluate the cytotoxicity of TPAP-BB. Cells were seeded in 96-wellplates (Costar, IL, USA) at a density of 60 000 cells per well. After anovernight incubation, the medium was replaced with 100 μL of freshmedium supplemented with different concentrations of TPAP-BB.After 24 h incubation, 10 μL of MTT (5 mg/mL in phosphatebuffered saline (PBS)) was added. After 4 h incubation, 100 μL ofdimethyl sulfoxide (DMSO) was then added. After 15 min, theabsorbance at 595 nm was measured using a plate reader(PerkinElmer Victor3). Each experiment (i.e., each TPAP-BBconcentration) was conducted in quintuplicate.

Cell Imaging. Cells grown in a 35 mm Petri dish with glass coverat 37 °C were treated with 5 μM TPAP-BB (1 μL of TPAP-BB stocksolution in DMSO was added to 2 mL of cell culture medium and thefinal concentration of DMSO was

The cells were washed three times with PBS prior to imaging. Theimaging was conducted at an excitation wavelength of 405 nm using alaser scanning confocal microscope with 0.2% laser power. Conductedin parallel, the cells were treated with 500 nM Nile Red (a commercialdye) and the imaging was carried out at an excitation wavelength of560 nm.Medaka Fish Imaging. Live Medaka fish were soaked in buffer

solution containing 5 × 10−5 M TPAP-BB for 30 min at roomtemperature. Prior to fluorescence imaging, the fish were washed threetimes with the buffer solution. The imaging was conducted using afluorescence microscope (Olympus BX41).

■ RESULTS AND DISCUSSIONSynthesis and Optical Properties. The synthetic route

to TPAP and TPAP-BB was outlined in Figure 1. TPAP wassynthesized in high yield by Suzuki coupling of 4-(diphenylamino)phenylboronic acid (1) and 2-bromopyridine(2) catalyzed by Pd(PPh3)4. Treatment of TPAP with borontribromide followed by the reaction with diphenylzincgenerated TPAP-BB in a moderate yield of 67%. It is worthnoting that the synthetic approach for TPAP and TPAP-BB issimple and facile, which facilitates large-scale synthesis forcommercialization purpose. The chemical structures of TPAPand TPAP-BB were then characterized and confirmed by 1HNMR, 13C NMR, and MALDI-TOF-MS spectroscopies withsatisfactory results (see Figures S1−S5, Supporting Informa-tion).The UV−vis spectra of TPAP and TPAP-BB in solution and

thin film were shown (see Figure S6, Supporting Information).In THF, TPAP absorbed at 343 nm due to ICT between theelectron-rich TPA unit and the electron-deficient pyridinering.47,48 TPAP-BB displayed a redder absorption than TPAP-BB caused by the enhanced ICT effect for the vacant pz orbitalof boron atom in backbone.49−52 The absorption of bothTPAP and TPAP-BB then red-shifted in the thin film state,suggestive of some sort of intermolecular interactions in thesolid state. When the THF solutions of TPAP and TPAP-BBwere photoexcited, strong photoluminescence at 415 and 485nm was then observed, giving Stocks’ Shift of 72 and 93 nm,respectively (Figure 2a,c). To further study whether thesemolecules are AIE-active, their PL in THF/water mixture wasstudied. The relative PL intensity (I/I0) and wavelength versusdifferent water fraction ( fw) in THF/water mixture for TPAPand TPAP-BB were also plotted and given in Figure 2b,d. Asobserved, for both compound TPAP and TPAP-BB, emissivefeatures can be observed in their solution state, but when waterwas added gradually, the fluorescence intensity was decreased,caused by the twisted intramolecular charge transfer (TICT)effect, which is a common phenomenon for the D−Astructured molecules, such as TPAP and TPAP-BB. However,it should be mentioned that the change of PL intensity whenmuch more water was added to form the nanoaggregates isgenerally the key criterion to estimate whether they are AIE-active or not. For TPAP, although the emission in THF/watersolution ( fw = 90%) is much weaker than that in THF, slight

enhancement was still observed when fw is larger than 70%,indicating that TPAP should be AIE-active, but with an inferiorAIE performance. This poor AIE feature for TPAP finally ledto the much lower fluorescence intensity in its aggregationstate than that in solution state. On the other hand, in contrastto TPAP, TPAP-BB exhibited much twisted molecularstructures to further prevent the emission quenched π−πstacking; therefore, the PL features for TPAP-BB wasstrengthened significantly during the aggregates formation( fw > 70%), finally leading to the large PL enhancement inaggregated states ( fw = 90%), which is actually much higherthan that in THF solution, indicating that TPAP-BB exhibiteda much more promising and much better AIE performancethan TPAP. Moreover, the corresponding emission wavelengthalso varied with the fw values due to the TICT effect.

53,54 Thiswas further proved by the PL spectra of TPAP-BB measured insolvents with different polarities, where a large emission redshift from 445 to 529 nm was observed when the solvent waschanged from nonpolar hexane to polar DMSO (Figure 2e).To further investigate and understand the emission of TPAP

and TPAP-BB, their PLQYs and transient decay spectra inboth solution and aggregate state were measured (Figure 2f).The data was summarized in Table 1. The PLQYs of TPAPwere moderate, being 14.6% and 49.0% in solution and thinfilm, respectively. Much higher PLQYs, however (69.4% insolution and 88.4% in thin film, respectively), were observed inTPAP-BB because of its higher molecular conjugation andbetter AIE performance. Accordingly, the emission lifetime (τ)of TPAP-BB was also larger than TPAP. Their radiative rates(kr) and nonradiative decay rates (knr) in solution and thin filmwere then calculated according to the equations kr = PLQYs/τand knr = 1/(τ − kr).55,56 As shown in Table 1, the large kr andsmall knr values of TPAP-BB lead to high PLQYs in solution.The much smaller knr value in thin film indicated that thenonradiative decay channel was efficiently blocked to endowthe TPAP-BB film with intense light emission. The kr/knr ratiowas also calculated and compared. As expected, the higher kr/knr values of TPAP-BB than TPAP resulted in its much higherPLQYs values in both solution and thin film. On the otherhand, it is known that the fluorescence lifetime for OLMs isinversely proportional to kr and knr values. Therefore, the muchsmaller kr value for TPAP in solution resulted in a much longerlifetime than that in solid state.

Theoretical Calculations and Crystal Structure Anal-ysis. The ground-state geometries of TPAP and TPAP-BBwere optimized using DFT at B3LYP/6-31G** level. Asshown in Figure 3, both TPAP and TPAP-BB exhibited twistedmolecular structures with large dihedral angles. This preventedthe π−π stacking between neighboring molecules in theaggregate state to avoid emission quenching. The highestoccupied molecular orbitals (HOMO) and the lowestunoccupied molecular orbitals (LUMO) of TPAP andTPAP-BB were also presented in Figure 3. The HOMO ofboth TPAP and TPAP-BB was contributed mainly by their

Table 1. Photophysical Properties of TPAP and TPAP-BB in Solution and thin Film Statesa

UV (sol/film) PL (sol/film)

compounds λabs (nm) λem (nm) PLQYs (%) τ (ns) kr (×108 s−1) knr (×10

8 s−1) kr/knr

TPAP 343/355 415/425 14.6/49.0 2.39/1.55 0.61/3.16 3.57/3.29 0.17/0.96TPAP-BB 392/415 485/490 69.4/88.4 5.42/5.80 1.28/1.52 0.56/0.20 2.28/7.60

aAbbreviation: sol = THF solution, film = solid thin film, λabs = absorption maximum, λem = emission maximum, PLQYs = photoluminescencequantum yields, τ = fluorescence lifetime, kr = radiative decay constant, knr = nonradiative decay constant.



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electron-donating unit, while the orbitals of their LUMO werelocated predominately on the electron-deficient buildingblock.57 However, although the orbitals of TPAP weresomewhat overlapped, those of TPAP-BB were betterseparated, indicative of more efficient ICT effect and redderemission in TPAP-BB. Moreover, the significant dense electroncloud in the boron atom of LUMO for TPAP-BB furtherdemonstrated the stronger ICT effect in TPAP-BB than inTPAP.58

To further confirm the molecular structures and understandtheir optical properties, single crystals of TPAP and TPAP-BBwere grown and analyzed crystallographically. As illustrated inFigure 4, the obtained crystal structures were similar to theirDFT-optimized ones with twisted molecular backbones and

also large dihedral angles. The distance between neighboringplanes of TPAP and TPAP-BB were calculated to be 6.646 and7.112 Å, respectively, which were long enough to prevent theoccurrence of determined π−π stacking. On the other hand,abundant interactions, such as C−H···π, were observed in thecrystal lattice of TPAP-BB to restrain its molecular rotations.59

All these factors made the molecules highly emissive in theaggregate state.

Piezo- and Acido-chromisms. As observed, TPAP-BBexhibited much superior fluorescent properties in contrast toTPAP; therefore, we choose this newly developed compoundto further investigate its piezo-chromic and acido-chromicperformances. Upon grinding of the pristine sample of TPAP-BB, its emission red-shifted significantly (Figure 5a). Analysis

by powder X-ray diffraction showed that the pristine sampleexhibited many sharp diffraction peaks, which disappearedupon grinding (Figure 5b). This suggested that the piezo-chromism of TPAP-BB was caused by the conformationalchange from crystalline to amorphous state.60 Interestingly, theground sample recrystallized upon heating to recover thecrystal-state emission. On the other hand, since organoboronfluorophores have been found that can undergo protonationeasily to quench their emission, a filter paper loaded withTPAP-BB was employed to study its acido-chromic property.The dye-doped filter paper exhibited a strong emission at 475nm but became nonluminescent upon fuming with vapor ofhydrochloric acid (Figure 5c). The emission was thensubsequently “turned on” when the filter paper was exposedto ammonia vapor for 30 s. Such “on/off” cycle could berepeated for more than 5 times without considerable intensitydecay (Figure 5d), indicating that TPAP-BB may be potentiallyused as a solid-state “on/off” luminescent switch.61,62

Lipid Droplets Imaging. Although TPAP-BB showsefficient solid-state emission, to serve as visualizing agent for

Figure 3. Optimized geometry and HOMO and LUMO of TPAP andTPAP-BB calculated at B3LYP/6-3/G** level using DFT.

Figure 4. Structures (a and d), twisting angles of aromatic units (band e), intermolecular stacking distances (c and f), and intermolecularinteractions (g) in crystal lattices of TPAP (a, b, and c) and TPAP-BB(d, e, and f).

Figure 5. Normalized PL spectra of pristine, ground, and ground +heating TPAP-BB powder (a, inset shows the piezo-chromicbehaviors of TPAP-BB); XRD diffractograms of pristine, ground,and ground + heating TPAP-BB powder (b). PL spectra of pristine,HCl-fumed, and (HCl + NH3)-fumed filter paper loaded with TPAP-BB (c). Reversible switching the light emission of TPAP-BB byrepeatedly HCl−NH3 fuming cycle (d).



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bioimaging, it should also exhibit low cytotoxicity. Therefore,the cytotoxicity of TPAP-BB to living cells was first evaluatedusing MTT assay.63 Because of that, HeLa cells have beendemonstrated as one of the most widely used model cells forbioimaging and process monitoring. Therefore, we chose thiseasily available cell here.14,59 As shown in Figure 6a, the HeLa

cells treated with TPAP-BB at a high concentration of 50 μMdisplayed a viability of 90%, indicating that TPAP-BB exhibitedlow toxicity to living cells. On the other hand, photostability isanother key factor for bioimaging. As shown (see Figure S7,Supporting Information), the emission of TPAP-BB in bothsolution and aggregate state remained nearly constant after anextended period of irradiation, suggestive of its high photo-stability. Then the photostability of TPAP-BB in living cellswas further studied and compared to that of Nile Red, acommercial bioprobe, by continuous scanning using a confocallaser scanning microscope (CLSM). While the signal of NileRed was attenuated to about 60% after 90 scans, that of TPAP-BB was virtually unchanged, indicating that TPAP-BBpossessed much higher photobleaching resistance and superiorphotostability than Nile Red (Figure 6b). TPAP-BB was then

successfully utilized in the imaging of HeLa cells using 3DCLSM (insert in Figure 6b).After confirmation of the cytotoxicity and photostability of

TPAP-BB, detailed cell-imaging experiments were thenconducted. As displayed in Figure 6c, bright fluorescencewas observed mainly in the lipid droplets of HeLa cells. Toconfirm TPAP-BB targets specifically to LDs, the HeLa cellswere co-stained with Nile Red (Figure 6d).64 High Pearson’scorrelation coefficiency of up to 97% was finally achieved(Figure 6e,f), which implied that TPAP-BB was highly specificto LDs. To gain further insight into such high specificity, theHeLa cells were respectively stained with TPAP-BB and NileRed and then observed under different emission intensity(Figure 7). LDs-specific images were first obtained from bothTPAP-BB and Nile Red at low emission intensity (Figure 7a).When the emission intensity was gradually increased,fluorescence signals associated with Nile Red were then alsoobserved in the cytoplasm of HeLa cells, while those associatedwith TPAP-BB were still confined in LDs (Figure 7b,c). Afterthat, the signal-to-noise ratio was calculated using the averageemission intensity in LDs and cytoplasm in three differentcases. As suggested (see Figure S8, Supporting Information)and Figure 7d, TPAP-BB exhibited a much higher signal-to-noise ratio than Nile Red. This result revealed that TPAP-BBwas a potential and promising fluorescence agent forbioimaging of LDs in biomedical study.TPAP-BB was further used to monitor the dynamic

movement of LDs using a confocal microscope. The imagescaptured at different times showed that TPAP-BB was able totrack the spatial distribution of LDs and their dynamicmovement in living cells (see Figure S9, SupportingInformation).65 On the other hand, it is important to realizethe emission behaviors of co-stained bioprobes in a biologicalenvironment to minimize the possible cross-talk. For TICT-featured molecules, such as TPA-BB, their tunable emissioncould also provide information on the polarity of thesurrounding environment. Therefore, the imaging of LDsusing TPAP-BB was further investigated using a confocalmicroscope operated in lambda mode. The fluorescent imagesof TPAP-BB-stained HeLa cells were taken at differentemission wavelengths (see Figure S10, Supporting Informa-tion) and the intensity of each image was plotted against thewavelength, from which an emission spectrum similar to thatmeasured in toluene was obtained (see Figure S11, SupportingInformation). This further indicated the nonpolar biologicalenvironment near the LDs in living cells.

Figure 6. Cell viability of HeLa cells treated with TPAP-BBdetermined by MTT assay (a). Attenuation of fluorescence forTPAP-BB- and Nile Red-stained HeLa cells as a function of numberof scan (b, inset shows the 3D CLSM image of TPAP-BB-stainedHeLa cells). CLSM images of HeLa cells incubated with 5 μM TPAP-BB (c) and Nile Red (d) in DMSO at 37 °C for 30 min. Dark-fieldimage (e) of merged (c) and (d). Bright-field image (f) of merged (c)and (d).

Figure 7. CLSM images of HeLa cells stained with TPAP-BB and Nile Red with different emission intensities of 1.0 (a), 2.5 (b), 3.0 (c), and theircorresponding signal-to-noise ratios (d).



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Apoptosis Monitoring. Cell apoptosis is generally closelyassociated with mitochondrial dysfunction to result inextensive formation of LDs.66 Therefore, the developed LDs-specific TPAP-BB was used to monitor the dynamic process ofcell apoptosis induced by reactive oxygen species generated byhydrogen peroxide (H2O2).

67 Commercial MitoTracker-Red(MT-Red) was also employed to track the mitochondrialchanges during apoptosis. As shown (see Figure S12a,Supporting Information), the LDs and mitochondria of HeLacells were respectively stained with TPAP-BB and MT-Red. Atlow H2O2 concentration (1 mM), no apoptosis occurred as themorphology of mitochondria only changed slightly after 2 h(see Figure S12b, Supporting Information). However,apoptosis was induced at high H2O2 concentration (5 mM;Figure S12c, Supporting Information). Afterward, theapoptosis process was real-time-monitored using TPAP-BBand MT-Red (Figure 8).

Initially, the emission of LDs and mitochondria wasdistinguishable (Figure 8a), and the morphology of mitochon-dria was gradually altered after 20 min (Figure 8b). At longertime (40 and 60 min), the mitochondria were found to showthe green color from the TPAP-BB-stained LDs (Figure 8c,d).As a control, TPAP-BB and MT-Red were used to co-stainHeLa cells in the presence of light irradiation for 60 min.Interestingly, the above phenomenon was not observed (seeFigure S13, Supporting Information), indicating that its causewas due to apoptosis induced by H2O2. On the other hand,because apoptosis can largely decrease the membrane potentialof mitochondrion to allow entry of neutral TPAP-BB,68 toprove that TPAP-BB observed in the CLSM images is boundto lipid droplets, rather than TPAP-BB’s entering intomitochondria, carbonyl cyanide 3-chlorophenyl-hydrazonewas employed to decrease the mitochondrial membranepotential.69 Although the morphology of mitochondria wasaltered, the green signals associated with TPAP-BB-stainedlipid droplets were still observed at the same positions (seeFigure S14, Supporting Information). These results furthersuggested that the formation of LDs during apoptosis played

roles here, indicating the great potential of TPAP-BB inapoptosis monitoring. Interestingly, the cell fragments denoted“apoptotic bodies” were also successfully monitored duringapoptosis process (see Figure S15, Supporting Information).

In Vivo Imaging of Medaka Fish. On the basis of theimpressive cell imaging results in HeLa cells, as a proof ofconcept, the developed TPAP-BB was then applied to in vivoimaging using Medaka fish for its high optical transparence.The images showed that TPAP-BB was able to stain the livingMedaka fish after 30 min incubation at a concentration of 5 ×10−5 M (Figure 9e−h). TPAP-BB was found to probably

localize in the fish’s excretory system. Like the H2O2-treatedHeLa cells, Medaka fish fed with H2O2-containing food alsoexhibited much stronger emission of TPAP-BB (Figure 9i−l),suggesting that some fish cells had undergone apoptosis. Thesefindings further demonstrate our developed TPAP-BB couldpotentially be applied for in vivo imaging, a technique used formonitoring of various biological processes.

■ CONCLUSIONNew AIEgens (TPAP and TPAP-BB) with strong lightemission in solution and aggregate state were preparedthrough a facile synthetic approach. Both molecules exhibitedTICT and AIE characteristics. Compared to TPAP, TPAP-BBshowed higher PLQYs of 69.4% and 88.4% in solution and thinfilm, respectively, with also impressive piezo- and acido-chromic properties. Moreover, TPAP-BB also showed lowtoxicity to living cells and impressive photostability and couldstain the lipid droplets in living HeLa cells specifically withhigh signal-to-noise ratio. TPAP-BB was also then successfullyapplied in the real-time monitoring of apoptosis in HeLa cellsand in vivo imaging of Medaka fish.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.chemma-ter.8b03495.

NMR spectra, high-resolution mass spectrum, UV−visspectra of TPAP and TPAP-BB, photostability of TPAP-BB in THF and THF/water mixture, and CLSM imagesof TPAP-BB-stained HeLa cells at different momentsand emission wavelengths (PDF)

Figure 8. CLSM images of TPAP-BB- and MT-Red-stained HeLacells treated with 5 mM H2O2 for 0 min (a), 20 min (b), 40 min (c),and 60 min (d).

Figure 9. Bright-field (a, c) and fluorescent images (b, d) of Medakafish as control; bright-field (e, g, i, and k) and fluorescent images (f, h,j, and l) of TPAP-BB-stained Medaka fish without (e−h) and with (i−l) treatment with 2 mM H2O2 for 2 h.



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■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] (B.Z.T.).ORCIDRyan T. K. Kwok: 0000-0002-6866-3877Ben Zhong Tang: 0000-0002-0293-964XAuthor Contributions#D. Dang and H. Liu contributed equally.

NotesThe authors declare no competing financial interest.Crystallographic data can be obtained free of charge by TheCambridge Crystallographic Data Centre (CCDC: 1842802and 1842801) via www.ccdc.cam.ac.uk/data_request/cif, or byemailing [email protected], or by contacting TheCambridge Crystallographic Data Centre, 12 Union Road,Cambridge CB2 1EZ, UK. Fax: +44 1223 336033.

■ ACKNOWLEDGMENTSThis work was supported by the National Science Foundationof China (21788102, 81372274, 81501591, and 8141101080),the Research Grant Council of Hong Kong (N_HKUST604/14, C6009-17G, A-HKUST605116, and C2014-15G), theInnovat ion and Technology Commiss ion (ITC-CNERC14SC01 and ITS/251/17), the Shenzhen Scienceand Technology Program (JCYJ20160509170535223,JCYJ20166428150429072, JCYJ20160229205601482, andJCY 20170818113602462), and the International Science &Technology Cooperation Program of Guangzhou(20170403069). D. Dang is also thankful for financial supportfrom the National Natural Science Foundation of China(51603165) and Young Talent Fund of University Associationfor Science and Technology in Shaanxi, China (20180601).

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Highly Emissive AIEgens with Multiple Functions: Facile Synthesis, Chromism…ias.ust.hk/ias/files/pdf/1568711643_b2.pdf · 2019. 9. 17. · emission (AIE). Moreover, TPAP-BB not