Highly Emissive AIEgens with Multiple Functions: Facile Synthesis

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Highly Emissive AIEgens with Multi-Functions: Facile Synthesis, Chromism, Specific Lipid Droplet Imaging, Apoptosis Monitoring and In Vivo Imaging Dongfeng 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 Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b03495 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 2, 2018

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Chemistry of Materials

Highly Emissive AIEgens with Multi-Functions: Facile Synthesis, Chromism, Specific Lipid Droplet Imaging, Apoptosis Monitoring and In Vivo Imaging Dongfeng Dang,a,b,‡ Haixiang Liu,a,‡ Jianguo Wang,a,c Ming Chen,a,c Yong Liu,a,c Herman H.-Y. Sung,a Ian D. Williams,a Ryan T. K. Kwok,a Jacky W. Y. Lam,a Ben Zhong Tang a,c,d,* a

Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Division of Life Science, Institute for Advanced Study, Institute of Molecular Functional Materials, and Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. b

School of Science, MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, Xi’an Jiaotong University, Xi’an 710049, China.

c

HKUST-Shenzhen Research Institute, No.9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen 518057, China.

d

NSCF Center for luminescence from molecular aggregates, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China.

ABSTRACT: The development of new luminescent materials has allowed us to gain new knowledge and opened a new opportunity to scientific achievement and social development. In this work, luminogens, namely (4-pyridinyl)phenyldiphenylamine (TPAP) and 3-diphenylamino-6-(2-pyridinyl)phenyldiphenylboron (TPAP-BB) were synthesized in satisfactory yields through convenient synthetic routes and their properties were systematically investigated. These luminogens exhibited a moderate fluorescence quantum yield in THF (14.6% for TPAP and 49.0% for TPAP-BB), whereas, their values in thin films were much higher (69.4% and 88.4%), demonstrating a phenomenon of aggregation-induced emission (AIE). Moreover, TPAP-BB not only exhibited piezo-chromism: its emission color could be tuned repeatedly by grinding-heating cycle due to the transformation between crystalline and amorphous phases, but also showed on-off light emission process by repeatedly fuming with acid and ammonia vapor. Furthermore, TPAP-BB exhibited impressive high photo-stability and low toxicity to living cells. It could specifically stain lipid droplets (LDs) in live HeLa cells with higher signal-to-noise ratio than Nile Red, a commercial LDs agent. This dye could also be applied for real-time monitoring of cell apoptosis and in vivo imaging in Medaka fish. Such results were expected to create enthusiasm to generate new AIE luminogens for further technological applications.

INTRODUCTION 1,2

Monitoring and localizing organelles, such as nucleus, mitochondria,3 lysosomes,4 and cytoplasm membrane5 in eukaryotic cells, have attracted tremendous attentions in the past few decades for their critical roles in cellular functions and health care. However, lipid droplets (LDs) are ignored and have been regarded just as lipid-rich organelles in cells for a long time. Interestingly, recent studies show that LDs are closely related to many physiological processes.6-8 For instance, cellular stress was found to trigger apoptosis to induce extensive formation of LDs in cells.9,10 Meanwhile, it should be mentioned that the accumulation of abnormal LDs can also lead to various diseases, such as Alzheimer’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 their biological functions but also for the early diagnosis of related diseases.13-15 To achieve this goal, tremendous works have been devoted to developing

various imaging techniques in recent years, including Raman microscopy and transmission electron microscopy.16,17 However, although these techniques have been widely known and well developed, their abilities in realtime detection and in situ monitoring are still low. Moreover, the high cost and time-consuming measurement of these techniques also limited their further applications in biomedical studies. Hence, new approaches for real-time detection of LDs and monitoring their dynamic biological processes, such as apoptosis, are yet to be developed. Currently, fluorescence imaging technique using highly emissive visualizing agents is emerging as one of the most powerful approaches in biomedical study for its advantages of fast response, high sensitivity, easy and noninvasive manner.18-27 Among these visualizing agents, organic luminescent materials (OLMs) possess the unique features of good chemical stability and excellent biocompatibility. The structural tailorability of OLMs also enables researchers to tune their properties easily to meet

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different needs.28-38 However, it is worth noting that although many OLMs have been developed and commercialized for fluorescence imaging, their drawbacks, such as inferior emission features in aggregate state,39 high background noise, poor photo-stability, small Stokes shifts, and complicated synthetic process, have prevented further realization of their potential for applications.

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tron-rich building blocks (D) and electron-deficient units (A) were facilely synthesized through a simple synthetic route (Figure 1). Owing to their D-A architectures and molecular rotors introduced in molecular backbones, both TPAP and TPAP-BB exhibited intramolecular charge transfer (ICT) and AIE characteristics (Figure 1). Interestingly, in contrast to the moderate photoluminescence quantum yields (PLQYs) of TPAP in THF solution (14.6%) and in thin film (49.0%), TPAP-BB exhibited more efficient light emission with higher PLQYs (69.4% in THF and 88.4% in thin film, respectively). In addition to its superior fluorescent performance, TPAP-BB also showed piezo- and acido-chromisms: its emission color and process could be turned by grinding-heating and acid-base vapor fuming cycles. Moreover, low toxicity to living cells and also good photo-stability were also observed for TPAP-BB to serve as visualizing agent for LDs specific imaging with much higher signal-to-noise ratio than the commercial LDs dyes of Nile Red. Finally, TPAP-BB was successfully applied in the real-time monitoring of apoptosis in HeLa cells induced by hydrogen peroxide (H2O2) and in vivo imaging of Medaka fish. Such highly emissive, multi-functional AIEgens with low cytotoxicity is thus expected to found wide applications, not only in biological area but also in other fields such as optics and sensors. EXPERIMENTAL SECTION

Figure 1. Schematic diagram for molecular structures and emission mechanism of TPAP and TPAP-BB (a, color code: gray= C; yellow= N; and purple= B); Synthetic route to TPAP and TPAP-BB (b). In 2001, Tang et al. discovered an unusual photophysical phenomenon of ‘‘aggregation-induced emission’’ (AIE) in some propeller-like molecules and found that these AIE luminogens (AIEgens) were generally nonemissive in solution but emit intensively in the aggregate state.40-42 Compared with commercially available OLMs, AIEgens usually exhibit larger Stokes shifts to minimize emission self-quenching to impart high sensitivity.43,44 Moreover, AIEgens also exhibit high photo-bleaching resistance and photo-stability, making them suitable for long-term process tracking and real-time monitoring. Thus, AIEgens are potential materials for bio-medical applications,45,46 such as lipid droplets imaging. Recently, several easily prepared AIEgens were developed and reported 13,14,19 with either photoactivatable characteristics 13 or two-photon and near infra-red (NIR) absorption features 14 for precise spatial and temporal imaging. For instance, Jiang and Tang et al. prepared TPA-BI to achieve the precise two-photon imaging of LDs with large Stokes shift and also large two-photon absorption cross-section.14 However, it should be mentioned that although significant progress has been achieved in this field currently, AIEgens with super brightness for the investigation of real time monitoring for lipid droplets and their related process, such as apoptosis monitoring, is seriously limited. Therefore, based on this consideration, in this work, new AIEgens abbreviated as TPAP and TPAP-BB with elec-

Instruments. 1H NMR and 13C NMR spectra were measured on a Bruker ARX 400 NMR using CDCl3 as solvent. Mass spectra were obtained on a GCT premier CAB048 mass spectrometer operating in MALDI-TOF mode. UVVis spectra were measured on a Milton Ray Spectronic 3000 array spectrophotometer. The PL spectra were collected on a PerkinElmer LS 55 spectrophotometer. The theoretical study was carried out on 6-31G** basis set in Gaussian09 using the density functional theory (DFT) approximated by B3LYP. Data collection from single crystals was conducted using a Bruker Smart APEXII CCD diffractometer equipped with graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). The absolute quantum yield was measured using an Edinburgh Instrument FLS980 Integrating sphere. The fluorescence lifetime was measured using a Hamamatsu Compact Fluorescence Lifetime Spectrometer C11367. Fluorescent images were acquired using Olympus BX 41 fluorescence microscope. Laser confocal scanning microscope images were collected using a Zeiss laser scanning confocal microscope (LSM7 DUO) and analyzed using ZEN 2009 software (Carl Zeiss). X-ray crystallography. Crystal data for TPAP: 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/°= 90, β/°= 99.9799(14), γ/°= 90, V/Å3 = 1669.20(4), Z= 4, ρcalcg/cm3 = 1.283, μ/mm-1= 0.581, F(000)= 680.0, 4806 measured reflections, 2935 independent reflections (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 of charge by The Cambridge Crystallographic Data Centre (CCDC: 1842802). Crystal data for TPAP-BB: C35H27BN2, MW= 486.39, monoclinic, P21/n, T/K= 220.00(10), a/Å=


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14.85425(18), b/Å= 10.61484(10), c/Å= 16.83686(18), α/°= 90, β/°= 94.4742(10), γ/°= 90, V/Å3= 2646.67(5), Z= 4, ρcalcg/cm3 = 1.221, μ/mm-1= 0.537, F(000)= 1024.0, 8124 measured reflections, 4619 independent reflections (Rint= 0.0138, Rsigma= 0.0188), GOF on F2= 1.002, R1= 0.0389, wR2= 0.0920 (all data). These data can be obtained free of charge by The Cambridge Crystallographic Data Centre (CCDC: 1842801). Synthesis of TPAP. To a solution of 4-(diphenylamino) phenylboronicacid (compound 1, 2.0 g, 6.9 mmol) and 2bromopyridine (compound 2, 1.1 g, 6.9 mmol) in a mixture of toluene (80 mL) and ethanol (10 mL) were added tetrakis(triphenylphosphine)palladium [Pd(PPh3)4] (150 mg) and potassium carbonate (K2CO3, 2M, 34 mL) under nitrogen atmosphere. After reflux for 20 h, the mixture was cooled and then extracted with dichloromethane (DCM) three times (3×50 mL). The obtained organic solution was washed with water (3×100 mL) and after solvent removal, the crude product was purified by column chromatography 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-diisopropylethylamine (20 mg) were dissolved in DCM and then boron tribromide solution in DCM (1.0 M, 10 mL) was added dropwise at 0-5 oC. Afterwards, the mixture was stirred overnight at room temperature. Then, saturated K2CO3 solution was added to quench the reaction and the resulting mixture was extracted with chloroform (3×80 mL). The collected organic layer was washed with water three times (3×100 mL) and then dried over anhydrous magnesium sulfate (MgSO4). Finally, the solvent was removed under reduced pressure to get compound 3 as an orange 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, 1.0 mmol) in toluene (30 mL) was added diphenylzinc (0.45 g, 2.0 mmol) under nitrogen atmosphere. Then the mixture was stirred at 70 oC for 12 h. After that, 20 mL water was added, and the obtained mixture was extracted with ethyl acetate (3×80 mL). The combined organic layer was washed with brine (3×100 mL) and after solvent removal, the crude product was purified by column chromatography 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 = 7.0 Hz, 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; found 486.2288. Cell Culture and Cytotoxicity Study. HeLa cells were cultured in minimal essential medium (MEM) supplemented with 10% FBS and antibiotics (100 units/mL peni-

cillin and 100 mg/mL streptomycin) at 37 °C and 5% CO2 humidity. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of TPAP-BB. Cells were seeded in 96-well plates (Costar, IL, USA) at a density of 60,000 cells per well. After an overnight incubation, the medium was replaced with 100 µL of fresh medium supplemented with different concentrations of TPAP-BB. After 24 h incubation, 10 µL of MTT (5 mg/mL in PBS) was added. After 4 h incubation, 100 µL of DMSO was then added. After 15 min, the absorbance at 595 nm was measured using a plate reader (Perkin-Elmer Victor3). Each experiment (i.e., each TPAP-BB concentration) was conducted in quintuplicate. Cell Imaging. Cells grown in a 35 mm petri dish with glass cover at 37 °C were treated with 5 μM of TPAP-BB (1 µL of TPAP-BB stock solution in DMSO was added to 2 mL of cell culture medium and the final concentration of DMSO was < 0.1 vol%) for a certain period. The cells were washed three times with PBS prior to imaging. The imaging was conducted at an excitation wavelength of 405 nm using a laser scanning confocal microscope with 0.2% laser power. Conducted in parallel, the cells were treated with 500 nM of Nile Red (a commercial dye) and the imaging was carried out at an excitation wavelength of 560 nm. Medaka Fish Imaging. Live Medaka fish was soaked in buffer solution containing 5×10-5 M of TPAP-BB for 30 min at room temperature. Prior to fluorescence imaging, the fish were washed three times with the buffer solution. The imaging was conducted using a fluorescence microscope (Olympus BX41). RESULTS AND DISCUSSION Synthesis and Optical Properties. The synthetic route to TPAP and TPAP-BB was outlined in Figure 1. TPAP was synthesized in a high yield by Suzuki coupling of 4(diphenylamino)phenylboronic acid (1) and 2bromopyridine (2) catalyzed by Pd(PPh3)4. Treatment of TPAP with boron tribromide followed by the reaction with diphenylzinc generated TPAP-BB in a moderate yield of 67%. It is worth noting that the synthetic approach for TPAP and TPAP-BB is simple and facile, which facilitates large scale synthesis for commercialization purpose. The chemical structures of TPAP and TPAP-BB were then characterized and confirmed by 1H NMR, 13C NMR, and MALDI-TOF-MS spectroscopies with satisfactory results (see Figures S1-S5, supporting information). 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 the electron-rich TPA unit and the electrondeficient pyridine ring.47,48 TPAP-BB displayed a redder absorption than TPAP-BB caused by the enhanced ICT effect for the vacant pz orbital of boron atom in backbone.49-52 The absorption of both TPAP and TPAP-BB then red-shifted in the thin film state, suggestive of some sort of intermolecular interactions in the solid state. When the THF solutions of TPAP and TPAP-BB were photo-excited, strong photoluminescence at 415 nm and 485 nm was then observed, giving Stocks’ Shift of 72 nm



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Figure 2. PL spectra of TPAP (a) and TPAP-BB (c) in THF solution with different water fractions (fw); Plots of relative PL intensity (I/I0) and emission wavelength versus fw in THF/Water mixture for TPAP (b) and TPAP-BB (d); PL spectra of TPAP-BB in hexane, toluene, THF and DMSO (e); Transient decay spectra of TPAP and TPAP-BB in THF and thin film (f). Table 1. Photo-physical properties of TPAP and TPAP-BB in solution and thin film states.[a] UV (sol/film)

PL (sol/film)

Compounds

λabs λem PLQYs τ kr knr kr/knr (nm) (nm) (%) (ns) (×108 s-1) (×108 s-1) TPAP 343/355 415/425 14.6/49.0 2.39/1.55 0.61/3.16 3.57/3.29 0.17/0.96 TPAP-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 [a] Abbreviation: sol= THF solution, film= solid thin film, λabs= absorption maximum, λem= emission maximum, PLQYs= photoluminescence quantum yields, τ= fluorescence lifetime, kr= radiative decay constant, knr= non-radiative decay constant. and 93 nm respectively (Figure 2a and 2c). To further study whether these molecules are AIE-active, their PL in THF/Water mixture was studied. The relative PL intensity (I/I0) and wavelength versus different water fraction (fw) in THF/Water mixture for TPAP and TPAP-BB were also plotted and given in Figure 2b and 2d. As observed, for both compound TPAP and TPAP-BB, emissive features can be observed in their solution state, but when water was added gradually, the fluorescence intensity was decreased caused by the twisted intramolecular charge transfer (TICT) effect, which is a common phenomenon for the D-A structured molecules, such as TPAP and TPAP-BB. However, it should be mentioned that the change of PL intensity when much more water was added to form the nano-aggregates is generally the key criterion to estimate whether they are AIE active or not. For TPAP, although the emission in THF/Water solution (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 inferior AIE performance. This poor AIE features for TPAP finally led

to the much lower fluorescence intensity in its aggregation state than that in solution state. On the other hand, in contrast to TPAP, TPAP-BB exhibited much twisted molecular structures to further prevent the emission quenched π-π stacking, therefore, the PL features for TPAP-BB was strengthened significantly during the aggregates formation (fw> 70%), finally leading to the large PL enhancement in aggregated states (fw= 90%), which is actually much higher than that in THF solution, indicating that TPAP-BB exhibited a much more promising and much better AIE performance than TPAP. Moreover, the corresponding emission wavelength also varied with the fw values due to the TICT effect.53,54 This was further proved by the PL spectra of TPAP-BB measured in solvents with different polarities, where a large emission redshift from 445 nm to 529 nm was observed when the solvent was changed from nonpolar hexane to polar dimethylsulfoxide (DMSO) (Figure 2e). To further investigate and understand the emission of TPAP and TPAP-BB, their PLQYs and transient decay


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spectra in both solution and aggregate state were measured (Figure 2f). The data was summarized in Table 1. The PLQYs of TPAP were moderate being 14.6% and 49.0% in solution and thin film, respectively. Much higher PLQYs, however, (69.4% in solution and 88.4% in thin film, respectively) were observed in TPAP-BB, due to its higher molecular conjugation and better 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 film were then calculated according to the equations: kr = PLQYs/τ and knr = 1/τ-kr.55,56 As shown in Table 1, the large kr and small knr values of TPAP-BB lead to high PLQYs in solution. The much smaller knr value in thin film indicated that the non-radiative decay channel was efficiently blocked to endow the TPAP-BB film with intense light emission. The kr/knr ratio was also calculated and compared. As expected, the higher kr/knr values of TPAP-BB than TPAP resulted in its much higher PLQYs values in both solution and thin film. On the other hand, it’s known that the fluorescence lifetime for OLMs is inversely proportional to kr and knr values. Therefore, the much smaller kr value for TPAP in solution resulted a much longer lifetime than that in solid state.

on the electron-deficient building block.57 However, although the orbitals of TPAP were somewhat overlapped, those of TPAP-BB were better separated, indicative of more efficient ICT effect and redder emission in TPAPBB. Moreover, the significant dense electron cloud in the boron atom of LUMO for TPAP-BB further demonstrated the stronger ICT effect in TPAP-BB than in TPAP.58 To further confirm the molecular structures and understand their optical properties, the single crystals of TPAP and TPAP-BB were grown and analyzed crystallographically. As illustrated in Figure 4, the obtained crystal structures were similar to their DFT-optimized ones with twisted molecular backbones and also large dihedral angles. The distance between neighboring planes of TPAP and TPAP-BB were calculated to be 6.646 Å and 7.112 Å, respectively, which were long enough to prevent the occurrence of determined π-π stacking. On the other hand, abundant interactions, such as C-H…π, were observed in the crystal lattice of TPAP-BB to restrain its molecular rotations.59 All these factors made the molecules highly emissive in the aggregate state.

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

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

Theoretical Calculations and Crystal Structure Analysis. The ground-state geometries of TPAP and TPAP-BB were optimized using DFT at B3LYP/6-31G** level. As shown in Figure 3, both TPAP and TPAP-BB exhibited twisted molecular structures with large dihedral angles. This prevented the π-π stacking between neighboring molecules in the aggregate state to avoid emission quenching. The highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO) of TPAP and TPAP-BB were also presented in Figure 3. The HOMO of both TPAP and TPAP-BB was contributed mainly by their electron-donating unit, while the orbitals of their LUMO were located predominately

Piezo- and Acido-chromisms. As observed, TPAP-BB exhibited much superior fluorescent properties in contrast to TPAP, therefore, we choose this newly developed compound to further investigate its piezo-chromic and acido-chromic performances. Upon grinding the pristine sample of TPAP-BB, its emission red-shifted significantly (Figure 5a). Analysis by powder X-ray diffraction showed that the pristine sample exhibited many sharp diffraction peaks, which disappeared upon grinding (Figure 5b). This suggested that the piezo-chromism of TPAP-BB was caused by the conformational change from crystalline to amorphous state.60 Interestingly, the ground sample recrystallized upon heating to recover the crystal-state



emission. On the other hand, since organoboron fluorophores have been found that can undergo protonation easily to quench their emission, a filter paper loaded with TPAP-BB was employed to study its acido-chromic property. The dye-doped filter paper exhibited a strong emission at 475 nm but became non-luminescent upon fuming with vapor of hydrochloric acid (Figure 5c). The emission was then subsequently “turned-on” when the filter paper was exposed with ammonia vapor for 30 s. Such “on/off” cycle could be repeated for more than 5 times without considerable intensity decay (Figure 5d), indicating that TPAP-BB may be potentially used as a solid-state “on/off” luminescent switch.61,62

Figure 5. The normalized PL spectra of pristine, ground, and ground+ heating TPAP-BB powder (a, inset shows the piezo-chromic behaviors 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 by repeatedly HCl-NH3 fuming cycle (d). Lipid Droplets Imaging. Although TPAP-BB shows efficient solid-state emission, to serve as visualizing agent for bio-imaging, it should also exhibit low cytotoxicity. Therefore, the cytotoxicity of TPAP-BB to living cells was firstly evaluated using MTT assay.63 Owing to that Hela cells have been demonstrated as one of the most widely used model cells for bio-imaging and process monitoring. Therefore, we chose this easily available cell here.14,59 As shown in Figure 6a, the HeLa cells treated with TPAP-BB at a high concentration of 50 μM displayed a viability of 90%, indicating that TPAP-BB exhibited low toxicity to living cells. On the other hand, photo-stability is another key factor for bio-imaging. As shown (see Figure S7, supporting information), the emission of TPAP-BB in both solution and aggregate state remained nearly constant after an extended period of irradiation, suggestive of its high photo-stability. Then, the photo-stability of TPAPBB in living cells was further studied and compared with that of Nile Red, a commercial bio-probe, by continuous

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scanning using a confocal laser scanning microscope (CLSM). While the signal of Nile Red was attenuated to about 60% after 90 scans, that of TPAP-BB was virtually unchanged, indicating that TPAP-BB possessed much higher photo-bleaching resistance and superior photostability than Nile Red (Figure 6b). TPAP-BB was then successfully utilized in the imaging of HeLa cells using 3D CLSM (insert in Figure 6b). After confirming the cytotoxicity and photo-stability of TPAP-BB, detailed cell-imaging experiments were then conducted. As displayed in Figure 6c, bright fluorescence was observed mainly in the lipid droplets of HeLa cells. To confirm TPAP-BB targets specifically to LDs, the HeLa cells were co-stained with Nile Red (Figure 6d).64 High Pearson’s correlation co-efficiency of up to 97% was finally achieved (Figure 6e and 6f), which implied that TPAPBB was highly specific to LDs. To gain further insight into such high specificity, the HeLa cells were respectively stained with TPAP-BB and Nile Red, then observed under different emission intensity (Figure 7). LDs-specific images were firstly obtained from both TPAP-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 also observed in the cytoplasm of HeLa cells, while those associated with TPAP-BB were still confined in LDs (Figure 7b and 7c). After that, the signal-to-noise ratio was calculated using the average emission intensity in LDs and cytoplasm in three different cases. 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-BB was potential and promising fluorescence agent for bio-imaging of LDs in biomedical study.

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


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Figure 7. CLSM images of HeLa cells stained with TPAP-BB and Nile Red with different emission intensity of 1.0 (a), 2.5 (b), 3.0 (c) and their corresponding signal-to-noise ratios (d). TPAP-BB was further used to monitor the dynamic movement of LDs using a confocal microscope. The images captured at different time showed that TPAP-BB was able to track the spatial distribution of LDs and their dynamic movement in living cells (see Figure S9, supporting information).65 On the other hand, it is important to realize the emission behaviors of co-stained bio-probes in biological environment to minimize the possible crosstalk. For TICT-featured molecules, such as TPA-BB, their tunable emission could also provide information on the polarity of the surrounding environment. Therefore, the imaging of LDs using TPAP-BB was further investigated using a confocal microscope operated in a lambda-mode. The fluorescent images of TPAP-BB stained HeLa cells were taken at different emission wavelengths (see Figure S10, supporting information) and the intensity of each image was plotted against the wavelength, from which an emission spectrum similar to that measured in toluene was obtained (see Figure S11, supporting information). This further indicated the non-polar biological environment near the LDs in living cells. Apoptosis Monitoring. Cell apoptosis is generally closely associated with mitochondrial dysfunction to result extensive formation of LDs.66 Therefore, the developed LDs-specific TPAP-BB was used to monitor the dynamic process of cell apoptosis induced by reactive oxygen species generated by hydrogen peroxide (H2O2).67 Commercial MitoTracker-Red (MT-Red) was also employed to track the mitochondrial changes during apoptosis. As shown (see Figure S12a, supporting information), the LDs and mitochondria of HeLa cells were respectively stained with TPAP-BB and MT-Red. At low H2O2 concentration (1 mM), no apoptosis occurred as the morphology 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, the apoptosis process was real-time monitored using TPAP-BB and MT-Red (Figure 8).

Figure 8. CLSM images of TPAP-BB- and MT-Red-stained HeLa cells treated with 5 mM of H2O2 for 0 min (a), 20 min (b), 40 min (c) and 60 min (d). Initially, the emission of LDs and mitochondria was distinguishable (Figure 8a), and the morphology of mitochondria was gradually altered after 20 min (Figure 8b). At longer time (40 min and 60 min), the mitochondria were found to show the green color from the TPAP-BB stained LDs (Figure 8c and 8d). As a control, TPAP-BB and MT-Red were used to co-stain HeLa cells in the presence of light irradiation for 60 min. Interestingly, the above phenomenon was not observed (see Figure S13, supporting information), indicating that its cause was due to apoptosis induced by H2O2. On the other hand, owing to that apoptosis can largely decrease the membrane potential of mitochondrion to allow entry of neutral TPAPBB,68 therefore, to prove that TPAP-BB observed in the CLSM images is bound to lipid droplets, rather than TPAP-BB’s entering into mitochondria, carbonyl cyanide 3-chlorophenyl-hydrazone was employed to decrease the mitochondrial membrane potential.69 Although the mor-



phology of mitochondria was altered, the green signals associated TPAP-BB-stained lipid droplets were still observed at the same positions (see Figure S14, supporting information). This results further suggested that it’s the formation of LDs during apoptosis played roles here, indicating the great potential of TPAP-BB in apoptosis monitoring. Interestingly, the cell fragments denoted “apoptotic bodies” were also successfully monitored during apoptosis process (see Figure S15, supporting information). In vivo imaging of Medaka fish. Based on the impressive cell imaging results in HeLa cells, as a proof of concept, the developed TPAP-BB was then applied to in vivo imaging using Medaka fish for its high optical transparence. The images showed that TPAP-BB was able to stain the living Medaka 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-treated HeLa cells, Medaka fish fed with H2O2containing food also exhibited much stronger emission of TPAP-BB (Figure 9i-l), suggesting that some fish cells had underwent apoptosis. These findings further demonstrate our developed TPAP-BB could potentially be applied for in vivo imaging, a technique used for monitoring of various biological processes.

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Supporting Information. The NMR spectra, High resolution mass spectrum, UV-Vis spectra of TPAP and TPAPBB, Photo-stability of TPAP-BB in THF and THF/Water mixture, CLSM images of TPAP-BB-stained HeLa cells at different moments and emission wavelengths were listed in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author B.Z. Tang: [email protected] Author Contributions ‡

D. Dang and H. Liu contributed equally.

Notes The authors declare no conflict of interest. ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (21788102, 81372274, 81501591 and 8141101080), the Research Grant Council of Hong Kong (N_HKUST604/14, C6009-17G, A-HKUST605116 and C2014-15G), the Innovation and Technology Commission (ITC-CNERC14SC01 and ITS/251/17), the Shenzhen Science and Technology Program (JCYJ20160509170535223, JCYJ20166428150429072, JCYJ20160229205601482 and JCY 20170818113602462), and the International Science & Technology Cooperation Program of Guangzhou (20170403069). D. Dang also thanks the financial supports by National Natural Science Foundation of China (51603165) and young talent fund of university association for science and technology in shaanxi, China (20180601). REFERENCES

Figure 9. Bright-field (a, c) and fluorescent images (b, d) of Medaka fish 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 of H2O2 for 2 h. CONCLUSION New AIEgens (TPAP and TPAP-BB) with strong light emission in solution and aggregate state were prepared through a facile synthetic approach. Both molecules exhibited TICT and AIE characteristics. Compared with TPAP, TPAP-BB showed higher PLQYs of 69.4% and 88.4% in solution and thin film, respectively, with also impressive piezo- and acido-chromic properties. Moreover, TPAP-BB also showed low toxicity to living cells, impressive photo-stability and could stain the lipid droplets in living HeLa cells specifically with high signal-to-noise ratio. TPAP-BB was also then successfully applied in the real-time monitoring of apoptosis in HeLa cells and in vivo imaging of Medaka fish. ASSOCIATED CONTENT

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Highly Emissive AIEgens with Multiple Functions: Facile Synthesis

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