Aggregation-Induced Emission Luminogens with the Capability of

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Biological and Medical Applications of Materials and Interfaces

Aggregation-Induced Emission Luminogens with the Capability of Wide Color Tuning, Mitochondrial and Bacterial Imaging, Photodynamic Anticancer and Antibacterial Therapy Na Zhao, Pengfei Li, Jiabao Zhuang, Yanyan Liu, Yuxin Xiao, Ruilin Qin, and Nan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01655 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019

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ACS Applied Materials & Interfaces

Aggregation-Induced Emission Luminogens with the Capability of Wide Color Tuning, Mitochondrial and Bacterial Imaging, Photodynamic Anticancer and Antibacterial Therapy Na Zhao,* Pengfei Li,† Jiabao Zhuang,† Yanyan Liu, Yuxin Xiao, Ruilin Qin and Nan Li*

Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, and School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710119, China. E-mail: [email protected]; [email protected].

†These

authors contributed equally to this work.

KEYWORDS: aggregation-induced emission, wide color tuning, mitochondrial imaging, bacterial imaging, photosensitizer

ABSTRACT: Recently, luminogens with aggregation-induced emission character (AIEgens) received much attention in the field of bioimaging and therapeutic application. However, development of AIEgens that derived from simple core skeleton with emission color tuning for

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imaging and therapy is still a formidable challenge. To address this constraint, we present a series of cationic AIEgens based on cyano-pyridinium salt (CP1 to CP5). The AIEgens can be facilely prepared by varying aromatic electron donor while fixing cyano-pyridinium group as the electron acceptor within single benzene ring. The obtained AIEgens possess wide color tunability, large Stokes shifts and bright emission in the condensed state. Due to their good biocompatibility and cationic nature, these AIEgens can be utilized for multiple-color imaging of intracellular mitochondria as well as Gram-negative and Gram-positive bacteria. Importantly, these AIEgens exhibit remarkable structure-dependent singlet oxygen generation ability under white light illumination (25 mW cm–2), and CP4 was optimized to serving as an excellent photosensitizer for photodynamic anticancer and antibacterial therapy.

1. INTRODUCTION The development of organic fluorescent materials that display intense and color tunable emission in the aggregated or solid state have received great interest due to their potential applications in the field of optoelectronic devices, full-color bioimaging, multi-target sensing, disease diagnosis and treatment and so on.1–13 However, the traditional organic fluorescent molecules undergo the process of aggregation-caused quenching (ACQ), namely high emission in solution state but faint emission in the condensed state, due to the intrinsic intermolecular π–π stacking interaction.14–17 ACQ problem significantly limited their practical applications.


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Recently, the discovery of an abnormal phenomenon, named aggregation-induced emission (AIE), has provided a new avenue to fabricating organic luminogens to overcome the ACQ effect.18,19 Different from the ACQ process, luminogens with AIE characteristics (AIEgens) display non-emission or weak emission in the molecular dissolved state, but emit intensely in the condensed state, which resulted by the restriction of intramolecular motion (RIM).20–22 In the light of fascinating advantages of AIEgens such as high luminescent efficiency, high photostability, large Stokes shifts and unique fluorescence “light-up” mode through flexible deaggregation/aggregation processes, AIEgens have been widely applied in the areas of the bioimgaing and biosensing.23–30 Furthermore, AIEgens were reported to provide increased ability of reactive oxygen species (ROS) generation in the aggregated state, which efficiently solve the reduction of ROS production for traditional photosensitizer once molecular aggregates formed. As a result, AIEgen is a kind of ideal photosensitizer for photodynamic therapy (PDT) of cancer cells and killing of bacteria.31–35 In order to extend AIE system with biomedical application, some elegant functionalized AIEgens with color tunability have been developed. For instance, imidazole-based AIEgens with blue to red emission were designed for mitochondria-targeted imaging and antifungal activity.36 Colorful AIEgens derived from diphenyl isoquinolinium with functions of mechanochromism, mitochondrial and bacterial imaging was reported.37 A kind of AIE probes with two-photon excitation based on tetraphenylethene was explored for multiple-color cell and microbe imaging.38 However, due to the difficulties of structural design and modification, the applications


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of most reported AIE systems were limited to specific aspects, development of novel AIEgens based on simple molecular backbone with wide emission color tunability and diverse biological applications, such as organelle-targeted imaging in living cells, photodynamic ablation of cancer cells and bacteria killing is still a great challenge. In this contribution, we design and synthesize a series of symmetric cyano-pyridinium (CP)based AIEgens (CP1 to CP5) with wide emission color tunability across the whole visible region by employing CP unit as the electron withdrawing group and diverse aromatic moieties as the electron donating groups within single benzene ring (Figure 1). The obtained AIEgens show faint emission in dissolved state, but turn into the strong emitters in the condensed state. The crystal packing analysis revealed that the AIEgens adopted highly twisted conformation, which is responsible for their special AIE feature. These AIEgens exhibit large Stokes shift as well as good biocompatibility, which significantly facilitate their biological applications. In view of the cationic nature, the AIEgens can serve as colorful fluorescent probes to stain the mitochondria in living cells and bacteria with high specificity. Furthermore, these AIEgens exhibit remarkable structure-dependent 1O2 generation ability under illumination of white light (25 mW cm–2). Owing to the most superior 1O2 generation ability of CP4, it was successfully utilized as an excellent photosensitizer for photodynamic anticancer and antibacterial. 2. RESULTS AND DISCUSSION 2.1. Design and Synthesis. Detailed synthesis of AIEgens CP1 to CP5 was depicted in Supporting Information. At the initial step, Suzuki cross-coupling reaction was conducted by


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using 2,5-dibromo-terephthalaldehyde and varied aromatic boronic acids to give diverse 2,5position functionalized terephthalaldehyde. Subsequently, the functionalized terephthalaldehyde condensed with 2-(pyridin-4-yl)acetonitrile at the alkaline condition to yield 2,5-position functionalized 1,4-phenylene-bis(2-(pyridin-4-yl) acrylonitrile). The desired pyridinium salt was produced by treating functionalized 1,4-phenylene-bis(2-(pyridin-4-yl)acrylonitrile) with methyliodide and then underwent the counterion exchange using KPF6. All pyridinium salts (CP1 to CP5) were characterized by NMR and high resolution mass spectrometry (HRMS).

Figure 1. Chemical structure and corresponding properties of AIEgens CP1–CP5. 2.2. Photophysical Property. The photophysical properties of CP1 to CP5 were examined using absorption and emission spectra (Figure 2A and Table 1). All AIEgens exhibited the highest absorption band at around 360 nm in DMSO solution, which could be ascribed to the inherent π– π* transition. Meanwhile, the distinct longest absorption peak varied from 428 to 457 nm for CP2, CP3 and CP5, which were probably due to the formation of intramolecular charge transfer


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(ICT) process from varied electron donors to the electron acceptor of cyano-pyridinium unit.39 The solvent effect of AIEgens was also investigated by taking CP2 and CP3 as examples. By increasing the polarity of the solvent (from toluene to methanol), the emission peak displayed an obvious red shift compared to their absorption peak (Figure S1), which indicated the existence of ICT process. For CP1 and CP4, the typical ICT absorption bands were not observed, which were probably embodied in the intense π–π* absorption band. Table 1. Optical properties and energy levels of CP1–CP5. solutiona)

solidb) HOMOe)

LUMOe)

Egf)

[eV]

[eV]

[eV]

1.08

–6.85

–4.09

2.76

23.7

2.28

–6.74

–4.04

2.70

519

54.9

3.17

–6.36

–3.83

2.53

0.96

570

14.8

1.94

–6.22

–3.74

2.48

0.69

614

3.9

1.53

–5.88

–3.67

2.21

λabs

λem

Φfc)

τd)

λem

Φfc)

τd)

[nm]

[nm]

[%]

[ns]

[nm]

[%]

[ns]

CP1

368

470

0.3

1.05

464

9.4

CP2

405

510

0.8

1.62

495

CP3

418

549

0.2

0.48

CP4

361

567

0.8

CP5

453

605

0.4

AIEgen

aIn

DMSO solution; bIn solid; cDetermined by using calibrated integrating sphere; dMean

fluorescence lifetime (τavg); eDetermined by cyclic voltammetry in acetonitrile; fCalculated based on the onset of absorption spectrum. Upon photoexcitation, the corresponding AIEgen gave weak emission in DMSO solution ranging from 470 to 605 nm with extremely low quantum yield (ФF < 1%). The weak emission


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was probably attributed to the consumption of the excited state energy by non-radiative decay due to the efficient intramolecular rotation in dissolved state. The emission spectra of AIEgens in DMSO/water mixture with varied water fraction (fw) were next investigated. The results were summarized in Figure S2–S5 and taking CP5 as an example (Figure 2B). When the fw increased from 0 to 80% in DMSO solution, the emission of CP5 did not change significantly. After increasing fw from 80 to 99%, however, the red-shifted emission peak at 600 nm was observed and the intensity enhanced remarkably. This bathochromic shift of emission could be ascribed to the more planar conformation when molecules were aggregated. Similar emission lighting up phenomenon with increasing of fw was also observed for other AIEgens, indicated their typical AIE property (Figure 2C). The emission enhancement was probably arised from the molecules aggregation that restricted the intramolecular rotation and opened the decay channel. The particle size distribution of AIEgens at fw 99% varied from 203.7 to 852.2 nm measured by using dynamic light scattering, which further confirmed the formation of nanoaggregates (Figure S6). It is noted that all AIEgens exhibited large Stokes shifts (> 100 nm), which can efficiently reduce the chance of self-absorption and is benefited to the bioimaging. Consistent with their AIE effect, the strong solid-state emission from 464 to 614 nm was also observed for CP1 to CP5 (Figure 2D). And the solid-state ФF of all AIEgens was obviously higher than that in solution state while the maximum ФF reached up to 54.9% for CP4 (Table 1). The solid-state lifetimes of all AIEgens were measured in the range of 1.08–3.17 ns (Table 1), which were slightly longer than those


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AIEgens in solution state (0.48–1.62 ns). It is clearly that a kind of AIEgens with wide emission tuning was obtained based on simple core skeleton by varying the type of electronic donor.

Figure 2. (A) UV-vis spectra of CP1–CP5 in DMSO (50 μM). (B) Emission spectra of CP5 in DMSO/water mixtures with varied water fractions (fw). (C) Plot of I/I0 of CP1–CP5 versus fw of the solvent mixture, I0 represents the emission intensity in DMSO. (D) Normalized emission spectra of CP1–CP5 in the solid state. Inset in B: photograph of CP5 in DMSO and DMSO/water mixtures with fw values of 99% under irradiation of 365 nm UV light. Inset in D: photograph of CP1–CP5 in solid state under irradiation of 365 nm UV light.


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2.3. Self-Assemblies of AIEgens. The intense solid-state emission inspired us to study the selfassembly property of these AIEgens due to their potential application as optical devices or probes.40,41 The self-assembled samples were prepared by classical precipitation method.42,43 Injection of corresponding AIEgens in acetonitrile solution into tetrahydrofuran solution under ultrasonic condition and then aged for several hours led to the aggregation of molecules. The resulted aggregates were characterized with scanning electron microscopy (SEM) and fluorescent microscopy (Figure 3 and Figure S7). CP1 self-assembled into microrods while other fluorogens assembled into microplates with different size. The emission color from bright blue to red (440 to 600 nm) could be observed under the fluorescent microscopy.

Figure 3. The SEM (A–E) and fluorescence microscopy images (F–J) of CP1–CP5 micromaterials. Scale bar for F–J: 20 μm. 2.4. Single Crystal Analysis. To gain further insight into their specific emission behavior, the crystals of all AIEgens were grown and the signal crystals of CP1, CP2 and CP3 were successfully obtained by slowly evaporating of their mixture of dichloromethane/n-hexane. Their structure was determined by using single crystal X-ray diffraction (Table S1) and the crystal


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conformations were inspected carefully. As shown in Figure 4, it’s clearly that all crystals showed nonplanar structure and adopted highly twisted molecular conformation. The torsion angle between the core benzene ring and substituted aromatic group (conjoint vinyl group) was 44.40o, 47.38o and 45.73o (42.38o, 42.57o and 42.57o) for CP1, CP2 and CP3, respectively. In addition, the torsion angle between the side pyridinium group and vinyl groups varied from 19.64o, 20.45o to 20.45o when the substituted groups changed from 4-trifluoromethylphenyl to 4methylphenyl. Such distorted conformation was able to effectively increase the distance between neighboring molecules (> 6.504 Å) in all crystals, and helped to avoid the intermolecular π–π stacking, which was recognized as the vital factor for the emission quenching in the condensed phase. Meanwhile, multiple C–H···F interactions (2.305 to 2.558 Å) between cationic and counter anion PF6¯ were found throughout the crystals while intermolecular interaction such as C–H···N bond (2.404 Å, 2.476 Å and 2.658 Å) existed in the crystal of CP1 and CP2 (Figure S8). Those weak interactions further helped to rigidify the configuration and result in the intense solid-state emission. The 3D conformation of CP4 and CP5 were expected to be similar to that of CP1–CP3 due to their analogical molecular skeleton. In brief, the AIE effect of CP1–CP5 could be originated from their twisted molecular conformation as well as multiple intermolecular interactions.


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Figure 4. The drawing of the crystal structures, distance between neighboring molecules and crystal packing of CP1 (A), CP2 (B) and CP3 (C). 2.5.

Electrochemical

Property

and

Theoretical

Calculation.

To

investigate

the

electrochemical behaviors of all AIEgens, the cyclic voltammetry experiment was carried out. The practical energy levels were estimated based on the onset oxidation potential and optical bandgaps. As exhibited in Figure 5A, all of the AIEgens experienced irreversible oxidation process and the oxidation potential was lowered (from 2.4 to 1.4 eV) in the order from CP1 to CP5. The highest occupied molecular orbital (HOMO) energy levels for CP1 to CP5 were calculated to be –6.85 to –5.88 eV, and the lowest unoccupied molecular orbital (LUMO) energy levels ranged from –4.09 to –3.67 eV (Figure 5B, Table 1). The introduction of stronger electronic donor dramatically increased the energy level of HOMO while raised the energy level


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of LUMO gently, which resulted in the decrease of energy gap between HOMO and LUMO from 2.76 to 2.21 eV for CP1 to CP5 (Figure 5B). These observations reproduced the trends derived from the absorption spectra.

Figure 5. (A) Cycle voltammograms of CP1–CP5 measured in acetonitrile with 0.1 M tetra-nbutylammonium hexafluorophosphate (TBAH). (B) The energy gap of CP1–CP5 determined from the onset of absorption spectrum. To better understand the relationship between electronic transitions and optical behavior, the theoretical calculations of all AIEgens were performed at the basis level of CAM-B3LYP/6-31G (d, p) using Gaussian 09 (Figure 6). The HOMOs of all AIEgens were primarily located on the central benzene and the aromatic electron-donating groups, whereas LUMOs were spread over the core benzene ring as well as the cyano-pyridinium moiety. It is noteworthy the distribution of HOMOs and LUMOs presented X shape and such separated frontier orbital distribution indicated that all AIEgens experienced the CT process.


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Figure 6. The amplitude plots of frontier molecular orbital of CP1–CP5 and ΔEst value, calculated at the basis set of CAM-B3LYP/6-31 G (d, p). 2.6. Mitochondrial Imaging. The cationic fluorescent dye was preferred to stain the mitochondria in living cells through electronic interaction due to the negative change of mitochondrial inner membrane.44,45 The cationic nature of these AIEgens inspired us to explore their application for the mitochondria-targeted imaging. Cytotoxicity studies were carried out at the initial stage by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate cell viability in order to assess their suitability as mitochondria probes. As illustrated in Figure S9 and 8C, after incubated HeLa cells with CP1 to CP5 over a concentration range of 0–20 μM, nearly no significant cytotoxic effects were observed. Meanwhile, more than 65% viability was still obtained when the concentration reached up to 50 μM, demonstrated their good biocompatibilities. The negligible cytotoxicity encouraged us to carry out the cellular uptake and localization studies of CP1 to CP5. After incubated AIEgens (20 μM) with HeLa cells, all AIEgens could


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enter and light up the cells with different colors that range from blue to red (Figure S10). To conclusively confirm the localization of these AIEgens at the mitochondria, HeLa cells were costained with 100 nM of commercial mitochondria-targeting reagent (Mito-tracker deep red, MTR) and the images were collected using confocal laser scanning microscopy (CLSM). As shown in Figure 7, the merged images revealed that location of AIEgens in HeLa cells were practically consistent with that of MTR and the overlap ratio was calculated to be 75.2%, 63.5%, 86.4%, 84.7% and 90.2% for CP1 to CP5, respectively, suggested all these AIEgens can selectively stain and light up the mitochondria in living cells. The excellent mitochondria-targeting specificity could be attributed to the suitable lipophilicity and cationic property of these AIEgens.46–50


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Figure 7. CLSM of HeLa cells stained with CP1–CP5 (20 μM) and MTR (200 nM). Scale bar: 20 μm. 2.7. Selective Photodynamic Ablation of Cancer Cells. Besides the mitochondrial imaging application, cationic AIEgens were frequently used as photosensitizers to generate the 1O2 under light irradiation.51,52 To understand the cell oxidative stress induced by the treatment of CP1 to CP5, the 1O2 generation capability was assessed by using the indicator of 9,10-anthracenediylbis(methylene)-dimalonic acid (ABDA). ABDA and different AIEgens were incubated in DMSO/water mixtures with 99% fw initially, and then exposed to the white light (25 mW cm–2). The variation of absorption spectra of ABDA was measured with elapse of time. As described in Figure 8A and Figures S11–S15, the absorption spectra of ABDA deceased gradually. After 200 s illumination, the absorbance intensity of ABDA at 378 nm reduced to 80% for CP1 while the value reduced to 50% and 45% in the case of CP2 and CP3 or CP5 (Figure 8B). It is noted that the intensity of ABDA dropped to around 30% in the presence of CP4, demonstrated that CP4 could generate 1O2 more effectively and the quantum yield of 1O2 for CP4 was determined to be 0.52 (Figure S16). Based on the previous literatures, the capability of 1O2 generation is directly proportioned to the intersystem crossing process of photosensitizer from the lowest excited singlet state (S1) to the lowest triplet state (T1).53–55 In order to assess the 1O2 generation capability, the energy level between S1 and T1 for all AIEgens were calculated using CAM-B3LYP/6-31G (d, p) and the


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value of energy gap between S1 and T1 (ΔEst) was calculated as 0.032 eV, 0.024 eV, 0.020 eV, 0.007 eV and 0.020 eV in order from CP1 to CP5, respectively (Figure 6). From CP1 to CP5 except CP4, the distribution of HOMO on –CN group were decreased gradually, led to the more separation of HOMO–LUMO with reduced ΔEst. Unexpectedly, the HOMO distribution of CP4 was entirely located over core benzene ring as well as the electron-donating moiety with no occupancy on –CN group, which resulted in a completely separation of HOMO–LUMO and gave extremely low ΔEst value of CP4. As a result, the 1O2 generation efficiency of CP4 is best. Subsequently, CP4 was utilized to exam its performance as a photosensitizer for PDT of cancer cells. The quantified therapeutic effect was conducted by MTT assay using cancer (HeLa) and normal (NIH-3T3) cells, respectively. Upon incubation both cell lines with CP4 under dark, more than 65% cell viability was observed when the concentration was up to 50 μM, suggested its relatively low dark cytotoxicity. However, upon the white light illumination (25 mW cm–2) for 30 min, a dose-dependent toxicity was observed for HeLa cells. The cell viability showed moderate decrease at the concentration of 5 μM (60%), whereas dropped to 20% when the concentration increased to 50 μM (Figure 8C). In contrast, dramatically reduced cytotoxicity was observed with light irradiation for NIH-3T3 cells (Figure 8D). The selectively ablate the cancer cells through PDT process of CP4 was attributed to the higher mitochondrial membrane potential of cancer cells than normal cells.32


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Figure 8. (A) Change of UV-vis spectra of ABDA (100 μM) in the presence of CP4 (50 μM) with different durations of light illumination (25 mW cm–2). (B) Plots of A/A0 of CP1–CP5 versus different illumination time. The cell viability of HeLa (C) and NIH-3T3 (D) cells after incubation with varied concentration of CP4 under white light illumination (25 mW cm–2, 30 min). The capability of CP4 for intracellular ROS production was further assessed using 2,7dichlorofluorescein diacetate (DCF-DA) as a ROS indicator. HeLa cells were firstly stained with


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CP4 for 30 min and then treated with DCF-DA (200 μM). After illuminated using white light (25 mW cm–2) for 30 min, the images of cell were collected using CLSM. As shown in Figure 9A, the signal of DCF-DA increased gradually with time elapsed while the bright green fluorescence was observed after 30 s. Conversely, negligible green fluorescence was detected for cells treated with DCF-DA alone, demonstrated the generation of ROS by CP4 under white light irradiation (Figure S17A). Next, the Annexin V-FITC and propidium iodide (PI) were employed to monitor the process of cell apoptosis that induced by PDT. The cell plasma membrane can be selectively labeled by green fluorescence of Annexin V-FITC during the early stage of apoptosis, while the nuclei of dying or dead cells was further specific labeled with red fluorescent PI. After incubated HeLa cells with CP4 and exposed to white light (25 mW cm–2) for 30 min, the cells were treated with Annexin V-FITC and PI, and collected their fluorescent images by CLSM. As shown in Figure 9B, nearly no green and red fluorescent signals were detected at the beginning. However, after 30 min incubation, weak green fluorescence was observed, indicated the cell apoptosis started. Continue incubated cells up to 60 min, the red fluorescent signal from PI appeared besides green fluorescence. When the incubation time prolonged to 90 min, both bright green and red fluorescent signals were observed, suggested the cells were dead. In sharp contrast, no significant fluorescent signal were obtained for control cells incubated for same time without light irradiation (Figure S17B), revealed that the cell apoptosis should be induced by the PDT process of CP4.


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Figure 9. (A) Intracellular ROS detection by CLSM after HeLa cells incubated with CP4 (20 μM) and DCF-DA (200 μM) under white light illumination (25 mW cm–2, 30 min). (B) Apoptosis observation of HeLa cells by CLSM after treated with CP4 (20 μM) under white light illumination (25 mW cm–2, 30 min). Scale bar: 20 μm. 2.8. Bacterial Imaging and Antibacterial Activity. In view of the good cell imaging performance, the imaging ability of AIEgens for bacteria was also investigated by using Gramnegative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) as model. After incubated E. coli with CP1 to CP5 (20 μM) for 10 min, the images were collected. As displayed in Figure 10, bright fluorescence signals of E. coli from blue to red were detected and similar phenomena were also observed after treated S. aureus with AIEgens under the same condition, indicated these AIEgens can successfully image both E. coli and S. aureus. It is worth


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mentioning that the wash-step after staining was not necessary in labeling procedure due to specific AIE effect of these fluorogens, which will simplify the process of bacterial staining. Based on the positive charge of AIEgens, the excellent bacterial imaging could be originated from the hydrophobic and electrostatic interactions between AIEgen and bacteria.56

Figure 10. CLSM of E. coli and S. aureus stained with CP1–CP5 (20 μM). Scale bar: 10 μm. Enlightened by the excellent performance in 1O2 generation, CP4 was also chosen as photosensitizer for antibacterial application. The plate-counting method was employed to estimate the ability of CP4 for killing of bacteria (Figure 11, Figure S18–S20). In the absence of


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CP4 and white light irradiation, E. coli grew healthy on LB-agar plate (Figure 11A). After treated E. coli with either white light (25 mW cm–2, 30 min) or CP4 (20 μM) under dark, the growth of E. coli on LB-agar plate suffered slight influence (Figure 11B, C). However, upon treatment E. coli with CP4 (20 μM) for 10 min and then exposed to white light (25 mW cm–2) for 30 min, almost no colonies were formed on plate, demonstrated E. coli were killed efficiently (Figure 11D). For S. aureus, similar photodynamic antibacterial effect was also observed at the same condition (Figure 11E–H). Above results elucidated that CP4 was considerably powerful in killing of both Gram-negative and Gram-positive bacteria.

Figure 11. Plates of E. coli and S. aureus without (A, E) and with (B, F) white light irradiation (25 mW cm2, 30 min) in the absence of CP4. Plates of E. coli and S. aureus treated with CP4 (20 μM) for 10 min without (C, G) and with (D, H) white light irradiation (25 mW cm–2, 30 min). 3. CONCLUSION


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In summary, we developed a series of cationic AIEgens (CP1 to CP5) with wide color tunability across the whole visible range by fixing the electron acceptor and varying the electron donor within single benzene ring. Single crystal analysis revealed that the twisted conformation with rotatable phenyl ring should be responsible for their AIE effect. In view of good biocompatibility and cationic nature, the AIEgens can specific accumulate and light up the mitochondria as well as image both Gram-negative and Gram-positive bacteria with a wash-free manner. Importantly, these AIEgens, particularly CP4, can efficiently generate the 1O2 after exposed to the white light irradiation. Further biological experiments suggested CP4 can be utilized as an ideal photosensitizer for photodynamic ablation of cancer cells and killing of bacteria. This work offers a new strategy to construct colorful AIEgens for potential cancer and bacteria diagnosis and therapy. 4. EXPERIMENTAL SECTION 4.1. Cytotoxicity Studies. HeLa cells were seeded in 96-well plate at the density of 10000 cells per well. After 48 h, the cells were incubated with CP1–CP5 in PBS at various concentrations for 30 min. After that, the suspension of CP1–CP5 was replaced by PBS and cultured for 2 h under dark. For photodynamic therapy, HeLa and NIH-3T3 cells were seeded at the density of 10000 cells per well, respectively. After 48 h, the cells were incubated with CP4 in PBS at various concentrations for 30 min. The suspension was replaced by PBS and chosen wells were irradiated using white light (25 mW cm–2, 30 min). Then the plate was cultured for 2 h


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under dark. After that, MTT was added and incubated for another 4 h. Finally, the lysate buffer was added to solubilize the formazan crystals, and the absorbance at 570 nm was collected. 4.2. Cell Imaging. HeLa cells were seeded in the 35-mm glass-bottom dish to reach 80% confluence. Then the medium was removed and the cells were washed with PBS. The cells was treated with CP1–CP5 (20 μM) and MTR (100 nM) in PBS for 30 min. After that, the cells was washed by PBS and used for imaging by CLSM. 4.3. Intracellular ROS Detection. The ROS generation within living cells was detected by using DCF-DA. HeLa cells were seeded in the 35-mm glass-bottom dish to reach 80% confluence, and then the medium was removed and washed with PBS. Following incubation was proceeded with CP4 (20 μM) and DCF-DA (200 μM) under dark. After 30 min, cells were washed with PBS and irradiated with white light (25 mW cm–2) for 30 min. For control experiments, same protocol was performed in the absence of CP4. The images were collected with the time elapsed. 4.4. Annexin V-FITC/Propidium Iodide Co-Stainning Assay. HeLa cells pre-treated with CP4 (20 μM) for 30 min were stained with both annexin V-FITC and PI following standard protocol (Life Technologies) and imaged after irradiation of white light (25 mW cm–2, 30 min). For control experiments, HeLa cells pre-incubated with CP4 (20 μM), annexin V-FITC and PI were imaged without light irradiation. The excitation wavelength for annexin V-FITC and PI was 488 and 559 nm, respectively, and the emission for annexin V-FITC and PI was collected at the range of 500–530 nm and 600–630 nm.


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4.5. Bacterial Staining. A single colony of E. coli or S. aureus that picked from LB or BHI plates was incubated in 2YT or BHI medium overnight. After that, the bacteria were washed with 0.9% NaCl solution. The resulted bacteria with density of 2×107/mL were transferred into an EP tube, and stained with CP1–CP5 (20 μM) for 10 min. Then 10 μL of the bacteria solution was transferred on to a glass slide with a coverslip and the fluorescence images were collected. 4.6. Killing of Bacteria. The antibacterial activity of CP4 were determined by incubating the bacterial suspensions with CP4 (20 μM) for 10 min under dark. The bacterial suspensions were either exposed to white light (25 mW cm–2) or incubated under dark for 30 min. Then the bacteria were transferred on to agar plate (1.2 % agar + LB for E. coli and 1.2 % agar + BHI for S. aureus) and incubated at 37 oC overnight. ASSOCIATED CONTENT

Supporting Information. Synthetic routes, optical spectra, crystal data, cell imaging and NMR spectra.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected], [email protected].

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21672135, 21402115, and 51403122), Natural Science Foundation of Shaanxi Province (2018JM5086), Young Talent fund of University Association for Science and Technology in Shaanxi (2017030021) and Fundamental Research Funds for the Central Universities (GK201702002, GK201902006). REFERENCES (1) Grimsdale, A. C.; Chan, K. L.; Martin, R. E.; Jokisz, P. G.; Holmes, A. B. Synthesis of Light-Emitting Conjugated Polymers for Applications in Electroluminescent Devices. Chem. Rev., 2009, 109, 897–1091. (2) Weil, T.; Vosch, T.; Hofens, J.; Peneva, K.; Müllen, K. The Rylene Colorant FamilyTailored Nanoemitters for Photonics Research and Applications. Angew. Chem., Int. Ed., 2010, 49, 9068–9093. (3) Pascal, S.; Haefele, A.; Monnereau, C.; Charaf-Eddin, A.; Jacquemin, D.; Guennic, B. L.; Andraud, C.; Maury, O. Expanding the Polymethine Paradigm: Evidence for the Contribution of a Bis-Dipolar Electronic Structure. J. Phys. Chem. A, 2014, 118, 4038–4047. (4) Zalar, P.; Henson, Z. B.; Welch, G. C.; Bazan, G. C.; Nguyen, T. Q. Color Tuning in Polymer Light-Emitting Diodes with Lewis Acids. Angew. Chem. Int. Ed., 2012, 51, 7495–7498.


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TOC:


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Aggregation-Induced Emission Luminogens with the Capability of

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