Organic Dots with Aggregation-Induced Emission (AIE Dots

Oct 1, 2013 - cytotoxicity of both the green and red AIE dots allow for the simultaneous discrimination of different populations of cancer cells both ...
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Organic Dots with Aggregation-Induced Emission (AIE Dots) Characteristics for Dual-Color Cell Tracing Kai Li,† Zhenshu Zhu,‡ Pingqiang Cai,§ Rongrong Liu,† Nikodem Tomczak,† Dan Ding,∥ Jie Liu,∥ Wei Qin,⊥ Zujin Zhao,# Yong Hu,∇ Xiaodong Chen,§ Ben Zhong Tang,⊥,# and Bin Liu*,†,∥ †

Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, 117602, Singapore ‡ Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China § School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore ∥ Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576, Singapore ⊥ Department of Chemistry, Division of Biomedical Engineering, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China # SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China ∇ National Laboratory of Solid State Microstructure, Department of Material Science and Engineering, Nanjing University, Nanjing 210093, China S Supporting Information *

ABSTRACT: Modern fluorescence imaging techniques have become essential tools to provide crucial insights in understanding complicated biological processes. Because of their unique optical properties (e.g., excellent photostability, high brightness, broad absorption, and narrow emission), inorganic quantum dots (QDs) have attracted great interest in fluorescence bioimaging. However, the intrinsic toxicity resulting from their heavy-metal components as well as the low-pH-induced fluorescence-quenching phenomenon has motivated researchers to explore novel fluorescent probes with the goal of overcoming these obstacles. In this work, we report the synthesis of two groups of organic fluorescent dots with aggregation-induced emission (AIE) characteristics that have a large Stokes shift, ensuring distinct emission spectra (green and red fluorescence) under singlewavelength excitation. Single-particle imaging experiments revealed the unique optical properties of such AIE dots, which outperform their commercially available inorganic QD counterpart in physical stability and brightness. Upon functionalization with a cell-penetrating peptide, the strong absorptivity, high brightness, good cellular-internalization efficiency, and low cytotoxicity of both the green and red AIE dots allow for the simultaneous discrimination of different populations of cancer cells both in culture medium and animal organs, which is of high importance for understanding cellular interactions during cancer metastasis. Considering the versatile surface functionalities endowed by the encapsulation matrix, a series of organic AIE dots with customized properties will provide prospective platforms to satisfy multifarious bioimaging tasks in the near future. KEYWORDS: aggregation-induced emission, fluorescence imaging, cell tracing, dual color, AIE dots, single-nanoparticle imaging

1. INTRODUCTION Fluorescence techniques have been extensively employed to develop noninvasive methodologies for tracking and understanding complex biological processes both in vitro and in vivo, which is of high importance in modern life-science research.1,2 To accomplish challenging biological imaging tasks with desired outputs, a variety of fluorescent probes, including discrete molecules and bulk materials, has been explored.3,4 As compared with discrete molecules (e.g., fluorescent proteins and organic chromophores), inorganic semiconductor quantum dots (QDs) have shown advantages in terms of better photostability, larger Stokes shift, and more feasible surface functionalization.5 More importantly, the broad excitation © 2013 American Chemical Society

range and narrow emission spectra of QDs are also beneficial for the simultaneous analysis of multibiotargets using a series of QDs upon a single laser excitation,6 showing the merits of a reduction of instrument complexity, test time, and overall cost as compared to the multiple laser setup.7 However, the release of free heavy-metal ions in an oxidative environment may cause potential toxicity,8 which still remains a major concern when applying QDs in vivo. Moreover, the unstable fluorescence of QDs at low pH greatly impedes the reliability of experimental Received: May 28, 2013 Revised: September 9, 2013 Published: October 1, 2013 4181

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suggest that AIE dots can act as a novel class of fluorescent probes in future advanced biological-imaging applications.

analysis in practice.9 Although efforts have been made to develop organic dye-doped nanoparticles with dramatically enhanced photostability that is comparable to inorganic QDs, these fluorescent nanoparticles share the same shortcoming of a small Stokes shifts with their isolated dye molecules. Another major challenge of conventional organic-dye-encapsulated nanoparticles is that their light emission is always weakened or annihilated in aggregates, which is known as aggregationcaused quenching (ACQ).10 As a result, novel fluorescent probes with excellent photostability, large Stokes shift, and good biocompatibility are highly desirable to satisfy the growing interest in sophisticated biological studies. Recently, we have developed several fluorogens with aggregation-induced emission (AIE) characteristics, which are directly opposite to ACQ.11 The AIE fluorogens are nonemissive in solution but become highly emissive upon aggregation as the restriction of intramolecular rotations (RIR) activates radiative decay channels. This unique fluorescent property makes AIE fluorogens promising candidates to construct fluorescent nanoparticles.12,13 The fact that the attachment of an ionic AIE unit, tetraphenylethene (TPE), to various ACQ dyes can transform them into AIE dyes has opened new opportunities to synthesize AIE fluorogens with tunable emission wavelengths and large Stokes shifts.14 Additionally, using AIE fluorogens as the fluorescent domain and biocompatible distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) derivatives as the encapsulation matrix, AIE dots that greatly outperform inorganic QD probes in in vitro and in vivo long-term celltracing applications have been successfully explored.15 The feasible synthetic routes and outstanding optical properties make AIE fluorogens promising candidates in the exploration of the next generation of organic fluorescent probes, especially for multitarget imaging applications. In this work, we aimed to synthesize QD-sized organic AIE dots and to demonstrate the application of such AIE dots with distinct emission colors for simultaneously monitoring the migrating behaviors and interactions between two populations of cancer cells, which is essential to elucidate cellular behavior during cancer metastasis. Two AIE fluorogens, with 2,1,3benzothiadiazole and 2,3-bis[4-(diphenylamino)phenyl]fumaronitrile as core building blocks, respectively, and TPE units as peripheries were employed to synthesize green and red AIE dots using DSPE-PEG derivative as the encapsulation matrix. Upon surface decoration using a cell-penetrating peptide (HIV-1 Tat peptide), both AIE dots show high cellular-internalization efficiency with low cytotoxicity. Singleparticle imaging results indicate that the AIE dots are ultrabright and “nonblinking” under continuous laser excitation, with a higher brightness as compared to the most widely used commercial QD probe. The UV−vis absorption spectra of the green and red AIE dots intersect at 455 nm with high molar extinctive coefficients, whereas the emission wavelengths are distinct with negligible overlap. In vitro studies suggest that these Tat-functionalized AIE dots are able to label and simultaneously track the migration and interaction of two populations of cancer cells. For in vivo studies, the mixture of cancer cells separately labeled by the green and red AIE dots was intravenously injected into mice to mimic metastasis at the postintravasation stage, and the performance of these new fluorescent probes in imaging different cell populations in organ tissues was evaluated through ex vivo analysis. These results

2. EXPERIMENTAL SECTION Materials. 4,7-Bis[4-(1,2,2-triphenylvinyl)phenyl]benzo-2,1,3-thiadiazole (BTPETD) and 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumaronitrile (TPETPAFN) were synthesized according to the literature. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Mal) were purchased from Avanti Polar Lipids, Inc. Tetrahydrofuran (THF), Dulbecco’s modified Eagle medium (DMEM), penicillin/streptomycin solution, fetal bovine serum (FBS), trypsin-EDTA solution, and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were provided by Sigma-Aldrich. A customized cell-penetrating peptide, HIV-1 Tat (49−57) with the C-terminus modified with cysteine (RKKRRQRRRC), was a commercial product by GenicBio, Shanghai, China. Milli-Q water used in the experiments was supplied by a MilliQ Plus System (Millipore Corporation, Bedford, MA, USA). C6 glioma cells and HT1080 fibrosarcoma cells were provided by the American Type Culture Collection. Characterization. UV−vis spectra were measured using a Shimadzu UV-1700 spectrometer. Fluorescence spectra were recorded on a fluorometer (LS-55, PerkinElmer, Waltham, MA, USA). Average particle sizes were determined by laser light scattering with a particlesize analyzer (90 Plus, Brookhaven Instruments Co., Holtsville, NY, USA) at a fixed angle of 90° at room temperature. High-resolution transmission electron microscope (HR-TEM) images were recorded using a JEM-2010F microscope (JEOL, Japan) with an accelerating voltage of 200 kV. Synthesis of Surface-Functionalized AIE Dots. BTPETD, DSPE-PEG2000, and DSPE-PEG2000-Mal (1 mg of each) were dissolved in 1 mL of THF to form a homogeneous solution. The mixture was quickly added into 9 mL of water followed by sonication using a microtip probe sonicator (XL2000, Misonix, Inc., Farmingdale, NY, USA) for 60 s at 12 W output. The obtained dot suspension was vigorously stirred at room temperature overnight in fumehood at 700 rpm to evaporate the organic solvent to yield green BTPETD dots (AIE dots) with surface maleimide groups in 8 mL of water. The AIE dots were filtered using a 0.2 μm syringe-driven filter to purify. To attach HIV-1 Tat peptides on the dots, AIE dots were mixed with HIV1-Tat peptide (0.3 μmol) and stirred overnight. The free peptides were removed by dialysis against Milli-Q water for 2 days using a 3.5 kDa molecular weight cutoff dialysis membrane to afford green TatAIE dots (GT-AIE dots). The red Tat-AIE dots (RT-AIE dots) were synthesized using TPETPAFN, DSPE-PEG2000, and DSPE-PEG2000Mal following the same procedure. Wide-Field Microscopy Imaging. The samples for single-dot imaging were prepared through serially diluting the solutions and depositing small liquid droplets on a glass cover slide using a fixed protocol. Upon decreasing the concentration of the dots in solution, the number of absorbed dots decreased linearly until a desired concentration of dots on the image was obtained. Below a certain concentration threshold, the intensity of the fluorescence originating from individual spots did not change, but the number of spots per image decreased. This indicated that we were in the regime suitable for single-dot imaging. Fluorescence imaging of individual GT-AIE dots, RT-AIE dots, and Qtracker 655 was performed with a wide-field microscope (WFM) based on a Nikon ECLIPSE Ti-U inverted microscope frame. Light from a CW multiline Ar ion laser (Melles Griot, Carlsbad, CA, USA) was fiber-coupled to a Nikon TIRF attachment and focused on the back aperture of a high-NA objective (Nikon TIRF Apo 100×, NA = 1.49, oil immersion). Immersion oil (nD = 1.4790, Cargille, Cedar Grove, NJ, USA) was added between the high-NA objective and the coverslip for index matching. The luminescence was collected by the same objective, and after passing through the dichroic mirror and the emission filter it was directed onto an iXonEM+897 EMCCD camera (512 × 512 pixels, 150 nm per pixel 4182

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Scheme 1. Schematic Illustration of Synthesis of Tat-Functionalized AIE Dots

resolution, Andor Technology, Northern Ireland) connected to the side port of the microscope. The filter set for 488 nm excitation wavelength consisted of a z488/10× excitation filter, Z488RDC dichroic mirror, and HQ500LP emission filter. The camera was connected to a computer furbished with camera-dedicated software to control the imaging parameters and data acquisition. The samples were prepared by depositing a droplet of nanoparticle solutions on a glass cover slide, waiting for 30 s for particle adsorption to the substrate, removing excess liquid, and drying under nitrogen. Fluorescence intensity time traces were obtained by acquiring 1000 consecutive frames at a rate of 10 frames per second (100 ms exposure time, 100 s time-traces), extracting the number of counts per particle at each frame using a 7 × 7 pixels region of interest, and subtracting the background. The images were analyzed using Andor Solis (ver. 4.14.30001.0, Andor Technology, Northern Ireland) and NIS Elements Ar 4.10.00 (Nikon, Japan) software. Statistical analysis was performed using OriginPro8 software (OriginLab Corporation, Northampton, MA, USA). In Vitro Cell Imaging. C6 glioma cells were precultured in 6-well plates (Costar, IL, USA) containing cell culture coverslips to achieve 80% confluence. The medium was then removed, and the cells were washed twice with 1× PBS buffer followed by addition of 2 nM GTAIE and RT-AIE dots in fresh DMEM for incubation at 37 °C, respectively. After 12 h, the cell monolayer was washed using 1× PBS buffer and fixed using 70% ethanol for 10 min. The coverslips were sealed with mounting medium, and the confocal images were obtained using Leica TCS SP 5X upon excitation at 458 nm (1 mW). The fluorescence signal was collected using 480−560 and 670−800 nm bandpass filters. Cytotoxicity Studies. The cytotoxicity of GT-AIE and RT-AIE dots was evaluated using C6 cells and HT1080 cells with an MTT assay. In brief, C6 cells were seeded in 96-well plates at a density of 4 × 104 cells/mL. After a 24 h incubation, the medium was discarded, and various concentrations of Tat-AIE dots in DMEM were added into each sample well for further incubation at 37 °C. To eliminate the UV absorption interference of AIE chromophores at 570 nm, the cells incubated with a series of Tat-AIE dots at the same doses but not posttreated by MTT were used as the control. After the designated time intervals, the sample wells were washed twice with 1× PBS buffer, and 100 μL of freshly prepared MTT (0.5 mg/mL) solution in culture medium was added into each well. After a 3 h incubation in the incubator, the MTT medium solution was carefully removed followed by addition of 100 μL of DMSO into each well, and the plate was gently shaken for 10 min at room temperature to dissolve all of the precipitates that formed. The absorbance of MTT at 570 nm was monitored by a microplate reader (Genios Tecan). Cell viability was expressed by the ratio of the absorbance of the cells incubated with a

Tat-AIE dot suspension to that of the cells incubated with culture medium only. Animals. Male SCID mice were obtained from the Biological Resource Centre (Biopolis, Singapore). Mice were housed in groups (5 per cage) and provided with standard mouse chow and water ad libitum. The cages were maintained in a room with controlled temperature (25 ± 1 °C) and a 12 h light/dark cycle (lights on at 7:00 am). All animal experiments were performed in compliance with guidelines set by the Institutional Animal Care and Use Committee (IACUC), SingHealth. In Vivo Cell Tracing. C6 glioma cells were cultured in culture flasks to achieve 80% confluence. After medium removal and washing with 1× PBS buffer, 2 nM GT-AIE or RT-AIE dots in DMEM medium were then added to the wells and incubated at 37 °C. After 12 h, the cells were washed twice using 1× PBS buffer and detached with 1× trypsin. Trypsin was discarded through centrifugation at 2000 rpm for 5 min, and the labeled cells were resuspended in culture medium. After cell counting, cells labeled with GT-AIE dots and that labeled with RTAIE dots (0.2 mL, 2 × 106 cells) were then separately injected into each mouse through the tail vein. At 3 h postinjection, the mice were imaged using the Maestro in vivo fluorescence imaging system (CRi Inc.) while under anesthesia. The time-dependent biodistribution of injected cells in mice was imaged using the Maestro in vivo fluorescence imaging system (CRi Inc.). Light with a central wavelength at 455 and 523 nm was selected as the excitation source for the GT-AIE and RT-AIE dots, respectively. The fluorescence signals from the GT-AIE and RT-AIE dots were collected in the range of 500 to 720 nm and 560 to 750 nm at 10 nm steps, respectively. Autofluorescence was removed using spectral unmixing software. Ex Vivo One-Photon Excited Fluorescence Imaging. C6 cells labeled with GT-AIE dots and those labeled with RT-AIE dots were mixed at the same concentration in fresh culture medium to achieve a final concentration of 1 × 107 cells/mL. The labeled C6 glioma cell mixture (0.2 mL, 2 × 106 cells) was then intravenously injected into each mouse. At 3 h postinjection, the mice were euthanized with CO2 to harvest the organs (heart, kindney, spleen, lung, and liver). The obtained organs were then fixed in 4% paraformaldehyde and imaged using confocal microscopy (Leica TCS SP 5X) upon excitation at 458 nm (1 mW), and the fluorescence signal was collected using 480−560 and 670−800 nm bandpass filters.

3. RESULTS AND DISCUSSION Synthesis and Characterization of AIE Dots. The two AIE fluorogens, BTPETD and TPETPAFN, were synthesized according to the literature.14,15 The green and red AIE dots were synthesized using a previously reported nanoprecipitation method as shown in Scheme 1.16,17 Two lipid-PEG derivatives, 4183

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25% using rhodamine 6G in ethanol (95%) and 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran in methanol (43%) as the references,18 respectively. The high fluorescence quantum yield, desirable molar extinction coefficient, and negligible emission overlap of the GT-AIE and RT-AIE dots will greatly benefit the simultaneous imaging of biotargets upon labeling by different dots under a single laser excitation. Single-nanoparticle fluorescence imaging of GT-AIE and RTAIE dots was performed upon excitation at 488 nm using a commercially available QD probe, Qtracker 655, as the benchmark. The intensity time-traces were obtained by integrating the emission from individual GT-AIE dots, RTAIE dots, and Qtracker 655 nanoparticles over 100 ms for 1000 consecutive frames. Fluorescence intensity time traces for individual dots reveal that Qtracker 655 shows pronounced fluorescence intermittency, referred as blinking,19 which is absent in the GT-AIE and RT-AIE dots (Figure S1 in the Supporting Information). It is noteworthy that the GT-AIE dots have good photostability upon continuous laser excitation. Although a gradual fluorescence decrease is observed in the photobleaching trajectory of the RT-AIE dots under the experimental conditions, they still holds great promise for use in fluorescence imaging because the total number of photons emitted in 100 s from a single RT-AIE dot is much higher than a single GT-AIE dot and Qtracker 655. The histograms of the numbers of photons emitted from GT-AIE dots, RT-AIE dots, and Qtracker 655 after statistical analysis are shown in Figure 2. The average number of total photons emitted by RT-AIE dots (4.44 × 107 counts) is the highest among the three fluorescent probes, which is ∼80 and ∼70 times higher than that of GTAIE dots and Qtracker 655, respectively. The degree of intermittency in Qtracker 655 emission greatly compromises the total number of photons emitted in a given period of time, restricting their applications in single-nanoparticle tracking studies because the decrease in the emission to the background level shortens the diffusion traces and increases the error in determining key parameters (e.g., diffusion coefficients). Moreover, the photostabilities of GT-AIE dots, RT-AIE dots, and Qtracker 655 in cells upon internalization were investigated after incubation with C6 glioma cells for 4 h at 37 °C. Upon continuous excitation at 458 nm (1 mW) for 10 min, both GTAIE and RT-AIE dots in cells show comparable photostability to Qtracker 655 without any obvious change in fluorescence intensities (Figure S2 in the Supporting Information). Overall, the high brightness and continuous emission behavior with negligible fluorescence blinking of Tat-functionalized AIE dots will be particularly valuable in various advanced bioimaging tasks, including single-molecule tracking applications. In Vitro Imaging. The cell-imaging performance of Tatfunctionalized AIE dots was examined using rat C6 glioma cells as a model. After separate incubation of the dots with C6 cells for 12 h at a dot concentration of 2 nM, the fluorescent signal interference of the two AIE dots was examined using confocal laser scanning microscopy (Figure 3). The samples were excited with a 458 nm laser, and the fluorescence signals were simultaneously collected with 480−560 and 670−800 nm bandpass filters. The bright-green fluorescence in Figure 3a suggests that the cells were successfully labeled by GT-AIE dots, whereas the negligible signal from Figure 3b indicates that no interfering fluorescence can be detected in the 670−800 nm range. However, the cells labeled with RT-AIE dots emit intense fluorescence in the range of 670−800 nm (Figure 3e),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyl(polyethylene glycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Mal), were used to encapsulate the AIE fluorogens to afford fluorescent dots with surface maleimide groups for further functionalization. Because of the amphiphilic property of DSPE-PEG derivatives, the hydrophobic DSPE backbones tend to entangle the AIE molecules to form the core, whereas the hydrophilic PEG segments extend into the aqueous phase. The presence of PEG chains on the dot surface will not only provide functional groups but also minimize nonspecific absorption during bioimaging studies. After THF evaporation, the dots were modified with HIV-1 Tat peptide (RKKRRQRRRC) through conjugation between maleimide groups on the dot surface and thiol groups at the C-terminus of the Tat peptide. The obtained Tat-functionalized green and red dots, named GT-AIE and RTAIE dots, were filtered using a 0.2 μm syringe filter and concentrated through freeze-drying for further in vitro and in vivo studies. The average sizes of the GT-AIE and RT-AIE dots in water suspensions were 32.2 ± 2.3 and 33.1 ± 1.7 nm, respectively, determined by laser-light scattering. High-resolution transmission electron microscopy (HR-TEM) images reveal that both GT-AIE and RT-AIE dots are spherical in shape (Figure 1a,b). The UV−vis and photoluminescence (PL) spectra of the

Figure 1. HR-TEM images of (a) GT-AIE and (b) RT-AIE dots. (c) UV−vis absorption (solid) and PL (dashed) spectra of GT-AIE (green) and RT-AIE (red) dots in water.

AIE dots in water are shown in Figure 1c. Although the water suspensions of GT-AIE and RT-AIE dots show different absorption maxima at 423 and 510 nm, they both have intense absorption at the intersection of 455 nm. On the basis of the dot concentration, the molar extinction coefficients at 455 nm are 3.7 × 107 M−1 cm−1 for GT-AIE dots and 4.2 × 107 M−1 cm−1 for RT-AIE dots. It is also noteworthy that the two AIE dots have different emission peaks at 539 and 670 nm with minimized fluorescence spectral overlap. The quantum yields of GT-AIE and RT-AIE dots in water were measured to be 58 and 4184

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Figure 2. Histograms of the total number of photons collected during 100 s for many individual (a) GT-AIE dots and Qtracker 655 nanoparticles as well as (b) RT-AIE dots.

Figure 3. Confocal images of C6 cells after incubation with (a−c) GT-AIE and (d−f) RT-AIE dots for 12 h at a dot concentration of 2 nM. The images were recorded under excitation at 458 nm with (a, d) 480−560 nm and (b, e) 670−800 nm bandpass filters. (c, f) Corresponding fluorescence/transmission overlay images.

whereas the fluorescence signal remains extremely low in the 480−560 nm channel (Figure 3d). The 3D confocal fluorescence images of cells labeled with GT-AIE and RTAIE dots were also obtained (Figure S3 in the Supporting Information), revealing that the intense fluorescence signals are mainly from the cell cytoplasm. The emission profiles of AIE dots in C6 cells were also obtained from confocal microscopy (Figure S4 in the Supporting Information), suggesting that both GT-AIE and RT-AIE dots have similar emission maxima in cells as compared to that in water suspensions, respectively. These results confirm that the green and red AIE dots can be efficiently internalized into living cells and that the separately labeled cells are able to be simultaneously discriminated without fluorescence interference upon excitation under a single wavelength. An important issue in cell tracking is to continuously study the cell migration and interaction. Therefore, a cancer cell line with high motility, HT1080 fibrosarcoma cells, was further employed to evaluate the ability of Tat-functionalized AIE dots to simultaneously track cell-interacting behaviors during cell migration. In addition, it also provides the opportunity to study the potential effect of the AIE dots on cell activity and motility. Two groups of HT1080 cells were preincubated with 2 nM of either GT-AIE or RT-AIE dots for 12 h. Upon trypsinization, the two groups of cells were mixed and cocultured for another

12 h to allow cells to attach to the bottom of the culture chamber to facilitate confocal imaging. As shown in Figure 4, green and red fluorescence signals can be clearly distinguished in the cytoplasm of the two populations of labeled cells without signal interference. The motility of these cells was then continuously monitored under laser excitation through recording fluorescence images every 10 min. The obtained images were edited and are shown in the video file in the Supporting Information, suggesting that the two groups of labeled cells still remain highly motile and invasive. These results indicate that the motility, adhesion, and invasion of HT1080 cells are not obviously affected by the uptake of AIE dots, which is beneficial for long-term cell-behavior studies with minimal side effects.20 Moreover, no obvious leak of internalized AIE dots is detected upon cell interaction. These results strongly indicate that the AIE dots hold great promise for simultaneously monitoring different populations of cells without signal interference or probe-exchang during cell contact, which offers a promising methodology to understand cell interactions in cell behaviors like caner metastasis, embryo development, and immune reaction. It is of high importance that the fluorescent probes have good biocompatibility with low toxicity to target biosubstrates. The metabolic viability of C6 glioma cells and HT1080 fibrosarcoma cells after incubation with GT-AIE or RT-AIE 4185

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can be observed in both of the mice injected with green- and red-dot-labeled cells, and the livers also emit weak signals. The preferential accumulation of injected cells in the lung tissue is consistent with the results from previously reported celltransplantation studies, suggesting that intravenously delivered cells are likely to be trapped inside the pulmonary system because of the pulmonary microvascular barrier.21 The much higher fluorescence intensity from mice treated with RT-AIE dot-labeled cells as compared to that treated with GT-AIE dotlabeled cells should be attributed to the minimized spectral overlap between the RT-AIE dots and tissue autofluorescence, revealing the advantages of far-red/near-infrared emissive probes in in vivo imaging studies.22 To evaluate further the ability of AIE dots to be used in the simultaneous identification of different cell populations in organs, the lung tissue of one mouse injected with the mixture of GT-AIE dots-labeled cells and RT-AIE dots-labeled cells was collected at 3 h postinjection. The lung tissue was imaged by confocal laser scanning microscopy upon excitation at 458 nm with two bandpass filters to collect fluorescence signals from the corresponding cell populations. As shown in Figure 6, upon excitation, fluorescence signals could be separately recorded from the green and red cells labeled with the corresponding AIE dots without inference, which should be attributed to the efficient cell internalization, desired absorption coefficients, and high quantum yields of the Tat-functionalized AIE dots.

Figure 4. Simultaneous monitoring of HT1080 fibrosarcoma cells labeled with 2 nM of either GT-AIE or RT-AIE dots after coculture for 12 h. Images are recorded under excitation at 458 nm with (a) 480− 560 and (b) 670−800 nm bandpass filters. (c) Transmission image. (d) Fluorescence/transmission overlay image.

dots was determined through methylthiazolyldiphenyltetrazolium bromide (MTT) assays to evaluate the cytotoxicity of the dots. The results suggest that the cell viability remains above 90% after a 48 h incubation with either GT-AIE or RT-AIE dots at different dot concentrations (Figure 5), indicating that the Tat-functionalized AIE dots have low cytotoxicity during the test period. In Vivo Cell Discrimination. The performance of the Tatfunctionalized AIE dots in in vivo cell-tracking studies was then evaluated using an animal model. C6 glioma cells were incubated with 2 nM of either GT-AIE and RT-AIE dots at 37 °C for 12 h. The cells treated with GT-AIE dots and those treated with RT-AIE dots were then trypsinized and intravenously injected into the mice through the tail vain (2 × 106 cells in 0.2 mL of culture medium). At 3 h postinjection, the mice were sacrificed, the chest skin was removed, and the mice were imaged using the Maestro in vivo fluorescence imaging system. The autofluorescence was diminished using the spectral unmixing software (Nuance, CRI), and the order of red, orange, yellow, green, and blue corresponds to the successive decrease in fluorescence intensity. As shown in Figure S5 in the Supporting Information, obvious fluorescence from the lungs

4. CONCLUSIONS In this work, we report a general strategy to synthesize quantum dot-sized organic fluorescent probes with aggregationinduced emission, and we demonstrate their application in the simultaneous tracking of two populations of cancer cells. Two AIE luminogens were employed to synthesize AIE dots (GTAIE and RT-AIE dots) with distinct emission wavelengths, large absorption coefficients, and high brightness. Upon separate incubation with GT-AIE and RT-AIE dots, the migration of two groups of a metastatic cell line with high motility (HT1080 cell) was continuously monitored with single wavelength excitation. Ex vivo fluorescence imaging studies indicate that the GT-AIE dot-labeled C6 cells and RT-AIE dotlabeled ones can be differentiated in the lung tissue of a mouse injected with a mixture of these two populations of labeled cells. These results suggest that organic AIE dots with tunable fluorescence wavelengths are promising fluorescent probes to study cell behaviors and the interactions among different tumor cells in cancer metastasis. Moreover, the AIE dots greatly outperform their commercial QD probes in single-particle

Figure 5. Viability of (a) C6 cells and (b) HT1080 cells after incubation with GT-AIE and RT-AIE dots for 48 h at different dot concentrations. 4186

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Figure 6. Confocal images of the lung tissue section collected from the mouse injected with the mixture of C6 cells labeled by either GT-AIE dots or RT-AIE dots. The images were recorded under excitation at 458 nm with (a) 480−560 and (b) 670−800 nm bandpass filters. (c) Overlay image. (9) Liu, Y. S.; Sun, Y. H.; Vernier, P. T.; Liang, C. H.; Chong, S. Y. C.; Gundersen, M. A. J. Phys. Chem. C 2007, 111, 2872. (10) Birks, J. B. Photophysics of Aromatic Molecules; WileyInterscience: London, 1970. (11) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Chem. Soc. Rev. 2011, 40, 5361. (12) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Chem. Commun. 2009, 4332. (13) Kim, S.; Pudavar, H. E.; Bonoiu, A.; Prasad, P. N. Adv. Mater. 2007, 19, 3791. (14) Zhao, Z.; Deng, C.; Chen, S.; Lam, J. W. Y.; Qin, W.; Lu, P.; Wang, Z.; Kwok, H. S.; Ma, Y.; Qiu, H.; Tang, B. Z. Chem. Commun. 2011, 47, 8847. (15) Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J.; Liu, R.; Liu, J.; Zhang, X.; Liu, H.; Liu, B.; Tang, B. Z. Sci. Rep. 2013, 3, 1150. (16) Li, K.; Jiang, Y. H.; Ding, D.; Zhang, X.; Liu, Y.; Hua, J.; Feng, S. S.; Liu, B. Chem. Commun. 2011, 47, 7323. (17) Li, K.; Ding, D.; Huo, D.; Pu, K.-Y.; Thao, N. N. P.; Hu, Y.; Li, Z.; Liu, B. Adv. Funct. Mater. 2012, 22, 3107. (18) Drake, J. M.; Lesiecki, M. L.; Camaioni, D. M. Chem. Phys. Lett. 1985, 113, 530. (19) Galland, C.; Ghosh, Y.; Steinbrü c k, A.; Sykora, M.; Hollingsworth, J. A.; Klimov, V. I.; Htoon, H. Nature 2011, 479, 203. (20) Wu, Y. L.; Putcha, N.; Ng, K. W.; Leong, D. T.; Lim, C. T.; Loo, S. C. J.; Chen, X. Acc. Chem. Res. 2013, 46, 782. (21) Schrepfer, S.; Deuse, T.; Reichenspurner, H.; Fischbein, M. P.; Robbins, R. C.; Pelletier, M. P. Transplant. Proc. 2007, 39, 573. (22) Li, L.; Daou, T. J.; Texier, I.; Chi, T. T. K.; Liem, N. Q.; Reiss, P. Chem. Mater. 2009, 21, 2422.

imaging analyses, revealing the great potential of such organic fluorescent dots to overcome the obstacles encountered with QDs for use in versatile imaging applications.

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ASSOCIATED CONTENT

* Supporting Information S

Fluorescence images of GT-AIE dots, RT-AIE dots, and Qtracker 655; photobleaching resistance of GT-AIE dots, RTAIE dots, and Qtracker 655; color-coded projections of z stacks of C6 glioma cells after incubation with GT-AIE and RT-AIE dots; fluorescence spectral profiles of GT-AIE and RT-AIE dots in C6 cells; and representative in vivo fluorescence images of mice intravenously injected with C6 cells labeled by GT-AIE or RT-AIE dots (PDF, AVI). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the support from the A*STAR Joint Council Office and Institute of Materials Research and Engineering of Singapore (IMRE/12-8P1103 and IMRE/138P1104), the Singapore National Research Foundation (R-279000-390-281), the Research Grants Council of Hong Kong (603509, HKUST2/CRF/10, 604711 and N_HKUST620/11), the National Natural Science Foundation of China (20974028 and 51173077), and the Guangdong Innovative Research Team Program (201101C0105067115). We thank Tong Yan from the confocal microscopy lab at the Centre for BioImaging Sciences (CBIS) in NUS for technical support with the confocal imaging experiments.

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REFERENCES

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dx.doi.org/10.1021/cm401709d | Chem. Mater. 2013, 25, 4181−4187