Ultrafast and Non-invasive Long-Term Bioimaging with Highly Stable

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Ultrafast and Non-invasive Long-Term Bioimaging with Highly Stable Red Aggregation-Induced Emission Nanoparticles Wei-Long Che, Liping Zhang, Yuanyuan Li, Dongxia Zhu, Zhigang Xie, GuangFu Li, Pengfei Zhang, Zhong-Min Su, Chuandong Dou, and Ben Zhong Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05024 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Analytical Chemistry

Ultrafast and Non-invasive Long-Term Bioimaging with Highly Stable Red Aggregation-Induced Emission Nanoparticles Weilong Che,† Liping Zhang,† Yuanyuan Li,‡ Dongxia Zhu,*,† Zhigang Xie,*,‡ Guangfu Li,† Pengfei Zhang,§ Zhongmin Su,*,†,|| Chuandong Dou,‡ Ben Zhong Tang*,§ †Key

Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, P. R. China ‡State key laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry Chinese Academy of Sciences, Changchun, 130022, P. R. China ||School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun, 130022, P. R. China §Department of Chemistry Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected] ABSTRACT: Strongly red luminescent and water soluble probes are very important for studying biological events and processes. Fluorescent nanoparticles (NPs) built from the aggregation-induced emission luminogen (AIEgen) and amphipathic polymeric matrices have been considered as promising candidates for bioimaging. However, AIE NPs with long-wavelength absorption suitable for in vivo application are still scarce. In this work, three AIE-active red-emissive BODIPY derivatives with longwavelength absorption were rationally designed and synthesized. Then three NPs based on these AIEgens exhibit bright red photoluminescence with high ﬂuorescence quantum yield in aqueous media. These NPs uniformly dispersed in water and showed excellent stability and good biocompatibility. They can be readily internalized by HeLa cells, and the staining process is performed by simply shaking the culture with cells for just a few seconds at room temperature, which indicates an ultrafast and easy-to-operate staining protocol. More importantly, long-term tracing in living cells and mouse over 15 days is successfully achieved. The strong fluorescence signals, ultrafast staining procedure and long-term tracing ablities indicate that these AIE NPs hold great potential for monitoring biological processes. and high photostability. So far, many BODIPY derivatives Non-invasive bioimaging has been demonstrated as an have been explored and applied to solid-state lasers,26,27 solar indispensable and powerful tool for clinical diagnostics, cells,28,29 light harvesting,30,31 electroluminescent devices,32 sketching the fine biological structures and monitoring fluorescent bioprobes,33,34 chemosensors,35,36 etc. However, complex processes in the intracellular environments.1-3 they often suffer from obvious ACQ effect due to the Continuous and long-term cellular tracing can offer dynamic relatively strong intermolecular stacking, which significantly information on a variety of intricate biological processes.4-6 limits their applications.32,37 In principle, the BODIPY core For this purpose, many bioimaging techniques have been with bulky groups can effectively avoide ACQ through explored, such as magnetic resonance imaging,7 single photon increased stacking distance between molecules.38 emission computing tomography,8 positron emission 9 10,11 Nevertheless, few of them emit red or NIR fluorescence.39 The tomography, and fluorescence technique. Among these red emissive BODIPY derivatives can be achieved through techniques, the fluorescence imaging possesses numerous introducing electrondonate substituents to BODIPY cores by advantages such as good biocompatibility, high contrast, high intramolecular charge transfer (ICT) process.40-43 In our sensitivity and real-time monitoring with higher 12,13 previous work, a red-emissive compound with both AIE and resolutions. In the past decades, various fluorescent twisted intramolecular charge transfer (TICT) properties has materials have been developed for bioimaging applications, been successfully developed as highly effective fluorescence such as carbon dots,14,15 quantum dots,16,17 fluorescent probe for sensing F- in biological system.44 proteins,18,19 and organic dyes.20,21 However, owing to the π-π stacking and other non-radiative pathways, the emission of Furthermore, most of the π-conjugated organic dyes are conventional fluorophores are usually quenched at high inherently hydrophobic, which limit their application in concentration or in the aggregation state. This phenomenon is physiological condition. The nanoparticle (NP) formulations, notoriously known as aggregation-caused quenching (ACQ) which could encapsulate fluorophore into amphiphilic effect.22,23 Fortunately, luminogens with aggregation-induced polymers, have been developed as a feasible strategy of emission (AIEgens) have been developed by Tang's group as bioprobes for bioimaging applications.45-50 Compared with promising solutions to solve the ACQ problem.24 The red or conventional fluorescent NPs, AIE NPs show great advantages NIR emissive fluorescent materials with AIE characteristics is including good water dispersibility, high brightness, excellent highly desirable but still scarce.25 photostability and low cytotoxicity.51 However, AIE NPs Boron dipyrromethene (BODIPY) and its derivatives have developed so far usually with short-wavelength absorption, attracted much attention owing to their excellent which is rarely over 550 nm and limits AIE NPs used in vivo.52 photophysical properties, such as intense photoluminescence To obtain AIE NPs with long-wavelength absorption is the (PL), high quantum yields, large molar extinction coefficients key point for their wide application in biological field.

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In this contribution, an easy-to-get method is realized to construct novel AIE NPs with long-wavelength absorption and red emission for non-invasive bioimaging application. Triphenylamine units is widely used in the design of luminescent molecules because it has the following advantages: (i) The three-bladed propeller-like structure is beneficial to avoid ACQ phenomenon caused by π-π stacking; (ii) Strong electron donating ability enables the absorption and emission wavelength could be adjusted through a charge transfer process; (iii) Small volume is suitable for the preparation of small size NPs. Thus, a series of BODIPY derivatives with long-wavelength absorption, red emission and AIE properties was designed and facilely synthesised by introducing triphenylamine units into methylated BODIPY core. And three AIE NPs with good water-dispersibility, excellent stability and prominent biocompatibility were fabricated by encapsulating them into polymeric micelles. The generated NPs can be endocytosed by cells, and image the cells in only a few seconds. More importantly, these fabricated NPs exhibited very bright red fluorescence in the cells and can remain for more than 15 days. Once applied to in vivo imaging of U14 tumor-bearing mouse, the strong red fluorescence in the tumor can also be maintained for more than 14 days.

EXPERIMENTAL SECTION Materials. (4-(carbazolyl)phenyl)boronic acid and (4(diphenylamino)phenyl)boronic acid were purchased from Aladdin (China). 2,4-Dimethyl-1H-pyrrole, Pd(PPh3)4, pchloranil, trifluoroacetic acid, boron trifluoride etherate, iodic acid, amiloride, genistein, sucrose and sodium azide were purchased from Energy Chemical Co., Ltd. (China). Iodine was purchased from Tocean Iodine Products Co., Ltd.. (China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxyl-(polyethylene glycol)-2000] (DSPE-PEG2000) was purchased from Shanghai Ponsure Biotech Inc Co., Ltd.. (China). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (California). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was obtained from Shanghai Beyotime Biotechnology Co., Ltd.. (China) Cell viability (live dead cell staining) assay kit was purchased from Jiangsu KeyGEN Biotechnology Co., Ltd.. (China) All of the other chemicals and reagents were acquired from commercial sources without further purification, unless otherwise noted. All the solvents were purified according to the standard methods whenever needed. Milli-Q water was collected from a Milli-Q system (Millipore, USA). Characterization. 1H NMR and 13C NMR spectra were measured at 25 ºC on a Varian 500 MHz spectrometer in CDCl3 using tetramethylsilane (TMS; δ = 0) as internal reference. The UV-vis spectra were measured on a Shimadzu UV-3100 spectrophotometer. The fluorescence spectra were obtained on a Shimadzu RF-5301PC spectrophotometer and Maya 2000Pro optical fiber spectrophotometer. The fluorescence quantum efficiencies of the organic compounds and NPs were measured with a calibrated integrating sphere (C-701, Labsphere Inc.), with a 365 nm Ocean Optics LLSLED as the excitation source, and the laser was introduced into the sphere through the optical fiber. The molecular weights of the complexes were obtained by using matrixassisted laser desorption-ionization time-of-flight (MALDITOF) mass spectrometry. Elemental analysis was performed on a Flash EA1112 analyser. Transmission electron

microscopy (TEM) images of the sample were taken by a TECNAI F20 microscope. The samples were prepared by placing microdrops of the solution on a holey carbon copper grid. The size distribution was performed using a Zeta-sizer Nano-ZS (Malvern Instruments Ltd.). The cell confocal images were obtained using Zeiss confocal laser microscope (ZEISS LSM 700). Synthesis of compounds TPA-BDP and Cz-BDP. The synthesis of TPA-BDP have been provided in our previous work.44 The synthetic procedure to Cz-BDP was analogous to that of TPA-BDP: In a round-bottom flask, a mixture of 1 (576 mg, 1.0 mmol), (4-(carbazolyl)phenyl)boronic acid (689 mg, 2.4 mmol) and Pd(PPh3)4 (60 mg, 0.05 mmol) was dissolved in CH3OH (10 mL) and THF (40 mL). Aqueous potassium carbonate solution (2 M, 10 mL) was added an under nitrogen atmosphere. The solution was stirred at 80 °C for 24 h, and thin layer chromatography (TLC) was used to monitor the reaction process. After cooling to room temperature, the reaction mixture was extracted by dichloromethane and the organic layer was dried over anhydrous Na2SO4. The extract was concentrated by rotary and evaporation and the crude product was purified by column chromatography on silica gel (eluent: ethyl acetate /petroleum ether (1:2, v/v) to obtain Cz-BDP (630 mg, 78% yield) as an orange-red solid. 1H NMR (500 MHz, CDCl3), δ (ppm): 8.15 (d, J = 6.5 Hz, 4H), 7.61 (d, J = 6.5 Hz, 4H), 7.53‒7.57 (m, 3H), 7.48 (d, J = 7.0 Hz, 4H), 7.42 (t, J = 7.0 Hz, 10H), 7.29 (t, J = 6.0 Hz, 4H), 2.69 (s, 6H), 1.45 (s, 6H). 13C NMR (125 MHz, CDCl3), δ (ppm): 12.95, 13.58, 109.80, 120.04, 120.34, 123.46, 125.96, 126.83, 128.04, 129.38, 131.58, 136.65, 140.77. MS: (MALDI-TOF) [m/z]: 806.3392 (M+). Anal. Calcd. for C55H41BF2N4: C 81.88, H 5.12, N 6.94. Found C 81.86, H 5.11, N 6.97. Synthesis of compound TPACz-BDP. TPACz-BDP was synthesized by using the above procedure except that intermediate 1 replaced by intermediate 2. (749 mg, 80% yield) as an orange-red solid. 1H NMR (500 MHz, CDCl3), δ (ppm): 8.16 (t, J = 6.5 Hz, 6H), 7.82 (d, J = 7.0 Hz, 2H), 7.63‒7.67 (m, 8H), 7.50 (d, J = 7.0 Hz, 3H), 7.42‒7.47 (m, 11H), 7.31 (t, J = 6.5 Hz, 8H), 2.71 (s, 6H), 1.74 (s, 6H). 13C NMR (125 MHz, CDCl3), δ (ppm): 13.34, 13.63, 109.80, 120.02, 120.09, 120.38, 123.46, 123.50, 124.52, 124.82, 125.98, 126.89, 127.41, 128.04, 128.20, 131.65, 131.80, 132.86, 136.73, 139.42, 140.80, 140.85, 146.76, 148.45, 154.33, 154.92. MS: (MALDI-TOF) [m/z]: 973.4127 (M+). Anal. Calcd. for C67H50BF2N5: C 82.62, H 5.17, N 7.19. Found C 82.65, H 5.13, N 7.20. Synthesis of compound 3TPA-BDP. 3TPA-BDP was synthesized by using the above procedure except that intermediate 1 and (4-(carbazolyl)phenyl)boronic acid replaced by intermediate 2 and (4(diphenylamino)phenyl)boronic acid, respectively. (791 mg, 81% yield) as a red solid. 1H NMR (500 MHz, CDCl3), δ (ppm): 7.49 (d, J = 7.0 Hz, 2H), 7.45 (d, J = 7.0 Hz, 2H), 7.26 (t, J = 6.5 Hz, 10H), 7.23 (d, J = 7.0 Hz, 2H), 7.16 (d, J = 7.0 Hz, 4H), 7.13 (d, J = 7.0 Hz, 12H), 7.09 (d, J = 7.5 Hz, 4H), 7.02 (d, J = 6.5 Hz, 6H), 2.58 (s, 6H), 1.59 (s, 6H). 13C NMR (125 MHz, CDCl3), δ (ppm): 13.22, 13.52, 122.93, 123.00, 123.10, 123.97, 124.40, 124.53, 124.68, 127.33, 127.46, 127.54, 129.18, 129.20, 129.28, 129.29, 130.91, 131.49, 131.60, 133.38, 134.37, 134.77, 135.62, 138.83, 146.06, 146.72, 146.97, 147.68. MS: (MALDI-TOF) [m/z]: 977.4440


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Analytical Chemistry (M+). Anal. Calcd. for C67H54BF2N5: C 82.28, H 5.57, N 7.16. Found C 82.27, H 5.56, N 7.19. Nanoparticle Preparation. AIE NPs were prepared by using a well-documented nanoprecipitation method. In a typical procedure, AIE compounds (1 mg) and DSPE-PEG2000 (2 mg) were dissolved in 1 mL of THF solution. The organic solution was poured into 10 mL of Milli-Q water. The THF/water mixture was then sonicated for 5 min using a ultrasound sonicator at 12 W output to ensure maximum agitation. The solution was stirred overnight to ensure evaporation of the THF. The NPs was obtained by filtration through a 220 nm syringe filter for further use. Cell culture. HeLa cells were propagated to confluence in DMEM medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 10% FBS, and maintained at 37 °C in a humidified atmosphere of 5% CO2 for further cell experiments. Cytotoxicity of AIE NPs. Cells harvested in a logarithmic growth phase were seeded in 96-well plates at a density of 8×103 cells per well and incubated in DMEM for 24 h. The old medium was then replaced by 200 µL of DMEM containing predetermined concentrations of AIE NPs, and then incubated for 24 h, followed by MTT assays to evaluate the cell viabilities, which were determined by reading the absorbance of the plates at 490 nm with a microplate reader. The cells incubated with only DMEM were used as the control. The cell viability (%) =A sample /A control ×100%. Cellular uptake. The cellular uptake measurement was investigated by CLSM. Cells harvested in a logarithmic growth phase were seeded in 6-well plates at a density of 2.5 × 105 cells/well and incubated in DMEM for 24 h. The medium was then replaced by 2 mL of DMEM containing 5 µg mL-1 of AIE NPs and incubated for different time at 37 °C, and further washed 3 times with PBS buffer. For the CLSM, the cells were fixed with 4% of paraformaldehyde solution for 10 min. After that, DAPI was added for another 5 min incubation to locate the nucleus. Later, the cells were washed with PBS and observed using confocal laser scanning microscopy (CLSM, Zeiss LSM 700). Endocytosis pathway detection. Cells harvested in a logarithmic growth phase were seeded in 6-well plates at a density of 2.5 × 105 cells/well and incubated in DMEM for 24 h. Then, serum-free DMEM as the control and various inhibitors including sucrose (clathrin-mediated endocytosis, 153.9 mg mL−1), genistein (caveolin-dependent endocytosis, 27.024 μg mL−1), amiloride (micropinocytosis, 13.3 μg mL−1) and sodium azide (energy-dependent, 1 mg mL−1) were used in serum-free DMEM for 1 h. Then, 5 µg mL-1 of AIE NPs were further added for 5 s incubation, respectively. Long-term cellular tracing. Cells harvested in a logarithmic growth phase were seeded in 6-well plates at a density of 2.5×105 cells/well and incubated in DMEM for 24 h. The medium was then replaced by 2 mL of DMEM containing 20 μg mL-1 of AIE NPs and incubated for 6 hours at 37 °C (Day 0). Then the cells were diluted and subcultured in 6-well plates for 0 to 15 day regeneration, respectively. Upon reaching designated day, the cells were washed with PBS buffer and then fixed with 4% of paraformaldehyde solution for 10 min. Later, the cells were washed with PBS and observed using confocal laser scanning microscopy (CLSM, Zeiss LSM 700).

Animal experiments. All animal experiments were performed complying with the NIH guidelines for the care and use of laboratory animals. U14 cells were administered by subcutaneous injection into the male BALB/c mice. We choose the mice to bear the tumor to carry out this study. In order to detect the imaging capacity, 3TPA-BDP NPs (100 µg/mL) was administrated into the mice via intratumor injection. Then, under anesthesia, the in vivo imaging was performed using an in vivo imaging system (excitation: 575605 nm, emission: 645-750 nm). Maestro software was used to remove the mouse background fluorescence.

RESULTS AND DISCUSSION Synthesis and Characterization. The molecular structures and synthetic routes of TPA-BDP, Cz-BDP, TPACz-BDP and 3TPA-BDP are illustrated in Scheme 1a and S1. The 1,3,5,7-tetramethyl-8-phenyl-BODIPY and 1,3,5,7tetramethyl-8-triphenylamino-BODIPY were synthesized from benzaldehyde and 4-diphenylaminobenzaldehyde, respectively,53,54 which were treated with iodine and iodic acid to produce the intermediates 1 and 2.55 The target compounds are comprised of triphenylamine (TPA) or carbazole (Cz) as the electron donor (D) and BODIPY (BDP) core as the electron acceptor (A), and they were prepared through the Suzuki reaction between the intermediates 1 or 2 and 4(diphenylamino)phenylboronic acid or 4(carbazolyl)phenylboronic acid using Pd(PPh3)4 as the catalyst. The chemical structures were validated by NMR, mass spectrometry and elemental analysis.

Scheme 1. (a) Molecular structures of TPA-BDP, Cz-BDP, TPACz-BDP and 3TPA-BDP; (b) Schematic illustration of AIE NPs preparation. Photophysical Properties. To investigate the AIE behaviours of these compounds, the photophysical properties of TPA-BDP, Cz-BDP, TPACz-BDP and 3TPA-BDP in the aggregate state were first studied by monitoring their emission spectra upon addition of different amounts of water in tetrahydrofuran (THF) solution. As shown in Figure 1a and Table S1, TPA-BDP exhibit weak red emission maxima at 645 nm with a low fluorescence quantum yield (ΦF) of about 7% in THF solution (1×10−5 M). With the increase of water content, the fluorescence intensity of TPA-BDP gradually



increased. And the ΦF increased to 23% in the aqueous mixture with fw of 90%. These results indicate that TPA-BDP is an AIE-active molecule. As well known, the 1,3,5,7tetramethyl-8-phenyl-BODIPY is a luminogenic molecule that emit bright emission with high fluorescence quantum yields in dilute solution.56 We wonder whether the introduction of triphenylamine with a propeller-like structure into the TPABDP molecule was the major factor to its AIE properties. Furthermore, we used the planar carbazole units instead of diphenylamine to synthesize the compound Cz-BDP. The CzBDP exhibits strong fluorescence at 569 nm in a dilute THF solution and has a high ΦF value of 70% (Figure 1b and Table S1). The emission of Cz-BDP bathochromically shifted to 582 nm and its emission intensity is significantly decreased after addition of water into THF. Meanwhile, the ΦF decreased to 12% in the solution with fw of 90%. The ACQ phenomenon of Cz-BDP indicate that the introduction of triphenylamine is the key to AIE property of TPA-BDP. Previously, the introduction of bulky groups such as mesityl, tetrephenylethene, tert-butylphenyl and trimethylsilylphenyl to the meso- position of the BODIPY core could effectively inhibited ACQ.57,58 Therefore, we introduced triphenylamine at the meso-position of Cz-BDP to form a new compound TPACz-BDP. TPACz-BDP emits intense fluorescence at 570 nm with a relatively high quantum efficiency of 15% in THF solution (Figure 1c and Table S1). This may be due to the presence of planar carbazole as an electron donor in the molecule, which reduces its intramolecular motion and energy loss in the THF solution. Upon gradual addition of water into THF (fw ≤ 30%), the emission of TPACz-BDP is weakened. With a further increase in fw, the emission of TPACz-BDP is intensified and bathochromically shifted from 570 nm to 586 nm, and the ΦF increased to 18%. These data confirm that TPACz-BDP possess the AIE features, and the introduction of triphenylamine is beneficial to the properties of AIE. Finally, we synthisized 3TPA-BDP, a new BODIPY derivative with three triphenylamine units. As depicted in Figure 1d, 3TPABDP has a faint red emission at 649 nm, and the emission is intensified with the water content increased. The ΦF of 3TPABDP is 8% in THF solution and 18% in the aqueous mixture with fw of 90%. The results indicated 3TPA-BDP is also AIEactive. In addition to Cz-BDP, the ΦF of TPA-BDP, TPACzBDP and 3TPA-BDP in the solid state are higher than those in THF solution (Table S2). These data can also indicate that they are AIE-active molecules The good AIE performance of these compounds benefits from the introduction of triphenylamine on the BODIPY core. The TPA-BDP and 3TPA-BDP exhibit strong absorption at 546 and 547 nm, respectively, in THF solution (Figure S13). But the absorption of TPACz-BDP is blue-shifted to 534 nm, which is mainly because of the weaker D-A interaction.40 The long-wavelength absorption of these BODIPY-derived AIEgens can avoid photodamage caused by UV excitation, making these compounds very suitable for fabrication of AIE NPs for bioimaging applications.

Figure 1. PL spectra of TPA-BDP (a), Cz-BDP (b), TPACzBDP (c) and 3TPA-BDP (d) in THF/water mixtures with different water fractions (fw). Preparation and Characterization of NPs. Three AIE compounds TPA-BDP, TPACz-BDP and 3TPA-BDP were encapsulated in lipid-PEG derivative of 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxyl-(polyethylene glycol)-2000] (DSPE-PEG2000) to afford the AIE NPs (Scheme 1b), respectively.59,60 The DSPE-PEG2000 was chosen as the encapsulation matrix due to its good encapsulation performance and biocompatibility.61-63 The encapsulation efficiencies were calculated to be ~96%, ~93% and ~97% for TPA-BDP NPs, TPACz-BDP NPs and 3TPA-BDP NPs, respectively (Figure S14).64 The size and morphology of these NPs were examined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). As shown in Figure 2a; Figures S15a and S16a, the average hydrodynamic diameter of 3TPA-BDP NPs, TPA-BDP NPs and TPACzBDP NPs are 89.97 ± 0.53 nm, 68.45 ± 0.91 nm and 77.36 ± 0.44 nm, respectively. The TEM images revealed that these NPs have similar quasi-spherical morphologies and uniform size distribution (Figure 2b; Figures S15b and S16b). The average particle size of 3TPA-BDP NPs, TPA-BDP NPs and TPACz-BDP NPs were determined to be 60, 54 and 58 nm, respectively. The slightly smaller size of these AIE NPs measured by TEM may be attributed to the formation of a hydrated layer on the NPs in aqueous system.65 The spherical NPs less than 100 nm in size are more easily endocytosed by cells.65

Figure 2. (a) DLS intensity-weighted diameter of 3TPA-BDP NPs; (b) TEM image of 3TPA-BDP NPs; (c) UV-vis


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Analytical Chemistry absorption spectra of 3TPA-BDP in THF, THF/water (v/v = 1/9), and 3TPA-BDP NPs in water; (d) The PL spectra of 3TPA-BDP in THF, THF/water (v/v = 1/9), and 3TPA-BDP NPs in water. The photophysical properties of these NPs were subsequently studied. The absorption and fluorescence spectra of these NPs in water were found to be similar to the spectra of the original AIE materials, indicating that they are weakly affected by the matrix. (Figure 2c,d; Figures S15c,d and S16c,d). The fluorescence intensity of these NPs was significantly enhanced compared with that of the original AIE material in THF solution. And the ΦF of TPA-BDP NPs, TPACz-BDP NPs and 3TPA-BDP NPs increased to 21%, 26% and 26%, respectively, in water solution (Table S1). The emission tails of these AIE NPs all extended to 850 nm, and they have a relatively large Stokes shift which was advantageous for bioimaging. Stability and Cytotoxicity of AIE NPs. Excellent stability of NPs is crucial and imperative for maintaining their shape and function in biological system. We first evaluated the stability of these NPs by monitoring their size, fluorescence and absorption spectra changes with different shelf time. As shown in Figure 3a,b,c, no significant size variation of 3TPABDP NPs, TPA-BDP NPs and TPACz-BDP NPs is observed in 14 days at room temperature, indicating the excellent colloidal stability. In addition, we measured the absorption and fluorescence spectra of these NPs in water during 7 days, respectively (Figure 3d,e; Figures S17-S19). As depicted in Figure 3d, the absorbance of 3TPA-BDP NPs and TPA-BDP NPs both retains more than 95% of its initial value within one week, while that of TPACz-BDP NPs decreased a little but remained above 90%. The result of fluorescence intensity change is similar to the absorbance (Figure 3e). These results indicate that these NPs all have excellent colloidal and optical stability, which is beneficial for further commercial application. The photostability of these NPs was also investigated by monitoring the fluorescence intensity upon continuous laser irradiation. As shown in Figure S20a, these NPs are exposed to excitation light for 30 min, the fluorescence intensity remained above 98% of their original value, indicative of the high photostability of these NPs.

Figure 3. Stability of size distribution of (a) 3TPA-BDP NPs, (b) TPA-BDP NPs and (c) TPACz-BDP NPs during 14 days; (d) The variation of absorbance intensity and e) fluorescence intensity of 3TPA-BDP NPs, TPA-BDP NPs and TPACzBDP NPs during seven days, respectively. In addition to excellent stability, biocompatibility is also very important for the application of fluorescent NPs in bioimaging. Therefore, we investigated the potential cytotoxicity of these NPs toward HeLa cells by MTT (3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Figure S20b shows the cell viability after incubation with varied concentrations of these NPs ranging from 0 to 20 µg mL−1, respectively. After incubation for 24 hours, HeLa cells still exhibited more than 95% cell viability even at very high NPs concentration of 20 µg mL−1. The cytotoxicity can also be determined by flow cytometry analysis (Figure S21). HeLa cells were double-labeled with annexin V−FITC (fluoresceine isothiocyanate) and PI (propidium iodide). It is can be seen that all of these NPs have high cell viability (Q4) above 95%. The results based on above discussion suggest that these NPs have negligible cytotoxicity, which is very important for longterm bioimaging. In Vitro Cell Imaging. The as-prepared AIE NPs have spherical morphology with uniform particle size, good biocompatibility and water-dispersibility, which is expected to be preferable for bioimaging. HeLa cells were incubated with 5 μg mL-1 AIE NPs for different times, and 4, 6-diamidino-2phenylindole (DAPI) was then used to stain the cell nuclei. As presented in Figure 4a, an intense homogeneous red fluorescence of 3TPA-BDP NPs can be clearly observed at the cytoplasm and the perinuclear region, indicating the 3TPA-BDP NPs can be internalized effectively by cancer cells. To a certain extent, some red emission is also observed from the nuclei region, indicating that some NPs might locate in the cell nuclei. This may be because some of them traverse the nuclear pore complex (NPC) to the nucleus by passive diffusion.66 Surprisingly, the cytoplasm was strongly lit up via the facile staining process of simply shaking the cell culture with 3TPA-BDP NPs for a few seconds at room temperature, revealing its ultrafast staining (at the second-level) characteristic. The fluorescence intensity increased obviously from 5 s to 5 min, indicating that the 3TPA-BDP NPs had a sustained cellular uptake and accumulation in HeLa cells.67 The TPA-BDP NPs and TPACz-BDP NPs showed similar results as evidenced in Figures S22a and S23a. These results confirmed that the as-prepared AIE NPs could be applied for ultrafast cellular imaging.

Figure 4. (a) CLSM images of HeLa cells incubated with 3TPA-BDP NPs for 5 min, 1 min and an extremely short incubation period (around 5 s). Cells are viewed in the blue channel for DAPI, the red channel for 3TPA-BDP NPs. Scale



bars represent 20 μm in all images; (b) CLSM images showing HeLa cells incubated with different endocytic inhibitors: none (Control); amiloride (+Amiloride, 13.3 µg mL−1); genistein (+Genistein, 27.024 µg mL−1); sucrose (+Sucrose, 153.9 mg mL−1) and sodium azide (+Sodium azide, 1 mg mL−1). The 3TPA-BDP NPs was 5 µg mL−1. The scale bars are 20 µm. To determine the internalization pathways of the 3TPABDP NPs, TPA-BDP NPs and TPACz-BDP NPs, three types of inhibitors were chosen to block macrocytosis, caveolaemediated and clathrin-mediated endocytosis, namely amiloride, genistein and sucrose. Another inhibitor of sodium azide was used to reveal that the endocytosis is an energydependent process. The experiment conditions were optimized in order to decrease the influence of inhibitors to HeLa cells.68 The CLSM images of HeLa cells pretreated with inhibitors are shown in Figure 4b; Figures S22b and S23b. The relatively weak fluorescence in HeLa cells was observed after pretreatment with genistein (Figure 4b), while the intracellular fluorescence after treatment with other inhibitors did not significantly weaken. This result indicates that the endocytosis of 3TPA-BDP NPs is primarily a caveolae-mediated endocytosis process. The similar phenomenon was also found for TPA-BDP NPs and TPACz-BDP NPs (Figures S22b and S23b). The internalization pathway of the three NPs was also determined in cancer cells (A549 cells). A similar phenomenon was observed in A549 cells (Figure S24). Therefore, we concluded that the caveolae-mediated endocytosis is the primary uptake pathway for 3TPA-BDP NPs, TPA-BDP NPs and TPACz-BDP NPs. Long-term imaging in vitro and in vivo. The strong fluorescence emission, favourable stability and excellent cytocompatibility of these NPs suggested that they could be used as fluorescent probes for non-invasive long-term cellular tracing. When the HeLa cells were incubated with the 3TPABDP NPs (20 μg mL-1) at 37 °C for 6 h (labeled as day 0, the first generation), the cell internal showed strong emission. Then the treated cells were digested and divided into two dishes for specified time intervals. One of the dishes was washed with PBS twice to remove the previous 3TPA-BDP NPs existing in the culture medium for further incubated, while the other one was used for CLSM imaging observation. The processes were repeated to proceed to the 6th generation. As shown in Figure 5a, bright red fluorescence signal from the 3TPA-BDP NPs can be clear observed at the initial stage (day 0). With the increase of incubation time, the cells stained by 3TPA-BDP NPs were still visible even after six generations (Figure 5). This result indicates that the 3TPA-BDP NPs can act as a fluorescent bioprobe for long-term cellular tracing. More importantly, this long-term tracing strategy is based on the proliferation of cells containing endogenous organic nanoprobes rather than the continuous addition of imaging agents during long-term monitoring.69 Similarly, the TPABDP NPs and TPACz-BDP NPs also showed obvious fluorescence after 15 days incubation (Figures S25 and S26). In order to investigated the imaging ability in vivo of 3TPABDP NPs, a U14 tumor-bearing mouse was administered by subcutaneously injection with 3TPA-BDP NPs at the tumor and then imaged by an in vivo optical imaging system. Figure 6 indicates the fluorescence emission form the tumor from 0 to 14 days. At the initial injection time, strong fluorescence was observed at the tumor. The intensity of fluorescence could maintain even after 14 days, and keep at 30% of the initial intensity, validating the excellent imaging ability of 3TPA-

BDP NPs (Figure S27). To further study the biodistribution of 3TPA-BDP NPs, the ex vivo fluorescence imaging was performed on the major organs and tumor tissue of the tumorbearing mouse sacrificed immediately post-injection with 3TPA-BDP NPs. As shown in Figure S28, a strong fluorescence could be clearly observed in tumor, which is extremely distinct from those of other organs. It reveals that 3TPA-BDP NPs could be accumulate and retained around the tumor. Based on these results, it is expected that these AIE NPs with excellent stabilities and non-invasive long-term tracing abilities are of great potential in the real-time and practical biological applications.

Figure 5. Long-term cell tracing images of the 3TPA-BDP NPs at 37 oC for 6 h and then subcultured for designated time intervals including (a) day 0; (b) day 3; (c) day 6; (d) day 9; (e) day 12; and (f) day 15. Scale bars represent 20 μm in all images.

Figure 6. In vivo fluorescence images of the tumor-bearing mouse subcutaneously injected with 3TPA-BDP NPs from day 0 to day 14.

CONCLUSIONS In summary, we successfully designed and synthesized a kind of new red luminogens with the AIE characteristic by attaching electron donor (triphenylamine units) to BODIPY core. The introduction of triphenylamine units could effectively reduce the emission quenching of the D-A fluorophores in the aggregated state. In addition, the corresponding functionalized AIE NPs were fabricated in the presence of DSPE-PEG2000. The formulated AIE NPs exhibit excellent colloidal and photo stability. It is worth noting that these NPs showed excellent cell image by ultrafast staining processes in a few seconds at room temperature. Cellular experiments have demonstrated that these NPs are


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Analytical Chemistry biocompatible and internalized by caveolae-mediated endocytosis. More importantly, these NPs exhibited excellent long-term imaging capabilities in vitro and in vivo. The superior performance of these NPs could be used as long-term cellular tracers for pathologic evolution, biological processes, monitoring drugs therapeutic effects and other unique biomedical applications. Our contribution may provide a feasibility strategy for the construction of efficient red AIE NPs to facilitate the exploration of bioprobes in biological applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis, 1H NMR,13C NMR, and MS characterization, spectroscopic characterization, calculation of fluorescence quantum yield, dynamic light scattering (DLS), TEM image, photostability, cytotoxicity, and confocal images (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The work was funded by NSFC (No.51473028), the key scientific and technological project of Jilin province (20150204011GX, 20160307016GX), the development and reform commission of Jilin province (20160058). The project was supported by Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

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