Highly Stable Near-Infrared Fluorescent Organic Nanoparticles with a

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Highly Stable Near-Infrared Fluorescent Organic Nanoparticles with a Large Stokes Shift for Noninvasive Long-Term Cellular Imaging Jinfeng Zhang, Rui Chen, Zelin Zhu, Chihaya Adachi, Xiaohong Zhang, and Chun-Sing Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08539 • Publication Date (Web): 12 Nov 2015 Downloaded from http://pubs.acs.org on November 17, 2015

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ACS Applied Materials & Interfaces

Highly Stable Near-Infrared Fluorescent Organic Nanoparticles with a Large Stokes Shift for Noninvasive Long-Term Cellular Imaging Jinfeng Zhang,† Rui Chen,† Zelin Zhu,† Chihaya Adachi,§,* Xiaohong Zhang,‡ Chun-Sing Lee†,* †

Center of Super-Diamond and Advanced Films (COSDAF) & Department of Physics and Materials

Science, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, P. R. China. §

Center for Organic Photonics and Electronics Research (OPERA), Kyushu University, 744 Motooka,

Nishi-ku, Fukuoka 819-0395, Japan. ‡

Functional Nano & Soft Materials Laboratory (FUNSOM) and Jiangsu Key Laboratory for

Carbon-Based Functional Materials & Devices, Soochow University, Suzhou, P. R. China.

ABSTRACT: Fluorescent organic nanoparticles based on small molecules have been regarded as promising candidates for bioimaging in recent years. In this study, we report a highly stable near infrared (NIR) fluorescent organic nanoprobes based on nanoparticles of an anthraquinone derivate with strong aggregation-induced emission (AIE) characteristics and a large Stokes shift (> 175 nm). These endow the nanoprobe with high fluorescent brightness and high signal-to-noise ratio. On the other hand, the nanoprobe also shows low cytotoxicity, good stability over a wide pH range, superior resistance against photodegradation and photobleaching comparing to typical commercial fluorescent organic dyes such as fluorescein sodium. Endowed with such merits in term of optical performance, biocompatibility and stability, the nanoprobe is demonstrated to be an ideal fluorescent probe for noninvasive long-term cellular tracing and imaging applications. As an example, it is shown that

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strong red fluorescence from the nanoprobe can still be clearly observed in A549 human lung cancer cells after incubation for six generations over 15 days.

KEYWORDS: fluorescent organic nanoparticles, aggregation-induced emission, large Stokes shift, noninvasive, long-term cellular imaging

1. INTRODUCTION Fluorescence imaging has been demonstrated as an indispensable and versatile tool for biomedical research and clinical diagnostics.1-3 As compared to other imaging modalities, fluorescence imaging offers high spatiotemporal resolution and can provide high sensitivity images with dynamic details and quantitative information at subcellular levels.4-8 In particular, many fluorescent nanoprobes have been shown to process improved optical properties such as broader absorption spectra, size-tunable emissions, larger Stokes shifts and better biocompatibility over conventional organic dyes.9-13 For example, inorganic semiconductor quantum dots (Qdots) represent one of the most popular nanoprobes due to their high brightness and size-tunable absorption/emission profiles.14-18 However, before wide clinic applications, further researches are needed to addresses issues such as photo-blinking and their possible cytotoxicity stemmed from the heavy metal ions.19-24 Alternatively, fluorescent polymers dots (Pdots) have recently emerged as a new generation of nanoprobes for biological applications owing to their high brightness, excellent photostability, non-blinking and nontoxic features.25-31 In the recent few years, fluorescent organic nanoparticles (FO-NPs) based on small molecules have also attracted much attention. Some of these FO-NPs not only share the mentioned merits of Pdots, they also show additional advantages such as easier synthesis and purification, better biodegradability and chemistry tailorability.32-42 However, a major challenge of FO-NPs is that fluorescent intensity of many molecules would be decreased or quenched upon


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aggregation to form nanoparticles.43 To address this problem, molecules with aggregation-induced emission (AIE) have been used for preparing high performance FO-NPs with excellent potential for molecules detection and bioimaging.41, 43-48 On the other hand, much effort has been made towards the development of Near-Infrared (NIR, 650-900 nm) fluorescent organic nanoparticles (NFO-NPs) which could minimize photo-damage to living cells, enable deep penetration in tissue and reduce interference from the background autofluorescence.39, 40, 49-51 Furthermore, large Stokes shift (typically over 80 nm) is desirable to minimize cross-talk between the excitation source and the fluorescent emission for cellular imaging with high signal-to-noise ratio.[52-55] However, typical organic NIR dyes generally exhibit small Stokes shifts which would reabsorb emitted photons leading to undesired background interferences.52, 55, 56

For example, regular Stokes shifts of BODIPY dyes is in the range of 7-15 nm.52 To address this

issue, introduction of energy transfer between organic dyes and other fluorophore has been developed.53, 55, 57 Although effective, such systems are structurally more complicated and require much synthetic effort. Last but not the least, long-term cellular tracking is of great significance for investigating biological processes, pathological pathways and therapeutic effects over long time spans.58-62 Given the above considerations, an AIE-based NIR fluorescent organic nanoparticle simultaneously with superior photostability, large Stokes shift, and good biocompatibility for long-term noninvasive cellular imaging is highly desirable. However, so far there are few reports on molecules or nanoparticle s with all these merits.63 In this work, we prepared a new NIR emissive NFO-NPs via reprecipitation based on a NIR emissive AIE compound, 2,6-Bis[4-(diphenylamino)phenyl]anthraquinone The AIE compound is non-emissive in solvent but become highly fluorescent upon aggregation which reduces



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non-radiative decay via intramolecular rotations.64 The NFO-NPs were prepared by the reprecipitation method without further surface modification which show excellent imaging ability in the NIR region with a large Stokes shift (177 nm). The NFO-NPs also exhibit good biocompatibility and potential for applications in direct long-term cellular imaging with superior photostability.

2. EXPERIMENTAL SECTION 2.1 Materials. 2,6-Bis[4-(diphenylamino)phenyl]anthraquinone was synthesized according to our previous published work.65 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 4, 6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich and used after drying in vacuo for 24 h. Dulbecco’s modiﬁed Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (PBS), trypsin-EDTA (0.5% trypsin, 5.3 mM EDTA tetra-sodium), and the antibiotic agents penicillin-streptomycin (100 U/ml) were purchased from Life Technologies. Deionized water with a resistivity greater than 18.4 MΩ·cm was collected from an in-line Millipore RiOs/Origin water purification system. All pH measurements were conducted with a Eutech PH 700 pH-meter. Unless otherwise noted, all chemicals were obtained from commercial suppliers and used as received. 2.2 Preparation of The NFO-NPs. NFO-NPs were prepared using a well-documented reprecipitation method. In a typical procedure, 1 mg/mL solution of NFO-molecules (TPAAQ) dissolved in THF was prepared. 200 µL of the TPAAQ THF solution was quickly dropped into 5 mL of deionized water under vigorous stirring at room temperature for 10 min, afterwards, the NFO-NPs suspension was sonicated for another 30 min under room temperature.


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2.3 Characterization of The NFO-NPs. Size and morphology of the NFO-NPs were investigated using SEM (Philips XL-30 FEG). The SEM samples were prepared by drying a dispersion of the NFO-NPs on a Si substrate followed by coating of a 2 nm gold layer. DLS measurements were carried out using a Malvern Zetasizer instrument employing a 4 mW He-Ne laser (λ= 632.8 nm) and equipped with a thermostatic sample chamber. 2.4 UV-vis, Fluorescence and Photostability Measurements. UV-vis spectra were recorded using a Cary 50Conc UV-Visible Spectrophotometer. Fluorescence spectra were recorded on a Cary Eclipse Fluorescence Spectrophotometer. Time-dependent photo-degradation and photo-bleaching experiments were carried out using a xenon lamp (150 W) equipped with a filter passing light from 400 to 700 nm (100 mW/cm2). 2.5 Cytotoxicity by MTT assay. The A549 cells were seeded on 96-well plates in DMEM (with 10% FBS, 1% penicillin/streptomycin). After growing overnight, the cells were used for experiments. After removing the original medium in each well, 200 µl of DMEM containing predetermined concentrations of NFO-NPs were added to the designated wells. The final concentration of these NPs on each plate ranged from 3.125 to 50 µg/mL. After 24 h incubation at 37 °C, the original medium in each well was removed. Subsequently, 180 µL of DMEM (without FBS) and 20 µL of MTT stock solution (5 mg/mL in PBS) were added and incubated for 4 hours. Then the medium containing MTT was completely removed, followed by adding 200 µL of DMSO (Acros) to each well. Cell viabilities were determined by reading the absorbance of the plates at 540 nm using a BioTek Powerwave XS microplate reader. The cells incubated with serum-supplemented medium represent 100% cell survival. Five replicate wells were run for each concentration.



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2.6 Cellular Uptake and Localization. A549 cells were cultured with DMEM (with 10% FBS, 1% penicillin/streptomycin) in 5% CO2 at 37 °C in a humidified incubator. The cells were trypsinized and resuspended on 60 mm culture plates, and 2 mL of the medium was combined with 1.0 mL cell suspension. The cells were then seeded on 6-well plates, which were then placed in an incubator at 37 °C overnight with 5% CO2 for 24 h. The NFO-NPs solutions (20 µg/mL) and PBS buffer for control were respectively added to each plate and carefully mixed. The treated cells were returned to the incubator (37 °C, 5% CO2) for 4 h. After incubation, the plates were washed thoroughly with sterile PBS. DAPI was used to stain the cell nuclei for 5 min before fixing the treated cells. Finally, fluorescent images of the cells were recorded with a Nikon ECLIPSE 80i fluorescent microscope. 2.7 Long-term cellular imaging. A549 cells were cultured in 6-well plates to achieve 80% confluence overnight with 5% CO2 for 24 h. After medium removal and washing with PBS buffer, 20 µg/mL NFO-NPs in DMEM were then added to the wells. The treated cells were returned to the incubator (37 °C, 5% CO2) for 6 h (Day 0). The cells were then diluted and subcultured in 6-well plates containing cell culture coverslips for designated passages (from Day 0 to Day 15). Upon reaching designated time points, the cells were washed with PBS buffer and then fixed by 4% paraformaldehyde for 5 min. The coverslips were sealed with mounting medium and used for fluorescent imaging.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of NFO-NPs


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Figure 1. a) Schematic illustration of preparation of the NFO-NPs by reprecipitation for cellular imaging; b) An SEM image of the NFO-NPs (Inset is the corresponding TEM image. Scale bar is 100 nm) ; c) Dynamic light scattering (DLS) and polydispersity index (PDI) measurements and d) Zeta potential of the NFO-NPs; e) Photographs of the TPAAQ molecules dissolved in THF (e1) under UV light and the NFO-NPs dispersed in deionized water under room light (e2) and UV light (e3) respectively.

The chemical structure of NFO-molecule: 2,6-Bis[4-(diphenylamino)phenyl]anthraquinone is shown in Figure 1a which is abbreviated as TPAAQ as it is an adduct of triphenylamine (TPA) and anthraquinone (AQ). In previous work, we applied this molecule to act as a high performance emitter in organic light-emitting diodes.65 In this study, NFO-NPs were prepared by the well-documented reprecipitation approach in which AIE compound TPAAQ dissolved in tetrahydrofuran (THF) solution was rapidly dropped into deionized water under vigorous stirring. The as-prepared NFO-NPs can be taken up by cancer cell via endocytosis for NIR imaging attributing to their AIE based character as illustrated in Figure 1a. Figure 1b shows an SEM image of the NFO-NPs in the form of well-defined and monodispersed nanospheres of ~100 nm in diameter. Dynamic light



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scattering measurement (DLS, Figure 1c) presents a hydrodynamic diameter of 92.8 nm and a polydispersity index (PDI) value of 0.145. The zeta potential of the NFO-NPs was also measured displaying negative charge of around -17.3 mV due to the protonation of oxygen atom in anthraquinone of the TPAAQ molecules, which will stabilize the NPs in aqueous medium by electrostatic repulsion (Figure 1d). Aggregation-induced fluorescence was demonstrated using a UV lamp with a wavelength of 365 nm. Figure 1e displays photographs of the TPAAQ molecules dissolved in THF solution (e1) under UV light and the NFO-NPs dispersed in water under room light (e2) and UV irradiation (e3) respectively. TPAAQ molecules in pure THF show no emission observable to naked eye (e1), while NFO-NPs in water emit intense red fluorescence (e3). These results confirm the AIE characteristics of TPAAQ. The presence of NFO-NPs in water was also confirmed by shining a red laser beam through the solution to clearly observe the Tyndall effect (e2).

Figure 2. SEM images of the NFO-NPs prepared by TPAAQ solutions with different concentrations including a) 4 mg/mL, b) 2 mg/mL, c) 1 mg/mL, d) 0.5 mg/mL, e) 0.25 mg/mL, f) 0.125 mg/mL.


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Left insets are photographs of the as-prepared NFO-NPs dispersed in DI water under room light. Right insets are corresponding size distribution and PDI data. Scale bar is 1 µm.

To investigate the influences of TPAAQ solutions with different concentrations on preparetion of the NFO-NPs. We optimize the condition for fabricating the NPs by changing the concentration of free TPAAQ dissolved in THF ranging from 0.125 to 4 mg/mL. We chose 4 mg/mL as a maximum concentration because TPAAQ molecules cannot be completely dissolved in THF when its concentration larger than that value. As shown in Figure 2, when the concentration is as large as 2 or 4 mg/mL, only cohesive big ribbon-like structures instead of mono-dispersed NPs can be observed. When the concentration is smaller than 1 mg/mL, the size of the as-prepared NFO-NPs is not uniform, for example, the PDI value is as high as 0.381 when the concentration of TPAAQ dissolved in THF is 0.125 mg/mL. Size and PDI of the NFO-NPs prepared by using 1 mg/mL solution are 93.5 nm and 0.148 respectively. Morphology of the NFO-NPs formed at this concentration is much more uniform comparing to the other groups. Therefore, we choose 1 mg/mL as the optimal concentration for the following preparation. 3.2. Optical Properties and AIE Behaviors of TPAAQ Molecular geometry of the NFO-molecule: TPAAQ is presented in Figure 3a, large disc-shaped planar molecules of TPAAQ make them suitable to form nanostructures through π-π stacking interaction. Due to their propeller-like nonplanar conformation, the fluorogen is AIE active. As shown in Figure 3b, the absorption and emission spectra of NFO-molecules dissolved in THF solution and NFO-NPs dispersed in deionized water were respectively measured. Here, the free NFO-molecules in the THF exhibit an absorption peak at 448 nm. In comparison, absorbance peak of the NFO-NPs exhibits a range of 200-550 nm with a red-shift of ~25 nm at the reddest absorption maximum which possibly due to the strong intermolecular π-π interactions. The emission maximum of NFO-NPs appears at 650 nm with intense emission tail extending to 800 nm which is beneficial to NIR fluorescence imaging, while the free NFO-molecules are almost non-fluorescent in the THF due



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to easy intramolecular rotations of the TPA phenyl rings in aqueous media.64 The fluorescence quantum yield of the NFO-NPs dispersed in water is determined to be ~9.6% by using rhodamine B as a reference system (the fluorescence quantum yield of rhodamine B in water is ~31%). It is noteworthy that the AIE NFO-NP shows a large Stokes shift of 177 nm (reddest absorption maximum: 473 nm; emission maximum: 650 nm). This greatly minimizes the self-absorption effect typically observed in other conventional organic dye molecules and thus improves the signal-to-noise ratio for bioimaging.

Figure 3. a) Molecular geometry of the NFO-molecule: TPAAQ which was optimized by the DFT calculations using B3LYP/6-31G(d) basis set in Gaussian 09 program (grey dots: C atoms, white dots: H atoms, red dots: O atoms, blue dots: N atoms; twisting angle shown in the picture: planar angle between TPA and AQ: 32.7°, linear angles between TPA phenyl rings: 120.4°, 119.3°, 20.3°);


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b) Normalized absorbance and fluorescence spectra of free NFO molecules dissolved in THF and NFO-NPs dispersed in deionized water respectively (reddest absorption maximum: 473 nm; emission maximum: 650 nm, Stokes shift: 177 nm); c) Emission spectra of TPAAQ molecules in THF-water mixtures with different water fractions at 0.01 mg/mL. Excitation wavelength: 470 nm; d) Plot of I650nm/I0 versus water content of the solvent mixture, where I0 is the PL intensity in pure THF solution. The insets are respectively photographs of the NFO-NPs powder and the fluorescein sodium powder taken under UV light.

The AIE behavior of NFO-NPs was further investigated in THF/water mixture solutions through varying the fraction of water by monitoring their fluorescence intensity. In pure THF, little emission from TPAAQ molecules can be observed. Upon addition of different amount of water in THF, the fluorescence intensity dramatically increased (Figures 3 c, d). When the water fraction reached to 90 vol %, the fluorescence intensity of TPAAQ was nearly 44-fold stronger than in 100% THF, indicating the AIE characteristic of TPAAQ. We also measured the DLS of the TPAAQ molecules in THF-water mixtures with different water fractions at 0.01 mg/mL. As displayed in Figure S1, when the water fraction ranging from 10% to 40 %, diameters change from 0.6487 to 1.594 nm, which indicates negligible aggregate formed, thus no obvious fluorescence intensity of TPAAQ detected in THF-water mixtures. When the water fraction larger than 40%, the size of the aggregate was larger than 100 nm. The formed aggregates induced the emission of TPAAQ in THF-water mixtures. These results can be well correlated with their optical properties in Figure 3d. The insets in Figure 3d are respectively photographs of the NFO-NPs powder and the fluorescein sodium powder taken under UV light. Bright red emission from NFO-NPs can be clearly observed.

3.3. Cytotoxicity and Cellular Imaging of the NFO-NPs



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Figure 4. Cell viability of the NFO-NPs in A549 (red) and Hela (blue) cell lines at various concentrations after 24 hours of incubation, indicating the as-prepared NPs have low cytotoxicity. Data represent mean values ± standard deviation, n = 5.

Cytotoxicities of the NFO-NPs after 24 hours of incubation with the A549 human lung cancer cells

and

Hela

human

cervical

cancer

cells

were

evaluated

using

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays (Figure 4). The results demonstrate that the as-prepared NPs have low cytotoxicity towards both cancer cell lines at different concentrations. Fluorescence imaging was then performed on A549 cells to verify the cellular uptake and localization of the NFO-NPs. 4, 6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei for 5 min before cell imaging studies. As presented in Figure 5, an intense homogeneous cytoplasmic red fluorescence around nuclei can be clearly observed after cultured with the NFO-NPs for 4 hours, indicating the NFO-NPs can be successfully taken up and localize in the cytoplasm and the perinuclear region within the cells. Only PBS treated A549 cells were severed as a control group, which show no red signal because of the absence of the NFO-NPs. These results proved that the as-prepared organic NPs could be applied for cellular imaging with high


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signal-to-noise ratio.

Figure 5. Subcellular imaging and localization of the NFO-NPs, monitored by fluorescence imaging in A549 cells. (a) Bright field channel; (b) DAPI channel (excited at 330-380 nm); (c) NPs channel (excited at 450-490 nm); (d) Overlap of the above images. Scale bar is 50 µm. Upper: control group (incubated with PBS only); Lower: the NFO-NPs group. 3.4. Long-Term Tracing To investigate the NFO-NP’s noninvasive long-term cellular tracking capability, fluorescence images after different incubation periods were captured as shown in Figure 6. The A549 cancer cells were first incubated with the NFO-NPs (20 µg\mL) for 6 h at 37 °C (labeled as Day 0, Generation one). The treated cells were then subcultured for specified time intervals and the fluorescence signals were monitored (from the first generation to the sixth generation during 15 days). For each cell passage, the old culture medium was extracted and A549 cells were washed with PBS twice to remove the previous NFO-NPs existing in the culture medium. At the initial stage (Day 0, Generation one), bright red fluorescence signal from the NFO-NPs can be clear observed in Figure 6a. At this stage (Day 0, Generation one), the cells are mother cells which would be divide into



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daughter cells in the following experiments. So the strongest red signal is observed at this initial stage. Along with the increase of incubation time (from Day 3 to Day 15, from Generation two to Generation six), the red fluorescence gradually decreases because of cell proliferation, in which the NFO-NPs divide into daughter cells. Interestingly, the red signal can still be clearly observed after 15 days (Generation six), which indicates that the NFO-NPs can act as a fluorescent probe for long-term cellular tracing and imaging applications. More importantly, this long-term tracking strategy is based on the cellular proliferation containing endogenous organic nanoprobes rather than continuous exogenous addition of imaging agents during the long-term monitoring period. This noninvasive nature makes the NFO-NPs suitable for potential clinical bioimaging. We also investigated the cell viability of these A549 cells which incubated with the NFO-NPs after 15 days. As shown in Figure S2, no shrinkage and deformation of the A549 cells after incubation with NFO-NPs can be observed when comparing to the control group which demonstrated negligible toxicity of the as-prepared NPs. Furthermore, subsequent MTT assay also proved the low cytotoxicity of the NFO-NPs to the A549 cells after 15 days.


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Figure 6. Long-term cell tracing images of the NFO-NPs at 37 °C for 6 h and then subcultured for designated time intervals including a) Day 0/ Generation one; b) Day 3/ Generation two; c) Day 6/ Generation three; d) Day 9/ Generation four; e) Day 12/ Generation five; f) Day 15/ Generation six. Scale bar is 50 µm. (All photos were taken under identical settings. Excitation wavelength: 450-490 nm; Exposure time: 2.1s; Gain: 4) 3.5. Stabilities To further confirm the potential of the NFO-NPs to serve as an effective imaging agent in biological environment, stabilities of the NFO-NPs in different conditions were further evaluated. As shown in Figure 7, firstly, we studied photostability comparison of fluorescence signals from A549 cells labeled with the NFO-NPs (Upper) and fluorescein sodium (Lower) which is a commercially available fluorescent organic dye under light irradiation for 15 min respectively. The fluorescence signals of the NFO-NPs exhibited bright signal and showed mild intensity decrease upon 15 min irradiation. While the intensity of fluorescein sodium rapidly diminished and became negligible after 5 min due to severe photo-bleaching. For further comparison, we monitored the absorbance and fluorescence of the NFO-NPs and fluorescein sodium upon irradiation respectively. As depicted in Figures 8 a, b and Figures S3, 4, there is little degradation in absorbance and less bleaching in fluorescence comparing with the commercial organic dye fluorescein sodium after 40 min irradiation with a Xeon lamp. For example, the absorbance of the NFO-NPs retained more than 98.1 % of the original intensity after continuously irradiating for 40 min while the absorbance of fluorescein sodium was degraded to 15.3 % (Figure 8a and Figure S3). In addition, the fluorescence intensity of the NFO-NPs decreased to 75 % while the fluorescein sodium was bleached to 13% within the same irradiation time (Figure 8b and Figure S4).



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Figure 7. Photostability comparison of fluorescence signals of A549 cells labeled by the NFO-NPs (Upper) and fluorescein sodium (Lower) under laser irradiation for 15 min respectively. Scale bar is 25 µm. (Excitation wavelength: 450-490 nm; Exposure time: 1.5s; Gain: 2)

We also investigated the photostability of the as-prepared nanoprobes in environments of different pHs obtained by mixing buffer solutions of citric acid and Na2HPO4 in different ratios. As displayed in Figure 8c and Figure S5, no obvious fluorescence quenching was observed at emission peak (650 nm) in both PBS (pH 7.4), DMEM and other buffer solutions with different pH ranging from 2 to 10 comparing with that in deionized water, demonstrating that the NFO-NPs exhibit superior photostability over a wide pH range, which is beneficial for their potential bio-imaging applications. Since size-stability of nanomaterials in physiological environments is of vital importance for their biomedical applications, we firstly studied the size-stability of the NFO-NPs in different pH buffers, Figure S6 shows that the as-prepared NPs are still in in nano dimension in different pH buffers ranging from 2 to 9. We next measured the mono-dispersed stability of the NFO-NPs in PBS and DMEM supplemented with 10 % FBS and 1% penicillin/streptomycin respectively to ensure that the


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NPs would not aggregate after a period of time. As shown in Figure 8d and Figure S7, the increase in size of the NFO-NPs is negligible and their PDI value remains below 0.2 within 12 days. All these results demonstrate that the NFO-NPs possess good water dispersibility and robust stability in a variety of bio-environments for bioimaging application.

Figure 8. a) Time-dependent photo-degradation (absorption intensity) and b) photo-bleaching (fluorescence intensity) of the NFO-NPs comparing with fluorescein sodium after 40 min irradiation; c) fluorescence intensity of the NFO-NPs at 650 nm in PBS (pH 7.4), DMEM and other buffer solutions with different pH ranging from 2 to 10 comparing with that in deionized water; d) Size-stabilities including diameters and PDI values of the NFO-NPs.

4. CONCLUSION In summary, highly stable NIR fluorescent organic nanoprobes was fabricated via well-documented reprecipitation method for in vitro long-term tracking. They show good monodispersity, low cytotoxicity, and significant aggregation-induced emission properties in aqueous



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solution, and were able to successfully enter A549 cancer cells via endocytosis. Furthermore, the as-prepared NFO-NPs exhibit superior photodegradation and photobleaching resistance comparing to the commercially available fluorescent organic dye fluorescein sodium, high photostability over a wide pH range, large Stokes shift (> 175 nm), and good biocompatibility. More importantly, the red signal from the NFO-NPs can still be clearly observed after 15 days (cells were incubated for six generations in these days). These merits make the AIE-based NFO-NPs promising fluorescent probe for noninvasive long-term cellular tracing and imaging applications.

ASSOCIATED CONTENT

Supporting Information. Description of materials and methods and Supporting Figures: (Figure S1) DLS of the TPAAQ molecules in THF-water mixtures with different water fractions at 0.01 mg/mL, (Figure S2) Bright field images of A549 cells incubated with a) PBS as a control group and b) the NFO-NPs after 15 days (six generations); c) Cell viability of the A549 cells incubated with the NFO-NPs after 15 days comparing with the PBS treated group, (Figure S3) Time-dependent photo-degradation of the NFO-NPs comparing with fluorescein sodium with continuous irradiation under xenon light for 40 min, (Figure S4) Time-dependent photo-bleaching of the NFO-NPs comparing with fluorescein sodium with continuous irradiation under xenon light for 40 min, (Figure S5) Photostability studies of the NFO-NPs treated with PBS, DMEM and pH 2 to 10 solutions, (Figure S6) Size-stability studies of the NFO-NPs in treated with pH 2 to 9 solutions, (Figure S7) Size-stability of the NFO-NPs dispersed in DMEM supplemented with 10 % FBS and 1% penicillin/streptomycin over 12 days. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION


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Corresponding Author * E-mail: [email protected]

* E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. T23-713/11) and JST, ERATO, Adachi Molecular Exciton Engineering Project.

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Thermally

Activated

Delayed

Fluorescence,

and

Electroluminescence. J. Am. Chem. Soc. 2014, 136, 18070-18081.


Highly

Efficient

Red


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Table of Contents Graphic


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