AIE active NIR Fluorescent Organic Nanoparticles for Noninvasive

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AIE active NIR Fluorescent Organic Nanoparticles for Noninvasive Long-Term Monitoring of Tumor Growth Qi Xia, Zikang Chen, Zhiqiang Yu, Lei Wang, Jinqing Qu, and Ruiyuan Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03861 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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ACS Applied Materials & Interfaces

AIE active NIR Fluorescent Organic Nanoparticles for Noninvasive Long-Term Monitoring of Tumor Growth a

a

a

c

c

Qi Xia , Zikang Chen , Zhiqiang Yu , Lei Wang , Jinqing Qu , Ruiyuan Liu

a,b,

*

a

School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, P.R. China.

b

School of Biomedical Engineering, Southern Medical University, Guangzhou 510515, P.R. China

c

School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, P.R.China

KEYWORDS : AIE, NIR, Fluorescent Organic nanoparticles, optical imaging, cell tracking, Long-Term monitoring, tumor growth ABSTRACT: Effective long-term monitoring of tumor growth is significant for the evaluation of cancer therapy. AIE

active NIR fluorescent organic nanoparticles (TPFE-Rho dots) are designed and synthesized for long-term in vitro cell tracking and in vivo monitoring of tumor growth. TPFE-Rho dots display the advantages of NIR fluorescent emission, large Stokes shift (~180 nm), good biocompatibility, and high photostability. In vitro cell tracing studies demonstrate that TPFE-Rho dots can track SK-Hep-1 cells over 11 generations. In vivo optical imaging results confirm that TPFE-Rho dots can monitor tumor growth for more than 19 days in a real-time manner. This work indicates that TPFE-Rho dots could act as NIR fluorescent nanoprobes for real-time long-term in situ in vivo monitoring of tumor growth. 1. INTRODUCTION Cancer remains one of the most challenging threats to human health in the world. Effective real-time monitoring of tumor growth is significant for the evaluation of cancer therapeutic results. Long-term in situ in vivo fluorescence imaging as a highly sensitive and non-invasive technology provides researchers an indispensable and versatile tool for diagnosis of cancer, monitoring of tumor growth, image-guided surgery, due to the ability to visualize the tumor location and assess the biological processes1-3. In particular, several types of fluorescent nanoprobes have been explored for long-term in situ in vivo fluorescent imaging of tumor4-6. For example, inorganic fluorescent nanoparticles (noble metal nanoparticles7-8, upconversion nanoparticles9-10, and inorganic quantum dots11-12) have been utilized to optical image cancer at both cellular and animal levels. However, the low biodegradability and potential cytotoxicity limited their applications13-15. In comparison to inorganic fluorescent nanoparticles, organic fluorescent nanoparticles should be an alternative because of their excellent photostability, easy functionality and good biocompatibility3, 16-19. Unfortunately, the fluorescent intensity of organic nanoparticles functionalized from many fluorescent molecules would be decreased or quenched in the aggregated state. To address this problem, researchers have fabricated fluorescent organic nanoparticles based on the aggregation-induced emission (AIE) active molecules, which exhibited excellent potential for sensing, bioimaging, and therapy applications20-26. To date, several kinds of AIE active fluorescent organic nanoparticles have been developed27-31. As compared to other visible light-emitting fluorogens, NIR fluorescent

probes have become the most promising materials for in vitro and in vivo fluorescent imaging of tumor due to their minimized autofluorescence interference, lowered damage to tissue, and increased penetration depth16, 32-38. Furthermore, fluorescent probe with lager Stokes shift can minimize the interference between the excitation source and the fluorescent emission, thus obtaining a fluorescence image with high signal to noise ratio39-40. As a consequence, AIE active NIR fluorescent organic nanoparticles with superior photostability, large Stokes shift, great biocompatibility and excellent cellular retention for longterm noninvasive in vivo optical imaging of tumor is highly desirable. Although some researchers have applied AIE near-infrared fluorescent probes or nanoprobes to longterm cell tracking and tumor imaging applications in recent years41-44, there have been few reports about organic molecules or nanoparticles having all these advantages so far42. In this paper, we prepared AIE active fluorescent probe (named TPFE-Rho) and fabricated AIE active NIR fluorescent organic nanoparticles for long-term non-invasive in vivo monitoring of tumor growth. The intense NIR fluorescent emission and large Stokes shift (~180 nm) render TPFE-Rho dots suitable for in vivo fluorescent imaging with low disturbed absorption, minimum background fluorescence, and deep tissue penetrability. The cytotoxicity, photostability, and cellular-internalization efficiency of TPFE-Rho dots were assessed. It was found that TPFE-Rho dots could trace SK-Hep-1 cells for 11 generations in vitro. SK-Hep-1 cells labeled TPFE-Rho dots were further transplanted into the nude mice to monitor the

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tumor growth, which exhibited that the optical signals can be tracked for 19 days. These results demonstrated that TPFE-Rho dots could be employed as NIR fluorescent nanoprobes for real-time long-term in vivo monitoring of tumor growth. OH HO B O Br

CHO O

S

CN

N

CN

O

NC N O

CHO PdCl2(PPh3)2 2M K2CO3, Toluene/EtOH

CN

S O

HAc NH4Ac TPFE-CHO

TPFE-Rho

Scheme 1 Synthetic routine to TPFE-Rho

2. MATERIALS AND METHODS 2.1. Materials. All chemicals and solvents were commercially available and used directly without further purification unless specified. All kind of solvents (including absolute ethanol, n-Hexane, ethyl acetate, N,Ndimethylformamide, dichloromethane, acetonitrile) were purchased from Guangdong Guanghua Sci-Tech Co. Ltd. Tetra(triphenylphosphine)palladium (Pd(PPh3)4), (2bromoethene-1,1,2-triyl)tribenzene, malononitrile, ethyl bromoacetate, phenyl isothiocyanate, K2CO3 were obtained from Admas China Co. Ltd. Bromotriphenylethylene and 5-formyl-2-furanboronic acid was purchased from TCI. 2.2. Characterization. NMR spectra were measured via a Bruker 400 MHz NMR with CDCl3. Infrared spectroscopy (IR) was performed with a Shimadzu FTIR-8100 spectrophotometer. High-resolution mass spectra was conducted on a Bruker Autoflex instrument. UV absorption spectra were recorded on Thermofisher Evolution 300 spectropolarimeter. FL spectra were obtained using a Thermofisher Lumina spectrofluorometer. 2.3. Synthesis of rhodanic-CN45. Malononitrile (1.32 g, 20 mmol), phenyl isothiocyanate (2.98 g, 22 mmol) and DBU(3.04 g, 20 mmol) were added to acetonitrile (70 ml) at room temperature. After stirred for 30 min, ethyl bromoacetate (5.65 g, 34 mmol) was added to the mixture. The mixture was refluxed overnight. The acetonitrile was evaporated. The solid was acidified with 1 M HCl (60 ml) and extracted with CHCl3 (60 ml × 2). The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by recrystallization in acetonitrile (20 ml) to yield a pale yellow solid (4.1 g, 85%).1H NMR (400 MHz, CDCl3), δ (ppm):7.59-7.45 (m, 5H), 4.35 (s, 2H). 13C NMR (100 MHz, CDCl3), δ (ppm): 174.67, 172.36, 133.98, 131.74, 130.21, 129.76, 114.11, 109.77, 53.36, 33.87. 2.4. Synthesis of TPFE-CHO. Bromotriphenylethylene (3.35 g, 10 mmol), 5-formyl-2-furanboronic acid (2.1 g, 15 mmol), PdCl2 (PPh3)2 (175 mg, 0.25mmol) , distilled toluene (37.5 ml), EtOH (25 ml), 2M K2CO3 aq. (3.46 g, 25 mmol/12.5 ml H2O) were added to the flask, which was degassed over 1h with N2. After reflux overnight, the reaction was quenched by water (50 ml) and extracted with

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CHCl3. The organic solvents were evaporated. The residue was purified by column chromatography on silica gel using n-Hexane/EA (9:1; v/v) as the eluent for obtaining TPFE-CHO as a pale yellow solid. 1H NMR (400 MHz, CDCl3), δ (ppm): 9.81~9.82(s, CHO, 1H), 7.41~7.52(m, Ar, 13H), 7.31~7.34(m, Ar, 3H), 6.83~6.86(d, Ar, 1H). 13C NMR (100 MHz, CDCl3), δ (ppm): 181.72, 162.61, 150.48, 141.52, 141.43, 141.27,131.24,131.07,129.88,129.51, 128.91,128.67,128.55 128.43, 125.87, 119.83, 114.33, 114.25. 2.5. Synthesis of TPFE-Rho. Rhodanic-CN (1.21 g, 5 mmol), TPFE-CHO (1.75 g, 5 mmol), and ammonium acetate(500 mg)were added to acetic acid (30 ml). The mixture was refluxed overnight. After cooling the solution, the crude precipitate was filtered and washed two times with cold MeOH. The solid was recrystallized from CH2Cl2 (5 mL) and ethanol (50 mL) to give TPFE-Rho as brick red solid. Yield: 2.23 g (78%). 1H NMR (400 MHz, CDCl3), δ (ppm): 7.57~7.64(m, Ar, 3H), 7.52(s, Ar, 1H), 7.37~7.40(t, Ar, 3H), 7.23~7.28(m, Ar, 11H), 7.11~7.13(m, 3H), 7.02~7.04(m, Ar, 1H),6.86~6.87(d,1H). 13C NMR (100 MHz, CDCl3), δ (ppm): 166.17, 165.66, 161.98, 147.46, 146.23, 143.06, 142.09, 139.85, 132.91, 131.56, 131.30, 131.24, 130.25, 130.01, 128.89, 128.66, 128.57, 128.23, 127.87, 127.61, 127.42, 127.35, 122.24, 120.68, 116.22, 113.14, 112.94, 109.89, 56.74. IR (ν-1, KBr): 3035, 2214, 1726, 1610, 1285, 1385, 1215, 1199, 1157, 1024, 754, 694. HR-MS (ESI): [M+H]+ calcd for C37H23N3O2S, found, 574.1571. 2.6. TPFE-Rho dots Preparation. TPFE-Rho dots were prepared via coprecipitation method with Pluronic F-127 as the encapsulation. Generally, 2 mL THF containing Pluronic F-127 (200mg) and TPFE-Rho (20mg) was drop added slowly into the DI water (10 mL) with vigorous stirring. The mixture was stirred overnight to completely remove THF. After that, the unbounded F-127 was completed removed by dialysis to form the TPFE-Rho dots. 2.7. Cell culture. TPFE-Rho (10 mM in DMSO) was diluted with culture medium to obtain a working solution (10 μM) for the subsequent cellular experiments. SK-Hep1 cells were gifted from Nanfang Hospital (Guangzhou, China). SK-Hep-1 cells were cultured in DMEM culture medium in a 5% CO2 incubator at 37 °C. 2.8. Cytotoxicity of TPFE-Rho. SK-Hep-1 cells were seeded in a 96-well plate with the density of 6500 cells per well overnight. After attachment, the old medium was replaced by TPFE-Rho of gradient concentrations (1.56, 3.13, 6.25, 12.5, 25, 50μM). After 24 h or 48 h incubation, the culture medium was removed, and 100 μL of 10 μM CCK-8 was added into each well and incubated for 4 h. The absorbance at 450nm of each well was measured using a BIOTEK ELX80 enzyme-linked immunosorbent assay reader. 2.9. Photostability Study. To evaluate the photostability of TPFE-Rho dots, the procedures were as follows: the imaging mode was time lapse, and the SK-Hep-1 cells stained by incubation with TPFE-Rho dots were ana-


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lyzed every 1 min while under continuous laser irradiation at 358 nm for DAPI and 530 nm for TPFE-Rho dots, respectively. The fluorescence intensity of each image was assessed by ImageJ software. 2.10. Real-time and Long-term cell tracking In Vitro. SK-Hep-1 cells were seeded in 48-well culture plates with the density of 3 × 104 cells/well or in confocal microscope dish with 5 × 104 cells. After attachment, the old medium was replaced by 10 µM TPFE-Rho dots. After incubation at 37 °C for 4 h, the cells were washed with PBS buffer, detached by EDTA-trypsin and resuspended in culture medium. Upon dilution, the cells were subcultured in wells containing cell culture coverslips for different cell generations. At specified time intervals, the cells were trypsinized, suspended and fixed with 4% paraformaldehyde for 15 min. The stained cells were imaged using a confocal fluorescence microscope (OLYMPUS FV 1000). The fluorescent intensities of corresponding cells were recorded by flow cytometry measurements using BD LSRFortessa. A batch of blank cells without any treatment was used as the control group. 2.11. Animals. Adult nude mice (18~20g) were obtained from animal center (Southern Medical University, Guangzhou, China). Imaging procedures were conducted under general anesthesia by injecting sodium pentobarbital (0.5 mL/0.03%). 2.12. Real-time and long-term tracking In vivo. SKHep-1 cells were incubated with 10 µM TPFE-Rho dots for 4 h at 37 °C. SK-Hep-1 cells (1 × 106 cells in 0.1 ml DMEM) were subcutaneously injected into the mice. At designated time intervals post-injection, the mice were imaged using an Bruker FX Pro with a 10 s exposure upon excitation at 530 nm and emission at 600 nm while under anesthesia with 1~2% of isoflurane in oxygen. The timedependent fluorescence intensity of regions of interest (ROI) in the tumor was recorded with a 10 s exposure upon excitation at 530 nm and emission at 600 nm. Scans were carried out on days 0, 3, 6, 12, 16, and 19. 2.13. Ex vivo study. The fluorescent imaging of organs were recorded with an in vivo imaging instrument. After exposure, the outlines of the organs were depicted in the Bruker MI SE software as regions of interest (ROI), and then the fluorescence intensities of each group were analyzed. 2.14. Hematoxylin and eosin (H&E) staining analysis. The physical conditions of the nude mice were monitored and observed, including their general status, e.g., skin and activities. At 19 day after tumor growth, the mouse was sacrificed, and then the liver, spleen, lung, kidney and heart were sectioned into slices for H&E staining. 3. RESULTS AND DISCUSSION 3.1. Synthesis and optical property of TPFE-Rho. TPFE-Rho was synthesized according to Scheme 1. The key intermediate TPFE-CHO was prepared from (2-

bromoethene-1,1,2-triyl)tribenzene and 5-formyl-2furanboronic acid in 83% yield. TPFE-CHO reacted with rhodanic-CN to obtain TPFE-Rho in 78% yields. The final product has been carefully purified and fully characterized by NMR, IR spectra and high-resolution MS which are given in Figure S1-S4. TPFE-Rho is soluble in common organic solvents such as THF, chloroform, toluene, acetonitrile, DMSO and DMF, but insoluble in solvents such as water, Hexane, methanol and ethanol. The absorption band and emission maximum of TPFE-Rho in good solvent such as THF is around 470 nm and 640 nm, respectively. Furthermore, the fluorescence intensity of TPFE-Rho in good solvent is very weak. The optical properties of TPFE-Rho were investigated in THF/water system. As depicted in Figure S5, the absorption maximum shows red-shift from 470 nm to 500 nm when fw (by volume %) increases from 10 to 99%. Meanwhile, the absorption intensity changes slightly when fw is lower than 50 %, but it decreases rapidly when fw is over 60%. This phenomenon can be explained by the formation of aggregates in high water fractions, which reduce the number of the molecules available for light absorption.

Figure. 1 (a) PL spectra of TPFE-Rho in water/THF mixture. (b) Fluorescent intensity in 654 nm in water/THF mixture. Insert of (b): PL image of TPFE-Rho in water/THF mixture.

As shown in Figure 1, TPFE-Rho shows very weak fluorescence in THF. Upon gradual addition of water into THF at fw 60 vol %) is admixed with THF, the resultant mixture emits evidently enhanced emission, it indicates that the nanoparticles are formed when fw > 60 vol %, and that intramolecular rotation is restricted due to physical constraint46. In solution with water fraction (fw) of 70%, the PL intensity of the mixture becomes stronger and red emission with a peak around 654 nm can be observed. At fw = 80 vol%, a 54-fold enhancement of PL intensity has been observed as compared to that in THF, this may be because TPFE-Rho can be aggregated in a slower crystallizing manner when the water content in the solution reaches 70% to 80%, and the fluorescence is enhanced. When the water content exceeds 80%, TPFE-Rho aggregates mainly in an amorphous manner, and the fluorescence is relatively weak47, we think TPFE-Rho has been



clustered into larger nanoparticles, it may cause the fluorescent molecules inside nanoparticles to be excused by excitation light, so the fluorescence intensity also decreases. These data confirm that TPFE-Rho is an AIEactive molecule. 3.2. Preparation and characterization of TPFE-Rho dots. Due to the insolubility of TPFE-Rho in water, TPFE-Rho cannot be directly employed as a fluorogen for cell labeling. We subsequently fabricated the organic fluorescent nanoparticles through coprecipitation method by using Pluronic F-127 as the encapsulation (Scheme 2). Generally, THF containing Pluronic F-127 and TPFE-Rho was drop added slowly into the water. Then, THF was removed completely. After that, the unbounded F-127 was completed removed by dialysis to obtain TPFE-Rho dots.

studied via DLS experiment. (b) Normalized UV and FL spectra of TPFE-Rho dots in water. Insert of (a): TEM image of TPFERho dots

To evaluate the photostability of TPFE-Rho dots, we have studied their PL intensity under different conditions. As shown in Figure S7, the PL intensity of TPFE-Rho dots remains 92% of its initial value after 7-day incubation in cell culture medium (DMEM) at room temperature. The optical stability of TPFE-Rho dots at different pH was also studied by incubating them in 1×PBS buffer with different pH varying from 3 to 12 (Figure S8). The stable fluorescence indicates that TPFE-Rho dots could maintain good stability in both acidic and basic environment. The fluorescence stability of TPFE-Rho dots in cells were subsequently evaluated after incubation with SKHep-1 cells for 4 h (37 °C, 5% CO2). Figure 3 shows that the photostability of TPFE-Rho dots in cells, investigated by measuring the fluorescent change of TPFE-Rho dots labelled SK-Hep-1 cells under continuous laser irradiation on a confocal fluorescence microscope. Upon continuous excitation for 8 min, the intracellular fluorescence intensity of TPFE-Rho dots shows little change. These results show TPFE-Rho dots possess superior photostability, which is promising in bioimaging applications.

Scheme 2 Schematic illustration of preparing TPFE-Rho dots for noninvasive long-term monitoring of tumor growth

TPFE-Rho dots were subsequently characterized by the DLS experiment and HR-TEM. Figure 2a shows TPFERho dots sample has a narrow size distribution and the average hydrodynamic diameter is around 110 nm. The transmission electron microscopy (TEM) image reveals that the nanoparticles have spherical morphology with mean diameter of ≈100 nm, which agrees with the DLS results. When TPFE-Rho dots were stored in PBS at room temperature for two weeks, no aggregation, precipitation or obvious size change can be observed, indicating their good colloidal stability (Figure S6). The absorption and emission spectra of TPFE-Rho dots in water were displayed in Figure 2b. As shown in Figure 2b, the emission maximum of TPFE-Rho dots is around 654 nm, which belongs to the near-infrared region. The Stokes shift is about 183 nm. These results are significantly beneficial for bioimaging.

Figure 2 (a) Particle-size distribution of TPFE-Rho dots in water

Figure 3 (a) Photostability of TPFE-Rho dots and DAPI under continuous scanning at 530 nm and 358 nm, where I0 is the initial fluorescence intensity and I is the fluorescence intensity of each sample at various time points; (b) Confocal microscopy images of SK-Hep-1 cells before (0 min, left) and after 8 min of laser irradiation (right), top: TPFE-Rho dots, bottom: DAPI. Concentration: 2 µM

3.3. Cytotoxicity and cellular uptake of TPFE-Rho dots. The cytotoxicity of TPFE-Rho dots first was investigated using CCK-8 assays. Figure S9 shows the metabolic activities of cells after 24 and 48 h incubation with TPFERho dots concentration ranges from 1.56 to 50 μM. The metabolic activities of cells remain above 90% even treated with 10 μM TPFE-Rho dots. Moreover, it still remains above 60% after being treated with 50 μM TPFE-Rho dots, indicating low cytotoxicity and good biocompatibility of TPFE-Rho dots. After verification of cytotoxicity, TPFE-Rho dots were used to label the living cells. The cellular uptake and localization of TPFE-Rho dots in SK-Hep-1 cells are shown in Figure 4. After SK-Hep-1 cells were incubated with 2 μM TPFE-Rho dots for 4 h (37 °C, 5% CO2), TPFE-Rho dots located in the cytoplasm of SK-Hep-1 cells and emitted intense red fluorescence. Moreover, the stained cells


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grow healthy and display their normal morphology, which confirm that TPFE-Rho dots are biocompatible.

Figure 4 Confocal microscopy images of SK-Hep-1 cells incubated with TPFE-Rho dots. Concentration: 2 µM

The fluorescence intensities of cells were analyzed by flow cytometry measurements to evaluate the cell internalization of TPFE-Rho dots by SK-Hep-1 cells. Cells in the absence of TPFE-Rho dots were used as the control group. After incubated with 2 μM TPFE-Rho dots for 4 h (Figure S10), the relative geometrical mean fluorescence intensity of the labeled cells was almost 10000-fold higher than that of control group, indicating that TPFE-Rho dots are readily internalized by SK-Hep-1 cells. These cytometry results were highly consistent with the confocal laser scanning microscopy (CLSM) images regarding the high cellular uptake of TPFE-Rho dots. 3.4. In vitro cell tracing. The low cytotoxicity, high photostablity, and biocompatibility of TPFE-Rho dots suggest that it may be applied in long-term cell tracking. SK-Hep-1 cells were incubated with 10 µM TPFE-Rho dots for 4 h as the first generation. The persistent fluorescence signals from the stained live cells were recorded by confocal microscopy and flow cytometry. As shown in Figure 5, the stained cells still showed red fluorescent signals, even for up to 11 generations. It seems that the subsequent cell division causes the distribution of TPFE-Rho dots into daughter cells, inevitably decreas-

ing TPFE-Rho dots concentration per cell and resulting in a progressive decrease in average fluorescence intensity per cell population. The flow cytometry results (Figure S11) are also in accordance with the results from the confocal images of the cell populations. The efficient intracellular redistribution of TPFE-Rho dots to daughter cells and its low leakage into the medium suggested that TPFE-Rho dots are capable of long-term cell tracing and maintaining its fluorescence signal after up to 11 cell divisions. These results demonstrated the superior cell tracking ability of TPFE-Rho dots, indicating TPFE-Rho dots as a promising long-term tracing probe.

Figure 5 Confocal microscopy images of TPFE-Rho dots stained Sk-Hep-1 cells at different generations

3.5. Tumor Growth Monitoring In Vivo. We envisaged that the remarkable properties of TPFE-Rho dots would enable its application to real-time and long-term tracking in living organisms. Accordingly, tumor growth monitoring was utilized to assess the long-term tracking ability of TPFE-Rho dots in vivo. TPFE-Rho dots -loaded

Figure 6 In vivo fluorescence imaging of a tumor by TPFE-Rho dots. (a) Representative in vivo fluorescence images of the mouse subcutaneously injected with 1×106 of SK-Hep-1 cells from day 0 to day 19; (b) Normalized time-dependent fluorescence intensity changes of regions of interest (ROI) in the tumors, n = 3; (c) Representative ex vivo fluorescent images of the isolated organs from the nude mice.



SK-Hep-1 cells (1×106) were subcutaneously implanted into nude mice. Tumor growth was monitored via in vivo optical imaging. Figure 6a shows that the injected site exhibits bright red fluorescence upon excitation at 530 nm. The fluorescence signals remain detectable even after 19 days. Quantitative analysis of the fluorescent signals at the injection site also reveals the consistent changes in fluorescence intensity over time (Figure 6b). These results clearly indicate that TPFE-Rho dots can act as an efficient long-term tracking probe in living organisms.

of tumor growth. The results in vitro demonstrate that TPFE-Rho dots labelled SK-Hep-1 cells can be tracked for up to 11 passages. Moreover, in vivo optical imaging results indicate that TPFE-Rho dots can monitor tumor growth for more than 19 d in a real-time manner. This study suggests that TPFE-Rho dots could act as NIR fluorescent nanoprobes for real-time long-term in vivo monitoring of tumor growth.

ASSOCIATED CONTENT

After 19 days of tumor growth monitoring, the nude mouse was sacrificed to collect its organs (tumor, heart, lung, spleen, kidney and liver). The ex vivo fluorescent imaging of organs (liver, heart, lung, spleen, kidney and tumor) was carried out. As presented in Figure 6c, fluorescence could be detected only in tumor, indicating that there were labelled cells throughout the tumor tissue.

Supporting Information. Synthetic procedure, NMR, FTIR absorbance spectrum and analysis characterization of TPFE-Rho; cytotoxicity of SK-Hep-1 cells, and fluorescence stability of TPFE-Rho dots. This material is available free of charge via the Internet at http://pubs.acs.org.

Subsequently, the isolated organs were analyzed by hematoxylin and eosin (H&E) staining. As shown in Figure 7, tumor treated with TPFE-Rho dots manifestated significant apoptosis or necrosis. On the contrary, other organs showed few apoptosis or necrosis indicating that TPFE-Rho dots have little side effects. To further prove it, we added a graph of H&E staining results in supporting information (Figure S12). We injected 200 μl of 20 μM TPFE-Rho dots solution into the tail vein of nude mice, observed the nude mice for one week, and then euthanized them and collected their organs (liver, heart, lung, spleen and kidney) for H&E staining. The organs showed no apoptosis or necrosis, which further indicating that TPFE-Rho dots have little side effects on nude mice. These data strongly suggested the great potential of TPFE-Rho dots in long-term cell tracing in living organism, which is of high guidance in tumor tracking and cell labeling.

Corresponding Author

Figure 7 H&E staining of the major organs (i.e., tumor, heart, liver, spleen, lung, and kidney) separated from the nude mice after 19 days of tumor tracking

4. CONCLUSIONS In summary, we designed and prepared AIE active nearinfrared fluorescent organic nanoparticles (TPFE-Rho dots). TPFE Rho dots have the advantages of near infrared fluorescence emission, large Stokes shift, good biocompatibility and high resistance to photobleaching. We further demonstrated the application of TPFE-Rho dots for long-term in vitro cell tracking and in vivo monitoring

AUTHOR INFORMATION * E-mail: [email protected] Ruiyuan Liu: 0000-0002-6696-8351

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (81671749), and the Natural Science Foundation of Guangdong Province, China (No. 2015A030313205).

REFERENCES (1) Cheng, Y.; Sun, C.; Ou, X.; Liu, B.; Lou, X.; Xia, F. Dualtargeted peptide-conjugated multifunctional fluorescent probe with AIEgen for efficient nucleus-specific imaging and long-term tracing of cancer cells. Chem. Sci. 2017, 8 (6), 45714578. (2) Weissleder, R.; Pittet, M. J. Imaging in the era of molecular oncology. Nature 2008, 452 (7187), 580-589. (3) Wang, Z.; Yong, T. Y.; Wan, J.; Li, Z. H.; Zhao, H.; Zhao, Y.; Gan, L.; Yang, X. L.; Xu, H. B.; Zhang, C. Temperaturesensitive fluorescent organic nanoparticles with aggregationinduced emission for long-term cellular tracing. ACS Appl. Mater. Interfaces 2015, 7 (5), 3420-3425. (4) Costa, C. R. M.; Feitosa, M. L. T.; Bezerra, D. O.; Carvalho, Y. K. P.; Olivindo, R. F. G.; Fernando, P. B.; Silva, G. C.; Silva, M. L. G.; Ambrosio, C. E.; Conde Junior, A. M.; Argolo Neto, N. M.; Costa Silva, L. M.; Carvalho, M. A. M. Labeling of adipose-derived stem cells with quantum dots provides stable and long-term fluorescent signal for ex vivo cell tracking. In Vitro Cell. Dev. Biol. Anim. 2017, 53 (4), 363-370. (5) Ding, D.; Mao, D.; Li, K.; Wang, X.; Qin, W.; Liu, R.; Chiam, D. S.; Tomczak, N.; Yang, Z.; Tang, B. Z.; Kong, D.; Liu, B. Precise and long-term tracking of adipose-derived


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stem cells and their regenerative capacity via superb bright and stable organic nanodots. ACS Nano 2014, 8 (12), 1262012631. (6) An, F. F.; Yang, Y. L.; Liu, J.; Ye, J.; Zhang, J. F.; Zhou, M. J.; Zhang, X. J.; Zheng, C. J.; Liang, X. J.; Zhang, X. H. A reticuloendothelial system-stealthy dye–albumin nanocomplex as a highly biocompatible and highly luminescent nanoprobe for targeted in vivo tumor imaging. RSC Adv. 2014, 4 (12), 6120-6126. (7) Wang, X.; Xia, J.; Wang, C.; Liu, L.; Zhu, S.; Feng, W.; Li, L. Preparation of Novel Fluorescent Nanocomposites Based on Au Nanoclusters and Their Application in Targeted Detection of Cancer Cells. ACS Appl. Mater. Interfaces 2017, 9 (51), 44856-44863. (8) Li, Y. J.; Yan, X. P. Synthesis of functionalized tripledoped zinc gallogermanate nanoparticles with superlong near-infrared persistent luminescence for long-term orally administrated bioimaging. Nanoscale 2016, 8, 14965-14970. (9) Zheng, B.; Wang, H.; Pan, H.; Liang, C.; Ji, W.; Zhao, L.; Chen, H.; Gong, X.; Wu, X.; Chang, J. Near-Infrared Light Triggered Upconversion Optogenetic Nanosystem for Cancer Therapy. ACS Nano 2017, 11 (12), 11898-11907. (10) Li, J.; Lee, W. Y.; Wu, T.; Xu, J.; Zhang, K.; Hong, W. D.; Li, R.; Li, G.; Bian, L. Near-infrared light-triggered release of small molecules for controlled differentiation and long-term tracking of stem cells in vivo using upconversion nanoparticles. Biomaterials 2016, 110, 1-10. (11) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat. Biotechnol. 2003, 21 (1), 47-51. (12) Yong, K. T.; Roy, I.; Ding, H.; Bergey, E. J.; Prasad, P. N. Biocompatible near-infrared quantum dots as ultrasensitive probes for long-term in vivo imaging applications. Small 2009, 5 (17), 1997-2004. (13) Kuno, M.; Fromm, D. P.; Hamann, H. F.; Gallagher, A.; Nesbitt, D. J. Nonexponential “blinking” kinetics of single CdSe quantum dots: A universal power law behavior. J. Chem. Phys. 2000, 112 (7), 3117-3120. (14) Yong, K. T.; Law, W. C.; Hu, R.; Ye, L.; Liu, L.; Swihart, M. T.; Prasad, P. N. Nanotoxicity assessment of quantum dots: from cellular to primate studies. Chem. Soc. Rev. 2013, 42 (3), 1236-1250. (15) Smith, W. E.; Brownell, J.; White, C. C.; Afsharinejad, Z.; Tsai, J.; Hu, X.; Polyak, S. J.; Gao, X.; Kavanagh, T. J.; Eaton, D. L. In vitro toxicity assessment of amphiphillic polymercoated CdSe/ZnS quantum dots in two human liver cell models. ACS Nano 2012, 6 (11), 9475-9484. (16) Zhang, J.; Chen, R.; Zhu, Z.; Adachi, C.; Zhang, X.; Lee, C. S. Highly Stable Near-Infrared Fluorescent Organic Nanoparticles with a Large Stokes Shift for Noninvasive Long-Term Cellular Imaging. ACS Appl. Mater. Interfaces 2015, 7 (47), 26266-26274. (17) Fischer, I.; Petkau-Milroy, K.; Dorland, Y. L.; Schenning, A. P. H. J.; Brunsveld, P. L. Self-Assembled Fluorescent Organic Nanoparticles for Live-Cell Imaging. Chem. Eur. J. 2013, 19 (49), 16646-16650. (18) Tang, F.; Wang, C.; Wang, J.; Wang, X.; Li, L. Fluorescent organic nanoparticles with enhanced fluorescence by selfaggregation and their application to cellular imaging. ACS Appl. Mater. Interfaces 2014, 6 (20), 18337-18343.

(19) An, F. F.; Ye, J.; Zhang, J. F.; Yang, Y. L.; Zheng, C. J.; Zhang, X. J.; Liu, Z.; Lee, C. S.; Zhang, X. H. Non-blinking, highly luminescent, pH- and heavy-metal-ion-stable organic nanodots for bio-imaging. J. Mater. Chem. B 2013, 1 (25), 31443151. (20) Zhang, J.; Li, C.; Zhang, X.; Huo, S.; Jin, S.; An, F. F.; Wang, X.; Xue, X.; Okeke, C. I.; Duan, G.; Guo, F.; Zhang, X.; Hao, J.; Wang, P. C.; Zhang, J.; Liang, X. J. In vivo tumortargeted dual-modal fluorescence/CT imaging using a nanoprobe co-loaded with an aggregation-induced emission dye and gold nanoparticles. Biomaterials 2015, 42, 103-111. (21) Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes based on AIE fluorogens. Acc. Chem. Res. 2013, 46 (11), 2441-2453. (22) Yan, L.; Zhang, Y.; Xu, B.; Tian, W. Fluorescent nanoparticles based on AIE fluorogens for bioimaging. Nanoscale 2016, 8 (5), 2471-2487. (23) Li, D.; Qin, W.; Xu, B.; Qian, J.; Tang, B. Z. AIE Nanoparticles with High Stimulated Emission Depletion Efficiency and Photobleaching Resistance for Long-Term SuperResolution Bioimaging. Adv. Mater. 2017, 29 (43), 1703643. (24) Zhu, Z.; Leung, C. W.; Zhao, X.; Wang, Y.; Qian, J.; Tang, B. Z.; He, S. Using AIE Luminogen for Long-term and Lowbackground Three-Photon Microscopic Functional Bioimaging. Sci. Rep. 2015, 5, 15189. (25) Battistelli, G.; Cantelli, A.; Guidetti, G.; Manzi, J.; Montalti, M. Ultra-bright and stimuli-responsive fluorescent nanoparticles for bioimaging. WIREs Nanomed Nanobiotechnol 2016, 8 (1), 139-150. (26) Wang, D.; Su, H.; Kwok, R. T. K.; Hu, X.; Zou, H.; Luo, Q.; Lee, Michelle. M. S.; Xu, W.; Lam, J. W. Y.; Tang, B. Z. Rational design of a water-soluble NIR AIEgen, and its application in ultrafast wash-free cellular imaging and photodynamic cancer cell ablation. Chem. Sci.2018, doi : 10.1039/c7sc04963c. (27) Wang, Z.; Chen, S.; Lam, J. W.; Qin, W.; Kwok, R. T.; Xie, N.; Hu, Q.; Tang, B. Z. Long-term fluorescent cellular tracing by the aggregates of AIE bioconjugates. J. Am. Chem. Soc. 2013, 135 (22), 8238-8245. (28) Cheng, Y.; Sun, C.; Ou, X.; Liu, B.; Lou, X.; Xia, F. Dualtargeted peptide-conjugated multifunctional fluorescent probe with AIEgen for efficient nucleus-specific imaging and long-term tracing of cancer cells. Chem. Sci. 2017, 8 (6), 45714578. (29) Liu, M.; Zhang, X.; Yang, B.; Deng, F.; Yang, Y.; Li, Z.; Zhang, X.; Wei, Y. Preparation and bioimaging applications of AIE dye cross-linked luminescent polymeric nanoparticles. Macromol. Biosci. 2014, 14 (12), 1712-1718. (30) Liu, Y.; Deng, C.; Tang, L.; Qin, A.; Hu, R.; Sun, J. Z.; Tang, B. Z. Specific detection of D-glucose by a tetraphenylethene-based fluorescent sensor. J Am Chem Soc 2011, 133 (4), 660-663. (31) Xue, X.; Zhao, Y.; Dai, L.; Zhang, X.; Hao, X.; Zhang, C.; Huo, S.; Liu, J.; Liu, C.; Kumar, A.; Chen, W. Q.; Zou, G.; Liang, X. J. Spatiotemporal drug release visualized through a drug delivery system with tunable aggregation-induced emission. Adv. Mater. 2014, 26 (5), 712-717. (32) Xiong, L.; Guo, Y.; Zhang, Y.; Cao, F. Highly luminescent and photostable near-infrared fluorescent polymer dots for long-term tumor cell tracking in vivo. J. Mater. Chem. B 2015, 4 (2), 202-206.



(33) He, X.; Wang, K.; Cheng, Z. In vivo near-infrared fluorescence imaging of cancer with nanoparticle-based probes. WIREs Nanomed Nanobiotechnol 2010, 2 (4), 349-366. (34) Chen, D.; Li, Q.; Meng, Z.; Guo, L.; Tang, Y.; Liu, Z.; Yin, S.; Qin, W.; Yuan, Z.; Zhang, X.; Wu, C. Bright Polymer Dots Tracking Stem Cell Engraftment and Migration to Injured Mouse Liver. Theranostics 2017, 7 (7), 1820-1834. (35) Liu, J.; Sun, Y. Q.; Zhang, H.; Shi, H.; Shi, Y.; Guo, W. Sulfone-Rhodamines: A New Class of Near-Infrared Fluorescent Dyes for Bioimaging. ACS Appl. Mater. Interfaces 2016, 8 (35), 22953-22962. (36) Liu, J.; Li, K.; Liu, B. Far-Red/Near-Infrared Conjugated Polymer Nanoparticles for Long-Term In Situ Monitoring of Liver Tumor Growth. Adv. Sci. 2015, 2 (5), 1500008. (37) An, F. F.; Chan, M.; Kommidi, H.; Ting, R. Dual PET and Near-Infrared Fluorescence Imaging Probes as Tools for Imaging in Oncology. AM J Roentgenol 2016, 207 (2), 266-273. (38) An, F. F.; Zhang, X. H. Strategies for Preparing Albuminbased Nanoparticles for Multifunctional Bioimaging and Drug Delivery. Theranostics 2017, 7 (15), 3667-3689. (39) Araneda, J. F.; Piers, W. E.; Heyne, B.; Parvez, M.; McDonald, R. High Stokes shift anilido-pyridine boron difluoride dyes. Angew. Chem. Int. Ed. Engl. 2011, 50 (51), 12214-12217. (40) Shcherbakova, D. M.; Hink, M. A.; Joosen, L.; Gadella, T. W.; Verkhusha, V. V. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J Am Chem Soc 2012, 134 (18), 7913-7923. (41) Wang, Z.; Chen, S.; Lam, J. W. Y.; Qin, W.; Kwok, R. T. K.; Xie, N.; Hu, Q.; Tang, B. Z. Long-Term Fluorescent Cellular Tracing by the Aggregates of AIE Bioconjugates. J. Am. Chem. Soc. 2013, 135 (22), 8238-8245. (42) Qin, W.; Li, K.; Feng, G.; Li, M.; Yang, Z.; Liu, B.; Tang, B. Z. Bright and Photostable Organic Fluorescent Dots with Aggregation-Induced Emission Characteristics for Noninvasive Long-Term Cell Imaging. Adv. Funct. Mater. 2014, 24 (5), 635-643. (43) Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J.; Liu, R.; Liu, J.; Zhang, X.; Liu, H.; Liu, B.; Tang, B. Z. Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing. Sci. Rep. 2013, 3, 1150. (44) Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T. D.; Tian, H.; Zhu, W. In vivo and in situ tracking cancer chemotherapy by highly photostable NIR fluorescent theranostic prodrug. J Am Chem Soc 2014, 136 (9), 3579-3588. (45) Zhang, F.; Di, Y.; Li, Y.; Qi, Q.; Qian, J.; Fu, X.; Xu, B.; Tian, W. Highly efficient Far Red/Near-Infrared fluorophores with aggregation-induced emission for bioimaging. Dyes and Pigments 2017, 142, 491-498. (46) Hong, Y.; Lam, J. W.; Tang, B. Z. Aggregation-induced emission: phenomenon, mechanism and applications. Chem. Commun. 2009, (29), 4332-4353. (47) Xu, B. J.; Chi, Z. G.; Li, X. F.; Li, H. Y.; Zhou, W.; Zhang, X. Q.; Wang, C. C.; Zhang, Y.; Liu, S. W.; Xu, J. R. Synthesis and properties of diphenylcarbazole triphenylethylene derivatives with aggregation-induced emission, blue light emission and high thermal stability. J. Fluoresc. 2011, 21 (1), 433441.


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AIE active NIR fluorescent organic nanoparticles (TPFE-Rho dots) are fabricated for real-time longterm in vivo monitoring of tumor growth. TPFE-Rho dots show NIR emission, large Stokes shift (~180 nm), good biocompatibility, and high photostability, which allow tracking cell for 11 generations, and monitoring tumor growth for 19 days.


AIE active NIR Fluorescent Organic Nanoparticles for Noninvasive

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