Celecoxib Conjugated Fluorescent Probe for Identification and

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Celecoxib conjugated fluorescent probe for identification and discrimination of cyclooxygenase-2 enzyme in cancer cells Bhaskar Gurram, Shuangzhe Zhang, Miao Li, Haidong Li, Yahui Xie, Hongyan Cui, Jianjun Du, Jiangli Fan, Jingyun Wang, and Xiaojun Peng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05337 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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Analytical Chemistry

Celecoxib conjugated fluorescent probe for identification and discrimination of cyclooxygenase-2 enzyme in cancer cells Bhaskar Gurram,† Shuangzhe Zhang,† Miao Li,† Haidong Li,† Yahui Xie,† Hongyan Cui,‡ Jianjun Du,† Jiangli Fan,† Jingyun Wang,‡ and Xiaojun Peng*† †State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, High-tech District, Dalian 116024, China. *Email: [email protected]. Tel/Fax: +86 -411- 84986306. ‡Department School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, High-tech District, Dalian 116024, China. ABSTRACT: Cyclooxygenase-2(COX-2) is an enzyme over expressed in most types of cancers and has been used for an excellent targetable biomarker. Celecoxib is an effective inhibitor of COX-2, used in anti-inflammation. Herein we report a one and two-photon fluorescence probe (NP-C6-CXB) for COX-2, based on the conjugation of naphthalamide with Celecoxib, by using flexible hexylene linker. NP-C6-CXB is non-fluorescent in buffer solution and normal cells, while it shows bright fluorescence in solutions and cancer cells in the presence of COX-2 with an excellent selectivity. Interestingly, NP-C6-CXB can discriminate cancer cells (MCF-7) from normal cells (COS-7) in the single culture medium under confocal microscopy. Due to the selective binding affinity of NP-C6-CXB with COX-2 enzyme, the intensity is proportional to the level of COX-2 enzyme in cancer cells. In vivo and in vitro experiments proved that NP-C6-CXB is a potential tool for identification of tumor and might be used in surgical resection of COX-2 expressed tumors.

INTRODUCTION In recent years, cancer is a deadly global disease. Mortality of cancer patients is increasing year by year. Therefore, it is necessary to overcome difficulties in detection of cancer in early stages. Currently, ionizing radiation technologies include positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI) and X-ray imaging are being used in clinical diagnosis. Nevertheless, these technologies detect only internal and absolute cancers and are of low resolution as well as expensive.1-3 The performance of surgical treatment depends on critical factors like accurate localization and visibility of tumor.4,5 Oversight resection of a tumor would cause effect on surrounding healthy parts and survival rate.6 Fluorescent probes have been used for enzyme detection which was highly expressive in all stages of cancers, for example glutamyl-transpeptidase (GGT), quinoneoxido-reductase isozyme-1 (NQO1) and cyclooxygenase-2 (COX-2) were used to distinguish tumor cells from normal cells.7-9 Likewise, fluorescence visualization of COX-2 in Golgi apparatus of cancer cells, discrimination of cancer from inflammation and normal tissue were also described in detail.10,11

Cancer cells express elevated level of enzymes or an enzyme frequently proteins.9,12,13 COX-2 is over-expressed in cancer cells and very less in normal cells.14-16 Prostaglandin synthesis was regulated by cyclooxygenases (COX-2 or COX-1) which plays a pivotal role in tumor expansion and development.17,18 COX-2 is present in various types of tumors including breast, colonic and cervical cancer cells13,18-20 (such as HT-29, HeLa, MCF-7 cells) and tumor tissues, but COX-2 expresses at low levels in healthy cells (HL-7702 and COS-7 cells), and normal tissues.8,18,21,22 Therefore, we selected the COX-2 as a suitable and excellent targetable biomarker to discriminate cancer cells from normal cells. Celecoxib (CXB) is an inhibitor with excellent selectivity to COX-2 was stated.23,24 Polyamine naphthalimide co-treatment with celecoxib has induced the apoptosis in colorectal cancer cells (HT29, Caco-2), those were COX-2 high expressive cells.25 Celecoxib inhibits cell proliferation in mouse hepatoma cells (HepG 2 cells).26 Also, CXB as a selective inhibitor binds to COX-2 side pocket tightly at Val523, Arg513 and Val434 in COX-2.27 Herein, CXB chose as recognition group for the first time, to the best of our knowledge, linking to a naphthalamide via a flexible hexylene linker28 (NP), constructed a targeted-COX-2 fluorescent probe NP-C6-CXB with excitation with one- (at 488 nm) and

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two-photon (at 840 nm) with high two-photon absorption cross section of 270 GM. (Figure 1)

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reference standard, whose two-photon property has been well characterized in the literature32. The intensities of the two-photon induced fluorescence spectra of the reference and sample emitted at the same excitation wavelength were determined. The TPA cross section was calculated by using equation as follow: δ

=

δr

(SsФrφrcr)

/

(SrФsφscs)

(1) Where the subscripts s and r stand for the sample and reference molecule. The fluorescence integral intensity of the signal collected by a CCD detector was denoted as S. Φ is the fluorescence quantum yield. φ is the overall fluorescence collection efficiency of the experimental apparatus. The number density of the molecules in solution was denoted as c. δr is the TPA cross section of the reference molecule. The measurements were performed as above while stirring the mixture with a small magnetic stirring bar at 25 ± 0.5°C. Data were obtained from replicate experiments (n = 5).

Figure 1. Chemical structures of NP-C6-CXB. a) NP, b) Celecoxib (CXB) and c) NP-C6-CXB.

Cell culturing and staining with NP-C6-CXB: The human breast cancer cells (MCF-7), Human cervical cancer cells (HeLa), African green monkey kidney cells (COS-7) and human normal liver cells (HL-7702) were obtained from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. Live cancer and normal cells were incubated with 10% fetal bovine serum, 1% streptomycin and penicillin medium was supplemented. The live cells were seeded in flat-bottomed flask and incubated at 37ºC under 5% CO2 for 24 h. Before imaging, the live cells were stained with NP-C6-CXB (2.0 µM) for additionally 10 min and washed with phosphate-buffered saline (PBS) for three more times. Fluorescence imaging was performed by an OLYMPUS FV-1000 confocal fluorescence microscope, 60 × objective lenses were used. The stained cell images were acquired by using excitation and emission wavelengths at 488 nm and 500-560 nm, separately.

Compared with indomethacin (IMC, IC50= 0.75 μM) as reorganization group14,29, CXB (IC50= 0.07 μM) has better selectivity to COX-2 and also shows anti-cancer effect8,23,30. We expect that NP-C6-CXB behave with high selectivity and sensitivity towards COX-2 expressive cancer cells. EXPERIMENTAL SECTION Materials and methods: All solvents and reagents used were reagent grade and were used without further purification. The solution of NP-C6-CXB was dissolved in dimethyl sulphoxide (DMSO) at a concentration of 5 mM as the stock solution. 1 H NMR and 13C NMR spectra were recorded on a Bruker Avance II 400 MHz spectrometer. Chemical shifts (δ) were reported as ppm (in DMSO or CDCl3, with TMS as the internal standard). Mass spectrometric data were carried out using TOF-MS instruments. Absorption spectra were measured on a Cary 60 UV-V spectrophotometer (Agilent technologies, USA). Fluorescence spectra were obtained with a Cary Eclipse Fluorescence (Agilent technologies, USA). The absolute fluorescence quantum yields of NP-C6-CXB in different solvents were determined by Absolute PL Quantum Yield Spectrometer (HAMAMATSU C11347). Excitation and emission slit widths were modified to adjust the fluorescence intensity to a suitable range. NBD-C6-CX (GREEN) and BODYPY FL-C5-ceramide (RED) were purchased from Life Technologies Co. (USA). Frozen tissue sections were prepared by Leica CM1860 UV (Germany). Mice with tumors (MDA-MB-231 and S180 sarcoma) were purchased from SLAC Laboratory Animal Co. (Shanghai, China).

Inhibition concentration (IC50): Human COX-2 enzyme (0.4 μg/ml) was taken into 96-well micro plate, NP-C6-CXB was added at different concentration (0-5 μM) to above micro plates. The measurements were taken after 30 min incubation at 25°C. COX-2 activity data were obtained micro-plate reader (Thermo Fisher Scientific). Native-PAGE: MCF-7 cells were incubated at 37ºC under 5% CO2 with NP-C6-CXB at varying concentrations (0, 5, 10 μM) for 30 min. For competitive experiments, cells were pre-incubated with 10 μM celecoxib for 1 h. and then added NP-C6-CXB (10 µM) were added to individual culture medium for another 30 min. When digested by trypsin, the cells fell off from culture flask, and then treated with 1 mL lysate and tissue proteomes. After concentrate through the over peed centrifugal, the protein extracts were obtained. Add 25 μL 5 × Native-PAGE buffers (0.2 M Tris/40% glycerol /0.4% bromophenol blue) into 100 μL protein supernatant for gel electrophoresis. Bands were visualized by staining with Coomassie blue staining. Fluorescent labeling, the protein bands were visualized

Two-photon properties of NP-C6-CXB: The two-photon cross section (δ) was determined by using femto second (fs) fluorescence measurement technique as described in the literature31. NP-C6-CXB were dissolved in Tris-HCl buffer pH 8.0 at concentrations of 5.0×10-3 M and then the two-photon induced fluorescence intensity was measured at 700-900 nm by using Rhodamine B as the 2


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Analytical Chemistry using a Night-OWL II LB983 small animal in vivo imaging system containing a sensitive Charge Coupled Device (CCD) camera, through excitation laser of 480 nm and an emission filter of 550 nm was used for NP-C6-CXB.

The cultured medium was washed sensibly with PBS and then DMSO (200 μL) was added, purple crystals were observed in the medium. The optical density was determined by the microplate reader (Thermo Fisher Scientific). Cell viabilities were expressed as percent of the control culture value.35,36

Fluorescence image of live cells co-stained with Golgi-tracker: NP-C6-CXB (2.5 μM) was added to cells and BODYPY FL C5-ceramide (Golgi-tracker, 2.5 μM) was used for co-stain the cells. Cells were incubated under 5% CO2 at 37ºC for 30 min and washed with PBS for trice. Confocal fluorescence images were acquired with inverted confocal fluorescence microscope (OLYMPUS FV-1000), by using a 60× objective lens. NP-C6-CXB (green channel) was excited at 488 nm and the emission spectra were scanned at 500-550 nm. The BODYPY FL-C5-ceramide probes (commercial dye) were excited at 630 nm and the emission spectra were taken at 640-700 nm (Red channel).

RESULT AND DISCUSSION Design of COX-2 selective two-photon fluorescent probe: Naphthalimide derivatives had been widely used in the field of biochemical sensing owing to its excellent photo-physical properties including high two-photon absorption cross section, photostability and spectral adjustable. Celecoxib (CXB) is an inhibitor with outstanding selectivity to COX-2. Hence, the fluorophore (Naphthalimide) was connected with inhibitor (CXB) through a flexible alkyl chain demonstrated in Figure 1. The probe NP-C6-CXB was inhibited due to PET process in solution. When it combined the target, the fluorescent intensity became strong become of PET process liberated. Spectral properties of NP-C6-CXB and response to COX-2, NP-C6-CXB (λex 488 nm) has very weak fluorescence in aqueous buffer solution, when interacted with COX-2, restored fluorescence for the reason that CXB binds to COX-2 side pocket of three amino acids(Val523, Arg513 andVal434), leads to fluorescence emission (Figure 3A). The IC50 values for NP-C6-CXB and CXB are 0.4 μM and 0.35 μM respectively (Figure 2a).23 NP-C6-CXB has selective binding ability towards COX-2.

Flow cytometry (Competition with Celecoxib): MCF-7 cells were cultured in DMEM as a supplemented medium with 10% fetal bovine serum (FBS) under an atmosphere of 5% CO2 and 95% air at 37°C. Only MCF-7 cells were regarded as a control group (figure S5 a, d). NP-C6-CXB (2.5 or 5.0 μM) were cultured in MCF-7 cells and incubated for 20 min (figure S5 b, e). Pre-incubated with Celecoxib (5 μM) for 3 hour and then added 2.5 or 5.0 μM of NP-C6-CXB, the cells were incubated for 20 min (figure S5 c, f). Cells were analyzed in a FAC Scan cytometer (Becton Dickinson Biosciences Pharmingen, USA), and all data were analyzed with Cell Quest software. Fluorescence imaging in vivo: All procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources. The tumor implants were established by sub skin injection of 1 × 106 to 2×106 cells suspended in 200 to 300 μL of PBS in BALB/c nude mice. Experiments with tumor bearing mice were performed about 8 days, when the tumor grew up approximately about to 0.5 cm in size. The subcutaneous injection of the NP-C6-CXB (200 μM/100 μL) was injected away 2 cm from the tumor region in tumor mice.33 The mice with tumors were given a, near the enterocoele. Afterwards injected the NP-C6-CXB for 40 min, the mice were imaged using a Night OWL II LB983 small animal in vivo imaging system with 488 nm excitation laser and 560 nm emission filter.34

Synthesis procedures of NP-C6-CXB and intermediates were characterized by the 1H-NMR, 13C-NMR and ESI-HRMS (Scheme S1and S6-S11). Two-photon fluorescent microscopy (TPM) has a crucial advantage that two near-infrared photons as the excitation source offers an excellent application over single-photon microscopy, including depth penetration into tissues, localization of excitation is higher and longer observation time, due to less photo bleaching and photo damage of sample.37,38 The cross section value (Φδ max) of NP-C6-CXB is 270 GM at 840 nm (1GM =10−50 cm4 s/photon), definitely high enough for TPM39-41 (Figure 2b).

Cytotoxicity experiments: Cell viability measurements were calculated by using MTT (3-(4, 5)-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide) to formazan crystals using mitochondrial dehydrogenases. MCF-7 were seeded in 96-well micro plates, density for each one is about 1×105 cells/mL in 100 μL of medium containing 10% FBS. After 24 h of cell attachment, the plates were washed twice with PBS (100 μL/well). The cells were then cultured in medium with 2.5 and 5.0 μM of NP-C6-CXB for 24 hours. Cells in culture medium without NP-C6-CXB were used as the control. Six replicate wells were used to make each one as a control and test concentrations. MTT (10 μL, 5 mg/mL) prepared in PBS was added to each well and the plates were incubated in 5% CO2 humidified incubator at 37ºC for another 4 hrs.

Figure 2. a) Dose-inhibition curves of NP-C6-CXB (0 - 5.0 μM) with COX-2 (0.5 μg/ml) enzyme; b) Two–Photon absorption cross section of NP-C6-CXB in PBS buffer. For the response to COX-2 in vitro, NP-C6-CXB (5.0 μM) showed non-fluorescent in Tris-HCl buffer solution (pH 8.0) at 250C due to electron transfer between CXB and 3



NP. The electron transfer process of the NP (fluorophore) and CXB (inhibitor) was analyzed by cyclic voltammetry (CV),42 we observed the HOMO energy level of NP-C6 and CXB to be -6.44 and -6.38 eV, accordingly, with LUMO level of NP-C6 was -3.49 eV, indicated as an electron transfer from CXB (donor-HOMO) to NP-C6 (acceptor-HOMO) (Figure S2a).36 In addition, the HOMO energy level of NP-C6 (-6.44 eV) is differentiating with HOMO level of NP-C6-CXB (-5.81 eV) (Figure S2 b, c), this energy variation of NP-C6 and NP-C6-CXB was assumed the electron withdrawing nature of naphthalimide moiety, which was determined the electron transfer process in NP-C6-CXB.42 While, adding or increasing concentration of COX-2 (0.05-0.4 µg/mL) enzyme (Figure 3A), increases the fluorescence intensity level of Tris-HCl buffer owing to the binding of CXB moiety to COX-2 and restraining of the interaction between NP and CXB moieties

concentration of COX-2 enzyme. NP-C6-CXB was a photo-stable probe with strong intensity profile with cancer cells (Figure S3).

Figure 4. Fluorescent images of live cells stained with NP-C6-CXB (2.5 μM) under excitation of one-photon (above, λex 488 nm) in and two-photon (below, λem 840 nm) with emission of 500-560 nm in MCF-7 (a and e), HeLa (b and f), HL-7702 (c and g), and COS-7 (d and h) cells.

The native polyacrylamide gel electrophoresis (native-PAGE) analysis was used to investigate whether NP-C6-CXB specifically binds to the COX-2 enzyme. MCF-7 cancer cell lines were treated with NP-C6-CXB (0, 5 and 10 μM) for 30 min. The fluorescence intensity bands observed in gel contains COX-2. Additionally, intensity depends on the concentration of probe. In difference, cells pre-treated with Celecoxib shows weak fluorescence intensity (Figure 3 B). These data prove that NP-C6-CXB selectively binds to the COX-2 enzyme.

Discrimination of cancer cells from normal cells in single medium: The living cancer and normal cell lines in a same culture dish were incubated with NP-C6-CXB (2.5 μM) for 10 min, and images were acquired by using excitation (λex 488 nm) and emission windows (λem 500–560 nm) for NP-C6-CXB. Cancer cell lines (MCF-7) expressed much stronger fluorescence intensity than normal (COS-7) cells with NP-C6-CXB (Figure 5).

Figure 3. Fluorescence emission spectra of NP-C6-CXB (λex 488 nm) in the presence of COX-2 (0 to 0.4 µg/ml ) in o Tris-HCl buffer at 25 C (A); and Native-PAGE analysis of NP-C6-CXB labeling (B): a, Coomassie brilliant blue staining; b, fluorescence image (λex 488 nm and λem 500-560 nm; Lane 1: purified COX-2; lane 2–4: protein extracts of MCF-7 cells incubated with various concentrations of NP-C6-CXB for 30 min; lane 5: Protein extracts of MCF-7 cells pre-incubated with 15 μM of Celecoxib for 1.5 h and then added 5.0 μM of NP-C6-CXB for another 30 min.

Figure 5. a) Cancer (MCF-7) and normal cells (COS-7) were stained with NP-C6-CXB (2.5 μM) in same culture medium (λex 488 nm, scan range λem 500-560 nm), b) The fluorescence intensity of probe NP-C6-CXB in different cells.

Imaging COX-2 in live cells and differentiating cancer cells: NP-C6-CXB (2.5 μM) was incubated for 10 min in different living cell lines. Images were acquired by using λex 488 nm (or 870 nm for two-photon) and λem 500–560 nm. Cancer cell lines (MCF-7 and Hela) expressed strong fluorescence (Figure 4a, 4b, 4e and 4f) with NP-C6-CXB, whereas COS-7 and HL-7702 cells expressed weak fluorescence (Figure 4c, 4d, 4g and 4h) due to the low

CXB competition experiments: CXB competition experiment had supported the selectivity for COX-2. When MCF-7 and Hela cells were pre-incubated with Celecoxib (0, 5 and 10 μM) for 1.5 h before adding NP-C6-CXB (2.5 μM), the fluorescence 4


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Analytical Chemistry intensity was inhibited by increasing concentration of Celecoxib (Figure 6 c and f). After MCF-7 cells were pre-treated with Celecoxib (5 μM) for 2 or 3 h, then added NP-C6-CXB (2.5 μM) and analyzed by flow cytometer, little fluorescence intensity was observed. Interesting, MCF-7 cells treated by NP-C6-CXB (2.5 μM) showed high fluorescence intensity (Figure S5). The flow cytometer experiments proved that NP-C6-CXB has excellent potential for differentiating cancer cells via high throughput analysis.

Figure 7. Co-localization Fluorescence images of NP-C6-CXB (2.5 μM) with commercial BODYPY FL-C5-ceramide (2.5 μM) in cancer cells (MCF-7). a) Green emission of NP-C6-CXB (λex 488 nm, λem 500-560 nm); b) Red emission of Golgi tracker (λex 635 nm, λem 640-700 nm); c) Overlay of the both green and red channels; d) Pearson's co-efficient graph of overlay, and e) intensity profile of cross co-stain image. The co-localization coefficient was measured by the Pearson’s co-relation factor.43 NP-C6-CXB (Figure 7a) and BODYPYFL-C5-ceramide (Figure 7b) were localized at Golgi complex in MCF-7 cells (Figure 7c) with Pearson’s co-relation factor 0.96 (Figure 7d) and cross co-stain intensity profile (Figure 7e) of both probes merged correctly. These results proved that NP-C6-CXB was particularly located in Golgi (where COX-2 is over-expressed). Similarly, HeLa (Figure S4) cancer cells also showed exact localization of NP-C6-CXB in Golgi.

Figure 6. Competition of CXB with NP-C6-CXB (2.5 μM) for COX-2 in MCF-7 (A): a) 0; b) 5; c) 10 μM pre-incubated CXB. and Hela (B): d) 0; e) 5; f) 10 μM pre-incubated CXB, for 1 hour and then added 2.0 μM of NP-C6-CXB (λex 488 nm and λem 500-560 nm); Quantitative image analysis of the average total fluorescence intensity of MCF-7 cell (C) and HeLa cell (D).

Fluorescence imaging in tissues slices and tumor mouse: The cancer tissue and healthy liver tissue from Balb/c nude mouse were incubated with NP-C6-CXB (2.5 μM) for 20 min, afterwards washed twice with phosphate-buffered saline (PBS).

Golgi-localization: The maximum COX-2 enzyme was expressed in Golgi complex, so that we selected commercial BODYPYFL-C5-ceramide (Golgi localization) for comparison. NP-C6-CXB (2.5 μM) and BODYPYFL-C5-ceramide (2.5 μM) co-stained with MCF-7 and Hela cells.

Figure 8. Fluorescence imaging of cancer and normal tissues. (A): a) bright field; b) overly; c) fluorescence of cancer tissue; (B): a) bright field; b) FL depth; c) 3D depth 5



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image of cancer tissue and (C): d) bright field; e) overly; d) fluorescence of normal mouse liver tissue; f) fluorescence of normal tissue. Images were acquired by NP-C6-CXB (2.0 μM) using excitation (λex 840 nm) and emission windows (λem 500–560 nm). Fluorescence images were acquired by the OLYMPUS FV-1000 inverted confocal fluorescence microscope, with a 60 × objective lenses. NP-C6-CXB (Green channel) was excited at 488 nm and the emission wavelengths were scanned at 500-550 nm. The cancer tissue expressed strong fluorescence with deep penetration (500 μm) into tissue (Figure 8c), although normal liver tissue has no fluorescence intensity observed (Figure 8f).

Figure 10. MTT assay of NP-C6-CXB in living MCF-7 cells for 24 h. Conclusions

NP-C6-CXB (50 μM) was dissolved in PBS (100 μM) and injected to MCF-7 tumor mouse by subcutaneous injection. The fluorescence excitation at 488 nm and emission at 500-550 nm was fixed in the small animal imaging system (Night OWL II LB983). The MCF-7 tumor bearing mouse expressed high-level fluorescence at a particular point of the tumor (Figure 9A). From the same tumor mouse, we collected tumor, liver, heart, kidney, spleen by resection (Figure 9B). The strong fluorescence emission observed particularly in tumor tissue (Figure 9Ba) and no fluorescence signal was observed in normal organs, which means there was no probe absorption in normal tissue and organs (Figure 9Bb-f).

We report a COX-2 active, one- and two-photon fluorescent probe (NP-C6-CXB). The probe exhibits strong one- and two-photon fluorescence emission in the presence of cancer cells expressing high level of COX-2, because of selective binding of COX-2 inhibitor (CXB). NP-C6-CXB can discriminate cancer cells from normal cells, even in the same culture medium. The experiments on tumor mouse and organs show excellent selective visualization on tumor site with high fluorescence intensity. Interestingly, MTT analysis showed no cytotoxicity to living cells. Live cell imaging shows selective fluorescence intensity to discriminate cancer from normal healthy cells and the intensity of NP-C6-CXB depends on the amount of COX-2 present in cancer cells. Flow cytometer analysis shows the COX-2 selectivity of NP-C6-CXB. In vitro and in vivo experiments proved that NP-C6-CXB is a potential tool for identification of a tumor, as we expected.

ASSOCIATED CONTENT Supporting Information Synthetic protocol and characterization data for NP-C6-CXB, living cell imaging by OPM and Photo-stability in cells were shown in Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.

Figure 9. Fluorescence imaging of tumor mouse and organs by using NP-C6-CXB. A (a) white light and (b) merged; B (a) tumor; b) liver; c) spleen; d) intestine ; e) heart and f) kidney.

AUTHOR INFORMATION Corresponding Author

*Email: [email protected]. Fax/Tel: +86 -411- 84986306.

Cytotoxicity

Notes

NP-C6-CXB (0, 2.5, 5.0, 10, 15 μM) was added to living MCF-7 cells and incubated for 24 h, analyzed by micro plate reader. The experimental results demonstrated that NP-C6-CXB has no cytotoxicity in live MCF-7 cells (Figure 10).

The authors declare no competing financial interests.

ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of China (21421005, U1608222, 21422601, 21576037 and 21406028).

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Celecoxib Conjugated Fluorescent Probe for Identification and

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