Indocyanine-Based Activatable Fluorescence Turn-On Probe for γ

Article pubs.acs.org/acssensors

Indocyanine-Based Activatable Fluorescence Turn-On Probe for γ‑Glutamyltranspeptidase and Its Application to the Mouse Model of Colon Cancer Seokan Park,† Soo-Yeon Lim,† Sang Mun Bae,‡,§ Sang-Yeob Kim,‡,§ Seung-Jae Myung,*,‡,∥ and Hae-Jo Kim*,† †

Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea Asan Institute for Life Sciences and ∥Department of Gastroenterology, Asan Medical Center, and §Department of Medicine, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea

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S Supporting Information *

ABSTRACT: An activatable fluorescent probe from indocyanine was developed for the detection of tumor-enriched γglutamyltranspeptidase (γGT). The probe exhibited a dramatic fluorescence enhancement (F/F0 = 10) as well as a bathochromic shift (>100 nm) upon the treatment of γGT with a low limit of detection of 0.15 unit/L and was further successfully applied as a sensitive probe for γGT in the mouse model of colon cancer. KEYWORDS: activatable fluorescent probe, colon cancer, cyanine dye, γ-glutamyltranspeptidase (>100 nm) as well as a fluorescence turn-on effect (F/F0 > 10 at 557 nm) in the presence of γGT in phosphate buffered saline (PBS). Further application to in vivo imaging was successfully carried out with the mouse model of colon cancer.

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uccessful oncologic operation involves accurate surgical resection or ablation of tumor sites, as well as early and specific diagnosis of the suspected cancer. Toward this aim, the optical signal for the specified tissue, especially such as tiny foci of cancer metastases or invading cells, is important because it helps surgeons easily detect the surgical resection sites from the other normal tissues by the naked eye or endoscope. Therefore, recent research has been focused on the molecular imaging probes, which induce large UV−vis and fluorescence spectral changes upon the reaction with a tumor biomarker. In particular, γ-glutamyltranspeptidase (γGT) is under intense research as a tumor-related enzyme, and a higher level of γGT in serum is reported as a risk factor of cancer.1−4 A pioneering study of the Urano group based on rhodamine−glutamic acid conjugate has inspired many researchers with γGT-induced fluorescence turn-on probes.5−7 However, there are only a few reports on real in vivo applications, because of a limited source of red or near-infrared (NIR) fluorophores available with good water solubility. Herein we report a new indocyanine-based fluorescent probe that is readily activated by a tumor-enriched enzyme, γGT. The activatable probe displayed a clear colorimetric change with a dramatic bathochromic shift © XXXX American Chemical Society

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EXPERIMENTAL SECTION

Synthesis. 1. To a solution of 3.0 mmol N-Boc-Glu-OtBu and 3.0 mmol 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) hydrochloride in 6 mL of CH2Cl2 was dropwise added a solution of 4aminobenzyl alcohol (2.97 mmol) in 4 mL of CH2Cl2 at 0 °C and the resulting solution was stirred for an additional 1.5 h. After the reaction was complete, all the volatiles were removed under reduced pressure and the crude product was purified by column chromatography on silica gel (EtOAc-Hexane-MeOH as eluent, 1:2:0.5 v/v) to afford 945 mg of 1 as a white solid (78% yield). 1H NMR (400 MHz, CD3OD): δ 7.54 (d, J = 8.4, 2H), 7.31 (d, J = 8.4, 2H), 4.57 (s, 2H), 4.05 (dd, J = 8.8, 4.8, 1H), 2.46 (t, J = 7.6, 2H), 2.23−2.15 (m, 1H), 2.03−1.94 (m, 1H), 1.49 (s, 9H), 1.45 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 171.8, 171.8, 156.7, 137.6, 137.1, 127.2, 119.8, 81.4, 79.2, 63.5, 54.1, Received: November 27, 2015 Accepted: March 15, 2016

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DOI: 10.1021/acssensors.5b00249 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors Scheme 1. Design Principle and Synthetic Scheme of AP−Glu

32.8, 27.3, 27.0, 26.9. LRMS (MALDI+, DHB): m/z found 431.21, calcd. 431.21 for [M + Na]+. 2. To a solution of 1 (4.4 mmol) and triethylamine (TEA, 4.8 mmol) in 10 mL of CH2Cl2 was dropwise added a solution of ptoluenesulfonyl chloride (TsCl, 4.8 mmol) in 10 mL of CH2Cl2 at 0 °C and the resulting solution was further stirred for 2.5 h. After the reaction was complete, all the volatiles were removed under reduced pressure and the crude product was purified by column chromatography on silica gel (EtOAc-Hexane, 1:2 v/v) to afford 304 mg of the chlorobenzyl intermediate as a white solid (32% yield). To a solution of the intermediate (1.4 mmol) in 12 mL of CH3CN were added 4hydrobenzylaldehyde (1.4 mmol), K2CO3 (7.1 mmol), and NaI (0.4 mmol). The resulting mixture was then heated to 85 °C and stirred for additional 5 h. After the reaction was complete, all the volatiles were removed under reduced pressure and the crude product was purified by column chromatography on silica gel (EtOAc-hexane, 1:2, v/v) to afford 668 mg of 2 as a white solid (92% yield). 1H NMR (400 MHz, CD3OD): δ 9.85 (s, 1H), 7.88 (d, J = 8.8, 2H), 7.60 (d, J = 8.4, 2H), 7.42 (d, J = 8.4, 2H), 7.17 (d, J = 8.8, 2H), 5.17 (s, 2H), 4.05 (dd, J = 9.2, 4.8, 1H), 2.50 (t, J = 7.6, 2H), 2.24−2.15 (m, 1H), 2.05−1.91 (m, 1H), 1.49 (s, 9H), 1.44 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 191.4, 171.8, 164.1, 156.7, 138.5, 131.9, 131.7, 130.1, 128.0, 119.8, 115.0, 81.4, 79.2, 69.7, 54.2, 54.1, 32.9, 27.4, 27.0, 26.9. LRMS (MALDI+, DHB): m/z found 535.26, calcd. 535.24 for [M + Na]+. 3. A solution of 2 (1.3 mmol) and 1-(5-carboxypentyl)-2,3,3trimethyl-3H-indolium bromide (1.8 mmol) in 10 mL of EtOH was refluxed at 80 °C overnight. After the reaction was complete, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2− MeOH, 10:1 v/v) to afford 714 mg of 3 as a dark orange solid (82% yield). 1H NMR (400 MHz, CD3OD): δ 8.46 (d, J = 16.0, 1H), 8.09 (d, J = 8.8, 2H), 7.82−7.77 (m, 2H), 7.66−7.61 (m, 4H), 7.51 (d, J = 16.0, 1H), 7.44 (d, J = 8.4, 2H), 7.22 (d, J = 8.8, 2H), 5.23 (s, 2H),

4.66 (t, J = 7.2), 4.06 (dd, J = 8.4, 4.4, 1H), 2.50 (t, J = 7.2, 2H), 2.29 (t, J = 7.2, 2H), 2.24−2.16 (m, 1H), 2.05−1.94 (m, 3H), 1.67 (s, 2H), 1.77−1.70 (m, 2H), 1.61−1.53 (m, 2H), 1.49 (s, 9H), 1.44 (s, 9H). 13 C NMR (100 MHz, CD3OD): δ 182.1, 171.9, 164.1, 156.8, 156.7, 155.2, 143.6, 140.8, 138.6, 132.3, 131.8, 129.3, 129.2, 128.1, 127.4, 122.7, 119.9, 115.9, 114.5, 109.2, 81.4, 79.2, 69.9, 54.1, 52.3, 46.3, 34.5, 32.8, 28.0, 27.4, 27.2, 27.0, 26.9, 25.9, 25.5, 24.7. 46.2, 39.9, 33.4, 32.5, 28.0, 26.3, 25.7, 25.4, 24.2. HRMS (FAB+, m-NBA): found 769.4302, calcd. 769.4310 for C45H59N3O8 ([M + H]+). AP−Glu. To a solution of 3 (0.10 mmol) in 1 mL of CH2Cl2 were added 1 mL anisole and 0.1 mL CF3CO2H in an ice bath. The resulting solution was cooled down to −10 °C and further stirred for 4 h. After the reaction was complete, all the volatiles were removed under reduced pressure and the crude solid was washed with acetone to afford 11 mg of AP−Glu as an orange solid (13% yield). 1H NMR (400 MHz, CD3OD): δ 8.46 (d, J = 16.0, 1H), 8.08 (d, J = 8.8, 2H), 7.83−7.77 (m, 2H), 7.66−7.62 (m, 4H), 7.51 (d, J = 16.0, 1H), 7.45 (d, J = 8.4, 2H), 7.22 (d, J = 8.8, 2H), 5.23 (s, 2H), 4.66 (t, J = 7.2), 2.66 (t, J = 7.2, 2H), 2.33 (t, J = 7.2, 2H), 2.23−2.17 (m, 3H), 2.05− 1.97 (m, 2H), 1.87 (s, 2H), 1.77−1.70 (m, 2H), 1.60−1.53 (m, 2H). 13 C NMR (100 MHz, CD3OD): δ 182.1, 172.0, 164.1, 155.0, 143.6, 140.8, 138.4, 132.9, 132.0, 129.4, 129.3, 129.2, 128.7, 128.1, 127.4, 122.7, 120.1, 119.8, 115.9, 114.5, 113.5, 109.1, 69.8, 54.3, 54.1, 52.3, 46.2, 39.9, 33.4, 32.5, 27.9, 26.3, 25.7, 25.4, 24.2. HRMS (FAB+, mNBA): found 612.3073, calcd. 612.3074 for C36H42N3O6 ([M]+). UV−vis and Fluorescence Spectral Measurement. A stock solution of AP−Glu in DMSO (10 mM) was prepared and used for UV−vis and fluorescence measurements by dilution with PBS buffer (1×, pH 7.4). In a typical experiment, the sample solution of AP−Glu (20 μM) was prepared by placing 4 μL stock solution of the probe (10 mM in DMSO) into a test tube, and adding PBS buffer to afford 2 mL of working solution of the probe. As usual, fluorescence spectra were obtained by excitation at the isosbestic point of AP−Glu. Both B

DOI: 10.1021/acssensors.5b00249 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors

Figure 1. (A) Time-dependent UV−vis, (B) γGT concentration-dependent fluorescence (λex 460 nm, λem 557 nm) spectra of AP−Glu (20 μM in PBS, pH 7.4) 4 h after treatment of γGT in the absence and presence of an enzyme inhibitor (GGsTop). (C) Naked eye images of AP−Glu (20 μM in PBS, pH 7.4) in the presence of γGT and (D) their epifluorescence images using an IVIS (λex 500 nm, λem 540−660 nm). excitation and emission slit widths were set to 3 nm × 3 nm. All the fluorescence spectra of AP−Glu were monitored 12 h after addition of γGT unless otherwise stated. Tumor Model and Ex Vivo Imaging. All the animal experiments and procedures were performed in compliance with the Principles of Laboratory Animal Care formulated by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea and were consistent with the Institute of Laboratory Animal Resources (ILAR) guidelines. Female mice (BALB/c, 5−6 weeks old) were given a single intraperitoneal injection of azoxymethane (AOM, 10 mg/kg body weight). One week later, the animals were fed with 2% dextran sulfate sodium (DSS, 36−50 kDa in molecular weight) for 7 days via the drinking water, followed by maintaining the basal diet and normal drinking water for 14 days. This administration of DSS in the drinking water was repeated. Colonoscopy (Karl Storz, Tuttlingen, Germany) was performed with an AOM/DSS mouse model to check whether the tumor cancers were grown up adequately in the colon. The mice were sacrificed at the end of the DSS administration (week 14). Fresh colon tissues were obtained from the AOM/DSS mouse model by surgically excising and longitudinally cutting along the main axis to expose the mucosa layer of colon immediately after the sacrifice. Two different types of whole colons, one from the AOM/DSS-treated mice and the other from normal mice as a control, were sprayed with AP−Glu (50 or 100 μM/100 μL PBS). The probe solution of AP− Glu was evenly sprayed on the surface of the mucosa layers of colons by micropipettes and sustained for 10 min. After incubation with the probe, the colon tissues were unfolded and washed off with PBS three times (3 × 500 μL) on a plate with the mucosa layer facing up before the imaging experiment and then their images were obtained in the eXplore Optix MX3 system (ART Advanced Research Technologies Inc., Montreal, Canada). The rainbow fluorescence signals, expressed at the colon tissue, were acquired using the optical system with excitation at 525 nm and emission at BP 575(±50) nm under the condition of 2 mm scan step and polygon selection.

luminescent because of its ether functionality, but was strongly fluorescent through an enzyme-mediated amide hydrolysis and subsequent ether cleavage reaction when exposed to γGT. For this aim, AP−Glu was straightforwardly prepared from 4hydroxybenzylamine by the EDC-mediated amide coupling reaction with N-Boc and O-tert-butyl ester protected γ-glutamic acid. The activation of the benzylic alcohol, followed by Oalkylation of 4-hydroxybenzaldehyde, afforded aldehyde 2. The aldol condensation reaction of 2 with an indolinium salt produced a tert-butyl protected precursor. Final deprotection of both the Boc and tert-butyl ester groups with trifluoroacetic acid allowed the desired activatable probe (AP−Glu) in 3% overall yield over five steps (Scheme 1). UV−vis and Fluorescence Spectra. First, UV−vis spectra of AP−Glu (20 μM in PBS, pH 7.4) was monitored in the presence of γGT (50 unit/L). The absorbance maximum slowly changed from 424 to 525 nm and was saturated in 4 h after incubation of γGT with a pseudoisosbestic point at 460 nm (Figure 1A). Next, we investigated the fluorescence change of AP−Glu (20 μM in PBS, pH 7.4, λex 460 nm) in the presence of γGT. The initial fluorescence of AP−Glu was very weak but its fluorescence became very strong with a maximum wavelength at 557 nm plausibly owing to the γGT-mediated formation of AP. The fluorescence intensity of AP−Glu was dependent on the concentration of the treated γGT and saturated at 30 unit/L of γGT (Figure 1B). The chromogenic and fluorogenic changes of AP−Glu were readily detectable by the naked eye. In the presence of even 1.0 unit/L of γGT, the red color of AP was distinguishable from the initial green color of AP−Glu (Figure 1C). Moreover, the epiluminescence microscopic images of AP−Glu displayed strong and clear fluorescence exclusively with the samples of AP−Glu in the presence of γGT (Figure 1D). Limit of Detection and pH Profile. The detection limit of γGT in PBS buffer (1×, pH 7.4) was determined from the linear range of a fluorescence titration graph of AP−Glu against γGT (λex/λem 460/557 nm) (Figures S10 and S11). The limit

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RESULTS AND DISCUSSION Design Principle. To design a red fluorescent activatable probe for enzyme γGT, a γGT-specific cleavable site was introduced into AP−Glu as an amide bond between arylamine and γ-glutamic acid. Initial AP−Glu was not expected to be C

DOI: 10.1021/acssensors.5b00249 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors

Figure 2. Time-dependent ex vivo fluorescence imaging of the cancerous (+) and normal colons (−) after incubation of 100 (A) or 50 μM AP−Glu (B) in PBS. Tumor sites detectable with the naked eye are designated with white arrows in the first rows.

Strong fluorescence signals in the cancerous colons were observed at the tumor-bearing polyps from 10 min after spraying a high dosage of AP−Glu (100 μM/100 μL PBS), and they remained intense over 60 min (Figure 2A), whereas the fluorescence signals at the low dose of AP−Glu-treated polyps (50 μM/100 μL PBS) are weakly fluorescent (Figure 2B). As expected, the normal colons did not exhibit any significant fluorescence over 60 min either in the high or low dose of AP− Glu. These experiments indicate that the fluorescence signal was dependent on the degree of dosage of AP−Glu. The probe was appreciably activated even at 0.05 mM AP−Glu (50 μM in PBS) by the cancerous enzyme within the tumor sites to produce strong fluorescence of AP. Reaction Mechanism. For a mechanistic study of the activatable probe for γGT, the reaction profile of AP−Glu (1.0 mM) was monitored in the presence of γGT (5 unit/mL) by high performance liquid chromatography on a reverse C18 column. The time-dependent HPLC analysis indicates that the reaction is readily initiated by the enzyme γGT, and the probe (AP−Glu at tR 15 min) undergoes a rapid enzyme-mediated glutamyl cleavage reaction to allow the evolution of the hemicyanine conjugated with phenol (AP at tR 9.0 min) (Figure S14). Furthermore, a selective inhibitor of γGT (GGsTop) was fed on AP−Glu to investigate whether AP−Glu is a suitable and good substrate for γGT.11,12 The introduction of GGsTop (2.0 mM) to a mixture of AP−Glu (20 μM) and γGT (50 unit/L) displayed very weak fluorescence which is comparable to that of AP−Glu without γGT (Figure S13), owing to the competitive inhibition of GGsTop to the enzyme action (Figure 1A, inset). These results indicate that AP−Glu is a good substrate for γGT and rapidly transformed into AP with strong fluorescence through the γGT-specific amide hydrolysis and a subsequent degradation reaction via an amine intermediate (I) (Scheme 1).

of detection of γGT was estimated to be 0.15 unit/L of γGT at 3σ/m, where σ and m represent the standard deviation of blank measurements of AP−Glu and the slope obtained from the linear plot of AP−Glu fluorescence against [γGT], respectively. This value of the limit of detection is among the lowest ones and is 5 times lower compared to the best one recently reported.8 We assumed that the resulting lower detection limit of γGT is attributable to the latent fluorescence of AP−Glu and the dramatic fluorescence turn-on property of AP evolved by the treatment of γGT. AP exhibited a strong and stable fluorescence response with a maximum wavelength at F557 nm in biological pH range (7 to 10), whereas the inherent fluorescence intensities of AP−Glu were negligible over a wide range of pH (3 to 10) (Figure S12). The weakly fluorescent probe (AP−Glu) turned into a strong fluorophore (AP) in the presence of γGT at biological pH 7.4. We assume this type of “Off−On” fluorescence property of AP−Glu to be beneficial for ex vivo application. Ex Vivo Imaging of a Mouse Model of Colon Cancer. A mouse model of colon tumors was grown using two carcinogens as reported: azoxymethane (AOM) and dextran sulfate sodium salt (DSS).9,10 Two groups of colons, one from AOM/DSS-treated mice and the other from normal mice, were excised and the mucosa layer of each colon was exposed by longitudinal cutting. Multiple adenomatous polyps were observable in the colons obtained from the AOM/DSS-treated mouse model, but no polyps in the colons from the AOM/ DSS-free normal mouse (Figure 2). Two whole colons from each group of mice were incubated with either 100 μM AP− Glu (Figure 2A, 100 μL PBS) or 50 μM AP−Glu (Figure 2B, 100 μL PBS) for 10 min. The resulting colons were imaged simultaneously using an eXplore Optix optical imaging system (λex 525 nm, λem BP 575(±50) nm). The fluorescence intensity changes of the cancerous colons were monitored over 60 min and compared to the normal colons. White light images were taken to visualize the adenomatous polyps of the cancerous colons, and overlaid with the fluorescence images of the AOM/ DSS-treated or free mouse colons.

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CONCLUSIONS We developed a latent fluorescence probe for the detection of tumor-enriched enzyme, γGT. The probe was readily activated by γGT with LOD of 0.15 unit/L and successfully applied for D

DOI: 10.1021/acssensors.5b00249 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors

(7) Ramsay, E. E.; Decollogne, S.; Joshi, S.; Corti, A.; Apte, M.; Pompella, A.; Hogg, P. J.; Dilda, P. J. Employing pancreatic tumor γ− glutamyltransferase for therapeutic delivery. Mol. Pharmaceutics 2014, 11, 1500−1511. (8) Hou, X.; Yu, Q.; Zeng, F.; Yu, C.; Wu, S. Ratiometric fluorescence assay for γ−glutamyltranspeptidase detection based on a single fluorophore via analyte−induced variation of substitution. Chem. Commun. 2014, 50, 3417−3420. (9) Greten, F. R.; Eckmann, L.; Greten, T. F.; Park, J. M.; Li, Z.-W.; Egan, L. J.; kagnoff, M. F.; Karin, M. IKKβ links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 2004, 118, 285−296. (10) Ishikawa, T. O.; Herschman, H. R. Tumor formation in a mouse model of colitis associated colon cancer does not require COX-1 or COX-2 expression. Carcinogenesis 2010, 31, 729−736. (11) Yamamoto, S.; Watanabe, B.; Hiratake, J.; Tanaka, R.; Ohkita, M.; Matsumura, Y. Preventive effect of GGsTop, a novel and selective g-glutamyl transpeptidase inhibitor on ischemia/reperfusion-induced renal injury in rats. J. Pharmacol. Exp. Ther. 2011, 339, 945−951. (12) Tuzova, M.; Jean, J.-C.; Hughey, R. P.; Brown, L. A. S.; Cruikshank, W. W.; Hiratake, J.; Joyce-Brady, M. Inhibiting lung lining fluid glutathione metabolism with GGsTop as a novel treatment for asthma. Front. Pharmacol. 2014, 5, 179.

the imaging of a mouse model of colon cancer. The result demonstrates that the enzyme-activatable fluorophore can be applied as a detection probe for cancerous colons and may be useful as a potential endoscopic surgery tool for cancer. Further studies are in progress and will be reported in the near future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.5b00249. NMR and mass spectra of compounds, enzymatic HPLC profiles of the probe, cell viability assays (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +82 2 3010 3917. Fax: +82 2 476 0824. *E-mail: [email protected]. Phone: +82 31 330 4703. Fax: +82 31 330 4566. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported from the National Research Foundation of Korea, Ministry of Science, ICT and Future Planning (NRF−2014R1A2A2A01002430 to H.J.K.) and by the Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by Ministry of Health & Welfare, Republic of Korea (No. HI06C0868 to S.J.M.).

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REFERENCES

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DOI: 10.1021/acssensors.5b00249 ACS Sens. XXXX, XXX, XXX−XXX