Development of a long-lived luminescence probe for visualizing β

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Development of a long-lived luminescence probe for visualizing #-galactosidase in ovarian carcinoma cells Wanhe Wang, Kasipandi Vellaisamy, Guodong Li, Chun Wu, Chung-Nga Ko, Chung-Hang Leung, and Dik-Lung Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03114 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017

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Development of a long-lived luminescence probe visualizing β-galactosidase in ovarian carcinoma cells

for

Wanhe Wang,†,§ Kasipandi Vellaisamy,†,§ Guodong Li,‡,§ Chun Wu,† Chung-Nga Ko,† ChungHang Leung,*,‡ and Dik-Lung Ma*,† †

Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China. *E-mail: [email protected]. Fax No.: (+852) 3411-7348. ‡ State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. *E-mail: [email protected]. Fax No.: (+853) 2884-1358 ABSTRACT: β-Galactosidase (β-gal) is an important biomarker for ovarian cancers. In this work, we designed and synthesized a novel iridium(III)-based probe 1 for discriminating ovarian carcinoma cell lines from normal cell lines. The probe could detect β-gal even in the presence of a highly autofluorescent background. The probe also showed a good linear response to β-gal between 0 and 30 U/mL, with a detection limit of 0.51 U/mL. Importantly, complex 1 could selectively “light up” ovarian carcinoma cells, while exhibiting negligible luminescence in normal cells. Overall, complex 1 could be potentially used as a useful probe for detecting β-gal expression in the context of ovarian cancer diagnostics.

β-Galactosidase (β-gal) is a glycoside hydrolase enzyme that cleaves the glycosidic bond of βgalactosides, forming monosaccharides.1 It plays a key role in Jacoband Monod’s development of the operon model for the regulation of gene expression.2 β-Gal has also been demonstrated as an important biomarker for cell senescence and primary ovarian cancer.3-5 Particularly, β-gal is an excellent biomarker for ovarian carcinoma.3,6 Therefore, methods for the sensitive detection of β-gal could aid in the monitoring of ovarian cancer development. Numerous detection tools have been developed to detect β-gal,7-11 such as colorimetric assays, immunostaining assays and histochemical methods. However, those methods suffer from drawbacks, such as low sensitivity, tedious sample preparation and high cost. Luminescence imaging has become a powerful tool to study biological molecules in living systems, due to its high sensitivity, compatibility with living cells and minimally invasive mode of detection.12-14 Up to now, a range of fluorescent probes have been developed for visualizing β-gal activity in living cells.15-27 However, fluorescent probes are limited by the high endogenous fluorescence of biological samples. Moreover, few probes have demonstrated the ability to detect β-gal activity in ovarian carcinoma cell lines,28-31 with some instead being demonstrated in cultured cells transfected with lacZ to express high levels of β-gal.15,16,19-27 Recently, Urano’s group reported a fluorescent probe to detect β-gal activity in different types of ovarian cancer cell lines.28

However, the ability of the probe to distinguish between ovarian carcinoma cells and normal cells was not explored. Iridium(III) complexes have emerged as alternative scaffolds for the development of luminescence probes for biomolecules due to their large Stokes shift that could limit interference between excitation and emission, and long-lived phosphorescent emission that could allow their detection in strongly autofluorescent samples.32-35 We herein designed and synthesized the cyclometalated iridium(III) complex 1 as a long-lived luminescent probe for β-gal activity in living cells (Scheme 1). The sensing mechanism of this probe is based on the fact that the photophysical properties of octahedral phenanthroline– iridium(III) cyclometalated complexes are highly sensitive to the substitution pattern on the phenanthroline scaffold and a change of the local environment of the complexes.36-39 Here, the galactose moiety on complex 1 acts both as an enzyme-active trigger as well as a quencher of luminescence. Upon hydrolysis by β-gal, the

Scheme 1. Chemical structures of iridium(III) complexes 1 and 2, and a schematic diagram showing the mechanism of β-gal detection.

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dealkylated complex 2 is formed which emits with a yellow and long-lived phosphorescence by binding to βgal or other proteins. To the best of our knowledge, this complex is the first light-up phosphorescent iridium(III) probe for β-gal that can also distinguish ovarian carcinoma cells from normal cells.

EXPERIMENTAL SECTION Materials and cell lines. 7-(diethylamino)-4-methyl-2H1-benzopyran-2-one (Cm460) was purchased from J&K Chemical Ltd. (China). Iridium chloride hydrate (IrCl3·xH2O) was purchased from Precious Metals Online (Australia). Bovine serum albumin (BSA, B9000S) was purchased from New England Biolabs (Massachusetts, USA). Human serum (H4522), rhodamine B, thioflavin T (ThT) and β-galactosidase (β-gal, G5635-5KU) and other reagents, unless specified, were purchased from Sigma Aldrich (St. Louis, MO) and used as received. SKOV3 and OVCAR3 cell lines were a generous gift from Dr. Wen-An Qiang. Other cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco BRL (Gaithersburg, MD, USA). Cells were cultured in DMEM supplemented with 10% FBS (20% FBS for OVCAR3) and in 5% CO2 in a 37 °C incubator. Synthesis of complex 1. Complex 1 was prepared according to (modified) literature methods.40,41 The precursor iridium(III) complex dimer Ir2(ppy)4Cl2 (where ppy = 2-phenylpyridine) was prepared as previously reported. Then, a suspension of Ir2(ppy)4Cl2 (0.1 mmol) and the corresponding N^N ligand S5 (2-((1,10phenanthrolin-5-yl)oxy)-6-(hydroxymethyl)tetrahydro2H-pyran-3,4,5-triol) (0.22 mmol) in a mixture of dichloromethane (CH2Cl2): methanol (MeOH) (1:1, 10 mL) was stirred overnight under a nitrogen atmosphere. The work-up procedure was the same as previously reported. Complex 1 was characterized by 1H-NMR (400 MHz, MeOD) δ 9.01 (d, J = 8.6 Hz, 1H), 8.56 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 4.9 Hz, 1H), 8.09 (d, J = 4.9 Hz, 1H), 8.03 (d, J = 8.2 Hz, 2H), 7.85 – 7.81 (m, 1H), 7.81 – 7.75 (m, 2H), 7.71 (dd, J = 11.2, 4.1 Hz, 2H), 7.35 (dd, J = 10.3, 5.4 Hz, 2H), 6.98 (t, J = 7.6 Hz, 2H), 6.83 (dd, J = 14.4, 8.2 Hz, 4H), 6.35 – 6.27 (m, 2H), 5.31 (d, J = 7.8 Hz, 1H), 4.82 (m, 1H), 4.75 (m, 1H), 4.73 (m, 1H), 3.89 (d, J = 9.8 Hz, 1H), 3.63 (dd, J = 15.8, 6.8 Hz, 3H), 3.55 – 3.45 (m, 1H), 3.45 – 3.31 (m, 2H). 13C NMR (101 MHz, MeOD) δ 160.07, 152.15, 151.07, 148.68, 143.99, 140.08, 138.19, 137.61, 133.72, 131.45, 130.09, 126.15, 124.62, 122.99, 119.58, 99.11, 76.48, 75.87, 73.62, 73.37, 69.95, 61.16. HRMS: Calcd. for C40H34F6IrN4O6P [M–PF6]+: 859.2104 Found: 859.2120. β-Gal detection. 1 mM of complex 1 stock solution was prepared in DMSO. Complex 1 (2.5 µM) and different concentrations of β-gal were incubated in PBS buffer (2×, pH 7) at 37 °C for 1.5 h. Luminescence emission spectra were recorded on a PTI QM-4 spectrofluorometer (Photo Technology International, Birmingham, NJ) at ambient

Figure 1. Synthetic route of complexes 1 and 2. Conditions: a) HClO, tetrabutylammonium hydrogensulfate, CH2Cl2/H2O, pH 8.2–8.3; b) H2SO4, 100 °C; c) 2,3,4,6-tetra-O-acetyl-alpha-Dgalactopyranosyl bromide, Cs2CO3, ACN, r.t.; d) NaOMe, MeOH/H2O, r.t.; e) and f) Ir2(ppy)2Cl2, CH2Cl2/MeOH, NH4PF6, r.t.. temperature, with the slits for both excitation and emission set at 1 nm. Confocal imaging. Cells were seeded into a glassbottomed dish (35 mm dish with 20 mm well). After 12 h, cells were incubated with complex 1 for the indicated time periods or concentrations, followed by washing with phosphate-buffered saline three times. The luminescence imaging of complexes in cells was carried out by a Leica TCS SP8 confocal laser scanning microscope system. The excitation wavelength was 405 nm. Measurement of β-gal activity. Experiments were performed using a β-galactosidase assay kit according to the manufacturer’s instructions (Beyotime, Shanghai, China). Cells were harvested in lysis buffer after incubation for 24 h at 37 °C in a CO2 incubator. To each 96-well were added 50 μL of cell lysate and 50 μL of Test Reagent. The plate was incubated at 37 °C for 3 h. To each well, 150 μL of Stop Solution was added to quench the reaction. The intensity of absorbance was determined by a SpectraMax M5 microplate reader at a wavelength of 420 nm.

RESULTS AND DISCUSSION Synthesis of cyclometalated iridium(III) complexes. The synthesis of complexes 1 and 2 is shown in Figure 1. The synthesis of complex 1 begins with commercially available 1,10-phenanthroline monohydrate S1, which is treated with aqueous hypochlorite (CLOROX) (with pH carefully maintained between 8.2 and 8.3) to obtain the racemic epoxide S2.42,43 The epoxide S2 is slowly added over 40 min with stirring to concentrated sulfuric acid at 0 °C, and the yellow reaction mixture was heated to 100 °C for 1 h. After completion of reaction, the reaction mixture was diluted with cold water and neutralized to pH 7, forming 1,10-phenanthrolin-5-ol S3 as a dark red solid.43 The O-alkylation of compound S4 was achieved by treating with commercially available 2,3,4,6-tetra-Oacetyl-alpha-D-galactopyranosyl bromide in the presence of cesium carbonate in acetonitrile to give the protected

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Figure 2. (a) Luminescence spectra and the relationship between luminescence intensity of the 1/β-gal incubation system at λ = 550 nm in response to various activity of β-gal: 0, 2, 4, 6, 8, 10, 13, 17, 20, 25, 30 U/mL. (b) Linear plot of the change in luminescence intensity at λ = 550 nm vs. β-gal activity. (c) Relative luminescence intensity of complex 1 (2.5 µM) at λ = 550 nm to 20 µg/mL BSA or 100 µM other interferences. The blue bars show the relative luminescence intensity of complex 1 and the addition of various interferences. The red bars show the relative luminescence intensity after the further addition of 20 U/mL β-gal. Excitation was at 282 nm. glycosylated compound S4 in good yield.28 Compound S4 was subsequently deprotected using sodium methoxide in methanol for 1 h, giving compound S5 after purification by column chromatography.28 Finally, compound S5 was treated with cyclometalated dichloro-bridged dimer Ir2(ppy)2Cl2 (where ppy = 2-phenylpyridine) in CH2Cl2/MeOH (1:1, v/v) overnight, followed by anion exchange with PF6–, producing the desired complex 1 in good yield.40 For comparative analysis, we also synthesized complex 2, which is the putative product formed after hydrolysis of the sugar moiety of 1 by β-gal. The complexes 1 and 2 were fully characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS) (Figure S1). Additionally, the stability of complex 1 was investigated by UV–Vis and 1H NMR experiments (Figure S2).44 The results showed that complex 1 was stable in 20 mM Tris–HCl buffer containing 20% acetonitrile (ACN) and 20 mM NaCl (pH = 7.4) or in 90% DMSO-d6/10% D2O at 298 K over at least 3 days. We also investigated the photostability of complex 1 using timebased luminescence spectroscopy (Figure S2C). The luminescence intensity of complex 1 showed almost no change over a course of 1800 s, while the luminescence of a well-known organic dye, ThT, decreased gradually under the same conditions. Complex 1 exhibited maximum excitation and emission at 282 nm and 586 nm, respectively (Figure S3). Other photophysical properties of complexes 1 and 2 are presented in Table S1.

Figure 3. Time-resolved spectra of complex 1 (5 μM) with or without 30 U/mL β-gal in the presence of Cm460 (0.1 µM) or rhodamine B (0.1 µM). Time gate (a) 0 ns delay or (b) 333 ns delay in the presence of Cm460, and time gate (c) 0 ns delay or (d) 333 ns delay in the presence of rhodamine B. λex = 355 nm. Luminescent response of cyclometalated iridium(III) complex 1 to β-gal. We first tested whether complex 1 responded to β-gal in PBS buffer. Remarkably, the luminescence of complex 1 was remarkably enhanced after the treatment with β-gal (Figure S4). β-Gal itself showed negligible luminescence, suggesting that the enhanced luminescence arose from its interaction with complex 1. To obtain the optimal performance of the probe, conditions such as incubation time, PBS concentration, pH and complex concentration were optimized (Figure S5). The results showed that the optimal conditions were 1.5 h incubation time, 2× PBS, pH 7, and 2.5 µM complex 1. Next, we incubated complex 1 with different concentrations of β-gal (Figure 2a). The luminescence of the system was enhanced with the increase of β-gal concentration ranging from 0 to 30 U/mL.The emission of the system showed good linear relationship with β-gal activity from 0 to 30 U/mL (R2 = 0.9924), with a detection limit of 0.51 U/mL to according to LOD = 3σ/s, where σ was standard deviation of six blank signals and s was the slope of the calibration curve. (Figure 2b). To investigate the selectivity of complex 1 for β-gal, the response of complex 1 against some potential interfering species in biological system was examined. There was negligible luminescence response upon the addition of 100 µM glutathione (GSH), cysteine (Cys), homocysteine (Hcy), magnesium ion (Mg2+), adenosine triphosphate (ATP), S-adenosyl methionine (SAM) and 20 µg/mL bovine serum albumin (BSA) (Figure 2c). Moreover, we investigated the luminescence response of complex 1 to β-gal in the co-existence of other interfering species. The results showed that the luminescence response of complex 1 to β-gal was similar in the absence and in the presence of the other species. Taken together, these results showed that the probe 1

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Figure 4. Confocal imaging of SKOV3 cell lines incubated with different concentrations of complex 1 (0, 3, 10, 30, and 60 μM) for 12 h at 37 °C. Excitation was at 405 nm, and luminescence images were recorded from 540 to 640 nm. The scale bar is 50 µm. had good selectivity for β-gal over other common interferences in cells. Time resolved emission spectroscopy (TRES) of complex 1 against β-gal. To verify that the luminescent response of 1 to β-gal could be distinguished from the autofluorescent background of biological samples, we tested the luminescence response of 1 in the presence of the fluorescent organic dyes Cm460 and rhodamine B as two model matrix interferences. For TRES measurement, the time gate was set to be either 0 ns delay time or 333 ns delay time. Cm460 has a distinctive emission peak at about 460 nm. With the time gate set to 0 ns delay time, the fluorescence of Cm460 clearly interfered with the emission response of complex 1 to β-gal (Figure 3a). However, the fluorescence of Cm460 was eliminated after 333 ns delay time, while the phosphorescence of complex 1 treated with β-gal remained (Figure 3b). This suggests that TRES could be used to enhance the sensitivity of the probe for β-gal even in the presence of a strongly autofluorescent background. Similarly, the fluorescence of rhodamine B at 570 nm substantially perturbed the signal response of complex 1 when the time gate was set at 0 ns delay time (Figure 3c), but not when it was at 333 ns delay time (Figure 3d). Furthermore, β-gal was quantitatively measure in human serum samples by timegated luminescence assay.45 Human serum was 100-fold diluted with the PBS buffer (2×, 7.0), and then used for the measurements. 333 ns Delay time was used for the detection in time-gated luminescence. The results showed that the time-gated luminescence intensity was increased with the increasing β-gal (Figure S6a), there was also good linear relationship between luminescence intensity and 0–30 U/mL (Figure S6b). These demonstrated the potential applicability of complex for the quantitative detection of β-gal in complicated serum samples under time-gated luminescence mode. Sensing mechanism of complex against β-gal. To confirm the sensing mechanism of complex 1, a highresolution mass spectrometry (HRMS) experiment was performed. Before incubation with β-gal, the peak of complex 1 was found at 859.2112, while after incubation with β-gal the mixture showed a peak at

Figure 5. Application of 1 to differentiate between ovarian cell lines. (a) β-Gal activity determined in SKOV3, OVCAR3, HUVEC, and HEK-293T cell lines. (b) Confocal imaging of SKOV3, OVCAR3, HUVEC, HEK-293T cell lines treated with complex 1 (30 μM). Excitation was at 405 nm, and luminescence images were recorded from 540 to 640 nm. The scale bar is 50 µm. 697.1611, which was consistent with molecular weight of complex 2 cation (Figure S7). Curiously, synthetically-prepared complex 2 showed little luminescence in the absence of β-gal, whereas the addition of β-gal triggered a strong luminescence (Figure S8). We therefore propose that complex 2 is non-emissive to due non-radiative interactions with the solvent leading to luminescence quenching. However, β-gal cleaves the galactose moiety of complex 1 to generate the dealkylated product 2, which binds to β-gal. Upon binding to β-gal, the complex is protected from the bulk solvent and hence becomes emissive via the triplet metal-to-ligand charge-transfer (3MLCT) excited state.36,37 However, we do not preclude the possibility that complex 2 may also bind to other proteins (if present) beside βgal to trigger an increase in luminescence (Figure. S8 shows a slight luminescence increase for complex 2 in the presence of BSA). However, as the conversion from complex 1 to complex 2 only occurs in the presence of β-gal, the probe 1 is still very highly specific for β-gal activity (as indicated in Fig. 2). Luminescence imaging of β-gal in living cells. Encouraged by the performance of complex 1 in aqueous media, we next investigated the ability of complex 1 to differentiate ovarian carcinoma cells from normal cells. The 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay was first employed to investigate the cytotoxicity of complex 1 (Figure S9). Encouragingly, complex 1 had minimal cytotoxicity towards the ovarian carcinoma

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cell line SKOV3 and the normal human umbilical vein endothelial cell line HUVEC over 12 or 24 h (IC50 > 100 μM), and only minimal toxicity after 48 h (IC50 values of 79.4 and 63.1 μM for SKOV3 and HUVEC respectively). These results indicate that complex 1 has sufficient biocompatibility under the experimental conditions. We next explored whether complex 1 could be used for the imaging of β-gal in ovarian carcinoma cells. SKOV3 cells were chosen as a model ovarian carcinoma cell line, as it has been reported to express high levels of β-gal.46 After incubation with complex 1 (0–60 μM) for 12 h in a CO2 incubator, SKOV3 cells displayed a luminescence that was enhanced with increasing concentrations of 1 as revealed by confocal imaging (Figure 4). A subsequent time-course experiment of showed that the emission of the probe increased with time (0–24 h), consistent with the enzymatic mechanism of luminescence enhancement (Figure S10). We next tested the luminescence response in HUVEC cells, which express very low levels of β-gal.28 Strikingly, no obvious luminescence was detected in HUVEC cells even after 12 h incubation with up to 60 μM of complex 1 (Figure S11). This suggests that the luminescence enhancement of complex 1 in SKOV3 cells could be attributed to the presence of β-gal. Moreover, the luminescence of the probe in SKOV3 cells decreased with preincubation with the competitive β-gal inhibitor, D-galactose (Figure S12). Taken together, these results indicate that complex 1 could detect β-gal activity in living cells, with the further potential to discriminate ovarian carcinoma cells from normal cells. Application to differentiate ovarian carcinoma cell lines from normal cell lines. The ability of complex 1 to distinguish between ovarian and normal cell lines was further validated using another ovarian carcinoma cell line, OVCAR3, and another normal cell line, human embryonic kidney HEK-293T. High levels of β-gal activity were detected in the two ovarian carcinoma cell lines SKOV3 and OVCAR3, while significantly less β-gal expression was detected in the two normal cell lines (Figure 5a), which were consistent with previous reports.28 Importantly, the two ovarian carcinoma cell lines (SKOV3 and OVCAR3) were “lighted up” after incubation with 30 μM complex 1 for 12 h at 37 °C, while the two normal cell lines (HUVEC and HEK-293T) displayed negligible luminescence under same conditions (Figure 5b). Therefore, this probe shows promising potential in discriminating ovarian carcinoma cell lines with high expression of β-gal from normal cell lines.

CONCLUSIONS As β-gal has been demonstrated as an important biomarker for ovarian cancers, the development of luminescent probes for β-gal detection is important in the

context of ovarian cancer diagnostics. We have developed herein the first long-lived iridium(III)-based probe for discriminating ovarian carcinoma cell lines from normal cell lines. The probe 1 was designed to contain an enzyme-cleavable bond, such that luminescence is only activated after the complex 1 is cleaved by β-gal. Meanwhile, the probe shows good stability and long lifetime, and could detect β-gal activity in the presence of an autofluorescent background typical of biological samples through the use of TRES. The probe also selectively responded to β-gal activity in a linear fashion from 0 to 30 U/mL, and exhibited a detection limit of 0.51 U/mL. Importantly, complex 1 could selectively “light up” ovarian carcinoma cells, while, exhibiting negligible luminescence in normal cells. Thus, complex 1 shows high potential for the diagnosis of ovarian cancer.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. General information, chemical structures and characterization data of compounds, 1H NMR, 13C NMR, and HRMS spectra of compounds, detailed protocols for assays, additional spectral data, confocal microscopy images (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] Author Contributions § These authors contributed equally to this paper. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We would like to thank Dr. Wen-An Qiang (Department of Obstetrics and Gynecology, Feinberg School of Medicine at Northwestern University, Chicago) for kindly providing SKOV3 and OVCAR3 cell lines. This work is supported by Hong Kong Baptist University (FRG2/1617/007), the Health and Medical Research Fund (HMRF/14130522 and 14150561), the Research Grants Council (HKBU/12301115 and HKBU/201913), the National Natural Science Foundation of China (21575121 and 21628502), the Guangdong Province Natural Science Foundation (2015A030313816), the Hong Kong Baptist University Century Club Sponsorship Scheme 2016, the Interdisciplinary Research Matching Scheme (RCIRMS/15-16/03), Innovation and Technology Fund (ITS/260/16FX), Matching Proof of Concept Fund (MPCF001-2017/18), the Science and Technology Development Fund, Macao SAR (007/2014/AMJ and 077/2016/A2), the University of Macau (MYRG2015-00137-ICMS-QRCM, MYRG2016-00151-ICMS-QRCM and MRG044/LCH/2015/ICMS).

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