A Fluorescent Probe for Early Detection of Melanoma and Its Metas

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A Fluorescent Probe for Early Detection of Melanoma and Its Metastasis through Specifically Imaging Tyrosinase Activity in Mouse Model Chenyue Zhan, Jiatian Cheng, Bowen Li, Shuailing Huang, Fang Zeng, and Shuizhu Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00594 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Analytical Chemistry

A Fluorescent Probe for Early Detection of Melanoma and Its Metastasis through Specifically Imaging Tyrosinase Activity in Mouse Model Chenyue Zhan ‡, Jiatian Cheng ‡, Bowen Li, Shuailing Huang, Fang Zeng*, Shuizhu Wu* State Key Laboratory of Luminescent Materials and Devices, College of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China. ABSTRACT: Melanoma is a type of highly malignant and metastatic skin cancer, and early detection of melanoma by analyzing the level of its biomarker may decrease the likelihood of mortality. In this study, a fluorescent probe NBR-AP for detecting tyrosinase (a biomarker of melanoma) has been created by incorporating a hydroxyphenylurea group (as a substrate for the enzyme) onto a fluorescent dye phenoxazine derivative (as activatable signal reporter). This probe can be activated to generate fluorescence through a tyrosinase-mediated oxidation followed by hydrolysis of the urea linkage. The probe is able to sensitively and selectively detect the endogenous tyrosinase level in live cells and in zebrafish. Moreover, by imaging the tyrosinase activity, NBR-AP has been successfully applied to diagnose the melanoma and its metastasis in xenogeneic mouse models established via subcutaneous injection of B16F10cells.

Melanoma (melanotic carcinoma) is a type of malignant cutaneous cancer with highly metastatic nature. The incidence rate of cutaneous melanoma has grown rapidly over the last two decades, making it one of the fastest rising cancers worldwide and the leading cause of death from skin diseases.15 Realizing the lethality of malignant melanoma, researchers have developed diversified methods to diagnose the primary and metastatic melanoma6-8. Traditional clinical diagnosis relys on broad clinical groups and long-term objective outcomes9, while recent researches have brought about some new diagnostic methods such as melanoma-targeting imaging10 or label-free photoacoustic detection.11 On the other hand, given the fast evolvement and metastasis of melanoma, early detection of this malignant disease can achieve earlier diagnosis and prognosis so as to decrease the likelihood of fatality. Assaying the reliable melanoma biomarkers is an effective approach for early detection of the disease; and currently a number of serum/tissue melanoma biomarkers have been developed for the diagnosis, prognosis and monitoring of treatment for this disease. 12, 13 Tyrosinase (EC 1.14.18.1) is a cytoplasmic melanocyte differentiation protein and it initiates the formation of melanin by mediating the oxidation of tyrosine to some quinones14-17. This enzyme is constitutively expressed in melanocytes and in malignant melanoma cells, and high level of tyrosinase and its mRNA have been detected in serum of melanoma patients, and thereby the serum tyrosinase was used as an independent biomarker for melanoma diagnosis and prognosis. 18, 19 Scientists have explored several methods for detecting tyrosinase activity, such as colorimetric, fluorometric, electrochemical and spectrophotometric method20-23. Due to the ease

of accessibility, high sensitivity as well as superior bioimaging capability of the fluorescent analysis24-44, a number of fluorescent probes have been successfully developed for tyrosinase assay; and the relevant fluorescent probes were fabricated based on quantum dots45-47, noble metal nanoclusters48, 49 , polymeric nanoparticles50-52 as well as molecular dyes53-56. Recently, Ma and co-workers created a molecular probe by incorporating a tyrosinase recognition element (3hydroxybenzyloxy) onto a hemicyanine skeleton, and applied it to tyrosinase imaging in cell and in zebrafish.57 Currently, serum assay remains the mainstream approach for biomarker detection. However, it has been found that, sometimes serum exhibits high fluctuation in tyrosinase level; and this is probably caused by sample processing as well as the very transient presence of metastasizing tumour cells in the blood.58 In addition, a recent study found that when comparing patients to healthy volunteers, no differences in serum tyrosinase level could be detected.59 Moreover, tyrosinase resides in some organs or tissues besides the melanoma focus, 60-62 this may compromise the detection specificity of serum assay. We anticipate that, by exploring in vivo fluorescent imaging strategy, we can spatially localize the elevation of the tyrosinase level (or activity) at the melanoma focus, thereby greatly reducing the risk of false-positive signals. In this context, we have developed a novel tyrosinasedetecting fluorescent probe NBR-AP, which has a (4hydroxyphenyl)urea group as a substrate for the enzyme and a fluorescent dye phenoxazine derivative as the activatable signal reporter, as shown in Figure 1. The presence of tyrosinase breaks the carbamide bond and leaves an amine group on the dye, thereby restoring its fluorescence and achieving the fluo-

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rescent detection of the enzyme. This fluorescent probe is sensitive and can be used to detect the tyrosinase in PBS buffers, cells and in live zebrafish. More importantly, this probe is successful in real-time early diagnosis of melanoma and its metastasis in mouse models.

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to afford compound 1 as a yellow semisolid (5.519 g, 78.4%). H NMR (600 MHz, CDCl3): δ 7.06 (t, J=7.8 Hz, 1H), 6.26 (s, 1H), 6.24 (s, 1H), 6.23 (s, 1H), 6.22 (s, 1H), 3.68 (s, 6H), 3.62 (t, J=7.2 Hz, 4H), 2.59 (t, J=7.2 Hz, 4H). HR-MS (ESI): calcd for C14H19NO5:282.1336 ([M+H]+), found: 282.1333. Synthesis of compound 2 (dimethyl 3, 3'-((3-hydroxy-4((4-nitrophenyl) diazenyl) phenyl) azanediyl) dipropionate). A solution of 4-nitroaniline (1.062 g, 8 mmol) was dissolved in a mixture of 5 mL of concentrated hydrochloric acid and 5 mL of water, and then was added into a flask pre-cooled with ice-bath. Sodium nitrite (531 mg, 7.70 mmol) dissolved in 5 mL of water was then slowly added into the ice-cold flask. The reaction mixture was stirred at 0 °C for another 30 min. After that, a solution of compound 1 (1.967 g, 7.0 mmol) in 2 mL of methanol was added to the reaction flask. The mixture was stirred for 60 min at room temperature. The resultant red precipitate was filtered and washed with cold ethanol. The crude product was recrystallized in ethanol. After drying, compound 2 (1.566 g, 52%) was obtained as an orange red solid. 1H NMR (600 MHz, CDCl3)：δ 14.88 (s, 1H), 8.29 (d, J=9 Hz, 2H), 7.76 (d, J=9.6 Hz, 2H), 7.50 (d, J=9 Hz, 1H), 6.47 (d, J=9.6 Hz, 1H), 6.04 (s, 1H), 3.81 (t, J=7.2 Hz, 4H), 3.72 (s, 6H), 2.71 (t, J=7.2 Hz, 4H) HR-MS (ESI): calcd for C20H22N4O7 :431.1561 ([M+H]+), found: 431.1564. Synthesis of compound 3 (9-(bis(3-methoxy-3oxopropyl)amino)-5H-benzo[a] phenoxazin-5-iminium) A mixture of 1-aminonaphthalene (272 mg, 1.90 mmol), compound 2 (731 mg, 1.70 mmol) and 30 mL of DMF containing perchloric acid (1 mL, 70%) was heated to 160 ℃ for 15 min under stirring. The color of the reaction mixture changed from brown to deep blue. The reaction was monitored by silica gel TLC. After cooling, DMF was evaporated to dryness under reduced pressure. The crude perchlorate salt was purified by column chromatography (silica gel, 1:40 methanol/dichloromethane) to afford compound 3 as a blue solid (429 mg, 58.1%).1H NMR (600 MHz, DMSO-d6): δ 10.09 (s, 2H), 8.80 (d, J=8.4 Hz,1H), 8.48 (d, J=8.4 Hz, 1H), 8.00 (t, J=7.8 Hz, 1H), 7.92 (t, J=7.2 Hz, 1H), 7.86 (d, J=9 Hz, 1H), 7.27 (d, J=2.4 Hz, 1H), 7.08 (d, J=3 Hz, 1H), 6.86 (s, 1H), 3.87 (t, J=7.2 Hz, 4H), 3.65 (s, 6H), 2.74 (t, J=7.2 Hz, 4H). HR-MS (ESI): calcd for C24H24N3O5+:434.1721 ([M-H]-), found: 431.1718. Synthesis of compound 4 (4-((tertbutyldimethylsilyl)oxy) aniline). 4-Aminophenol (1.0 g, 9.17 mmol) and imidazole (0.936 g, 13.75 mmol) were dissolved in 30 mL of tetrahydrofuran, and tert-butyldimethylsilyl chloride (1.797 g, 11.92 mmol) was added into the flask with rapid stirring for 30 minutes. The solvent of the reaction mixture was evaporated under reduced pressure. The resultant residual was purified by column chromatography (silica gel, 10:1 petroleum ether/ethyl acetate) to afford compound 4 (1.842g, 90.1%) as a colorless viscous semisolid. 1H NMR (600 MHz, DMSO-d6): δ 6.41 (d, J=9 Hz, 1H), 6.33 (d, J=9 Hz, 1H), 4.50 (s, 2H), 0.82 (s, 9H), 0.00 (s, 6H). HR-MS (ESI): calcd for C12H21NOSi: 224.1465 ([M+H]+), found: 224.1463. Synthesis of compound 5 (dimethyl 3,3'-((5-(3-(4-((tertbutyldimethylsilyl) oxy)phenyl)ureido) -5Hbenzo[a]phenoxazin-9-yl)azanediyl) dipropionate). Under nitrogen atmosphere, a solution of N,N-diisopropylethylamine (959 µL) in 15 mL of dichloromethane was added dropwise to 15 mL dichloromethane solution of compound 4 (0.210 g, 0.94 1

Scheme 1. Schematic diagram for detection of tyrosinase by probe NBR-AP and melanoma imaging in mouse model. EXPERIMENTAL SECTION Chemicals and material. 4-Aminophenol, tbutyldimethylchlorosilane, 3-aminophenol, methyl acrylate, 1naphthylamine, p-nitroaniline , acetic acid, hydrochloric acid, Kojic acid, perchloric acid, triphosgene (BTC), N,NDiisopropylethylamine (DIPEA), dichloromethane (DCM), methanol, tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and tetrabutylammonium fluoride, triethylamine, NaNO2 and NaBr were purchased from Aladdin Reagents. Tyrosinase (from purified mushroom), carboxylesterase and leucine aminopeptidase were obtained from Sigma. The reagents for Western blotting were purchased from KeyGen Biotech Co., Ltd. The water used herein was the double-distilled water upon being treated by ion exchange columns. All the other reagents and solvents were of analytical grade and used without further purification unless noted otherwise. Cells (B16F10 and Hela) were purchased from KeyGen Biotech Co. Ltd. Measurements. 1H NMR spectra were determined on a Bruker Avance 600 MHz NMR spectrometer operated at 600 MHz with tetramethylsilane (TMS) as a reference for chemical shifts. High resolution mass spectra were obtained on AB Sciex Triple TOF 5600+ mass spectrometer. UV-vis spectra were obtained on a Hitachi U-3010 UV-vis spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. Cell and zebrafish imaging was obtained on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD. HPLC analyses were performed on Agilent 1260 equipped with DAD detector. Mouse imaging was performed on AMI small animal imaging system (Spectral Instruments Imaging). Synthesis of compound 1(dimethyl 3,3'-((3hydroxyphenyl) azanediyl) dipropionate ). Methyl acrylate (12.931 g, 150.30 mmol), 3-aminophenol (2.730 g, 25.03 mmol), sodium bromide (0.618 g, 6.05 mmol) and acetic acid (3.1 mL) were added to a flask under nitrogen atmosphere. The mixture was refluxed at 95 °C for 19 h. Afterwards, the mixture was diluted with 150 mL water, and neutralized by sodium bicarbonate and extracted by ethyl acetate. The organic layer was dried by Na2SO4, filtered, and then evaporated under reduced pressure. The product was purified by column chromatography (silica gel, 2:1 petroleum ether/ethyl acetate)


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mmol) and triphosgene (1.160 g, 3.91 mmol) at 0 ℃ over a period of 1 h, the mixture was stirred at room temperature for 2 h. The mixture was then dried under reduced pressure to afford a yellowish solid. Afterwards, under nitrogen atmosphere, a 15 mL dichloromethane solution of the yellowish solid was added dropwise to 15 mL dichloromethane solution containing compound 3 (0.449 g, 1.03 mmol) and triethylamine (170 mL) at 0 °C over a period of 30 minutes. The mixture was stirred at room temperature for an additional 24 h. The mixture was dried under reduced pressure, and the collected residue was subjected to silica column chromatography (methanol, dichloromethane, v/v = 1:40) to afford purple solid product (0.463 mg, 76.2%). 1H NMR (600 MHz, DMSO-d6)： δ 9.54 (s, 1H), 8.35 (d, J=6.6 Hz, 1H), 8.23 (d, J=7.8 Hz, 1H), 7.59 (t, J=7.2 Hz, 1H), 7.54 (t, J=7.8 Hz, 6.6 Hz, 1H), 7.38 (d, J=8.4 Hz, 2H), 6.62 (d, J=8.4 Hz, 2H), 6.58 (s, 1H), 6.42 (s, 2H), 3.52 (s, 4H), 3.43 (s, 6H), 2.44 (t, J=7.2 Hz, 6.6 Hz, 4H), 0.77 (s, 9H), 0.00 (s, 6H) HR-MS (ESI): calcd for C37H44N4O7Si: 683.2900 ([M-H]-), found: 683.2904. Synthesis of compound 6 (dimethyl 3,3'-((5-(3-(4hydroxyphenyl)ureido)-5Hbenzo[a]phenoxazin-9yl)azanediyl) dipropionate ). Compound 5 (0.1 g, 0.15 mmol) was dissolved in 15 mL tetrahydrofuran, and 73 µL of tetra-butyl ammonium fluoride was added into the solution, followed by stirring at room temperature for 30 minutes. The solvent of the resultant mixture was evaporated under reduced pressured, and the residue was washed with 3×30 cm3 water, and the solvent of the mixture was removed under reduced pressured. The resultant solid was purified by column chromatography (silica gel, 1:50 methanol/dichloromethane) to afford compound 6 (0.055 g, 66%). 1H NMR (600 MHz, DMSO-d6) ：δ 9.60 (s, 1H), 9.14 (s, 1H), 8.56 (d, J=7.8 Hz, 1H), 8.43 (d, J=8.0 Hz, 1H), 7.79 (t, J=6.6 Hz, 7.2 Hz 1H), 7.74 (t, J=6.6 Hz, 7.2 Hz, 1H), 7.58 (d, J=9.0 Hz,1 H), 7.46 (d, J=9 Hz, 2H), 6.79 (d, J=2.40 Hz, 1H), 6.72 (d, J=8.4 Hz, 2H), 6.65 (d, J=2.4 Hz, 1H), 6.61(s, 1H), 3.71 (t, J=7.2 Hz, 4H), 3.61 (s, 6H), 2.63 (t, J=7.2 Hz, 4H). HR-MS (ESI): calcd for C31H30N4O7: 569.2042 ([M-H]-), found: 569.2036. Detection of tyrosinase in PBS buffers. Unless otherwise specified, all the measurements were performed according to the following procedure. In a test tube, 4 mL of PBS (pH 7.4) and 25 µL of probe (NBR-AP) was mixed, followed by addition of an appropriate volume of tyrosinase sample solution. The final volume was adjusted to 5 mL with PBS and the reaction solution was well-mixed. After incubation at 37℃ for 0 to 2 h, a 3-mL portion of the reaction solution was transferred to a quartz cell of 1-cm optical length to measure the spectral properties with λ ex/em = 580/660 nm (excitation slit widths were set to 5 nm, and emission slit widths were set to 10 nm). Under the same conditions, a control solution containing no tyrosinase was used for comparison. Cell viability assay. To examine the cytotoxicity of the probe NBR-AP in living cells, B16F10 cells were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS) within 96-well microtiter plates. Plates were firstly maintained in a 5% CO2 incubator at 37 ℃ for 24 h. Then, B16F10 cells were incubated with various concentrations of NBR-AP respectively for another 24 h. The cytotoxicity of the probe against the cells was assessed by MTT assay according to ISO 10993-5. Three independent experiments were per-

formed, and for each independent experiment, the assays were performed in eight replicates. And the statistic mean and standard derivation were utilized to estimate the cell viability. Cell incubation and imaging. Cells (B16F10 and HeLa) were grown on the glass-bottom culture dishes (MatTek CO.) in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin at 37℃ in a humidified 5% CO2 incubator. Before use, the adherent cells were washed three times with FBS-free DMEM. The cells were incubated with NBR-AP (0 µM, 5 µM or 10 µM) in FBSfree DMEM (containing 1% DMSO) at 37 ℃ for different periods of time (0 h, 1 h or 2 h), and then washed twice with DMEM to remove the free probe. The fluorescent signal was collected in the range of 650-750 nm. For kojic acid-involved experiments, the cells were incubated with 200 µM of kojic acid for 2 h at 37 ℃ before fluorescence imaging. Imaging processing and analysis was performed on Olympus IX 71 fluorescence microscope. The flow cytometry analyses were performed on a BD Accuri C6 flow cytometer and the obtained data were analyzed using the BD Biosciences software. Zebrafish maintenance and imaging. All zebrafish experiments were in full compliance with international ethics guidelines. The wild-type zebrafish larvae (provided by School of Life Sciences, Sun Yat-sen University, China) were cultured at 28 ℃and maintained at optimal breeding conditions. For the imaging experiments, 3-day-old larvae were transferred into a 96-well microplate by using a disposable transfer pipette. The larvae were incubated with NBR-AP (0 µM, 5 µM or 10 µM, in E3 culture medium containing 1% DMSO) for 1 h, respectively. After that, the fishes were washed for three times with 100 µL E3 media to remove the remaining probe for subsequent fluorescence imaging. For kojic acid-involved experiments, the fishes were incubated with E3 media containing 400 µM kojic acid for 2 h at 28 ℃ before imaging. The fluorescence images for zebrafish larvae were observed on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD. All the photographs were taken under identical exposure condition. Animal Experiments and in vivo Imaging. Balb/c nude mice (4 wk, 20-25g) were provided by Guangdong Experimental Animal Centre, and they were kept under SPF condition with free access to standard food and water. All animal experiments were performed in Laboratory Animal Centre of South China Agricultural University in accordance with international ethical principles and guidelines for experiments on animals. And all animal experimental protocols were approved by the Ethics Committee of Laboratory Animal Centre of South China Agricultural University. For tumour imaging experiments, tumour-bearing mouse models were established by subcutaneous injection of B16F10 (106 cells/0.1 mL/flank) and/or HeLa (107 cells/0.1 mL/flank) in both flanks of each mouse (n=5 per experimental group). After tumour cells were injected into mice for given times, mice were then injected with the probe NBR-AP solution via tail vein, and the mice were imaged upon 1 hour after i.v. injection on an AMI small animal imaging system of SI Imaging Co. The in vivo fluorescent images were quantified by measuring fluorescent signal intensity at the region of interest (ROI) using Image J software.



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For metastatic melanoma, the mouse model was established by subcutaneous injection of B16F10 cells. 14 days upon injection, mice were dissected and the fluorescence imaging was conducted.

in (D) displays the plot of the fluorescence intensity at 660 nm versus reaction time. λex/em = 580/660 nm. All the measurements were conducted at 37 °C in 10 mM phosphate buffer (pH 7.4) containing 2% (v/v) DMSO.

Western blot. The tissue/organs were treated with RIPA buffer (Cell Signaling), and then homogenized using GentleMACS Mtubes (Miltenyi Biotech) with Protein 1.1 program. Proteins were concentrated using Amicon Ultra 10k centrifugal filter units. Protein concentration was determined by BCA kit (Pierce). Samples were denatured at 99 ℃ for 5 minutes before being loaded on to 420% Tris-HCl precast Criterion gels (Bio-Rad). Tyrosinase or GAPDH (loading control) were detected by primary antibodies TYR-pAB (1:5000, ABclonal) or GAPDH-pAB (1:5000, ABclonal) respectively followed by goat anti-rabbit IgG HRPlinked (1:2000, ABclonal). A ChemiDoc imaging system (BioRad) was used to detect chemiluminescence after using Supersignal west Dura ECL kit (ThermoFisher). Intensity of TYR and GAPDH bands were quantified using ImageJ gel analysis function. In all cases, the samples being compared were exposed equivalently for a given protein target.

Spectral properties of probe and fluorescent response of NBR-AP toward tyrosinase Figure 1 shows spectroscopic properties of NBR-AP before and after incubation with tyrosinase. As depicted in Figure 1A, the probe exhibited a weak absorption peak around 510 nm. Upon addition of tyrosinase, the absorption peak red-shifted to about 580 nm accompanying a remarkable enhancement in absorbance. Accordingly, almost no fluorescent emission could be detected for the probe alone; whereas 7-fold increase in fluorescent intensity at about 660 nm (Figure 1B) was observed under the maximum excitation of 580 nm upon incubation with tyrosinase. The quantum yield of the probe NBR-AP (0.02) and the reporter NBR (0.23) were determined and the results are shown in Table S1, and the quantum yield value for NBR is moderate compared to some tyrosinase probes, as shown in Table S2. Moreover, the dose- and time- dependence for the fluorescent emissions for the probe are shown in Figure 1C and 1D respectively. Under normal physiological condition (37 ℃, pH 7.4), the probe displayed a good linear fluorescence response to tyrosinase in the concentration range from 0 to 200 U/mL (Figure 1C). As for the time-dependent fluorescence response, the probe solution exhibited higher fluorescent intensities at longer incubation time, until they reached a saturation point. This sharp fluorescence off-on response is desirable for sensitive imaging of tyrosinase activity.

RESULTS AND DISCUSSION Synthesis of the probe. The probe was synthesized by linking the tyrosinase recognition element ((4hydroxyphenyl)urea) with the phenoxazine skeleton by forming the urea linkage (Scheme S1). Phenoxazine was first synthesized according to previous literature63, and a TBS-Cl protected 3-aminophenol was conjugated onto phenoxazine through the urea bond. Finally, TBAF was utilized in the deprotection of TBS-Cl and yielding final product. The intermediates and the final product were characterized by 1H NMR and MS, as shown in Figures S1 -S12.

The pH stability of the probe and the reporter were determined by recording the fluorescent intensities at 660 nm, as shown in Figure S13A. Compared to some previously reported tyrosinase detection systems (shown in Table S2), the current probe exhibits relatively good pH stability. The pH stability is important for the in vivo imaging of melanoma, since the microenvironment in tumors is generally more acidic than in normal tissues, and the extracellular pH is also different from the intracellular pH for tumor cells. A fluorescent probe with higher pH stability can afford more accurate imaging. The effect of temperature on the fluorescent emission was also determined. The fluorescence exhibited an enhanced intensity at about 37 ℃ (Figure S13B), which is in accordance with the fact that enzymes usually have a maximum activity at 37 ℃. The above results indicate that NBR-AP functions well under the normal physiological conditions. The detection selectivity for the probe towards tyrosinase over some potential interferant species was also investigated and the results are given in Figure S14A. As we can see from the figure, only the reactive oxygen species (ROS) caused slight increase in fluorescence. The other potential interfering substances, such as some inorganic salts, glucose and some enzymes (including xanthine oxidase, an oxidase highly expressed in some cancer cells64, its fluorescent response to the tyrosinease is given in Figure S15), could not interfere with the detection. To investigate the fluorescence generation caused by the ROS, we recorded fluorescent intensities at 660 nm over time for the probe upon incubation with some ROS (Figure S14B), which indicate that upon incubation with the ROS, the fluorescent intensities increased slowly and then stabilized at certain low values. We also de-

Figure 1. Changes in absorptivity (A) and fluorescence (B) spectra of NBR-AP (10 µM) with or without incubation of tyrosinase (100 U/mL) for 1 h; (C) Fluorescence response of NBR-AP (10 µM) to tyrosinase at varied concentrations from 0 to 200 U/mL in 20 min. (D) Fluorescence response of NBR-AP (10 µM) to tyrosinase (200 U/mL) from 0 to 120 min. The inset in (C) is the curve of ∆F against the concentration of tyrosinase. The inset


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Analytical Chemistry taining 1% DMSO) for 1 h, cells incubated with probe NBR-AP (10 µM, in DMEM medium containing 1% DMSO) for 1 h and cells pretreated with kojic acid (200 µM) for 2 h and then incubated with probe NBR-AP (5 µM, in DMEM medium containing 1% DMSO) for 1 h. The excitation wavelength was 510-550 nm, the emission signal was collected in the range of 650-750 nm, the exposure time was 50ms. Scale bar: 15 µm. (B) Relative intensity values obtained from (A). The results are expressed as the mean ± standard deviation (n=3).

termined the mass spectrum for the probe after incubation with a ROS (H2O2). As shown in Figure S16, the peak for the reporter NBR (at 434.17 Da) and the probe NBR-AP (at 569.20 Da) can be observed after the probe had been incubated with H2O2 for 2 h. This result indicates that small amount of NBR was produced after ROS treatment. These results indicate that, despite of slight interference from ROS, the probe exhibits high selectivity towards tyrosinase over many potential interfering substances. Furthermore, we performed the Michaelis– Menten kinetics assay for the tyrosinase-catalyzed reaction, and some parameters such as Km, Vmax, kcat, and kcat/Km were also obtained, as shown in Figure S17.

Imaging of tyrosinase in living cells. Before cell imaging experiments, the potential cytotoxicity of the probe was evaluated by using MTT assay. When B16F10 melanoma cells were treated with NBR-AP at the concentration (up to 10 µM) for 24 h, only slightly lower cell viability was observed as compared to the control (Figure S19), indicating the low cytotoxicity of the probe.

To verify the mechanism of the tyrosinase-mediated reaction, the reaction products for the probe were detected by HPLC analysis. Before reaction, NBR-AP gave chromatographic peaks at 2.1 min (curves c, Figure S18); whereas phenoxazine (NBR) dye exhibited a chromatographic peak at 4.2 min (curve b). Upon reaction (curve a), the chromatographic peak at 2.1 min (corresponding to NBR-AP) decreased significantly, along with the emergence of the NBR’s peak at 4.2 min. Besides, the new peak at 1.4 min was proved to be 4aminoorthoquinone by comparing the chromatogram (curve a) of the tyrosinase-catalyzed oxidation products with 4aminoorthoquinone (curve d). These data clearly indicate that the fluorescence response of the probe is attributed to the oxidization-induced cleavage of the urea linker, and the subsequent generation of NBR.

Figure 3. (A) Images for 3-day-old zebrafish: untreated zebrafish (the control), zebrafish incubated with probe NBR-AP (5 µM, in E3 culture medium containing 1% DMSO) for 1 h, zebrafish incubated with probe NBR-AP (10 µM) for 1 h, and zebrafish treated with 400 µM kojic acid for 2 h and then incubated with probe NBR-AP (5 µM, 1% DMSO in E3 culture medium) for 1 h. The excitation wavelength was 510-550nm, the emission signal was collected in the range of 650-750 nm, the exposure time was 50 ms. Scale bar: 200µm. (B) Relative intensity values obtained from images in (A) and calculated using the Image J software. The results are expressed as the mean ± standard deviation (n=5). The ability of NBR-AP to detect the tyrosinase activity in living cells was then examined by using fluorescence microscopy and flow cytometry, and the results are given in Figure 2, Figure S20-S22. It is reported that B16F10 melanoma cells have a high

Figure 2. (A) Images of B16F10 cells: cells only (control), cells incubated with probe NBR-AP (5 µM, in DMEM medium con-



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Diagnosing melanoma by imaging tyrosinase activity in mouse models. The successful application of NBR-AP in monitoring the endogenous tyrosinase activity in living zebrafish inspired us to use it to diagnose early melanoma in rodent models. For the optical imaging in vivo, the scattering and absorption of light by tissue limit the imaging depth. The longer absorption wavelength is beneficial to the in vivo imaging. In this study, although the maximum absorption of NBRAP lies out of the NIR range (wavelength >650 nm), melanoma is the skin cancer and lays shallow in tissue, which reduces the light scattering effect. On the other hand, the possible major interferents in tissue around the melanoma include water, hemoglobin (Hb) and oxyhemoglobin (HbO2). By comparing the absorption spectrum of the probe to that of water (Figure S25), Hb and HbO2,72 we can see at 580 nm (the excitation wavelength for the probe), water exhibits very low absorption; and Hb and HbO2’s molar absorptivity (molar extinction coefficients) are around 1× 10 4 L⋅mol -1⋅cm-1, lower than that of the probe (5.0 × 10 4 L⋅mol -1⋅cm-1, as shown in Figure 1A). Hb and HbO2 are highly abundant in blood vessels, but in tissue outside the blood vessels, their presence is scarce. Thus the excitation light can pass by the blood vessels to excite NBR in tumor tissue. These analyses suggest the interference from water, Hb and Hb02 is not significant. Furthermore, the activity level of tyrosinase in healthy skin cells of the mice was relatively weak.73 These all favor the successful imaging of tyrosinease by using our probe.

level of tyrosinase, whereas HeLa cells show low tyrosinase expression.65,66 Thus, B16F10 and HeLa cells are chosen as the model cell line and the negative control, respectively. The timeand dose-dependent response of the two cell lines to the probe are presented in Figure 2, Figure S21 and Figure S22. In both cases the B16F10 cells exhibited strong intracellular fluorescence, while HeLa cells displayed almost no fluorescence in the presence of 5 µM of probe and very weak fluorescence at 10 µM of probe (Figure S22). The flow cytometry result also revealed the stronger fluorescence in B16F10 than HeLa cells (Figure S20). To verify whether the fluorescence generation in B16F10 cells originates from the action of tyrosinase, inhibition experiments were conducted by pretreating B16F10 cells with a tyrosinase inhibitor, kojic acid, as carried out by some other groups 67-69. As expected, much weaker intracellular fluorescence was observed for B16F10 cells pretreated with kojic acid, as shown in Figure 2. In consideration of the fact that kojic acid is not a very specific inhibitor for tyrosinase, we also applied deoxyarbutin70, a specific inhibitor of tyrosinase to further evaluate the selectivity of the probe towards tyrosinase. To rule out the possible interference from deoxyarbutin, we recorded the emission spectra for 5 µM NBR alone and that of the NBR with 10 or 100 µM deoxyarbutin. The result is given in Figure S23, which indicates that deoxyarbutin does not pose any interference to the detection. Before incubated with the probe NBR-AP, B16-F10 cells were pre-treated with 10 µM or 100 µM of deoxyarbutin for 2 hours. As shown in Figure S24, the pretreatment of deoxyarbutin could reduce the intracellular fluorescence, and the higher concentration of deoxyarbutin decreased the fluorescence more significantly, further proving that the intracellular fluorescence is due to the production of NBR catalyzed by the tyrosinase. Considering that deoxyarbutin is a more specific inhibitor of tyrosinase than kojic acid, we can conclude that NBR-AP is a robust probe for imaging tyrosinase in cells.

To verify the capability of the probe in detecting melanoma in mouse, two kinds of tumors were established in BALB/c nude mice by subcutaneously injecting melanoma cell (B16F10) in left flank and HeLa cells in right flank of the mice, respectively. The mice were then subject to in vivo imaging on a small animal optical imaging system, and the result is given in Figure 4A. The probe solution was intravenously injected into the mice 7 days upon cancer cell injection, and the fluorescence images were obtained 1 h later. We can see from Figure 4A and 4B that, the left flank injected with melanoma cells (B16F10) exhibited strong fluorescence; while the right flank injected with HeLa cells showed very weak fluorescence. This result suggests that the probe could selectively detect melanoma by imaging the over-expressed tyrosinase in melanoma tissue. The probe was then used to detect early melanoma in the mouse model. The mice were intravenously injected with the probe solution after they were subject to B16F10 cell injection at right flank for different time periods (1, 2, 5 and 7 days respectively). As shown in Figure 4C and Figure S26, the control group (0 day), which was not injected with the melanoma cells, displays no fluorescence at right flank. The melanoma is a kind of fast-evolving malignant cancer, and in this study the mice exhibited detectable fluorescence at their right flank only one day after injection of B16F10 cell. However, at this time period there was no visible sign of the melanin pigmentation (a characteristic of melanoma) at right flank of the mouse. The fluorescence signal at the injection site of B16F10 cell became more and more distinct

Real-time imaging of tyrosinase in zebrafish. We then used a type of transparent fish (zebrafish) to verify the probes capability of imaging the tyrosinase activity in vivo. Zebrafish expresses high level of tyrosinase since its embryo period, and tyrosinase disperses to whole epidermis as zebrafish growing up, according to previous report.71 As shown in Figure 3A, without NBR-AP treatment, the 3-day-old zebrafish larvae displayed nearly no fluorescence; while for the larvae treated with 5 µM or 10 µM NBR-AP, bright fluorescence could be observed in fish bodies. This implies that the probe is tissuepermeable and zebrafish contains tyrosinase at detectable level. Interestingly, the fluorescence in the zebrafish was not uniformly distributed; and the digestive system and skin showed much stronger fluorescence, suggesting that the probe mainly permeated into the fish via skin and digestive tract and underwent the oxidation reaction mediated by the endogenous tyrosinase. Moreover, an inhibition experiment was performed to further demonstrate the tyrosinase-mediated fluorescence generation from NBR-AP in zebrafish. As seen in Figure 3, the zebrafish pretreated with kojic acid showed weak fluorescence and 400 µM of kojic acid could causes a significant decrease in fluorescence by about 75% (Figure 3B). These results indicate that NBR-AP is capable of monitoring the endogenous tyrosinase activity in living bodies.


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with the elapse of time. At 5 days upon melanoma cell injection, a pigmented spot could be visually identified at the right flank, as shown in Figure 4C. In contrast, the mice injected with HeLa cells at the same site (right flank) exhibited weak fluorescence even at day 7 upon cell injection, as shown in Figure S27. This result suggests that the fluorescent signal can be detected earlier than the emergence of pigmentation, and this in vivo imaging of tyrosinase could be a promising approach for early detection of malignant melanoma. Melanoma is a highly metastatic cancer, the circulating tumor cells (CTC) have been found in patients with metastatic melanoma and are associated with advanced melanoma stage and poor patient outcome, since CTC corresponds to cancer “seeds” that initiates metastatic relapse.74-76 To further test if the probe could detect the metastatic behavior, a mouse model was established by subcutaneous injection of B16F10 cells. The injected melanoma cells can lead to formation of a mass and can subsequently metastasize to other sites. In this study, after 14 days upon injection, mice were dissected and the fluorescence imaging was conducted; while for the mouse injected with B16F10 cells (lower panel in Figure 5A), its tumour and lung displayed strong fluorescence, and the spleen displayed relatively weak fluorescence, indicating the fast metastasis of melanoma to these organs after subcutaneous injection of melanoma cells. With this property, the probe may be used in detecting metastatic melanoma during cancer surgery. Moreover, to verify the over-expression of tyrosinase in tumour and the metastatic organs, the tumour and organs were lysed and analyzed by western blot. As shown in the inset of Figure 5B, the level of tyrosinase in the tumour, lung and spleen were found much higher than that in other unaffected organs, which is in accord with the fluorescence intensity result (Figure 5B). Among them, the tyrosinase activity in lung ranks only second to tumour, suggesting the lung is the main metastatic target of malignant melanoma.

Figure 4. (A) Typical images (BF+FL) for the mouse injected with the probe (100µL, 200µM, in saline containing 1% DMSO) upon injection with B16F10 (left) or HeLa (right) cells for 7 days. λex/em = 570/650 nm. The exposure time was 2 s. (B) Relative intensity values obtained from (A) and calculated using the Image J2x software. The results are expressed as the mean ± standard deviation (n=5.) (C) Typical fluorescent and bright field images of 4-week-old mice injected with the probe upon injection with B16F10 for 0 (control), 1, 2, 5 or 7 days. λex/em = 570/650 nm. The exposure time was 2 s. The successful imaging of melanoma using NBR-AP implies that, the use of a fluorophore with high extinction coefficient and selection of a suitable excitation wavelength to avoid the co-absorption by interferents can also achieve sensitive in vivo imaging. And in this context, using a fluorophore 57 with longer absorption band (670 nm) can undoubtedly reduce the light scattering, but may also suffer from higher co-absorption from the water, since the molar absorption of water quickly increases above 670 nm (Figure S25).



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cent microscopy images, cytotoxicity assay, flow cytometry analysis, determination of quantum yield, mice images as well as tables summarizing probe properties. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

Author Contributions ‡These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support by NSFC (21788102， 51673066, 21574044 and 21474031), the Science and Technology Planning Project of Guangzhou (Project No. 201607020015), the Science and Technology Planning Project of Guangdong (Project No. 2014A010105009) and the Natural Science Foundation of Guangdong Province (2016A030312002).

REFERENCES (1) (2)

Figure 5. (A) Typical Images of dissected organs of the mice injected with the probe upon injection with B16F10 cells for 14 days. Organs of lower panel were dissected from mice subcutaneously injected with B16F10, upper panel of untreated mice as control. λex/em = 570/650 nm. The exposure time was 1 s. (B) Relative intensity values (n=3) obtained from (A) and calculated using Image J2x software. The relative intensity from tumour is defined as 1.0. The results are expressed as the mean ± standard deviation.The inset in (B) display relative tyrosinase content in tumour and organs of metastatic mouse model measured by western blot.

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CONCLUSION In summary, we have successfully developed a fluorescence probe NBR-AP for the assay of tyrosinase activity. The probe is able to respond to tyrosinase through an oxidizationcleavage reaction. By exploiting this unique property, we have imaged the activity of endogenous tyrosinase in B16F10 cells as well as in live zebrafish. Further, the probe has been successfully utilized in early diagnosis of melanoma and metastasis in mouse model by imaging the tyrosinase activity. To our best knowledge, it is the first investigation that a fluorescent probe is used to diagnosing early melanoma in rodent model.

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ASSOCIATED CONTENT

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Supporting Information (15)

Additional information includes the synthetic scheme, 1H NMR spectra, mass spectra, effect of pH and temperature, selectivity experiment, HPLC analysis, enzymatic reaction kinetics, fluores-

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A Fluorescent Probe for Early Detection of Melanoma and Its Metas

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