Melanosome-Targeting Near-Infrared Fluorescent Probe with Large

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Melanosome-Targeting Near-Infrared Fluorescent Probe with Large Stokes Shift for in Situ Quantification of Tyrosinase Activity and Assessing Drugs Effects on Differently Invasive Melanoma Cells Manshu Peng, Yan Wang, qiang fu, Feifei Sun, Na Na, and Jin Ouyang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00734 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Analytical Chemistry

Melanosome-Targeting Near-Infrared Fluorescent Probe with Large Stokes Shift for in Situ Quantification of Tyrosinase Activity and Assessing Drugs Effects on Differently Invasive Melanoma Cells Manshu Peng, Yan Wang, Qiang Fu, Feifei Sun, Na Na, and Jin Ouyang* Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China * E-mail: [email protected]. Phone: +86-10-58805373

ABSTRACT: Tyrosinase (TYR) plays a vital role in melanin biosynthesis and is widely regarded as a relatively specific marker for melanocytic lesions which involve vitiligo, malignant cutaneous melanoma, Parkinson’s disease (PD), etc. However, the detection of TYR in living cells with fluorescent probes is usually interfered by diverse endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS). Herein, we synthesized a melanosome-targeting near-infrared (NIR) fluorescent probe (HB-NP) with a large Stokes shift (195 nm), achieving a highly sensitive and selective in situ detection for intracellular TYR, by incorporating mhydroxybenzy moiety that recognizes TYR specifically and the morpholine unit which facilitates the probe accumulating in melanosome into salicyladazine skeleton. When treated with TYR, the probe itself with weak fluorescence is lighted up via an inhibited photo-induced electron transfer (PET) effect and HB-NP shows a strong fluorescent signal (nearly 48-fold enhancement) with a low detection limit of 0.5 U mL-1. HB-NP has been successfully applied in visualizing and in situ quantification of the intracellular TYR activity. Moreover, owing to the different expression levels of TYR, two human uveal melanoma cells with different invasive behaviors are distinguished by means of bioimaging and the effects of the inhibitor, kojic acid and the up-regulating treatment, psoralen/ultraviolet A on TYR activity of the two melanoma cells are evaluated. HB-NP is expected to be a useful tool to monitor diseases associated with the abnormal level of melanin and screen medicines for TYR disorder more effectively.

Melanin, as an essential pigment which determines the color of eyes, skins and hairs, is widespread in animals, plants, fungi and bacteria.1-3 Tyrosinase (TYR), a ubiquitous coppercontaining metallo-glycoprotein, is the crucial and ratelimiting enzyme involved in the biosynthesis of melanin.4 In the presence of molecular oxygen, TYR catalyzes the synthesis of melanin by converting the monophenolic derivatives to the corresponding ortho-quinones that followed by oxidative polymerization in melanocytes.5,6 TYR has been widely regarded as a relatively specific marker for melanocytic lesions because the dysfunction of the TYR expression will lead to the disorder of melanin, resulting in various diseases.7-9 For example, people who are deficient in melanin will suffer from albinism or vitiligo, and consequently are sensitive to UV exposure and at a high risk of developing skin cancer. The excessive expression of TYR means a high level of melanin which probably forecast a malignant melanoma. Moreover, recent researches have shown that TYR is associated with immune response, wound healing and dopamine toxicity exacerbating which gives rise to neurodegenerative diseases, such as Parkinson’s disease (PD) and schizophrenia.10,11 Therefore, it makes senses to develop a highly sensitive and selective probe for TYR that could be applied in clinical diseases monitoring and medicine screening. A great deal of efforts has been focused on developing a highly efficient and sensitive analysis method for the detection of TYR activity. Compared with electrophoresis, 12 radiometric,13 electrochemical14 and colorimetric methods15 which

are either time-costing, labor-consuming or only valuable for the invasive detection, fluorescence strategies offer more advantages in biological and clinical applications. Fluorescence techniques have aroused considerable interest as easyoperating, reliable and noninvasive tools for imaging and detection of biological analytes in vivo and in vitro.16-19 In recent studies, the major fluorescence sensing systems fall into two general categories: based on nanometer materials or organic fluorescent molecules. For the first sensing platform, the TYR detection mechanism is mostly based on fluorescence quenching. Gao et al.20 modified the Quantum dots (QD) with tyrosine residue, and the tyrosine as a substrate would be oxidized into dopachrome by TYR, leading to the fluorescence decreasing of QD conjugates because of nonradiative electron transition process. Based on that, they established the linear relationship between the TYR activity and the fluorescence quenching. In a similar principle, Jiang et al.21 reported a novel g-C3N4 nanosheet-based nanosensor whose fluorescence could be quenched by the catalytic oxidation product of the reaction that TYR acting on tyrosine, for the detection of TYR activity and its inhibitor. However, those “turn-off” fluorescent sensors usually are accompanied by various shortcomings, such as low signal-to-background contrast and vulnerability to external quenchers. The second sensing platform is more popular with researchers because it is a smart and general approach to design various “turn-on” fluorescent probes by incorporating recognizing groups into fluorophore scaffolds.22-24 The fluorescence response mechanisms of TYR assays performed with organic molecules are mainly based on two ways:

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The first is oxidization-cleavage mechanism25-28 that hydroxyphenyl moiety is oxidized to ortho-quinone by TYR and leaves from parent moiety with fluorophore releasing and fluorescence intensity increasing. The second is the inhibited photo-induced electron transfer (PET) process inducing the fluorescence enhancement.29,30 TYR triggers the weak fluorescent probe lighting up owing to the fact that the initial hydroxyphenyl group of the probe displays a PET effect towards parent moiety but the effect is interrupted by the conversion of hydroxyphenyl to ortho-quinone. To date, several convenient and sensitive fluorescent probes in a turn-on mode have been reported for monitoring TYR activity,25-27,29 but they are not the best choice when applied in analyzing and bioimaging intracellular TYR in living systems. Those probes have the defects of bad tissue penetration, high excitation light interference and autofluorescence disturbance due to their short-wavelength emission and small Stokes shifts. The development of a near-Infrared (NIR) fluorescent probe with large Stokes shift would overcome above drawbacks with many advantages such as low phototoxicity, deeper penetration imaging of tissue and minimum interference from autofluorescence.31,32 In addition, probes containing phydroxyphenyl or o-dihydroxyphenyl as the interaction site are still faced with an inevitable problem that the detection of TYR in living cells is usually interfered by diverse endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS) which weakens the specificity of the probes.33 Recently, it was reported that the introduction of the mhydroxyphenyl group to replace p-hydroxyphenyl and odihydroxyphenyl into the probe as a TYR recognition moiety would solve this issue.33 On the other hand, TYR is mainly located in the melanosomes of melanocytes and the stimulation from drugs will induce the misdistribution of TYR,34 so the development of a melanosome-targeting probe is of great importance to better understand the role of TYR in melanin synthesis and the drugs action mechanism in living cells. The morpholine moiety has been identified as a targeting group in melanosome,25 and would contribute to melanosome labelling. Inspired by the excellent works of predecessors, herein we synthesized a new TYR-targeting probe HB-NP as shown in Scheme 1A. To combine the outstanding properties of selectivity and melanosome-targeting, m-hydroxybenzy moiety that recognizes TYR specifically and the morpholine unit which facilitates probe accumulating in melanosome are incorporated into salicyladazine skeleton to construct ultrasensitive and highly selective fluorescent probe in subcellular level. The probe itself shows weak fluorescence due to that mhydroxybenzy moiety has a PET effect towards the parent structure of the molecule, but the process is inhibited by the oxidation of the phenol unit to the corresponding benzoquinone via TYR-catalyzed reaction, resulting in an intense “turnon” fluorescent response (Scheme 1C). As expected, HB-NP exhibits high selectivity towards TYR among various biologically relevant ROS/RNS. Furthermore, HB-NP shows a good NIR characteristic (λabs/λem = 480/675 nm) with a large Stokes shift (195 nm), which effectively reduces self-quenching of fluorescence and the interference caused by excitation light and scattered light. Thus, the new probe is more suitable for the detection of intracellular endogenous TYR activity avoiding the possible disturbances in living systems and HB-NP has been successfully applied in visualizing and in situ quantitative detection of the intracellular TYR activity in various living cells. The accurate melanosome-targeting property of HB-

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NP was demonstrated by colocalization experiment. To our knowledge, HB-NP was the first NIR melanosome-targeting probe with high selectivity for the detection of TYR activity and the probe showed promising potential for diseases monitoring and medicine screening. Scheme 1. Synthesis of probes (A) HB-NP and (B) HB-MP; (C) Proposed fluorescence turn-on mechanism for TYR activity detection with HB-NP

EXPERIMENTAL SECTION Synthesis of m-(bromomethyl)phenol. The material was prepared according to the reported literature.35 Synthesis of N-1. To a solution of 2-hydroxy-1naphthaldehyde (2.07 g, 12 mmol) in anhydrous DMF (20 mL), Cs2CO3 (3.91 g, 12 mmol) was added with stirring under a N2 atmosphere. Then, a solution of m-(bromomethyl)phenol (2.43 g, 13 mmol) in 5 mL DMF was dropped into the suspension, followed by stirring at 60 °C for 3 h. The resulting mixture was diluted with dichloromethane (40 mL). Afterwards, the organic layer was separated, washed with water (50 mL×3) and brine (50 mL×3), and then dried over anhydrous MgSO 4. The solvent was removed by evaporation, and the residue was purified by silica gel chromatography with petroleum ether/ethyl acetate (v/v, 2:1) as eluent, resulting in a white solid. Yield: (1.87 g, 56%). 1H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H), 9.46 (s, 1H), 9.08 (d, J = 8.72 Hz, 1H), 8.25 (d, J = 9.16 Hz, 1H), 7.92 (d, J = 7.84 Hz, 1H), 7.65-7.61 (m, 2H), 7.47-7.45 (m, 1H), 7.18 (t, J = 8.02 Hz, 1H), 6.91-6.89 (m, 2H), 6.71-6.69 (m, 1H), 5.38 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 191.8, 163.7, 158.1, 138.3, 138.2, 131.2, 130.3, 130.2, 129.1, 128.8, 125.3, 124.4, 118.6, 116.6, 115.7, 115.6, 114.8, 71.3. ESI-MS (m/z): calcd for C18H13O3, 277.0865 [M H]-; found, 277.1415 (ESI). Synthesis of HB-NP. N-1 (0.56 g, 2 mmol) and 4morpholinoaniline (0.36 g, 2 mmol) were dissolved into a minimal amount of ethanol, then the mixture was refluxed at 75 °C for 3 h and allowed to cool. A yellow solid precipitated out and filtered off, and then it was recrystallized from ethanol for purifying. Yield: (0.76 g, 87%). 1H NMR (600 MHz, DMSO-d6): δ 9.46 (d, J = 8.94 Hz, 1H), 9.41 (s, 1H), 9.23 (s, 1H), 8.02 (d, J = 9.12 Hz, 1H), 7.86 (d, J = 8.10 Hz, 1H), 7.56-7.53 (m, 2H), 7.41-7.38 (m, 1H), 7.21-7.19 (m, 2H), 7.14 (t, J = 7.74 Hz, 1H), 7.00-6.98 (m, 2H), 6.87 (d, J = 7.56 Hz, 1H), 6.84 (m, 1H), 6.67 (dd, J = 8.10, 1.86 Hz, 1H), 5.30 (s, 2H), 3.72 (t, J = 4.80 Hz, 4H), 3.11 (t, J = 4.80 Hz, 4H). 13C


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NMR (100 MHz, DMSO-d6): δ 158.8, 158.1, 155.6, 150.2, 144.8, 138.8, 133.8, 131.8, 130.1, 129.4, 128.9, 128.6, 126.1, 124.8, 122.2, 118.6, 117.5, 116.2, 115.6, 115.4, 114.8, 71.3, 66.6, 49.1. ESI-MS (m/z): calcd for C28H27N2O3, 439.2022 [M + H]+; found, 439.2978 (ESI). Synthesis of M-1 and HB-MP. The detailed experimental procedures are shown in Supporting Information. General procedures for tyrosinase detection. Unless otherwise noted, all the spectrophotometric measurements were made in PBS (10 mM, pH 6.8) buffer solution containing 0.5% DMSO as the co-solvent according to the following procedure. 2 mL of PBS and 15 μL of the stock solution of the probe (HB-NP or HB-MP) in DMSO were mixed in a tube, followed by adding an appropriate volume of tyrosinase sample that was prepared by dissolving in PBS. The final volume was adjusted to 3 mL with PBS to make the final concentration of the probe be 30 μM and the reaction solution was mixed well. After incubation at 37 °C for 12 h, the reaction solution was transferred to a quartz cell to measure absorbance or fluorescence with λex/em = 500/675 nm. Under the same conditions, the control sample, a blank solution containing 30 μM probes without tyrosinase was measured for comparison. The reactivity of the probe, HB-NP, with a wide variety of reactive oxygen species, ions, and enzymes was examined as above and the preparation or generation of ROS were prepared referring to the literature.36,37 Fluorescence microscopy imaging of tyrosinase in living cells. B16, HeLa, HepG2, A549, CCC-HPF-1 and CCC-HSF1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin at 37 °C in a humidified 5% CO2 incubator. OCM-1A and M619 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin at 37 °C in a humidified 5% CO2 incubator. For fluorescence imaging, the cells were seeded into confocal dishes and incubated for 24 h for cell attachment, then the probe (10 μM) was added to co-incubate for 12 h. Afterwards, the cells were washed twice with PBS to remove the free probe and fixed with methanol for 15 min for fluorescent confocal imaging. Fluorescence imaging was conducted with an excitation wavelength of 488 nm and the fluorescence was collected in the ranges of 580-700 nm. To evaluate the effects of the drugs on the enzyme activity, the adherent cells were co-incubated with kojic acid for 3 h, or the cells were treated with 8-methoxypsoralen for 12 h, then exposed to ultraviolet A with a dose of 1.0 J/cm2. The medium containing drugs was removed and washed twice with PBS, followed by incubating with the probe (10 μM) for 12 h. The fluorescence images were obtained as above. Detection of tyrosinase activity in cell extract. 1.5 × 106 B16 cells in the exponential phase of growth were collected and washed twice with ice cold PBS, then lysed in 200 µL of ice-cold RIPA buffer. The mixture was incubated for 30 min on ice and centrifuged at 12000 rpm and 4 °C for 20 min. The supernatant was collected and diluted to 200 µL as cell extract for detection. The tyrosinase activity in 10 µL cell extract was determined by ELISA kits and calculated to be 3.66 × 10 -4 U, thus, a single B16 cell averagely contained 4.88 × 10 -9 U tyrosinase. Quantification of Intracellular Tyrosinase Activity. The calibration curve for quantification of intracellular tyrosinase

activity was obtained by treating B16 cells with different amounts of kojic acid for 3 h, and then detecting the tyrosinase-triggered fluorescence intensity with the probe by fluorescence imaging and tyrosinase activity of a single B16 cell with ELISA kit analysis. Immunofluorescence Assays. Referring to the procedures reported in literatures,38,39 B16 cells were seeded on confocal dishes and incubated for 24 h, then treated with probe HB-NP (10 µM) for 12 h at 37 °C. Then, the cells were rinsed with PBS buffer and fixed with 4% paraformaldehyde for 10 min at room temperature, treated with 10 mM ammonium-chloride solution for 10 min to reduce autofluorescence, followed by permeabilization with 0.1% triton X-100 for 5 min, and incubation with blocking solution (10% normal goat serum, 1% BSA in PBS buffer) for 1 h at room temperature. Cells were then incubated with primary antibodies (mouse-anti-TRP-1 (TA-99)) in blocking solution, 1:50 dilution for 1 h at room temperature, followed by wash with PBST buffer, and then incubated with secondary antibodies (Alexa Fluor 488 conjugated) for 1 h at room temperature. Immunofluorescence images were acquired on the confocal laser scanning microscope.

RESULTS AND DISCUSSION Spectroscopic Properties of Probe HB-NP for TYR Detection. The novel NIR probe HB-NP was synthesized via aldimine condensation for TYR detection (Scheme 1A). At the same time, a probe with similar structure, HB-MP was also synthesized (Scheme 1B). Both of them showed strong fluorescence responses for TYR detection, but HB-NP had a better performance exhibiting a stronger response compared to HBMP. That might be due to the fact that slightly greater πconjugated system of HB-NP increases the emission quantum yield, and the improved signal-to-noise ratio increases the detection sensitivity. Figure 1 shows absorption and fluorescence spectra of HB-NP (Figure 1A and 1B) and HB-MP (Figure 1C and 1D) before and after reaction with TYR. HBNP showed a sharp absorption band with peaks at 303 nm and a weak shoulder band located at around 360 nm. After TYR was introduced into the HB-NP solution and incubated for 12 h, a broad absorption band around 480 nm appeared and the previous sharp band had a blue-shift to about 290 nm. In addition, the color altered dramatically from nearly colorless to red violet. Similarly, a new absorption band arose at around 400 nm for the co-incubation solution of HB-MP and TYR accompanied with a distinct color change from almost colorless to bluish violet. The obvious color change indicates the capacity of these two probes for sensing TYR activity through nakedeye. HB-NP and HB-MP were incubated with various concentrations of TYR ranging from 0 to 100 U mL-1 for 12 h. As increasing concentration of TYR, both of them showed gradual enhanced fluorescence with peak at 675 nm, while HB-NP kept higher fluorescent signals under the same conditions. At the excitation wavelength of 500 nm, nearly 48-fold enhancement emission intensity was achieved when HB-NP was incubated with 100 U mL-1 TYR, in contrast with the 9-fold enhancement of HB-MP. Therefore, HB-NP was developed for detection of TYR activity in the following experiments.



Figure 1. Absorption spectra of (A) HB-NP (1), TYR (2), the mixture of HB-NP with TYR (3) and (C) HB-MP (1), TYR (2), the mixture of HB-MP with TYR (3); the insert photographs show the corresponding solutions under daylight. Fluorescence spectra of (B) TYR and HB-NP incubated with different concentrations of TYR (0-100 U mL-1) and (D) TYR and HBMP incubated with different concentrations of TYR (0-100 U mL-1); the insert photographs are the corresponding solutions in (A) and (C) under 365 nm light irradiation. Detection of TYR Activity with HB-NP. First, the optimal reaction conditions, including temperature, pH and time were explored. The probe, HB-NP is almost non-fluorescent in the temperature and pH range of 25-42 °C and pH 3.0-11.0, while the fluorescence is enhanced in the presence of TYR. Figure S1 shows at the temperature from 30 to 42 °C, the fluorescence intensity of the reaction system fluctuates slightly and the maximum value is achieved at around 37 °C. Meanwhile, in the pH studies, a high fluorescence response appears in the pH range of 5.8-7.2, nearly same as in the physiological pH scale. It has been reported that the intramelanosomal pH will switch from pH 5 to 6.8 during the completion of melanosome maturation.40,41 Above data indicates it is extremely appropriate to use HB-NP as a melanosome-targeting probe for TYR detection in living cells. Furthermore, reaction time and kinetics of HB-NP were studied in the PBS buffer (pH 6.8, 10 mM) at 37 °C. The fluorescence signal of the reaction system increases as time goes on and nearly reaches a plateau after 12 h while HB-NP treated without TYR shows the negligible fluorescence during the same time (Figure S2A and B). The kinetic parameters of the enzymatic reaction with HB-NP were determined by Lineweaver-Burk plot (Figure S2C). The Michaelis constant (Km) and maximum of initial reaction rate (Vmax) were tested to be 87.35 μM and 1.07 μM min-1, respectively. The TYR activity assays with HB-NP were implemented the under the optimized conditions of pH 6.8 PBS buffer at 37 °C. As shown in Figure 2A, the fluorescence intensity at 675 nm was obviously positive correlation with the concentrations of TYR. The linear relationship was fitted in the TYR concentration range of 0.5 to 60 U mL-1, with an equation of ΔF = 38.34 × C (U mL-1) - 4.71 (R2 = 0.995), where ΔF is the fluorescence enhanced intensity of HB-NP after and before reaction with TYR (Figure 2B). The detection limit (3σ/k) was calculated to be as low as 0.5 U mL-1, which indicates a highly sensitive respond to TYR. Then, the selectivity of the probe HB-NP was investigated (Figure 2C). The probe was treated with the representative

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ROS, biological cations with oxidizing activity and some oxidases, which is widely distributed in human and animals and can cause false positive results in the process of TYR in situ detection, at a concentration much higher than their physiological levels, but no significant fluorescence enhancement was observed compared with the free probe. HB-NP only generated distinct fluorescence response in the presence of TYR, which indicates the high specificity for TYR detection. Kojic acid is reported as a standard inhibitor of TYR.42 To demonstrate the fluorescence enhancement of the probe was triggered by TYR, a series of kojic acid solutions from 0 to 200 μM was added to the reaction system. And the fluorescence signal of the probe decreased substantially as the concentration of kojic acid increasing, which means a dosedependent inhibiting effect of kojic acid on TYR (Figure 2D). The result highlights the potential of HB-NP for TYR activity detection as well as for inhibitors screening.

Figure 2. (A) Fluorescence spectra of HB-NP (30 μM) upon treatment with a serial of TYR (concentrations: 0-100 U mL-1). (B) Linear relationship between ΔF and the TYR concentration (0.5-60 U mL-1). ΔF is the fluorescence intensity difference after and before reaction. (C) Fluorescence responses of (1) the free probe (30 μM) and the probe (30 μM) exposure to (2) H2O2 (100 μM), (3) ·OH (100 μM), (4) 1O2 (100 μM), (5) ·O2- (100 μM), (6) ClO- (100 μM), (7) NO·(100 μM), (8) NO2- (100 μM), (9) ONOO- (100 μM), (10) TBHP (100 μM), (11) TBO· (100 μM), (12) Cu2+ (100 μM), (13) Fe3+ (100 μM), (14) Zn2+ (100 μM), (15) glucose oxidase (100 mU mL-1), (16) galactose oxidase (100 mU mL-1), (17) ascorbate oxidase (100 mU mL-1) and (18) TYR (60 U mL-1). (D) Fluorescence spectra of the free probe (30 μM) and the probe (30 μM) with TYR (60 U mL-1) reaction systems added different concentrations of inhibitors (0-200 μM). The measurements were carried out in PBS buffer (pH 6.8, 10 mM) at 37 °C after 12 h incubation with λex/em = 500/675 nm. Each test was repeated three times and the error bars represent standard deviations. Reaction Mechanism. Initially, we thought the mhydroxybenzy moiety (HB) would be hydroxylated at adjacent position by TYR and leave via 1, 6-rearragement-elimination, then the fluorescent signal molecule 1-(((4morpholinophenyl)imino)methyl)naphthalen-2-ol (MNP) was released. So MNP was synthesized (ESI) and incubated with TYR for 24 h at 37 °C. However, we found the maximum emission wavelength of the mixture was 578 nm rather than


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675 nm in the reaction system of HB-NP and TYR (Figure S3). It indicated that the red emission was not from MNP. We therefore speculated ortho-quinone did not leave from the probe. The new mechanism postulated of the TYR-catalyzed reaction is illustrated in Scheme 1C. We hypothesized that HB has a PET effect towards the parent structure (NP) of the molecule, resulting in fluorescence quenching, but the process is inhibited by the oxidation of HB to the corresponding benzoquinone (QB) via TYR-catalyzed reaction, triggering the fluorescence response. The formation of ortho-quinone was verified by the 3-methyl-2-benzothiazolinone hydrazone (MBTH) color test (Figure S4).28 The ortho-quinone produced in the enzymatic reaction will react with MBTH subsequently through a Michael reaction to form a dark-pink product. The product of the probe HB-NP and TYR was analyzed by using electrospray ionization mass spectroscopy (ESI-MS). The peak at m/z 451.3055 [M-H]- was found (Figure S5), which was attributed to QB-NP. Then, the frontier orbital energy diagram was used to further understand the mechanism of the “turn-on” fluorescent response (Figure S6).43 The time-dependent density functional theory (TD-DFT) calculation was carried out to calculate the energy of the excited state of the probe fragments with the B3LYP/6-31G(d,p) method basis set using the Gaussian 09 program and solvent effect is included using the polarizable continuum model (PCM). The HOMO energy level of the HB (-5.41 eV) was higher than that of NP (-5.63 eV), hence, the fluorescence of probe HB-NP was quenched through a PET process. After the TYR-catalyzed reaction, HB was oxidized to the corresponding benzoquinone. The HOMO energy level of the QB (-6.57 eV) was lower than that of the NP, leading to the inhibition of the PET process and the emission of strong fluorescence. In Situ Imaging and Quantitative Detection of Endogenous TYR Activity in Living Cells. B16 cells were chosen as the central experimental model due to their known high expression of TYR.20,33 To exploit the excellent sensing properties of the probe in living cells, we assessed its cytotoxicity by MTT assays at first. The average viability of B16 cells in each group was above 90% after incubation with different concentrations of HB-NP in the range of 0-100 μM for 24 h (Figure S7), confirming the low cytotoxicity of the probe to cells. Then, the probe was applied in detecting the endogenous TYR activity in living cells, such as melanoma cells B16, cancer cells (HepG2, A549 and HeLa) and normal cells (CCC-HPF-1 and CCC-HSF-1). The significant differences of fluorescence intensity suggested the different expression levels of TYR in different types of cells (Figure 3A). Melanoma cells generated the strongest fluorescence signals compared to other cancer cells and normal cells, probably because the relatively high TYR activity in B16 cells and the low abundance of TYR in other cells. Under the stimulation of different quantity of TYR inhibitor, kojic acid, the variation of intracellular TYR activity was detected. The calibration curve was established to reflect the TYR activity quantitatively by combining fluorescence images with TYR activity of a single B16 cell analyzed with ELISA kit. Figure 3B and Figure S8 show the dose-dependent inhibiting effect of kojic acid on intracellular TYR activity in melanoma cells. B16 cells were pretreated with 0-200 μM kojic acid for 3 h and then incubated with 10 μM probe for 12 h before performed the confocal imaging. Along with the in-

crease of dosage of kojic acid, the fluorescence emitted from HB-NP in cells became weaker and weaker. The TYR activity in a single B16 cell treated with different concentrations of kojic acid were detected and calculated via enzyme-linked immunosorbent assay (ELISA) kit analysis of the cell extracts (Figure 3D) and a standard curve (Figure 3C). To construct the linear relationship between the fluorescence intensity and the intracellular TYR activity in a single cell, we obtained the mean fluorescence intensity from random 20 cells with Adobe Photoshop software by reading the average red channel intensity value in a single cell area (Figure 3E),44 then the calibration curve corresponding to figure 3B was established (Figure 3F). By virtue of the calibration curve, we investigated the feasibility of HB-NP for in situ quantification of TYR activity in various types of cells via fluorescence imaging (Figure S9). The TYR activities in a single HepG2, HeLa, B16, CCC-HSF1 and CCC-HPF-1 cells were estimated to be 1.96 × 10−9, 1.85 × 10−9, 4.89 × 10−9, 2.06 × 10−9 and 2.21 × 10−9 U, respectively. Above studies indicate that the probe HB-NP can be used to quantify endogenous TYR activity in living cells and distinguish the different cells because of the relative different expression levels of TYR.

Figure 3. (A) Intracellular imaging of TYR in B16, HepG2, A549, HeLa, CCC-HPF-1 and CCC-HSF-1 cells after incubation with 10 μM probe for 12 h, respectively. (B) Intracellular imaging of TYR in B16 cells pretreated with 0, 20, 50, 100 and 200 μM kojic acid for 3 h (from a to e) and then incubated with 10 μM probe for 12 h, and confocal images of B16 cells only (f). (C) Standard curve for in vitro detection of TYR standards with the ELISA kit. (D) Absorbance of tyrosinase ELISA kit for cell extracts collected from 1.5×106 B16 cells treated with 0, 20, 50, 100 and 200 μM kojic acid for 3 h (from a to e). (E) Relative fluorescence intensity after background subtracting of the corresponding fluorescence images in panel B from a to e. (F) Calibration curve for detection of intracellu-



lar TYR activity in a single cell with probe HB-NP via fluorescence imaging. Scale bars: 20 µm. Visualizing Uveal Melanoma Cells Invasion and the Evaluation of Drugs Effect on TYR Activity of These Cells. Malignant melanoma is a life-threatening disease and hard to cure due to invasion and metastasis.45 It is an urgent need to develop a simple and effective method for identifying the metastatic potential in clinical diagnosis. The expression of TYR in melanoma cells is closely associated with their invasive and metastasizing behaviors, thus TYR could be a prognostic maker to understand the transition in the progression of invasion from poor to high. Two uveal melanoma cells, weakly invasive OCM-1A and highly invasive M619, were chosen to investigate the differential expression of TYR in the same type of cells with different invasive behaviors. After incubation of the two uveal melanoma cells with the probe HB-NP for 12 h, the red fluorescence detected in OCM-1A was stronger than M619 (panel a in Figure 4A). The experiment of in situ quantitative detection showed TYR activity in OCM-1A was about 1.2-fold higher than M619 under the same conditions (compare the values of OCM-1A-a and M619-a in Figure 4B). We found weakly invasive uveal melanoma cells possess higher TYR activity. It may be the suppression of invasion induces increased expression of TYR.45,46 Psoralens combined with the exposure to ultraviolet A have been reported could upregulate the TYR activity and were used for curing vitiligo and psoriasis. 25 The TYR inhibitor, kojic acid were often used in the cosmetics industry for skinwhitening. Therefore, we further evaluated the effects of psoralen/ultraviolet A and kojic acid on TYR activity of two uveal melanoma cells with different invasive behaviors. As shown in Figure 4, under the combined stimulation of 8methoxypsoralen (a typical psoralen) and ultraviolet A, the fluorescent signals of HB-NP in OCM-1A and M619 cells both enhanced (image b), and the fluorescence enhancement rates of TYR activity were about 16.8% (compare the values of OCM-1A-a and OCM-1A-b in Figure 4B) and 12.9% (compare the values of M619-a and M619-b in Figure 4B), respectively. When OCM-1A and M619 were pretreated with 100 μM kojic acid for 3 h then incubated with 10 μM HB-NP, the inhibitory effect on TYR activity from kojic acid with the fluorescence reduction has been observed obviously in image c. The fluorescence intensity decreased by about 62.8% (compare the values of OCM-1A-a and OCM-1A-c in Figure 4B) in OCM-1A, which was much higher than the about 30.7% drop in M619 (compare the values of M619-a and M619-c in Figure 4B). Following addition of kojic acid to the psoralen/ultraviolet A treated OCM-1A cells and M619 cells for 3 h, the cells were incubated with 10 μM probe and performed on laser confocal fluorescence microscopy. The overall fluorescence of the two kinds of cells became weak (image d) in comparison to those cells without drugs treatment (image b) but brighter than cells only treated by inhibitors (image c). These results demonstrated that both of psoralen/ultraviolet A and kojic acid had a more marked effect on weakly invasive OCM-1A, which indicated the efficacy of drugs could be fluctuant on the same type of cells with different invasion. At the same time, above experiments demonstrated the probe HB-NP could be used for medicine screening and contribute to finding the best remedy for diseases related to melanin.

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Figure 4. (A) Confocal images of OCM-1A and M619 cells treated under different conditions with probe HB-NP. (a) Cells were incubated with 10 μM probe. (b) Cells were pretreated with 50 μM 8-methoxypsoralen for 12 h, then exposed to ultraviolet A with a dose of 1.0 J/cm2 and finally incubated with 10 μM probe. (c) Cells were stimulated with 100 μM kojic acid for 3 h and incubated with 10 μM probe. (d) Cells were pretreated with 50 μM 8-methoxypsoralen for 12 h and exposed to ultraviolet A with a dose of 1.0 J/cm2, then stimulated with 100 μM kojic acid for 3 h, and finally incubated with 10 μM probe. (B) TYR activity in a single cell corresponding to (A). Scale bars: 20 µm. Then colocalization experiments were performed to confirm the capability of HB-NP in subcellular localization (Figure 5). Melanosomes of B16 cells were stained with HB-NP, followed by tagged with Alexa Fluor 488 (a commercial fluorescent dye) via immunofluorescence technique. The significant fluorescence in the red channel generated by HB-NP in B16 cells is well overlapped with the fluorescence in the green channel displayed by Alexa Fluor 488, with a good Pearson’s correlation coefficient of 0.88, reflecting the accurate melanosome-targeting property of HB-NP.


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Analytical Chemistry J. Ouyang gratefully acknowledges the support from the National Natural Science Foundation of China (21475011 and 21675014) and the Fundamental Research Funds for the Central Universities. N. Na gratefully acknowledges the support from the National Natural Science Foundation of China (21675015).

REFERENCES (1) (2) (3)

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Figure 5. Colocalization of HB-NP and Alexa Fluor 488 in B16 cells. The cells were stained with HB-NP and then tagged with Alexa Fluor 488 by immunostaining. (A) Bright-field image of the B16 cells; (B) fluorescence image of the red channel for HB-NP (λex = 488 nm, λem = 580-700 nm; (C) fluorescence image of the green channel for Alexa Fluor 488 (λex = 488 nm, λem = 500-555 nm); (D) the merged image of (A)-(C). (E) Intensity correlation plot of HB-NP and Alexa Fluor 488. (F) Intensity profile of the linear ROI across the cell (yellow line in images B-D). Scale bars: 20 µm.

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CONCLUSIONS In summary, a NIR melanosome-targeting fluorescent probe was designed for specific detection of TYR activity by incorporating the TYR-recognizing moiety, m-hydroxyphenyl and melanosome-targeting unit, morpholine into salicyladazine skeleton. The fluorescent probe in turn-on mode exhibits good selectivity and sensitivity toward TYR via the inhibited PET process. By virtue of the excellent properties, the probe has been successfully applied in bioimaging and in situ quantitative detection of the intracellular TYR activity in diverse living cells, and two uveal melanoma cells with different invasive behaviors have been distinguished because of the different expression levels of TYR. Moreover, the distinct efficacy of TYR-upregulating treatment, psoralen/ultraviolet A and TYR inhibitor, kojic acid on these two uveal melanoma cells were imaged with the probe, which validated the capability of the probe for medicine screening. We expect that our probe will be a powerful tool for clinical diagnosis and monitoring the endogenous TYR activity in living systems.

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ASSOCIATED CONTENT Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. reagents; apparatus; synthesis of M-1 and HB-MP; studies on the optimum reaction conditions; kinetics studies; reaction mechanism studies; cytotoxicity assay; fluorescence imaging; NMR spectra and MS of N-1, M-1, HB-NP, HB-MP and MNP (PDF).

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AUTHOR INFORMATION

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Corresponding Author * E-mail: [email protected]. Phone: +86-10-58805373

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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Scheme 1. Synthesis of probes (A) HB-NP and (B) HB-MP; (C) Proposed fluorescence turn-on mechanism for TYR activity detection with HB-NP 33x27mm (600 x 600 DPI)



Figure 1. Absorption spectra of (A) HB-NP (1), TYR (2), the mixture of HB-NP with TYR (3) and (C) HB-MP (1), TYR (2), the mixture of HB-MP with TYR (3); the insert photographs show the corresponding solutions under daylight. Fluorescence spectra of (B) TYR and HB-NP incubated with different concentrations of TYR (0-100 U mL-1) and (D) TYR and HB-MP incubated with different concentrations of TYR (0-100 U mL-1); the insert photographs are the corresponding solutions in (A) and (C) under 365 nm light irradiation. 30x24mm (600 x 600 DPI)


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Figure 2. (A) Fluorescence spectra of HB-NP (30 µM) upon treatment with a serial of TYR (concentrations: 0100 U mL-1). (B) Linear relationship between ∆F and the TYR concentration (0.5-60 U mL-1). ∆F is the fluorescence intensity difference after and before reaction. (C) Fluorescence responses of (1) the free probe (30 µM) and the probe (30 µM) exposure to (2) H2O2 (100 µM), (3) ·OH (100 µM), (4) 1O2 (100 µM), (5) ·O2- (100 µM), (6) ClO- (100 µM), (7) NO·(100 µM), (8) NO2- (100 µM), (9) ONOO- (100 µM), (10) TBHP (100 µM), (11) TBO· (100 µM), (12) Cu2+ (100 µM), (13) Fe3+ (100 µM), (14) Zn2+ (100 µM), (15) glucose oxidase (100 mU mL-1), (16) galactose oxidase (100 mU mL-1), (17) ascorbate oxidase (100 mU mL-1) and (18) TYR (60 U mL-1). (D) Fluorescence spectra of the free probe (30 µM) and the probe (30 µM) with TYR (60 U mL-1) reaction systems added different concentrations of inhibitors (0-200 µM). The measurements were carried out in PBS buffer (pH 6.8, 10 mM) at 37 °C after 12 h incubation with λex/em = 500/675 nm. Each test was repeated three times and the error bars represent standard deviations. 30x23mm (600 x 600 DPI)



Figure 3. (A) Intracellular imaging of TYR in B16, HepG2, A549, HeLa, CCC-HPF-1 and CCC-HSF-1 cells after incubation with 10 µM probe for 12 h, respectively. (B) Intracellular imaging of TYR in B16 cells pretreated with 0, 20, 50, 100 and 200 µM kojic acid for 3 h (from a to e) and then incubated with 10 µM probe for 12 h, and confocal images of B16 cells only (f). (C) Standard curve for in vitro detection of TYR standards with the ELISA kit. (D) Absorbance of tyrosinase ELISA kit for cell extracts collected from 1.5×106 B16 cells treated with 0, 20, 50, 100 and 200 µM kojic acid for 3 h (from a to e). (E) Relative fluorescence intensity after background subtracting of the corresponding fluorescence images in panel B from a to e. (F) Calibration curve for detection of intracellular TYR activity in a single cell with probe HB-NP via fluorescence imaging. Scale bars: 20 µm. 39x51mm (600 x 600 DPI)


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Figure 4. (A) Confocal images of OCM-1A and M619 cells treated under different conditions with probe HBNP. (a) Cells were incubated with 10 µM probe. (b) Cells were pretreated with 50 µM 8-methoxypsoralen for 12 h, then exposed to ultraviolet A with a dose of 1.0 J/cm2 and finally incubated with 10 µM probe. (c) Cells were stimulated with 100 µM kojic acid for 3 h and incubated with 10 µM probe. (d) Cells were pretreated with 50 µM 8-methoxypsoralen for 12 h and exposed to ultraviolet A with a dose of 1.0 J/cm2, then stimulated with 100 µM kojic acid for 3 h, and finally incubated with 10 µM probe. (B) TYR activity in a single cell corresponding to (A). Scale bars: 20 µm. 46x76mm (600 x 600 DPI)



Figure 5. Colocalization of HB-NP and Alexa Fluor 488 in B16 cells. The cells were stained with HB-NP and then tagged with Alexa Fluor 488 by immunostaining. (A) Bright-field image of the B16 cells; (B) fluorescence image of the red channel for HB-NP (λex = 488 nm, λem = 580-700 nm; (C) fluorescence image of the green channel for Alexa Fluor 488 (λex = 488 nm, λem = 500-555 nm); (D) the merged image of (A)-(C). (E) Intensity correlation plot of HB-NP and Alexa Fluor 488. (F) Intensity profile of the linear ROI across the cell (yellow line in images B-D). Scale bars: 20 µm. 24x16mm (600 x 600 DPI)


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Melanosome-Targeting Near-Infrared Fluorescent Probe with Large

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