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A Red-emitting Fluorescent Probe for Detection of #Glutamyltranspeptidase and Its Application of RealTime Imaging under Oxidative Stress in Cells and In Vivo Feiyan Liu, Zhen Wang, Wenli Wang, Jian-Guang Luo, and Lingyi Kong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00994 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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Analytical Chemistry

A Red-emitting Fluorescent Probe for Detection of γGlutamyltranspeptidase and Its Application of Real-Time Imaging under Oxidative Stress in Cells and In Vivo Feiyan Liu, Zhen Wang, Wenli Wang, Jian-Guang Luo*, Lingyi Kong* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China. Correspondence Author: Lingyi Kong and Jian-Guang Luo (E-mail: [email protected] and [email protected]). Abstract: γ-Glutamyltranspeptidase (GGT) plays critical roles in regulating various physiological/pathophysiological processes including the intracellular redox homeostasis. However, an effective fluorescent probe for dissecting the relationships between GGT and oxidative stress in vivo remains largely unexplored. Herein, we present a light-up fluorescent probe (DCDHF-Glu) with long wavelength emission (613 nm) for the highly sensitive and selective detection of GGT using dicyanomethylenedihydrofuran derivative as the fluorescent reporter and γ-glutamyl group as the enzyme-active trigger. DCDHF-Glu is competent to real-time image endogenous GGT in live cells and mice. In particular, DCDHF-Glu enables the direct real-time visualization of the upregulation of GGT under drug-induced oxidative stress in the HepG2 cells and the LO2 cells, as well as in vivo, vividly implying its excellent capacity in elucidation of GGT function in GGT-related biological events.

INTRODUCTION γ-Glutamyltranspeptidase (GGT, EC 2.3.2.2) is a type of surface-bound enzyme that catalyzes the transfer of the γglutamyl group from glutathione (GSH) or other γ-glutamyl compounds to acceptors like amino acids and dipeptides.1,2 The aberrant production of GGT is involved in cancer progression, invasion, and chemotherapy resistance.3-6 Notably, the pathologic states of oxidative stress could lead to increased GGT levels and GGT plays a significant role in mitigating the effects of oxidative stress by maintaining cellular glutathione metabolism and homeostasis.7-9 As an integral component of endogenous antioxidant system in cells, GGT is crucial to understand major pathophysiological resist mechanism to oxidative injury in mediating many disease states. Therefore, efficient tools to determine endogenous GGT activity should be invaluable in further exploring GGT’s underlying pathological function even for the diagnosis of GGT-related diseases. Recently, fluorescence imaging has received wide attention due to the superiorities of simplicity, facile visualization, high sensitivity/selectivity, high spatial resolution, and nondestructive detection.10-12 Many small-molecule probes have been developed for the detection of GGT in biological systems.13-28 In vivo imaging is a reliable approach for the determination of specific enzyme activity in the complex and dynamic living system because it can provide visual information in real time with excellent temporal-spatial resolution. However, GGT-

probe for in vivo imaging to understand the precise role of GGT in oxidative stress status is still lacking. To date, only one fluorescent probe (Np-Glu) has been presented to monitor GGT in oxidative stress model of cells and tissues.29 Nonetheless, the fluorescence imaging in the blue (450-495 nm) or green region (495-570 nm) can be interfered by background autofluorescence caused by intrinsic biomolecules like riboflavin and hemoglobin, rendering it difficult to be utilized for further applications in living animals.30, 31 By contrast, the red emission (> 600 nm) is favorable for in vivo bioimaging because of the long wavelength lights possess preeminent superiorities of low photodamage, little autofluorescence interference, and excellent light penetrability.32-34 Taking these situations into consideration, it is indeed much needed to develop a suitable fluorescent probe for exploring the GGT variation under oxidative stress in vivo with long emission wavelength.

Scheme 1. Structure of DCDHF-Glu and its reaction with GGT

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Herein, we report a new turn-on fluorescent probe (DCDHF-Glu) for tracking the in vivo behavior of GGT and elucidating its physiological functions. As shown in Scheme 1, a dicyanomethylenedihydrofuran derivative (DCDHF-NH2) was chosen as a fluorescence reporter due to its prominent photophysical properties such as red emission, and excellent photochemical stability.35, 36 Our strategy is based on the following considerations. Firstly, the fluorogen with longwavelength emission should have a large π-conjugated system. 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran is a favorable conjugated system,37-40 and incorporated it to the aniline ring through C=C double bond can extend the πconjugation and trigger on a red shifted fluorescence emission. Secondly, the electron-rich amine moiety is an electron donor (D) and the electron-withdrawing cyano group is an electron acceptor (A), thereby the D-A interaction in the conjugated system could further redshift the emission spectrum. On the basis of this design, red-emitting fluorogen DCDHF-NH2 was synthesized. Specifically, a γ-glutamyl amide group was introduced as an enzyme active trigger of GGT for its specific response and high sensitivity. For construction of the target probe, the Boc-protected glutamic acid moiety was grafted onto the fluorescence reporter, following deprotection under the acidic condition (Scheme S1). As a result, the electronwithdrawing glutamyl amide moiety could quench the fluorescent emission of DCDHF-Glu. The enzyme-triggered cleavage reaction induced by GGT caused the release of electrodonating amine moiety, accompanying by the generation of compound DCDHF-NH2, thereby obtaining a “Turn-On” fluorescent response with a long emission wavelength (613 nm). The versatile utility of DCDHF-Glu in biological contexts was evaluated by real-time tracking endogenous GGT in living cells and animals. Moreover, DCDHF-Glu was utilized as a powerful tool to disclose that GGT was up-regulated during drug-induced oxidative stress at both cell and animal levels, corroborating the close relationship between GGT and oxidative stress process. EXPERIMENTAL SECTION Materials and General Instruments. . All chemicals and solvents used for synthesis were purchased from commercial suppliers and used without further purification. γGlutamyltranspeptidase (GGT), monoamine oxidase A (MAOA), monoamine oxidase B (MAO B), 6-diazo-5-oxo-Lnorleucine (DON), N-acetyl cysteine (NAC), nitroreductase and GGT activity fluorometric assay kit (MAK090) were obtained from Sigma-Aldrich. Oxidation-sensitive fluorescent probe DCFH-DA (S0033-1) was purchased from Beyotime Biotechnology Co. Ltd. (Nanjing, China). 1 H NMR and 13C NMR spectra were recorded on a Bruker spectrometer (500 and 600 MHz) with tetramethylsilane as an internal standard. High resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded using Mariner ESI-TOF spectrometer. UV-Vis absorption spectra were recorded on a Shimadzu UV-2450 spectrometer. Fluorescence spectra were determined using a Shimadzu FR-6000 luminescence spectrometer. HPLC analyses were performed on Agilent 1200 high performance liquid chromatography. Cells imaging was performed with an ImageXpress Micro Confocal analysis. In vivo fluorescence images were collected by a Caliper IVIS Spectrum small animal in vivo imaging system.

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Synthesis of DCDHF-Glu. DCDHF-NH2 (Compound 3) and probe DCDHF-Glu were synthesized as outlined in Scheme S1 of the Supporting Information. The chemical structures of the intermediate products and DCDHF-Glu were confirmed by 1H NMR, 13C NMR and HR-ESI-MS. DIPEA (0.58 mL), HATU (1.28 g, 3.36 mmol), and BocGlu-OtBu (1.02 g, 3.36 mmol) were dissolved in dry DMF (20 mL) with stirring at room temperature for 30 min. Then, DCDHF-NH2 (0.68 g, 2.24 mmol) in DMF (5 mL) was added to the above solution and the reaction mixture was further stirred at room temperature for 24 h. After that, the mixture was concentrated under vacuum. The residue was dissolved in ethyl acetate and washed with water, finally dried by anhydrous Na2SO4. After the solvent was removed, the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate, 3:1) to give compound 4 as yellow solid (0.42g, 32.2%). 1H NMR (600 MHz, CDCl3): δ 9.68 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.62 (d, 8.4 Hz, 2H), 7.59 (d, J = 16.4 Hz, 1H), 6.94 (d, J = 16.4 Hz, 1H), 5.43 (d, J = 7.7 Hz, 1H), 4.18 (t, J = 7.56 Hz, 1H), 2.47 (t, J = 5.82 Hz, 2H), 2.29 – 2.23 (m, 1H), 1.86-1.85 (m, 1H), 1.78 (s, 6H), 1.48 (s, 9H), 1.45 (s, 9H). 13C NMR (150 MHz, CDCl3): δ 175.63, 174.13, 171.38, 171.15, 157.28, 147.18, 143.35, 130.70, 129.69, 129.23, 120.12, 113.45, 111.97, 111.21, 110.60, 97.72, 83.40, 81.25, 57.61, 53.21, 34.69, 31.48, 28.55, 28.19, 26.78. HR-ESI-MS m/z ﹣ calcd for C32H36N5O6 [M﹣H] , 581.2671; found 586.2670. Probe DCDHF-Glu was prepared as follows. Compound 4 (0.35 g, 0.60 mmol) was stirred in CH2Cl2 (10 mL) containing trifluoroacetic acid (10 mL) at room temperature for 5 h. After the removal of solvent, the residue was purified by preparative HPLC to afford DCDHF-Glu as brown powder (0.24 g, yield 91.5%). 1H NMR (600 MHz, MeOD) δH 7.80 (d, J = 16.4 Hz, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 16.4 Hz, 1H), 3.55 (t, J = 6.0 Hz, 1H), 2.57 (t, J = 7.1 Hz, 2H), 2.08 (m, 2H), 1.72 (s, 6H). 13C NMR (150 MHz, DMSOd6) δC 177.23, 175.52, 171.53, 170.39, 147.45, 143.46, 130.90, 128.90, 119.15, 113.34, 112.87, 112.04, 111.13, 99.26, 97.85, 53.80, 53.28, 32.87, 26.66, 25.25. HR-ESI-MS m/z calcd for ﹣ C23H20N5O4 [M﹣H] , 430.1521; found 430.1522. General Procedure for Spectra Measurements. If there are no special instructions, both absorption and fluorescence spectra were measured in 10 mM PBS buffer (pH 7.4, 0.1% DMSO) at 37 oC. A 10 mM stock solution of DCDHF-Glu was prepared in DMSO and the final test solution of DCDHFGlu (10 µM) was obtained by mixing 1 µL of stock solution with 999 µL of PBS buffer. The stock solutions of various physiologically important species were prepared from Ca2+, Fe3+, K+, Mg2+, Mn2+, Na+, Zn2+, H2O2, NH4+, CO32-, H2PO42-, HCO32-, NO3-, SO32-, GSH, Cys, Hcy, MAO-A, MAO-B, nitroreductase using ultrapure water. In fluorescence selectivity experiment, the test samples were prepared by placing appropriate amounts of the stock solutions of the analytes into probe solution. The UV-vis spectra were acquired from 350 nm to 600 nm, and the fluorescence spectra were recorded at emission wavelength range from 550 to 700 nm with excitation wavelength of 510 nm (λem = 613 nm, slit widths: 10 nm/10 nm). Cell Culture and Fluorescence Imaging. HepG2 cells and LO2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 oC in a humidified 5% CO2 incubator. Before


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Analytical Chemistry imaging, the cells were initially seeded into a 96-well plate in 100 µL DMEM medium containing 10% FBS. For imaging of exogenous GGT, the HepG2 cells were incubated with DCDHF-Glu (10 µM) for 0, 15, 30, 45, and 60 min. As a negative control, some HepG2 cells were pretreated with DON (GGT inhibitor, 1 mM) for 60 min, followed by treatment with DCDHF-Glu (10 µM) for another 60 min. After rinsing with sterile PBS buffer, the fluorescence images of the cells were acquired with an ImageXpress Micro Confocal analysis. For the cell oxidative stress damage imaging, the two types of cells were treated with different concentrations of menadione (0, 10, 20, 30, 40, and 50 µM) for 8 h, and then incubated with DCDHF-Glu (10 µM) for 1 h before imaging. For the comparative investigation, the cells were pretreated with menadione (50 µM) in the presence of N-acetyl cysteine (NAC, 1 mM) for 8 h before treatment with DCDHF-Glu. In Vivo Visualization of GGT in Tumor-bearing Nude Mice. All of the animal experiments were approved by the Animal Ethical Committee of China Pharmaceutical University, and the procedures were performed strictly in accordance with the guidelines of the National Institutes of Health on the use of experimental animals (China). For in vivo imaging, about 3×106 HepG2 cells were grafted into the 6-week-old BALB/c nude mice to establish tumor model. Tumors were allowed to grow to 8-10 mm in diameter before they were ready for the experiments. Then, the realtime in vivo imaging was obtained at different time internals (5, 10, 20, 30, 40, 50, 60, 70, and 80 min) after orthotopic injection of DCDHF-Glu (50 µM, 50 µL) by using a Caliper IVIS Spectrum small animal in vivo imaging system with an excitation filter of 460-560 nm. For inhibitor experiment, DON (2 mM, 50 µL) in PBS was intratumoral injected into a nude mouse to restrain GGT activity in tumor. After 6 h, DCDHFGlu (50 µM, 50 µL) was injected on tumor region of mouse through intratumoral injection. For in vivo oxidative stress experiment, a nude mouse was given an in situ injection of menadione (0.5 mM) to induce the overproduction of reactive oxygen species (ROS). After 6 h, the mouse was injected with DCDHF-Glu (50 µM, 50 µL). RESULTS AND DISCUSSION Spectral Properties of DCDHF-Glu to GGT. As shown in Figure 1, free DCDHF-Glu (10 µM) displayed a faint yellow color with a maximum absorption band at 425 nm and a very weak emission at 572 nm (Φ = 0.00071 in PBS solution), which was attributed to the substitution of the amino group of DCDHF-NH2 by glutamic acid. After treatment with GGT (40 U/L), the solution changed to pink with a decrease in the absorption at 425 nm and formation of a new red-shifted band at 506 nm. Simultaneously, the maximum fluorescence wavelength was shifted to 613 nm (24.29-fold emission increment), accompanied by a colour change from colourless to red. This long wavelength emission is favorable for in vivo bioimaging, because of minimum photo-damage, deep tissue penetration, and minimum interference from auto-fluorescence of indigenous biomolecules. Notably, the remarkable 107 nm Stokes shift could facilitate the efficient monitoring the fluorescence signal of DCDHF-Glu in biological samples, as such a large Stokes shift is advantageous to minimize the fluorescence background interference and increasing the signal fidelity.41 This strong fluorescence enhancement was ascribed to the

cleavage of the γ-glutamyl group and the further formation of DCDHF-NH2.

Figure 1. (A) Absorption and (B) fluorescence emission spectra of DCDHF-Glu (10 µM) before and after reaction with GGT (40 U/L) in PBS buffer. Note: Excitation wavelength is 510 nm, the inset shows the corresponding color change toward GGT.

Figure 2. (A) Time dependent fluorescence spectra of DCDHFGlu (10 µM) upon addition of GGT (40 U/L). Inset: the plot of fluorescence intensity at 613 nm depending on time. (B) The fluorescence spectra change of DCDHF-Glu (10 µΜ) to GGT (0-60 U/L) in PBS buffer. Inset: Linear correlation between the intensity (613 nm) and the GGT concentration.

To verify the enzymatic cleavage reaction mechanism depicted in Scheme 1, the reaction mixture of DCDHF-Glu with GGT was examined by HR-ESI-MS and obtained a major peak at m/z 301.1096 (Figure S1), corresponding to DCDHF﹣ NH2 ([M﹣H] , 301.1095), clearly confirming that the enzymatic reaction caused the release of DCDHF-NH2 (Figure S1). HPLC analyse also verified the generation of DCDHF-NH2 as a major product (Figure S2), which exhibited a chromatographic peak at 49.150 min. These results corroborated the fact that the distinct fluorescence enhancement of DCDHF-Glu for GGT was resulted from the conversion of DCDHF-Glu into DCDHF-NH2 upon the enzyme-triggered cleavage reaction. The fluorescence quantum yield of DCDHF-NH2 was 0.0078 in PBS buffer, while its quantum yield in DMSO was determined to be 0.17. Like other push-pull fluorophores bearing dicyanomethylene-based acceptors, the quantum yield increases in a more viscous environment.42, 43 This dependence on media can be advantageous for biological imaging, because the viscous cellular environment and the interaction with intracellular macromolecules could increase the quantum yield of fluorophore.43 Next, we investigated the pH-dependent and temperature-dependent responses of DCDHF-Glu toward GGT (Figure S3). There were almost no change of fluorescence signal in the pH range from 5.0 to 9.0, and temperature from 25 to 40 oC. Upon reaction with GGT, the reaction solution exhibited remarkable fluorescence enhancement, especially kept a maximum intensity at pH 7.4 and 37 oC, suggesting that DCDHF-Glu responded to GGT well under the normal physiological conditions. Moreover, the reaction kinetic was investigated through the time-dependent fluorescence response (Figure 2A). The fluorescence intensity increased to its maximum within about 60 min, indicating its promising capacity for rapid detection of GGT. Thus, all the following spectro-



scopic tests were conducted in PBS buffer (pH 7.4) at 37 oC for 60 min. The concentration-dependent spectral changes of the probe for GGT (0-60 U/L) were carried out to investigate the fluorescence sensing sensitivity of DCDHF-Glu (Figure 2B). The fluorescence intensity progressively increased upon a stepwise addition of GGT. A linear relationship between the fluorescence enhancement and GGT concentration in the range of 0-40 U/L can be obtained with R2 as 0.9946. The calculated limit of detection (3σ/slope) for GGT was 0.0379 U/L, which is lower than some mentioned probes,15-22 implying that this probe system possessed high sensitivity for GGT under physiological condition. In addition, the Michaelis constant (Km) of DCDHF-Glu and GGT was assessed to be 11.48 µM according to the Michaelis-Menten equation (Figure S4).

Figure 3. (A) Fluorescence response of DCDHF-Glu (10 µΜ) to GGT (40 U/L), as well as other various analytes in PBS buffer. (B) The fluorescence intensity of DCDHF-Glu toward GGT in the presence of other analytes in PBS buffer. 0. Blank; 1. GGT; 218. Ca2+, Fe3+, K+, Mg2+, Mn2+, Na+, Zn2+, H2O2, NH4+, CO32-, H2PO42-, HCO32-, NO3-, SO32-, ClO-, Cys, Hcy: 50 µΜ; 19. GSH: 10 mM; 20-22. MAO-A, MAO-B, nitroreductase: 10 µg/mL. Error bars represent ± SE, n = 3.

cence enhancement was indeed arisen from the enzymecatalyzed reaction of GGT. The capability of DCDHF-Glu for detecting the GGT expression level was further evaluated in HepG2 cells and LO2 cells under the same imaging condition (Figure S6). HepG2 cells exhibited a 5.7 times higher fluorescence intensity than LO2 cells, suggesting the higher level of GGT in HepG2 cells than LO2 cells, which agreed well with the fact that GGT expression is elevated in human tumors.44 Altogether, these results demonstrated that DCDHF-Glu was capable of visualizing endogenous GGT in living cells with high cell membrane permeation.

Figure 4. Fluorescence images of endogenous GGT in HepG2 cells. (A) The HepG2 cells were incubated with different concentrations of DCDHF-Glu (image a-d, 0, 2.5, 5, 10 µM) for 60 min; image e: the cells were pretreated with the inhibitor of DON (1 mM) for 60 min, and then incubated with DCDHF-Glu (10 µM) for 60 min. (C) The HepG2 cells were treated with DCDHF-Glu (10 µM) at different time points (images f-j, 0, 15, 30, 45, 60 min). Scale bar, 20 µm. (B and D) The relative intensity levels of the cells shown in the above corresponding images by using an ImageXpress Micro Confocal analysis. The intensity from image d and j were defined as 1.0 respectively. The bars are shown as mean ± standard deviation (SD), n = 3.

Selectivity and Interference. The fluorescence responses of DCDHF-Glu toward some biologically relevant species (including Ca2+, Fe3+ , K+, Mg2+, Mn2+, Na+, Zn2+, H2O2, NH4+, CO32-, H2PO42-, HCO32-, NO3-, SO32-, ClO-, Cys, Hcy, 50 µΜ; GSH, 10 mM; MAO-A, MAO-B, Nitroreductase, 10 µg/mL) were performed to evaluate the specificity of this probe. As shown in Figure 3A, DCDHF-Glu possessed pronounced selectivity toward GGT which ascribed to the specific cleavage of the the γ-glutamyl group only by GGT. Moreover, the anti-interference experiments showed that the fluorescent turn-on response of DCDHF-Glu to GGT was not interfered by other biologically relevant analytes (Figure 3B). This result also cleraly indicated that DCDHF-Glu had prominent specificity toward GGT under physiological conditions. Fluorescence Imaging of GGT in Cells and in Mice. Encouraged by the desirable fluorescence response of DCDHFGlu to GGT under simulated physiological conditions, we further evaluated the capability of the probe for fluorescence imaging of GGT activity in human hepatocarcinoma cell line HepG2 cells and normal hepatocyte cell line LO2 cells. Initially, the MTT assay was performed and revealed no significant effects on cell viability at the concentration of 5-50 µM after incubation for 48 h (Figure S5), implying the low cytotoxicity and good biocompatibility of DCDHF-Glu. Thereafter, the cell imaging experiments were performed by utilizing the ImageXpress Micro Confocal analysis system. After incubated with DCDHF-Glu, the dose- and time-dependent fluorescence responses were clearly observed (Figure 4 and Video S1). In contrast, the HepG2 cells pretreated with inhibitor DON (1 mM) exhibited a largely suppressed fluorescence (Figure 4A, image e).27 This result verified that the intracellular fluores-

Figure 5. (A) Time-dependent fluorescence images (pseudocolor) of endogenous GGT in nude mice with skin-pop (s.p.) injection of 106, 105 HepG2 cells. (B) In vivo imaging of nude mice with skinpop injection of probe DCDHF-Glu. The injection position (A1, A2) were for LO2 and HepG2 grafted cell positions respectively.

With the prominent performance of DCDHF-Glu in cellular fluorescence imaging, we further applied this probe to realtime track GGT activity in HepG2 cells lines in situ in living mice. 106 and 105 HepG2 cells were injected into the nude mice, respectively, and incubated for 6 h, followed with in situ injection of probe DCDHF-Glu (50 µM, 50 µL). The timedependent fluorescence responses were imaged with a Caliper IVIS Spectral imaging system. As shown in Figure 5A, a significant fluorescence signal in the cell-injected region was observed at 10 min in both groups. After 40 min, the remarka-


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Analytical Chemistry ble fluorescence signal can be clearly detected for 105 and 106 HepG2 cells in living nude mice, and the 106 cell-group exhibited stronger fluorescence than the 105 cell-group at each time point, indicating high sensitivity of DCDHF-Glu for GGT imaging. For comparison purposes, a mouse was injected with 106 LO2 and HepG2 cells, and DCDHF-Glu was applied to image the two types of cell lines at 6 h after injection. As shown in figure 5B, the fluorescence intensity of HepG2 cells injection region was higher than that of the LO2 cells region at each time point, which implied that the fluorescence enhancement difference between the HepG2 and LO2 cells reflected the different expression levels of GGT in the two types of cells. Therefore, the preceding observations clearly demonstrated that probe DCDHF-Glu could be utilized as a facilitative tool to directly visualize endogenous GGT in living cells and in mice.

is documented that glutathione (GSH) plays key roles in protection against oxidative stress and acts as an antioxidant in the maintenance of cellular redox status.7 Increasing evidence strongly suggests that GGT is critical in the homeostasis of GSH by breaking down extracellular GSH and supplying cysteine, the rate-limiting substrate for intracellular GSH biosynthesis and GGT deficiency results in oxidative stress and cellular susceptibility to oxidant injury.9, 48, 49 As such, GGT has an important role in antioxidant defense. Therefore, convenient and sensitive detection of GGT level is great importance in disease diagnosis and pathophysiology elucidation.

Figure 7. Effects of menadione on the GGT activity in HepG2 cells and LO2 cells. (A and C) Fluorescent imaging of GGT in cells with DCDHF-Glu (10 µM). Images a-f and i-n: the cells were pretreated with various concentrations of menadione (0, 10, 20, 30, 40, and 50 µM) for 8 h; images g and o: the cells were firstly treated with menadione (50 µM) for 8 h, and then incubated with DON (1 mM) for 1 h; images h and p: the cells were pretreated with menadione (50 µM) in the presence of NAC (1 mM) for 8 h. Scale bar, 20 µm. (B and D) The relative fluorescence intensity of corresponding images a-h, and i-p, the fluorescence intensity levels of image f and n were defined as 1.0.

Figure 6. (A and C) Flow cytometry analysis of ROS changes in the cells exposed to different menadione concentrations (0-50 µM) for 8 h and incubated with a ROS probe DCFH-DA (10 µM) for 1 h. (B and D) Flow cytometry analysis of GGT changes in the cells after incubated with different concentrations of menadione (0-50 µM, 8 h), followed by treatment with DCDHF-Glu (10 µM, 1 h). For inhibition experiment, the cells were pretreated with menadione (50 µM, 8 h), and then treated with DON (1 mM, 1 h) before incubation with probe DCDHF-Glu (10 µM, 1 h). For the comparative investigation, the cells were pretreated with menadione (50 µM, 8 h) in the presence of NAC (1 mM) before treatment with DCDHF-Glu (10 µM, 1 h).

Detection of GGT in Drug-induced Oxidative Stress Model. Oxidative stress is exerted by the increased formation of reactive oxygen species (ROS) which results from the imbalance between ROS generation and antioxidant defenses and has been considered as the main risk factor of numerous diseases (e.g., cancer, inflammation, Alzheimer’s disease).45-47 It

According to the preliminary imaging studies in living cells and mice, DCDHF-Glu was further applied to dissect the relationships between the GGT expression level and the oxidative stress. We constructed the oxidative stress cell model by using the oxidative stress stimulant, menadione, which can cause severe oxidative stress due to the generation of endogenous ROS.50, 51 Fow cytometry analysis was firstly performed to quantify menadione-induced oxidative stress. The HepG2 cells or LO2 cells were incubated with different concentrations of menadione (0-50 µM) for 8 h before loading with commercial ROS probe DCFH-DA (10 µM) or probe DCDHF-Glu (10 µM) for 1 h. As exprcted, the fluorescence signal intensity (FL1-A detector channel) increased as the concentration of menadione increased, implying that the ROS concent was dose-dependently increased by induction of menadione in both cells (Figure 6A and 6C). Simultaneously, the flow cytometry analysis showed that the fluorescence intensity (FL3-A detector channel) also increased in the DCDHF-Glu-treated cells with menadione in a dosedependent manner (Figure 6B and 6D), indicating upregulation of GGT level in menadione-induced oxidative stress. This upreguation was further verified by the commercial GGT fluorometric assay kit (Figure S7). In addition, the 50 µM menadione pretreated cells with DON



showed a markedly decreased fluorescence (Figure 6B and 6D), which revealed that the fluorescence response was induced by GGT, and the intensity increment was attributed to the upregulation of GGT. Furthermore, fluorescence imaging of GGT in the two types of cells was conducted to monitor GGT changes under oxidative stress status reguled by menadione (Figure 7, Figure S8 and S9). The HepG2 cells and LO2 cells were incubated with menadione at different concentrations (0-50 µM, 8 h) prior to treatment with DCDHF-Glu (10 µM, 1 h). The menadione-treated cells displyed a dose- and time-dependent fluorescence enhancement and this increase can be efficiently restrained by DON, which also revealed the upregulation of GGT in the the menadione-induced oxidative stress. This finding could be attributed to the oxidative stress stimulator, which induced the overproduction of ROS, and caused significant increase in the cellular oxidative stress. Since GGT is critical in maintaining GSH and cysteine homeostasis for cellular redox regulation.7, 49 The cells have to upregulate GGT to induce sufficient cysteine/GSH and thus counteract the ROS level for the maintenance of redox balance. To further confirm this mechanism, the comparative experiments were conducted by using a cysteine prodrug, N-acetyl cysteine (NAC) which is the pharmacological source of cysteine for GSH synthesis.51, 52 The cells were pretreated with menadione (50 µM, 8 h) in the presence of NAC (1 mM), subsequently treated with DCDHF-Glu (10 µM, 1 h). As expected, the strong fluorescence intensity could be effectively attenuated by NAC (Figure 7, images h and p). The fluorescence intensity decrease was further verified by flow cytometry (Figure 6B and 6D). The above results indicate that the upregulation of GGT under the oxidative stress injury is closely associated with the level of GSH, and this probe can be employed to detect the endogenously generated GGT level under oxidative stress.

site and diffused to the whole tumor region in 50 min. Comparatively, for the menadione pretreated tumor-bearing mouse, the tumor region (group B) displayed a much strong fluorescence signal, and the intensity of group B was higher than group A at every time point, suggesting the up-regulation of endogenous GGT in drug-induced oxidative stress model. Meanwhile, pre-injection of inhibitor DON significantly decreased the intratumoral fluorescence (group C), and the intensity of group C was lower than that of group A at every time point, implying that the fluorescence response of the tumor was arise from the activity of GGT. These in vivo results demonstrated that DCDHF-Glu could be utilized as a prominent bioimaging tool for real-time visualization of GGT formation under drug-induced oxidative stress status in living animals. To our konwledge, DCDHF-Glu is the first redemitting probe has been used for the real-time tracking GGT activity under drug-induced oxidative stress in vivo. CONCLUSIONS In conclusion, by combining the strategies of enzymetriggered reaction mechanism and optimal-screening, we developed a red-emissive fluorescent probe DCDHF-Glu based on dicyanomethylenedihydrofuran derivative for detecting GGT with a detection limit of 0.0379 U/L. Combining its favorable light-up fluorescence feature, high selectivity, long wavelength emission, large Stokes shift, low cytotoxicity, and good membrane permeability, we have applied DCDHF-Glu to achieve the real-time visualization of GGT in living cells and mice. Notably, by utilizing DCDHF-Glu, the fluorescent imaging studies revealed a higher level of GGT in HepG2 cells than in LO2 cells. Moreover, this probe is capable of monitoring the upregulation of endogenous GGT under druginduced oxidative stress in live cells and in vivo, and supplementary N-acetyl cysteine could suppress this upregulation, directly revealing a close relationship between oxidative stress injury and GGT generation, and the GGT increase is connected with the level of GSH. These findings demonstrate that this probe could be served as a promising tool to disclose GGT function in oxidative stress injury and elucidation of the underlying pathological mechanism.

ASSOCIATED CONTENT Supporting Information Figure 8. Time based in vivo fluorescence imaging (pseudocolor) of endogenous GGT in HepG2 cells tumor-bearing mice after intratumoral injection of 50 µM DCDHF-Glu in PBS (image A), or menadione (0.5 mM, 6 h), and then injected with 50 µM DCDHF-Glu (image B); or DON (2 mM) followed by a intratumor injection of 50 µM DCDHF-Glu (image C), the fluorescence images were acquired at 5, 10, 20, 30, 40, 50, 60, 70, and 80 min.

Encouraged by the above-mentioned results of fluorescence imaging GGT in drug-induced oxidative stress model of cells, we further investigated the suitability of DCDHF-Glu in tracking GGT level in living tumor-bearing mice. As shown in Figure 8, after intratumor injection of DCDHF-Glu, a significant fluorescence enhancement could be clearly observed in the tumor region (group A) within 5 min, and the increased fluorescence intensity gradually extended from the injection

The Supporting Information is available free of charge on the ACS Publications website at DOI: *** The synthesis and structural characterization of compounds, additional experimental details, data for response mechanism, the pHdependent and temperature-dependent spectra, Lineweaver-Burk plot for the enzyme-catalyzed reaction, cell viability, the comparisons of HepG2 and LO2 cells about the fluorescence intensity, GGT fluorometric assay kit analysis, time-dependent fluorescence imaging of endogenous GGT in menadione-treated cells, the spectrums of 1H NMR, 13C NMR and HR-ESI-MS (PDF). Video S1 of HepG2 cells stained by DCDHF-Glu (ZIP).

AUTHOR INFORMATION Corresponding Author * Lingyi Kong: E-mail: [email protected]. * Jian-Guang Luo: E-mail: [email protected].

Author Contributions


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Analytical Chemistry All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported financially by the National Natural Science Foundation of China (No. 81573570), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63).

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