Efficient Two-Photon Fluorescent Probe for Glutathione S-Transferase

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Efficient Two-Photon Fluorescent Probe for Glutathione S-transferase Detection and Imaging in Drug-induced Liver Injury Sample Jing Zhang, Zhen Jin, Xiao-Xiao Hu, Hong-Min Meng, Jin Li, XiaoBing Zhang, Hong-Wen Liu, Tanggang Deng, Shan Yao, and Lili Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01659 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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Analytical Chemistry

Efficient Two-Photon Fluorescent Probe for Glutathione S-transferase Detection and Imaging in Drug-induced Liver Injury Sample Jing Zhang2, Zhen Jin* 1, Xiao-Xiao Hu2, Hong-Min Meng3, Jin Li2, Xiao-Bing Zhang* 2, Hong-Wen Liu2, Tanggang Deng2, Shan Yao4, and Lili Feng2 1

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development

and Safety Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China. 2

Molecular Science and Biomedicine Laboratory, State Key Laboratory of

Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China. 3

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou

450001, China. 4

The People's Hospital of Dangshan County, Dangshan 235300, China.

* To whom correspondence should be addressed. E-mail: [email protected], [email protected].

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Abstract Drug-induced liver injury (DILI) is a potential complication of any prescribed medication. So far, the diagnosis of DILI is still a clinical challenge due to the lack of efficient diagnosis method. Glutathione S-transferase (GST), with a high concentration in liver cytosol, can reduce toxicity and facilitate urinary excretion by catalyzing the conjugation of glutathione (GSH) with reactive metabolites in liver. When liver is seriously damaged, GST and GSH will be released into plasma from liver cytosol, which caused a lower GST activity in liver cytosol. Therefore, monitoring the level of GST activity in liver tissue may be a potential strategy for diagnosis of DILI. Here, we reported a two-photon probe P-GST for GST activity detection for the first time. In the proposed design, a donor-π-acceptor (D-π-A) structured naphthalimide derivative with efficient two-photon properties was chosen as the fluorescent group, and a 2, 4-dinitrobenzenesulfonate group was employed as the GST recognition unit, which also acted as the fluorescence quencher. In the present of GST and GSH, the recognition unit was removed and the fluorophore was released, causing a 40-fold enhancement of fluorescence signal with a detection limit of 35 ng/mL. At last, P-GST was successfully applied in two-photon imaging of GST in cells and DILI samples, which demonstrated its practical application in complex biosystems as a potential method for diagnosis of DILI.


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Introduction Drug-induced liver injury (DILI) is a potential complication of any prescribed medication1-3, for the reason that liver plays a central role in drug metabolism and elimination4. And about one-half of acute liver failure were induced by drug5, which may lead to death or require liver transplantation6. So, in order to avoid poor prognoses which are often caused by continued ingestion of the drug, it is crucial for the early diagnosis of hepatotoxicity7. DILI is still a clinical challenge because the lack of efficient diagnosis method 6. Therefore, the development of efficient methods to detect of drug-induced liver injury is urgently needed. Glutathione S-transferase (GST), with a high concentration in liver cytosol8, plays an important role in reducing toxicity and facilitates urinary excretion by catalyzing the conjugation of glutathione (GSH) with reactive metabolites9-11. When liver is seriously damaged, hepatocyte damage will make cell membrane stability decline and permeability increase, and then GST and GSH can be released into plasma from liver cytosol12 13, leading to a lower GST activity in liver cytosol. Therefore, estimating the level of GST activity in liver may be a potential strategy for diagnosis of drug-induced liver injury. Recently, several methods have been reported for GST activity detection. They include 2, 4-dinitrochlorobenzene (CDNB)14-20, UV spectroscopy

21

, bioluminescent

methods22. However, these methods were limited due to their low operability in organisms. Fluorescence imaging method is one of the most attractive method for detection of biorelated species in living systems for its high sensitivity, real-time spatial imaging, and low-damaging to biosamples23,24. Many fluorescence probes for detection of GST have been developed22,25-29. In these probes, an electrophilic 3, 4-dinitrobenzamide group or a sulfonamide group induces an electron-transfer quenching of the emission from the fluorophore. In the present of GST, the sulfhydryl group of GSH will attack the electrophilic centers p-nitro group or the sulfonamide group, and the electrophilic group will be removed, and a large fluorescence



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enhancement is obtained25-29. However, all these GST probes are excited by one-photon laser, which limited their biological applications resulting from the photodamage to biosamples, photobleaching, and interference from autofluorescence caused by short excitation wavelength (usually < 500 nm) 30-37. Two-photon (TP) fluorescent probe, which is excited by two near-infrared photons with low energy, can well solve the abovementioned problems. Two-photon imaging can provide better three-dimensional spatial localization, higher imaging resolution, and increased penetration depth with extended observation time38-44. In this work, by employing a naphthalimide derivative, with Donor-π-acceptor (D-π-A) structured and efficient two-photon properties45,46 as the TP fluorophore, and a 2, 4-dinitrobenzenesulfonate group as the GST recognition unit, the TP fluorescence probe

4-(6-amino-1,

3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)

phenyl

2,

4-dinitrobenzenesulfonate (P-GST) was first reported for GST detection and imaging in living cells and tissues. The emission of the fluorophore will be quench by 2, 4-dinitrobenzenesulfonyl group through the photon-induced electron-transfer (PET) mechanism. In the present of GST and GSH, the sulfhydryl group of GSH was catalyzed by GST to attack the electrophilic centers of 2, 4-dinitrobenzenesulfonyl group, and the recognition unit was removed and the TP fluorophore would be released, causing a large enhancement of fluorescence signal (~40-fold) with the detection limitation of 35 ng/mL observed. P-GST was then successfully applied in imaging of GST in cells and DILI samples.


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Scheme 1. Structure and Response Mechanism of P-GST to GST. EXPERIMENTAL SECTION Reagents and Apparatus. All chemicals were used without any purification. GST and GSH were purchased from Sigma. The enzyme powder and GSH powder were dissolved into ultrapure water to obtain stock solution respectively. All enzyme stock solution was kept in -80 ºC for keeping the activity47. A stock solution of probe (0.1 mM) was prepared by dissolving an appropriate amount of P-GST into DMSO. Milli-Q reference system (Millipore) was used to get ultrapure water which was use throughout. A Fluoromax-4 spectrofluorometer (HORIBA JobinYvon) system was used to collect fluorescence signal excited by one-photon. Column chromatography and thin layer chromatography (TLC) were carried out with silica gel (200-300 mesh) and silica gel 60 F254 respectively. All silica gel (200-300 mesh) and silica gel 60 F254 were gotten from Qingdao Ocean Chemicals (Qingdao, China). All fluorescence imagings were carried out by an Olympus FV1000-MPE confocal microscope. Synthesis of Compound 2. 4-Amino-1, 8-naphthalenedicarboxylic anhydride (0.40 g, 2 mmol) and 4-aminophenol (0.20 g, 2 mmol) dissolved in acetic acid (100 ml) were added into a 150 mL round-bottomed flask, and the mixed solution was refluxed at N2-atmosphere for 10 h. After cooling to room temperature, the solution was poured into ice water (150 mL). The pH of the solution was adjusted to 7 by NaHCO3. Then, 5 ACS Paragon Plus Environment


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the solution was extracted by CH2Cl2 (3 × 250 mL). The organic layers were dried by MgSO4 and concentrated under vacuum, and the residue was purified by the silica gel chromatography (DCM) to get compound 2 as a yellow solid (0.52 g, 75%). 1H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.56 (dd, J = 7.5, 1.5 Hz, 1H), 8.38 (dd, J = 7.4, 1.5 Hz, 1H), 8.25 (d, J = 7.5 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.20 - 7.14 (m, 2H), 6.90 - 6.84 (m, 2H), 6.78 (d, J = 7.5 Hz, 1H), 4.84 (s, 2H). 13C NMR δ 161.78, 157.35, 144.72, 133.59, 131.28, 129.86, 128.40, 128.09, 126.91, 125.88, 121.56, 115.82, 110.68, 109.82. MS (ESI): [M + H]+, calcd 305.2 Synthesis of P-GST. Compound 2 (0.30 g, 1 mmol) dissolved by dry triethylamine (30 ml), dropped into 2, 4-dinitrobenzenesulfonyl chloride (0.52 g, 2 mmol) dissolved into dry DMF (70 ml), with an ice water bath for 20 min. Then, the solution was refluxed at N2-atmosphere for 8 h, and poured into ice water (100 mL). The pH was adjusted by HCl to 7, and the solution was extracted by CH2Cl2 (3 × 200 mL). The organic layers were dried by MgSO4 and concentrated under vacuum. the residue was purified by the silica gel chromatography (DCM) to get P-GST as a yellow solid (0.25 g, 50%). 1H NMR (500 MHz, Chloroform-d) δ 8.83 (d, J = 2.0 Hz, 1H), 8.75 (dd, J = 7.4, 1.9 Hz, 1H), 8.60 (dd, J = 7.5, 1.5 Hz, 1H), 8.51 (d, J = 7.4 Hz, 1H), 8.43 (dd, J = 7.5, 1.6 Hz, 1H), 8.29 (d, J = 7.5 Hz, 1H), 7.80 (t, J = 7.5 Hz, 1H), 7.41 – 7.34 (m, 2H), 7.20 – 7.14 (m, 2H), 6.82 (d, J = 7.5 Hz, 1H), 4.84 (s, 2H). 13C NMR δ 161.78, 151.88, 147.37, 146.84, 144.72, 139.05, 136.20, 133.59, 130.38, 129.09, 128.40, 128.14, 128.04, 126.91, 126.03, 125.88, 123.67, 122.85, 121.56, 110.68, 109.82. MS (ESI): [M + H]+, calcd 534.1041, [M + Na]+, calcd 557.0876.


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Scheme 2. Synthetic Route for P-GST. Spectrophotometric Experiments. 10 mM PBS (pH 7.4) solution was used to carry out all spectroscopic measurements. Different concentrations of GST were added into the probe which contained 5 µL of P-GST DMSO stock solution and 10 µL of GSH stock solution (1 mM) and PBS solution was used to adjust the total volume of mixed solution to 0.1 mL. All these operations were carried out in an ice box at 0 ºC. After vigorous shaking, these solutions were all kept at 37 ºC for 40 min, and then their fluorescence signals were measured. The investigated of TP action absorption cross-section (ϕδ). Rhodamine B48, whose TP property has been reported, was used as the reference for the TP excited fluorescence intensity measurement. After being dissolved in PBS buffered DMSO solution (10 mM, pH =7.4, H2O/DMSO = 19:1, v/v), the TP excited fluorescence spectra of P-GSTand compound 2 were collected, respectively. The TP absorption cross-section (δ) was calculated by using the following formula 1: ϕδ = δr(SsϕrΦrCr)/(SrΦsCs)

(1).

Here the subscripts of s denotes the sample, and the r denotes Rhodamine B. The S, Φ, ϕ and C denote the fluorescence intensity, fluorescence quantum, overall fluorescence collection efficiency, and sample concentration respectively. Cytotoxicity Study of P-GST on MCF-7 Cells. The cytotoxicity of P-GST on MCF-7 cells was determined by MTT assays. First, MCF-7 cells were seeded at 1 × 7 ACS Paragon Plus Environment


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105 cells per well and grown for 24 h on a 96-well plate. Various concentrations of P-GST (5, 10, 15, 20, 25, and 30 µM) were added into. After 0.5 h, all the cell medium was removed followed 200 µL of fresh DMEM cell medium being added into. Then, these cells were kept at 37 °C for 48 h. 20 µL of MTT was added into very well, and incubated for 1.5 h at 37 °C. The absorbance was recorded. Fluorescence Microscopy Imaging. GST-MCF-7 cells was transfected with a vector for overexpressing GST, and cultured as reported before27. MCF-7 cells and GST-MCF-7 cells were incubated with N-Methylmaleimide (NMM, 2 mM) or Dulbecco’s phosphate buffered saline (DPBS) only for 1 h, and then washed with DPBS for three times. After all cells were incubated with P-GST (5 µM) for 30 min at 37 °C, and washed with DPBS for three times. They were imaged with an Olympus FV1000-MPE with a mode-locked titanium-sapphire laser source. Two-Photon Fluorescence Imaging of DILI Model. DILI Model was established according to the method reported previous49-51. Male C57BL/6 mice at the age of 8-9 weeks, weighing 25-30 g were gotten commercial suppliers and housed in a room maintained at 24 °C with 40-80% humidity. All procedures and care were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory animals. After adjustment to the environment, mice were randomly divided into two groups and received intraperitoneally acetaminophen (400 mg/kg) dissolved 100 µL 0.9% saline or saline only as control respectively. Mice were euthanized, and the liver samples were collected at 10 h after injection. These liver samples were incubated P-GST (20 µM) at 37 °C for 1 h and then washed with DPBS three times. TP fluorescence images were carried out. Voltammetry. Voltammetric measurement was carried out to assess the thermodynamic feasibility fluorescence quenching of P-GST52 by a CHI 660A electrochemistry workstation (Shanghai, China) with a three-electrode cell. ITO electrode, Ag/AgCl (saturated KCl) electrode, and platinum wire were used as working electrodes, reference electrode, and counter electrode respectively. Also the spontaneity of Compound 2 (donor) quenching by the 2, 4-dinitrobenzenesulfonyl group (acceptor) was determined by Rehm-Weller formula 253: 8 ACS Paragon Plus Environment

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∆GPET = eED – eEA – ∆G00 – C

(2)

the ∆GPET, e, ED, EA, ∆G00 and C denote free energy of PET, electronic electricity, oxidation potential of the compound 2, reduction potential of the acceptor, singlet excited energy of compound 2 and the Coulombic interaction energy of the ion pair respectively. ED and EA can be estimated by voltammetric measurements. ∆G00 can be estimated according to UV absorption 54, and C is known as 0.06 eV 55. RESULTS AND DISCUSSION Design and Synthesis of P-GST. In this work, a TP probe for GST was designed, since the TP imaging technique showed better spatial localization, deeper penetration and extended observation time. Due to its excellent photophysical properties, including high fluorescence quantum yield, large TP excitation action cross section, and good photostability, a naphthalene derivative with D-π-A structure (Compound 2) was selected as the TP fluorophore. A 2, 4-dinitrobenzenesulfonyl group was designed as substrate for the GST-catalyzed reaction22,27. 2, 4-dinitrobenzenesulfonyl group was induced into the probe as the GST recognition unit. As expected, almost no fluorescence signal was detected by P-GST, because of the outstanding fluorescence quenching effect of the 2, 4-dinitrobenzenesulfonyl group through PET process. In the presence of GST and GSH, the recognition unit will be removed, releasing free Compound 2 to afford a “turn-on” fluorescent response. The synthetic route of P-GST is listed in Scheme 2, and its structure was confirmed by 1H NMR, 13C NMR and MS. The PET process causing fluorescence quenching thermodynamic feasibility was assessed by voltammetric measurements. ED and EA were determined to be 1.0 eV and -0.6 eV (see the Supporting Information, Figure S1) respectively, and ∆G00 was estimated to be 3.05 eV. The ∆GPET is calculated to be -1.51 eV, meaning the PET process is thermodynamic feasible. Spectroscopic Properties and Analytical Performance of P-GST. The spectroscopic experiments were carried out at 37 °C in PBS buffer solution (pH = 7.4, 10 mM). In the present of P-GST and GSH only, P-GST exhibited an absorption peak



at the wavelength of 450 nm. After reacting with GST, the absorption peak blue-shifted to 437 nm. P-GST itself only exhibited almost no fluorescence emission. After reacting with GST, a ∼40-fold fluorescence enhancement was observed. All the change in the UV absorbance and fluorescence are caused by the remove of 2, 4-dinitrobenzenesulfonyl group and end of the PET process52, 53. A

Absorbance

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380

400 420 440 460 Wavelength (nm)

480

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B

50000 40000 30000 20000 10000 0

500 520 540 560 580 600 620 640

Wavelength (nm)

Figure 1. (A) Absorption and (B) fluorescence emission of spectra of P-GST (5 µM) (black line) before and (red line) after reaction with GST (12 µg/mL) in the presence GSH (0.1 mM). at 37 °C for 40 min. The effect of GSH concentration on the GST activity was first investigated. As is shown in Figure S2, the GST activity was increased with increased GSH concentration presented, 0.1 mM of GSH was added in the system to keep the high activity of GST. The fluorescence signal of P-GST with different concentrations of GST were then examined. As shown in Figure 2, in the presence of GSH, with the increasing of the GST concentration, enhanced fluorescence intensity at 550 nm was observed. Moreover, the fluorescent intensities are found to be linearly proportional to GST concentration at the range of 0-0.5 µg/mL. The detection limit (S/N = 3) for GST was calculated to be 35 ng/mL.


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Figure 2. (A) In the presence GSH (0.1 mM), fluorescence response of P-GST (5 µM) to GST at varied concentrations in PBS buffered (pH 7.4) aqueous DMSO solution. B) Calibration curve of P-GST to GST, the curve was plotted with the fluorescence intensity vs GST concentration after incubation of them for 40 min. λex = 420 nm. Kinetic Investigations of P-GST to GST. The catalytic activity of GST toward the reaction of P-GST was assessed. Fluorescence kinetic curves of P-GST with GST in the presence of GSH (0.1 mM), at varied concentrations (0, 0.06, 0.6, 6 µg/mL) was depicted in Figure 3. Higher concentration of GST induced faster reaction and a larger fluorescence enhancement. When the GST concentration was 0.6 µg/mL, the fluorescence signal could reach a plateau in about 25 min. But no obvious fluorescence signal change was observed when the GST absent which demonstrated that P-GST is stable in the reaction system. At the same condition, the kinetic parameters of the GST-catalyzed reaction of P-GST were also assessed. A Lineweaver-Burke plot of 1/V (V is the initial reaction rate) versus the reciprocal of the P-GST concentration is shown in Figure S3. With Michaelis-Menten equation, the Michaelis constant (Km) and maximum of initial reaction rate (Vmax) were assessed to 11 ACS Paragon Plus Environment


be 84 µM and 0.064 µM/s, respectively.

Fluorescence Intensity

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Time (min) Figure 3. In the presence of GSH (0.1 mM), plot of fluorescence intensity of P-GST (5 µM) vs the reaction time in the presence of varied concentrations of GST (0, 0.06, 0.6, 6 µg/mL). The measurements were performed at 37 °C in 10 mM PBS (pH 7.4). λex/em = 420/550 nm. 25000


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Figure 4. Fluorescence responses of P-GST (5 µM), in the presence of GSH (0.1 mM), to various species, (1) blank, (2) CaCl2 (2.5 mM), (3) MgCl2 (2.5 mM), (4) ZnCl2 (2.5 mM), (5) CuCl2 (2.5 mM), (6) NaNO3 (2.5 mM), (7) glucose (1 mM), (8) vitamin C (1 mM), (9) homocysteine (1 mM), (10) glutamic acid (1 mM), (11) cysteine (1 mM), (12) nitroreductase (1 µg/mL), (13) γ-glutamyl transpeptidase (1 µg/mL), (14) alkaline phosphatase (1 µg/mL), (15) β-galactosidase (1 µg/mL), (16) GST (0.75 µg/mL) λex/em = 420/550 nm.

Selectivity and Effect of pH and Temperature. The selectivity of P-GST to GST was investigated. P-GST was treated with various potential interfering species, such as inorganic salts (CaCl2, MgCl2, ZnCl2, CuCl2, NaNO3), glucose, vitamin C, 12 ACS Paragon Plus Environment

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homocysteine, glutamic acid, cysteine, nitroreductase, γ-glutamyl transpeptidase, alkaline phosphatase, β-galactosidase, and the signal was recorded. As is shown in Figure 4, no obvious fluorescence signal changes were observed in the present of these interfering species. The result indicated that P-GST is highly selective for GST over the other biologically related species. The pH and temperature effect was also assessed. (see the Supporting Information, Figure S4), which indicated that P-GST responded to GST well under physiological conditions (at 37 °C and pH = 7.4). Response Mechanism. The response mechanism of P-GST to GST was also investigated. The reaction mixture solution of P-GST with GST was injected to HPLC analyses. As shown in Figure S5 (see the Supporting Information), after reaction with GST, the peak at 9.4 min belonging to P-GST decreased, and a new peak at 8.05 min belonging to compound 2 appeared. These results indicated that the fluorescence signal enhancement of the P-GST for GST was resulted from the release of Compound 2. The response mechanism of P-GST was further verified. NMM, a typical thiol-depleting agent56, was adde into the reaction solution to remove GSH in solution and evaluate its impact on the reaction of P-GST to GSH. As is shown in Figure S6 (see the Supporting Information), the fluorescence intensity decreased with the addition of NMM (1 mM), and a greater decreasement was detected with for the reaction solution with the addition of 2 mM NMM. The result indicated that in the present of GSH the enzyme-catalyzed reaction caused the fluorescence enhancement of P-GST to GST. TP Fluorescence Properties of P-GST and Compound 2. The TP fluorescence properties of P-GST and Compound 2 were assessed. The TP excitation action crosssection (Φδ) is an important parameter for TP probes. Compound 2 and P-GST were all dissolved into buffered aqueous DMSO solution (10 mM PBS, pH = 7.4; H2O/DMSO = 19:1, v/v) respectively, and the Φδ was evaluated. Exciting by 820 nm femtosecond pulses, the maximum Φδ value of Compound 2 and probe is estimated to be 95.2 GM and 2.21 GM respectively (Figure 5).


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b

30 20 10 0 700

a 750 800 850 Wavelength (nm)

900

Figure 5. (A) TP excitation spectra of P-GST (red) and Compound 2 (blank) in buffered (pH 7.4) aqueous DMSO solution. λex = 820 nm (B) TP fluorescence properties of P-GST and Compound 2 in buffered (pH 7.4) aqueous DMSO solution.

Fluorescence Imaging of GST in MCF-7 Cells. MTT assays was carried out to evaluate the cytotoxic effect of P-GST on MCF-7 cells. As shown in Figure S7 (see the Supporting Information), after incubation with P-GST for 48 h, the viability of MCF-7 cells had no significant change, even the MCF-7 cells incubated with 30 µM of P-GST. The result showed the P-GST possess good biocompatibility. The application of P-GST in cells was also carried out. The cells were incubated with P-GST for 30 min at 37 °C. As shown in Figure 6A, MCF-7 cells incubated with P-GST showed weak fluorescence signal, due to the low GST activity3,27,57-59. In contrast, in the GST-MCF-7 cells, the fluorescence signal enhanced obviously. To further prove that the fluorescence signal in the cells incubated with P-GST was caused by GST, NMM (2 mM) was used to remove the GSH in cells. As shown in Figure 6A, the fluorescence intensity decreased sharply, when NMM was added into the MCF-7 cells and GST-MCF-7 cells. The results indicated that intracellular fluorescence indeed caused by the GST enzymatic reaction. The evidence for the GST 14 ACS Paragon Plus Environment

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overexpression in GST-MCF-7 cells was also provided by western blot assay. As shown in Figure S8, the results proved the overexpression of GST in the GST-MCF-7 cells. The pixel signal intensity of cells TP imaging was further analyzed by Olympus software to compare the GST activity level in the cells quantitatively. The pixel signal intensity of 5 cells was calculated, and the result was shown in Figure 6B. The fluorescence intensity of fluorescence images increased with the increasing of GST activity. These result indicated that P-GST was capable of measuring the level of GST activity in the cells.

Figure 6. (A) Images of MCF-7 cells and GST-MCF-7 cells. (a) TP imaging of MCF-7 cells with NMM, (b) TP imaging of GST-MCF-7 cells with NMM, (c), TP imaging MCF-7 cells without NMM, (d) TP imaging GST-MCF-7 cells without NMM. (e), the DIC imaging of MCF-7 cells with NMM, (f) DIC imaging of GST-MCF-7 cells with NMM, (g) DIC imaging of MCF-7 cells without NMM (h) DIC imaging of GST-MCF-7 cells without NMM (i-l) Merge imaging of DIC and TP imaging. (B) Relative pixel fluorescence intensity of the a, b, c and d images. The strongest pixel signal intensity from the images of GST-MCF-7 cells was defined as 1. λex = 820 nm. Scale bar: 40 µm.

Application in DILI samples. DILI causing one-half of acute liver failure is still a clinical challenge because the rarity of its diagnosis and make determination of 15 ACS Paragon Plus Environment


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causality difficultly. When liver is damaged seriously, GST and GSH will be released into plasma from liver cytosol, and low the GST activity in liver60. Male C57BL/6 mice were injected with acetaminophen and the control group treated with saline only. After 10 h, the liver samples were collected and incubated with P-GST (20 µM) at 37 °C for 1 h, As shown in Figure 7, the DILI samples showed a significantly decrement fluorescence caused by the decrease in the intracellular level of GSH and GST12,13. The fluorescence signal of the control samples at different depths was collected in the Z-scan mode, with results shown in Figure S9. The results indicated that P-GST was successfully applied for imaging in tissue, with imaging depths of 48-114 µm. These results showed outstanding tissue penetrating and staining abilities of P-GST.

Figure 7. TP fluorescence imaging of control samples (a), and DILI samples (b). λex= 820 nm, Scale bar: 40 µm. CONCLUSIONS A TP fluorescent probe P-GST was designed by introducing a 2, 4-dinitrobenzenesulfonyl group, as recognition unit for GST, to a naphthalimide derivative for GST activity detection. In the present of GST and GSH, the recognition unit will be removed and the TP fluorophore will be released, causing enhancement of fluorescence signal (~40-fold) with the GST concentration changing from 0 to 12 µg/mL. The detection limitation of P-GST is 35 ng/mL. P-GST was then successfully applied in imaging of GST in cells and DILI samples. All of these features demonstrate that P-GST is promising for practical applications in biological systems


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ACKNOWLEDGMENTS This work was supported by NSFC (Grants 21405051, 21605038, 21325520, 21327009, J1210040), the Foundation for Innovative Research Groups of NSFC (Grant 21521063) and the science and technology project of Hunan Province (2016RS2009, 2016WK2002). SUPPORTING INFORMATION AVAILABLE Supplementary data. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES (1) Chitturi, S.; Farrell, G. C. Curr. Treat. Options. Gastroenterol. 2000, 3, 457-462. (2) Khetani, S. R.; Bhatia, S. N. Nat. Biotechnol. 2008, 26, 120-126. (3) Weinander, R.; Mosialou, E.; DeJong, J.; Tu, C. P.; Dypbukt, J.; Bergman, T.; Barnes, H. J.; Hoog, J. O.; Morgenstern, R. Biochem. J. 1995, 311 ( Pt 3), 861-866. (4) Lee, W. M. N. Engl. J. Med. 1995, 333, 1118-1127. (5) Kaplowitz, N. Drug Saf. 2001, 24, 483-490. (6) Kaplowitz, N. Nat. Rev. Drug Discov. 2005, 4, 489-499. (7) Farrell, G. C. J. Gastroenterol. Hepatol. 1997, 12, S242-S250. (8) Beckett, G. J.; Chapman, B. J.; Dyson, E. H.; Hayes, J. D. Gut 1985, 26, 26-31. (9) Beckett, G. J.; Foster, G. R.; Hussey, A. J.; Oliveira, D. B.; Donovan, J. W.; Prescott, L. F.; Proudfoot, A. T. Clin. Chem. 1989, 35, 2186-2189. (10) Hayes, J. D.; Pulford, D. J. Crit Rev Biochem Mol Biol 1995, 30, 445-600. (11) Mannervik, B.; Danielson, U. H. CRC Crit Rev Biochem 1988, 23, 283-337. (12) Becketta, G. J.; Dyson, E. H.; Chapman, B. J.; Templeton, A. J.; Hayes, J. D. Clin. Chim. Acta 1985, 146, 11-19. (13) Yan, H.; Cui, Z. Z.; Wang, B. C. Pak. J. Pharm. Sci. 2011, 24, 1-5. (14) Misquitta, S. A.; Colman, R. F. Biochemistry 2005, 44, 8608-8619. (15) Ralat, L. A.; Colman, R. F. J. Biol. Chem. 2004, 279, 50204-50213.



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Efficient Two-Photon Fluorescent Probe for Glutathione S-Transferase

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