Bioluminescent Probe for Hydrogen Peroxide Imaging in Vitro and in

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Bioluminescent Probe for Hydrogen Peroxide Imaging in Vitro and in Vivo Wenxiao Wu,† Jing Li,† Laizhong Chen,† Zhao Ma,† Wei Zhang,† Zhenzhen Liu,† Yanna Cheng,‡ Lupei Du,† and Minyong Li*,† †

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China ‡ Department of Pharmacology, School of Pharmacy, Shandong University, Jinan, Shandong 250012, China S Supporting Information *

ABSTRACT: Reactive oxygen species (ROS) often have significant roles in mediating redox modifications and other essential physiological processes, such as biological process regulation and signal transduction. Considering that H2O2 is a substantial member of ROS, detection and quantitation of H2O2 undertakes important but urgent responsibility. In this report, a bioluminescent probe for detecting H2O2 was well designed, synthesized, and evaluated. This probe was designed into three parts: a H2O2-sensitive aryl boronic acid, a bioluminescent aminoluciferin moiety, and a self-immolative linker. After extensive evaluation, this probe can selectively and sensitively react with H2O2 to release aminoluciferin. It should be pointed out that this probe is a potential bioluminescent sensor for H2O2 since it can provide a promising toolkit for real-time detection of the H2O2 level in vitro, in cellulo, and in vivo.

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bioluminescence are the most used methods. Moreover, laser confocal imaging and living imaging technology contribute to accomplishing active substances visualized in cells but understanding distinctive characteristics in in vivo biology to be realtime online.8−10 Bioluminescent imaging (BLI) is a reliable, sensitive, appropriate, and noninvasive imaging technique and has been enormously practical on imaging various processes envisioned in life sciences. Generally, BLI employs firefly luciferase and its high-specific substrate luciferin or aminoluciferin to produce light in the presence of ATP, O2, and Mg2+.11 It has well been recognized that the 6′-amino (or 6′-hydroxyl) group of Daminoluciferin (or luciferin) is essential for enzyme combinations. Consequently, the caging at 6′-position of aminoluciferin (or luciferin) can hinder its recognition with firefly luciferase as well as quench the release of bioluminescence.12,13 For example, we have successfully developed several APN bioluminogenic probes by conjugating D-aminoluciferin with the APN recognition amino acids.14 On the basis of the same strategy, Chang and co-workers recently have presented a highly efficient reaction-based approach by utilizing the unique reaction between H2O2 and the boronate moiety: a H2O2 Dluciferin probe has been devised and used for biological application.15

eactive oxygen species (ROS) refer to the oxygen containing substances whose chemical character is active that exist in the living organisms or the natural environment. So far, they are classified into four groups: (a) excited state oxygen molecule (singlet state oxygen molecule, 1O2); (b) oxygenrelated free radicals (superoxide anion free radical O2−•; hydroxyl radical, HO•; hydroperoxyl radical, HO2•); (c) hyperoxides (hydrogen peroxide, H2O2; lipid peroxide, ROO•); (d) nitrogen oxide (nitric oxide, NO). H2O2 is a vital member for cell signaling, energy synthesis, cell growth, living material production, cell metabolism, and so on.1−3 However, the imbalance induced by H2O2 production can impair tissues and organ systems and then lead to aging, injuries, and diseases, such as malignant tumor, cardiovascular disease, Alzheimer’s disease, Parkinson’s syndrome, etc.4−6 Therefore, H2O2 is a strategic attention of study into the molecular mechanisms underlying the development and progression of a disease. Given the short lifetime, low concentration of the steady state, and high reactivity of H2O2, it is challenging to capture and detect H2O2 in vivo. In this context, developing a sensitive, selective, and biocompatible probe for detecting H2O2 in cells and tissues is one of the thought-provoking topics.7 So far, there are various methods for detecting H2O2, including electron spin resonance, chemiluminescence, spectrophotometry, fluorescence, bioluminescence, and so on. In view of the steadfast sensitive, convenient, and noninvasive advantages for understanding in vivo biology, fluorescence and © 2014 American Chemical Society

Received: June 30, 2014 Accepted: September 5, 2014 Published: September 5, 2014 9800

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pH 7.4, with 10 mM MgCl2 and 0.1 mM ZnCl2 at 37 °C. The bioluminescence images of the cells were captured by using a Zeiss Observer A1 microscopy implemented with a cooled CCD camera (Hamamatsu ORCA-R2). The bioluminescence spectra were determined with an IVIS Kinetic (Caliper Life Sciences) equipped with a cooled CCD camera that was used for bioluminescent imaging. Luciferase was purchased from Promega. ATP and catalase were purchased from Aladdin. The pseudocolored bioluminescent images (in photons/s/cm2/scr) were superimposed over the gray scale photographs of the animals. Circular ROIs were drawn over the areas and quantified using Living Image software. The results were reported as total photon flux within an ROI in photons per second. Kinetic Analysis. A volume of 50 μL of probe 3 (10 μM) solution was added to 50 μL of ROS (1 mM) solution in the Tris buffer (50 mM, pH 7.4) and incubated at 37 °C for 30 min in 96-well plate. The fluorescent properties of probe 3 and aminoluciferin were recorded. The secondary rate constant (k) was determined by following the methods described in the literature. A volume of 50 μL of H2O2 probe 3 (10 μM) and 50 μL of H2O2 between 1 and 4 mM were used. The assays were performed in triplicate. Selectivity Measurement. A volume of 50 μL of probe 3 (20 μM) and the same volume of ROS (200 μM) (H2O2, O2−, TBHP, t-BuO•, ClO−, •OH) added into a white 96-well plate, buffer solution as a control. After various time (10, 20, 30, 40, 50, 60 min) incubation, added commercially available luciferase (2 μg/100 μL) and ATP (2 mM), the mixture solutions came out to initiate bioluminescence and then measured the bioluminescence intensity. As a blank control, Tris buffer was added instead of ROS solution under the same condition. Relative total photon flux for each condition was calculated by dividing the total photon flux for the experimental condition by the total photon flux for the blank control. Measurement for Different H2O2 Concentration. A volume of 50 μL of H2O2 probe 3 (20 μM) was added to 50 μL of various concentrations of H2O2 (0, 10, 20, 50, 100, 200, 500 μM) in the Tris buffer (pH 7.4) and incubated at 37 °C for 5, 20, 40, and 60 min in a white 96-well plate, then added 100 μL of luciferase (100 μg/mL) with ATP (2 mM), and measured the bioluminescence intensity. Influence of ROS to the Properties of Aminoluciferin and Luciferase Bioluminescence. A volume of 50 μL of aminoluciferin (20 μM) was added to 50 μL of various ROS (200 μM) in the Tris buffer (pH 7.4) and incubated at 37 °C for 0, 5, 20, 30, 40, 50, and 60 min in a white 96-well plate, then added 100 μL luciferase (100 μg/mL) with ATP (2 mM), and measured the bioluminescence intensity. Cell Culture. ES-2 cells (human ovarian cancers cell line) were purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences. ES-2 cells expressing firefly luciferase (Fluc) were supplied by Cellcyto. The ES-2-luc cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere in a 5% CO2 incubator. Cell Bioluminescence Imaging of Exogenous H2O2. Cells were grown in black 96-well plates (4 × 105 cells per well). After a 24-h incubation period, the medium was removed and cells were treated with 100 μL of various concentrations of H2O2 (range 2.5 to 500 μM) and 100 μL of probe 3 (100 μM), luciferase activity was measured 15 min later using a Xenogen IVIS Spectrum imaging system. Luminescent signal (photons

The bioluminescence emission peak for D-luciferin and aminoluciferin were 565 and 584 nm, respectively. It should be underlined that the above-mentioned red-shifted bioluminescence profile for aminoluciferin is of significance for penetrating tissues in live animal imaging.13 In the current study, we designed a H2O2 bioluminescent probe (probe 3) by coupling boronic acid group with D-aminoluciferin at the 6′-position by a self-immolative linker. This probe is a poor substrate for firefly luciferase, which leads to the weak bioluminescence; however, in the presence of H2O2, the caging group is proposed to be cleaved to release the free D-aminoluciferin, simultaneously. The free D-aminoluciferin will experience a subsequent recognition with the firefly luciferase to produce the bioluminescence (Scheme 1). The quantitative relationship between the bioluminescence intensity and the concentration of H2O2 has been confirmed herein, as well. Scheme 1. Design Strategy for H2O2-Mediated Release of Aminoluciferin from Probe 3

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EXPERIMENTAL SECTION Synthesis. The preparation of probe 3 (Figure 1) was conveniently reached by three steps. Briefly, the synthesis starts

Figure 1. Structure of the probe 3.

with the preparation of intermediate 1 using an one-pot reaction: 5-aminobenzo[d]thiazole-2-carbonitrile as the starting materials by reacting with bis(trichloromethyl)carbonate (BTC) and 4-bromobenzyl alcohol. Compound 1 reacted with bis(pinacolato)diboron via the catalysis of Pd(dppf)Cl2 under a dry and anaerobic atmosphere to obtain the pure boronate intermediate 2 after column separation. Probe 3 was provided with the cross-coupling reaction of 2 and D-cysteine hydrochloride under N2 atmosphere without light. The detailed preparation of these probes is described in the Supporting Information. Bioluminescent Assay Instrumentation. Millipore water was used to prepare all aqueous solutions. Measurements for bioluminescent assays were performed in 50 mM Tris buffer, 9801

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anesthetized with isoflurane and intratumorally injected with 50 μL of probe 3 (5.3 mM), followed immediately by bioluminescent imaging every 5 min for 30 min. The bioluminescent signal changed with the time. Day 2: The mice were intratumorally injected with 50 μL of cisplatin (333 μM, diluted by normal saline) followed by intratumorally injected with 50 μL of probe 3 (5.3 mM, diluted by normal saline−DMSO 1:1) 12 h later. Controls for the cisplatin experiment were completed by intratumorally injecting vehicle (50 μL) followed by the probe 3, 12 h later. Subsequently, light production was measured 10 min after the injection of probe 3. The relative total photon flux for each condition was calculated by dividing the total photon flux after the injection of cisplatin or vehicle by the total photon flux before the injection of cisplatin or vehicle.

per second) for each well was measured and plotted as average values (experiments conducted in triplicate). Cell Bioluminescence Imaging of Endogenous H2O2. Cells were grown in black 96-well plates (4 × 105 cells per well). After a 24-h incubation period, the medium was removed, and cells were treated with 100 μL of various concentrations of cisplatin (range 0 to 400 μM). After incubating for 12 h in the incubator, 100 μL of probe 3 (100 μM) was added to each well and the luciferase activity was measured 5 min later using a Xenogen IVIS Spectrum imaging system. Luminescent signal (photons per second) for each well was measured and plotted as average values (experiments conducted in triplicate). Subcellular Localization Study. Cells were grown in glassbottom dishes (4 × 105 cells). The bioluminescence images of the cells were captured by using a Zeiss Observer A1 microscopy implemented with a cooled CCD camera (Hamamatsu ORCA-R2). (A) After a 36-h incubation period, the medium was removed, cells were washed with RPMI 1640 (without FBS) twice, 1 mL of probe 3 (20 μM) was added to the dish, and luminescence was recorded for 6 s. (B) After a 36-h incubation period, the medium was removed and cells were treated with 3 mL of H2O2 (30 μM) for 30 min, the medium was removed, cells were washed with RPMI 1640 (without FBS) twice, 1 mL of probe 3 (20 μM) was added to the dish, and luminescence was recorded for 6 s. (C) After a 24-h incubation period, the medium was removed and cells were treated with 3 mL of cisplatin (30 μM). After incubating for 16 h in the incubator, the medium was removed, cells were washed with RPMI 1640 (without FBS) twice, 1 mL of probe 3 (20 μM) was added to the dish, and luminescence was recorded for 6 s. Mice Model. Balb/c-nu male mice, 8 weeks of age, were purchased from the Animal Center of China Academy of Medical Sciences (Beijing, China). To generate tumor xenografts in mice, ES-2-luc cells (1 × 107) were implanted subcutaneously under the right armpit region of each 4-weekold female nude mouse. Mice were single or group-housed on a 12:12 light−dark cycle at 22 °C with free access to food and water. Tumors were allowed to grow for 2 weeks before imaging. All animal studies were approved by the Ethics Committee of Qilu Health Science Center, Shandong University, and conducted in compliance with European guidelines for the care and use of laboratory animals. In Vivo Bioluminescence Imaging of Exogenous H2O2. Day 1: Mice bearing ES-2-luc subcutaneous tumors were anesthetized with isoflurane and intraperitoneally injected with 0.2 mL of probe 3 (5.3 mM), followed immediately by bioluminescent imaging every 5 min for 30 min. The bioluminescent signal changed with the time. Day 2: The mice were intraperitoneally injected with 0.2 mL of H2O2 (98 mM, diluted by normal saline) followed by an intraperitoneal injection of 0.2 mL of probe 3 (5.3 mM, diluted by normal saline−DMSO 1:1). Controls for the exogenous H2O2 experiment were completed by intraperitoneally injecting vehicle (0.2 mL) followed by the probe. Subsequently, light production was measured 15 min after the injection of probe 3. The relative total photon flux for each condition was calculated by dividing the total photon flux after the injection of exogenous H2O2 or vehicle by the total photon flux before the injection of exogenous H2O2 or vehicle In Vivo Bioluminescence Imaging of Endogenous H2O2. Day 1: Mice bearing ES-2-luc subcutaneous tumors were

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RESULTS AND DISCUSSION Kinetic Analysis. The reactions were monitored by the change in fluorescence wavelength of the probe before and after reacting with various ROS (Figure 2). Compared with the

Figure 2. (a) Fluorescent property of aminoluciferin and probe 3, 5 μM, λex = 345 nm. (b) The fluorescent intensity of probe 3 added in various ROS, probe 3, 5 μM; ROS, 500 μM; λex = 345 nm.

fluorescent property of the aminoluciferin, this evidence indicated that the caged group of probe 3 was removed upon reaction with ROS and free aminoluciferin was released, in the meanwhile the maximum emission peak experienced a red shift from 450 to 525 nm. As a result, fluorescence intensity was changed significantly before and after the addition of ROS. 9802

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than the control in bioluminescent signal over an hour; there was little to no increase in signal when the boronic acid probe reacted with the other ROS. These attractive results indicate that probe 3 has the reasonable activity and selectivity to identify H2O2. Measurement for Different H2O2 Concentration. To further confirm the sensitivity of the probe, the bioluminescence of the probe was measured by adding diverse concentrations of H2O2. Figure 5a,b suggested that the

The secondary rate constant (k) was measured based on the change of the fluorescent property of probe 3 before and after degradation. A volume of 50 μL of H2O2 whose concentration was from 1 to 4 mM was added to 50 μL of probe 3 (10 μM), then the fluorescence intensity was measured every 5 s for 20 min. As time passed, the fluorescent intensity of the mixture at 450 nm was decreased and that at 525 nm was increased clearly. Pseudo first order reaction rate eq 1 of the fluorescence is ln(Ft /Fmax ) = −k′t

(1)

Ft, the fluorescence intensity (450 nm) at t seconds; Fmax, the fluorescence intensity (450 nm) at the beginning; k′, pseudo first-order reaction rate constant. The second order reaction rate constant can be obtained through eq 2 (Figure 3):

k′ = KM

(2)

Figure 3. Linear relationship between pseudo first-order rate constant (k′) and the concentration of H2O2.

M, the concentration of H2O2; K = 0.52 ± 0.08 M−1 s−1. As we know, the smaller the K, the slower the reaction. The small K of the probe makes the contribution to real-time H2O2 detection. Selectivity Measurement. Next, the activity and selectivity of the probe was examined via a comparison of the bioluminescence intensities of the products achieved by the probe reacted with different ROS. As a result, the solution mixture came out to initiate bioluminescence. Figure 4 exhibited that addition of H2O2 presented a 11-fold increase

Figure 5. (a) Change of the probe 3 (10 μM) relative bioluminescence intensity after added diverse concentrations H2O2; (b) linear relationship between the relative bioluminescence intensity of the probe 3 and the concentration of H2O2 (R2 = 0.993); (c) change of aminoluciferin (10 μM) relative bioluminescence intensity after added diverse ROS at a different point.

Figure 4. Relative total photon flux of probe 3 with ROS for various time incubation after adding luciferase and ATP. 9803

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concentrations of cisplatin for different incubation times. As a result, 12 h is the optimum (Figure S1 in the Supporting Information). Bioluminescence intensity of the cells incubated with various concentrations of cisplatin for 12 h was measured at different time points, and the peak of luminescent signal is from 4 to 7 min (Figure S2 in the Supporting Information). As depicted in Figure 7, the bioluminescence intensity was

bioluminescence intensity of probe 3 increased following the increased concentration of H2O2 within a certain range. Moreover, varying from 5 to 100 μM of hydroperoxide, the relative total photon flux could reflect a good “linear” growth (R2 = 0.993), which can be used to quantify the concentration of H2O2. Influence of ROS to the Properties of Aminoluciferin of Luciferase Bioluminescence. To determine if the ROS can affect aminoluciferin and luciferase, their bioluminescence activities were collected using the same method as above. Most of the ROS have no obvious influence on aminoluciferin and luciferase; however, after adding 1O2, the bioluminescence intensity decreased in a time-dependent manner (Figure 5c). We speculated that 1O2 might disturb the excited electron intermediate that was produced during the bioluminescence process; thus the bioluminescence character changed. Cell Bioluminescence Imaging. On the basis of our in vitro result, the activity of probe 3 was further verified for detecting exogenous H2O2 in living cells. As depicted in Figure 6, the

Figure 7. Imaging the endogenous H2O2 activity in ES-2-luc cells. (a) Bioluminescence imaging of ES-2-luc cells incubated with probe 3 and various concentrations of cisplatin and (b) quantification of bioluminescent signal from part a.

increased with the induction of cisplatin, especially in the concentration range between 17 and 50 μM; the intensity with induction is two times than that without induction. However, at the high concentration of cisplatin (50−400 μM), the bioluminescence signal was gradually decreased, which may be caused by the strong cytotoxicity of cisplatin that diminishes the cell number. Therefore, the change of the concentration of endogenous H2O2 in living cells can be detected by probe 3, even if the H2O2 was produced by cells in normal condition. If H2O2 was removed by free radical scavenging agents, the bioluminescence intensity is weaker than the control (Figure S3 in the Supporting Information). All results indicate that the probe has potential to quantify the amount of H2O2 in a real time manner. The bioluminescence imaging ability of probe 3 was well examined for detecting cell-based exogenous or endogenous H2O2. Figure 8 exhibits the imaging potential of probe 3 without and with exogenous H2O2 and endogenous H2O2 in the ES-2-luc cells, respectively. These interesting results indicate that the lipid solubility of probe 3 could lead to the reasonable membrane permeability. This property can ensure that this probe can easily move across into cells and release the bioluminescence signal upon reacting with H2O2, which implies the various concentrations of H2O2 in the cell.

Figure 6. Imaging the exogenous H2O2 activity in ES-2-luc cells (a) bioluminescence imaging of ES-2-luc cells incubated with probe 3 and various concentrations of H2O2; (b) quantification of bioluminescent signal of part a.

activity of probe 3 did not change in the living cells; what’s more, the bioluminescence intensity is dose-dependent on the concentration of H2O2. Given the above results, we do believe that our probe 3 can be employed to trace H2O2 (as low as 2.5 μM) in the living cell. We next examined whether the probe could have detection efficacy for the endogenous H2O2 in living cells. It is wellknown that cisplatin is a potent and broad-spectrum antitumor agent, especially for ovarian cancer. Considering that cisplatin can induce the oxidative stress to produce endogenous H2O2 for killing tumor cells,16 we choose cisplatin as the cell stimulus for the endogenous H2O2. The cells were treated with various 9804

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Figure 8. Subcellular localization study with probe 3 of ES-2-luc cells: (A) probe 3 (20 μM); (B) probe 3 with exogenous H2O2; (C) probe 3 with endogenous H2O2. (a) Bright-field images, expose time,10 ms; (b) cells stained by probe 3, expose time, 6 s; (c) merged images. Objective lens: 40×.

In Vivo Bioluminescence Imaging. To investigate whether this probe has detection efficacy for H2O2 in vivo, a living animal imaging test was well conducted. In such a case, the tumor xenografts in nude mice were used as the animal model. A volume of 0.2 mL of H2O2 (98 mM) solution was intraperitoneally injected as the exogenous H2O2, and then 0.2 mL of probe 3 (5.3 mM) was intraperitoneally injected. Controls for the exogenous H2O2 experiment were completed by intraperitoneally injecting vehicle (0.2 mL). After 15 min, light production was measured. The relative total photon flux for each condition was calculated by dividing the total photon flux after the injection of H2O2 or vehicle by the total photon flux before the injection of H2O2 or vehicle. The relative total photon flux increased significantly after the injection of H2O2. The results in Figure 9 further confirmed that our probe could image exogenous H2O2 activity in xenografted breast cancer tumors in mice. Interestingly, mice treated with only probe in the absence of exogenous H2O2 also displayed modest but measurable bioluminescence in xenografted breast cancer tumors, which suggests that H2O2 may detect basal levels of H2O2 produced in these living animals. Therefore, to determine whether this emission signal was due in part to the detection of endogenous H2O2, we intratumorally injected 50 μL of cisplatin as the external stimulation for endogenous H2O2 before the probe was intratumorally injected. It is observed that the cisplatin-treated animals exhibited a significantly increasing bioluminescent signal compared to vehicle control animals (Figure 10). The above results suggest that probe 3 can release a timedependence bioluminescence in vivo.

Figure 9. Bioluminescence imaging of exogenous H2O2 activity with ES-2-luc tumors in nude mice. (a) Integrated bioluminescence emission for mice with probe 3, in the presence or absence of the exogenous H2O2. (b,c) Representative time-course images from one mouse treated with H2O2 or the control.

CONCLUSIONS In conclusion, a novel bioluminescent probe was well designed, prepared, and evaluated for H2O2 imaging herein. After careful evaluation, our probe paraded high selectivity and sensitivity for hydroperoxide in vitro, in cellulo, and in vivo in the presence of firefly luciferase. The boronic acid recognition group in this probe can respond to the H2O2 quickly and then the structure and optical property of the probe change timely, and as a result, probe 3 can identify the H2O2 in a real time manner. In addition, the reasonable histocompatibility and long bio-

luminescence wavelength of this probe may contribute to the feasible application in the animals. Overall, all experimental results suggest that this bioluminescent probe stipulates a selective and sensitive platform for real-time imaging of both exogenous and endogenous hydrogen peroxide in living mice. Furthermore, the influence of the levels of hydrogen peroxide on health, aging, and disease may be illuminated in the upcoming future by utilizing such a bioluminescent approach. We do believe that this bioluminescence probe could provide a convenient toolkit for the longitudinal noninvasive monitoring

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone/fax: +86-531-8838-2076. Author Contributions

W.W. and J.L. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Program on Key Basic Research Project (Grant No. 2013CB734000), the Program of New Century Excellent Talents in University (Grant No. NCET-11-0306), the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT13028), the Shandong Natural Science Foundation (Grant No. JQ201019), and the Independent Innovation Foundation of Shandong University, IIFSDU (Grant Nos. 2014JC008 and 2012JC002).

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Figure 10. Bioluminescence imaging of endogenous H2O2 activity with ES-2-luc tumors in nude mice. (a) Integrated bioluminescence emission for mice with probe 3, in the presence or absence of the cisplatin. (b,c) Representative time-course images from one mouse treated with cisplatin or the control.

of oxidative stress caused by the excess amount of H2O2 in diagnostic and therapeutic fields.

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ASSOCIATED CONTENT

S Supporting Information *

The details for preparation of probe and their NMR and HRMS spectra. This material is available free of charge via the Internet at http://pubs.acs.org. 9806

dx.doi.org/10.1021/ac502396g | Anal. Chem. 2014, 86, 9800−9806