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A mitochondria-targeted fluorescent probe for imaging hydrogen peroxide in living cells Jian Xu, Yan Zhang, Hui Yu, Xudong Gao, and Shijun Shao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04424 • Publication Date (Web): 22 Dec 2015 Downloaded from http://pubs.acs.org on December 25, 2015

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Analytical Chemistry

A Mitochondria-targeted Fluorescent Probe for Imaging Hydrogen Peroxide in Living Cells Jian Xu,† Yan Zhang,‡ Hui Yu,† Xudong Gao,† and Shijun Shao*,† †

Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural

Medicine of Gansu Province, Lanzhou institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, P. R. China. ‡

College of Physics and Electronic Engineering, Northwest Normal University, Lanzhou 730070,

China.

ABSTRACT: Hydrogen peroxide (H2O2), as a type of reactive oxygen species (ROS), can be endogenously produced from the mitochondrial electron transport chain in aerobic respiration, and plays important roles in several physiological processes. However, the design and synthesis of fluorescent probes which can detect mitochondrial H2O2 in living cells still remain rare. Herein, we report the preparation of a novel cationic probe 1 (Mito-H2O2), which targets the mitochondria in living cells and is sensitive to the presence of H2O2. The probe Mito-H2O2 displays desired properties such as high specificity, “Turn-On” fluorescence response with suitable sensitivity, appreciable water solubility and rapid response time (within 5min). The sensing mechanism was confirmed by high-resolution mass spectroscopy analysis and the mechanism of “Turn-On” fluorescent response was also determinated using a density functional

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theory (DFT) calculation method. Moreover, as a biocompatible molecule, the probe Mito-H2O2 has been successfully applied for the detection of the intrinsically generated intracellular H2O2 in living cells, and the fluorescence co-localization studies indicate that the probe localizes solely in the mitochondria of HeLa cells.

■ INTRODUCTION As a type of reactive oxygen species (ROS), hydrogen peroxide (H2O2) is an essential oxygen metabolite in living systems, and mounting evidence supports its role as an oxidative stress marker and a messenger in cellular signal transduction.1-4 In this regard, aberrant production or accumulation of H2O2 from the mitochondrial electron transport chain can lead to the accumulation of oxidative stress and the subsequent functional decline of organ systems, which is connected to serious human diseases including cancer, diabetes, neurodegenerative Alzheimer’s, Parkinson’s, and Huntington’s diseases.5-10 In addition, it is reported that overexpression of catalase, a mitochondrial targeted peroxide-detoxifying enzyme, can increase life span in mouse models.11 Considering the far-ranging impacts of H2O2 homeostasis on human health and disease, it is necessary to devise new imaging methods that allow visualization of localized production and accumulation of H2O2 in living cells, especially in specific subcellular organelle such as mitochondria. In the past decade, fluorescence sensing and imaging has emerged as one of the most powerful techniques and has been used in diverse fields including biology, clinical diagnosis, and drug discovery.12-14 Moreover, some of well-designed fluorescent probes can be targeted to precise subcellular locations,15-18 which provides a powerful and versatile method to monitor the level, localization, and transportation of bio-molecules within the context of living systems and


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especially in certain cells and organelles. To date, quite a lot of fluorescent probes have been developed for H2O2 detection and most of them were based on a reaction-based approach by utilizing the unique reactivity between H2O2 and boronate moiety.19-23 However, to the best of our knowledge, just one example has been reported to quantify the mitochondrial H2O2 in living cells and thus mitochondrial-targeted small molecules for detection of specific ROS remain rare.24 Recently, Chang et al. used a mitochondrial-targeting triphenylphosphonium (TPP) moiety for H2O2 and Cu+ detection.24,25 Tang et al. also embedding triphenylphosphonium group into tetraphenylethene fluorophore for mitochondrial imaging and tracking.17 However the toxicity of triphenylphosphonium is a particular concern for biological applications. Not long ago, quaternarized pyridine or indolium moiety has been confirmed to target mitochondria specifically. Guided by this principle, Peng et al. prepared a pyridine based BODIPY derivative for mitochondrial location26,27 and Yu et al. presented several mitochondria targeted fluorescent probes for in vitro and in vivo imaging ClO- and SO2 derivatives.28,29 Very recently, a Nalkylated BODIPY fluorescent probe was applied by our group for monitoring and imaging of H2O2 both in living cells and living angelfish.30 Inspired by these findings, a novel “Turn-On” fluorescent probe 1 (Mito-H2O2) was presented here for the detection of mitochondrial H2O2 (Scheme 1). In our design, a carbazole group was introduced as the fluorogen due to its excellent photophysical and photochemical properties such as high fluorescence quantum yield, high extinction coefficient, excellent photochemical stability and low biological toxicity.31,32 Meanwhile, a p-pinacolborylbenzyl moiety has been chosen as the reaction site for H2O2, because of the high oxidative activity of H2O2 for boronate.30,33 In addition, the quaternarized quinoline unit here not only acts as a


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mitochondria-targeted carrier but also improves the water solubility of the probe. As expected, probe Mito-H2O2 showed weak fluorescence due to the photo-induced electron transfer (PET) process and such an extremely low background signal is rather favorable to affording high detection sensitivity for H2O2. Reaction of Mito-H2O2 with H2O2 under physiological conditions causes the oxidation of the boronate moiety, followed by the 1,6-rearrangement elimination reaction and thereby the release of dye 2.30 As a result, a “Turn-On” fluorescent response is obtained, which leads to the development of a highly sensitive and selective method for monitoring H2O2 activity in biological systems. The probe exhibited high selectivity toward H2O2 among the various reactive oxygen species (ROS), reactive nitrogen species (RNS) and biologically relevant species. Most notably, the probe displayed excellent mitochondria targeted properties and has been successfully applied for imaging the mitochondrial H2O2 in Hela tumor cells.

Scheme 1 Design of Probe Mito-H2O2 and Its Reaction with H2O2


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■ EXPERIMENTAL SECTION Materials and Measurements. All chemicals were purchased from Sigma and Aladdin reagent company without further purification except especial instruction. All the organic solvents were of analytical grade. Water was purified by a Milli-Q system. 1H and 13C NMR spectra were measured on a Varian INOVA 400 M spectrometer. ESI mass spectra were recorded out on an Agilent 1100 series LC/MSO Trap of MS spectrometer. Hela cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All pH measurements were made with a Sartorius basic pH-Meter. UV-visible spectral studies were performed on a Perkin Elmer Lambda-35 UV-visible double beam scanning spectrophotometer. Solution fluorescence spectra were measured on a Perkin Elmer LS 55 scanning spectrofluorometer equipped with a Xenon flash lamp. Samples for absorption and fluorescence measurements were contained in 1 cm×1 cm quartz cuvettes. Transmission electron microscopy (TEM) images were collected on a JEOLJEM-2010 instrument with a 100 keV accelerating voltage. Fluorescence imaging experiments were performed on an Olympus Fluoview 1000 confocal laser scanning microscope with excitation at 488 nm. Measurement of Fluorescence Quantum Yields. For determination of the fluorescence quantum yields (Φfl), we used a Perkin Elmer LS 55 instrument, which fluorescein in 0.1 M NaOH as a fluorescence standard. Fluorescence quantum yields (Φfl) were obtained with the following equation (F denotes fluorescence intensity at each wavelength and Σ[F] was calculated by summation of fluorescence intensity). Φflsample = Φflstandard Absstandard Σ[Fsample] / Abssample Σ[Fstandard] Determination of the Detection Limit. The detection limit was calculated based on the fluorescence titration. In the absence of H2O2, the fluorescence emission spectrum of probe


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Mito-H2O2 was measured by five times and the standard deviation of blank measurement was achieved. To gain the slop, the fluorescence intensity at 527 nm was plotted to the concentration of H2O2. So the detection limit was calculated with the following equation: Detection limit = 3σ/k Where σ is the standard deviation of blank measurement, k is the slop between the fluorescence intensity versus H2O2 concentration. Computational study. The density functional theory (DFT) calculations were performed for gas-phase molecules by using the Gaussian 09 package. The adopted exchange-correlation functional was B3LYP with Becke’s three parameter form,34,35 in which the nonlocal correlation is expressed by the Lee, Yang, and Parr functional,36 and the local correlation part is by the Vosko, Wilk, and Nusair III functional.37 The 6-31G(d) basis set was used in the DFT calculations. The synthetic routes of probe Mito-H2O2 are shown in Scheme 2. Synthesis of N-ethylcarbazole. NaH (60% in oil, 2.8 g, 70 mmol) was gradually added to a solution of carbazole (10 g, 60 mmol) in DMF (100 mL) under stirring for 1.5 h. The reaction temperature was kept below 30 °C. After hydrogen evolution closed, iodoethane (9.4 g, 60 mmol) was added dropwise at room temperature. After 2 h, the reaction mixture was added to water (200 mL), and the solution was extracted with ether. The solvent was removed and purified by silica gel column chromatography (hexane/ethyl acetate, 10/1, v/v, as eluent) to afford white product (10.7 g, 92%). Synthesis of Compound 3. Phosphorus oxychloride (78.5 g, 513 mmol) was added dropwise to 20 mL DMF at 0 °C. The solution was allowed to warm to room temperature, and Nethylcarbazole (10 g, 51.3 mmol) in 50 mL CH2Cl2 was added. The reaction mixture was heated


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to 90 °C and kept at this temperature for 24 h. It was poured into water and extracted with chloroform. The chloroform extract was washed with water, and the solvent was removed to yield a deeply colored product which was purified by silica gel column chromatography (hexane/ethyl acetate, 7/3, v/v, as eluent) to afford the desired compound 3 (2.9 g, 26%). 1H NMR (CDCl3, 400 MHz): δ=10.11 (s, 1H, CHO), 8.62 (s, 1H, carbazole H), 8.18 (d, 1H, carbazole H), 8.01 (d, 1H, carbazole H), 7.56 (t, 1H, carbazole H), 7.48 (d, 2H, carbazole H), 7.35 (t, 1H, carbazole H), 4.42 (q, 2H, CH2), 1.49 (t, 3H, CH3). 13C NMR (CDCl3, 400 MHz): δ 13.8, 37.9, 108.7, 109.2, 120.3, 120.8, 123.1, 124.0, 126.8, 127.2, 128.5, 140.7, 143.6, 191.8. MS (ESI): Calcd for C15H13NO: 223.10, found: m/z 224.10 (M+H)+. Synthesis of Compound 4. 4-methylchinoline (7 mmol, 1.0 g) and 4-(Bromomethyl)benzeneboronic acid pinacol ester (8.4 mmol, 2.5 g) were dissolved in toluene, and then the mixture was refluxed at 110 °C for 12 h. The obtained white powdery solid was filtered, washed with toluene and dried in vacuo to afford pure compound 4 (2.4 g, 34%). 1H NMR (CD3CN, 400 MHz): δ=9.21 (d, 1H, chinoline H), 8.44 (d, 1H, chinoline H), 8.25 (m, 1H, chinoline H), 8.06 (m, 1H, chinoline H), 7.91-7.96 (m, 2H, chinoline H), 7.74 (d, 1H, ArH), 7.67 (d, 1H, ArH), 7.26 (d, 2H, ArH), 6.18 (s, 2H, CH2), 2.99 (s, 3H, CH3), 1.25(s, 12H, CH3). 13C NMR (CD3CN, 400 MHz): δ 20.7, 25.0, 61.5, 75.6, 85.2, 118.8, 120.3, 123.9, 127.4, 130.9, 136.1, 137.4, 138.4, 149.5, 161.5. MS (ESI): Calcd for [C23H27BNO2]+: 360.21, found: m/z 360.21 (M)+. Synthesis of Probe 1. Compound 3 (1 mmol, 0.22 g) and compound 4 (1 mmol, 0.28 g) were mixed in ethanol (20 ml), and then piperidine (0.05 ml) was added to the solution. The reaction mixture was refluxed with stirring for 1 h and then evaporated in vacuo. The resulting solid was dissolved in CH2Cl2, and the organic layer was washed three times with water, dried over anhydrous MgSO4, and evaporated in vacuo. The residue was purified by silica gel column


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chromatography (CH2Cl2/MeOH, 10/1, v/v, as eluent) to afford compound 1 (0.12 g, 21%) as a red solid. 1H NMR (CD3CN, 400 MHz): δ=9.07 (d, 1H, chinoline H), 8.93 (d, 1H, chinoline H), 8.76 (s, 1H, carbazole H), 8.24-8.28 (m, 2H, chinoline H), 8.18 (d, 1H, carbazole H), 8.07 (m, 2H, chinoline H), 7.98 (m, 1H, carbazole H), 7.75 (d, 1H, ArH), 7.68 (d, 1H, ArH), 7.58-7.62 (m, 2H, carbazole H), 7.46 (d, 1H, carbazole H), 7.36-7.39 (m, 1H, carbazole H), 7.32-7.35 (m, 2H, ArH), 6.91 (d, 2H, vinylic), 6.12 (s, 2H, benzyl-CH2), 4.49 (q, 2H, CH2), 1.45 (t, 3H, ethyl-CH3), 1.32 (s, 12H, CH3). 13C NMR (CD3CN, 400 MHz): δ 14.2, 22.7, 44.1, 73.9, 84.3, 110.3, 115.9, 120.2, 121.1, 122.7, 123.7, 126.8, 127.2, 128.3, 129.4, 133.4, 135.5, 135.6, 138.3, 140.8, 141.9, 148.2, 154.6. MS (ESI): Calcd for [C38H38BN2O2]+: 565.30, found: m/z 565.30 (M)+.

Scheme 2. Synthesis of Probe Mito-H2O2 (1)

■ RESULTS AND DISCUSSION Spectroscopic Property of Probe Mito-H2O2 and Its Response to H2O2. Initially, spectroscopic evaluation of probe Mito-H2O2 and its response to H2O2 was carried out in


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DMSO/phosphate buffer (1:99 v/v, 20 mM, pH 7.4) at room temperature (25°C). Probe MitoH2O2 exhibits a maximum absorbance at 490 nm and its reaction with H2O2 induces a new blueshift band at 376 nm (Figure 1A), which was reasonably attributed to the H2O2-induced oxidation of probe Mito-H2O2 to release fluorophore 2. Meanwhile, a prominent solution color change from light-red to colourless was observed, thereby suggesting H2O2 can be detected with the “naked-eye” when using probe Mito-H2O2. In the fluorescence emission spectrum, probe Mito-H2O2 features a very weak emission at 527 nm, with a quantum yield of Φfl = 0.003. This low background signal is mainly due to the photo-induced electron transfer (PET) process and is rather desirable for sensitive detection. However, treatment of probe Mito-H2O2 with H2O2 triggers a dramatic increase of fluorescence intensity at 527 nm and accordingly an obvious bright green-colored fluorescence was clearly observed (Figure 1B). 33-fold increase in its fluorescence intensity suggested that probe Mito-H2O2 is one of the most sensitive probes for H2O2 detection in bionic systems.38,39

Figure 1. Absorption (A) and fluorescence (B) spectra of probe Mito-H2O2 (5 µM, black line), and the reaction mixture (red line) of 5 µM probe Mito-H2O2 with 50 µM H2O2 in DMSO/phosphate buffer (1:99 v/v, 20 mM, pH 7.4) for 5 min. The inset shows the color changes (A) and fluorescence changes (B) of probe MitoH2O2 in the absence and presence of H2O2 under visible or UV light at 365nm.


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Time Response and Linearity Test. The time course of the fluorescent emission of probe Mito-H2O2 (5µM) in the absence and presence of H2O2 (10 equiv) was studied, and the results are shown in Figure 2A. The fluorescent emission (527nm) of probe Mito-H2O2 itself exhibits no noticeable changes in DMSO/phosphate buffer (1:99 v/v, 20 mM, pH 7.4), which implies that the probe is stable in the detection system. By contrast, upon addition of H2O2, a sharp fluorescence enhancement at 527 nm was observed at first and the fluorescent emission essentially reached a maximum in about 4 min. The rapid response time is favorable for mitochondria targeted detection and imaging in the complex biological systems.

Figure 2. (A) Fluorescence response of probe Mito-H2O2 (5 µM) toward H2O2 (50 µM). Spectra shown were acquired 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 min after H2O2 was added. Inset: a plot of fluorescence intensity at 527 nm depending on time. (B) Fluorescence response of probe Mito-H2O2 (5 µM) to H2O2 at varied concentrations (0, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10 µM). Inset: Linear plot of the emission intensity (527 nm) against H2O2 concentration.

The detailed emission titration experiments of probe Mito-H2O2 (5µM) with various concentrations of H2O2 were also carried out in DMSO/phosphate buffer (1:99 v/v, 20 mM, pH 7.4) system. As shown in Figure 2B, the fluorescence intensity is increased with increasing H2O2 concentration, and a good linearity is obtained in the concentration range of 0.2–10.0 µM, with a


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detection limit of 0.04 µM (3 S/m, in which S is the standard deviation of blank measurements, n = 11, and m is the slope of the linear equation). Effects of pH and Selectivity Studies. The effects of pH on the reaction system were investigated. As shown in Figure 3A, the fluorescence intensity dramatically increased when pH value was higher than 7.0 and reached a peak value about at pH=10. The pH-dependent property of probe Mito-H2O2 is mainly because that arylboronic acids can only react with H2O2 under mild alkaline conditions and the phenomenon has been demonstrated by other groups.40 Besides, the fluorescent emission of the probe alone was unchanged at various pH, which convinced the stability of probe Mito-H2O2.

Figure 3. (A) Fluorescence intensity of 5 µM probe Mito-H2O2 with the addition of 10 µM H2O2 at various pH values. (B) Fluorescence responses of 5 µM probe Mito-H2O2 to 50 µM various reactive oxygen species (ROS), reactive nitrogen species (RNS), GSH, ascorbic acid, glucose and HSO3−. Every data point was the mean of three measurements. The error bars are the standard deviation.

To further verify the selectivity, probe Mito-H2O2 was incubated with various reactive oxygen species (ROS), reactive nitrogen species (RNS) and the representative biologically relevant species in DMSO/phosphate buffer (1:99 v/v, 20 mM, pH 7.4). As shown in Figure 3B,


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only H2O2 induces a dramatic fluorescence enhancement, while other reactive oxygen species (HOCl, O2•−, •OtBu, •OH, TBHP), reactive nitrogen species (NO, ONOO−), GSH, ascorbic acid, and glucose trigger no or very minor changes. Notably, some other similar indolium-containing systems have been utilized for HSO3− detection.41-43 Whereas, no obvious fluorescence change was found when HSO3− was added in our determination, which means that probe Mito-H2O2 could not respond to HSO3−. And the result is accordance with one recent report.29 Mechanism Studies and Density Functional Theory (DFT) Calculation. To confirm the sensing mechanism, the reaction of probe Mito-H2O2 and H2O2 was conducted using highresolution mass spectroscopy analysis. The peak at m/z 349.1691 [M+H]+ was clearly observed, which was attributed to compound 2 (Figure S9 of the Supporting Information).

Figure 4. The comparison of calculated LUMO energy level between probe Mito-H2O2 and product 2.


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In order to further understand the mechanism of the “Turn-On” fluorescent response of probe Mito-H2O2 to H2O2, a density functional theory (DFT) calculation was carried out with the B3LYP/6-31G (d) method basis set using the Gaussian 09 program (Figure 4 and Figure S1 of the Supporting Information). Before the reaction with H2O2, the LUMO energy level (-6.12 eV) of the 4-(pinacolboryl)benzyl quinoline cation moiety was much lower than that of Nethylcarbazole unit (-0.63 eV) and thus the fluorescence of probe Mito-H2O2 was quenched through a PET process (Φfl = 0.003). After reaction, the LUMO energy level of the quinoline moiety (-1.38 eV) was comparable to that of the N-ethylcarbazole unit. Therefore, the PET process was inhibited and product 2 displayed stronger fluorescence (Φfl = 0.47). Further, probe Mito-H2O2 was tested for its ability to both target the mitochondria and respond to H2O2 in living biological systems. Cervical cancer HeLa cells loaded with 10µM Mito-H2O2 for 30 min at 37°C show faint fluorescence prior to treatment with the stimulants (Figure 5A). Treatment of probe-loaded cells with 100 µM H2O2 for 90 min triggers a striking bright-green fluorescence increase, consistent with H2O2-mediated boronate cleavage occurring within these cells (Figure 5B). In addition, Mito Tracker Deep Red (MT DeepRed, commercially available mitochondrial dye) was employed for a co-localization study to prove the subcellular localization of Mito-H2O2 (Figure 5C). As illustrated in Figure 5D, the Mito-H2O2 signal overlaid very well with the fluorescence of MT DeepRed, which confirms that the dye is located in the mitochondria and can detect localized rises in H2O2 concentrations. The brightfield measurements indicate that the cells are viable throughout the imaging experiments (Figure 5E). Because exposure of cells to stimuli such as lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) will activate the generation of ROS,22,44 in the follow-up experiment, PMA (1 µg/mL) was added to test the ability of Mito-H2O2 for detecting endogenous bursts of H2O2


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produced within living cells. The image of Figure 5G-5I showed clear increases in green emission localized within mitochondrial subcellular regions. Taken together, these data establish that Mito-H2O2 is targeted to cellular mitochondria, where it can respond to exogenous and endogenous changes of H2O2 levels in living samples.

Figure 5. (A) Confocal fluorescence images of HeLa cells incubated with 10µM probe Mito-H2O2 for 30 min at 37 °C. (B) Probe-stained HeLa cells treated with 100 µM H2O2 for 90 min. (C) Co-staining and imaged with 50 nM MT DeepRed. (D) Merged images of (B) and (C). (E) Bright field of (B). (F) HeLa cells incubated with 10µM Mito-H2O2 for 30 min at 37 °C. (G) Probe-stained HeLa cells stimulated with 1 µg/mL PMA for 90 min. (H) Co-staining and imaged with 50 nM MT DeepRed. (I) Merged images of (G) and (H). (J) Bright field of (G). Scale bar: 20 µm.

■ CONCLUSIONS In summary, we have described the design and synthesis of a new mitochondria-targeted fluorescent probe Mito-H2O2 for rapid and convenient detection of H2O2. The probe exhibited high H2O2 selectivity over other reactive oxygen species (ROS), reactive nitrogen species (RNS) and biologically relevant species, which was ascribed to the high oxidative activity of H2O2 for boronate. The reaction mechanism was confirmed by high-resolution mass spectroscopy analysis and the sensing mechanism was further studied by a density functional theory (DFT) calculations


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using Gaussian 09 program. Due to the admirable properties such as “Turn-On” fluorescence response, rapid response time, high specificity and sensitivity, the probe Mito-H2O2 has been successfully applied for monitoring and imaging of H2O2 in HeLa cells under physiological conditions. Moreover, fluorescence co-localization studies indicate that the probe localizes specifically in the mitochondria. Therefore, this unique organelle-targeted fluorescent probe is hopeful to serve as a practical tool for H2O2-related biological studies and inspire the production of new organelle specific fluorescence sensor devices.

■ ASSOCIATED CONTENT Supporting Information Additional information as noted in text.

■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +86-931-4968209. Fax: +86-931-8277088.

■ ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21275150, 21505145 and 21572239), the Funds for Distinguished Young Scientists of Gansu (1210RJDA013) and the top priority program of “One-Three-Five” Strategic Planning of Lanzhou Institute of Chemical Physics, CAS. We acknowledge National Supercomputing Center in Shenzhen for providing the computational resources and the Gaussian 09 package.


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