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Observation of the generation of ONOO- in mitochondria under various stimuli with a sensitive fluorescence probe Hongyu Li, Xiaohua Li, Xiaofeng Wu, Wen Shi, and Huimin Ma Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 25, 2017

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Analytical Chemistry

Observation of the Generation of ONOO– in Mitochondria under Various Stimuli with a Sensitive Fluorescence Probe Hongyu Li,ab Xiaohua Li a*, Xiaofeng Wu, a Wen Shi,a Huimin Ma ab* a

Key Laboratory of Analytical Chemistry for Living Biosystems, CAS Research/Education

Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. E-mail: [email protected]; [email protected] b

University of Chinese Academy of Sciences, Beijing 100049, China.

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Peroxynitrite (ONOO–), an important reactive oxygen species (ROS), may be generated in the mitochondria under various physiological and pathological conditions. In this work, we designed and synthesized a mitochondria-targetable fluorescence probe RPTPP by using rhodamine as the fluorophore, phenylhydrazine as the recognition moiety, and triphenylphosphonium cation as the mitochondria-targeting moiety. Upon reaction of the probe with ONOO–, the oxidation of phenylhydrazine by ONOO– and the subsequent hydrolysis opens the non-fluorescent spirocyclic structure and thus triggers a fluorescence turn-on response, which provides a sensitive and selective method for the detection of ONOO–. The mitochondria-targeting property of RPTPP was confirmed by the co-localization experiments as well as the mitochondria uncoupling treatments. Moreover, the applications of the probe for imaging intracellular ONOO– were performed in living cells, which reveals that the ONOO– level in RAW264.7 cells undergoes an about 9-fold increase with the stimulation of LPS and IFN-γ for 15 h.

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INTRODUCTION Peroxynitrite (ONOO–) is an important reactive oxygen species (ROS), and may be produced endogenously in living biosystems by a diffusion-limited reaction of the free radicals nitric oxide (NO) and superoxide (O2·–).1-4 It can damage many critical biomolecules such as proteins, lipids, transition-metal enzyme centers and nucleic acids. These biochemical properties make ONOO– a vital biological pathogenic factor for many diseases (e.g., cardiovascular, neurodegenerative or inflammatory disorders, Alzheimer’s disease and cancer).5-7 On the other hand, the oxidization and nitration activity of ONOO– also plays a beneficial role in many cell signal transduction pathways.8 Moreover, immune cells such as macrophages can produce a large quantity of ONOO– (reckoned to be ca. 100 µM per min) to kill the invading microorganisms.9-12 Therefore, it is very important to develop an efficient method for the detection of ONOO– in biological systems. Small molecular fluorescence probes have become a powerful tool for detecting and monitoring biologically active species due to their high sensitivity, non-invasiveness and, in particular, great temporal and spatial resolution.13-20 In the past several years, a number of fluorescence probes were developed to detect ONOO– by usually taking advantage of the strong oxidizability and nucleophilicity of ONOO–. They mainly include the phenyl boronic ester-based,21,22 hydrazine-based,23,24 selenium or tellurium-based25-27 and methyl(4hydroxyphenyl)amino-based probes.28-30 Most of these probes were well utilized for sensitive and real-time detection of ONOO– in biological systems. For living cells, ONOO– is mainly produced within the mitochondria,31 therefore monitoring mitochondrial ONOO– would provide a more direct perspective for its formation and functions. Except the recent

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examples,32,33 however, the fluorescence probes that could sensitively and selectively detect ONOO– in mitochondria are still rare,34,35 which encourages us to develop a low background fluorescence probe for sensitive monitoring of mitochondrial ONOO–. Herein, we report such a new mitochondria-targetable ONOO– fluorescence probe RPTPP (Scheme 1 and Scheme S1). This probe is designed by using rhodamine as a stable and high quantum yield fluorophore, which is known to exhibit a fluorescence off-on response via the opening of its spirocyclic structure.36-38 In addition, mitochondria is a bilayer organelle with a large membrane potential (∆ψ, 150~180 mV) across the lipid bilayer, and thus lipophilic cations like rhodamine 123, triphenylphosphonium (TPP) and pyridinium cations are easily uptaken and accumulated into the mitochondrial matrix.39-41 Therefore, the TPP cation was chosen as the mitochondria-targetable moiety and linked to rhodamine. Moreover, phenylhydrazine as the reaction moiety24 was conjugated to rhodamine, forming a nonfluorescent spirocyclic structure. Reaction of RPTPP with ONOO– leads to the selective oxidation and then hydrolysis of the phenylhydrazine moiety, accompanied by the fluorescence recovery of rhodamine (Scheme 1). This highly sensitive fluorescent response can be used to monitor the change of mitochondrial ONOO–.

Scheme 1. Reaction of RPTPP with ONOO–.

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EXPERIMENTAL SECTION Reagents. 2-(4-Diethylamino-2-hydroxybenzoyl)benzoic acid was obtained from Adamas. 3-(1-Piperazzynyl) phenol, trifluoroacetic acid (TFA), phenylhydrazine, and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Alfa Aesar. (4-Iodobutyl)triphenylphosphonium procedure.42

(IBTP)

3-Morpholinosydnonimine

was

prepared

hydrochloride

following (SIN-1),

the

reported

aminoguanidine

hydrochloride (AG), lipopolysaccharide (LPS), phorbol-12-myristate-13-acetate (PMA), rhodamine 123, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Fetal bovine serum was obtained from Invitrogen Corporation and new-born calf serum from Zhejiang Tianhang Biological Technology Co., LTD. Minocycline hydrochloride was obtained from Tokyo Chemical Industry Co., LTD. Murine recombinant interferon-γ (IFN-γ) was purchased from ProSpec-Tany TechnoGene LTD. Lyso Tracker Green DND-26 (DND-26) was obtained from Cell Signaling Technology, Inc. The cell lines (HeLa and RAW264.7), Dulbecco’s modified Eagle’s media (DMEM) and RPMI 1640 media were purchased from KeyGEN BioTECH Co., LTD, Nanjing, China. The stock solution (1 mM) of RPTPP was prepared in DMSO (note that RPTPP is stable in DMSO for at least one month, but should be used within 2 h in the phosphate buffer due to its slight hydrolysis). Ultrapure water (over 18 MΩ·cm) from a Milli-Q reference system (Millipore) was employed throughout. Apparatus. 1H NMR and

13

C NMR spectra were measured on Bruker Fourier-300

spectrometers. Electrospray ionization mass spectra (ESI-MS) were measured with a Shimadzu LC-MS 2010A instrument (Kyoto, Japan). High-resolution electrospray 5



ionization mass spectra (HR-ESI-MS) were performed on an APEX IVFTMS instrument (Bruker, Daltonics). UV-vis absorption spectra were recorded by a TU-1900 spectrophotometer (Beijing, China) in 1-cm quartz cells. Fluorescence spectra were determined on a Hitachi F-4600 spectrophotometer in 1×1 cm quartz cells with both excitation and emission slit widths of 10 nm and a 400 V PMT voltage. The incubations were performed on a shaker water bath (SKY-100C, Shanghai Sukun Industry & Commerce Company). Confocal fluorescence images were recorded on an FV 1200-IX83 confocal laser scanning microscope (Olympus, Japan) and image processing was carried out with Olympus software (FV10-ASW). The absorbance measurements in MTT analysis were made on a microplate reader (BIO-TEK Synergy HT, USA). Synthesis of RPTPP. RPTPP was synthesized according to the route shown in Scheme S1 through 3 steps. First, compound 3 was prepared according to our previous procedure41 with some modifications. A solution of 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid (1; 3 g, 9.6 mmol) and 3-(piperazin-1-yl)phenol (2; 1.7 g, 9.6 mmol) in TFA (30 mL) was heated to reflux for 3 h. After cooling down, the solvent was evaporated under reduced pressure. The crude product was purified with silica-gel column chromatography (CH2Cl2/MeOH, 9:1, v/v), obtaining 3 (2.74 g, 6.02 mmol, 63%) as a red solid, which was used directly in the next step. Second, compound 3 (682 mg, 1.5 mmol), IBTP (1.33 g, 3 mmol) and K2CO3 (414 mg, 3 mmol) were dissolved in 20 mL acetonitrile. The mixture was refluxed under an Ar atmosphere for 24 h, then the insoluble K2CO3 was filtered and the solvent was removed

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under reduced pressure. Separation of the reaction products with silica-gel column chromatography (CH2Cl2/MeOH, 10:1, v/v) afforded RTPP as a dark red solid (686 mg, 0.89 mmol, 59%). The 1H-NMR and 13C-NMR spectra of RTPP are shown in Figure S1 and S2 in the Supporting Information, respectively. 1H-NMR (300 MHz, 298 K, CD3OD): δ 8.24 (t, 1H, J=8.8 Hz), 7.92-7.75 (m, 18H), 7.32 (t, 1H, J=8.1 Hz), 7.25-7.16 (m, 2H), 7.12 (d, 1H, J=7.5 Hz), 7.07 (d, 1H, J=7.7 Hz), 7.00 (d, 1H, J=2.0 Hz), 3.74-3.67 (m, 8H), 3.66-3.51 (m, 2H), 3.15 (s, 4H), 3.02 (t, 2H, J=7.1 Hz), 2.01 (t, 2H, J=7.0 Hz), 1.80 (t, 2H, J=7.8 Hz), 1.30 (t, 6H, J=6.9 Hz). 13C-NMR (75 MHz, 298 K, CD3OD): δ 171.76, 158.33, 157.22, 156.08, 155.51, 134.94, 134.90, 133.66, 133.53, 133.42, 131.88, 131.05, 130.90, 130.30, 130.24, 130.13, 129.77, 129.17, 118.90, 117.75, 114.61, 114.25, 114.18, 113.87, 98.20, 95.94, 55.64, 51.46, 45.67, 44.69, 25.18, 24.94, 21.60, 20.91, 19.68, 19.63, 11.58. HR-ESI-MS: m/z calcd for RTPP (C50H51N3O3P+, [M]+), 772.3665; found, 772.3662. Finally, to a stirred solution of RTPP (154 mg, 0.2 mmol) in dry CH2Cl2 (10 mL), 0.5 mL dry DMF and 0.5 mL oxalyl chloride were added dropwise and the mixture was stirred for 4 h at room temperature. After removing the solvent under reduced pressure, the obtained rhodamine acid chloride was dissolved in 5 mL of acetonitrile and added dropwise into a solution of phenylhydrazine (0.5 mL, 5 mmol) in 5 mL of acetonitrile. The reaction solution was refluxed for 2 h, and then cooled down for filtration. The filtrate was evaporated under reduced pressure and the crude product was purified with silica-gel column chromatography (CH2Cl2/MeOH, 25:1, v/v), affording RPTPP (78 mg, 0.093 mmol 46%) as a brown solid. 1H-NMR and 13C-NMR spectra of RPTPP are shown in Figure S3 and S4 in the Supporting Information, respectively. 1H-NMR (300 MHz, 298 K, CD3OD): δ

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8.07 (t, 1H, J=6.8 Hz), 7.97 (d, 1H, J=7.2 Hz), 7.94-7.71 (m, 21H), 7.20 (d, 1H, J=7.2 Hz), 7.06 (t, 1H, J=7.7 Hz), 6.85 (t, 1H, J=7.9 Hz), 6.70 (d, 1H, J=7.7 Hz), 6.60 (d, 1H, J=7.5 Hz), 6.42 (t, 2H, J=8.1 Hz), 3.38-3.33 (m, 4H), 3.16 (s, 4H), 2.61 (s, 4H), 2.50(s, 2H), 1.77 (s, 4H), 1.28 (s, 2H), 1.12 (t, 6H, J=7.03 Hz).

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C-NMR (75 MHz, 298 K, DMSO-d6): δ

165.21, 162.98, 153.23, 151.86, 150.26, 148.25, 148.18, 147.86, 142.36, 134.89, 134.25, 133.58, 133.44, 133.30, 132.29, 131.48, 130.35, 130.09, 130.01, 129.95, 129.68, 129.52, 128.74, 128.66, 128.51, 128.20, 127.93, 127.63, 124.39, 124.10, 122.52, 118.80, 118.41, 117.66, 112.47, 111.31, 108.47, 104.99, 97.41, 96.99, 64.88, 57.48, 43.57, 34.24, 19.22, 18.53, 12.25. HR-ESI-MS: m/z calcd for RPTPP (C56H57N5O2P+, [M]+), 862.4246; found, 862.4244. General Procedure for Spectral Measurements. Unless otherwise noted, all the spectral measurements were performed in 50 mM phosphate buffer (pH 7.4) according to the following procedure. To a test tube, 0.5 mL of 0.2 M phosphate buffer was added, followed by addition of 20 µL RPTPP stock solution (1 mM) and appropriate volume of reactant (ONOO–, or other ROS and biological active species) solution, and the final volume was adjusted to 2 mL by ultrapure water. After incubation on shaker water bath at 37 °C for 0.5 h, the absorption and/or fluorescence spectra were measured. Preparation of ROS. NO was generated by dissolving sodium nitroferricyanide(III) dihydrate in deoxygenized ultrapure water and stock at 4 °C. H2O2 was diluted from 33% H2O2 by ultrapure water and the concentration was determined based on the absorbance at 240 nm (43.6 M−1 cm−1). OCl– was diluted from commercial NaOCl solution by ultrapure water and the concentration was determined based on the absorbance at 292 nm (391 M−1

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cm−1).41 ONOO– was prepared according to the reported method and the concentration was determined based on the absorbance at 302 nm (1670 M−1 cm−1).43 •OH was generated from the Fenton reaction of Fe2+ and H2O2. 1O2 was prepared through the reaction of OCl– and H2O2. O2·– was prepared by dissolving KO2 into dry DMSO. NO2– was prepared by dissolving NaNO2 in ultrapure water. tert-Butyl hydroperoxide (TBHP) was diluted from 70% aqueous solutions. TBO• were generated by the Fenton reaction of TBHP with Fe2+. Cytotoxicity Assay. The cytotoxicity of RPTPP to RAW264.7 and HeLa cells was examined by standard MTT assay.15 Cell Culture and Fluorescence Imaging. RAW264.7 cells (mouse leukemic monocyte macrophage) were cultured in RPMI 1640 media supplemented with 10% heat-inactivated new-born calf serum and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The growth media were replaced every day and cells were grown to 70% confluence prior to experiment. HeLa cells were cultured following the previous method.41 Fluorescence imaging experiments of ONOO– in living cells were made by using either the exogenous ONOO– donor SIN-1 (for both RAW264.7 and HeLa cells) or the endogenous production of ONOO– (for RAW264.7 cells only) under the stimulation with bacterial products lipopolysaccharide (LPS, bacterial endotoxin) and interferon-gamma (IFN-γ, pro-inflammatory cytokine)44,45 in the presence of phorbol-12-myristate-13-acetate (PMA, which can promote the overproduction of superoxide and is thus favorable for the generation of ONOO–).27 Co-localization Fluorescence Imaging of RAW264.7 and HeLa cells. Co-localization experiments were performed according to our previous method.41 In brief, cells were

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seeded in glass bottom dishes and allowed to adhere for 24 h, followed by co-staining with RPTPP (10 µM) and rhodamine 123 (500 nM) or DND-26 (500 nM) for 0.5 h at 37 °C. After washed with serum-free media for three times, the cells were treated with the ONOO– donor SIN-1 (100 µM) for 0.5 h, or under the stimulation of LPS, IFN-γ and PMA. In the fluorescence imaging experiments, rhodamine 123 and DND-26 were excited at 488 nm and the emissions were collected at 500-545 nm; RPTPP was excited at 559 nm and emissions were collected at 570-670 nm. For mitochondria uncoupling experiments, cells were treated as above, followed by an additional incubation with CCCP (10, 20 or 50 µM) for 0.5 h. Imaging exogenous ONOO– in RAW264.7 and HeLa cells. Cells were seeded in glass bottom dishes and allowed to adhere for 24 h. Then the cells were incubated with 10 µM RPTPP for 0.5 h at 37 °C and washed with serum-free media for three times. After the cells were treated with SIN-1 (100 µM) for 0.5 h in serum-free media at 37 °C, fluorescence imaging was made with excitation at 559 nm and emission collected from 570 to 670 nm. For scavenging assays, the RPTPP loaded cells were treated with SIN-1 (100 µM) and minocycline (100 µM), and then the fluorescence images were recorded. Imaging endogenous ONOO– in RAW264.7 cells. RAW264.7 cells were seeded in glass bottom dishes and adhered, followed by stimulation with LPS (1 µg/mL) and IFN-γ (100 ng/mL) for 4 h, 10 h or 15 h, and then with PMA (10 nM) for 0.5 h. The as-treated cells were washed with serum-free media for three times and incubated with 10 µM RPTPP in serum-free media for 0.5 h at 37 °C, followed by fluorescence imaging as above. For inhibiting assays, RAW264.7 cells were pretreated with LPS (1 µg/mL) and IFN-γ (100

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ng/mL) in the presence of AG (5 mM), then with PMA (10 nM), and finally washed and loaded with RPTPP.

RESULTS AND DISCUSSION Spectroscopic Response of RPTPP to ONOO–. The absorption and fluorescence spectra of RPTPP before and after reaction with ONOO– are shown in Figure 1. As is seen, RPTPP exhibits a very weak absorption in the visible region (Figure 1A), but its reaction with ONOO– produces a strong one at about 550 nm, which is similar to that of RTPP (Figure S5), with a distinct color change from nearly colorless to pink (see the inset of Figure 1A). Moreover, RPTPP itself displays an extremely low background fluorescence (Φ < 0.01), owing to the formed spirocyclic rhodamine lactam structure. However, the reaction with ONOO– leads to an over 80-fold fluorescence enhancement with the maximum emission peak at 578 nm. The fluorescence recovery is attributed to the oxidation of phenylhydrazine by ONOO– and the subsequent release of RTPP (Φ = 0.21), which is verified by ESI-MS analysis (m/z = 772.5 [M]+; Figure S6). (a) (b)

(a)

B

0.2

0.1

(a) 0.0 450

500

(b)

(b)

(b) Fluorescence Intensity

A Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


550

600

Wavelength (nm)

450

300

150

0 550

(a) 600

650

700

750

Wavelength (nm)

Figure 1. Absorption spectra (A) and fluorescence spectra (B) of RPTPP (10 µM) in phosphate buffer (pH 7.4) before (a) and after (b) reaction with ONOO– (100 µM) for 0.5 h, λex = 530 nm. 11



The reaction conditions such as pH and reaction time were optimized. As shown in Figure S7, the fluorescence intensity of the reaction solution is independent of pH over a broad range from 3.0 to 8.0 in the presence of either 50 or 100 µM ONOO–, which covers well the physiological pH of mitochondria (ca. 8.0). This suggests that the probe is suitable for fluorescence detection in mitochondria. Studies on the effect of reaction time revealed that the fluorescence reaches a maximum in about 20 min and remains unchanged at least for 1 h. Thus, a reaction time of 0.5 h may be chosen for the following measurement. Under the optimized conditions, the fluorescence response of RPTPP to varied concentrations of ONOO– (0-100 µM) was examined. As shown in Figure 2, the fluorescence exhibits a good linear relationship in the concentration range of 0.5-60 µM ONOO– with an equation of F = 6.92 × [ONOO–] (µM) + 7.16 (R2 = 0.997), where F represents the fluorescence intensity of the reaction solution. The detection limit (k = 3) was calculated to be 55 nM ONOO–. Considering the weakly alkaline environment in mitochondria, the fluorescence response of RPTPP to ONOO– at pH 8.0 was also examined (Figure S8), which showed similar results to that at pH 7.4 with a linear equation of F = 7.00 × [ONOO–] (µM) + 7.24 (R2 = 0.990) and the detection limit of 53 nM ONOO–.

B

A 100µM 450

300

450

0

300 F

Fluorescence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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150

0 550

150

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600 650 700 Wavelength (nm)

750

0

20

12


40

60 ONOO (µM)

80

100

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Figure 2. (A) Fluorescence response of RPTPP (10 µM) toward ONOO– at varied concentrations (0-100 µM) in phosphate buffer (pH 7.4). (B) Linear fitting curve of F against the concentration of ONOO–. λex/em = 530/578 nm. The specificity of RPTPP at both pH 7.4 and 8.0 was investigated for ONOO– over other ROS, including OCl–, NO, H2O2, •OH, 1O2, O2•–, NO2–, TBHP, and TBO•. The results (Figure S9) demonstrated that RPTPP can react selectively with ONOO– instead of the other ROS. The fluorescence responses of RPTPP to inorganic salts and other biological active species, such as amino acids, proteins, glutathione, and glucose, were also examined at both pH 7.4 and 8.0. As shown in Figure S10, no obvious interference is produced in the presence of these species, either. Moreover, the cytotoxicity of RPTPP toward RAW264.7 and HeLa cells were assessed by standard MTT assay (Figure S11). The cell viability was still over 85% after incubation even with a high concentration of 30 µM RPTPP, indicating the good biocompatibility of RPTPP to cells. Evaluating the Mitochondria-targeting Property of RPTPP in Living Cells. To examine the mitochondria-targeting ability of RPTPP, co-localization experiments were performed by co-staining the living RAW264.7 or HeLa cells with RPTPP and a widely-used mitochondria-targeting dye rhodamine 123. As depicted in Figure 3, the resulting confocal fluorescence images show that the fluorescence from the rhodamine 123 channel (Figure 3a) overlaps very well with that from the RPTPP channel (Figure 3b). A high Pearson's coefficient of 0.97 and an overlap coefficient of 0.98 were observed according to the intensity correlation plot (Figure 3e) by Olympus software FV10-ASW, indicating that RPTPP possesses good mitochondria-targeting ability. The intensity profiles

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within the linear ROI 1 (white line across the RAW264.7 cell in Figure 3a-3c) vary in close synchrony (Figure 3f). In contrast, the control experiment performed by co-staining RAW264.7 cells with 500 nM DND-26 (a lysosome tracker) and 10 µM RPTPP presents a poor overlap behavior with a Pearson's coefficient of 0.34 and an overlap coefficient of 0.38 as well as the irrelevant change in the intensity profiles of the linear ROI 1 (Figure S12). In the same way, the mitochondria-targeting property of RPTPP was examined by the co-localization experiment in HeLa cells, and similar phenomena were observed (Figure S13 and S14). The above results demonstrated the good mitochondria-targeting ability of RPTPP in living cells.

a

b

c

d

e

f

Rhodamine 123 RPTPP

4000

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2000

0

0

5

10

Length (µm)

Figure 3. Co-localization experiments with RAW264.7 cells. Cells were co-stained with rhodamine 123 (500 nM) and RPTPP (10 µM), and then treated with exogenous SIN-1 (100 µM). (a) Rhodamine 123 channel (λex = 488 nm, λem = 500–545 nm). (b) RPTPP channel (λex = 559 nm, λem = 570–670 nm). (c) Merged image of image (a) and image (b). (d) Corresponding differential interference contrast (DIC) image. Scale bar: 20 µm. (e) Intensity correlation plot of rhodamine 123 and RPTPP (Pearson’s coefficient 0.97 and overlap coefficient 0.98). (f) Intensity profiles of rhodamine 123 and RPTPP within the linear ROI 1.

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On the other hand, co-localization experiments were also made by the endogenous production of ONOO– in living RAW264.7 cells under the stimulation with LPS, IFN-γ, and PMA. As shown in Figure 4, the fluorescence from the rhodamine 123 channel (Figure 4a) also overlaps well with that from the RPTPP channel (Figure 4b). A high Pearson's coefficient of 0.96 and an overlap coefficient of 0.97 were determined according to the intensity correlation plot (Figure 4e). Moreover, the intensity profiles within the linear ROI 1 (white line across the RAW264.7 cell in Figure 4a-4c) change synchronously (Figure 4f), clearly indicating that RPTPP possesses good mitochondria-targeting ability.

a

b

c

d

e

f 4000 Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Rhodamine 123 RPTPP

2000

0

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Length (µm)

Figure 4. Co-localization experiments with RAW264.7 cells. Cells were pretreated with LPS (1 µg/mL) and IFN-γ (100 ng/mL) for 15 h, then with PMA (10 nM) for 0.5 h, and finally co-stained with rhodamine 123 (500 nM) and RPTPP (10 µM). (a) Rhodamine 123 channel (λex = 488 nm, λem = 500–545 nm). (b) RPTPP channel (λex = 559 nm, λem = 570–670 nm). (c) Merged image of image (a) and image (b). (d) Corresponding DIC image. Scale bar: 20 µm. (e) Intensity correlation plot of rhodamine 123 and RPTPP. (f) Intensity profiles of rhodamine 123 and RPTPP within the linear ROI 1.

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It is known that lipophilic cations such as rhodamine 123 and triphenylphosphonium cations tend to accumulate into the mitochondrial matrix depending on the large mitochondrial

membrane

potential.

Meanwhile,

the

uncoupler

cyanide

m-chlorophenylhydrazone (CCCP) can act as a protonophore, translocating protons across the mitochondrial inner membranes to abolish the mitochondrial membrane proton gradient, which leads to the dissipation of membrane potential.46 Herein, an uncoupling experiment was further performed. After incubation with rhodamine 123, RPTPP and SIN-1, the RAW264.7 cells were then treated with CCCP (10, 20 or 50 µM). As shown in Figure 5, the fluorescence from both rhodamine 123 and RPTPP channels with CCCP reduces greatly compared with that without CCCP, and more fluorescence reduction is observed when the concentration of CCCP increased from 10 to 50 µM, indicating that CCCP eliminated the membrane potential of mitochondria, and the damaged mitochondria as well as the dyes rhodamine 123 and RPTPP may be removed out of the cells. In addition, the uncoupling experiments on HeLa cells provided similar results (Figure S15). Taken together, these phenomena further confirm the excellent mitochondria-targeting ability of RPTPP.

CCCP (µM)

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RPTPP

DIC image

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Figure 5. Uncoupling experiment with RAW264.7 cells. Cells were treated with rhodamine 123 (500 nM) and RPTPP (10 µM), then with SIN-1 (100 µM), and finally with CCCP (0, 10, 20 and 50 µM). First row, rhodamine 123 channel (λex = 488 nm, λem = 500–545 nm); second row, RPTPP channel (λex = 559 nm, λem = 570–670 nm); third row, DIC image. Scale bar: 20 µm. Evaluation of RPTPP for Imaging Exogenous ONOO– in Living Cells. The potential ability of RPTPP to image the exogenous ONOO– in live cells was evaluated. Here, RAW264.7 was chosen as a model, and SIN-1 was employed as the exogenous source of ONOO–. As shown in Figure 6, there is no or little fluorescence signal in the images of the untreated RAW264.7 cells (Figure 6a) or those incubated with RPTPP (Figure 6b). Nevertheless, as expected, a dramatic fluorescence increase is observed in the cells treated with 100 µM of SIN-1 (Figure 6c), and such a fluorescence increase can be efficiently eliminated in the presence of the ONOO– scavenger minocyline (Figure 6d). In addition, similar results (Figure S16) were observed in HeLa cells (the most widely used model cell line47,48). These results validates the potential of RPTPP as a fluorescence probe for imaging the exogenous ONOO– in different cells.

a

b

c

d

e Relative Pixel Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0

0.5

0.0

a

b

c

d

Figure 6. Confocal fluorescence images of RAW264.7 cells under different conditions. (a) Cells only. (b) Cells treated with RPTPP (10 µM) for 0.5 h. (c) RPTPP-loaded cells incubated with 100 µM SIN-1 for 0.5 h. (d) RPTPP-loaded cells incubated with 100 µM 17



SIN-1 for 0.5 h in the presence of 100 µM minocyline. The second row shows the DIC images. λex = 559 nm, λem = 570–670 nm. Scale bar: 20 µm. (e) Relative pixel intensity of fluorescence images a-d (the pixel intensity from image c is defined as 1.0). The results are presented as mean ± standard deviation (n = 3). Imaging Endogenous ONOO– in RAW264.7 Cells. Further experiments were carried out to test whether RPTPP could be used to monitor the endogenous ONOO– change in living cells. Here, RAW264.7 cells were chosen again as a macrophage model since they are known to generate the endogenous ONOO– under the stimulation with LPS and IFN-γ usually in the presence of PMA. As shown in Figure 7, when RAW264.7 cells were incubated with LPS (1 µg/mL) and IFN-γ (100 ng/mL) for 4, 10 or 15 h, and then with PMA (10 nM) for another 0.5 h, a gradually enhanced fluorescence signal is observed with the incubation time, suggesting the ONOO– generation. On the basis of the relative pixel intensity change of fluorescence images (Figure 7g), the ONOO– level is estimated to increase by about 9-fold for the stimulation time of 15 h. To further prove whether the fluorescence enhancement was caused by the intracellular ONOO– generation, another control experiment with aminoguanidine (AG, a nitric oxide synthase inhibitor) was made. As can be seen from image f in Figure 7, the fluorescence signal is largely attenuated when RAW264.7 cells were incubated with LPS and IFN-γ in the presence of AG and then with PMA, indicating that the production of ONOO– is inhibited efficiently. Moreover, an additional experiment was performed to validate that AG itself didn’t affect the fluorescence in the reaction of ONOO– and RPTPP (Figure S17). The above results clearly indicate that RPTPP is capable of imaging the formation of the endogenous ONOO– in live cells. 18


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a

b

c

d

e

g

f Relative Pixel Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0

0.5

0.0

a

b

c

d

e

f

Figure 7. Confocal fluorescence images of RAW264.7 cells under different conditions. (a) Cells only. (b) Cells treated with RPTPP (10 µM) for 0.5 h. (c-e) Cells were pretreated with LPS (1 µg/mL) and IFN-γ (100 ng/mL) for 4 h (c), 10 h (d) and 15 h (e), respectively, then with PMA (10 nM) for 0.5 h, and finally stained with RPTPP (10 µM) for 0.5 h. (f) Cells were pretreated with LPS (1 µg/mL), IFN-γ (100 ng/mL) and AG (5 mM) for 15 h, then with PMA (10 nM) for 0.5 h, and finally stained with RPTPP (10 µM) for 0.5 h. The second row shows the DIC images. λex = 559 nm, λem = 570–670 nm. Scale bar: 20 µm. (g) Relative pixel intensity of fluorescence images a-f (the pixel intensity from image e is defined as 1.0). The results are presented as mean ± standard deviation (n = 3).

CONCLUSIONS In

summary,

we

have

reported

the

design

and

preparation

of

a

new

mitochondria-targetable fluorescence probe RPTPP by conjugating rhodamine with the TPP cation and phenylhydrazine. RPTPP exhibits good mitochondria-targeting property, and high sensitivity and selectivity for the mitochondrial ONOO– over other ROS, with a detection limit of 55 nM ONOO–. The probe has been successfully utilized to image the exogenous and endogenous ONOO– in mitochondria by confocal laser scanning microscope. The good analytical performance of the probe may enable it to be widely used in the detection of ONOO– in mitochondria as well as the diagnosis of the ONOO–-associated diseases.

19



ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.XXXX. Synthetic route of RPTPP, 1H-NMR and

13

C-NMR spectra of RTPP and RPTPP,

additional fluorescence tests in vitro and cell imaging experiments. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected].

ACKNOWLEDGMENT We are grateful for the financial support from the 973 Program (Nos. 2015CB856301 and 2015CB932001), the NSF of China (Nos. 91432101, 21535009, and 21621062), and the Chinese Academy of Sciences (XDB14030102)

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