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A Bioluminescent Probe for Imaging Endogenous Peroxynitrite in Living Cells and Mice Jun-Bin Li, Lanlan Chen, Qianqian Wang, Hong-Wen Liu, Xiao-Xiao Hu, Lin Yuan, and Xiaobing Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00198 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Analytical Chemistry

A Bioluminescent Probe for Imaging Endogenous Peroxynitrite in Living Cells and Mice Jun-Bin Li,†, ‡ Lanlan Chen,†, ‡ Qianqian Wang,† Hong-Wen Liu,†, ǁ, * Xiao-Xiao Hu,† Lin Yuan,† and Xiao-Bing Zhang†, * †

Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of

Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, Hunan University, Changsha 410082, China ǁ

Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry

of Education, College of Chemistry, Xiangtan University, Xiangtan 411105, P. R. China *To whom correspondence should be addressed. E-mail: [email protected]

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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 extremely reactive nitrogen species (RNS), is implicated in diverse pathophysiological conditions, including cancer, neurodegenerative diseases and inflammation. Sensing and imaging of ONOO- in living systems remains challenging due to the high autofluorescence and the limited light penetration depth. In this work, we developed a bioluminescent probe BP-PN, based on luciferase-luciferin pairs and the ONOO--responded group α-ketoamide, for highly sensitive detection and imaging of endogenous ONOO- in living cells and mice for the first time. Attributed to the BL without external excitation, the probe BP-PN exhibits high signal-to-noise ratio with relatively low autofluorescence. Furthermore, we examine the application of probe BP-PN using mice model of inflammation and BP-PN shows high sensitivity for imaging endogenous ONOO- in inflamed mice. This newly developed bioluminescent probe would be potentially useful tool for in vivo imaging of ONOO- in wider physiological and pathological processes.

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Introduction Peroxynitrite (ONOO-) is known as an extremely reactive nitrogen species (RNS), playing important roles in many physiological and pathological processes. ONOO- is formed from the diffusion-controlled reaction of superoxide radicals (O2·–) and nitric oxide (NO) in living systems.1 Abnormally high concentrations of ONOO- may oxidize DNA, proteins and lipids resulting in cell necrosis and apoptosis. The early cell necrosis causes the release of a number of pro-inflammatory mediators and ONOO- may profoundly influence inflammatory responses at multiple levels.2,3 ONOO- can nitrate tyrosine, cysteine and methionine thereby influencing cellular processes.4 ONOO- can also nitrate the T-cell receptor, promoting antigen-specific tolerance and impairing recruitment of cytotoxic T cells to tumors. Thus, ONOO- is closely associated with diverse pathophysiological conditions, including cancer, neurodegenerative diseases and inflammation.5 Sensing and bioimaging of ONOO- in vivo is so significant not only for understanding its role in pathophysiological processes but also for early diagnosis of potentially related diseases. Currently, several methods have been reported for ONOO- detection, including microchip electrophoresis,6 immunohistochemistry,7 electron

spin

resonance and

liquid chromatography mass spectrometry,8 which show high selectivity and sensitivity toward ONOO- but are limited by complicated pre-processing or destruction of the intact specimens and not suitable for in vivo bioimaging. The method of molecular probes, including fluorescent probes, bioluminescent probes and so on, plays an important role in chemical analysis, biology and medicine studying 3



because of its fast response time, noninvasive and real-time visualization.9-14 Although many fluorescent ONOO- probes have been developed,15-26 they always suffer from high autofluorescence interference, photobleaching or phototoxicity, limiting the application of in vivo imaging.27 Bioluminescence (BL) imaging, a sensitive, reliable, convenient and noinvasive imaging method has attracted more and more attention. BL imaging is based on the light generated by the enzymatic reaction.28 Since no external excitation is required to produce light signal, BL imaging avoids high background signal from tissue autofluorescence and thus exhibits highly sensitive detection and imaging.29 The fireﬂy luciferase-luciferin pair is one kind of classical BL systems.30 In the presence of adenosine triphosphate (ATP), oxygen and magnesium ions (Mg2+), the fireﬂy luciferase (fLuc) catalyzes the decarboxylation of the luciferin or aminoluciferin substrate emitting visible photons (green to yellow or red).31 Notably, the BL has good tissue penetration of its red-shifted emission, rendering BL very suitable for in vivo imaging. To date, many bioluminescent probes have been developed for monitoring a variety of biological targets,32-34 such as caspase,35 β-galactosidase,36 hydrogen peroxide,37,38 hypochlorite,39 copper,40,41 hydrogen sulfide and so on.42,43 With advantages of very low background signal and deep tissue penetration, BL imaging provides excellent opportunity to achieve highly sensitive imaging of ONOO- in living systems, and to our best knowledge, no bioluminescent probes for selective detection and imaging of ONOO- have been reported yet. Herein, we reported a novel bioluminescent probe BP-PN for highly selective and 4


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sensitive detection and imaging of endogenous ONOO- in vivo for the first time. We constructed BP-PN by installing an ONOO- sensitive group α-ketoamide onto a luciferase substrate.17,44 With reacting with ONOO-, as shown in scheme 1a BP-PN can be reduced to aminoluciferin, generating bioluminescence. BP-PN exhibited high sensitivity and selectivity to ONOO- in vitro. We applied BP-PN to image endogenous ONOO- in living cancer cells with high signal-to-noise ratios. Furthermore, the probe was successfully used for BL imaging of ONOO- in tumors and inflammation of living mice.

Scheme 1. (a) Proposed mechanism of bioluminescent probe BP-PN for the detection of ONOO-. (b) Synthesis route of probe BP-PN.

Experimental Section Reagents and Apparatus. All chemicals were obtained from Aladdin or Sigma-Aldrich. A stock solution of probe (10.0 mM) was prepared by dissolving an appropriate amount of BP-PN into DMSO. Ultrapure water purified by a Milli-Q 5



reference system (Millipore) was used to prepare all solutions. Luminescence emission spectra were conducted on a HORIBA Fluoromax-4 spectrofluorometer (JobinYvon) with the Xe lamp shut off and emission slits set at 10.0 nm. UV-vis absorption spectra were plotted using a Shimadzu UV-2450 spectrophotometer. Mass spectra were carried out with a LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). 1HNMR and

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CNMR spectra were obtained via a Bruker DRX-400

spectrometer (Bruker). Thin layer chromatography analysis was performed on silica gel plates, and column chromatography was conducted over silica gel (200-300 mesh) columns, both of them were obtained from Yantai Jiangyou Silica Gel Development Co., Ltd. (Shandong, China). High Performance Liquid Chromatography (HPLC) analyses were performed on an Agilent 1260 HPLC system using an InetrSustain AQ-C18 column with water (0.1% of trifluoroacetic acid (TFA)) and CH3CN (0.1% of TFA) as the eluent. The pH was verified by a Mettler-Toledo Delta 320 pH meter. The BL images were obtained via an IVIS Lumina XR Imaging System (Caliper, America) equipped with a cooled charge coupled device (CCD) camera. Circular ROIs were drawn over the areas and quantified by Lumina XR Living Image software, version 4.3. Synthesis of Compound 1. A mixture of 2-(4-nitrophenyl)-2-oxoacetic acid (390.0 mg, 2.0 mmol), oxalyl chloride (530 µL, 6.0 mmol) and DMF (2 drops) in dichloromethane (5.0 mL) was refluxed for 1 h and then evaporated. To this crude product, 10 mL dichloromethane was added, and then 2-cyano-6-aminobenzothiazole (87.5 mg, 0.5 mmol) and triethylamine (60 µL, 2.0 mmol) were added. The reaction 6


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mixture was stirred at room temperature for 30 min. After that, the mixture was concentrated under vacuum, and the residue was purified by silica chromatography to afford Compound 1 as a yellow solid (72.7 mg, 41.3% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1 H), 8.92 (s, 1 H), 8.41 (d, J = 8.5 Hz, 2 H), 8.35 (d, J = 8.4 Hz, 2 H), 8.29 (d, J = 9.0 Hz, 1 H), 8.00 (d, J = 9.0 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 187.57, 162.27, 151.05, 148.99, 138.48, 138.02, 137.08, 136.71, 132.20, 131.20, 125.45, 124.35, 122.16, 113.59. MS (ESI): [M-H]- found, 350.9; calcd for C16H7N4O4S-, 351.0. Synthesis of BP-PN. To the solution of Compound 1 (35.2 mg, 0.1 mmol) in 2 mL dichloromethane/MeOH (1:1, v/v) was added the mixture of D-cysteine hydrochloride (D-Cys, 35.1 mg, 0.20 mmol) and potassium carbonate (27.6 mg, 0.20 mmol) dissolved in 2 mL H2O/MeOH (1:1, v/v) under a nitrogen atmosphere. The reaction was stirred vigorously for 20 min at room temperature. Dichloromethane and MeOH were then removed from the flask under low pressure prior to the addition of 0.1 M aqueous HCl solution until the pH became 2-3. Precipitate formed immediately. The precipitate was filtered off and washed with ice water. The precipitate was dried under reduced pressure to provide BP-PN as a yellow solid (36.6 mg, 80.3% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.41 (s, 1 H), 8.79 (s, 1 H), 8.41 (d, J = 8.3 Hz, 2 H), 8.35 (d, J = 8.4 Hz, 2 H), 8.19 (d, J = 8.8 Hz, 1 H), 7.88 (d, J = 8.6 Hz, 1 H), 5.46 (t, J = 8.9 Hz, 1 H), 3.76 (dt, J = 19.4, 10.7 Hz, 2 H). 13C NMR (101 MHz, DMSO-d6) δ 187.73, 171.53, 164.89, 162.23, 160.56, 151.02, 149.98, 137.99, 137.26, 136.70, 132.14, 124.88, 124.34, 121.04, 113.59, 78.62, 35.27. MS (ESI): [M-H]- found, 454.8, 7



calcd for C19H11N4O6S2-, 455.0. Spectrophotometric Experiments. Both the luminescence and UV-vis absorption measurements were performed in PBS (10 mM, pH 7.4) with 10 mM MgCl2 at 37 °C. A 20 µM final concentration solution of BP-PN in PBS (10 mM, pH 7.4) was prepared by diluting a 10.0 mM DMSO stock solution of BP-PN into pre-warmed PBS (37 °C) in a tube. ONOO- solution (synthesized according to literature report)20 was added into the tube. The luminescence emission spectra (λem = 500-700 nm) were immediately collected at 50 µL of luciferase (10 µg/mL) containing 2 mM ATP added, with emission slits set at 10 nm. In Vitro BL Measurements. The BP-PN solution was diluted to a final concentration of 20 µM in PBS (10 mM, pH 7.4) with 10 mM MgCl2 and incubated for 30 min at room temperature with various concentrations of ONOO-. Subsequently, 50 µL of luciferase containing 2 mM ATP was added. The BL was immediately measured at 60 s integration time. Circular ROIs were drawn over the each well and luminescent signals were quantified by Living Image software. Cytotoxicity Study of BP-PN for MDA-MB-231 Cells. The cytotoxicity of BP-PN to MDA-MB-231 cells was determined by MTT assays (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide). In 96-well plates, MDA-MB-231 cells were seeded at 1×105 cells per well and grown for 24 h. Then, these cells were incubated with various concentrations of BP-PN (0, 12.5, 25, 50, 100, 200 µM) and cultured for a further 24 h or 48 h. Then 20 µL of MTT solution was added to each well. After 4 h incubation at 37 °C, removed the medium and added in 100 µL dimethyl sulfoxide to 8


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fully dissolve the crystals. The absorbance at 570 nm was recorded by a microplate reader (BioTek). The following formula was used to calculate the viability of cell growth: Cell viability (%) = (mean of a value of treatment group / mean of a value of control) × 100. Living Cell BL Imaging. The MDA-MB-231 cells were obtained from the biomedical engineering center of Hunan University (Changsha, China). And the MDA-MB-231 cells expressing firefly luciferase were obtained by fLuc-transfected MDA-MB-231 cells. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere in a 5% CO2 incubator. We treated cells in black 96-well plates (4×105cells per well). After a 24 h incubation period, the medium was removed, and cells were treated with or without uric acid (an ONOO- scavenger, 100 µM, 2 h). Then, same concentrations of BP-PN (20 µM) and various concentrations of SIN-1 (as the ONOO- donor) were added to each well and the BL intensity was measured using an IVIS Lumina XR Imaging System. Circular ROIs were drawn over the each well and luminescent signals were quantified by Living Image software. In Vivo BL Imaging. All living cells and animal operations were conformed to the regulations of cells and animal use and care, according to protocol No. SYXK (Xiang) 2008-0001, approved by Laboratory Animal Center of Hunan. Mice were purchased from Hunan SJA Laboratory Animal Co.，Ltd. (Changsha, China). To generate tumor xenografts in mice, a total of 2×106 fLuc-transfected MDA-MB-231 were subcutaneously implanted into each nude mouse. Mice were single or group housed 9



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on a 12:12 light-dark cycle at 22 °C with free access to food and water. Mice bearing fLuc-transfected MDA-MB-231 subcutaneous tumors were anesthetized with isoflurane and injected i.p. with SIN-1 (1 mM in 100 µL of PBS, pH 7.4) or vehicle (100 µL of PBS, pH 7.4), after 2 min, mice were injected i.p. with probe (2.5 mM in 100 µL of 1:1 PBS: DMSO). Following, mice were imaged with an IVIS Spectrum. For inflamed mice, 200 µL of 1 mg/mL bacterial endotoxin lipopolysaccharide (LPS) was injected on right leg of mice. After 12 h, the BP-PN (20 µL, 100 µM) and fLuc (20 µL, 0.3 mg/mL, containing 1 mM ATP and 2.5 mM Mg2+) were injected on left and right leg of mice through subcutaneous injection. The anesthesia mice were imaged

with

an

IVIS

Spectrum.

The

pseudocolored

BL

images

(in

photons/s/cm2/steradian) were superimposed over the gray scale photographs of the animals. Circular ROIs were drawn over the tumor or the normal and inflamed tissue areas and quantified by Living Image software. The results were reported as total photon flux within an ROI in photons per second. Results and Discussion Sensitivity and Selectivity of BP-PN for ONOO- in Vitro. The BP-PN probe is successfully synthesized by the route shown in Scheme 1b, and all the new compounds were structurally confirmed by 1H NMR,

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C NMR and MS. The BL

sensing of BP-PN for ONOO- was investigated in PBS (10 mM, pH 7.4) with 10 mM MgCl2 at 37 °C. As expected, in the absence of ONOO-, almost no BL signal was observed in the BL spectrum of BP-PN. With the addition of 50 µM ONOO- to BP-PN solution resulted in a significant increase of BL intensity (Figure 1a). Figure 10


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1b showed a new absorption peak at 345 nm appearing in the UV-vis spectrum of the mixture of BP-PN with ONOO-. Then, 20 µM probe solution and different concentrations of ONOO- solution were mixed in black 96-well plate, incubated at 37 °C for 30 min, then luciferase solution (containing 2 mM ATP) was added. As shown in Figure 2, with the addition of 20 µM ONOO-, the BL intensity showed about 14-fold BL signal (total flux) increase compared with that of the no ONOOaddition. BP-PN also showed a good linearity between the total flux and ONOOconcentrations in the range of 0.5 to 10 µM (Figure 2b). On the basis of the 3σ method, the detection limit of BP-PN for ONOO- was estimated to be 200 nM. The abnormally high production rate of endogenous ONOO- has been estimated up to 100 µM min-1 and the steady-state concentration of ONOO- is assessed to be in the nanomolar levels.45,46 The high sensitivity of our probe may allow for the detection of endogenous ONOO-.

Figure 1. (a) Bioluminescent wavelength scan for BP-PN (20 µM) and luciferase in the presence (red) or absence (black) of ONOO- (50 µM) in PBS (10 mM, pH 7.4) with 10 mM MgCl2. (b) UV-vis spectra of 20 µM BP-PN (black) and the reaction product (red) of BP-PN with ONOO(50 µM) after incubation of them for 30 min at 37 °C in PBS (10 mM, pH 7.4). Inset of (a): Photograph of BP-PN in the absence (left) and presence (right) of ONOO- after adding luciferase and ATP.

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Figure 2. (a) BL imaging of BP-PN (20 µM) with various concentrations of ONOO- (0, 0.5, 1, 2, 5, 10, 20, 50 µM) solution after adding luciferase and ATP. (b) Quantification of the total flux (photon/s) in the various concentrations of ONOO-. Inset of (b): The linear curve derived from the total flux and the ONOO- concentration. All assays were performed in triplicate and expressed as the mean ± SD.

We tested the selectivity of BP-PN in vitro by comparing the total flux of the reaction solution between our probe and the biologically relevant species. Note that even when treated with 200 µM of H2O2, BP-PN displayed no obvious BL enhancement. But when incubated with 20 µM ONOO-, BP-PN exhibited a high turn-on BL signal. These results suggested that, BP-PN is capable of recognizing ONOO- without the interference of H2O2. Similarly, our BP-PN showed an excellent selectivity toward ONOO- over other testing species (Figure 3), indicating that BP-PN possesses an excellent selectivity towards ONOO-. In addition, detection of ONOO- in PBS buffer containing 10% fetal bovine serum (FBS) was achieved by using BP-PN 12


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as a probe.

Figure 3. (a) BL imaging of the selectivity of BP-PN (20 µM) toward various species (3-10: 100 µM): (1) blank, (2) H2O2 (200 µM), (3) NaClO, (4) H2S, (5) H2S2, (6) SO32−, (7) NO, (8) O2•−, (9) HNO, (10) uric acid, (11) glutathione (5 mM), (12) ONOO- (20 µM) and (13) ONOO- (20 µM) in 10% fetal bovine serum. (b) Quantification of the total flux (photon/s) of BP-PN with various bioanalytes. The data were recorded after the incubation of BP-PN with various bioanalytes for 30 min at 37 °C in PBS (10 mM, 7.4) after adding luciferase and ATP. All assays were performed in triplicate and expressed as the mean ± SD.

Response Mechanism. With the reaction of ONOO-, the BP-PN can be reduced to aminoluciferin, emitting a visible photon in the presence of luciferase, ATP, oxygen and Mg2+. The reaction mechanism of BP-PN with ONOO- was confirmed by HPLC, mass and fluorescence spectra. As shown in Figure 4, two absorption peaks at 3.0 min and 11.8 min represent aminoluciferin and BP-PN, respectively. The mass peak at m/z = 318.3 in the mass spectrum of the reaction mixture corresponded to the characteristic peak of aminoluciferin (Figure S1). As shown in Figure S2, the 13



fluorescence spectra of the reaction mixture is consistent with that of aminoluciferin, confirming the generation of aminoluciferin. These results validated the response mechanism of BP-PN to ONOO-.

Figure 4. HPLC traces of BP-PN, aminoluciferin, and reaction product of BP-PN with ONOOafter incubation of them for 30 min at 37 °C in PBS (10 mM, pH 7.4) solution. The blue, black, and red lines represent BP-PN, aminoluciferin, and reaction product of BP-PN with ONOO-, respectively. Wavelength for detection: 320 nm.

Cell BL Imaging of Exogenous and Endogenous ONOO-. To investigate the biocompatibility of BP-PN, the cytotoxic effects of probe BP-PN on cell were examined by the MTT assays. As shown in Figure S3, after incubation with BP-PN for 24 h and 48 h, the cell viability showed no significant change treated with BP-PN even at high concentration used (200 µM). The results indicated that BP-PN possesses low cytotoxicity and is suitable for biological applications. Then, we applied it to visualize the changes of ONOO- in living cells. We treated fLuc-transfected MDA-MB-231 cells grown in black 96-well plates. After a 24 h incubation period, the medium was removed, and cells were treated with PBS, SIN-1 or uric acid (an ONOO- scavenger), respectively. Then, the same concentrations of BP-PN (20 µM) were added to each well and the BL intensity was measured. As shown in Figure 5a, 14


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the cells only incubated with BP-PN exhibited obvious BL signal, indicating the detection of endogenous ONOO- in living cells. With increasing of incubation time, the BL signal increased and reached the maximum at 90 min and then decreased with further increasing time. To confirm that the observed BL signal was induced by endogenous ONOO-, ONOO- donor SIN-1 and ONOO- scavenger uric acid were added into cells, respectively. In the case of adding SIN-1, enhanced BL signal was observed compared with that of adding the probe only, as shown in Figure 5b. It is explained that SIN-1 promoted more ONOO- generating in cells and the probe responded the elevated level of ONOO- resulted in enhanced BL signal. With adding of uric acid, the elimination of ONOO- leads to the decreased BL signal, as shown in Figure 5b inset. The results demonstrated the ability of BP-PN to detect exogenous and endogenous ONOO- in living cells.

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Figure 5. (a) Time-course BL imaging of fLuc-transfected MDA-MB-231 cells incubated with 20 µM BP-PN (top row), preincubated with 1.0 mM SIN-1 and then with 20 µM BP-PN (middle row), preincubated with 100 µM uric acid and then with 20 µM BP-PN (bottom row) acquired at 0.5, 15, 30, 45, 60, 90, 120, 150, and 180 min in serum-free culture medium at 37 °C. (b) Quantification of the total flux (photon/s) for the cells BL images. The black, blue, and red lines represent top, bottom and middle row, respectively. All assays were performed in triplicate and expressed as the mean ± SD.

BL Imaging of ONOO- In Vivo. We further applied it for BL imaging of ONOOin living mice. Each nude mouse was xenografted with fLuc-transfected MDA-MB-231 tumor. The nude mice were randomly divided into 2 groups. Within the 60 min observation time, the mice injected with both SIN-1 and BP-PN showed increases of BL signal compared to the cases of injected with BP-PN only (Figure 6a, 16


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6b), suggesting BP-PN is capable of bioimaging ONOO- in living mice. As we known, ONOO- is related to the inflammatory process.2 Then, we applied BP-PN to image endogenous ONOO- in inflamed mouse model. 200 µL of LPS (1 mg/mL) was subcutaneously injected into right leg of mouse to cause inflammation (Figure 6c). After 12 h, BP-PN and the fLuc were subcutaneously injected in situ. As shown in Figure 6d, an obviously enhanced BL was observed in LPS-stimulated inflammation tissues compared to that in the normal tissues indicating that endogenous ONOOcould be detected in living inflamed mice by bioluminescent probe BP-PN.

Figure 6. (a) Time-course BL imaging of nude mice after i.p. injection of 100 µL BP-PN at 2.5 mM (top row), 100 µL SIN-1 at 1 mM followed by 100 µL BP-PN at 2.5 mM (bottom row) in saline at 0, 30, and 60 min. (b) Quantification of the total flux (photon/s) from the tumor regions for the mouse images. The black and red columns represent top and bottom row, respectively. (c) 200 µL of LPS (1 mg/mL) was subcutaneously injected into right leg of mouse to cause inflammation. After 12 h, the BP-PN and fLuc were subcutaneously injected in situ. (d) BL imaging of mice before (left) and after (right) injection of BP-PN and fLuc. (e) Quantification of the total flux (photon/s) from the normal and inflamed tissues in panel d (right). All assays were performed in triplicate and expressed as the mean ± SD. 17



Conclusions In summary, we have reported a bioluminescent probe BP-PN for highly selective detection of ONOO- in vitro and imaging ONOO- in vivo for the first time. Upon installing a group α-ketoamide onto a luciferase substrate, BP-PN is able to release aminoluciferin after reaction with ONOO-, and then generate BL in the presence of firefly luciferase. Attributed to the BL without external excitation, the probe exhibits relatively low background signal and shows high signal-to-noise ratios imaging of ONOO- in living cells and tumors. Furthermore, we applied BP-PN for imaging endogenous ONOO- in living cells and living mice model of inflammation. With these conclusions, we envision that BP-PN may be applied to elucidate the biological roles of ONOO- in wide physiological and pathological processes such as drug-induced organism injury in the near future. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website, http://pubs.acs.org. NMR, mass spectra, and additional spectroscopic data AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions ‡

J.-B. Li and L. Chen contributed equally.

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Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank Pro. G. L. Liang at University of Science and Technology of China for generous donation of fLuc-transfected MDA-MB-231 cells. This work was supported by the National Natural Science Foundation of China (Grants 21521063, 21325520, 21327009, J1210040, 31701249), the science and technology project of Hunan Province (2016RS2009, 2016WK2002) and the keypoint research and invention program of Hunan Province (2017DK2011).

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