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A two-photon near-infrared fluorescent probe for imaging endogenous hypochlorite in cells, tissue and living mouse
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Dyes and Pigments journal homepage: http://www.elsevier.com/locate/dyepig
A two-photon near-infrared fluorescent probe for imaging endogenous hypochlorite in cells, tissue and living mouse Xiangpeng Lin a, 1, Yunling Chen a, 1, Luo Bao a, Shoujuan Wang a, Keyin Liu a, *, Wei-dong Qin b, **, Fangong Kong a, *** a
State Key Laboratory of Biobased Material and Green Papermaking, Key Laboratory of Pulp & Paper Science and Technology of Shandong Province/Ministry of Education, Qilu University of Technology, Shandong Academy of Sciences, Jinan, 250353, China Department of Critical Care Medicine, Qilu Hospital of Shandong University, Jinan, Shandong, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Hypochlorite detection Two-photon Near infrared emission Endogenous In vivo imaging
ABSTRACT: Hypochlorite (HClO/ClO ) plays an important role in the body’s immune function system and helps destroy invading bacteria and pathogens. Therefore, real-time and visual detection of hypochlorous acid in the living body is necessary. Here, we describe the first example of a two-photon excitation near-infrared lightemitting fluorescent probe (Nil-ClO) for HClO/ClO based on Nile Red derivatives. Nil-ClO offers a large fluorescence response near 650 nm and good selectivity to HClO/ClO in solution. The probe showed an approximately 8-fold increase in fluorescence within 1 min when it was detected at 650 nm in the presence of 30 equivalents of HClO/ClO . The probe was further used for the fluorescence-based detection of HClO/ClO in cells, tissues, and mice in a liver injury model. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) experiments showed that the probe had almost no cytotoxicity at the concentrations needed for im aging. The Nil-ClO can response to HClO/ClO in both HeLa cells and RAW cells quickly. In tissue imaging experiments, Nil-ClO was used to detect HClO/ClO with a significant near-infrared fluorescence response down to 100 μm of depth. Finally, the obvious fluorescence enhancement in liver injury mouse model indicated a burst of endogenously generated HClO/ClO in mouse during in vivo imaging experiments.
1. Introduction Hypochlorite (HClO/ClO ) is an important reactive oxygen species (ROS) in the organism [1]. It is widely distributed in various biological environments. In vivo, it is produced by myoglobin (MPO) catalyzing the production of hydrogen peroxide and chloride ions and plays an important role in the human immune system [2,3]. Under normal con ditions, hypochlorite level remains relatively high to destroy invading bacteria and pathogens. Excessive hypochlorous acid or hypochlorite can cause a series of diseases, such as arteriosclerosis and arthritis [4]. Therefore, real-time detection of hypochlorous acid in organisms is critical for studies of the physiological effects and early diagnosis of diseases caused by hypochlorous acid [5]. Recently, many methods for detecting hypochlorous acid have been developed, including iodine reduction droplets, spectrophotometry,
chemiluminescence analysis, and coulometry [6]. However, these analytical methods are expensive and cumbersome. Therefore, the development of an effective, inexpensive, and simple hypochlorous acid detection method is an important research topic [7]. Fluorescence is particularly suitable for analysing biologically active small species: It is non-invasive and highly sensitive and selective in biological samples [8, 9]. In recent years, many fluorescent probes have been developed for hypochlorite detection based on traditional dyes, such as rhodamine [10,11], coumarin [12,13], fluorescein [14,15], naphthalimide [16,17], bodipy [18,19], and cyanine dyes [20,21]. These systems exhibit excellent photophysical properties and good selectivity for ClO . They have been used to detect ClO in cells, tissues, and animals [22]. Two-photon probes offer a high spatial discrimination rate and deep tissue penetration [23–25]. However, the fluorescence of most two-photon probes is in the visible region, and developing two-photon
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (K. Liu), [email protected] (W.-d. Qin), [email protected] (F. Kong). 1 These authors contributed equally to this works. https://doi.org/10.1016/j.dyepig.2019.108113 Received 10 October 2019; Received in revised form 6 December 2019; Accepted 6 December 2019 Available online 9 December 2019 0143-7208/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xiangpeng Lin, Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2019.108113
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Fig. 1. Design of a two-photon near-infrared fluorescent probe Nil-ClO for hypochlorite and its detection mechanism.
probes with near-infrared emission is still challenging [26,27]. Recently, Nile Red derivatives were demonstrated to be a unique platform with excellent two-photon excitation fluorescence performance, good pho tostability, high fluorescence quantum yield, and low cytotoxicity [28]. For example, a Nile Red-palladium-based fluorescent probe was re ported and used to measure endogenous CO in vivo [29]. Herein, we report the first example of a two-photon excitation, near-infrared emission fluorescent probe for HClO/ClO and used it to study the function of HClO/ClO in mice via a liver injury model. In our design (Fig. 1), Nile Red derivative dye (DyeA) has many advantages over conventional dyes, including two-photon excitation and near-infrared light emission. The remaining hydroxyl groups can be used for further chemical modification. The fluorescent probe (Nil-ClO) was constructed by reacting the hydroxyl group of DyeA with 1-fluoro4-nitrobenzene and then reducing the nitro group. Finally, we studied the photophysical properties of Nil-ClO and the bioimaging capabilities toward endogenous HClO in cells, tissues, and a liver injury mice model induced by lipopolysaccharide and D-galactosamine (LPS/D-GalN) [30–32]. Scheme 1. Synthesis of the fluorescent platform Nile Red derivative DyeA and the fluorescent probe Nil-ClO for detecting HClO.
2. Experimental 2.1. Reagents, materials, and apparatus
Olympus laser confocal micro-scope.
All of the solvents and reagents are available from commercial sources unless otherwise stated. Solvents were purified according to standard methods prior to use; deionized water was used throughout all the experiments. TLC plates and silica gels were purchased from Qing dao Ocean chemicals. The reactions were monitored by thin layer chromatography (TLC plate, silica gel GF254) and further purified by silica column chromatography (silica gel 200–300 mesh) TLC plates. The HRMS (ESI) spectra were recorded on a Bruker Apex-Ultra mass spec trometer ((Bruker Daltonics Corp., USA). The 1H NMR, and 13C NMR spectra were collected on a Bruker Avance III 400 MHz spectrometer (Bruker Daltonics Corp., USA), and tetramethylsilane (TMS) was used as an internal reference. The absorption spectra were recorded on a Shi madzu UV-2700 spectrophotometer (Shimadzu Suzhou Instruments Mfg. Co. Ltd.) and the fluorescence spectra were recorded on a Hitachi F7100 spectrofluorimeter (Hitachi High-Tech Science) with a 10 mm quartz cuvette, respectively. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) assays were measured on a micro MTT instrument (Thermo Fisher). Cell imaging were performed used an
2.2. Fluorescence detection The probe Nil-ClO was formulated into a 1 mM stock solution using dimethyl sulfoxide (DMSO). The Nil-ClO (1 � 10 5 mol/L) test solution was made in PBS (10 mM phosphate buffer, pH 7.4, 5% DMSO as cosolvent), and added an aliquot of each test substance solution, and small aliquots of each test substance solution were added. The resulting solution is fully oscillated at room temperature before measuring the spectrum [33]. 2.3. Synthesis procedures The synthesis of Nil-ClO is shown in Scheme 1. Synthesis of Dye A The synthesis and characterization of Dye A was performed based on our previous reports [29]. 1,6-Dihydroxynaphtha lene (0.176 g, 1.1 mmol) and 3-hydroxy-4-nitroso-N,N-diethylaniline (0.194 g, 1.0 mmol) were added to a 100 mL round bottom flask with 2
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Fig. 2. Fluorescence spectral changes of Nil-ClO titrated with ClO . (a) Fluorescence spectral changes of 10 μM Nil-ClO in PBS (pH 7.4, containing 5% DMSO) were titrated with increasing amounts of ClO . (b) Linear fitting of probe response to fluorescence signal at 650 nm by varying the concentration of ClO .
magnetic beads and DMF (10 mL). The mixture was degassed and heated to 140 � C in a magnetic stirrer to form a deep red solution. The reaction was monitored once per hour with a silica gel TLC plate (eluent: dichloromethane/methanol ¼ 50:1, v/v) until the starting material was consumed completely. The solution was cooled to room temperature. The organic layer was washed with deionized water, extracted with methylene chloride, dried with anhydrous magnesium sulfate and the DMF removed. The crude material was further purified with silica gel column and eluted with methylene chloride: methanol ¼ 50:1 (v/v). This led to Dye A, which was a dark red solid (0.22 g, 0.66 mmol). The yield was 60%. m.p.: 270–272 � C. IR (KBr) ν: 2976, 2932, 1567, 1409, 1117 cm 1. 1H NMR (400 MHz, DMSO‑d6) δ 10.44 (s, 1H), 7.98 (d, J ¼ 8.5 Hz, 1H), 7.89 (d, J ¼ 2.2 Hz, 1H), 7.59 (d, J ¼ 9.1 Hz, 1H), 7.10 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.81 (d, J ¼ 6.6 Hz, 1H), 6.66 (s, 1H), 6.16 (s, 1H), 3.51 (q, J ¼ 7.0 Hz,4H),1.17 (t, J ¼ 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 181.99, 161.04, 155.95, 153.83, 151.96, 151.09, 146.77, 139.23, 134.17, 131.17, 127.85, 127.19, 124.29, 124.13, 118.76, 117.37, 117.25, 110.28, 108.95, 108.62, 105.72, 104.57, 96.48, 44.84, 12.88. HRMS(ESI) calcd. for C20H18N2O3 [M]þ: 335.1396, found: 335.1385. Synthesis of compound 2 Dye A (65 mg, 0.19 mmol), 1-fluoro-4nitrobenzene (60 mg, 0.42 mmol), and potassium carbonate (60 mg, 0.43 mmol) were added to DMF (10 mL) and allowed to react at 40 � C overnight and then cooled the solution to room temperature. The solu tion was washed with deionized water and then extracted with methy lene chloride (20 mL) three times through a separation funnel. The organic layers were combined, dried with anhydrous sodium sulfate, then it was concentrated and purified by silica gel column (elution with dichloromethane and methanol, v/v ¼ 100:1). Compound 2 was received as a dark red solid (83 mg, 0.182 mmol) with a yield of 45%. m. p.: 256–257 � C. IR (KBr) ν: 3442, 3070, 2974, 2928, 1583, 1500, 1380, 1112 cm 1. 1H NMR (400 MHz, DMSO‑d6) δ 8.33 (d, J ¼ 9.2 Hz, 2H), 8.25 (d, J ¼ 8.6 Hz, 1H), 8.17 (d, J ¼ 2.5 Hz, 1H), 7.60 (d, J ¼ 9.2 Hz, 1H), 7.53 (dd, J ¼ 8.6, 2.5 Hz, 1H), 7.33 (d, J ¼ 9.2 Hz, 2H), 6.87–6.82 (m, 1H), 6.71 (d, J ¼ 2.7 Hz, 1H), 6.32 (s, 1H), 3.93 (d, J ¼ 5.9 Hz, 1H), 3.57–3.49 (m, 4H), and 1.17 (t, J ¼ 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 181.44, 163.62, 162.34, 161.64, 157.79, 152.59, 151.66, 151.35, 147.12, 143.49, 142.14, 134.45, 131.63, 128.73, 128.59, 126.81, 124.68, 122.33, 119.03, 113.62, 110.93, 104.75, 96.47, 44.98, and 12.92. HRMS (ESI) calcd. for C26H21N3O5 [M]þ: 456.1559, found: 456.1547. Synthesis of HClO fluorescent probe Nil-ClO Compound 2 (38 mg, 0.08 mmol) and iron powder (132 mg, 2.35 mmol) were added to a 25mL round-bottom flask with 10 mL glacial acetic acid. The mixture was stirred at room temperature for 16 h under nitrogen protection, and the
reaction was monitored with TLC plate every 4 h. Saturated sodium bicarbonate solution was added to the solution to neutralize the glacial acetic acid, and then methylene chloride (20 mL) was added and extracted three times through a separation funnel. We then combined the organic layers, washed them with saturated brine and anhydrous sodium sulfate, concentrated the product with an evaporator. Finally, the crude products were purified by silica gel column (eluted with dichloromethane and methanol, v/v ¼ 50:1). The product Nil-ClO was a dark red solid (22 mg, 0.057 mmol) with a yield of 60%. m.p.: 310–312 � C. IR (KBr) ν: 3414, 2927, 2851, 1583, 1495, 1407, 1117, 915 cm 1. 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J ¼ 8.7 Hz, 1H), 8.12 (d, J ¼ 2.2 Hz, 1H), 7.58 (d, J ¼ 9.0 Hz, 1H), 7.19 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.97 (d, J ¼ 8.6 Hz, 2H), 6.76 (d, J ¼ 8.6 Hz, 2H), 6.65 (dd, J ¼ 9.1, 2.4 Hz, 1H), 6.47 (d, J ¼ 2.4 Hz, 1H), 6.33 (s, 1H), 3.50–3.44 (m, 4H), and 1.25 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 183.06, 161.74, 152.18, 150.81, 147.95, 146.84, 143.22, 139.98, 134.19, 131.19, 127.94, 124.80, 121.43, 119.20, 116.36, 110.77, 109.50, 105.43, 96.38, 45.01, 29.64, and 12.57. HRMS (ESI) calcd. for C26H24N3O3 [M]þ: 426.1818 found: 426.1802. 2.4. Sample preparation and measurements A stock solution of Nil-ClO was prepared in dimethyl sulfoxide at a concentration of 1 mM, and a 10 mM NaClO stock solution was prepared in deionized water. Fluorescence measurements used PBS (phosphate buffer (10 mM, pH 7.4), 5% DMSO as a cosolvent). In fluorescence with pH change measurements, the PBS was used at a pH of 4.0–10.0. In the fluorescence spectral measurements, the Nil-ClO was 1 � 10 5 M, the excitation wavelength was at 560 nm, and the emission spectrum was 580–900 nm. In selective experiments, solutions for various test species, including TBHP (tert-butyl hydroperoxide), Glu, Cys, S2O23 , S2 , SO23 , SO24 , HSO23 , ClO , NO2 , H2O2, Vc , Zn2þ, Fe3þ, Mg2þ, and Ca2þ, were prepared according to the literature report. Nil-ClO interacted with 30 eq. of each of the test species for about 30 min before fluorescence selectivity measurements. 2.5. Cell culture and viability assay HeLa cells and RAW 264.7 cells were obtained from Shandong University School of Pharmacy and cultured in a minimal essential medium (MEM) supplemented with 10% calf serum at 37 � C, 5% CO2 in a humid environment. Cell viability was determined using MTT [34]. The HeLa cells were first incubated for 24 h in a 37 � C incubator, then they were incubated into a 96-well plate and further incubated with different concentrations of probes for 12 h. Followed by 10 μL MTT was 3
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Fig. 3. Fluorescence spectra of Nil-ClO versus ClO as a function of time. (a) Fluorescence spectral changes of 10 μM Nil-ClO and 30 equivalents of ClO in PBS over time. (b) Fluorescence intensity of 10 μM of Nil-ClO and 30 equivalents of ClO as a function of time at 650 nm; excitation wavelength: λ ¼ 560 nm.
added to each well of the plate, and further incubated for an additional 4 h, and then DMSO (100 μL) was added. The uptake of cells in each well plate was measured using a Herm Fisher Scientific plate reader. In cell imaging experiments of exogenous hypochlorous acid, HeLa cells were preincubated for 12 h in a 37 � C incubator, incubated with 5 μM probe for 30 min, washed with PBS, and further incubated with 30 μM NaClO for 20 min. In a control experiment, cells were incubated with only 5 μM probe and further studied using confocal microscopy [35]. 2.6. Imaging of HClO in tissue and in vivo Animal models and in vivo imaging. 4-Week-old Balb/c mice were obtained from animal center of Shandong University. All animal ex periments were performed in accordance with the guidelines issued by The Ethical Committee of Shandong University. Preparation of the fresh mouse liver tissue slices and two-photon fluorescence imaging. Slices were prepared from the liver of 4-week-old Balb/c mice. Slices were cut to 400 μm thickness by using a vibrating blade microtome in 10 mM PBS (pH 7.4). Slices were then incubated with Nil-ClO, then washed with PBS, and observed under a two-photon confocal microscope (Leica SP8). For in vivo imaging experiment, the mice were divided into two groups, in a control experiment, the mice were administered by tail vein injec tion of Nil-ClO. In experimental group, mice were given LPS/D-GalN stimulation to induce a burst of endogenous HClO/ClO , then admin istered by tail vein injection of Nil-ClO. Fluorescence images were taken by IVIS Lumina III living animal imaging instrument PerkinElmer.
Fig. 4. Fluorescence intensity changes of the Nil-ClO (10 μM) at different pH values in the absence (■) or presence ( ) of ClO (30 eq).
3.2. Fluorescence spectra changes of Nil-ClO versus HClO as a function of time
3. Result and discussion
Fluorescence changes of Nil-ClO over time during the assay were also investigated. When 30 equivalents of ClO were present in PBS (pH 7.4), the fluorescence of Nil-ClO increased significantly, and the fluo rescence intensity reached the maximum within 1 min after addition of Nil-ClO indicating that the response speed was very fast (Fig. 3b). The fluorescence intensity increased by about 8-fold after adding sodium hypochlorite for 1 min (Fig. 3a). We then tested the kinetics of the re action under pseudo-first-order conditions in which a large excess of ClO (30 equivalents) was reacted with Nil-ClO (10 μM) in PBS buffer (pH 7.4, 10 mM, 5% DMSO) at 37 � C. The pseudo-first-order rate con stant of ClO was k ¼ 0.0961 s 1 (Fig. S3). We further investigated the effect of pH on the fluorescence response of Nil-ClO to ClO . In the wide pH range of 5.0–9.0 without ClO , only very small changes in fluores cence intensity were observed in PBS with only probes, indicating that the free probes were stable over a wide pH range. (Fig. 4, Fig. S4). When ClO was added, a large fluorescence intensity increase was observed and the fluorescence intensity almost did not change across pH values from 5.0 to 9.0. Therefore, the pH change in solution has almost no
3.1. Fluorescence spectral changes of Nil-ClO titrated with ClO The spectral characteristics of Nil-ClO were investigated. Nil-ClO shows a maximum absorption peak at 588 nm and an emission peak near 650 nm (Fig. S1, Fig. S2 in Supporting information), and the Stokes shift of Nil-ClO is about 62 nm. Fig. 2a shows that the fluorescence of Nil-ClO (fluorescence quantum yield: 0.37%, rhodamine B in ethanol was used as standard in Supporting information) at 650 nm increased significantly upon addition of ClO . Based on the linear fit of fluorescence intensity to ClO concentration, the detection limit of Nil-ClO for ClO was about 4.37 � 10 6 M (Fig. 2b). Hypochlorous acid (HClO/ClO ) plays a major role in the immune system of living organisms. And it has a certain destructive effect on invading bacteria and pathogens [36]. The detec tion limit of Nil-ClO is lower than the normal concentration of HClO in the biological environment, thus it can be potentially applied to detect endogenously generated HClO/ClO in living organisms.
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3.4. Selective experiment of Nil-ClO on HClO The selectivity of Nil-ClO for HClO/ClO was investigated next, common interferents such as Glu, Cys, (CH3)3COOH (TBHP), S2O23 , S2 , SO23 , SO24 , HSO23 , ClO , NO2 , H2O2, Vc , Zn2þ, Fe3þ, Mg2þ, and Ca2þ, were added to PBS buffer containing Nil-ClO to monitor changes in fluorescence spectra. The effect of other factors such as BSA and tem perature toward the detection process were also measured (Fig. S8 and Fig. S9). Significant changes in the fluorescence spectra were observed only when ClO was added (Fig. 5), and other small molecules and changes in temperature do not interfere with the fluorescence spectra. Fluorescence changes at 650 nm also clearly indicate that Nil-ClO shows high selectivity for ClO . The high selectivity of Nil-ClO enables it to detect ClO in complex biological samples. 3.5. Imaging exogenous HClO in HeLa cells For the bioimaging experiments, the Nil-ClO was applied to assess ClO detection in biological environments. The standard MTT assay was performed to evaluate the effect of Nil-ClO on cell proliferation prior to HeLa cell imaging (Fig. S10). At a Nil-ClO concentration of 30 μM, the cell viability was maintained above 90%. However, at the probe con centration required for cell imaging, the cell viability remained above 95% even after incubation for 24 h [38]. Therefore, the MTT result indicated that the probe had relatively low cytotoxicity in the concen tration and incubation time required for fluorescence imaging experi ments in cells [39]. We then applied Nil-ClO to the fluorescence imaging of HClO in cells. Fig. 6 shows that fluorescent signal almost invisible in the TRITC channel during imaging when HeLa cells were incubated with only 5 μM probe. However, when HeLa cells were pretreated with 30 μM ClO and then incubated with 5 μM Nil-ClO, significant increase in fluorescence intensity was detected in the TRITC channel during imaging. Within 1 min, the fluorescence intensity increased sharply and the fluorescence enhancement tend to increase slowly with incubation times longer than 15 min (Fig. 6 m). The experiments show that Nil-ClO performs well in the fluorescence detection of HClO/ClO in living cells.
Fig. 5. A comparison of changes in fluorescence intensity after the addition of different small biomolecules. The excitation wavelength is 560 nm, and the fluorescence at 650 nm is slightly contrasted; 1–16 represent the biologically active small molecules (CH3)3COOH, Glu, Cys, S2O23 , S2 , SO23 , SO24 , HSO23 , ClO , NO2 , H2O2, Vc , Zn2þ, Fe3þ, Mg2þ, and Ca2þ, respectively. Error bar: n ¼ 3.
interference to the detection of HClO/ClO , however, the detection process was affected by solution pH in extreme acid (pH 1.0) condition (Fig. S5). 3.3. Characterization of the sensing mechanism The mechanism of hypochlorite action on the fluorescence of NilClO was investigated by 1H NMR and HRMS. An excessive amount of sodium hypochlorite was added to the PBS buffer containing Nil-ClO, and the reaction was monitored by HR-MS and TLC plate. When a sig nificant fluorescence increase at 650 nm was detected, the product was purified using flash column chromatography and characterized by 1H NMR (Fig. S6, Fig. S7). The results indicated that Dye A was obtained from the reaction. The detection mechanism was probably that the an iline group was oxidized by ClO and then cleaved (Scheme S1) from Dye A, the process was also supported by literature [37]. Thus, the fluorescence change is because that the fluorescence quenching aniline group of Nil-ClO was oxidized by hypochlorite and removed from the fluorescence platform leading to a fluorescence increase [37].
3.6. Imaging endogenous HClO in RAW 264.7 cells We then investigated the effect of the probe on the detection of endogenous HClO/ClO induced by LPS (lipopolysaccharide) in RAW 264.7 cells. It is well known that endogenous hypochlorous acid can be produced by stimulating RAW264.7 cells with LPS and phorbol myr istate acetate (PMA) [40,41]. Therefore, RAW 264.7 macrophages and Fig. 6. Fluorescence imaging of exogenous HClO in HeLa cells treated with 5 μM Nil-ClO and 30 μM ClO . (a–c) The TD, TRITC, and merged im ages of HeLa cells incubated with 5 μM of the probe only. (d–l) The TD, TRITC, and merged images of HeLa cells preincubated with 30 μM ClO and then incubated with 5 μM of the probe for different time. (m) The bar graph of fluores cence intensity change with time in TRITC channel, n ¼ 3. Excitation: ¼ 561 nm; collection: 580–650 nm. Scale bar: 20 μm.
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Fig. 7. Fluorescence of RAW 264.7 cells treated with various stimuli: (a–c) First, RAW 264.7 cells were imaged only after incubation with Nil-ClO for half an hour. (d–f) Second, RAW 264.7 cells were first treated with LPS (0.5 μg/mL), PMA (1 μg/mL) for 2 h, then incubated with Nil-ClO (5 μM) for half an hour, and then imaged by confocal microscopy. (g–i) Third, RAW 264.7 cells were first treated with LPS (0.5 μg/mL), PMA (1 μg/mL), and ABH (200 μM) for 2 h and then incubated with Nil-ClO (5 μM) for half an hour and then imaged by confocal microscopy. (j) The bar graph of fluorescence intensity change in TRITC channel, n ¼ 3. Excitation: ¼ 561 nm; collection: 580–650 nm. Scale bar: 20 μm.
fluorescence signal was observed in the near infrared region when excitation by 800 nm light as shown in Fig. 8. The penetration depth can reach about 100 μm with two-photon excitation (800 nm). These data indicate that Nil-ClO has deep tissue penetration and good HClO/ClO sensing ability under two-photon excitation in living tissue. 3.8. Imaging of endogenous HClO in a mouse model of liver injury Finally, we performed fluorescence imaging of endogenous HClO/ ClO in live mice. It is well known that administration of lipopolysac charide and D-galactosamine (LPS/D-GalN) to mice results in severe liver damage leading to an increase in hypochlorous acid concentration at the site of inflammation [44,45]. Here, Nil-ClO was administered to image a burst of endogenous HClO/ClO under LPS/D-GalN stimula tion. Both LPS/D-GalN and Nil-ClO were administered by tail vein in jection (Fig. 9 (a)). Fig. 9 (b) shows that versus the control group (LPS/D-GalN ( )), the mice induction of acute liver injury (LPS/D-GalN (þ) group) resulted in a significant fluorescence change in the near infrared channel (650–720 nm) in vivo by 560 nm excitation. Therefore, due to its high selectivity to hypochlorite and near-infrared emission wavelength and high fluorescence intensity, Nil-ClO is particularly suitable for fluorescence imaging of hypochlorite in living animals.
Fig. 8. Liver sections of the mice were first treated with the probe Nil-ClO (10 μM) and then subjected to two-photon Z-scan confocal fluorescence imaging after incubation with ClO (20 μM) for 30 min. Fluorescence images from the top layer to the end layer are the depth of 0–120 μm, respectively. The red channel (650–720 nm) is excited at 800 nm. Scale bar ¼ 50 μm. (For inter pretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4. Conclusions The probe, Nil-ClO, is capable of real-time visibility detection of hypochlorite (HClO/ClO ) in living organisms. Advantages of Nil-ClO include excellent two-photon excitation fluorescence, high light stabil ity, high selectivity, low cytotoxicity, and stable fluorescence out-put over a large pH range. These features make the probe an ideal indica tor for detecting and tracking HClO/ClO . The probe can image endogenous and exogenous HClO/ClO in cells, tissues, and living ani mals. The two-photon excitation near-infrared emission feature makes the probe an effective molecular tool for studying the role of HClO/ClO in physiological environments; it is very important to the diagnosis and treatment of diseases modulated by oxidative stress.
LPS (0.5 μg/mL) and PMA (1 μg/mL) were incubated together for 2 h and then incubated with the probe. As shown in Fig. 7(d–f), RAW 264.7 cells stimulated by LPS (0.5 μg/mL) and PMA (1 μg/mL) showed strong fluorescence; fluorescence emission was hardly observed in the control group (Fig. 7 a-c). Further, in the third group, 4-aminobenzoyl hydra zide (ABH) can reduce cellular HClO/ClO levels by inhibiting the ac tivity of MPO. The fluorescence intensity was also significantly reduced after the addition of ABH (Fig. 7 g-j). This result indicates that Nil-ClO can measure endogenous HClO/ClO in RAW 264.7 cells. 3.7. Two-photon fluorescence imaging of exogenous HClO in tissues
Author statement
Here Nil-ClO was used for HClO/ClO imaging in rat liver cry osection tissue. The fluorescence intensity was measured by confocal microscopy in the Z-scan mode as a function of scan depth [42,43]. We first treated the liver tissue with Nil-ClO (10 μM) and then incubated with ClO (20 μM) for half an hour at 37 � C and then imaged. Intense
Xiangpeng Lin: Conceptualization; Data curation; Yunling Chen: Formal analysis; Investigation; Methodology; Luo Bao: Methodology; Resources; Software; Shoujuan Wang: Software; Supervision; Keyin Liu: Writing – review & editing; Wei-dong Qin Validation; 6
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Fig. 9. In vivo imaging of endogenous HClO/ClO in an acute liver injury mouse model. (a) Schematic of the production and detection of endogenous HClO/ClO by Nil-ClO in a mice model. In the control group, the mice were injected with PBS only and Nil-ClO (50 μg) i.v. In the experimental group, mice were pretreated with LPS (10 μg/kg)/D-GalN (700 mg/kg) for 6 h (group: n ¼ 5). (b) In vivo imaging of endogenous HClO/ClO production due to LPS/D-GalN. Representative images of mice intravenously treated with Nil-ClO (50 μg) for 1 h, PBS (left), and LPS/D-GalN (right). λex ¼ 560 nm, λem ¼ 650–720 nm.
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acquisition;
Project
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Declaration of competing interest The authors declare no conflict of interest. Acknowledgements This work was financially supported by NSFC (Nos. 61605060, 31600472, 31570566, and 31800499), and the Key Research and Development Program of Shandong Province (Nos. 2019GSF107052). The Natural Science Foundation of Shandong Province (ZR2017LEM009), the Foundation of Key Laboratory of Pulp and Paper Science and Technology of Ministry of Education/Shandong Province of China (Nos. ZR201707 and ZR201710), the Foundation of Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control of China (No. KF201717), and the Undergraduate Innovation and Entrepreneur ship Program. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2019.108113.
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Contents lists available at ScienceDirect
Dyes and Pigments journal homepage: http://www.elsevier.com/locate/dyepig
A two-photon near-infrared fluorescent probe for imaging endogenous hypochlorite in cells, tissue and living mouse Xiangpeng Lin a, 1, Yunling Chen a, 1, Luo Bao a, Shoujuan Wang a, Keyin Liu a, *, Wei-dong Qin b, **, Fangong Kong a, *** a
State Key Laboratory of Biobased Material and Green Papermaking, Key Laboratory of Pulp & Paper Science and Technology of Shandong Province/Ministry of Education, Qilu University of Technology, Shandong Academy of Sciences, Jinan, 250353, China Department of Critical Care Medicine, Qilu Hospital of Shandong University, Jinan, Shandong, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Hypochlorite detection Two-photon Near infrared emission Endogenous In vivo imaging
ABSTRACT: Hypochlorite (HClO/ClO ) plays an important role in the body’s immune function system and helps destroy invading bacteria and pathogens. Therefore, real-time and visual detection of hypochlorous acid in the living body is necessary. Here, we describe the first example of a two-photon excitation near-infrared lightemitting fluorescent probe (Nil-ClO) for HClO/ClO based on Nile Red derivatives. Nil-ClO offers a large fluorescence response near 650 nm and good selectivity to HClO/ClO in solution. The probe showed an approximately 8-fold increase in fluorescence within 1 min when it was detected at 650 nm in the presence of 30 equivalents of HClO/ClO . The probe was further used for the fluorescence-based detection of HClO/ClO in cells, tissues, and mice in a liver injury model. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) experiments showed that the probe had almost no cytotoxicity at the concentrations needed for im aging. The Nil-ClO can response to HClO/ClO in both HeLa cells and RAW cells quickly. In tissue imaging experiments, Nil-ClO was used to detect HClO/ClO with a significant near-infrared fluorescence response down to 100 μm of depth. Finally, the obvious fluorescence enhancement in liver injury mouse model indicated a burst of endogenously generated HClO/ClO in mouse during in vivo imaging experiments.
1. Introduction Hypochlorite (HClO/ClO ) is an important reactive oxygen species (ROS) in the organism [1]. It is widely distributed in various biological environments. In vivo, it is produced by myoglobin (MPO) catalyzing the production of hydrogen peroxide and chloride ions and plays an important role in the human immune system [2,3]. Under normal con ditions, hypochlorite level remains relatively high to destroy invading bacteria and pathogens. Excessive hypochlorous acid or hypochlorite can cause a series of diseases, such as arteriosclerosis and arthritis [4]. Therefore, real-time detection of hypochlorous acid in organisms is critical for studies of the physiological effects and early diagnosis of diseases caused by hypochlorous acid [5]. Recently, many methods for detecting hypochlorous acid have been developed, including iodine reduction droplets, spectrophotometry,
chemiluminescence analysis, and coulometry [6]. However, these analytical methods are expensive and cumbersome. Therefore, the development of an effective, inexpensive, and simple hypochlorous acid detection method is an important research topic [7]. Fluorescence is particularly suitable for analysing biologically active small species: It is non-invasive and highly sensitive and selective in biological samples [8, 9]. In recent years, many fluorescent probes have been developed for hypochlorite detection based on traditional dyes, such as rhodamine [10,11], coumarin [12,13], fluorescein [14,15], naphthalimide [16,17], bodipy [18,19], and cyanine dyes [20,21]. These systems exhibit excellent photophysical properties and good selectivity for ClO . They have been used to detect ClO in cells, tissues, and animals [22]. Two-photon probes offer a high spatial discrimination rate and deep tissue penetration [23–25]. However, the fluorescence of most two-photon probes is in the visible region, and developing two-photon
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (K. Liu), [email protected] (W.-d. Qin), [email protected] (F. Kong). 1 These authors contributed equally to this works. https://doi.org/10.1016/j.dyepig.2019.108113 Received 10 October 2019; Received in revised form 6 December 2019; Accepted 6 December 2019 Available online 9 December 2019 0143-7208/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xiangpeng Lin, Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2019.108113
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Fig. 1. Design of a two-photon near-infrared fluorescent probe Nil-ClO for hypochlorite and its detection mechanism.
probes with near-infrared emission is still challenging [26,27]. Recently, Nile Red derivatives were demonstrated to be a unique platform with excellent two-photon excitation fluorescence performance, good pho tostability, high fluorescence quantum yield, and low cytotoxicity [28]. For example, a Nile Red-palladium-based fluorescent probe was re ported and used to measure endogenous CO in vivo [29]. Herein, we report the first example of a two-photon excitation, near-infrared emission fluorescent probe for HClO/ClO and used it to study the function of HClO/ClO in mice via a liver injury model. In our design (Fig. 1), Nile Red derivative dye (DyeA) has many advantages over conventional dyes, including two-photon excitation and near-infrared light emission. The remaining hydroxyl groups can be used for further chemical modification. The fluorescent probe (Nil-ClO) was constructed by reacting the hydroxyl group of DyeA with 1-fluoro4-nitrobenzene and then reducing the nitro group. Finally, we studied the photophysical properties of Nil-ClO and the bioimaging capabilities toward endogenous HClO in cells, tissues, and a liver injury mice model induced by lipopolysaccharide and D-galactosamine (LPS/D-GalN) [30–32]. Scheme 1. Synthesis of the fluorescent platform Nile Red derivative DyeA and the fluorescent probe Nil-ClO for detecting HClO.
2. Experimental 2.1. Reagents, materials, and apparatus
Olympus laser confocal micro-scope.
All of the solvents and reagents are available from commercial sources unless otherwise stated. Solvents were purified according to standard methods prior to use; deionized water was used throughout all the experiments. TLC plates and silica gels were purchased from Qing dao Ocean chemicals. The reactions were monitored by thin layer chromatography (TLC plate, silica gel GF254) and further purified by silica column chromatography (silica gel 200–300 mesh) TLC plates. The HRMS (ESI) spectra were recorded on a Bruker Apex-Ultra mass spec trometer ((Bruker Daltonics Corp., USA). The 1H NMR, and 13C NMR spectra were collected on a Bruker Avance III 400 MHz spectrometer (Bruker Daltonics Corp., USA), and tetramethylsilane (TMS) was used as an internal reference. The absorption spectra were recorded on a Shi madzu UV-2700 spectrophotometer (Shimadzu Suzhou Instruments Mfg. Co. Ltd.) and the fluorescence spectra were recorded on a Hitachi F7100 spectrofluorimeter (Hitachi High-Tech Science) with a 10 mm quartz cuvette, respectively. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) assays were measured on a micro MTT instrument (Thermo Fisher). Cell imaging were performed used an
2.2. Fluorescence detection The probe Nil-ClO was formulated into a 1 mM stock solution using dimethyl sulfoxide (DMSO). The Nil-ClO (1 � 10 5 mol/L) test solution was made in PBS (10 mM phosphate buffer, pH 7.4, 5% DMSO as cosolvent), and added an aliquot of each test substance solution, and small aliquots of each test substance solution were added. The resulting solution is fully oscillated at room temperature before measuring the spectrum [33]. 2.3. Synthesis procedures The synthesis of Nil-ClO is shown in Scheme 1. Synthesis of Dye A The synthesis and characterization of Dye A was performed based on our previous reports [29]. 1,6-Dihydroxynaphtha lene (0.176 g, 1.1 mmol) and 3-hydroxy-4-nitroso-N,N-diethylaniline (0.194 g, 1.0 mmol) were added to a 100 mL round bottom flask with 2
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Fig. 2. Fluorescence spectral changes of Nil-ClO titrated with ClO . (a) Fluorescence spectral changes of 10 μM Nil-ClO in PBS (pH 7.4, containing 5% DMSO) were titrated with increasing amounts of ClO . (b) Linear fitting of probe response to fluorescence signal at 650 nm by varying the concentration of ClO .
magnetic beads and DMF (10 mL). The mixture was degassed and heated to 140 � C in a magnetic stirrer to form a deep red solution. The reaction was monitored once per hour with a silica gel TLC plate (eluent: dichloromethane/methanol ¼ 50:1, v/v) until the starting material was consumed completely. The solution was cooled to room temperature. The organic layer was washed with deionized water, extracted with methylene chloride, dried with anhydrous magnesium sulfate and the DMF removed. The crude material was further purified with silica gel column and eluted with methylene chloride: methanol ¼ 50:1 (v/v). This led to Dye A, which was a dark red solid (0.22 g, 0.66 mmol). The yield was 60%. m.p.: 270–272 � C. IR (KBr) ν: 2976, 2932, 1567, 1409, 1117 cm 1. 1H NMR (400 MHz, DMSO‑d6) δ 10.44 (s, 1H), 7.98 (d, J ¼ 8.5 Hz, 1H), 7.89 (d, J ¼ 2.2 Hz, 1H), 7.59 (d, J ¼ 9.1 Hz, 1H), 7.10 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.81 (d, J ¼ 6.6 Hz, 1H), 6.66 (s, 1H), 6.16 (s, 1H), 3.51 (q, J ¼ 7.0 Hz,4H),1.17 (t, J ¼ 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 181.99, 161.04, 155.95, 153.83, 151.96, 151.09, 146.77, 139.23, 134.17, 131.17, 127.85, 127.19, 124.29, 124.13, 118.76, 117.37, 117.25, 110.28, 108.95, 108.62, 105.72, 104.57, 96.48, 44.84, 12.88. HRMS(ESI) calcd. for C20H18N2O3 [M]þ: 335.1396, found: 335.1385. Synthesis of compound 2 Dye A (65 mg, 0.19 mmol), 1-fluoro-4nitrobenzene (60 mg, 0.42 mmol), and potassium carbonate (60 mg, 0.43 mmol) were added to DMF (10 mL) and allowed to react at 40 � C overnight and then cooled the solution to room temperature. The solu tion was washed with deionized water and then extracted with methy lene chloride (20 mL) three times through a separation funnel. The organic layers were combined, dried with anhydrous sodium sulfate, then it was concentrated and purified by silica gel column (elution with dichloromethane and methanol, v/v ¼ 100:1). Compound 2 was received as a dark red solid (83 mg, 0.182 mmol) with a yield of 45%. m. p.: 256–257 � C. IR (KBr) ν: 3442, 3070, 2974, 2928, 1583, 1500, 1380, 1112 cm 1. 1H NMR (400 MHz, DMSO‑d6) δ 8.33 (d, J ¼ 9.2 Hz, 2H), 8.25 (d, J ¼ 8.6 Hz, 1H), 8.17 (d, J ¼ 2.5 Hz, 1H), 7.60 (d, J ¼ 9.2 Hz, 1H), 7.53 (dd, J ¼ 8.6, 2.5 Hz, 1H), 7.33 (d, J ¼ 9.2 Hz, 2H), 6.87–6.82 (m, 1H), 6.71 (d, J ¼ 2.7 Hz, 1H), 6.32 (s, 1H), 3.93 (d, J ¼ 5.9 Hz, 1H), 3.57–3.49 (m, 4H), and 1.17 (t, J ¼ 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 181.44, 163.62, 162.34, 161.64, 157.79, 152.59, 151.66, 151.35, 147.12, 143.49, 142.14, 134.45, 131.63, 128.73, 128.59, 126.81, 124.68, 122.33, 119.03, 113.62, 110.93, 104.75, 96.47, 44.98, and 12.92. HRMS (ESI) calcd. for C26H21N3O5 [M]þ: 456.1559, found: 456.1547. Synthesis of HClO fluorescent probe Nil-ClO Compound 2 (38 mg, 0.08 mmol) and iron powder (132 mg, 2.35 mmol) were added to a 25mL round-bottom flask with 10 mL glacial acetic acid. The mixture was stirred at room temperature for 16 h under nitrogen protection, and the
reaction was monitored with TLC plate every 4 h. Saturated sodium bicarbonate solution was added to the solution to neutralize the glacial acetic acid, and then methylene chloride (20 mL) was added and extracted three times through a separation funnel. We then combined the organic layers, washed them with saturated brine and anhydrous sodium sulfate, concentrated the product with an evaporator. Finally, the crude products were purified by silica gel column (eluted with dichloromethane and methanol, v/v ¼ 50:1). The product Nil-ClO was a dark red solid (22 mg, 0.057 mmol) with a yield of 60%. m.p.: 310–312 � C. IR (KBr) ν: 3414, 2927, 2851, 1583, 1495, 1407, 1117, 915 cm 1. 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J ¼ 8.7 Hz, 1H), 8.12 (d, J ¼ 2.2 Hz, 1H), 7.58 (d, J ¼ 9.0 Hz, 1H), 7.19 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.97 (d, J ¼ 8.6 Hz, 2H), 6.76 (d, J ¼ 8.6 Hz, 2H), 6.65 (dd, J ¼ 9.1, 2.4 Hz, 1H), 6.47 (d, J ¼ 2.4 Hz, 1H), 6.33 (s, 1H), 3.50–3.44 (m, 4H), and 1.25 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 183.06, 161.74, 152.18, 150.81, 147.95, 146.84, 143.22, 139.98, 134.19, 131.19, 127.94, 124.80, 121.43, 119.20, 116.36, 110.77, 109.50, 105.43, 96.38, 45.01, 29.64, and 12.57. HRMS (ESI) calcd. for C26H24N3O3 [M]þ: 426.1818 found: 426.1802. 2.4. Sample preparation and measurements A stock solution of Nil-ClO was prepared in dimethyl sulfoxide at a concentration of 1 mM, and a 10 mM NaClO stock solution was prepared in deionized water. Fluorescence measurements used PBS (phosphate buffer (10 mM, pH 7.4), 5% DMSO as a cosolvent). In fluorescence with pH change measurements, the PBS was used at a pH of 4.0–10.0. In the fluorescence spectral measurements, the Nil-ClO was 1 � 10 5 M, the excitation wavelength was at 560 nm, and the emission spectrum was 580–900 nm. In selective experiments, solutions for various test species, including TBHP (tert-butyl hydroperoxide), Glu, Cys, S2O23 , S2 , SO23 , SO24 , HSO23 , ClO , NO2 , H2O2, Vc , Zn2þ, Fe3þ, Mg2þ, and Ca2þ, were prepared according to the literature report. Nil-ClO interacted with 30 eq. of each of the test species for about 30 min before fluorescence selectivity measurements. 2.5. Cell culture and viability assay HeLa cells and RAW 264.7 cells were obtained from Shandong University School of Pharmacy and cultured in a minimal essential medium (MEM) supplemented with 10% calf serum at 37 � C, 5% CO2 in a humid environment. Cell viability was determined using MTT [34]. The HeLa cells were first incubated for 24 h in a 37 � C incubator, then they were incubated into a 96-well plate and further incubated with different concentrations of probes for 12 h. Followed by 10 μL MTT was 3
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Fig. 3. Fluorescence spectra of Nil-ClO versus ClO as a function of time. (a) Fluorescence spectral changes of 10 μM Nil-ClO and 30 equivalents of ClO in PBS over time. (b) Fluorescence intensity of 10 μM of Nil-ClO and 30 equivalents of ClO as a function of time at 650 nm; excitation wavelength: λ ¼ 560 nm.
added to each well of the plate, and further incubated for an additional 4 h, and then DMSO (100 μL) was added. The uptake of cells in each well plate was measured using a Herm Fisher Scientific plate reader. In cell imaging experiments of exogenous hypochlorous acid, HeLa cells were preincubated for 12 h in a 37 � C incubator, incubated with 5 μM probe for 30 min, washed with PBS, and further incubated with 30 μM NaClO for 20 min. In a control experiment, cells were incubated with only 5 μM probe and further studied using confocal microscopy [35]. 2.6. Imaging of HClO in tissue and in vivo Animal models and in vivo imaging. 4-Week-old Balb/c mice were obtained from animal center of Shandong University. All animal ex periments were performed in accordance with the guidelines issued by The Ethical Committee of Shandong University. Preparation of the fresh mouse liver tissue slices and two-photon fluorescence imaging. Slices were prepared from the liver of 4-week-old Balb/c mice. Slices were cut to 400 μm thickness by using a vibrating blade microtome in 10 mM PBS (pH 7.4). Slices were then incubated with Nil-ClO, then washed with PBS, and observed under a two-photon confocal microscope (Leica SP8). For in vivo imaging experiment, the mice were divided into two groups, in a control experiment, the mice were administered by tail vein injec tion of Nil-ClO. In experimental group, mice were given LPS/D-GalN stimulation to induce a burst of endogenous HClO/ClO , then admin istered by tail vein injection of Nil-ClO. Fluorescence images were taken by IVIS Lumina III living animal imaging instrument PerkinElmer.
Fig. 4. Fluorescence intensity changes of the Nil-ClO (10 μM) at different pH values in the absence (■) or presence ( ) of ClO (30 eq).
3.2. Fluorescence spectra changes of Nil-ClO versus HClO as a function of time
3. Result and discussion
Fluorescence changes of Nil-ClO over time during the assay were also investigated. When 30 equivalents of ClO were present in PBS (pH 7.4), the fluorescence of Nil-ClO increased significantly, and the fluo rescence intensity reached the maximum within 1 min after addition of Nil-ClO indicating that the response speed was very fast (Fig. 3b). The fluorescence intensity increased by about 8-fold after adding sodium hypochlorite for 1 min (Fig. 3a). We then tested the kinetics of the re action under pseudo-first-order conditions in which a large excess of ClO (30 equivalents) was reacted with Nil-ClO (10 μM) in PBS buffer (pH 7.4, 10 mM, 5% DMSO) at 37 � C. The pseudo-first-order rate con stant of ClO was k ¼ 0.0961 s 1 (Fig. S3). We further investigated the effect of pH on the fluorescence response of Nil-ClO to ClO . In the wide pH range of 5.0–9.0 without ClO , only very small changes in fluores cence intensity were observed in PBS with only probes, indicating that the free probes were stable over a wide pH range. (Fig. 4, Fig. S4). When ClO was added, a large fluorescence intensity increase was observed and the fluorescence intensity almost did not change across pH values from 5.0 to 9.0. Therefore, the pH change in solution has almost no
3.1. Fluorescence spectral changes of Nil-ClO titrated with ClO The spectral characteristics of Nil-ClO were investigated. Nil-ClO shows a maximum absorption peak at 588 nm and an emission peak near 650 nm (Fig. S1, Fig. S2 in Supporting information), and the Stokes shift of Nil-ClO is about 62 nm. Fig. 2a shows that the fluorescence of Nil-ClO (fluorescence quantum yield: 0.37%, rhodamine B in ethanol was used as standard in Supporting information) at 650 nm increased significantly upon addition of ClO . Based on the linear fit of fluorescence intensity to ClO concentration, the detection limit of Nil-ClO for ClO was about 4.37 � 10 6 M (Fig. 2b). Hypochlorous acid (HClO/ClO ) plays a major role in the immune system of living organisms. And it has a certain destructive effect on invading bacteria and pathogens [36]. The detec tion limit of Nil-ClO is lower than the normal concentration of HClO in the biological environment, thus it can be potentially applied to detect endogenously generated HClO/ClO in living organisms.
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3.4. Selective experiment of Nil-ClO on HClO The selectivity of Nil-ClO for HClO/ClO was investigated next, common interferents such as Glu, Cys, (CH3)3COOH (TBHP), S2O23 , S2 , SO23 , SO24 , HSO23 , ClO , NO2 , H2O2, Vc , Zn2þ, Fe3þ, Mg2þ, and Ca2þ, were added to PBS buffer containing Nil-ClO to monitor changes in fluorescence spectra. The effect of other factors such as BSA and tem perature toward the detection process were also measured (Fig. S8 and Fig. S9). Significant changes in the fluorescence spectra were observed only when ClO was added (Fig. 5), and other small molecules and changes in temperature do not interfere with the fluorescence spectra. Fluorescence changes at 650 nm also clearly indicate that Nil-ClO shows high selectivity for ClO . The high selectivity of Nil-ClO enables it to detect ClO in complex biological samples. 3.5. Imaging exogenous HClO in HeLa cells For the bioimaging experiments, the Nil-ClO was applied to assess ClO detection in biological environments. The standard MTT assay was performed to evaluate the effect of Nil-ClO on cell proliferation prior to HeLa cell imaging (Fig. S10). At a Nil-ClO concentration of 30 μM, the cell viability was maintained above 90%. However, at the probe con centration required for cell imaging, the cell viability remained above 95% even after incubation for 24 h [38]. Therefore, the MTT result indicated that the probe had relatively low cytotoxicity in the concen tration and incubation time required for fluorescence imaging experi ments in cells [39]. We then applied Nil-ClO to the fluorescence imaging of HClO in cells. Fig. 6 shows that fluorescent signal almost invisible in the TRITC channel during imaging when HeLa cells were incubated with only 5 μM probe. However, when HeLa cells were pretreated with 30 μM ClO and then incubated with 5 μM Nil-ClO, significant increase in fluorescence intensity was detected in the TRITC channel during imaging. Within 1 min, the fluorescence intensity increased sharply and the fluorescence enhancement tend to increase slowly with incubation times longer than 15 min (Fig. 6 m). The experiments show that Nil-ClO performs well in the fluorescence detection of HClO/ClO in living cells.
Fig. 5. A comparison of changes in fluorescence intensity after the addition of different small biomolecules. The excitation wavelength is 560 nm, and the fluorescence at 650 nm is slightly contrasted; 1–16 represent the biologically active small molecules (CH3)3COOH, Glu, Cys, S2O23 , S2 , SO23 , SO24 , HSO23 , ClO , NO2 , H2O2, Vc , Zn2þ, Fe3þ, Mg2þ, and Ca2þ, respectively. Error bar: n ¼ 3.
interference to the detection of HClO/ClO , however, the detection process was affected by solution pH in extreme acid (pH 1.0) condition (Fig. S5). 3.3. Characterization of the sensing mechanism The mechanism of hypochlorite action on the fluorescence of NilClO was investigated by 1H NMR and HRMS. An excessive amount of sodium hypochlorite was added to the PBS buffer containing Nil-ClO, and the reaction was monitored by HR-MS and TLC plate. When a sig nificant fluorescence increase at 650 nm was detected, the product was purified using flash column chromatography and characterized by 1H NMR (Fig. S6, Fig. S7). The results indicated that Dye A was obtained from the reaction. The detection mechanism was probably that the an iline group was oxidized by ClO and then cleaved (Scheme S1) from Dye A, the process was also supported by literature [37]. Thus, the fluorescence change is because that the fluorescence quenching aniline group of Nil-ClO was oxidized by hypochlorite and removed from the fluorescence platform leading to a fluorescence increase [37].
3.6. Imaging endogenous HClO in RAW 264.7 cells We then investigated the effect of the probe on the detection of endogenous HClO/ClO induced by LPS (lipopolysaccharide) in RAW 264.7 cells. It is well known that endogenous hypochlorous acid can be produced by stimulating RAW264.7 cells with LPS and phorbol myr istate acetate (PMA) [40,41]. Therefore, RAW 264.7 macrophages and Fig. 6. Fluorescence imaging of exogenous HClO in HeLa cells treated with 5 μM Nil-ClO and 30 μM ClO . (a–c) The TD, TRITC, and merged im ages of HeLa cells incubated with 5 μM of the probe only. (d–l) The TD, TRITC, and merged images of HeLa cells preincubated with 30 μM ClO and then incubated with 5 μM of the probe for different time. (m) The bar graph of fluores cence intensity change with time in TRITC channel, n ¼ 3. Excitation: ¼ 561 nm; collection: 580–650 nm. Scale bar: 20 μm.
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Fig. 7. Fluorescence of RAW 264.7 cells treated with various stimuli: (a–c) First, RAW 264.7 cells were imaged only after incubation with Nil-ClO for half an hour. (d–f) Second, RAW 264.7 cells were first treated with LPS (0.5 μg/mL), PMA (1 μg/mL) for 2 h, then incubated with Nil-ClO (5 μM) for half an hour, and then imaged by confocal microscopy. (g–i) Third, RAW 264.7 cells were first treated with LPS (0.5 μg/mL), PMA (1 μg/mL), and ABH (200 μM) for 2 h and then incubated with Nil-ClO (5 μM) for half an hour and then imaged by confocal microscopy. (j) The bar graph of fluorescence intensity change in TRITC channel, n ¼ 3. Excitation: ¼ 561 nm; collection: 580–650 nm. Scale bar: 20 μm.
fluorescence signal was observed in the near infrared region when excitation by 800 nm light as shown in Fig. 8. The penetration depth can reach about 100 μm with two-photon excitation (800 nm). These data indicate that Nil-ClO has deep tissue penetration and good HClO/ClO sensing ability under two-photon excitation in living tissue. 3.8. Imaging of endogenous HClO in a mouse model of liver injury Finally, we performed fluorescence imaging of endogenous HClO/ ClO in live mice. It is well known that administration of lipopolysac charide and D-galactosamine (LPS/D-GalN) to mice results in severe liver damage leading to an increase in hypochlorous acid concentration at the site of inflammation [44,45]. Here, Nil-ClO was administered to image a burst of endogenous HClO/ClO under LPS/D-GalN stimula tion. Both LPS/D-GalN and Nil-ClO were administered by tail vein in jection (Fig. 9 (a)). Fig. 9 (b) shows that versus the control group (LPS/D-GalN ( )), the mice induction of acute liver injury (LPS/D-GalN (þ) group) resulted in a significant fluorescence change in the near infrared channel (650–720 nm) in vivo by 560 nm excitation. Therefore, due to its high selectivity to hypochlorite and near-infrared emission wavelength and high fluorescence intensity, Nil-ClO is particularly suitable for fluorescence imaging of hypochlorite in living animals.
Fig. 8. Liver sections of the mice were first treated with the probe Nil-ClO (10 μM) and then subjected to two-photon Z-scan confocal fluorescence imaging after incubation with ClO (20 μM) for 30 min. Fluorescence images from the top layer to the end layer are the depth of 0–120 μm, respectively. The red channel (650–720 nm) is excited at 800 nm. Scale bar ¼ 50 μm. (For inter pretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4. Conclusions The probe, Nil-ClO, is capable of real-time visibility detection of hypochlorite (HClO/ClO ) in living organisms. Advantages of Nil-ClO include excellent two-photon excitation fluorescence, high light stabil ity, high selectivity, low cytotoxicity, and stable fluorescence out-put over a large pH range. These features make the probe an ideal indica tor for detecting and tracking HClO/ClO . The probe can image endogenous and exogenous HClO/ClO in cells, tissues, and living ani mals. The two-photon excitation near-infrared emission feature makes the probe an effective molecular tool for studying the role of HClO/ClO in physiological environments; it is very important to the diagnosis and treatment of diseases modulated by oxidative stress.
LPS (0.5 μg/mL) and PMA (1 μg/mL) were incubated together for 2 h and then incubated with the probe. As shown in Fig. 7(d–f), RAW 264.7 cells stimulated by LPS (0.5 μg/mL) and PMA (1 μg/mL) showed strong fluorescence; fluorescence emission was hardly observed in the control group (Fig. 7 a-c). Further, in the third group, 4-aminobenzoyl hydra zide (ABH) can reduce cellular HClO/ClO levels by inhibiting the ac tivity of MPO. The fluorescence intensity was also significantly reduced after the addition of ABH (Fig. 7 g-j). This result indicates that Nil-ClO can measure endogenous HClO/ClO in RAW 264.7 cells. 3.7. Two-photon fluorescence imaging of exogenous HClO in tissues
Author statement
Here Nil-ClO was used for HClO/ClO imaging in rat liver cry osection tissue. The fluorescence intensity was measured by confocal microscopy in the Z-scan mode as a function of scan depth [42,43]. We first treated the liver tissue with Nil-ClO (10 μM) and then incubated with ClO (20 μM) for half an hour at 37 � C and then imaged. Intense
Xiangpeng Lin: Conceptualization; Data curation; Yunling Chen: Formal analysis; Investigation; Methodology; Luo Bao: Methodology; Resources; Software; Shoujuan Wang: Software; Supervision; Keyin Liu: Writing – review & editing; Wei-dong Qin Validation; 6
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Fig. 9. In vivo imaging of endogenous HClO/ClO in an acute liver injury mouse model. (a) Schematic of the production and detection of endogenous HClO/ClO by Nil-ClO in a mice model. In the control group, the mice were injected with PBS only and Nil-ClO (50 μg) i.v. In the experimental group, mice were pretreated with LPS (10 μg/kg)/D-GalN (700 mg/kg) for 6 h (group: n ¼ 5). (b) In vivo imaging of endogenous HClO/ClO production due to LPS/D-GalN. Representative images of mice intravenously treated with Nil-ClO (50 μg) for 1 h, PBS (left), and LPS/D-GalN (right). λex ¼ 560 nm, λem ¼ 650–720 nm.
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Declaration of competing interest The authors declare no conflict of interest. Acknowledgements This work was financially supported by NSFC (Nos. 61605060, 31600472, 31570566, and 31800499), and the Key Research and Development Program of Shandong Province (Nos. 2019GSF107052). The Natural Science Foundation of Shandong Province (ZR2017LEM009), the Foundation of Key Laboratory of Pulp and Paper Science and Technology of Ministry of Education/Shandong Province of China (Nos. ZR201707 and ZR201710), the Foundation of Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control of China (No. KF201717), and the Undergraduate Innovation and Entrepreneur ship Program. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2019.108113.
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