Article Cite This: ACS Sens. XXXX, XXX, XXX−XXX
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Mitochondria-Accessing Ratiometric Fluorescent Probe for Imaging Endogenous Superoxide Anion in Live Cells and Daphnia magna Zhen Zhang, Jiangli Fan,* Yuhui Zhao, Yao Kang, Jianjun Du, and Xiaojun Peng State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P.R. China S Supporting Information *
ABSTRACT: Superoxide anion (O2•−), as the precursor of other reactive oxygen species (ROS), is significantly important in the maintenance of redox homeostasis and various cellular signaling pathways. Here we present a ratiometric mitochondria-accessing fluorescent probe (NA-T) based on nucleophilic substitution mechanism for real-time measuring O2•−. By regulating the intramolecular charge of 1,8-naphthalimide, a ratiometric response model was obtained, which evinced 18fold enhancement of fluorescence ratio (I540 nm/I475 nm) in the presence of O2•− over other ROS with rapid response (132 s), high sensitivity (DL = 0.370 μM) and selectivity. Confocal fluorescence images demonstrated that the probe could well permeate through plasma membrane for visualizing endogenous O2•− changes in mitochondria of living cells and in inflammatory Daphnia magna, indicating NA-T a potential tool for the diagnosis and research of corresponding diseases. KEYWORDS: superoxide anion, mitochondria-accessing, ratiometric bioimaging, fluorescent probe, fast response, inflammatory Daphnia magna uperoxide anion radical (O2•−) is one of the potent effector molecules and intracellular messengers.1,2 Generally, only considerably less than 1−2% of consumed oxygen molecules undergo the conversion to superoxide anion at two discrete points (Complex I and Complex III) in mitochondrial respiratory chain.3−5 And the diffusing superoxide anion in biological systems is typically converted to other ROS like hydrogen peroxide,6,7 peroxynitrite8 as well as perhydroxyl redical9,10 within a few tens of micrometers after leaving its generation sites.11,12 Accordingly, superoxide anion can directly or indirectly regulate innate immune defense against invading organisms and trigger various signaling pathways in cellular growth and metabolism.13,14 Despite the low concentration and short diffusion distance (or short lifetime), superoxide anion at excessive levels was suggested to induce increasing oxidative stress and mitochondrial dysfunction implicated in Alzheimer’s disease, rheumatoid arthritis, heart disease, diabetes mellitus, and even cancer.15−20 Hence, in suit specific tracking and measurement of superoxide anion in complex biological systems can be meaningful for elucidating the exact pathogenesis of related diseases. Comparing to electron paramagnetic resonance (EPR), high performance liquid chromatography (HPLC), electrochemical sensors, and mass spectrometry (MS), fluorescence imaging has allowed for a noninvasive method to specifically study superoxide anion associated processes in vitro and in vivo with high spatial and temporal resolution.21,22 To date, a number of fluorescent probes for superoxide anion visualization
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have been reported via redox mechanism23−25 and nucleophilic substitution mechanism.26−32 Actually, very few biological compounds can be oxidized by superoxide anion since it has a high reduction potential (+0.94 V),33 and the innate anionic charge of superoxide anion limits its reactivity with electronrich centers,2,34 making it a good nucleophile reagent. 2,4Dinitrobenzenesulfonate,26,27 trifluoromethanesulfonate28,30 as well as diphenyl-phosphinate29,31,32 are the most representative reactive groups via the nonredox mechanism, and several fluorescent sensors based on these groups have achieved highly selective and sensitive detecting of superoxide anion in vitro and in vivo. A pity is that almost all of them belong to “turn on” type whose fluorescence signal can be prone to be disturbed by other variables such as the excitation and emission efficiency, the background fluorescence, the concentration of probe itself and the microenvironmental conditions. Advances in ratiometric approaches provide efficient solutions for aforementioned problems and achieve more reliable quantitative detection. Recently an AIE-active fluorescent probe was developed to selectively detect superoxide anion with two channel outputs, and was used for imaging endogenous superoxide anion in HepG2 cells.31 However, more strides should be made in developing ratiometric approaches to map Received: January 24, 2018 Accepted: March 6, 2018 Published: March 6, 2018 A
DOI: 10.1021/acssensors.8b00082 ACS Sens. XXXX, XXX, XXX−XXX
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ACS Sensors the subcellular distribution and in vivo changes of superoxide anion under different circumstances. Herein, we report a mitochondria-accessing ratiometric fluorescent probe (NA-T) for superoxide anion based on the nonredox strategies. A diphenylphosphinate group was introduced as the trigger site, and an electron-withdrawing carbamate group was linked to 1,8-naphthalimide to regulate the ICT effect. Incorporation of triphenylphosphonium (TPP) made NA-T effective and led to preferential accumulation in mitochondria. NA-T shows rapid ratiometric response, good sensitivity, and good selectivity toward superoxide anion over latent interferents in vitro, and is subsequently applied to imaging endogenous superoxide anion in mitochondria of living cells with different treatments. Moreover, NA-T is successfully used in vivo to study the superoxide anion changes in inflammatory Daphnia magna stimulated by PMA/LPS.
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Scheme 1. Synthetic Approaches to Crucial Intermediates and NA-T
added. The solvent was evaporated after keeping reflux for 5 h, the solid residue was then dissolved in THF, and PPh3 (787 mg, 3 mmol) was carefully added and stirred at room temperature for 1 h. A volume of 10 mL of acetic acid solution was added then and stirred for another 30 min and then poured into ice water (100 mL). The emulsion formed was subsequently mixed with saturated sodium chloride aqueous solution to obtain the precipitated solid, which was further purified by silica gel chromatography to afford compound 3 as a yellow solid (442 mg, 64.5%). 1H NMR (400 MHz, DMSO) δ (ppm): 8.62 (d, J = 8.4 Hz, 1H), 8.37 (d, J = 8.2 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.91−7.81 (m, 3H), 7.81−7.67 (m, 12H), 7.65−7.57 (m, 1H), 7.50 (s, 2H), 6.82 (d, J = 8.4 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 3.72 (t, J = 15.1 Hz, 2H), 2.01−1.83 (m, 2H). MS (ESI): m/z 515.32 (M+), calcd for C33H28N2O2P+: 515.18. Syntheses of Compound 4. Compound 4 was synthesized according to previous work with some modifications.31 Briefly, 4hydroxybenzyl alcohol (372 mg, 3 mmol) and triethylamine (0.5 mL) were dissolved in distilled THF (10 mL), and phosphinic chloride (711 mg, 3 mmol) was added dropwise under nitrogen at 0 °C. After stirring at room temperature for overnight followed by addition of 15 mL of water to quench the reaction, the solvent was evaporated under reduced pressure, and the resulting residue was extracted with dichloromethane (DCM) for three times and purified by silica gel chromatography with DCM/MeOH (50:1) as eluent to afford the desire product as a white solid (641 mg, yield 65.9%). 1H NMR (400 MHz, DMSO) δ (ppm): 7.97−7.86 (m, 4H), 7.65−7.58 (m, 2H), 7.55 (m, 4H), 7.24 (s, 4H), 5.17 (t, J = 5.7 Hz, 1H), 4.41 (d, J = 5.7 Hz, 2H). HRMS (ESI-TOF) (m/z): 325.0989 (M + ), calcd for C19H18O3P+: 325.0988. Synthesis of NA-T. A DCM solution of triphosgene (150 mg, 0.5 mmol) was added dropwise to the mixture of compound 3 (297 mg, 0.5 mmol), DIPEA (387 mg, 3 mmol), DMAP (15 mg), and dry DCM in an ice bath. After stirring for 4 h under nitrogen atmosphere, the resulting solution was warmed to ambient temperature and stirred for another 6 h. A DCM solution of compound 4 (194 mg, 0.6 mmol) was then added into the mixture. The reaction was quenched with water and extracted three times with DCM after being stirred overnight, and the combined organic layer was dried, filtered, concentrated, and chromatographed on a neutral alumina column (eluent: 100/1−100/ 10). Pure product was harvested as a yellow solid (155 mg, yield 31.2%). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.89 (s, 1H), 8.57 (d, J = 8.2 Hz, 1H), 8.02 (dd, J = 17.0, 7.8 Hz, 2H), 7.90−7.82 (m, 5H), 7.82−7.68 (m, 10H), 7.63 (td, J = 7.5, 3.3 Hz, 6H), 7.50 (td, J = 7.5, 1.3 Hz, 2H), 7.41 (ddd, J = 12.4, 7.5, 3.7 Hz, 6H), 7.18 (d, J = 8.4 Hz, 2H), 5.15 (s, 2H), 4.20 (t, J = 6.6 Hz, 2H), 3.98−3.80 (m, 2H), 2.01 (s, 2H). 13C NMR (100 MHz, CDCl3) δ (ppm): 162.93, 162.33, 161.59, 153.54, 149.61, 149.55, 134.09, 132.65, 132.57, 131.79, 131.47, 130.81, 130.73, 129.57, 129.47, 129.31, 128.60, 127.67, 127.57, 119.71, 119.68, 117.44, 116.75, 65.34, 35.46, 30.42, 20.29. HRMS (FTMSESI) (m/z): 865.2605 (M+), calcd for C53H43N2O6P2+: 865.2591. Living Cells Imaging. RAW264.7 cells, HepG2 cells, and MCF-7 cells were typically seeded in culture dishes at a concentration of 2 × 104 cells/mL and cultured for 24 h in DMEM medium at 37 °C under 5% CO2 for confocal microscopy imaging. For detecting endogenous superoxide anion, cells were first washed with PBS buffer and then
EXPERIMENTAL SECTION
Materials and Instruments. 4-Bromo-1,8-naphthalic anhydride, triphenylphosphline, triphosgene, N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), diphenylphosphinyl chloride, 4-hydroxybenzyl alcohol, 3-bromopropylamine hydrobromide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Energy Chemical. Potassium superoxide (KO2) was purchased from Sigma-Aldrich. Lipopolysaccharide (LPS), 2-methoxyestradiol (2-ME), Tiron, xanthine oxidase (XOD), and xanthine (XA) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Phorbol 12-myristate 13-acetate (PMA) was purchased from J&K. MitoTracker Deep Red FM was purchased from Invitrogn. All chemicals and solvents used were analytically pure and used without further purification unless otherwise stated. Water was purified and doubly distilled by using a Milli-Q system. Column chromatography was performed using silica gel (HaiYang, Qingdao) 200−300 mesh or using neutral alumina oxide (Sinopharm Chemical Regent Co., Ltd.) 100−200 mesh. 1H NMR and 13C NMR spectra of the intermediates and target compound were recorded on the Bruker Avance spectrometer (400 MHz for 1H NMR; 100 MHz for 13C NMR). Chemical shifts (δ) were expressed as parts per million (ppm, in CDCl3 or DMSO, with TMS as the internal standard). Electrospray ionization mass spectrometry (ESI-MS) was performed with a H2100LC/MSD MS instrument, while high resolution mass spectrometry (HRMS) was obtained from ESI-TOF and FTMS-ESI. Fluorescence spectra were performed on the Varioskan LUX Multimode microplate reader or on a VARIAN CARY Eclipse fluorescence spectrophotometer, and absorption spectra were measured on a PerkinElmer Lambda 35 UV−vis spectrophotometer. The pH values of sample solutions were measured with a precise pHmeter pHS-3C. Fluorescence quantum yield was achieved from a C11347-11 Absolute PL Quantum Yield Spectrometer. MTT assays were conducted on the Varioskan LUX Multimode Microplate Reader. Flow cytometry analyses were performed on a Beckman Coulter CytoFLEX LX apparatus. The instrument used for imaging living cells and D. magna was an Olympus FV 1000 confocal microscope purchased from Olympus. Determination of the Detection Limit. According to the fluorescence titration data, a linear relationship between the logarithm of fluorescence ratio (I540 nm/I475 nm) and superoxide anion concentrations was observed; the detection limit was calculated with the following equation: Detection limit = 3σ/k, where σ is the standard deviation of log(ratio) for blank measurements (n = 13), k is the slop between the log(ratio) versus the concentrations of superoxide anion. Approaches to Crucial Intermediates and NA-T. The synthesis route of NA-T is illustrated in Scheme 1, and the details of experiments are described as follows. Intermediates 1 and 2 were first synthesized according to previous literature.35,36 Synthesis of Compound 3. Compound 2 (239 mg, 1 mmol) and (3-ammoniopropyl) triphenyl phosphonium bromide (479 mg, 1 mmol) were dissolved in MeOH, and 3 mL of triethylamine was B
DOI: 10.1021/acssensors.8b00082 ACS Sens. XXXX, XXX, XXX−XXX
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Figure 1. (A) Fluorescence response of NA-T (5 μM) toward different levels of O2•− generated by various concentrations of O2•− (from 0 to 60 μM). (B) Linear relationship between log(ratio) and concentration of O2•−. (C) Time-dependent fluorescence ratio changes of NA-T toward 60 μM of superoxide anion. (D) Fluorescence responses (I540nm/I475nm) of NA-T (5 μM) toward various biothiols (100 μM), O2•− (60 μM), and other ROS (100 μM). All data were obtained in HEPES buffer solution (20 mM, V/V, DMSO/HEPES = 1/1, pH = 7.4). Ex = 415 nm.
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incubated with PMA (1 μg/mL) for 2 h and LPS (10 μg/mL) or 2ME (10 μg/mL) for another 2 h, followed by addition of NA-T (2 μM). After 30 min of coincubation, cells were washed for twice with PBS buffer before imaging to reduce background interference. NA-T was excited at 405 nm and the correspondent emissions were collected at blue channel (455−495 nm) and green channel (520−560 nm). To investigate the subcellular distribution of NA-T, cells were first incubated with NA-T for 30 min, and further stained with MitoTracker Deep Red FM (100 nM) for 10 min, NA-T and the commercial dyes were excited at 405 and 635 nm respectively with the separated emissions collected at 455−495 nm (blue channel) and 645−685 nm (red channel). Flow Cytometry Analysis. HepG2 cells collected in logarithmic growth phase were incubated in 6-well plates at a density of 105 cells/ mL and cultured to about 80% confluence in DMEM medium at 37 °C under 5% CO2. Cells were washed with PBS buffer twice and treated as follows: (1) Incubated with PBS; (2) incubated with only NA-T (2 μM, 30 min); (3) incubated with PMA (1 μg/mL, 2 h), LPS (10 μg/mL, 2 h) and NA-T (2 μM, 30 min); (4) incubated with PMA (1 μg/mL, 2 h), 2-ME (10 μg/mL, 2h), and NA-T (2 μM, 30 min); (5) incubated with PMA (1 μg/mL, 2 h), 2-ME (10 μg/mL, 2h), Tiron solution (100 μM, 30 min) and NA-T (2 μM, 30 min). After completing the incubation, cells were collected by tyrosination, followed by repeated centrifugation (1000 r/min, 5 min) and dispersion with PBS solution. Cells were finally resuspended in 500 μL of PBS and analyzed using a flow cytometer (BEKMAN COULTER CytoFLEX LX). In Vivo Fluorescence Imaging. D. magna (age < 72 h) were cultured in clean nonchlorinated tap water for overnight before use, and were incubated with PMA (1 μg/mL) for 4 h, LPS (10 μg/mL) for 4 h and finally stained with NA-T (2 μM) for 2 h in Milli-Q water at room temperature. After updating the culture medium twice, the D. magna were loaded on cell dishes with very little water for fluorescence imaging. NA-T was excited at 405 nm, and the correspondent emissions were collected at blue channel (455−495 nm) and green channel (520−560 nm).
RESULTS AND DISCUSSION Design and Synthesis of NA-T. Inspired by the large negative potential of the mitochondrial membrane (ΔΨ = −150 to −180 mV),37 delocalized lipophilic cation, TPP, was induced to target the mitochondria for in situ sensing of superoxide anion. 1,8-Naphthalimide with easily regulated intramolecular charge transfer (ICT) was chosen as the fluorophore core to obtain the ratiometric response model. An electron-withdrawing carbamate group together with a cresol-like structure was introduced to weaken the ICT effect, so as to switch the fluorescence to blue. Diphenylphosphinate at the phenolic end worked as the reaction site; once triggered by superoxide anion, a self-immolative process occurred, resulting in the cleavage of carbamate and the release of the amino group. Enhanced ICT effect then triggered the green fluorescent signal and ratiometric changes were thereby induced. NA-T was synthesized according to the routes described in Scheme 1, the target compound and the intermediates were well characterized by NMR, ESI-MS, and HRMS. Spectral Properties and Titration Experiments. Absorption spectra and fluorescence spectra of NA-T in different solvents were first obtained to evaluate the solvation effect generally suffered by an ICT-based structure. As shown in Figure S1 and Table S1, the maximum absorption wavelengths remain unchanged and the fluorescence spectra exhibit small red shifts with the increment of solvent polarities, illustrating that the solvation effect has less impact on NA-T. Quantum yields of NA-T in different solvents were also measured and are listed in Table S1. With the basic information in hand, the spectroscopic properties of NA-T toward superoxide anion were investigated in 20 mM HEPES-DMSO (1:1, pH 7.4). Upon the addition of superoxide anion, the maximum C
DOI: 10.1021/acssensors.8b00082 ACS Sens. XXXX, XXX, XXX−XXX
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ACS Sensors absorption wavelength shifted from 375 nm (7658 M−1 cm−1) to 431 nm (5972 M−1 cm−1) accompanied by the red shift of fluorescent spectra from 475 to 540 nm excited by 415 nm (Figure S3). Different concentrations of superoxide anion, generated by enzymatic reaction of xanthine (XA) and xanthine oxidase (XOD) at 37 °C, were added to the test system containing 5 μM NA-T to construct the relationships with the fluorescence responses. As depicted in Figure 1A, the fluorescent intensity at 475 nm gradually decreased while the fluorescent intensity at 540 nm underwent corresponding enhancements with increasing superoxide anion concentrations. A good linear relationship can be found between the logarithm of fluorescence ratio (I540 nm/I475 nm) and superoxide anion concentrations as following: log(ratio) = 0.02057 × C(O2•−) − 0.4810, with a high correlation coefficient r = 0.9988 (Figure 1B). According to the well-known equation LOD = 3σ/k, the detection limit was determined to be 0.370 μM. In addition, the time-course spectral changes toward superoxide anion were investigated by monitoring the fluorescence intensities at 475 and 540 nm. As summarized in Figure 1C, with the addition of 60 μM superoxide anion, the fluorescence ratio (I540nm/I475nm) of NA-T changed rapidly and reached saturation within about 132 s, demonstrating that NA-T responses quickly to superoxide anion and may be used as an indicator to monitor it in real-time. Selectivity Estimation and pH Influence. Outstanding selectivity is necessary for a good fluorescent sensor, so the specific sensing performance was carefully examined by comparing the fluorescence responses of NA-T to superoxide anion and other various analytes, including H2O2, HOCl, •OH, TBHP, TBO•, 1O2, NO•, ONOO−, biothiols (Cys, Hcy, and GSH), as well as several ions (Na+, K+, Ca2+, Mg2+, Fe2+, Zn2+, HSO3−, SO32−). Figures 1D and S4 display the fluorescence ratios of NA-T under different treatments: negligible ratio changes of NA-T could be observed in the presence of the possible interfering substances. However, the ratio aroused by superoxide anion was 18-fold, suggesting that NA-T performed superior selectivity toward superoxide anion and had potential capacity for superoxide anion detection in complex biological environments with a ratiometric manner. Considering the mitochondrial pH maintaining at 7.9−8.0,38,39 a pH titration was then implemented to investigate the suitable pH range for superoxide anion sensing via NA-T. Clearly, the fluorescence ratios (I540nm/I475nm) kept basically constant before and after the addition of superoxide anion from pH 6 to 9 (Figure S5), implying that NA-T was appropriate for the measurement of superoxide anion in mitochondria. Study on the Sensing Mechanism. Taking consideration from previous literature,29,36 the proposed reaction mechanism was inferred as Scheme 2. Once attacked by superoxide anion, NA-T underwent deprotection of the diphenylphosphinate group first and a following self-immolation procedure to finally expose the amidogen. In order to confirm the sensing mechanism, NA-T (5 μM) in 20 mM HEPES-DMSO solution was treated with 6 and 20 equiv of superoxide anion, and the corresponding product was analyzed by HPLC by monitoring the absorbance at 370 and 435 nm (Figure S6). A new absorption peak that appeared at 8.211 min indicated that the reaction product matched well with compound 3 at retention time. After identifying the main reaction product with LCHRMS, the ion peak located at m/z 515.1877 was further deduced as compound 3.
Scheme 2. Proposed Sensing Mechanism of NA-T toward O2•−
Endogenous Superoxide Anion Detection. Having figured out the capacity of NA-T in solution, we took a further step to explore its applications in living cells. MTT assays were first conducted to look into the potential cytotoxicity of NA-T. A549 cells, HepG2 cells and RAW264.7 cells were incubated with NA-T (2.5 μM) for 12 and 24 h, more than 80% of cell viability indicated that the probe can be used safely in the course of living cell imaging (Figure S7). We next investigated the subcellular localization of NA-T by employing the colocalization assay with the commercial mitochondrial targetable dye (MitoTracker Deep Red FM) in HepG2 cells and MCF-7 cells. As shown in Figures 2 and S8, the blue channel (455−495 nm, λex = 405 nm) belongs to the NA-T and the red channel (645−685 nm, λex = 635 nm) exhibits the
Figure 2. Colocalization images of NA-T with MitoTracker Deep Red FM in HepG2 cells. Cells were treated with NA-T (2 μM) for 30 min and subsequently with MitoTracker Deep Red FM (100 nM) for 10 min. Fluorescence of NA-T and MitoTracker Deep Red FM were, respectively, collected from 455 to 495 nm (λex = 405 nm, A) and from 645 to 685 nm, (λex = 635 nm, B). (C) Bright field and (D) overlay image of (A)−(C). (E) Intensity correlation plot of stain NA-T and MitoTracker Deep Red FM. (F) Intensity profile of ROI across a HepG2 cell. Scale bar: 20 μm. D
DOI: 10.1021/acssensors.8b00082 ACS Sens. XXXX, XXX, XXX−XXX
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superoxide anion level similarly triggered on the increases of green fluorescence intensity and ratiometric fluorescent signal. However, if PMA/2-ME loaded cells were further incubated with Tiron (an O2•− scavenger)50 for 30 min before being stained with NA-T, almost no changes can be found both in blue and green fluorescent channels. Similar responses were also observed when NA-T was incubated with living HepG2 cells treated with PMA/2-ME or PMA/LPS (Figure S9). Subsequently, an analysis to further verify the above results was conducted on HepG2 cells by flow cytometry. The total green fluorescence (525/40 nm) of 10 000 cells for each sample was measured and counted as shown in Figure 4: upon being
MitoTracker Deep Red FM imaging of mitochondria. NA-T could present the filamentous morphology of mitochondria as good as the commercial one, and the intensity of correlation plots revealed a high Pearson’s coefficient (Figure 2E, Rr = 0. 96), confirming that NA-T was an excellent mitochondriontargeted probe. Since superoxide anion is unstable, its in vivo concentration balances dynamically at the lower nanomolar level. However, an outburst to higher micromolar range of superoxide anion usually appears when some “stresses” exist, such as apoptosis and inflammation. Here, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) were used to induce inflammation in RAW264.7 cells.26,40−44 As shown in Figure 3,
Figure 4. Flow cytometry analysis of endogenous O2•− in HepG2 cells exposed to different stimulation. (A) Incubated with only NA-T (2 μM, 30 min). (B) Incubated with PMA (1 μg/mL, 2 h), LPS (10 μg/ mL, 2 h), and NA-T (2 μM, 30 min). (C) Incubated with PMA (1 μg/ mL, 2 h), 2-ME (10 μg/mL, 2 h), and NA-T (2 μM, 30 min). (D) Incubated with PMA (1 μg/mL, 2 h), 2-ME (10 μg/mL, 2 h), Tiron solution (100 μM, 30 min), and NA-T (2 μM, 30 min).
Figure 3. Confocal fluorescence imaging without (top) and with endogenous O2•− using probe NA-T (2 μM) for 30 min at 37 °C. (A1−A3) Cells were loaded with only NA-T. (B1−B3) Cells were treated with PMA/LPS and stained with NA-T. (C1−C3) Cells were treated with PMA/2-ME and stained with NA-T. (D1−D3) Cells were first treated with PMA/2-ME and then Tiron before being stained with NA-T. (E) Mean fluorescence ratios of RAW264.7 cells with different treatments were quantified. Fluorescence was collected in blue channel (455−495 nm) and green channel (520−560 nm) respectively; ratio images were generated from green/red channel. λex = 405 nm. Scale bar: 20 μm.
treated with PMA/2-ME and PMA/LPS, the corresponding fluorescence enhanced remarkably (from 19.3% to 73.0% and 42.5%, respectively). However, when PMA/2-ME loaded cells were further treated with Tiron, only a slight fluorescence enhancement could be observed (from 19.3% to 23.5%). All the results demonstrated conclusively that NA-T could be used for the in suit monitoring of endogenous superoxide anion changes in living cells. Encouraged by the in vitro behavior of NA-T, its feasibility for in vivo imaging of superoxide anion was evaluated in live D. magna. D. magna is an arthropod with a short life cycle and fast breeding. It is very sensitive to chemical substances in the water environment and has long been widely used as standard test organisms in studying ecological evolution as well as ecotoxicology.51,52 Here, D. magna was randomly divided into two groups: one was for control and incubated only with NAT; the other one was first treated with PMA and LPS to construct the inflammation model and was subsequently incubated with NA-T at 25 °C. As shown in Figure 5, internalized fluorescence signals were gathered mainly from the regions of guts of D. magna. D. magna from experimental group exhibited lower blue fluorescence intensity but higher green fluorescence intensity compared with the control, demonstrat-
RAW264.7 macrophages loaded with only NA-T presented observable blue fluorescence but very weak green fluorescence. In contrast, a slightly weaker blue fluorescence and much stronger green fluorescence were observed within cells treated with PMA and LPS, and the corresponding ratiometric fluorescent signals also exhibited a distinct enhancement, indicating that NA-T can image endogenous superoxide anion in inflammation cells with a ratiometric response model. Superoxide dismutase (SOD) is widely distributed as the natural killer of oxygen free radicals in the biological community; it accelerates the conversion of superoxide anion to hydrogen peroxide, which is further converted to water by catalase and glutathione peroxidase.45−48 Accordingly, 2-ME was used to inhibit SOD for superoxide anion accumulation in PMA-stimulated living RAW264.7 cells,49 and the elevated E
DOI: 10.1021/acssensors.8b00082 ACS Sens. XXXX, XXX, XXX−XXX
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ACS Sensors Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by National Science Foundation of China (Projects 21421005, 21422601, 21576037, 21576039, and U1608222).
Figure 5. In vivo imaging of O2•− changes stimulated by PMA/LPS in living D. magna. (A−D) D. magna were incubated with NA-T (2 μM) for 2 h at 25 °C. (E−H) D. magna were treated with PMA (1 μg/mL, 4 h) and LPS (10 μg/mL, 4 h) before being incubated with NA-T (2 μM) for 2 h. Scale bar: 300 μm.
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ing that NA-T can be potentially used for in vivo superoxide anion imaging in living inflammation D. magna.
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CONCLUSION In summary, an efficient ratiometric fluorescent probe (NA-T) for bioimaing of superoxide anion has been successfully designed and synthesized. The ICT-based sensor was developed via the nonredox strategies, little affected by solvation effect. Upon addition of superoxide anion, the original blue-emitting probe (NA-T) decomposed within 132 s to result in a green-emitting product (compound 3). NA-T displayed 18-fold enhancement of the fluorescence emission ratio with high sensitivity and high selectivity over other latent interfering oxidative species, biothiols, and ions. Confocal fluorescence imaging experiments on several types of living cells demonstrated that NA-T was mitochondria-targeted and could be used for the in suit monitoring of endogenous superoxide anion under different treatments. In addition, NA-T was successfully applied to study the superoxide anion changes in inflammatory D. magna stimulated by PMA/LPS. All results suggested NA-T could potentially serve as an effective tool for investigation of superoxide anion levels in complex biosystems, and further for the diagnosis and research of corresponding diseases.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.8b00082. General information, additional spectroscopic investigations, HPLC and LC-HRMS results of NA-T sensing, cell culture and cytotoxicity assay, mitochondrial colocalization assay in MCF-7 cells, imaging of endogenous superoxide anion in HepG2 cells, and NMR and MS data for compounds (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jiangli Fan: 0000-0003-4962-5186 Jianjun Du: 0000-0001-7777-079X Xiaojun Peng: 0000-0002-8806-322X F
DOI: 10.1021/acssensors.8b00082 ACS Sens. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acssensors.8b00082 ACS Sens. XXXX, XXX, XXX−XXX
