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A BODIPY-based ratiometric probe for sensing and imaging hydrogen polysulfides in living cells
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117295
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A BODIPY-based ratiometric probe for sensing and imaging hydrogen polysulfides in living cells Changli Zhang a, Qian Sun d, Liming Zhao c, Shuwen Gong c, Zhipeng Liu b,d,⁎ a
Key Laboratory of Advanced Functional Materials of Nanjing, School of Environmental Science, Nanjing Xiaozhuang University, Nanjing 211171, China College of Materials Science and Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, China d Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China b c
a r t i c l e
i n f o
Article history: Received 24 April 2019 Received in revised form 11 June 2019 Accepted 18 June 2019 Available online 19 June 2019 Keywords: BODIPY Hydrogen polysulfides Fluorescent sensing Ratiometric probe Cell imaging
a b s t r a c t Hydrogen polysulfides (H2Sn, n N 1) have attracted increasing attention in biological systems due to its redox signaling effect. To illustrate the process of the physiological and pathological roles played by H2Sn, accurate detection is highly desired. In this work, we report a BODIPY-based fluorescent probe (BDP-PHS) for ratiometric H2Sn sensing. BDP-PHS shows higher sensitivity and selectivity ratiometric response toward H2Sn than various biological related species. Moreover, BDP-PHS has been successfully applied in imaging of H2Sn in living cells. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Hydrogen polysulfides (H2Sn, n N 1) are hydrogen sulfide (H2S) derived signaling molecules that are produced primarily by reacting H2S with reactive oxygen species/reactive nitrogen species (RNS) [1–4]. H2S has been recognized as a gaseous molecule and known as a critical cell signaling molecule, much like nitric oxide. The study in the past several years indicates that H2S can mediate many physiological and/or pathological processes, especially in cardiovascular systems [5–8]. Recently, polysulfides have attracted a lot of interest in comparison with H2S, as they are believed to be the real regulators in signal transmission [9,10]. Research results show that polysulfides have the properties of antioxidant, cytoprotection, and redox signaling in tissues and organs [11–13]. Meanwhile, polysulfides coexist with H2S and they work together to regulate sulfur redox balance in living systems [14,15]. More evidence suggests polysulfides also possess their own enzymemediated biosynthetic routes. For instance, polysulfides could be generated not only from the cystathionine g-lyase-mediated cysteine metabolism, [16] but also from 3-mercaptopyruvate by 3-mercaptopyruvate sulfur transferase [17]. Besides, polysulfides are more effective than H2S in activating TRPA1 channels and inducing Ca2+ influx in astrocytes by S-sulfhydration [4]. Moreover, polysulfides are gaining attraction in ⁎ Corresponding author at: College of Materials Science and Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China. E-mail address: [email protected] (Z. Liu).
https://doi.org/10.1016/j.saa.2019.117295 1386-1425/© 2019 Elsevier B.V. All rights reserved.
chemical biology research due to their immense physiological and pathophysiological functions in the human body [18,19]. Although the important progress made in recent years in studying polysulfides, there are still some problems to be solved such as their distribution in living organisms, degradation pathways and regulation mechanisms [20]. Thus, the development of novel techniques to monitor polysulfides in living systems is vital in order to understand their contribution to physiology and pathology. Some traditional techniques including colorimetry, gas chromatography, and metal-induced sulfide precipitation are available for the detection of polysulfides [21,22]. However, these technologies often require post-mortem processing and destruction of tissues or cells. Compared with these detection technologies, fluorescence assays may be the most feasible technique for detection of polysulfides in living systems, due to their numerous advantages, including real-time spatial imaging, high sensitivity, and minimal harm to biological samples [23,24]. To date, various “turn-on” type fluorescent probes for polysulfides detection have been elegantly developed and applied for imaging of polysulfides in living cells and in vivo [25–36]. However, this “turnon” polysulfides imaging still suffer from the interference induced by altered experimental conditions, such as the probe distribution, autofluorescence, etc. In contrast, self-calibration of the aforementioned factors can be achieved by measuring the ratio changes of the two excitation/ emission peaks, enabling more accurate polysulfides analysis using ratiometric fluorescent probes [37]. For example, Liu's group reported probe NRT-HP, based on a two-photon fluorophore, 1,8-naphthalimide,
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Fig. 1. (a) Time-dependent UV–vis absorption spectra of BDP-PHS (5 μM) reacting with Na2S2 (50 μM) in DMSO/phosphate buffer (3:97, v/v, 10 mM, pH 7.4, 0.4% Tween 80). Inset, timedependent absorbance at 563 nm and 576 nm. (b) Emission spectra of BDP-PHS (5 μM) in the presence of increasing concentrations of Na2S2 in buffer. Inset, the titration profile based on the emission ratio at 618 and 574 nm, F618/F574. (λex = 525 nm, slit width = dex = dem = 0.8 nm, PMT voltage = 950 V).
displayed both one- and two-photon ratiometric fluorescence changes toward polysulfides via polysulfides-mediated benzodithiolone formation [38]. Zhang's group reported a FRET-based ratiometric twophoton fluorescent probe TPR-S, producing a large emission shift in the presence of polysulfides [39]. However, few ratiometric polysulfides probes have been reported, [38–43] which make the development of new ratiometric polysulfides probes is highly desired. In this paper, a new BODIPY-based ratiometric fluorescent polysulfides probe, BDP-PHS, was designed and synthesized. This probe shows highly sensitive and selective ratiometric response toward polysulfides. The intracellular polysulfides ratiometric imaging ability of the probe has been demonstrated.
spectrophotometer. Fluorescence measurements were performed on a HORIBA Scientific spectrofluorimeter. The probe was dissolved in dimethyl sulfoxide (DMSO) to make 5 mM stock solutions. Na2Sn and other reactive bio-relevant species were prepared according to previous literature. [25,44] All photophysical spectra were carried out at ambient temperature in DMSO/phosphate buffer solution (3:97, v/v, 10 mM, pH 7.4, 0.4% Tween 80), the concentration of the BDP-PHS is 5 μM. The mixture was allowed standing in the dark for desired time and the fluorescence spectra were then recorded under excitation at 525 nm except otherwise indicated. Quantum yields were determined using Rhodamine 6G (Фstandard = 0.95 in ethanol) as a standard according to a published method. [45]
2. Experimental section
2.2. Synthesis of BDP-PHS
2.1. General procedures
To a solution of BDP1 [46] (370 mg, 0.86 mmol) and Et3N (174 mg, 1.72 mmol) in 10 mL of anhydrous dichloromethane (DCM), 2-fluoro-5nitrobenzoyl chloride (260 mg, 1.29 mmol, dissolved in 5 mL of DCM) was added dropwise at 0 °C. After stirring 30 min at 0 °C, the mixture was warmed to room temperature (r. t.) and further reacted for 8 h. The solution was diluted with DCM (10 mL), washed with H2O (30 mL × 3) and dried over anhydrous MgSO4. The solvent was dried in vacuo and the crude product was purified by column chromatography on silica gel (eluted with dichloromethane: petroleum ether, 4:3, v/v) to afford BDP-PHS as purple solid (110 mg, 29%). 1H NMR (500 MHz, CDCl3) δ 9.02 (dd, J = 3.0, 2.5 Hz, 1H), 8.52–8.47 (m, 1H), 7.70–7.65 (m, 3H), 7.50 (d, J = 6.0 Hz, 3H), 7.41 (t, J = 9.0 Hz, 1H), 7.34–7.30 (m, 2H),
All reagents were purchased from commercial suppliers without further purification. All reactions were carried out under nitrogen atmosphere without otherwise indicated. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were obtained on a Bruker AV-500 spectrometer. All NMR spectra were calibrated using residual solvents as internal references (for CDCl3: 1H NMR = 7.26, 13C NMR = 77.16). All chemical shifts were reported in parts per million (ppm) and coupling constants (J) in Hertz (Hz). High resolution mass spectra (HRMS) were recorded on a Finnigan LCQ mass spectrometer, a Shimadzu LC-IT-TOF spectrometer. Absorption spectra were acquired using a Shimadzu UV-2500
Fig. 2. (a) Time-dependent emission fluorescence spectra of BDP-PHS (5 μM) with 50 μM of Na2S2 (λex = 525 nm, slit width = dex = dem = 0.8 nm, PMT voltage = 950 V) in DMSO/ phosphate buffer (3:97, v/v, 10 mM, pH 7.4, 0.4% Tween 80) at room temperature. b) Plot of F618/F574 as a function of time upon addition of various equivalents of Na2S2.
C. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117295
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Fig. 3. Emission spectra (a) and fluorescence ratio (b) changes of BDP-PHS (5 μM) with 20 μM of Na2S2 and other analytes (•NO, 1O2, NaClO, H2O2, ROO•, •OH, ONOO−, GSH, Cys, Hcy, SO2− 3 , 2+ 2− 2− Na+, K+, Ca2+, Mg2+, Fe2+, SO2− , S and HS−) (λex = 525 nm, slit width = dex = dem = 0.8 nm, PMT voltage = 950 V) in DMSO/phosphate buffer (3:97, v/v, 10 mM, pH 7.4, 4 , CO3 , Zn 0.4% Tween 80).
7.28 (s, 1H), 6.61 (s, 1H), 6.03 (s, 1H), 2.61 (s, 3H), 1.53 (s, 2H), 1.42 (d, J = 17.5 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 166.5, 164.3, 160.4, 156.2, 151.9, 150.4, 144.1, 143.5, 142.3, 140.8, 135.1, 135.0, 134.2, 132.8, 132.2, 130.2, 130.1, 129.1, 129.0, 128.5, 128.3, 121.7, 119.9, 119.4, 118.8, 118.6, 117.5, 14.7, 14.5, 14.4. HRMS (m/z): C33H25BF2N3O4, Calculated: [M-F]+ = 576.1906, found: [M-F]+ = 576.1923. 2.3. MTT assay The cellular toxicity of BDP-PHS to HeLa cells was tested by standard MTT assays. HeLa cells were incubated in 96-well plates and pretreated with various concentrations of BDP-PHS (0, 0.01, 0.1, 0.5, 5, 10 and 20 μM) containing 2‰ DMSO in 100 μL culture medium (DMEM + 10% FBS). The control group was added the same volume of culture medium contained 2‰DMSO. All groups were incubated in incubator (5% CO2, 37 °C) for 24 h. Next, MTT solution (Final concentration, 0.5 mg/mL) was added into each well, followed by incubation for another 4 h under the same conditions. Then removed the supernatant and added 150 μL DMSO. After shaking for 10 min, the absorbance at 490 nm was measured by microplate reader (Synergy 2, BioTek Instruments Inc.). Cell viability was calculated by A/A0 × 100% (A and A0 are the absorbance of experimental group and control group, respectively).
polysulfides receptor in many reported polysulfides probes. [25,26] After reacting with polysulfides, 2-fluoro-5-nitrobenzoate will be removed via nucleophilic aromatic substitution reaction (SNAr), resulting in the recovery of the intermolecular charge transfer (ICT) from styryl group to the boron-difluoride center of BODIPY of BDP1. Therefore, ratiometric response through an obvious red shift in emission wavelengths can be expected. BDP-PHS was facile synthesized by reacting of 2-fluoro-5-nitrobenzoyl chloride with BDP1, and it was fully characterized by 1H, 13C NMR, and HRMS.
3.2. Photophysical properties of BDP-PHS Photophysical measurements of BDP-PHS were carried out in phosphate buffer solutions (PBS, 10 mM, pH 7.4, containing 3% DMSO and 0.4% Tween 80) (Fig. 1a). In these experiments, freshly prepared solutions of Na2S2 were used as the equivalent of polysulfides. BDP-PHS exhibits a main absorption band at 563 nm (ε = 1.08 × 105 M−1 cm−1) and a shoulder peak at ∼529 nm. After adding 10 equivalents of Na2S2, the absorption band at 563 nm of BDP-PHS decreased gradually and reached equilibrium within 30 min, accompanied by the concomitant increase of a new band at 576 nm (ε = 9.78 × 104 M−1 cm−2). The
2.4. Cell culture methods and confocal imaging HeLa cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin (100 units/ mL), streptomycin (100 mg/mL) and 5% CO2 at 37 °C. After removing the incubation media and rinsing with 1× PBS for three times, the HeLa cells were incubated with BDP-PHS (20 μM) for 20 min at 25 °C. Then the cells were washed three times with 1× PBS and imaged with Zeiss LSM-710 microscope equipped with a 63× oil-immersion objective. Background signals of all images were verified to be nearly zero by imaging the same cells treated with PBS solution containing 2‰ DMSO. The excitation wavelength of BDP-PHS used in the experiment was 488 nm, whereas the emissions were collected in the ranges of 550–580 nm (green) and 600–700 nm (red), respectively. 3. Results and discussion 3.1. Synthesis of BDP-PHS In BDP-PHS, BODIPY was selected as fluorophore due to its advantages of high molar extinction coefficient, high quantum yield, and excellent light stability [47]. 2-Fluoro-5-nitrobenzoic ester was integrated into the BODIPY core because of its high reliability as
Fig. 4. HPLC traces of the reaction of BDP-PHS with Na2S2. (a) BDP-PHS (100 μM); (b and c) BDP-PHS (100 μM) treated with Na2S2 (50 μM and 100 μM, respectively) for 2 min; (d) BDP1 (100 μM). HPLC conditions: 0.1 mL/min flow rate, 10% water + 90% methanol for 20 min. All samples were diluted to 100 μM before the test. Wavelength for detection: 563 nm.
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Scheme 1. Synthesis of BDP-PHS and proposed response mechanism of BDP-PHS toward polysulfides.
Na2S2-induced absorption red-shift leads to a clear isobestic point at 570 nm, implying the conversion of BDP-PHS to BDP1. The shoulder absorption at 529 nm also shows a minor red-shift to 537 nm. Free BDP-PHS exhibits one strong emission band centered at 574 nm and one relatively weak emission band centered at 618 nm, with λex of 525 nm (Fig. S1). After addition of Na2S2, the emission at 574 nm decreased distinctly, accompanied by gradually emission intensity increment of λem at 593 and 650 nm, which make λem at 618 nm cannot be distinguished. Meanwhile, emission color of the solution changes from orange to yellow. This red-shift should be attributed to the recovery of ICT effect from the hydroxyl group to BODIPY. The fluorometric titration of BDP-PHS with various concentrations of Na2S2 ([Na2S2]) was carried out to evaluate the sensing behavior of BDP-PHS. With the addition of [Na2S2] from 0 to 50 μM, the fluorescence intensity ratio value between 618 and 574 nm (F618/F574) gradually increased from 0.2 to 1.0 (Fig. 1b). Based on the emission intensity at 574 nm, the limit of detection of BDP-PHS for H2S2 is calculated to be 57 nM according to the equation of LOD = 3σ/k (n = 3), where σ is the standard deviation of a blank measurement, and k is the slope of the fitting straight line (Fig. S2 and Table S1).
3.3. Dynamic response of BDP-PHS to H2Sn To investigate the sensitivity of BDP-PHS, the reaction kinetics of BDP-PHS with various concentrations of Na2S2 were measured (Fig. 2). After treating BDP-PHS with low concentrations of Na2S2 (5, 10, 20, 30, 40 μM), the ratio of F618/F574 slowly increased and reached a balance in 20 min. However, after adding 50 μM of Na2S2 to the solution of BDP-PHS, the fluorescence intensity ratio rapidly increased with time in the first 10 min and reached the maximum after 30 min, accompanied with the fluorescent quantum yield (Фf) increment from 4.3% to 13.6% (Fig. 2b). The pseudo-first-order rate constant for the reaction of BDP-PHS with Na2S2 can be observed to be 0.071 min−1 (Fig. S3). This result suggests that BDP-PHS can be applied for rapid and sensitive sensing of Na2S2 in an aqueous solution at physiological pH.
3.4. Selectivity and pH effect of BDP-PHS There are many reactive oxygen species in vivo, which may affect the detection of polysulfides by BDP-PHS. To investigate the selectivity,
Fig. 5. Fluorescence images of probe BDP-PHS in HeLa cells by confocal fluorescence images. (a, b, c) Cells were treated with probe BDP-PHS (20 μM, 20 min), then imaged; (d, e, f) cells were pretreated with probe BDP-PHS (20 μM, 20 min), subsequently incubated with Na2S2 (200 μM, 30 min) then imaged. The fluorescence images were captured from the green channel of 550–580 nm and red channel of 600–700 nm with an excitation at 488 nm. Third row: Fred/Fgreen ratiometric images. (g) Bright-field transmission images; (h) average fluorescence intensity ratios (Fred/Fgreen) in c and f. Data are mean ± S.E.M., n = 3. Data are expressed as mean ± SD of three experiments. Scale bar: 20 μm, respectively. Ratio 58 and 89, for c and f, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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BDP-PHS was treated with a series of RSS (GSH, Cys, Hcy, S2−, HS− and − 1 HSO− 3 ), RNS (•NO and ONOO ), ROS (H2O2, NaClO, •OH and O2) and + + 2+ 2+ 2+ 2+ 2− 2− other ions (Na , K , Ca , Mg , Fe , Zn , SO3 , SO4 and CO2− 3 ). The results shown in Fig. 3 indicated that the addition of Na2S2 into BDP-PHS solution induced an obvious red-shift (Fig. 3a) and N8-fold increment in the fluorescence ratio of F618/F574 (Fig. 3b). However, no significant fluorescence ratio (F618/F574) increase was observed for any other tested analytes. The pH effects of the response of BDP-PHS to Na2S2 were also investigated, and the tested range of pH is from 4 to 10 (Fig. S4). The fluorescence intensity ratio (F618/F574) of BDP-PHS was quite stable within a wide pH range from 4 to 10, indicating excellent stability of BDP-PHS at physiological pH conditions. In the presence of Na2S2, ratio enhancement was observed in the pH range of 7–10. The low ratio increment could be due to the conversion of S2− n to H2Sn at pH 4–6. The pH effect investigation demonstrates that BDP-PHS can be applied into physiological environment over a wide pH range. All these results clearly indicate that BDP-PHS possesses high sensing ability toward polysulfides without interference from other biologically related species and pH effects, and it functions well under physiological conditions.
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specific ratiometric response toward polysulfides over other biologically related species such as RNS, ROS, RSS and other ions. The observed pseudofirst-order rate constant for the reaction of BDP-PHS with polysulfides was calculated to be 0.071 min−1, and the detection limit of BDP-PHS was determined to be 57 nM. Moreover, the intracellular polysulfides imaging ability of BDP-PHS was demonstrated in HeLa cells via confocal fluorescence microscopy imaging. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (81672508, 61505076 and 21501085), Natural Science Foundation of Jiangsu Province (BK20140951), Key University Science Research Project of Jiangsu Province (17KJA150004) and Scientific Research Projects of Nanjing Xiaozhuang College (2018NXY49). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117295.
3.5. Response mechanism The selective response mechanism of BDP-PHS to Na2S2 was further studied by high performance liquid chromatography (HPLC). After incubating BDP-PHS with Na2S2 for 2 min, an aliquot of the reaction mixture was subjected to HPLC at ambient temperature. The HPLC chromatogram of free BDP-PHS (100 μM) in MeOH showed a single peak with a retention time of 8.06 min (Fig. 4a). After adding Na2S2 (50 μM) into BDP-PHS solution for 2 min, the signal at 8.06 min decreased, whereas a new peak with a retention time of 4.33 min was observed (Fig. 4b), indicating the formation of BDP1. The addition of 100 μM Na2S2 to the solution of BDP-PHS induced the disappearance of the peak at 8.06 min, suggesting that BDP-PHS was almost completely consumed (Fig. 4c). The change in chromatographic peaks manifested that BDP1 was generated from the reaction between BDP-PHS and Na2S2, which was in accordance with the mechanism discussed above (Scheme 1).
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3.6. Imaging of living cells The cellular toxicity of BDP-PHS to HeLa cells was tested by standard MTT assays. After incubation with BDP-PHS for 24 h, no significant change of the viability of HeLa cells was observed. When the concentration of BDP-PHS was up to 20 μM, the cell viability was still near 100% (Fig. S5). These results demonstrate that BDP-PHS exhibits low cytotoxicity and good biocompatibility. Next the capability of BDP-PHS for fluorescent ratiometric imaging of polysulfides in living cells was investigated. As shown in Fig. 5, after incubating HeLa cells with BDP-PHS (20 μM) for 20 min, fluorescence could be observed in both green (550–580 nm, Fig. 5a) and red (600–700 nm, Fig. 5b) channels, which could be ascribed to the unreacted probe, indicating that BDP-PHS can penetrate the cell membrane and distribute in the cytoplasm. The average fluorescence intensity ratio (Fred/Fgreen) in ratiometric pseudo-colored image (Fig. 5c) was determined to be 58.1 ± 3.6 (Fig. 5h). When HeLa cells were treated with Na2S2 (200 μM) for 30 min and then incubated with BDP-PHS, the fluorescence intensity in green channel decreased (Fig. 5d), accompanied with fluorescence enhancement in red channel (Fig. 5e), resulting in distinct increase in the ratio (89.6 ± 6.8, Fig. 5f). These results demonstrate that BDP-PHS possesses good membrane permeability and is suitable for imaging of polysulfides in living cells. 4. Conclusion In summary, a new ratiometric fluorescent polysulfides probe, BDPPHS, was designed and synthesized. BDP-PHS showed rapid and
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A BODIPY-based ratiometric probe for sensing and imaging hydrogen polysulfides in living cells Changli Zhang a, Qian Sun d, Liming Zhao c, Shuwen Gong c, Zhipeng Liu b,d,⁎ a
Key Laboratory of Advanced Functional Materials of Nanjing, School of Environmental Science, Nanjing Xiaozhuang University, Nanjing 211171, China College of Materials Science and Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, China d Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China b c
a r t i c l e
i n f o
Article history: Received 24 April 2019 Received in revised form 11 June 2019 Accepted 18 June 2019 Available online 19 June 2019 Keywords: BODIPY Hydrogen polysulfides Fluorescent sensing Ratiometric probe Cell imaging
a b s t r a c t Hydrogen polysulfides (H2Sn, n N 1) have attracted increasing attention in biological systems due to its redox signaling effect. To illustrate the process of the physiological and pathological roles played by H2Sn, accurate detection is highly desired. In this work, we report a BODIPY-based fluorescent probe (BDP-PHS) for ratiometric H2Sn sensing. BDP-PHS shows higher sensitivity and selectivity ratiometric response toward H2Sn than various biological related species. Moreover, BDP-PHS has been successfully applied in imaging of H2Sn in living cells. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Hydrogen polysulfides (H2Sn, n N 1) are hydrogen sulfide (H2S) derived signaling molecules that are produced primarily by reacting H2S with reactive oxygen species/reactive nitrogen species (RNS) [1–4]. H2S has been recognized as a gaseous molecule and known as a critical cell signaling molecule, much like nitric oxide. The study in the past several years indicates that H2S can mediate many physiological and/or pathological processes, especially in cardiovascular systems [5–8]. Recently, polysulfides have attracted a lot of interest in comparison with H2S, as they are believed to be the real regulators in signal transmission [9,10]. Research results show that polysulfides have the properties of antioxidant, cytoprotection, and redox signaling in tissues and organs [11–13]. Meanwhile, polysulfides coexist with H2S and they work together to regulate sulfur redox balance in living systems [14,15]. More evidence suggests polysulfides also possess their own enzymemediated biosynthetic routes. For instance, polysulfides could be generated not only from the cystathionine g-lyase-mediated cysteine metabolism, [16] but also from 3-mercaptopyruvate by 3-mercaptopyruvate sulfur transferase [17]. Besides, polysulfides are more effective than H2S in activating TRPA1 channels and inducing Ca2+ influx in astrocytes by S-sulfhydration [4]. Moreover, polysulfides are gaining attraction in ⁎ Corresponding author at: College of Materials Science and Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China. E-mail address: [email protected] (Z. Liu).
https://doi.org/10.1016/j.saa.2019.117295 1386-1425/© 2019 Elsevier B.V. All rights reserved.
chemical biology research due to their immense physiological and pathophysiological functions in the human body [18,19]. Although the important progress made in recent years in studying polysulfides, there are still some problems to be solved such as their distribution in living organisms, degradation pathways and regulation mechanisms [20]. Thus, the development of novel techniques to monitor polysulfides in living systems is vital in order to understand their contribution to physiology and pathology. Some traditional techniques including colorimetry, gas chromatography, and metal-induced sulfide precipitation are available for the detection of polysulfides [21,22]. However, these technologies often require post-mortem processing and destruction of tissues or cells. Compared with these detection technologies, fluorescence assays may be the most feasible technique for detection of polysulfides in living systems, due to their numerous advantages, including real-time spatial imaging, high sensitivity, and minimal harm to biological samples [23,24]. To date, various “turn-on” type fluorescent probes for polysulfides detection have been elegantly developed and applied for imaging of polysulfides in living cells and in vivo [25–36]. However, this “turnon” polysulfides imaging still suffer from the interference induced by altered experimental conditions, such as the probe distribution, autofluorescence, etc. In contrast, self-calibration of the aforementioned factors can be achieved by measuring the ratio changes of the two excitation/ emission peaks, enabling more accurate polysulfides analysis using ratiometric fluorescent probes [37]. For example, Liu's group reported probe NRT-HP, based on a two-photon fluorophore, 1,8-naphthalimide,
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Fig. 1. (a) Time-dependent UV–vis absorption spectra of BDP-PHS (5 μM) reacting with Na2S2 (50 μM) in DMSO/phosphate buffer (3:97, v/v, 10 mM, pH 7.4, 0.4% Tween 80). Inset, timedependent absorbance at 563 nm and 576 nm. (b) Emission spectra of BDP-PHS (5 μM) in the presence of increasing concentrations of Na2S2 in buffer. Inset, the titration profile based on the emission ratio at 618 and 574 nm, F618/F574. (λex = 525 nm, slit width = dex = dem = 0.8 nm, PMT voltage = 950 V).
displayed both one- and two-photon ratiometric fluorescence changes toward polysulfides via polysulfides-mediated benzodithiolone formation [38]. Zhang's group reported a FRET-based ratiometric twophoton fluorescent probe TPR-S, producing a large emission shift in the presence of polysulfides [39]. However, few ratiometric polysulfides probes have been reported, [38–43] which make the development of new ratiometric polysulfides probes is highly desired. In this paper, a new BODIPY-based ratiometric fluorescent polysulfides probe, BDP-PHS, was designed and synthesized. This probe shows highly sensitive and selective ratiometric response toward polysulfides. The intracellular polysulfides ratiometric imaging ability of the probe has been demonstrated.
spectrophotometer. Fluorescence measurements were performed on a HORIBA Scientific spectrofluorimeter. The probe was dissolved in dimethyl sulfoxide (DMSO) to make 5 mM stock solutions. Na2Sn and other reactive bio-relevant species were prepared according to previous literature. [25,44] All photophysical spectra were carried out at ambient temperature in DMSO/phosphate buffer solution (3:97, v/v, 10 mM, pH 7.4, 0.4% Tween 80), the concentration of the BDP-PHS is 5 μM. The mixture was allowed standing in the dark for desired time and the fluorescence spectra were then recorded under excitation at 525 nm except otherwise indicated. Quantum yields were determined using Rhodamine 6G (Фstandard = 0.95 in ethanol) as a standard according to a published method. [45]
2. Experimental section
2.2. Synthesis of BDP-PHS
2.1. General procedures
To a solution of BDP1 [46] (370 mg, 0.86 mmol) and Et3N (174 mg, 1.72 mmol) in 10 mL of anhydrous dichloromethane (DCM), 2-fluoro-5nitrobenzoyl chloride (260 mg, 1.29 mmol, dissolved in 5 mL of DCM) was added dropwise at 0 °C. After stirring 30 min at 0 °C, the mixture was warmed to room temperature (r. t.) and further reacted for 8 h. The solution was diluted with DCM (10 mL), washed with H2O (30 mL × 3) and dried over anhydrous MgSO4. The solvent was dried in vacuo and the crude product was purified by column chromatography on silica gel (eluted with dichloromethane: petroleum ether, 4:3, v/v) to afford BDP-PHS as purple solid (110 mg, 29%). 1H NMR (500 MHz, CDCl3) δ 9.02 (dd, J = 3.0, 2.5 Hz, 1H), 8.52–8.47 (m, 1H), 7.70–7.65 (m, 3H), 7.50 (d, J = 6.0 Hz, 3H), 7.41 (t, J = 9.0 Hz, 1H), 7.34–7.30 (m, 2H),
All reagents were purchased from commercial suppliers without further purification. All reactions were carried out under nitrogen atmosphere without otherwise indicated. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were obtained on a Bruker AV-500 spectrometer. All NMR spectra were calibrated using residual solvents as internal references (for CDCl3: 1H NMR = 7.26, 13C NMR = 77.16). All chemical shifts were reported in parts per million (ppm) and coupling constants (J) in Hertz (Hz). High resolution mass spectra (HRMS) were recorded on a Finnigan LCQ mass spectrometer, a Shimadzu LC-IT-TOF spectrometer. Absorption spectra were acquired using a Shimadzu UV-2500
Fig. 2. (a) Time-dependent emission fluorescence spectra of BDP-PHS (5 μM) with 50 μM of Na2S2 (λex = 525 nm, slit width = dex = dem = 0.8 nm, PMT voltage = 950 V) in DMSO/ phosphate buffer (3:97, v/v, 10 mM, pH 7.4, 0.4% Tween 80) at room temperature. b) Plot of F618/F574 as a function of time upon addition of various equivalents of Na2S2.
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Fig. 3. Emission spectra (a) and fluorescence ratio (b) changes of BDP-PHS (5 μM) with 20 μM of Na2S2 and other analytes (•NO, 1O2, NaClO, H2O2, ROO•, •OH, ONOO−, GSH, Cys, Hcy, SO2− 3 , 2+ 2− 2− Na+, K+, Ca2+, Mg2+, Fe2+, SO2− , S and HS−) (λex = 525 nm, slit width = dex = dem = 0.8 nm, PMT voltage = 950 V) in DMSO/phosphate buffer (3:97, v/v, 10 mM, pH 7.4, 4 , CO3 , Zn 0.4% Tween 80).
7.28 (s, 1H), 6.61 (s, 1H), 6.03 (s, 1H), 2.61 (s, 3H), 1.53 (s, 2H), 1.42 (d, J = 17.5 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 166.5, 164.3, 160.4, 156.2, 151.9, 150.4, 144.1, 143.5, 142.3, 140.8, 135.1, 135.0, 134.2, 132.8, 132.2, 130.2, 130.1, 129.1, 129.0, 128.5, 128.3, 121.7, 119.9, 119.4, 118.8, 118.6, 117.5, 14.7, 14.5, 14.4. HRMS (m/z): C33H25BF2N3O4, Calculated: [M-F]+ = 576.1906, found: [M-F]+ = 576.1923. 2.3. MTT assay The cellular toxicity of BDP-PHS to HeLa cells was tested by standard MTT assays. HeLa cells were incubated in 96-well plates and pretreated with various concentrations of BDP-PHS (0, 0.01, 0.1, 0.5, 5, 10 and 20 μM) containing 2‰ DMSO in 100 μL culture medium (DMEM + 10% FBS). The control group was added the same volume of culture medium contained 2‰DMSO. All groups were incubated in incubator (5% CO2, 37 °C) for 24 h. Next, MTT solution (Final concentration, 0.5 mg/mL) was added into each well, followed by incubation for another 4 h under the same conditions. Then removed the supernatant and added 150 μL DMSO. After shaking for 10 min, the absorbance at 490 nm was measured by microplate reader (Synergy 2, BioTek Instruments Inc.). Cell viability was calculated by A/A0 × 100% (A and A0 are the absorbance of experimental group and control group, respectively).
polysulfides receptor in many reported polysulfides probes. [25,26] After reacting with polysulfides, 2-fluoro-5-nitrobenzoate will be removed via nucleophilic aromatic substitution reaction (SNAr), resulting in the recovery of the intermolecular charge transfer (ICT) from styryl group to the boron-difluoride center of BODIPY of BDP1. Therefore, ratiometric response through an obvious red shift in emission wavelengths can be expected. BDP-PHS was facile synthesized by reacting of 2-fluoro-5-nitrobenzoyl chloride with BDP1, and it was fully characterized by 1H, 13C NMR, and HRMS.
3.2. Photophysical properties of BDP-PHS Photophysical measurements of BDP-PHS were carried out in phosphate buffer solutions (PBS, 10 mM, pH 7.4, containing 3% DMSO and 0.4% Tween 80) (Fig. 1a). In these experiments, freshly prepared solutions of Na2S2 were used as the equivalent of polysulfides. BDP-PHS exhibits a main absorption band at 563 nm (ε = 1.08 × 105 M−1 cm−1) and a shoulder peak at ∼529 nm. After adding 10 equivalents of Na2S2, the absorption band at 563 nm of BDP-PHS decreased gradually and reached equilibrium within 30 min, accompanied by the concomitant increase of a new band at 576 nm (ε = 9.78 × 104 M−1 cm−2). The
2.4. Cell culture methods and confocal imaging HeLa cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin (100 units/ mL), streptomycin (100 mg/mL) and 5% CO2 at 37 °C. After removing the incubation media and rinsing with 1× PBS for three times, the HeLa cells were incubated with BDP-PHS (20 μM) for 20 min at 25 °C. Then the cells were washed three times with 1× PBS and imaged with Zeiss LSM-710 microscope equipped with a 63× oil-immersion objective. Background signals of all images were verified to be nearly zero by imaging the same cells treated with PBS solution containing 2‰ DMSO. The excitation wavelength of BDP-PHS used in the experiment was 488 nm, whereas the emissions were collected in the ranges of 550–580 nm (green) and 600–700 nm (red), respectively. 3. Results and discussion 3.1. Synthesis of BDP-PHS In BDP-PHS, BODIPY was selected as fluorophore due to its advantages of high molar extinction coefficient, high quantum yield, and excellent light stability [47]. 2-Fluoro-5-nitrobenzoic ester was integrated into the BODIPY core because of its high reliability as
Fig. 4. HPLC traces of the reaction of BDP-PHS with Na2S2. (a) BDP-PHS (100 μM); (b and c) BDP-PHS (100 μM) treated with Na2S2 (50 μM and 100 μM, respectively) for 2 min; (d) BDP1 (100 μM). HPLC conditions: 0.1 mL/min flow rate, 10% water + 90% methanol for 20 min. All samples were diluted to 100 μM before the test. Wavelength for detection: 563 nm.
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Scheme 1. Synthesis of BDP-PHS and proposed response mechanism of BDP-PHS toward polysulfides.
Na2S2-induced absorption red-shift leads to a clear isobestic point at 570 nm, implying the conversion of BDP-PHS to BDP1. The shoulder absorption at 529 nm also shows a minor red-shift to 537 nm. Free BDP-PHS exhibits one strong emission band centered at 574 nm and one relatively weak emission band centered at 618 nm, with λex of 525 nm (Fig. S1). After addition of Na2S2, the emission at 574 nm decreased distinctly, accompanied by gradually emission intensity increment of λem at 593 and 650 nm, which make λem at 618 nm cannot be distinguished. Meanwhile, emission color of the solution changes from orange to yellow. This red-shift should be attributed to the recovery of ICT effect from the hydroxyl group to BODIPY. The fluorometric titration of BDP-PHS with various concentrations of Na2S2 ([Na2S2]) was carried out to evaluate the sensing behavior of BDP-PHS. With the addition of [Na2S2] from 0 to 50 μM, the fluorescence intensity ratio value between 618 and 574 nm (F618/F574) gradually increased from 0.2 to 1.0 (Fig. 1b). Based on the emission intensity at 574 nm, the limit of detection of BDP-PHS for H2S2 is calculated to be 57 nM according to the equation of LOD = 3σ/k (n = 3), where σ is the standard deviation of a blank measurement, and k is the slope of the fitting straight line (Fig. S2 and Table S1).
3.3. Dynamic response of BDP-PHS to H2Sn To investigate the sensitivity of BDP-PHS, the reaction kinetics of BDP-PHS with various concentrations of Na2S2 were measured (Fig. 2). After treating BDP-PHS with low concentrations of Na2S2 (5, 10, 20, 30, 40 μM), the ratio of F618/F574 slowly increased and reached a balance in 20 min. However, after adding 50 μM of Na2S2 to the solution of BDP-PHS, the fluorescence intensity ratio rapidly increased with time in the first 10 min and reached the maximum after 30 min, accompanied with the fluorescent quantum yield (Фf) increment from 4.3% to 13.6% (Fig. 2b). The pseudo-first-order rate constant for the reaction of BDP-PHS with Na2S2 can be observed to be 0.071 min−1 (Fig. S3). This result suggests that BDP-PHS can be applied for rapid and sensitive sensing of Na2S2 in an aqueous solution at physiological pH.
3.4. Selectivity and pH effect of BDP-PHS There are many reactive oxygen species in vivo, which may affect the detection of polysulfides by BDP-PHS. To investigate the selectivity,
Fig. 5. Fluorescence images of probe BDP-PHS in HeLa cells by confocal fluorescence images. (a, b, c) Cells were treated with probe BDP-PHS (20 μM, 20 min), then imaged; (d, e, f) cells were pretreated with probe BDP-PHS (20 μM, 20 min), subsequently incubated with Na2S2 (200 μM, 30 min) then imaged. The fluorescence images were captured from the green channel of 550–580 nm and red channel of 600–700 nm with an excitation at 488 nm. Third row: Fred/Fgreen ratiometric images. (g) Bright-field transmission images; (h) average fluorescence intensity ratios (Fred/Fgreen) in c and f. Data are mean ± S.E.M., n = 3. Data are expressed as mean ± SD of three experiments. Scale bar: 20 μm, respectively. Ratio 58 and 89, for c and f, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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BDP-PHS was treated with a series of RSS (GSH, Cys, Hcy, S2−, HS− and − 1 HSO− 3 ), RNS (•NO and ONOO ), ROS (H2O2, NaClO, •OH and O2) and + + 2+ 2+ 2+ 2+ 2− 2− other ions (Na , K , Ca , Mg , Fe , Zn , SO3 , SO4 and CO2− 3 ). The results shown in Fig. 3 indicated that the addition of Na2S2 into BDP-PHS solution induced an obvious red-shift (Fig. 3a) and N8-fold increment in the fluorescence ratio of F618/F574 (Fig. 3b). However, no significant fluorescence ratio (F618/F574) increase was observed for any other tested analytes. The pH effects of the response of BDP-PHS to Na2S2 were also investigated, and the tested range of pH is from 4 to 10 (Fig. S4). The fluorescence intensity ratio (F618/F574) of BDP-PHS was quite stable within a wide pH range from 4 to 10, indicating excellent stability of BDP-PHS at physiological pH conditions. In the presence of Na2S2, ratio enhancement was observed in the pH range of 7–10. The low ratio increment could be due to the conversion of S2− n to H2Sn at pH 4–6. The pH effect investigation demonstrates that BDP-PHS can be applied into physiological environment over a wide pH range. All these results clearly indicate that BDP-PHS possesses high sensing ability toward polysulfides without interference from other biologically related species and pH effects, and it functions well under physiological conditions.
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specific ratiometric response toward polysulfides over other biologically related species such as RNS, ROS, RSS and other ions. The observed pseudofirst-order rate constant for the reaction of BDP-PHS with polysulfides was calculated to be 0.071 min−1, and the detection limit of BDP-PHS was determined to be 57 nM. Moreover, the intracellular polysulfides imaging ability of BDP-PHS was demonstrated in HeLa cells via confocal fluorescence microscopy imaging. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (81672508, 61505076 and 21501085), Natural Science Foundation of Jiangsu Province (BK20140951), Key University Science Research Project of Jiangsu Province (17KJA150004) and Scientific Research Projects of Nanjing Xiaozhuang College (2018NXY49). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117295.
3.5. Response mechanism The selective response mechanism of BDP-PHS to Na2S2 was further studied by high performance liquid chromatography (HPLC). After incubating BDP-PHS with Na2S2 for 2 min, an aliquot of the reaction mixture was subjected to HPLC at ambient temperature. The HPLC chromatogram of free BDP-PHS (100 μM) in MeOH showed a single peak with a retention time of 8.06 min (Fig. 4a). After adding Na2S2 (50 μM) into BDP-PHS solution for 2 min, the signal at 8.06 min decreased, whereas a new peak with a retention time of 4.33 min was observed (Fig. 4b), indicating the formation of BDP1. The addition of 100 μM Na2S2 to the solution of BDP-PHS induced the disappearance of the peak at 8.06 min, suggesting that BDP-PHS was almost completely consumed (Fig. 4c). The change in chromatographic peaks manifested that BDP1 was generated from the reaction between BDP-PHS and Na2S2, which was in accordance with the mechanism discussed above (Scheme 1).
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3.6. Imaging of living cells The cellular toxicity of BDP-PHS to HeLa cells was tested by standard MTT assays. After incubation with BDP-PHS for 24 h, no significant change of the viability of HeLa cells was observed. When the concentration of BDP-PHS was up to 20 μM, the cell viability was still near 100% (Fig. S5). These results demonstrate that BDP-PHS exhibits low cytotoxicity and good biocompatibility. Next the capability of BDP-PHS for fluorescent ratiometric imaging of polysulfides in living cells was investigated. As shown in Fig. 5, after incubating HeLa cells with BDP-PHS (20 μM) for 20 min, fluorescence could be observed in both green (550–580 nm, Fig. 5a) and red (600–700 nm, Fig. 5b) channels, which could be ascribed to the unreacted probe, indicating that BDP-PHS can penetrate the cell membrane and distribute in the cytoplasm. The average fluorescence intensity ratio (Fred/Fgreen) in ratiometric pseudo-colored image (Fig. 5c) was determined to be 58.1 ± 3.6 (Fig. 5h). When HeLa cells were treated with Na2S2 (200 μM) for 30 min and then incubated with BDP-PHS, the fluorescence intensity in green channel decreased (Fig. 5d), accompanied with fluorescence enhancement in red channel (Fig. 5e), resulting in distinct increase in the ratio (89.6 ± 6.8, Fig. 5f). These results demonstrate that BDP-PHS possesses good membrane permeability and is suitable for imaging of polysulfides in living cells. 4. Conclusion In summary, a new ratiometric fluorescent polysulfides probe, BDPPHS, was designed and synthesized. BDP-PHS showed rapid and
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