A viscosity sensitive azide-pyridine BODIPY-based fluorescent dye for ...

Dyes and Pigments 159 (2018) 166–172

Contents lists available at ScienceDirect

Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig

A viscosity sensitive azide-pyridine BODIPY-based fluorescent dye for imaging of hydrogen sulfide in living cells

T

Qi Zhaoa, Caixia Yina,∗, Jin Kanga, Ying Wena, Fangjun Huob,∗∗ a

Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China b Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Fluorescent probe BODIPY H2S Viscosity Cell imaging

A viscosity sensitive BODIPY fluorescent probe BDP-N3 with high selectivity for detecting H2S was synthesized and designed. The probe exhibited weak fluorescence in non-viscous media. With the increase of viscosity, the fluorescence intensity and decay time of the probe was enhanced significantly, which proved that it acted as a molecular rotor and could be utilized for the detection of changes in viscosity. Additionally, the probe showed a fast ‘turn-on’ fluorescence response to H2S with high selectivity. The H2S concentration was directly proportional to fluorescence intensity at 515 nm. Furthermore, cellular imaging experiments revealed that the probe was cell membrane permeable and could be applied for sensing of both H2S and viscosity in a biological system.

1. Introduction Among the reactive sulfur species family, hydrogen sulfide (H2S) is connected with various physiological and pathological functions. As the third gaseous signal molecule, H2S was regarded as the biologically active gas to regulate immune, endocrine, cardiovascular, neuronal, and gastro-intestinal systems [1–4]. Generally, in most mammalian tissues and organisms, H2S can be endogenously metabolized by at least certain enzymes such as cystathionine γ-lyase (CSE), cystathionine βsynthase (CBS), and 3-mercaptopyruvate sulfur-transferase (MST) [5]. Abnormally low or high endogenous levels of H2S will result in severe diseases like diabetes, Alzheimer's disease, Down syndrome, liver cirrhosis, and hypertension [6–16]. In addition, H2S is a famous inducer of apoptosis. It initiates the vital apoptotic enzymes caspase-3, caspase-8, and caspase-9 and induces cell death by causing cytoplasmic shrinkage and nuclear condensation which finally result in an increase of intracellular viscosity. From another point, as an important factor in the process of diffusion controlled processes, viscosity plays a primary role in varied biological activities, as well as in chemistry and other fields, and normally dominates the effective of mass transport of reagents [17,18]. In biological systems, viscosity as an important fundamental and structural sensitive physical parameter is related to diffusionmediated cellular processes, such as signal transduction and transportation of small solutes, macromolecules, protein-protein interactions, and other cellular organelles in living cells [19]. It has been described

∗

Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Yin), [email protected] (F. Huo).

∗∗

https://doi.org/10.1016/j.dyepig.2018.06.029 Received 19 May 2018; Received in revised form 18 June 2018; Accepted 19 June 2018 Available online 20 June 2018 0143-7208/ © 2018 Published by Elsevier Ltd.

that local viscosity in cells varies from 1 to 400 cP, abnormal change in the microviscosity of biological systems affects normal cellular functions and triggers a range of diseases [20], such as atherosclerosis [21], and even cell malignancy [22]. While methods to measure the bulk macroscopic viscosity are well developed, imaging local microscopic viscosity remains a challenge. Thus, simultaneous detection of viscosity and H2S needs further investigation in the biological and medical fields. Numerous studies have been carried out to explore the detection of H2S including electrochemical, colorimetric and gas chromatographic techniques (GC), monobromobimane method (MBB) [23–27]. However, many of these techniques required complicated sample preparation or destroying the biological tissues, and thus could not be applied in the biological systems. Therefore, fluorescent techniques with their simplicity, high sensitivity, selectivity, and real-time capability to detect intracellular H2S raised lots of interest. The design strategies of H2S fluorescent probes is mainly based on its nucleophilicity and strong reducing property, such as reducing nitro/azanol to amines [28–31], Michael addition reactions, and thiolysis reactions [32–34]. Recently a tremendous number of fluorescent probes have been reported for the detection of H2S but most of them are limited to the single function, and their practical application only detected the exogenous of H2S in solution. Furthermore, few probes can test a biological sample of both H2S and viscosity [35–43]. Accordingly, we were then interested to design a probe which can detect exogenous and endogenous H2S as well as measure viscosity in cellular systems.


Q. Zhao et al.

Scheme 1. Synthesis of the probe.

2.2. Synthesis of BDP-N3

To address this question, we assumed that if we could design a compound that has effective H2S reaction site and molecular rotation properties then it will be able to perform dual functions. Induction of apoptosis with dexamethasone in turn reflects the molecular rotation properties restricted in the viscous medium created during apoptosis process. Meanwhile, we recognized that BODIPY (4,4-difluoro-4-bora3a, 4a-diaza-s-indacene) dyes are well-known to the excellent photophysical and photochemical properties, extremely stable and particularly insensitive to the polarity of solvents as well as to pH [44]. BODIPY analogue have been applied in the fields of probes and protein marker [45]. An azide group is introduced in pyridine BODIPY derivative, as it is known to undergo reduction in the presence of H2S. Herein, we synthesized such a novel fluorescent probe BDP-N3 (Scheme 1) based on a BODIPY derivative incorporating quaternized 4-pyridinium group for multiply detecting intracellular H2S and viscosity. Because H2S related to gaseous signal transduction, and viscosity influenced diffusion in biological processes, we anticipate that the current probe BDP-N3 can be used to detect H2S and viscosity in living systems for more insight into the role of both in medical issue.

Compound 1: 4-Pyridinecarboxaldehyde (9.0 mmol, 0.96 g) was stirred with 2,4-dimethylpyrrole (19.4 mmol, 1.85 g) in deoxygenated CH2Cl2 (150 mL). One drop of TFA was added and the mixture was stirred overnight under Ar at room temperature. The red solution was treated with DDQ (9.0 mmol, 2.04 g), stirring was continued for 4 h followed by the addition of Et3N (15 mL). After 15 min, BF3⋅Et2O (15 mL) was added at 0 °C, and the mixture was stirred at room temperature for further 3 h. After washing with saturated aqueous NaHCO3, the organic phase was separated, dried with Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/Petroleum ether, 1/1, v/v, as eluent) to afford the desired 5,5-difluoro-1,3,7,9-tetramethyl-10-(pyridin-4-yl)-5H-4λ4,5λ4dipyrrolo [1,2-c:2′,1′-f] [1–3]diazaborinine (compound 1) as red powder (0.35 g, 12%). m.p. 241–243 °C. Elemental Analysis: Found C, 66.3; H, 5.4; N, 12.7%. Molecular formula: C18H18BF2N3, requires: C, 66.5; H, 5.6; N, 12.9%. 1H NMR (CDCl3, 600 MHz): δ (ppm): δ 8.79 (d, J = 5.8 Hz, 2H), 7.35 (d, J = 5.8 Hz, 2H), 6.01 (s, 2H), 2.56 (s, 6H), 1.41 (s, 6H). 13C NMR (CDCl3, 150 MHz): δ (ppm): 156.6, 150.1, 144.2, 142.5, 137.3, 130.2, 123.5, 121.8, 14.7 (Fig. S1). Compound BDP-N3: Compound 1 (0.06 mmol, 0.02 g) and 1-azido4-(bromomethyl)benzene (0.6 mmol, 0.12 g) were dissolved in toluene (20 mL), and then the mixture was refluxed at 110 °C for 12 h. After filtration, the precipitate was filtered, washed with toluene and dried in vacuo to afford deep red powder 1-(4-azidobenzyl)-4-(5,5-difluoro1,3,7,9-tetramethyl-5H-4λ4, 5λ4-dipyrrolo [1,2-c:2′,1′-f] [1–3]diazaborinin-10-yl)pyridin-1-ium bromide (compound BDP-N3) (0.018 g, 62%). m.p. > 300 °C. Elemental Analysis: Found C, 65.4; H, 5.1; N, 18.2%. Molecular formula: C25H24BF2N6, requires: C, 65.6; H, 5.3; N, 18.4%. 1H NMR (CDCl3, 600 MHz): δ (ppm): δ 9.40 (d, J = 6.2 Hz, 2H), 8.48 (d, J = 6.7 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 6.27 (s, 2H), 5.95 (s, 2H), 2.50 (s, 6H), 1.35 (s, 6H). 13C NMR (CDCl3, 150 MHz): δ (ppm):156.7, 151.1, 145.9, 142.2, 140.5, 134.4, 130.7, 130.5, 128.8, 128.6, 122.3, 119.8, 62.9, 14.5, 14.2 (Fig. S2). MS (ESI): found: m/z 457.2131; Molecular formula: [C25H24BF2N6] +, requires: 457.2118, (Fig. S3).

2. Experimental 2.1. Materials and instruments All reagents and solvents were purchased from commercial suppliers and used without further purification. Deionized water was used throughout all experiments. The solutions of anions were prepared from their sodium salts. The stock solutions of probe BDP-N3 were prepared in DMSO. SNP solution (20 mM) was prepared by dissolving SNP (8.8 g) in DMSO (2 mL). Chromatography was carried out on silica gel using silica gel GF254 plates with a thickness of 0.20–0.25 mm. A pH meter (Mettler Toledo, Switzerland) was used to determine the pH. Ultraviolet–visible (UV–vis) spectra were recorded on a Cary 50 Bio UV–visible spectrophotometer. Fluorescence spectra were measured on Hitachi F-7000 fluorescence spectrophotometer. All fluorescence and UV–vis spectra data were recorded at 2 min after the analytes addition. A PO-120 quartz cuvette (1 cm) was purchased from Shanhai Huamei Experiment Instrument Plants, China. 1H NMR and 13C NMR experiments were performed with a Bruker AVANCE-600 MHz NMR spectrometer, respectively (Bruker, Billerica, MA). Coupling constants (J values) are reported in hertz. ESI determinations were carried out on AB Triple TOF 5600plus System (AB SCIEX, Framingham, USA). FLIM images were obtained using FL920 transient fluorescence spectrometer purchased from Edinburgh Instruments Co., Ltd, United Kingdom. The ability of BDP-N3 reacting to H2S in the living cells was measured by a Zeiss LSM880 Airyscan confocal laser scanning microscope.

2.3. Solutions preparation and UV–vis and fluorescence measurements The stock solution of the probe, BDP-N3, was prepared with a concentration of 2 mM in DMSO. The stock solutions of H2S (2 mM) was prepared in deionized water, sodium hydrosulfide solid was added to aqueous solution to prepare a H2S solution. Reagents with analytical grades and demineralized water were used for preparing the solutions. Stock solutions (2 mM) of F−, Cl−, Br−, I−, NO3−, NO2−, AcO−, HCO3−, CO32−, SCN−, SO32−, SO42−, S2O32−, ClO4−, PO43−, Cys, Hcy, GSH, were prepared by direct dissolution of proper amounts of sodium salts. A test solution was prepared by placing appropriate 167


Q. Zhao et al.

Table 1 Spectral data of the probe in different solvents. Solvents

Dielectric constant

Viscosity at 20 °C(cP)

λabs (nm)

λem (nm)

ε × 10 (mol−1cm−1L)

Stokes' shift (nm)

Φf

lifetimes τavg(ns)

DCM EtOH MeOH DMSO MeCN H2O

8.93 24.9 32.6 46.8 37.5 78.4

0.43 1.20 0.595 2.0 0.35 1.01

514 508 508 509 508 508

525 515 525 526 520 523

0.86 1.1 0.76 0.66 0.76 0.76

11 7 17 17 12 15

0.17 0.16 0.19 0.46 0.31 0.24

2.18 1.87 1.59 2.90 2.35 2.07

amount of probe stock solution into a quartz cuvette upon the addition of various analytes and diluting the solution to 2 mL with DMSO at room temperature. UV–Vis and fluorescence spectra of BDP-N3 were obtained in DMSO solution. And any changes in the fluorescence intensity are monitored using a fluorescence spectrometer (λem = 515 nm, λex = 475 nm, slit: 5 nm/5 nm). For all measurements of fluorescence spectra, the scan speed is 1200 nm min−1. Statistical analysis of the data was carried out using Origin 9.1.

in a 5% CO2 incubator for 24 h. Different concentrations of BDP-N3 (0, 1, 2.5, 5, 10, 20, 30 and 50 μM) were then added to the wells. After incubation for 5 or 10 h, CCK-8 (10% in serum free culture medium) was added to each well, and the plate was incubated for another 1 h. The absorbance of each well was measured at 450 nm on a microplate reader.

2.4. Calculation of fluorescence quantum yield

3.1. Spectral properties of BDP-N3

The relative fluorescence quantum yields were determined by using fluorescein as reference in 0.1 M NaOH excited at 475 nm (Φflu = 0.79) and was calculated through the following equation [46]:

When the pyridine group of compound 1 was alkylated to form the N-methyl-substituted derivative or N-benzyl-substituted derivative, as might be expected, the water solubility of total molecule was elevated in a large part due to the newly formed cationic quaternary ammonium salt [48]. As shown in Table 1, we also studied the spectral properties of BDP-N3 (5 μM) in different solvents. Through the computation, the fluorescence quantum yield of BDP-N3 is relatively low in these solvents, which confirms the practical application of this probe because of the low background fluorescence.

3. Results and discussion

sample sample reference sample reference ⎛ F ⎞⎟ ⎛ Abs ⎞ ⎞ ⎛⎜ η φflu = φflu reference reference sample F η Abs ⎝ ⎠⎝ ⎠ ⎠⎝ ⎜

⎟

⎜

⎟

Where F represents the integrated emission area of fluorescent spectrum, η represents the refractive index of the solvent, and Abs represents the absorbance at excitation wavelength selected for standards and samples.

3.2. Response to hydrogen sulfide 2.5. Viscosity analysis and fluorescence lifetime As shown in Fig. 1A, treatment BDP-N3 (0.3 μM) with H2S triggers a remarkable increase of fluorescence intensity at 515 nm when excited at 475 nm. Correspondingly, a distinct bright green-colored fluorescence was clearly observed after H2S addition in DMSO. Fluorescence intensities enhanced about ten times suggested that BDP-N3 is one of the most sensitive H2S test kit available in abiotic systems. A linear correlation existed between the fluorescence intensity and the concentration of H2S within the range from 0 to 280 μM, as illustrated in Fig. 1B. The limit of detection was estimated to be 2.05 μM (Fig. S4). The Stokes shift of the probe is calculated to be 8 nm. The UV–Visible spectrum response of BDP-N3 (1 μM) toward H2S (1 mM) was studied in DMSO. After the addition of H2S for 15 min, UV–vis spectra of the probe recorded as Fig. 2A. We can observe that the absorption peak of the probe at 508 nm decreased, meanwhile, a new absorption peak at 430 nm appeared with a distinct isosbestic point at 480 nm. This change showed that the π-conjugation structure of BDP-N3 was shortened after reaction with H2S. Meanwhile, the reaction products were identified by high resolution mass spectrometry. As seen in Fig. S5, ESI-MS spectrum showed the presence of a peak at m/z 325.1671 and one noticeable isotopic peak at 326.1643, which correspond to the characteristic MS peaks of compound 1. As pH dependence is important for applications in biochemical systems, the fluorescence intensity changes of BDP-N3 induced by H2S were investigated at pH 2–9 in PBS: DMSO = 1:1 (v/v) solution. As shown in Fig. 2B, BDP-N3 gave obvious fluorescence enhancement responses at pH 7.4 with the addition of H2S, which indicated that the fluorescence probe to H2S works fine in physiological pH 7.4. In order to study its special recognition ability, we carried out the experiment by fluorescence spectrometer Fig. S6 shows the fluorescence spectral changes upon addition of various analytes in DMSO, including SO32−, S2O32−,SCN−, PO43−, NO3−, NO2−, HSO3−,

Viscous solutions were prepared by mixing methanol and glycerol in different volume proportions. Viscosity studies were carried out in 0.5 μM of BDP-N3 in different methanol: glycerol fractions at excitation wavelength of 475 nm by fluorescence spectrophotometer. We observed that with increasing glycerol fraction the fluorescence intensity increases. The fluorescence decays from solution samples were recorded using FL920 transient fluorescence spectrometer. The methanol/glycerol solutions of various viscosities were prepared in cuvette and the resulting values for fluorescence lifetime tested. The attenuation curve was obtained by scanning, and the fluorescence lifetime value was obtained by fitting the software with transient fluorescence instrument. 2.6. Cell culture and imaging HepG-2 cells were grown in Dulbecco's Modified Eagle's medium supplemented with 12% Fetal Bovine Serum and 1% antibiotics at 37 °C in humidified environment of 5% CO2. The cells were stained with BDP-N3 (5 μM) for 30 min. For the detection of H2S, the stained cells were incubated with H2S of different concentrations for another 30 min at 37 °C. Before imaging, the stained cells were washed three times with phosphate buffer. Study of fluorescence imaging was carried by Zeiss LSM880 Airyscan confocal laser scanning microscope. 2.7. Cell viability assay HepG-2 cells have been applied in study the toxic effect of BDP-N3. The cell viability experiment was evaluated by Cell Counting Kit-8 (CCK-8), and the absorbance at 450 nm was measured to determine the cell survival rate [47]. HepG-2 cells were seeded on a 96-well microtiter to a total volume of 100 μL/well, then the cells were incubated at 37 °C 168


Q. Zhao et al.

Fig. 1. (A) The emission spectra of BDP-N3 (0.3 μM) in DMSO with different concentrations of H2S (0–280 μM), λex = 475 nm. Inset: The color changes of BDP-N3 without and with addition of H2S under a 365 nm lamp illumination; (B) Dependence of fluorescence intensity at 515 nm on H2S concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

HCO3−,AcIO−, F−, Cl−, Br−, I−, CO32−, SCN−, SO42−, ClO−, Cys, GSH, Hcy. The changes in fluorescence intensity indicated that fluorescence augmentation occurred only upon reaction with H2S and BDPN3 exhibits excellent selectivity toward H2S among the various analytes in abiotic systems. In addition, time-dependent modulations in the fluorescence spectra of BDP-N3 (0.3 μM) were detected in the presence of 30 μM H2S. The time course showed that the reaction was complete within 15 min for H2S, indicating that BDP-N3 can achieve detection of H2S in abiotic systems (Fig. S7). 3.3. Response to viscosity BDP-N3 has low fluorescence in most routine solvents, such as methanol, ethanol and dichloromethane. The dye molecule consists of two parts: the BODIPY moiety and the benzene ring. It is assumed that the two parts of the molecule will rotate freely occurs in low viscosity solvents like methanol (Scheme 2), where the energy of the excited state is released through nonradiative transitions, causing a quite low fluorescence [49,50]. Then we studied the molecular fluorescence intensity changes in viscous media, Fig. 3A shows the change of BDP-N3 (0.3 μM) in different methanol: glycerol fractions, a significant increase in fluorescence intensity was observed with increasing glycerol fractions. The fluorescence intensity increasing with viscosity is attributed to the fact that high viscosity restricts the rotation of phenyl group, which makes the fluorescence emission by non-radiative pathway decrease.

Scheme 2. Schematic illustration of the possible rotation between two parts of BDP-N3.

In addition, lifetime studies were carried out in 5 μM of BDP-N3 in different methanol: glycerol fractions at excitation wavelength 475 nm. As shown in Fig. 3B, with the gradual increase in viscosity from 0.6 to 950 cP, the fluorescence lifetimeτ also increases from 1.59 ns to 3.58 ns The average life of the excited state is calculated by formula calculan n tion: τavg = ∑i = 1 Ai τi, when ∑i= 1 Ai = 1. The fluorescence lifetime value obtained is as shown in Table 2, the fluorescence decay exhibits a multiexponential decay. The lifetimes of BDP-N3 in various viscosities methanol/glycerol solutions are as below. The increase in lifetime with

Fig. 2. (A) UV–vis absorption spectra of BDP-N3 (1 μM) in DMSO addition of H2S (1 mM). (B) Fluorescence intensity at 515 nm of probe (0.3 μM) at different pH values in the presence of H2S (280 μM) and in the absence of H2S. 169


Q. Zhao et al.

Fig. 3. (A) Fluorescence emission spectra of BDP-N3 (0.3 μM) at λex = 475 nm with the variation of solution viscosity (methanol/glycerol system). Inset figure showing increase in fluorescence intensity with increase in viscosity. The image was taken under 365 nm lamp illumination. (B) Fluorescence lifetime spectra of BDPN3 (5.0 μM) in methanol-glycerol system with varying proportions to adjust the viscosity at 25 °C.

BDP-N3 towards HepG-2 cells at a concentration of 50 μM (82.9% viability). In general, at the staining concentration of BDP-N3, no evident cytotoxic responses were observed for all of the tested cells after incubation for 10 h. These results showed that BDP-N3 exhibited good biocompatibility and low cytotoxicity for luminescence cell imaging under the applied conditions. Intracellular imaging experiments were conducted using living HepG-2 cells (Fig. 4). As shown in Fig. 5B, a weak fluorescence was observed when HepG-2 cells were incubated with only 5.0 μM BDP-N3 for 20 min at 37 °C. In addition, to detect endogenously generated H2S in living cells, the cells were stimulated with sodium nitroprusside (SNP) for 30 min. Then incubated with BDP-N3 for 20 min, the obvious green fluorescence was observed and cell imaging was performed (Fig. 4A). The cell imaging results showed the enhancement of the fluorescence intensity is due to the fact that SNP treatment stimulated cells to generate H2S. Then HepG-2 cells (pre-incubated with probe) were incubated with H2S for different concentration intervals of 50, 100, 200 μM for 20 min, and green fluorescence increased gradually was observed (Fig. 4C, D, E). From the experimental result, the enhance

Table 2 The lifetimes of BDP-N3 in various viscosities methanol/glycerol solutions. system

τ1(ns)

A1(%)

τ2(ns)

A2(%)

τ3(ns)

A3(%) τavg(ns) x2

0%gly 50%gly 100%gly

1.2129 0.9638 1.0024

93.60 12.70 13.64

7.2442 2.4242 4.4161

6.4 82.71 76.34

\ 80.780 0.76

\ 1.59 1.113 4.58 2.63 1.163 10.03 3.58 1.102

increase in viscosity describes the rotation restriction in high viscous medium which proves the molecular rotation property. Thus BDP-N3 is truly a fluorescence molecular rotor which displays both fluorescence intensity and lifetime sensitivity to viscosity of the environment.

3.4. Cell experiment To further explore the biological application of BDP-N3, cytotoxicity of the probe for human HepG-2 cells was studied by the CCK-8 method (Fig. S8). Cytotoxicity experiments displayed minimal cytotoxicity of

Fig. 4. Cell images of HepG-2 cell lines. (A): cells were stimulated with SNP for 30 min at 37 °C and then treated with probe (5.0 μM) for 20 min (B)cells were treated with probe (5.0 μM) for 20 min at 37 °C. (C–D): cells were treated with probe (5.0 μM) for 20 min and then incubated with H2S (50, 100, 200 μM) for 20 min. Images were taken λex = 488 nm and λem range 500–550 nm.

170


Q. Zhao et al.

Fig. 5. Cell images of HepG-2 cell lines. (A–C): cells were treated with probe (5.0 μM) for 20 min and then incubated with 4 μM Dex for 20 min, 40 min and 60 min at 37 °C. (D–F): cells were treated with probe (5.0 μM) for 20 min, 40 min and 60min. Images were taken λex = 488 nm and λem range 500–550 nm.

of fluorescence intensity in HepG-2 cells, occurs not only because of the increase in hydrogen sulfide concentration, but also high concentrations of exogenous H2S inducing apoptosis in cells as a result of which the viscosity of cells increase leads to enhancement in fluorescence emission of BDP-N3. Viscosity increases during apoptosis in biological systems, hence, we carried out HepG-2 cells apoptosis experiment induced by dexamethasone without removing endogenous H2S. To confirm that the fluorescence turn on response due to viscosity changes in dexamethasone inducing apoptosis rather than endogenous H2S, we performed a group of controlled experiments. As shown in Fig. 5A, B, C, the cells were first incubated with BDP-N3 for 20 min at 37 °C and then treated with 4 μM dexamethasone for a longer period then apoptosis was initiated (e.g., cell deformation and cytoplasmic vacuolization) in cells and as a result of which, the intracellular viscosity gradually increased leading to enhancement in fluorescence emission of BDP-N3 via restriction of rotation. Another group of HepG-2 cells were incubated with BDP-N3 for 60 min at 37 °C, there was no prominent change of fluorescence in Fig. 5D, E, F, which may be due to the endogenous H2S concentration of cells being quite low when without SNP stimulated. Study revealed that the basal H2S level is about 10–15 nM in murine liver and brain [51–53]. In short, endogenous H2S concentration of cells was not large enough to enhance fluorescence when without SNP stimulation. Thus, from the above results, we propose that BDP-N3 could be a good candidate for detecting intracellular H2S and viscosity.

Moreover, upon addition of H2S to BDP-N3 in DMSO, markedly enhanced fluorescence was displayed. Since the catabolism of H2S is very fast, the fast response times, and low toxicity of BDP-N3 show its importance for determining the fluctuating concentrations of H2S in biological systems. Furthermore, cellular imaging experiments manifested BDP-N3 was cell membrane permeable and could be applied for sensing of both H2S and viscosity in a biological system. Acknowledgments We thank the National Natural Science Foundation of China (No. 21672131, 21775096, 21705102), Talents Support Program of Shanxi Province (2014401), Shanxi Province Foundation for Returness (No. 2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061) and Scientific Instrument Center of Shanxi University (No. 201512). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.dyepig.2018.06.029. References [1] Yu FB, Li P, Song P, Wang BS, Zhao JZ, Han KL. An ICT-based strategy to a colorimetric and ratiometric fluorescence probe for hydrogen sulfide in living cells. Chem Commun 2012;48:2852–4. [2] Liu CY, Wu HF, Han BJ, Zhu BC, Zhang XL. A highly selective fluorescent chemodosimeter for imaging hydrogen sulfide in living cells. Dyes Pigments 2014;110:214–8. [3] Paul BD, Snyder SH. H2S signalling through protein sulfhydration and beyond. Nat Rev Mol Cell Biol 2012;13:499–507. [4] Fukuto JM, Carrington SJ, Tantillo DJ, Harrison JG, Ignarro LJ, Freeman BA, Chen A. Wink, DA. Small molecule signaling agents: the integrated chemistry and biochemistry of nitrogen oxides, oxides of carbon, dioxygen, hydrogen sulfide, and their derived species. Chem Res Toxicol 2012;25:769–93. [5] Liu CR, Chen W, Shi W, Peng B, Zhao Y, Ma HM, Xian M. Rational design and bioimaging applications of highly selective fluorescence probes for hydrogen polysulfides. J Am Chem Soc 2014;136:7257–60.

4. Conclusions In summary, we have developed a viscosity sensitive BODIPY fluorescent probe BDP-N3 with high selectivity for detecting H2S. A dye BDP-N3 is weakly fluorescent in non-viscous media. We studied the fluorescence emission and fluorescence lifetime of BDP-N3 in different ratios of methanol: glycerol. It was observed that the fluorescence intensity and decay time enhanced with an increase in viscosity of the medium, which proved that BDP-N3 acts as a molecular rotor. 171


Q. Zhao et al.

[30] Wang BS, Li P, Yu FB, Song P, Sun XF, Yang SQ, et al. A reversible fluorescence probe based on Se-BODIPY for the redox cycle between HClO oxidative stress and H2S repair in living cells. Chem Commun 2013;49:1014–6. [31] Lou ZR, Li P, Pan Q, Han KL. A reversible fluorescent probe for detecting hypochloric acid in living cells and animals: utilizing a novel strategy for effectively modulating the fluorescence of selenide and selenoxide. Chem Commun 2013;49:2445–7. [32] Qian Y, Karpus J, Kabil O, Zhang SY, Zhu HL, Banerjee R, et al. Selective fluorescent probes for live-cell monitoring of sulphide. Nat Commun 2011;2:495–501. [33] Liu CR, Peng B, Li S, Park CM, Whorton AR, Xia M. Reaction based fluorescent probes for hydrogen sulfide. Org Lett 2012;14:2184–7. [34] Liu TY, Zhang XF, Qiao QL, Zou CY, Feng L, Cui JN, et al. A two-photonfluorescent probe for imaging hydrogen sulfide in living cells. Dyes Pigments 2013;99:537–42. [35] Zhu H, Fan JL, Li M, Cao JF, Wang JY, Peng XJ. A“Distorted-BODIPY”Basedfluorescent probe for imaging of cellular viscosity in live cells. Chem Eur J 2014;20:4691–6. [36] Raut S, Kimball J, Fudala R, Doan H, Maliwal B, Sabnis N, et al. A homodimeric BODIPY rotor as a fluorescent viscosity sensor for membrane-mimicking and cellular environments. Phys Chem Chem Phys 2014;16:27037–42. [37] Yang Z, He Y, Lee JH, Chae WS, Ren WX, Lee JH, et al. A Nile Red/BODIPYbased bimodal probe sensitive to changes in the micropolarity and microviscosity of the endoplasmic reticulum. Chem Commun 2014;50:11672–5. [38] Wang L, Xiao Y, Tian WM, Deng LZ. Activatable rotor for quantifying lysosomal viscosity in living cells. J Am Chem Soc 2013;135:2903–6. [39] Ding YB, Zhu WH, Xie YS. Development of ion chemosensors based on porphyrin analogues. Chem Rev 2017;117:2202–56. [40] Bu LL, Chen JQ, Wei XD, Li X, Agren H, Xie YS. An AIE and ICT based NIR fluorescent probe for cysteine and homocysteine. Dyes Pigments 2017;136:724–31. [41] Wei XD, Bu LL, Tang WQ, Zhao SL, Xie YS. Selective and sensitive fluorescence "turn-on" Zn2+ probes based on combination of anthracene, diphenylamine and dipyrrin. Sci China Chem 2017;60:1212–8. [42] Wang Q, Wei XD, Li CJ, Xie YS. A novel p-aminophenylthio- and cyano- substituted BODIPY as a fluorescence turn-on probe for distinguishing cysteine and homocysteine from glutathione. Dyes Pigments 2018;148:212–8. [43] Wang Q, Ma FT, Tang WQ, Zhao SL, Li CJ, Xie YS. A novel nitroethylene-based porphyrin as a NIR fluorescence turn-on probe for biothiols based on the Michael addition reaction. Dyes Pigments 2018;148:437–43. [44] Zhang J, Zhou JL, Dong XC, Zheng X, Zhao WL. A near-infrared BODIPY-based fluorescent probe for the detection of hydrogen sulfide in fetal bovine serum and living cells. RSC Adv 2016;6:51304–9. [45] Tanmoy S, Dnyaneshwar K, Pinaki T. A colorimetric and fluorometric BODIPY probe for rapid, selective detection of H2S and its application in live cell imaging. Org Biomol Chem 2013;11:8166–70. [46] Su DD, et al. Synthesis and systematic evaluation of dark resonance energy transfer (DRET)-Based library and its application in cell imaging. Chem Asian J 2015;10:581–5. [47] Yeonju B, Sang JP, Xin Z, Gyungmi K, Hwan MK, Juyong Y. A viscosity sensitive fluorescent dye for real-time monitoring of mitochondria transport in neurons. Biosens Bioelectron 2016;86:885–91. [48] Xu J, Li Q, Yue Y, Guo Y, Shao SJ. A water-soluble BODIPY derivative as a highly selective “Turn-On” fluorescent sensor for H2O2 sensing in vivo. Biosens Bioelectron 2014;56:58–63. [49] Knut R, Matthias K, Ute R, Jörg D. A selective and sensitive fluoroionophore for HgII, AgI, and CuII with virtually decoupled fluorophore and receptor units. J Am Chem Soc 2000;122:968–89. [50] Wang X, Song FL, Peng XJ. A versatile fluorescent probe for imaging viscosity and hypochlorite in living cells. Dyes Pigments 2016;125:89–94. [51] Mark CW. Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 1999;47:1101–9. [52] Furne J, Saeed A, Levitt MD. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values. Am J Physiol Regul Integr Comp Physiol 2008;295:R1479–85. [53] Levitt MD, Abdel-Rehim MS, Furne J. Free and acid labile hydrogen sulfide concentrations in mouse tissues: anomalously high free hydrogen sulfide in aortic tissue. Antioxidants Redox Signal 2011;15:373–8.

[6] Yang GD, Wu LY, Jiang B, Wei Y, Qi JS, Cao K, Meng QH, Mustafa AK, Mu WT, Zhang SM. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. Science 2008;322:587–90. [7] Li L, Bhatia M, Zhu YZ, Zhu YC, Ramnath RD, Zhong JW, Farhana BMA, Matthew W, Manuel ST, Philip K. Hydrogen sulfide is a novel mediator of lipopolysaccharideinduced inflammation in the mouse. FASEB J 2005;19:1196–8. [8] Andreas P, Anastasia P, Zaid A, Yang GD, Antonia M, Zhou ZM, Mark G, Ludwik K, David N, Wang R, Csaba S. Hydrogen sulfide is an endogenous stimulator of angiogenesis. Proc Natl Acad Sci Unit States Am 2009;106:21972–7. [9] Yang GD, Wu LG, Wang R. Pro-apoptotic effect of endogenous H2S on human aorta smooth muscle cells. FASEB J 2006;20:553–5. [10] Miller TW, Isenberg JS, Roberts DD. Molecular regulation of tumor angiogenesis and perfusion via redox signaling. Chem Rev 2009;109:3099–124. [11] Kamoun P, Belardinelli MC, Chabli A, Lallouchi K, Chadefaux-Vekemans B. Endogenous hydrogen sulfide overproduction in down syndrome. Am J Med Genet 2003;116:310–1. [12] Eto K, Asada T, Arima K, Makifuchi T, Kimura H. Brain hydrogen sulfide is severely decreased in Alzheimer's disease. Biochem Biophys Res Commun 2002;293:1485–8. [13] Stefano F, Elisabetta A, Eleonra D, Giovanni R, Andrea M, Stefano O, Renata Z, Benata Z, Barbara R, Moses DS, Antonion M, Giuseppe C, John L. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs. Gastroenterology 2005;129:1210–24. [14] Fiorucci S, Antonelli E, Mencarelli A, Orlandi S, Renga B, Rizzo G, Distrutti E, Shah V, Morelli A. The third gas: H2S regulates pressure in both the isolated and perfused normal rat liver and in cirrhosis. Hepatology 2005;42:539–48. [15] Wei HL, Zhang CY, Jin HF, Tang CS, Du JB. Hydrogen sulfide regulates lung tissueoxidized glutathione and total antioxidant capacity in hypoxic pulmonary hypertensive rats. Acta Pharmacol Sin 2008;29:670–9. [16] Yang W, Yang GD, Jia XM, Wu LG, Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms. J Physiol 2005;569:519–31. [17] Neha G, Shahi IR, Vandana B, Muskan G, Gurcharan K, Manoj K. A bodily based dual functional probe for the detection of hydrogen sulfide and H2S induced apoptosis in cellular systems. Chem Commun 2015;51:10875–8. [18] Yang ZG, Cao JF, He YX, Yang JH, Kim T, Peng XJ, Kim GS. Macro-/micro-environment-sensitive chemosensing and biological imaging. Chem Soc Rev 2014;43:4563–601. [19] Kuimova MK. Mapping viscosity in cells using molecular rotors. Phys Chem Chem Phys 2012;14:12671–86. [20] Peng XJ, Yang ZG, Wang GY, Fan JL, He YX, Song FL, et al. Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity. J Am Chem Soc 2011;133:6626–35. [21] Deliconstantinos G, Villiotou V, Stavrides JC. Modulation of particulate nitric oxide synthase activity and peroxynitrite synthesis in cholesterol enrichedendothelial cell membranes. Biochem Pharmacol 1995;49:1589–600. [22] Moriarty PM, Gibson CA. LDL apheresis and its effect on viscosity. Cardiovasc Rev Rep 2003;24:321–5. [23] Tsai D-M, Kumar AS, Zen J-M. A highly stable and sensitive chemically modified screen-printed electrode for sulfide analysis. Anal Chim Acta 2006;556:145–50. [24] Jimnez D, Martinez-Manez R, Sancenon F, Ros-Lis JV, Benito A, Soto J. A new chromo-chemodosimeter selective for sulfide anion. J Am Chem Soc 2003;125:9000–1. [25] Furne J, Saeed A, Levitt MD. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values. Am J Physiol Regul Integr Comp Physiol 2008;295:R1479–85. [26] Péter N, Zoltán P, Attila N, Barna B, Imre T, Anita V. Chemical aspects of hydrogen sulfide measurements in physiological samples. Biochim Biophys Acta 2014;1840:876–91. [27] Leticia AM, Shen XG, James JM, Christopher GK, Michael DP. Mechanistic investigations reveal that dibromobimane extrudes sulfur from biological sulfhydryl sources other than hydrogen sulfide. Chem Sci 2015;6:294–300. [28] Lippert AR, New EJ, Chang CJ. Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells. J Am Chem Soc 2011;133:10078–80. [29] Chen S, Chen ZJ, Ren W, Ai HW. Reaction-based genetically encoded fluorescent hydrogen sulfide sensors. J Am Chem Soc 2012;134:9589–92.

172