A mitochondria-targeted two-photon fluorescent probe for sensing and imaging pH changes in living cells

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117435

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A mitochondria-targeted two-photon fluorescent probe for sensing and imaging pH changes in living cells Xueqin Jiang a, b, 1, Zengjin Liu c, 1, Youzhe Yang b, Hao Li b, Xiaoyi Qi a, d, Wen Xiu Ren b, d, Mingming Deng b, Muhan Lü b, *, Jianming Wu a, *, Sicheng Liang b, a, d, ** a

The Pharmacy School of Southwest Medical University, Luzhou, China The Affiliated Hospital of Southwest Medical University, Luzhou, China The Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University, Luzhou, China d Nuclear Medicine and Molecular Imaging Key Laboratory of Sichuan Province, Luzhou, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 June 2019 Received in revised form 22 July 2019 Accepted 27 July 2019 Available online 29 July 2019

A novel two-photon pH probe, 3-benzimidazole-7-hydroxycoumarin (BHC), was designed and synthesized based on the structures of hydroxycoumarin and benzimidazole. BHC showed good linearity in the pH ranges of 3.30e5.40 (pKa ¼ 4.20) and 6.50e8.30 (pKa ¼ 7.20) at a maximum emission wavelength of 480 nm. BHC in acidic and alkaline media could be distinguished by an obvious spectral shift of the maximum absorption wavelength from 390 nm to 420 nm. In addition, BHC was well localized to mitochondria and successfully applied to one-photon and two-photon imaging of pH changes in the mitochondria of HeLa cells. The findings presented herein suggest that BHC can serve as an excellent fluorescent probe for selectively sensing mitochondrial pH changes with remarkable photostability and low cytotoxicity. © 2019 Elsevier B.V. All rights reserved.

Keywords: Benzimidazole 7-Hydroxycoumarin Fluorescent probe Two-photon Mitochondrial pH

1. Introduction The pH is a key factor for the regulation of cellular microenvironments, and plays an important role in the processes of cell growth and apoptosis [1,2], endocytosis [3], enzymatic activity [4], and ion transport [5]. In addition, intracellular pH is organellespecific. It is well known that lysosomes and endosomes have acidic compartments (pH 4.5e6.5), while a neutral pH is noted for mitochondria and cytosol (pH 6.8e7.4) [6,7]. In contrast, abnormal pH values in these organelles were reported to be associated with cellular dysfunctions [8], thereby leading to many diseases, such as cancer, neurodegenerative disorders and Alzheimer's disease [9e11]. For example, acidification in mitochondria can cause mitophagy and depolarization, and lead to serious damage to cells and tissues ultimately [12]. Therefore, quantitative determination of changes in intracellular pH is of great importance for cellular analysis and diagnosis. Fluorescent detection has become an indispensable tool to investigate biological events due to its simple operation, fast

* Corresponding authors. ** Correspondence to: S. Liang, The Affiliated Hospital of Southwest Medical University, Luzhou, China. E-mail addresses: [email protected] (M. Lü), [email protected] (J. Wu), [email protected] (S. Liang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.saa.2019.117435 1386-1425/© 2019 Elsevier B.V. All rights reserved.

response, high selectivity, and unparalleled spatiotemporal resolution [13,14]. With high sensitivity and visibility, fluorescencebased techniques have been widely applied for imaging and sensing of intact and subcellular pH levels [15,16]. In recent years, a number of pH fluorescent probes have been developed on the basis of different fluorophores (e.g., hemicyanine, coumarin, curcumin, perylene and naphthalene) [17e23]. However, many challenges still need to be overcome. Above all, probes with a single proton dissociation or binding site have difficulty in achieving wide-range pH sensing [22]. Additionally, intracellular pH sensing in live cells could be interfered by background emissions [24e27]. Therefore, probes with near-infrared emissions or two-photon excited emissions could be highly desirable. With a pKa value of 5.80, the benzimidazole group could be appropriate for the development of acidic pH fluorescent probes [28,29]. For acidic pH detection, 7-hydroxycoumarin (pKa ¼ 7.50) could be a suitable structure responsible for proton transformation [30]. More importantly, it has been reported that the introduction of a benzimidazole group into a coumarin molecule not only can drive intramolecular charge transfer (ICT), but also lead to a twophoton fluorescence profile [28,29,31,32]. In addition, an improvement in the fluorescence intensity could be predicated when the protonation at the benzimidazole/benzothiazole nitrogen or the deprotonation at the phenolic hydroxyl oxygen is occurred [28,33,34]. Inspired by these observations, we assumed that it is

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possible to design and develop a novel two-photon fluorescence probe for wide-range detection of pH by combining the merits of the benzimidazole group and 7-hydroxycoumarin. Herein, we have reported a two-photon fluorescence probe, BHC, for pH detection in aqueous solution and living cells. As a novel pH-sensitive probe, BHC has a large stokes shift, a good quantum yield, and promising specificity. In particular, it is capable of measurements over the two pH ranges of 3.30e5.40 and 6.50e8.30. More importantly, BHC was found to have a good mitochondria-targeting ability, and it was successfully applied for one- and two-photon imaging and sensing of pH changes in the mitochondria of living cells. The present results strongly demonstrated that BHC could serve as an excellent fluorescence probe to monitor mitochondrial pH fluctuations in living biological samples. 2. Experimental section

dihydroxybenzaldehyde (0.76 g, 5.5 mmol), and 2-(1H-benzo[d] imidazol-2-yl) acetonitrile (0.79 g, 5 mmol) in 15 mL mixed liquids of piperidine, ethanol, and con. HCl (v/v/v, 16/3/1) was heated with stirring at 70  C for 24 h. After removing the mixed liquids by distillation, the resulting mixture was treated with 2.0 mL ice water, and the precipitates were collected by filtration. Crude BHC was recrystallized from ethanol to afford a fine product (1.3 g) in 94% yield as a pale yellow solid. The structure of BHC was fully characterized by 1H NMR and 13C NMR. 1H NMR (400 MHz, DMSO‑d6): dH (ppm) ¼ 6.86 (d, 1H, H3), 6.89e6.92 (dd, 1H, J ¼ 12, 4 Hz, H1), 7.18e7.22, 7.65 (m, brs, 4H, H18e21), 7.83e7.86 (d, 1H, J ¼ 12 Hz, H6), 9.06 (s, 1H, H10), 10.98 (brs, 1H, NH), 12.44 (s, 1H, OH). 13C NMR (100 MHz, DMSO‑d6): d (ppm) 163.1, 160.2, 156.0, 146.9, 143.5, 143.3, 135.2, 131.7, 122.8, 122.4, 118.7, 114.6, 113.1, 112.3, 112.1, 102.5 þ (Fig. S1). HRMS (ESI) m/z calcd. for C16H11N2Oþ 3 (M) 279.0764,  found 279.0705; HRMS (ESI) m/z calcd. for C16H9N2O 3 (M) 277.0607, found 277.0618 (Fig. S2).

2.1. Materials and methods 2.3. UVeVis absorption and fluorescence spectroscopy

All materials and reagents were commercially available and used without purification. Ultrapure water was used throughout the experiments. Trypsin-EDTA solution, penicillin, streptomycin, and Mito-Tracker Green (MTG) were purchased from Beyotime Biotechnology (Nantong, China). Mito-Tracker Red (MTR) for imaging was acquired from Feiyu Biotechology (Nantong, China). Dulbecco's modified Eagle medium (DMEM) and Fetal Bovine Serum (FBS) were acquired from Thermo Fisher Scientific. Nigericin and monensin were purchased from Yuanye Biotechnology (Shanghai, China). Other reagents, including metal ions, amino acids, buffer solutions preparation, and chemicals for synthesis were obtained from Cologne Chemical Co. (Chengdu, China). The Britton-Robinson (BR) buffers were prepared by mixing 40 mM acetic acid, boric acid, and phosphoric acid. The pH value was adjusted with small amounts of diluted NaOH or HCl solutions by a pHS-3E acidity meter. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained on a Bruker AMX-400. The high resolution mass spectrometer (HRMS) spectra were measured with the X500R Q-TOF system AB Sciex (Danaher, Framingham, MA).

The relative quantum yield of BHC was determined according to the following equation:

2.2. Synthesis and characterization of BHC

Fx ¼ Fst  ðDx =Dst ÞðAst =Ax Þ hx 2 hst 2

As depicted in Scheme 1, BHC was synthesized according to the previous report with slight modification [35]. A mixture of 2,4-

which calculated the quantum yield of a compound under methanol and acid-alkali conditions, where F, D and A refer to the

A stock solution of BHC was prepared in DMSO. Metal ions (Naþ, K , Ca2þ, Zn2þ, Mg2þ, Mn2þ, Cu2þ, Co2þ, Ni2þ,Cd2þ, Pb2þ, Fe2þ, and Fe3þ), thiols (GSH, Cys, and Hcy) and related analytes of some amino acids were prepared in water solution. All experiments were conducted in DMSO/BR buffer (40 mM, 1/99, v/v) to prepare buffer solutions of different pH levels. The actual pH was determined by adding a solution of 0.2 M NaOH or 0.2 M HCl to the initial buffer solutions. All absorption and fluorescence spectra were measured on a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek) at room temperature. The excitation was performed at 390 nm (pH 3.0e6.0) and 420 nm (pH 6.0e9.0), and the excitation and emission slit widths were set at 5 nm. þ

2.4. Calculation of the quantum yield




.



Scheme 1. (a) The synthetic route of BHC. (b) The proposed mechanism of equilibrium for BHC and its protonated and deprotonated forms.

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quantum yield of the standard, the area under the emission spectra and the absorbance at the excitation wavelength. h represents the refractive index of the solvent used. The subscripts x and st represent the sample and the standard, respectively. Rhodamine B (Fst ¼ 0.69 in MeOH) was used as the reference standard.

NMR and TOF-MS (Figs. S1eS2). The signal 6.86e7.86 ppm in 1H NMR showed that there was seven AreH in the molecule, and the signal at 12.44, 10.98 and 9.06 ppm can be ascribed to NH, OH, and C]CH (H4), respectively. All these firmly identified the structure of compound BHC.

2.5. Cell viability

3.2. Spectroscopic properties of BHC to pH

To evaluate the cytotoxic effect of BHC in HeLa cells, a CCK-8 (Cell Counting Kit-8, Dojindo, Japan) assay was performed according to the manufacture's protocol. The cells with a density of 5  103 cells/well were cultured in a 96-well microplate to a total volume of 200 mL per well at 37  C in a 5% CO2 atmosphere. After overnight cultivation, different concentrations of BHC (1, 5, 10, 25, 50, 100 and 200 mM) were further incubated with HeLa cells in fresh medium for 24 h. Next, the cells were washed with phosphate buffered saline (PBS, pH 7.4) three times. 100 mL of 10% CCK-8 solution (CCK-8/ DMEM ¼ 1/9) was added to each well for 1 h. Then, the absorbance was measured at 450 nm wavelength with a microplate reader. The cell viability was determined by the following equation:

where Ai is the absorbance of different concentrations of the probe (1, 5, 10, 30, 50, 100 and 200 mM). Acontrol is the average absorbance of the control well where the probe was absent, and A0 is the absorbance of different concentrations of the probe in the absence of cells.

As shown in Fig. 1, the spectra of 2 mM BHC had a great difference with acid-alkali media. When the pH was changed from 3.0 to 6.0, BHC showed a significantly large stokes shift of 90 nm with an absorption maximum (labs) of 390 nm and an emission maximum (lem) of 480 nm. Interestingly, both intensity of absorption at 390 nm and fluorescence at 480 nm were dramatically reduced along with the pH being to 6.0. In contrast, when the pH was changed from 6.0 to 9.0, BHC displayed a stokes shift of 60 nm with labs of 420 nm and lem of 480 nm. Compared with those observations in the pH range of 3.0e6.0, an opposite trend for the intensity at labs and lem was noted. Upon excitation at 365 nm UV light, bluegreen fluorescence could easily be observed at pH of 3.0 and 9.0 compared with the colorless response at pH 6.0. In addition, the fluorescence quantum yield of BHC was also measured and the fluorescence quantum yield was large (F ¼ 0.646) relative to rhodamine B (Fst ¼ 0.69 in MeOH). By comparing the performance of BHC with that of other pH probes, it is suggested that BHC has several advantages over other pH probes, such as high fluorescence quantum yield and suitable range for mitochondrial pH detection (Table S1) [18-21,29].

2.6. Cell culture and imaging

3.3. pKa Values of BHC

HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (WelGene, Daegu, Korea), penicillin (100 units/mL), and streptomycin (100 mg/mL), and incubated at 37  C in a 5% CO2 humidified atmosphere. Then, the cells were seeded in a 20 mm diameter round glass Petri dish at a density of 1.5  105 cells/well. After being incubated for 24 h, the cells were washed three times with PBS (pH 7.4), and BHC (10 mM) dissolved in DMSO was added to the cell medium (1 mL) at a final concentration of 10 mM. The cells were washed three times with PBS before confocal microscope imaging. To evaluate biological applications of BHC, the subcellular distribution and pH fluorescence imaging of BHC were further tested. Cells were seeded on Petri dish at a density of 1.5  105 cells/well for 24 h. The medium was replaced with BHC and incubated for 45 min. Excess BHC was removed by gently rinsing the Petri dish with PBS (pH 7.4) three times, and then it was treated with the commercial mitochondrial dye MTG or MTR for another 45 min. Fluorescence imaging was acquired with a Nikon C1 confocal laserscanning microscope. To further observe the pH changes in live cells, about 1.5  105 HeLa cells were seeded onto a 20 mm diameter round glass Petri dish and incubated for 24 h. The medium was then removed and incubated with BHC (10 mM) and MTR (75 nM) for 45 min. Then, the medium was replaced with different PBS buffers (pH 4.0, 6.0, and 8.0) containing 10 mM nigericin and monensin. After incubation for another 25 min, the medium was subsequently removed and fluorescence images were collected with a Nikon C1 confocal laserscanning microscope.

To obtain the pKa values of BHC, the fluorescence intensities of pH 2.0e6.0 (lex ¼ 390 nm) and pH 6.0e9.0 (lex ¼ 420 nm) at 480 nm were measured by sigmoidal nonlinear fitting. As shown in Fig. 2, pKa values were 4.20 for pH 2.0e6.0 and 7.20 for pH 6.0e9.0. Moreover, the molar extinction coefficients of the maximum absorption wavelengths of 390 nm and 420 nm were measured under the conditions of pH 2.0e5.8 and pH 5.8e9.0, respectively, according to Lambert-Beer's law. By the nonlinear regression analysis, the pKa values were calculated to be 4.13 for pH 2.0e5.8 and 7.27 for pH 5.8e9.0, suggesting that BHC is capable of assessing both acidic and weakly alkaline media (Fig. S3). The possible mechanism for BHC with two pKa values could be ascribed to the nitrogen atom in the imidazole as a proton acceptor and the oxygen atom of the hydroxyl group as a proton donor [26,28]. Moreover, absorption and emission spectral shifts of BHC could be due to the function of protonation and deprotonation occurred in acid-alkali media (Scheme 1). To verify the proposed mechanism, the 1H NMR spectra of BHC in different pH solution were measured in DMSO‑d6. As shown in Fig. S4, a protonated form of BHC was observed in pH 3.0 acid media with downfield chemical shifts of AreH close to the protonable nitrogen atom in the imidazole ring. In contrast, a deprotonated form of BHC was observed in pH 9.0 alkali media with upfield chemical shifts of AreH close to the deprotonable oxygen atom in the hydroxyl group.

Viability% ¼ ½Ai  A0 =½Acontrol  A0   100%

3. Results and discussion 3.1. Synthesis of BHC Scheme 1 shows the synthetic route adopted for the preparation of BHC, whose structure was further characterized by 1H NMR, 13C

3.4. Response to pH within the acid-alkali range The pKa of the probe must be well matched to the pH range because each probe has its usable quantitative pH response in an approximate pKa. Interestingly, the probe also displayed good linearity between the fluorescence intensity and the pH in the ranges of 3.36e4.98 and 6.51e8.24, with correlation coefficients (R2) of 0.992 and 0.978, respectively (Fig. 2). The findings demonstrated that BHC could be qualified to quantitatively detect a wide range of pH changes.

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Fig. 1. Absorption (a) and fluorescence (b) spectra of BHC (2 mM) in BR buffer solutions at pH varying from 3.0 to 6.0. Absorption (c) and fluorescence (d) spectra of BHC (2 mM) in BR buffer solutions at pH varying from 6.0 to 9.0. Insets in (b) and (d) show the photograph and fluorescence images of the BHC solution under UV light (365 nm), respectively. The data in (b) and (d) were obtained in BR buffer solutions from excitation at 390 and 420 nm, respectively.

3.5. Photostability and reversibility of BHC The photostability of BHC was determined by monitoring changes in the fluorescence intensity for 2 h. As depicted in Fig. S5, the fluorescence intensity was stable within 2 h at pH of 3.0, 4.5, 6.0 and 7.3. This indicated that the probe not only has excellent photostability and photobleaching indices, but can instantly respond to changes in the proton concentration. As reported, the reversibility of the probe is highly required for monitoring the dynamic changes in pH levels in living cells [36]. In this study, the fluorescence intensity of BHC was measured repeatedly in BR buffers over pH 3.0, 6.0 and 9.0. From pH 3.0e6.0 (Fig. 2), the fluorescence intensity of BHC was gradually quenched. With respect to a pH of 6.0e9.0 (Fig. 2), the fluorescence intensity obviously recovered from a trough to a peak. The underlying mechanism for a recovery of the fluorescence intensity of BHC from pH 6.0 to 9.0 could be due to ICT induced by deprotonation of the hydroxyl group in coumarin [32,33]. On the other hand, the hindrance of electron transfer could occur when the amino atom in imidazole is protonated, thus in turn leading to the fluorescence enhancement from pH 6.0 to 3.0 [37]. Furthermore, the solution color changed repeatedly between colorless (pH 6.0) and blue-green (pH 9.0), indicating that BHC exhibits an excellent pH-reversibility.

3.6. Selectivity of BHC It is important to characterize the selectivity of a given probe to resist various interference factors, such as metal ions in solutions and the complexity of intracellular environments. Thus, experiments were conducted to investigate whether BHC had a preferential interaction with protons over other kinds of cations in different pH media. As shown in Fig. 3, little interference was observed for the fluorescence intensity of BHC in pH 6.0 and 7.3 media when it was

incubated with different physiologically ubiquitous metal cations (e.g., Kþ, Naþ, Ca2þ and Mg2þ), other metal cations (e.g., Fe3þ, Cu2þ and Ni2þ) or common amino acids (e.g., Cys, Ala and Gly). The results demonstrated that BHC has excellent selectivity toward protons over different metal cations and amino acids.

3.7. Cell viability test The cytotoxicity of BHC was evaluated using a CCK-8 assay, and the results showed that BHC had little effect on the viability of HeLa cells. Of note, the cell viabilities were higher than 80% even when the concentration of BHC was as large as 200 mM (Fig. S6). This suggested that BHC could be used for sensing the changes of intracellular pH with negligible cytotoxicity.

3.8. Two-photon fluorescence microscopy and cross section Two-photon fluorescence microscopy images of BHC-labeled cells were obtained with confocal and multiphoton microscopes. In this study, BHC (10 mM) was coincubated with HeLa cells, and the confocal fluorescence images were recorded with one-photon and two-photon modes. As shown in Fig. 4, cells loaded with BHC (10 mM) for 45 min exhibited strong blue fluorescence for the onephoton image, and green fluorescence for the two-photon image. These results indicated that BHC could be used for imaging of living cells with two-photon excited fluorescence. The two-photon cross section (d) was determined by using femtosecond (fs) fluorescence measurement techniques according to a previous report [38]. As shown in Fig. S7, the two-photon cross-sectional area was 11.3 GM at the top site of 800 nm.

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Fig. 2. Sigmoidal fitting of the fluorescence intensity for 2 mM BHC at 480 nm with lex at 390 nm (a) and lex at 420 nm (b), respectively. Reversible fluorescence changes of BHC between pH 3.0 and 6.0 at lex 390 nm (c), and between pH 6.0 and 9.0 at lex 420 nm (d).

3.9. Intracellular fluorescence imaging To evaluate the potential biological applications of the probe, cell imaging experiments were performed in HeLa cells with a confocal laser scanning microscope. As displayed in Fig. 4, BHC focused on the cytoplasm and dispersed blue fluorescence emissions, revealing a good cell membrane permeability of BHC. Due to the richness of mitochondria existing in the cytoplasm, colocalization experiments were conducted to study the mitochondrial

staining ability of the probe. Surprisingly, BHC had the same distributions as MTR on the basis of fluorescence imaging (Fig. 4). Moreover, the image of BHC merged well with the MTG when they were coincubated with HeLa cells (Fig. S8). The findings demonstrated that BHC has a robust staining ability for mitochondria. To further estimate cellular pH values in HeLa cells, BHC was first coincubated with MTR. Then, media was replaced with various PBS buffers containing nigericin and monensin, which are known as Hþ/Kþ ionophores for cell imaging [39]. As reported, the

Fig. 3. Fluorescence response of the BHC (2 mM) to different potential interferents in BR buffer solutions at pH 6.0 and 7.3: (1) Blank; (2) Kþ; (3) Naþ; (4) Ca2þ; (5) Mg2þ; (6) Fe3þ; (7) Zn2þ; (8) Cu2þ; (9) Pb2þ; (10) Mn2þ; (11) Co2þ; (12) Ni2þ; (13) Cd2þ; (14) Fe2þ; (15) HCO23; (16) Tyr; (17) Ala; (18) Lys; (19) Gly; (20) Met; (21) Phe; (22) Thr; (23) Tyr; (24) Iso; (25) Arg; (26) Ser; (27) Leu; (28) Glu; (29) His; (30) GSH; (31) Cys; (32) Hcy. The concentration was 1 mM for (2)e(5), 0.2 mM for (6)e(15) and 0.1 mM for others. The wavelengths were set at 420 nm (lex) and 480 nm (lem).

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Fig. 4. One-photon and two-photon confocal fluorescence images of HeLa cells. (a) One-photon imaging of cells costained with BHC (10 mM) for 45 min (lex ¼ 405 nm, lem ¼ 425e475 nm). (b) Two-photon imaging of cells costained with BHC (10 mM) for 45 min (lex ¼ 800 nm, lem ¼ 475e540 nm). (c) HeLa cells costained with BHC (10 mM) and MTR (75 nM) (lex ¼ 559 nm, lem ¼ 575e620 nm). (d) Merged image. Scale bar: 10 mm.

Fig. 5. Confocal microscopy images of HeLa cells costained with BHC (10 mM) and MTR (75 nM) in buffers at pH 4.0 (a), 6.0 (b), and 8.0 (c). For blue channel (a1, b1, c1, lex ¼ 405 nm, lem ¼ 425e475 nm), red channel (a2, b2, c2, lex ¼ 559 nm, lem ¼ 575e620 nm), and merged (a3, b3, c3). (d) Comparison of the fluorescence intensity ratio of blue (BHC) and red (MTR) channel from at different pH buffers. Scale bar: 10 mm.

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Fig. 6. Mitochondrial pH changes in nutrient-deprived (NP) and intact HeLa cells. Confocal microscopy images of HeLa cells pretreated with BHC (10 mM) and MTR (75 nM) in growth medium (for intact cells) and in serum-free medium (for nutrient-deprived cells). For blue channel (a1, b1, lex ¼ 405 nm, lem ¼ 425e475 nm), and red channel (a3, b3, lex ¼ 559 nm, lem ¼ 575e620 nm). a2 and b2 were the corresponding bright images of a1/a3 and b1/b3. Scale bar: 10 mm.

fluorescence intensity of MTR is little affected by changes in pH. Thus, MTR could be a good reference when quantitatively determining mitochondrial pH changes [21]. As shown in Fig. 5d, among the pH values that were tested, the fluorescence intensity ratio of BHC and MTR at pH 4.0 and 8.0 showed a higher fluorescence intensity than that at pH 6.0. In addition, confocal fluorescence images revealed emissions from both BHC and MTR that had an original location in the mitochondria of HeLa cells. The findings suggested that BHC has capacity to track pH fluctuations in mitochondria. Starvation model was employed to verify whether probe BHC can be used to identify mitochondrial damage associated with cell dysfunctions or cell apoptosis. It has been reported that nutrient deprivation could damage mitochondria through metabolic inhibitions and lead to mitochondrial acidification, which in turn is associated with increased levels of phagocytic cells [40]. Thus, HeLa cells were coincubated with BHC and MTR in serum-free medium. Changes to the fluorescence intensity were obtained using confocal microscopy. As shown in Fig. 6, the fluorescence intensity was significantly enhanced after starvation-induced mitochondrial acidification. Additionally, the pH of cells subjected to nutrient deprivation was lower than that of normal cells. These results clearly demonstrated that BHC could service as a probe for monitoring mitochondrial pH changes and corresponding pathogenic states in living cells. Despite having a mitochondrion-targeted ability, there is no triphenylphosphonium in the structure of BHC, which is widely used as a mitochondria-targeting substitute [41]. Thus, the location of BHC in mitochondria could be ascribed, at least partly, to the structure of 7-hdroxycoumarin. Coincidentally, studies have shown

that 7-hdroxycoumarin-containing probes tend to locate in the mitochondria [33,42,43]. Further studies are needed to study the underlying mechanism of 7-hdroxycoumarin substitute with mitochondrial localization. 4. Conclusions In conclusion, a two-photon probe (BHC) was developed for selectively and sensitively sensing mitochondrial pH changes in live cells. With a benzimidazole group in a 7-hydroxycoumarin structure, BHC shows a suitable pKa value in response to mitochondrial pH changes from 6.0 to 9.0. Additionally, it exhibits promising specificity, high sensitivity, low cytotoxicity, and a good mitochondrial location and is therefore capable of detecting mitochondrial pH in live cells. Moreover, mitochondrial pH imaging in a starvation-induced cell model has been successfully performed using BHC as pH sensor. These findings demonstrated that BHC could act as a powerful imaging tool for investigation of mitochondrial pH in biomedical research. Acknowledgements This work was supported by the National Natural Science Foundation (81672458), the Grants from the Science and Technology Planning Project of Sichuan Province (2016JY0101, 2018JY0237, 2019JDPT0010), the Educational Commission of Sichuan Province (18ZA0528, 18TD0051), the Health Commission of Sichuan Province (18PJ019), the Joint Fund of Luzhou City and Southwest Medical University (2016LZXNYD-T03, 2018LZXNYD-ZK18, 2018LZXNYDZK28), the Talent Introduction Startup Fund of Affiliated Hospital of

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Southwest Medical University (18052, 18054), and the Open Program of Nuclear Medicine and Molecular Imaging Key Laboratory of Sichuan Province (HYX18010, HYX18002). Declaration of competing interest

[21]

[22]

The authors declare that they have no conflict of interest. [23]

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117435.

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