Ratiometric imaging of mitochondrial pH in living cells with a colorimetric fluorescent probe based on fluorescein derivative

Accepted Manuscript Title: Ratiometric imaging of mitochondrial pH in living cells with a colorimetric fluorescent probe based on fluorescein derivative Authors: Guli Li, Bei Zhang, Xinbo Song, Ying Xia, Haibo Yu, Xinfu Zhang, Yi Xiao, Youtao Song PII: DOI: Reference:

S0925-4005(17)31086-9 http://dx.doi.org/doi:10.1016/j.snb.2017.06.065 SNB 22536

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

30-9-2016 8-6-2017 10-6-2017

Please cite this article as: Guli Li, Bei Zhang, Xinbo Song, Ying Xia, Haibo Yu, Xinfu Zhang, Yi Xiao, Youtao Song, Ratiometric imaging of mitochondrial pH in living cells with a colorimetric fluorescent probe based on fluorescein derivative, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ratiometric imaging of mitochondrial pH in living cells with a colorimetric fluorescent probe based on fluorescein derivative Guli Li,a† Bei Zhang,b† Xinbo Song,c† Ying Xia,a Haibo Yua*, Xinfu Zhang,c Yi Xiaoc* and Youtao Song,a* a.College of Environmental Sciences, Liaoning University, Shenyang 110036, P.R.China, email: [email protected] b.Department of Chemistry, University of Kentucky, Lexington, Kentucky, 40506, USA, email: [email protected] c.State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012,P .R. China. email: [email protected]

*Corresponding authors. Email addresses: [email protected] (H. Yu), [email protected] (Y. Xiao) and [email protected] (Y. Song)

ARTICLEINFO Article history: XXXXXX

Graphical abstract

TOC:

Highlights: 1. A novel strategy used to design ratiometric fluorescent probe (FDI) based on fluorescein fluorophore. 2. FDI provided ratiometric detection by CLSM in dual excitation/dual emission mode. 3. FDI exhibited excellent and fast cell-membrane permeability and mitochondrial-targetability. 4. It would be a potential tool to assess pH fluctuation in mitochondria of live cells.

AB STRACT 1

Mitochondrial pH plays a pivotal role in the regulation of physiological process. Developing ratiometric fluorescent probes for real-time detection of mitochondrial pH fluctuation is still highly demanded yet challenging. Herein, we present a novel strategy to design a ratiometric probe (FDI) by broadening absorption spectrum (360-700 nm) of fluorescein fluorophore. Unsaturated dialkene/indole quaternary ammonium moiety has broadened absorption and emission spectrum of fluorescein fluorophore to provide ratiometric detection by CLSM in dual excitation/dual emission mode, as well as directionally accumulated in mitochondria in living cells. Superior to commercially mitochondrial tracker, Carboxy-SNARF, probe FDI exhibits excellent and fast cell-membrane permeability and mitochondrial-targetability. It has been demonstrated that FDI could permit real-time monitoring of pH alkalization of mitochondria stimulated by chloroquine. Owing to nondestructive process and reversible ratiometric response to pH, image acquisition can be repeated frequently to trace and monitor the time course of mitochondrial pH responses. It is clearly confirmed that FDI would be a promising probe for real-time tracking of mitochondrial pH changes in the biomedical and biological fields. Keywords: Fluorescent probe Fluorescein Mitochondrion-targetable pH monitoring Ratiometric imaging

1. Introduction Mitochondria play pivotal roles in eukaryotic aerobic cells because they serve as the primary generators of aerobic energy production ATP,[1] regulators of ions exchange (Na+/Ca2+, K+/H+),[2,3] producers of reactive oxygen/nitrogen species (H2O2, NO, NOO-, etc.) [4] and the main regulated place of programmed cell death.[5,6] The disfunction of mitochondria involves in many pathogenesis of human diseases, such as oxidative phosphorylation (OxPhos) disease,[7] lactic acidemia,[8] exercise intolerance or neurological disorders (Parkinson disease and Alzheimer disease)[9,10] Of the numerous and complex clusters, e. g. cytochrome c oxidase, complex III, complex II, ATP synthase, photosystem I, mitochondrial pH gradient resulting from the proton motive force of the electron transport chain or mitochondrial respiratory chain is an important factor in the process of energy metabolism and modulation of mitochondrial apoptosis.[11] To synthesize ATP from ADP and phosphate via mitochondrial ATP synthase, mitochondria must generate a proton gradient and proton-motive potential across the inner membrane, which can further regulate Ca2+ homeostasis and other mitochondrial functions involved in the apoptosis process.[11a] In addition, mitochondrial pH also supports other energy-requiring reactions such as ion transport and NAD(P) transhydrogenase reaction.[11a] It is no doubt that any slight changes of mitochondrial pH will make a great influence on mitochondrial biology and associated diseases. Therefore, quantitative spatial and temporal monitoring of mitochondrial pH changes is urgently necessary for mitochondrial and cellular physiology 2

at the individual mitochondrial level in situ. Several methods such as microelectrodes developed to measure average mitochondrial pH in isolated mitochondria have been popularly used in the late 1990’s.[12] However, the pH measurement of individual mitochondria in intact cells has been much more difficult to achieve, since the experimental result obtained using isolated mitochondria has been reported to be different from results obtained in situ.[11b] Compared with the method of isolated mitochondria and microelectrodes, a combination of fluorescent probes and Confocal Laser Scanning Microscopy (CLSM) provides a rapid, facile, and sensitive method to monitor pH fluctuation in suit through the changes of fluorescent intensity, owing to their high resolution, high sensitivity and non-invasive damage for cells or tissues specimen.[13] Recently, our group have reported a ’turn-on’ fluorescent probe for neutral pH based on rhodamine derivatives and illustrated the ability of monitoring pH fluctuation in mitochondria of living cells.[14] Although significant fluorescence enhancement contributes to monitoring mitochondrial pH in cellular imaging, accurate measurement of mitochondrial pH is difficult because both its concentration and fluorescence change as a function of pH value. To overcome this problem, a few work were focused on designing ratiometric probes for mitochondrial pH detection.[15] Carboxy-SNARF,[16] a typical and commercial ratiometric probe, utilizes the ratios of intensity at dual emission wavelengths (580nm/610nm) as signal outputs which could be capable of self-calibration and independent of both excitation light and sample concentrations. Acetoxymethyl (AM) ester form of Carboxy-SNARF has to be chosen in the actual using process, in order to promote permeability through membrane. However, AM-Carboxy-SNARF does not have specifically mitochondrial targetability, since the ultimate intracellular distribution of AM form is dependent on the activity of cytosolic and organelle esterases and the rate of uptake of this form into cytosol or organelles.[17] As we know, fluorescent proteins (FPs) have also been employed to monitor mitochondrial pH in living cells.[18] Nevertheless, compared with organic fluorophores, FPs emit on average ten times less photons than organic fluorescent probes, and an additional disadvantage of FPs are that they may be more perturbative to biology systems than organic fluorescent probes.[19] Moreover, the extremely time-cost consuming preparation of FPs would be higher than that of organic fluorescent probes. Therefore, developing ratiometric fluorescent probes for monitoring mitochondrial pH in living cells continues to be a strong demand and challenge. In this work, a novel strategy has been used to design ratiometric fluorescent probe (FDI) based on fluorescein fluorophore (Scheme 1). In the design of FDI, three components such as fluorescein fluorophore, unsaturated dialkene moiety and substituted indole quaternary ammonium moiety were employed and carried out their duties 3

accordingly. Firstly, the fluorescein fluorophore is pH sensitive to serve as a fluorescent reporter. Secondly, unsaturated dialkene moiety can broaden absorption and emission spectrum of FDI to provide ratiometric detection by CLSM in dual excitation/dual emission mode. Finally, indole quaternary ammonium moiety bearing positive charge works as an anchor to accumulate in mitochondria. The synthetic route is illustrated in Scheme 1. Fluorescein unsaturated monoaldehyde was prepared via an olefination of fluorescein monoaldehyde synthesized by Reimer–Tiemann formylation reaction.[20,21] Via a condensation of 1,3,3-trimethyl-2-methylideneindole and Fluorescein unsaturated monoaldehyde in ethanol under N2 atmosphere, Ratiometric probe FDI was obtained as a black solid with 42% yield.

2. Experimental 2.1 Materials and methods 15-crown-5 and triphenylphosphoranylidene acetaldehyde were purchased from Ouhe Chem (Beijing, China). 1,3,3-trimethyl-2-methylidene- indole and Fluorescein were purchased from Aladdin (Shanghai, China). Fluorescein

monoaldehyde

was

obtained,

according

to

the

method

reported

in

literature.[20,21]

3,6-diamino-9-[2-(methoxy-carbonyl)phenyl]-xanthylium chloride (Rh 123) was purchased from GIBCO (Invitrogen). RPMI 1640 culture medium with L-glutamine and FBS (fetal calf serum) was also purchased from GIBCO (Invitrogen, USA). MCF-7 (human breast carcinoma) was obtained from Institute of Basic Medical Sciences (IBMS) of Chinese Academy of Medical Sciences (CAMS). All other regents such as CHCl3, ethanol and so on were purchased from commercial suppliers and used without further purification. Column chromatography was performed with silica gel (300-400 mesh). 1

H-NMR and

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C-NMR were measured on Varian MERCURY 400 spectrometer in DMSO with TMS as internal

reference. Mass spectra were measured on LTQ Orbitrap XL Mass spectrometers. Fluorescence spectra were measured on Spectrofluorophotometer (Cary Eclipse). Absorbance spectra were recorded on a UV-vis Spectrophotometer (TU-1901). An inverted confocal fluorescent microscopy (IX81, Olympus FV1000, Japan) equipped with an objective lens (×100 oil, 1.4 Numerical Aperture (NA), Scan mode XY) was used in the imaging of living cells. 2.2 General procedure for synthesis of FDI Fluorescein unsaturated monoaldehyde was prepared as reported in literature.[20,21] Briefly, fluorescein 4

monoaldehyde (0.106 g，0.294 mmol) was firstly dissolved in CHCl3 (25 mL), then triphenylphosphoranylidene acetaldehyde (0.111g，0.363mmol) was added into the solution. The mixture was refluxed under N2 for 24 h, cooled to room temperature and the solvent was removed in vacuum. Chromatography on silica gel (1:9 MeOH:CH2Cl2, Rf 0.42) yielded fluorescein unsaturated monoaldehyde (72 mg, 63%) as a red solid. Fluorescein unsaturated monoaldehyde (72 mg, 0.18mmol) and 1,3,3-trimethyl-2-methylidene- indole (57 mg， 0.18 mmol) were stirred in ethanol (5 mL) at 80 °C for 7 h under N2. The resulting mixture was concentrated by evaporation and was purified by silica gel column chromatography with MeOH:CH2Cl2 (1:10, Rf 0.28), affording FDI as a brown solid (41 mg), Yield 42%. FTMS+pESI(+): C35H28NO5(+) Caculed 542.1962, Found 542.1961. 1H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 10.21 (s, 1H), 8.45 (dd, 1H), 8.24 (d, 1H), 8.03

(m, 2H), 7.86 (m,

2H), 7.82 (d, 1H), 7.75 (t, 1H), 7.62 (t, 2H), 7.40 (d, 1H), 7.33 (d, 1H), 7.07 (s, 1H), 6.77 (d, 1H) 6.68 – 6.57 (m, 3H), 4.06 (s, 3H), 1.81 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 181.15, 168.56, 159.70, 159.59, 156.81, 152.11, 151.48, 150.59, 143.36, 141.89, 139.59, 135.69, 132.65, 131.13, 130.23, 128.93, 126.11, 124.71, 124.06, 122.80, 115.71, 114.81, 113.20, 112.49, 110.56, 110.03, 109.36, 102.93, 82.74, 51.73, 45.71, 33.98, 25.45, 8.59. 2.3 Preparation of the test solutions and spectral measurements A 1.0×10-4 M stock solution of probe FDI was prepared in ethanol. To 10 mL volumetric flask, proper amounts of the solution of FDI was added, and then diluted with ethanol to 10 mL. During testing of solvent effect, 300 uL FDI stock solution was added into 10 mL glass bottle containing different solvents, such as dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate, methanol, ethanol, dimethylsulfoxide (DMSO), Dimethyl Formamide (DMF). During pH titration, 5mL the stock solution of FDI was added into the 50 mL volumetric flask, and then diluted with aqueous solution (C2H5OH/H2O (v:v 1:1)). The pH titration of FDI solution was adjusted by conc. HCl and NaOH. Absorption and emission spectra were recorded on UV-Vis Spectrophotometer and Spectroflurophotometer, respectively. 2.4 Cell culture and fluorescence imaging MCF-7 is cultured in RPMI 1640 supplemented with 10% FBS (fetal bovine serum) in an atmosphere of 5% CO2 and 95% air at 37 °C. Grow MCF-7 Cells in the exponential phase of growth on 35-mm glass-bottom culture dishes (Φ 20 mm) for 1-2 days to reach 70-90% confluency. The cells are washed three times with RPMI 1640, and then incubated for 10 min in an atmosphere of 5% CO2 and 95% air at 37 °C with 2 mL RPMI 1640 containing a certain 5

concentration of FDI. Wash cells twice with 2 mL PBS at room temperature, and then add 1 mL RPMI 1640 culture medium and observe under a confocal microscopy. Confocal imaging of cells is performed using 635 nm excitation of a helium-neon laser. For ratiometric imaging of cells, excitation can be performed with 488 nm and 559 nm line of an argon-krypton laser. 2.5 Colocalization experiments MCF-7 cells were incubated with FDI (5 μM) in an atmosphere of 5% CO2 and 95% air at 37 °C for 20 minutes, and then the cells were washed three times with phosphate buffered saline (PBS,pH 7.4). 2 μM Rhodamine 123 (Rh123) was added and co-incubate for another 10 min, and then these cells were washed three times with PBS. Fluorescence images of MCF-7 cells were obtained using a confocal microscope (IX81, Olympus FV1000, Japan) under excitation wavelengths of 488 nm and 635 nm. Emission was collected at 500-535 nm (green channel) and 655-755 nm (red channel), respectively. 2.6 Real-time and ratiometric monitoring of mitochondrial pH in MCF-7 cells stimulated by chloroquine MCF-7 cells were incubated with FDI (5 μM) in an atmosphere of 5% CO2 and 95% air at 37 °C for 20 minutes. Confocal imaging of these cells was performed using 488-nm and 559 nm excitation of lasers, and adjusted the instrumental parameters (laser intensity, sensitivity of detector (HV), gain) to obtain Channel 1 (Ch1, band filter: 500-535 nm) and Channel 2 (Ch2, band filter: 565-665 nm) imaging. After background subtraction, the image of Channel 2 was divided by Channel 1 on a pixel-by-pixel basis. Instrument settings should be the same during stimulation experiment. Next, choroquine (50 μM) was added into these cells, and time-course of ratio imaging of MCF-7 cells was implemented.

3. Results and Discussion 3.1 Emission spectra of FDI excited at different wavelengths The absorption and emission spectra of FDI were measured in C2H5OH/H2O (v:v 1:1) at pH 7.4 (Tris-HCl buffer). As shown in Fig. 1a, FDI showed broad absorption spectra (360-700 nm) with two absorption peaks at 488 and 608 nm. Absorption peak at 488 nm was characteristic absorption of fluorescein moiety in FDI. Absorption band at 608 nm was ascribed to the ‘π-π*’ based transition between unsaturated dialkene and fluorescein moiety in FDI. It’s convinced that the combination of unsaturated dialkene group and fluorescein fluorophore in FDI could

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extremely broaden the absorption band of FDI, which was suitable for ratiometric detection by CLSM in dual excitation/dual emission mode. Then, upon excitation at different wavelengths, the emission spectra of FDI in aqueous solution (pH 7.4) were obtained (Fig. 1a). Excited at 488 nm, FDI showed a narrow and asymmetric emission peak at 520 nm. Through Lorenz fitting (R2=0.998), it could be found that the emission band at 520 nm was composed of two emission peaks at about 518 nm and 547 nm, respectively (Fig. 1b). When excited at 545, 559, 608, 635 nm, respectively, FDI exhibited a wide emission band in the range from 550 to 750 nm. These spectra revealed that FDI possessed the characteristic of multi-emission upon excitation at different wavelengths, and would be a powerful tool for fluorescent imaging in living cells. 3.2 Absorption spectra of FDI vs various pH Absorption spectra of FDI in C2H5OH/H2O (v:v 1:1) at various pH were recorded on UV-Vis Spectrophotometer (Fig. 2). Under acidic conditions (pH 3.1), FDI displayed an absorption band at 458 nm and a shoulder peak at 608 nm. With the pH value increasing (pH 3.10-7.10), the absorption peak at 458 nm was firstly decreased, subsequently increased and red-shifted to 488 nm, attributing to the absorption band of fluorescein fluorophore. Meanwhile, the absorption peak at 608 nm in accordance with ‘π-π*’ based transition of unsaturated dialkene moiety in FDI, appeared at with gradual increase from pH 3.10 to 7.10 (Fig. 2a). In the range of pH between 7.10 and 11.14, the absorption spectra of FDI did not change (Fig. 2b). The absorption intensity at 608 nm of FDI vs a wide pH range was also investigated (Fig. 2c), and the pKa value of the equilibrium of FDI was 5.78 (+ 0.03). In the pH range from 1.99 to 13.76, the colour of FDI solution changed from orange through dark green and finally to pink (Fig. 2d). 3.3 The proposed mechanism of FDI response to pH The mechanism of FDI response to pH was proposed, as shown in Scheme 2. Fluorescein fluorophore in FDI exhibited two absorption bands at 458 and 608 nm in acidic conditions (pH < 7.0). With increasing of pH values, the proton of phenolic group in fluorescein moiety would dissociate and result in an enhancement of ‘π-π*’ based transition of unsaturated dialkene, in accordance with the increasing of absorption peak at 608 nm. When pH was up to the range between 7.0 and 11.0, the protons of phenolic and benzoic acid groups in fluorescein moiety dissociated completely. As a result, no changes of absorption spectra of FDI were found in the pH range from 7.0 to 11.0. With further increasing of pH value (pH>11.0), the absorption peak at 608 nm decreased strongly, which indicated that the ‘π-π*’ based transition of unsaturated dialkene group remained present but diminished, because 7

absorption maximum at 488 nm simultaneously red-shifted to 503 nm and decreased slowly. It was assuming that the nucleophilic characteristic of phenoxy ion may be cyclized with unsaturated dialkene/indole quaternary ammonium moiety of FDI, illustrated in Scheme 2. In that case, the conjugated system would be larger than that of fluorescein fluorophore but less than that of FDI.[20] Although indole quaternary ammonium moiety was prone to addition reaction in the presence of OH-, the nucleophilic addition production of FDI and OH- didn’t find by FTMS-pESI (Fig. S1). In addition, FDI exhibited a good reversibility to pH, which was verified by absorption spectral changes of FDI in pH range from alkalinity to acidity. The reversible spectral responses to various pH were shown in Fig. S2. Adjusted the pH range between 3.5 and 10.0, colour changes of FDI solution from orange to dark green were significantly visible. Absorption intensity at 488 nm was reversible between pH 3.5 and 10 (Fig. 3a). Adjusted pH between 8.0 and 13.5, colour of FDI solution obviously changed from dark green to pink. The addition of OH- (pH 13.5) could induce the decrease of absorption intensity at 608 nm of FDI (Fig. S2). When H+ was added again (pH 8.0), intensity at 608 nm was recovered (Fig. 3b). 3.4 Emission spectra of FDI vs various pH excited at single and dual wavelengths Emission spectra of FDI in C2H5OH/H2O (v:v 1:1) at different pH were also investigated, as shown in Fig. 4. The emission spectra of FDI upon excitation at 488 nm exhibited emission peak centred at 530 nm and the emission band at 672 nm (Fig. 4a). A distinct enhancement of the peak (16 fold) at 530 nm was observed upon increasing of pH from 3.10 to 12.10. Meanwhile, the emission band at 672 nm was firstly increased and decreased subsequently, in accordance with that upon excitation at 635 nm (Fig. 4b, 4c). The plot of FDI intensity at 672 nm vs pH value from 1.99 to 13.17 was a roller-coaster shape, from which the pKa 6.49 (+0.09) was obtained in agreement with the pKa (6.5) of fluorescein. The ratio intensity of 570 and 530 nm at various pH (2.05-12.95) was plotted (Fig. 5), after excited at 488 and 559 nm, respectively (Fig. S3). This dual excitation/dual emission ratio was curvilinear function over the pH range from 4.0 to 8.0. The pKa value calculated from this plot was 6.60 (±0.07), which was consistent with that calculation of intensity at 672 nm upon excitation at 635 nm (Fig 4d). To confirm that FDI could measure the pH value efficiently among the complicated endocellular environment, the fluorescent response of FDI (1 μM) to different biological species was also investigated in Tris-HCl buffer (pH 7.40) (Fig. S4). In Tris-HCl buffer solution with pH 7.4 (C2H5OH:buffer, 1/1, v/v), the presence of metal ions (K+, Ca2+, Na+, Mg2+, Fe2+, Fe3+, Cu2+, Zn2+, Cr3+), anions(S2-, SH-, Cl-, SO42-, I-, HPO42-, H2PO4-), reactive oxygen 8

species (NaOCl, H2O2, hydroxyl radical) and biological relevant species (methinione, Glycine, Glu, Cys) didn’t cause any observable spectral changes (Fig. S4), indicating that the spectral response of FDI was not brought about by presence of these species at neutral pH. FDI displayed excellent spectral properties such as multi-wavelength excitation or emission, moderate pKa value and specific response to pH at acidic and basic conditions. All these results suggested that FDI would be a suitable fluorescent probe for ratiometric intracellular pH imaging. 3.5 Solvent effect of FDI in various solvents

The solvent effect of FDI was measured in solvents such as dichloromethane (DCM), chloroform, tetrahydrofuran (THF), ethyl acetic (EtOAc), methanol, ethanol, Dimethyl Formamide (DMF), dimethylsulfoxide (DMSO)(Fig. 6). Stokes Shift and the other spectral data were demonstrated in Tab. 1. In protic solvents, e. g. methanol and ethanol, there were two speaks of FDI in absorption or emission spectra (Fig. 6a, 6b). For example, in methanol absorption peaks at 488 nm and emission at 524 nm was ascribed to the characteristic peak of fluorescein fluorophore, while absorption and emission peaks at 608 nm and 692 nm should be assigned to the ‘π-π*’ based transition between unsaturated dialkene moiety and fluorescein fluorophore in FDI. Stokes shifts were 36 nm and 72 nm, respectively. Fluorescent quantum yield in methanol was up to 0.21 with reference to fluorescein (pH 8.0). On the contrary, in aprotic solvents the absorption intensity of the ‘π-π*’ based transition between unsaturated dialkene moiety and fluorescein fluorophore decreased dramatically, or even disappeared. Correspondingly, the molar extinction coefficient of the absorption reduced from 4.3×104 to 0.7×104 M-1cm-1. This weakening absorption of FDI was due to the ring-opening/closing transformer of fluorescein fluorophore in protic or aprotic solvents, as shown in Scheme 3. 3.6 Time-course of FDI permeating into MCF-7 cells As all know, to promote permeability through membrane, Carboxy-SNARF should be transformed to Acetoxymethy (AM) ester form in the actual using process. Moreover, in order to promote better mitochondrial 9

uptake, Carboxy-SNARF-AM usually needs to be loaded into cells at a cooler temperature (4–12°C) for a longer time.[17] Superior to commercial mitochondrion-targetable and ratiometric pH probe Carboxy-SNARF, FDI exhibited excellent and fast cell-membrane permeability. As shown in Fig. 7, the time-course of FDI stained into MCF-7 cells was monitored in CLSM. After stained with FDI 5 μM (containing 0.1% DMSO) for 5 minutes, the cell membrane partially displayed intense red fluorescence. Meanwhile, the other part of cell membrane showed low fluorescence. This observation may be due to the different existent form of FDI in protic and aprotic environment, as shown in Scheme 3. In protic environment of cell membrane the spirolactone of FDI occurred a ring-opening transformation, resulting that a red fluorescence was observed upon excitation at 635 nm. In aprotic environment of cell membrane spirolactone state of FDI was the common type of membrane permeability. When MCF-7 cells were stained with FDI for 10 minutes, the fluorescence of cell membrane appeared at with gradual decrease. Simultaneously, the cytoplasm of cells showed significant red fluorescence. With additional incubation time (30 min), the fluorescence of cytoplasm grew even further, while that of membrane dwindled continually. These results suggested that FDI with positive/negative electric charges could translocate across membrane of MCF-7 cells into cytoplasm. 3.7 Mitochondrion-targetable fluorescent imaging of FDI In protic solvents the characteristic positive and negative electric charges of FDI would make it tend to accumulate in mitochondria of cells. To verify this point, FDI was applied for biological imaging in cultured MCF-7 by using a CLSM. Upon excitation at 635 nm, red intracellular fluorescence (red channel BF: 655-755nm) was distributed in discrete subcellular locations of cells (Fig. S5). In order to further validate whether FDI can be directionally accumulated in mitochondria of cells, co-localization experiments were performed by co-staining MCF-7 cells with FDI and 2 μM 3,6-diamino-9-[2-(methoxy-carbonyl)phenyl]-xanthylium chloride (rhodamine 123, Rh123), a mitochondrial tracker. MCF-7 cells showed green and red fluorescence in Channel 1 and 2(Fig. 8a, Ch1 and Ch2), respectively, after staining with 5 μM FDI and 2 μM Rh123. The image of Ch2 merged well with the image staining with Rh123 (Ch1) indicating that FDI can specifically localize in mitochondria of living cells. High Pearson’s coefficient and overlap coefficient were 0.416 and 0.644, respectively, evaluated using conventional dye-overlay method. However, surface plot of MCF-7 cells stained with Rh123 and FDI varied in asynchrony (Fig. 8b, 8c). The main reason was that FDI was pH sensitive and emitted low red fluorescence in pH at neutral condition. We also investigated the intensities plot of staining FDI against Rh123 for each pixel. The staining in 10

Ch1 and Ch2 resulted in a highly correlated plot (Figure. 8d), and the intensity correlation analysis (ICA) plots for the two stains generate an unsymmetrical hourglass-shaped scatterplot that is markedly skewed toward positive values (Figure 8e, 8f). Moreover, intensity correlation quotient (ICQ) for the two dyes is 0.187, suggesting a positive result of the staining intensities associates in a dependent manner. From PDM (product of the differences from the mean) image with positive values in the pixels, we can easily identify those mitochondria with high intensity distribution of the two dyes (Figure 8g). In addition, the cytotoxic effect of FDI on MCF-7 cells was also evaluated using standard cell viability protocols (MTT assay) (Figure S6). When MCF-7 cells are treated with FDI (0-10 μM) for 12 h, survival rate was higher than 90% in 1.0 × 104 cells/well, which indicated that FDI has low cytotoxicity within the concentration range from 2 to 10μM. The low cytotoxic effect of FDI was well suited to being applied in mitochondrial imaging of living cells. 3.8 Ratiometric imaging of mitochondria in living cells Fluorescence ratio imaging is a quantitative monitoring of intracellular objects in presence of fluorescent indicator in living cells. To illustrate ratiometric monitoring of pH fluctuation in mitochondria, the ratio imaging of MCF-7 cells stained with FDI was recorded in CLSM (Fig S7). Living MCF-7 cells were incubated with FDI (5 uM) in culture medium for 30 minutes at 37 °C, and simultaneously exhibited green fluorescence in Ch 1 (Ex 488 nm, Em 500-535 nm) and red fluorescence in Ch 2 (Ex 559 nm, Em 565-665 nm) (Fig. 9). By ratioing the intensities between Ch2 and Ch1, it can be constructed a map showing the local pH values throughout the field of view. Chloroquine as a stimulant is usually used to cause the variety of pH in living cells.[22] Stimulated by addition of chloroquine (50 μM), the mitochondrial pH of cells fluctuated over time (Fig. 9). After stimulated by chloroquine for 10 minutes, the fluorescent intensity of Ch1 and Ch2 did not change obviously. However, the ratio of Ch2/Ch1 in the range from 0.75 to 3.0 was slightly larger, compared with cells unstimulated by chloroquine. When stimulated for 20 minutes, the ratio of intensity in two channels significantly increased up to 5.0. This demonstrates that probe FDI was highly sensitive to pH change in the mitochondria of MCF-7 cells. Since the monitoring process was nondestructive, image acquisition would be repeated frequently to trace and monitor the time course of cellular pH responses.

4. Conclusions In summary, we have developed a novel ratiometric fluorescent probe (FDI) composed of fluorescein 11

fluorophore, unsaturated dialkene moiety and substituted indole quaternary ammonium moiety. These three components perform their respective duties well in the properties of FDI responding to pH. FDI adopted pH sensitive fluorophore, fluorescein, to serve as a fluorescent reporter, as well as employed unsaturated dialkene/indole quaternary ammonium moiety, which can not only broaden absorption and emission spectrum of FDI, but also directionally accumulate in mitochondria in living cells, owing to positive charge of ammonium group. As such, FDI displayed multi-wavelength emission upon excitation at different wavelengths, such as 488, 545, 559, 608, 635nm. With the pH varying from 2.0-13.0, FDI exhibited excellent pH sensitivity and significant changes of solution color. The pKa value of FDI calculated from the ratio at 570/530 was 6.54 (±0.12). Probe FDI also showed extraordinary anti-interference capability with some cations, anions, reactive oxygen species and biological relevant species. Moreover, this probe also illustrated a good reversible response to pH in the range pH 3.5-10.0 and pH8.0-13.5. Additionally, FDI exhibited excellent and fast cell-membrane permeability, and mitochondrion-targetable localization. With good properties, FDI was successfully used for ratiometric fluorescent imaging of mitochondria in living cells. To our knowledge, FDI represents the first fluorescein probe bearing with the characteristics of multi-emission upon excitation at different absorption peaks, and for non-invasive ratiometric monitoring mitochondrial pH in living cells. We demonstrated that FDI could permit the real time monitoring of pH alkalization of mitochondria stimulated by chloroquine. Therefore, this work provides a promising probe for the effective detection and quantification of the pH value in mitochondria of living cells. Superior to Carboxy-SNARF, it is anticipated that FDI would be a hopeful candidate for real-time tracking of mitochondrial pH changes in the biomedical and biological fields. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 21302080), Program Funded by Liaoning Province Education Administration (No. L2014010) and National Water Pollution Control and Treatment Science and Technology Major Project (2015ZX07202012). Appendix A. Supplementary data Supplementary data: absorption and emission spectra of FDI, response of FDI to other distractions, ratiometridc imaging, colocalization experiment, ESI-MS and NMR spectra would be found in the online version. http://dx.doi.org/10.1016/XXXX

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Biographies Guli Li is currently studying her master degree at the laboratory of Associate Professor Haibo Yu in the College of Environmental Sciences at Liaoning University (Shenyang). Her research interests are focused on the design and 14

preparation of fluorescent probes for metal ions and biological sensing. Bei Zhang received his master’s degree from State Key Laboratory of Fine Chemicals at Dalian University of Technology in 2010. In 2012, he joined Prof. Watson’s research group of Department of Chemistry, University of Kentucky. His main research focuses on the design, synthesis of conjugated polymers for electrochromic device. Xinbo Song joined the research group of Prof. Yi Xiao in 2011 and received his MS and PhD degrees from Dalian University of Technology in 2017. His research interests are focused on the design and preparation of fluorescent probes for chemical and biological sensing. Ying Xia is currently studying his master degree at the laboratory of Associate Professor Haibo Yu in the college of Environmental Sciences, Liaoning University (Shenyang). Her research interests are focused on the design and preparation of fluorescent probes for reactive oxygen species and biological sensing. Haibo Yu received his MS degrees from Dalian University of Technology in 2008. In 2009, he joined the research group of Prof. Yi Xiao and received his PhD degrees from Dalian University of Technology in 2012. He then moved to the College of Environmental Sciences of Liaoning University as an Associate professor in 2013. His research interest includes supramolecular host–guest chemistry, fluorescent probes and super-resolution fluorescent materials. Xinfu Zhang received his BS, MS and PhD degrees from Dalian University of Technology in 2008, 2010 and 2015, respectively. He then became a postdoctoral researcher in College of Pharmacy at the Ohio State University from 2015 till now. His research interests focus on bioorganic chemistry and engineering related to fluorescent sensors and antitumor agents. Yi Xiao received his BS and MS degrees from Tianjin University in 1996 and 1999, respectively. He then moved to Dalian University of Technology (DLUT) where he received his PhD in 2003 with Prof. Xuhong Qian. After 10 months’ working as a postdoctoral researcher in AIST, Tsukuba, Japan, he returned to DLUT. Since 2010, he has been a professor of applied chemistry. He is interested in sensors and semiconductors based on fluorescent dyes. Youtao Song received his Master’s degree from Yamaguchi University in 1999, and then received his Ph. D from Tottori University in 2002. From 2002 to 2005 he worked as a postdoctoral fellow in the National Institutes of Health. Her research interests focus on landscape genetics, ecological restoration and environmental toxicology.

15

Measured Curve Fitted Curve Fitted Peak 1 Fitted Peak 2

488nm 1.0

abs Ex 488 nm Ex 545 nm Ex 559 nm Ex 608 nm Ex 635 nm

Normalized

608nm 0.6

Fitted Model: Lorentz Equation: y = y0 + (2*A/PI)*(w/(4*(x-xc)^2 + w^2)) Chi^2/DoF = 0.00013 R^2 = 0.99867 peak Area Center Width Height ----------------------------------------------------1 41.200 518.69 43.803 0.59879 2 81.355 547.74 98.099 0.52796

0.8

Intensity

0.8

1.0

0.6

0.4

0.4

0.2

0.2

0.0

0.0 500

400 450 500 550 600 650 700 750 800 850

550

600

Wavelength(nm)

650 700 750 Wavelength(nm)

800

850

Fig. 1 (a) Normalized absorption and emission spectra of FDI in C2H5OH/H2O (v:v 1:1) at pH 7.4 (Tris-HCl buffer). Emission spectra were excited at 488nm, 545nm, 559nm, 608 nm and 635 nm, respectively. (b) Lorenz fitting of emission spectra of FDI excited at 488 nm.

pH 7.10 6.40 ...... 5.81 5.33

0.7

pH 3.10 .... 5.33

0.6 0.5

pH 11.14 10.74 .... 7.10

0.8 0.7

Absorption

0.8

Absorption

486nm

0.9

0.9

pH 7.10 6.40 ..... 3.10

0.4 0.3

0.6

608nm

0.5 0.4 0.3 0.2

0.2

0.1

0.1 0.0 300

400

500 600 Wavelength( nm)

700

0.0 300

800

400

500

600

700

800

Wavelength(nm)

0.50 0.45 0.40

A608 nm

0.35 0.30 2

R = 0.998 pKa=5.77+0.03

0.25 0.20 0.15 0.10 0.05 3

4

5

6

7

8

9

10

11

pH

Fig. 2 (a), (b) Absorption spectral changes of FDI in C2H5OH/H2O (v:v 1:1) at various pH values (3.10-11.14). (c) Absorption intensity at 608 nm vs different pH (3.10-11.14). (d) The colour changes of FDI in C2H5OH/H2O (v:v 1:1) in the pH range from 1.99 to 13.76.

9.94

9.82

10.16

0.30

0.08

0.25

0.06

0.20 0.15 3.60 0

2

7.90

7.91

7.97

7.95

0.04 0.02

3.49 0.10

8.07

0.10

A608nm

A488nm

9.87

9.81

0.35

3.65

3.71 4

6 cycle

3.67 8

13.42

0.00 10

0

13.35

13.43 2

4

6 cycle

13.38

13.33 8

10

Fig. 3 (a) Absorption intensity at 488nm of FDI in C2H5OH/H2O (v:v 1:1) at pH 3.5-10.0. (b) Absorption intensity at 608 nm of FDI in C2H5OH/H2O (v:v 1:1) at pH 8.0-13.5.

16

530nm

700

500 400

600

pH 5.81 5.33 .... 672nm 3.10

300 200 100

pH 5.81 6.40 ..... 12.10

400 300 200 100 0

0 500

550

600 650 700 Wavelength(nm)

750

650

800

672nm

700

700

750 800 wavelength(nm)

850

900

700

600

600

pH 5.34 5.77 .... 13.17

500 400

500

I672

Intensity

pH 5.34 4.72 ... 2.66 1.99

500

Intensity

Intensity

600

672nm

700

pH 12.1 11.6 .... 3.10

300

pKa 6.49+0.09 2 R =0.996

400 300 200

200 100

100

0 650

700

750 800 Wavelength(nm)

0

850

2

4

6

pH

8

10

12

Fig. 4 (a), (b), (c) Emission spectra of FDI in C2H5OH/H2O (v:v 1:1) vs various pH (Ex 488 and 635 nm, respectively). (d) Emission intensity at 672 nm vs various pH.

3.0 2.5

570/530

2.0 2

R =0.994 pKa=6.60+0.07

1.5 1.0 0.5 0.0 2

4

6

8

10

12

pH

Fig. 5 Ratio of intensity at 570 and 530 nm of FDI vs various pH excited at 488 nm and 559 nm, respectively.

800

DMF DMSO DCM MeOH CHCl3 THF C2H5OH EtOAC

Absorption

0.6 0.5 0.4 0.3

600 500 400 300

0.2

200

0.1

100

0.0 300

DMF DMSO DCM MeOH CHCl3 THF C2H5OH EtOAC

700

Intensity

0.7

0

400

500 600 700 Wavelength(nm)

800

500

550

600

650 700 750 Wavelength(nm)

800

850

Fig. 6 (a) Absorption spectra, (b) emission spectra and (c) the colour of FDI in DCM, THF, EtOAc, Methanol, ehanol, DMF, DMSO and CHCl3, respectively. 17

Fig. 7 Time-course of FDI permeating into MCF-7 cells incubated for 5, 10, 15, 30 minutes, respectively.

Fig. 8 FDI colocalizes to mitochondria in MCF-7 cells. (a) (left) MCF-7 was stained with 2.0 μm Rh123 for 5 min at 37 °C (Channel 1 (Ch 1): λex 488 nm, λem 500-535 nm). (middle) MCF-7 was stained with 5.0 μm FDI (Channel 2 (Ch 2): λex 635 nm, λem 655-755 nm). (right) Overlay of (a) and (b). (b) and (c) Surface plot of Ch1 and Ch2. (d) Intensity correlation plot of stain FDI and Rh123. (e) ICA plot of cells stained with Rh123. (f) ICA plot of cells with stained FDI. (g) PDM image with positive PDM values in the pixels.

18

Fig. 9 Time course of fluorescence imaging of MCF-7 cells stained with FDI (5 μM) and choroquine (50 μM) at different time.

Scheme 1 Synthetic route of FDI

Scheme 2 Proposed mechanism of FDI response to pH

Scheme 3 Ring opening/closing changes of FDI in aprotic or protic solvents

19

Tab. 1 Absorption and fluorescent data of FDI (1x10-5 M) in solvents Stokes

λabsa

λemb

(nm)

(nm)

488/620

524/692

36/72

6.6(488)/4.3(620)

0.21

492/626

540/702

38/76

4.2(492)/2.9(626)

0.20

CH2Cl2

485/635

550/744

65/99

2.5(485)/1.8(635)

0.18

CHCl3

493/645

544/699

51/54

3.6(493)/1.8(645)

0.15

THF

480

541

48

0.3

0.06

EtOAc

479

535

47

0.4

0.07

DMF

462/668

560

98

0.7(462)/0.28(668)

0.05

DMSO

475/670

566

91

0.45(475)/0.7(670)

0.05

Solvent

CH3OH CH3CH2 OH

Shift (nm)

ε×10-4 (M-1·cm-1)c

ΦFLd

a λabs: absorption maximum. b λem emission maximum. c ε molar extinction coefficient. d ΦFL fluorescent quantum yield.

20