Pinpoint Diagnostic Kit for Heat Stroke by Monitoring Lysosomal pH

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A pinpoint diagnostic kit for Heat Stroke by Monitoring Lysosomal pH Ying Wen, Weijie Zhang, Tao Liu, Fangjun Huo, and Caixia Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03612 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017

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Analytical Chemistry

A pinpoint diagnostic kit for Heat Stroke by Monitoring Lysosomal pH Ying Wen,a Weijie Zhang,a Tao Liu,a Fangjun Huo,b and Caixia Yina,* 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. Email: [email protected]; Fax: (+86) 351-7011022. b Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China. ABSTRACT: Heat stroke is one of the most serious causes of mortality. To prevent from the happening of situation, it is fundamental to research the mechanism of heat cytotoxicity. The preliminary results revealed that heat stroke and the change of lysosome acidity had some certain correlation. To further clarify their relationship, herein, we report a highly selective and sensitive fluorescence probe (NT1) for turn-on sensing of pH value. NT2 was synthesized as control compound. Compared to NT2, NT1 showed accurate lysosome target ability. In addition, the suitable pKa value (5.67) allows NT1 to response to the changes of lysosomal pH values. Most importantly, NT1 could be used to study the correlation between the change of lysosomal pH and heat stroke. It was showed that the lysosomal pH value increasing with temperature during heat stroke. Thus, NT1 was an excellent candidate for research the complex biological mechanism about heat stroke..

Lysosome, an acidic environment organelle of pH 4.5-5.5, is vital for maintaining cellular homeostasis.1,2 It is responsible for degradation macromolecules and cell components in the biological processes of phagocytosis, endocytosis and autophagy.3,4 The minor pH fluctuation of lysosome may influence its normal function and even the fate of the cells.5 It is reported that the change of lysosomal pH may be associated with many serious diseases,6 such as lipid storage disorders, mucolipidoses, and even cancer, heat stroke. Heat stroke, defined as hyperthermia with a body temperature greater than 40.6 °C, is a life threatening condition.7 Classic heat stroke victims usually suffer dizziness, mental confusion, unconsciousness, organ failure, and even death. However, it is not yet fully clear about the mechanism of heat cytotoxicity.8 For example, the research about the change of lysosomal acidic level in heat stroke was only a few. The main reason was lacking of effective and reliable tools. Fluorescence labelling is a powerful technique for this purpose, because of its non-disruptive and real-time features.9-16 So far, many small molecular fluorescent probes for detecting lysosomal pH in living cells have been reported.17-22 However, few of them were reported to be used to study the relationship between the change of pH values and heat stroke. In 2014, Ma’s group developed a ratiometric pH probe and for the first time examined the trends of lysosomal pH variations upon heat stroke.23 To provide more optional tools to study the above mentioned relationship, the new fluorescence probes, such as turn-on ones, are still needed to develop. Here, a turn-on fluorescent pH probe (named NT1, Scheme 1) was synthesized based on a naphthalimide fluorophore tagged with a classic lysosome-targeting morpholine moiety.2426 1,8-Naphthalimide, a fluorescent chromophore with excel-

lent spectroscopic properties, was used previously as a sensor system by our group.27-31 In addition, for comparison, NT2 (Scheme 1) without morpholine group was also developed. By spectral experiments, NT1 and NT2 were found to be promising candidates for response to the change of pH values in vitro. In cell imaging, NT1 exhibited an accurate lysosome-targeting ability. While, owning to lacking of morpholine moiety, NT2 diffused to in cytoplasm. Therefore, we selected NT1 as suitable research tool to testify the conclusion of the lysosomal pH value increasing during heat stroke.

Scheme 1. Chemical structure of NT1 and NT2

EXPERIMENTAL SECTION Materials. All of the starting materials were obtained from commercial suppliers. H2O2, NaOCl, Cys, Hcy, and GSH were purchased from Sigma-Aldrich (St. Louis, MO, U.S.). Lysotracker red was supplied by Shanghai Beyotime Co., Ltd (Shanghai, China). Mitotracker red was purchased from Keygen Biotechnology Co., Ltd (Jiangsu, China). All organic solvents were supplied from Sinopharm Chemical Reagent Company (Shanghai, China).

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Figure. 1 (a) Absorption (Inset: the colour change) and (b) fluorescence emission spectra of NT1 (50 µM or 5 µM) in phosphate buffer at different pH values. (c) The fluorescence intensity at 550 nm (F550) of NT1 in responding to other biologically relevant species (200 µM) in phosphate buffer at pH 4.5. 1 was the F550 of only the probe in the media of pH 4.5. 2 to 22 represents additional Ag+, Al3+, Ba2+, Ca2+, Cu2+, Fe2+, K+, Na+, Zn2+, Cys, Lys, Hcy, GSH, H2O2, HClO, HCO3-, HSO3-, F-, CN-, CH3COO- and NaOH (pH 10.0), respectively. Spectra were acquired in phosphate buffer at different pH values (1% DMSO), λex = 447 nm. (d) Plot of F550 versus pH values. Inset: the linear relationship between F550 and pH values in the range pH 5.0–6.2.

Instruments. A pH meter (Mettler Toledo, Switzerland) was used to determine the pH. 1H NMR spectra were recorded with a Bruker Avance III HD spectrometer at 600 MHz. Proton chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS). The high-resolution mass spectra (HR-MS) were measured on a Bruker Micro TOF II 10257 instrument. UV-visible spectra were recorded on a Cary 50 Bio spectrometer. Steady-state emission experiments at room temperature were measured on a Hitachi F-7000 spectrometer with a Xe lamp as an excitation source. The cell imaging experiments were performed by a Leica DMi8 fluorescence inversion microscope system. Cell culture. The HeLa and A549 cells were the gift of Professor Fuyou Li and Tao Yi (Department of Chemistry, Fudan University, Shanghai, China). Cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum at 37ºC in a humidified atmosphere containing 5% CO2. Confocal laser scanning microscopy (CLSM) imaging. Cells were plated on 14 mm glass coverslips and were incubated overnight. After washing with PBS, the cells were incubated with 10 µM NT1 or NT2 in DMSO/RPMI 1640 (0.5%, v/v) for 20 min at room temperature. The channel of getting the green fluorescence signal of the two probes was set at 550 ± 25 nm, λex = 405 nm. For the co-localization experiments, the cells loaded on probes were incubated with Lysotracker (500 nM) or Mitotracker (1 µM) for another 15 min. The

emission of these trackers was collected at 650 ± 20 nm, under excitation at 561 nm. For the special experiments of monitoring lysosomal acidic change during heat stroke, the cells preloaded on NT1 were incubated at 37℃, 41℃ and 45℃ for 20 min. After washing three times, the cells were subjected to CLMS imaging.

RESULTS AND DISCUSSION Capability of NT1 and NT2 to response to pH values in the solution. The details of synthesis and characterization of NT1 and NT2 are provided in the Electronic Supplementary Information (ESI). The spectroscopic properties of the two probes were examined under in vitro phosphate buffer at different pH values (Figure 1 and S1). NT1 itself exhibited an absorption maximum at 381 nm (ε =10 860 M-1 cm-1, Figure 1a), in pH 4.0 media. When the pH value of the media changed from 4.0 to 7.0, the band decreased and red-shifted to 447 nm (ε = 13 320 M-1 cm-1, Figure 1a). The isoabsorptive point was 406 nm. Under excitation at 447 nm, NT1 did not emit any fluorescence, in pH 2.0 media. A green fluorescence


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Figure. 2 CLSM images of A549 cells co-labeled with 10 µM NT1 or NT2 with Lyso/Mitotracker. (a, g, m, s) the green channel image; (b, h, n ,t) the red channel image; (c, i, o, u) the brightfield image; and (d, j, p, v) the overlay images of red and green channels. (e, k) The correlation of Lyso/Mitotracker and NT1 intensities; (q, w) The correlation of Lyso/Mitotracker and NT2 intensities. (f, l, r, x) Cross-sectional analysis along the pink line in of the insets. The insets: the fluorescence images of a single cell. Green fluorescence: 550 ± 25 nm for NT1 and NT2, λex = 405 nm; red fluorescence: 650 ± 20 nm for Lyso/Mitotracker, λex = 561 nm. Scale bar = 20 µm.

peaked at 550 nm appeared and drastically increased with the increase of the system pH values (Figure 1b). The fluorescence intensity at 550 nm (F550) increased a 89.13-fold from pH 3.0 to 8.0. Compared to NT1, NT2 had similar photophysical property (Figure S1) in vitro. In the reversible fluorescent responses experiments (Figure S2), F550 of these two probes displayed negligible decrease after three cycles. The change of absorption and fluorescence spectra should be attributed to that the hydroxyl group was replaced by a negative oxygen moiety in alkaline environment. To verify the specificity of NT1 and NT2 for pH detection, the fluorescence response of the probes to other biologically relevant species was determined (Figure 1d and S1d). These biomolecules contained metal ions (Ca2+, Ag+, Al3+, Ba2+, Cu2+, Fe2+, K+, Na+, Zn2+), amino acid (Cys, Lys, Hcy), protein (GSH), reactive oxygen species (H2O2, HClO) and weak acid anion (HCO3-, HSO3-, F-, CN-, CH3COO-). Considering the lysosomal acidic environment 4.5–5.5, the media at pH 4.5 was selected to perform the specificity study. We found that these biologically relevant species did not induce any changes in the fluorescence intensities at 550 nm of the probes, in contrast to

a 14.07-fold (NT1) or 19.42-fold (NT2) increase after the addition of NaOH (at pH 10.0). These results indicated that NT1 and NT2 had high selectivity for pH detection. A plot of F550 of NT1 or NT2 vs pH is linear over the pH range of 5.0 to 6.2 (R2 = 0.99832 for NT1, R2 = 0.99802 for NT2). The pKa of these probes were calculated to be 5.67 and 5.69, respectively (Figure 1c and S1c), using the HendersonHasselbalch equation. The pKa values were suitable to allow these probes to monitor the changes of lysosomal pH values. Fluorescence imaging in living cells. Next, practical imaging applications of NT1 and NT2 in living cells were tested by using confocal laser scanning microscopy (CLSM). A549 cells incubated with 10 µM NT1 and NT2 at 37 °C for 20 min showed strong green fluorescence (550 ± 25 nm, Figure 2) in cytoplasm as observed, suggesting that the two probes could penetrate cell membrane. To evaluate the specific directed performance of the two probes, co-localization experiments were performed using commercially available



Figure. 3 CLSM images in HeLa (a) and A549 (b) cells pre-loaded on NT1 (10 µM, 20 min) and further incubated in buffers with various pH values (4.0, 6.0 and 8.0) in the presence of 10 µM of nigericin for 30 min at 37 ℃. Green channel: 550 ± 25 nm, λex = 405 nm, Scale bar = 20 µm.(c) and (d) Quantified relative fluorescence intensity of images. Statistical analyses were performed with Student’s t-test (n = 3). **p < 0.01, ***p < 0.001 and error bars are ± S.D. targeting dyes. NT1-loaded A549 cells were co-stained with 500 nM Lysotracker (a lysosomal indicator) or 1 µM Mitotracker (a mitochondrial indicator) for another 15 min, respectively. The merged images (Figure 2d) and the correlation mapping of the fluorescent intensities (Figure 2e) showed a good co-localization of NT1 and Lysotracker, indicating that NT1 was predominantly accumulated in the lysosome. The high Pearson’s correlation coefficient between green and red fluorescence images were calculated to 0.90 (with Lysotracker, compared to 0.49 with Mitotracker), equally that confirming NT1 had lysosome-targeted property. Similarly, the comparable co-localization analysis of NT2 was performed. A low Pearson’s coefficient of 0.49 (with Lysotracker) and 0.43 (with Mitotrakcer) demonstrated that NT2 did not mainly target to lysosome and mitochondria. Thus, only NT1 could be used as a turn-on probe for labelling lysosome.

Then, NT1 was used to monitor acidic changes in A549 cells and Hela cells (Figure 3). Cells were firstly incubated with NT1 (10 µM) in PBS buffer (pH 7.4) for 20 min at 37 °C and then incubated in buffers with various pH values (4.0, 6.0 and 8.0) in the presence of 10 µM of nigericin for 30 min at 37 °C. Nigericin, an H+/K+ ionophore to homogenize the intraand extracellular pH.32-34 As shown in Figure 3a and 3b, the green fluorescence signal gradually increased with the pH of the system. In Hela cells, the average fluorescence intensities from green channel were dependent of the pH value: 34.2 (pH 4.0), 44.9 (pH 6.0) and 70.8 (pH 8.0), which was consistent

with the fluorescence behaviour of NT1 in aqueous media. In A549 cells, a significant pH-dependent increase of cells was similarly observed. The fluorescence intensities were 22.3 (pH 4.0), 41.0 (pH 6.0), and 47.6 (pH 8.0), respectively. These results 5h

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Concentration (µM) Figure. 4 Cell viability values (%) estimated by CCK-8 assay in A549 cells, which were cultured in the presence of 0−100 µM NT1 for 5 and 10 h.


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Figure. 5 Relationship between lysosomal pH and heat stroke in HeLa (a) and A549 (b) cells. CLSM images of NT1-loaded cells under heat stroke at 37℃, 41℃, and 45 ℃ (left to right) for 20 min. Green channel: 550 ± 25 nm, λex = 405 nm, Scale bar = 20 µm.(c) and (d) Quantified relative fluorescence intensity of images. Statistical analyses were performed with Student’s t-test (n = 3). *p < 0.05, ***p < 0.001 and error bars are ± S.D.

demonstrated that NT1 could be served as a sensitive detector to measure changes of lysosomal pH values. In addition, NT1 has no marked acute toxicity to A549 cells. The viabilities were estimated to be > 90% at 5 h or 10 h in the presence of 1-100 µM NT1, by CCK-8 assay (Figure 4). Thus, NT1 had the potential to be used in biological applications. Monitoring the lysosomal pH changes during heat stroke. Next, NT1 was used to investigate the change of lysosomal acidity in heat stroke by fluorescence imaging (Figure 5). 41℃ and 45℃ were used as the heat stroke temperatures. NT1loaded (10 µM) Hela and A549 cells were incubated at 37℃, 41℃ and 45℃ for another 20 min. Compared to the control group (37 ℃ ), the fluorescence intensities from the green channel clearly increased with the temperature increase. The average fluorescence intensities were dependent of incubation temperature of Hela or A549 cells: 22.1 (Hela, 37℃), 44.9 (Hela, 41℃) and 48.2 (Hela, 45℃); 29.8 (A549, 37℃), 56.0 (A549, 41℃), and 78.4 (A549, 45℃), respectively.

crease of temperature (Figure 6). Thus, the change of fluorescence intensities in cells (Figure 5) was attributed to the small increase of the lysosomal pH value. These results clearly indicate that heat stroke can lead to an increase of lysosomal pH values.

CONCLUSIONS In summary, we synthesized a turn-on sensitive fluorescent probe, NT1, for lysosomal pH measurements upon heat stroke. The probe showed excellent optical properties and lysosometargeting ability. More importantly, using this probe, the change of lysosomal pH with temperature is demonstrated. The development and application of the fluorescence probe will help us provide a better understanding of the complicated mechanisms between lysosome pH change and heat stroke.

At the same time, the influence of temperature from 37℃ to 45℃ was determined on the fluorescence intensity of NT1. We found that F550 of this probe did not change with the in-



4200 pH = 8

FL. Intensity (a.u.)

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Temperature (? ) Figure. 6 Effects of temperature on the fluorescence intensity at 550 nm of NT1 (10 µM) in phosphate buffer at pH 4.0, 6.0 and 8.0, λex = 447 nm.

ASSOCIATED CONTENT Supporting Information The synthetic details, additional spectra and characteristics of compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

ACKNOWLEDGMENT 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 (2017-026), Shanxi Province Science Foundation for Youths (201701D221061) and Scientific Instrument Center of Shanxi University. We also thank Dr J. J. Wang for her assistance in confocal laser scanning microscopy imaging.

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Pinpoint Diagnostic Kit for Heat Stroke by Monitoring Lysosomal pH

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