An endoplasmic reticulum-targeted two-photon fluorescent probe for bioimaging of HClO generated during sleep deprivation

Journal Pre-proof An endoplasmic reticulum-targeted two-photon fluorescent probe for bioimaging of HClO generated during sleep deprivation

Qineng Xia, Xiaoyan Wang, Yanan Liu, Zhangfeng Shen, Zhigang Ge, Hong Huang, Xi Li, Yangang Wang PII:

S1386-1425(19)31391-5

DOI:

https://doi.org/10.1016/j.saa.2019.117992

Reference:

SAA 117992

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

18 October 2019

Revised date:

18 December 2019

Accepted date:

22 December 2019

Please cite this article as: Q. Xia, X. Wang, Y. Liu, et al., An endoplasmic reticulumtargeted two-photon fluorescent probe for bioimaging of HClO generated during sleep deprivation, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2019), https://doi.org/10.1016/j.saa.2019.117992

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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.

© 2019 Published by Elsevier.

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An endoplasmic reticulum-targeted two-photon fluorescent probe for bioimaging of HClO generated during sleep deprivation

Qineng Xia 1, Xiaoyan Wang 2, Yanan Liu 1, Zhangfeng Shen 1, Zhigang Ge 1, Hong Huang 1,*, Xi Li 1, Yangang Wang 1,* College of Biological, Chemical Sciences and Engineering, Jiaxing University,

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1

Jiaxing 314001, China.

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Zhejiang Sian International Hospital, Jiaxing 314031, China.

*Corresponding author:

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[email protected] (H. Huang);

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2

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[email protected] (Y. Wang).

1

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Abstract With the development of social society, sleep deprivation has become a serious and common issue. Previous studies documented that there is a correlation between sleep deprivation and oxidative stress. However, the information of sleep deprivation related ROS has rarely been obtained. Also, it has been demonstrated that sleep

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deprivation can induce endoplasmic reticulum (ER) stress. As such, for a better understanding of sleep deprivation as well as its related diseases, it is important to

ro

develop probes with ER-targeting ability for detecting ROS generated in this process.

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Herein, a novel two-photon fluorescent molecular probe, JX-1, was designed for

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sensing HClO in live cells and zebrafish. The investigation data showed that in

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addition to real-time response (about 150 s), the probe also exhibited high sensitivity

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and selectivity. Moreover, the probe JX-1 demonstrated two-photon fluorescence, low cytotoxicity and ER targeting ability. These prominent properties enabled the

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utilization of the probe for monitoring exogenous and endogenous HClO in both live cells and zebrafish. Using this useful tool, it was found that sleep deprivation can induce the generation of HClO in zebrafish.

Keywords: Endoplasmic reticulum targeted; Two-photon fluorescence; Exogenous; Endogenous; Sleep deprivation

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Introduction Sleep is essential for life maintenance, as it is a key modulator of immune system, hormone release, cardiovascular function and brain development [1, 2]. However, for a wide variety of reasons, ranging from lifestyle factors and work or family demands to psychological problems, chronic sleep deprivation is increasingly prevalent in

of

modern society. Sleep deprivation has strong adverse impacts on neurological and physiological processes [3]. Recent studies have documented that there exists a link

ro

between sleep deprivation and oxidative stress, which occurs in cells or tissues when

-p

there is an imbalance between the levels of reactive oxygen species (ROS) and the

re

antioxidant capability of the cells [4, 5]. This behavior can contribute damage to many

lP

bioactive species, resulting in the functional decline of organ systems and serious

na

human diseases [6-8]. Unfortunately, the knowledge about sleep deprivation related ROS, i.e., types and/or quantities, has barely been obtained. Therefore, to

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comprehensively understand sleep deprivation and its related diseases, it is of great value to exploit novel tools for elucidating the role of ROS generated in this process. Among the various ROS, hypochlorous acid (HClO), produced from the peroxidation reaction of Cl- with H2O2 under the catalysis of myeloperoxidase (MPO), plays a crucial role in innate immunity and preventing inflammation [9-13]. On the other hand, owing to its high oxidation ability, excess HClO can rapidly react with multiple biological molecules lead to various diseases, including diabetes, atherosclerosis, obesity, and even cancers [14, 15]. In this regard, it is important to trace the generation of HClO in situ. 3

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Fluorescent probes have emerged as effective tools for monitoring biological species in vitro and in vivo [16-21]. Compared with one-photon fluorescence imaging technique, two-photon imaging technique is superior for applications in the area of in vivo imaging because it has several prominent advantages, such as deep imaging depth, minimal phototoxicity, and low interference from autofluorescence of

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biomolecules [22-28]. To date, a host of two-photon fluorescent probes for fluorescence bioimaging of intracellular HClO have been designed [29-32]. For

ro

example, Ye and coworkers synthesized a lysosome-targeted probe with two-photon

-p

fluorescence for HClO sensing [33]. Xu et al. designed an off-on two-photon

re

fluorescent probe for recognizing HClO in mitochondria of living cells [34]. However,

lP

fluorescent probes with endoplasmic reticulum (ER) targeting ability for HClO

na

sensing in ER region have rarely been developed [35]. In this work, an ER targetable two-photon fluorescent molecular probe, JX-1, was

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synthesized for in situ analysis and visualization of HClO (Scheme 1). The developed probe exhibited fast response and high sensitivity towards HClO with a broad detection range of 1.0-160 μM and a low limit of detection 0.12 μM. The probe JX-1 also showed excellent selectivity for HClO sensing over other ROS, metal ions, and amino acids. Moreover, JX-1 demonstrated very low cellular toxicity and ER targetable ability. By virtue of these superior properties, JX-1 was resoundingly applied for in situ and real time two-photon bioimaging of HClO in ER region, an organelle intertwines with sleep deprivation [36], and could help us understand the metabolism of HClO during sleep deprivation. With the assistance of the probe JX-1, 4

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we had found that sleep deprivation can induce the generation of HClO for the first time.

2. Experimental section 2.1 Chemicals

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All the regents and solvents were of analytical grade and used as received. 4-Bromo-1,8-naphthalic anhydride, 4-aminobenzoic acid hydrazide (ABH), and (NAC)

were

purchased

from

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N-acetylcysteine

TCI

Company.

-p

N-Tosylethylenediamine, S-nitroso-N-acetyl-DL-penicillamine (SNAP), potassium

re

superoxide (KO2), phorbol 12-myristate 13-acetate (PMA), methanethiol sodium salt

lP

(CH3SNa), leucine (Leu), proline (Pro), histidine (His), isoleucine (Lle),

aspartic

acid

na

phenylalanine (Phe), alanine(Ala), lysine (Lys), cysteine (Cys), threonine (Thr), (Asp),

methionine

(Met),

and

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma-Aldrich. N,N-dimethylformamide (DMF), anhydrous dimethylsulfoxide (DMSO), triethylamine (Et3N), ascorbic acid (AA), NaNO2, NaClO, Na2SO4, NaNO3, NaCl, NaBr, NaI, Na2S, and H2O2 were purchased from Aladdin Chemistry Co. Ltd. ER-Tracker Red was purchased from Beyotime Biotechnology. High glucose Dulbecco’s modified Eagle’s media (DMEM) were received from Gibco BRL. All the metal salts were received from Sinopharm Chemical Reagent Co. Ltd. Silica gel employed for flash column chromatography was received from Qingdao Haiyang Chemical Co., Ltd (200-300 mesh). 5

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ROS solutions were obtained as follows. HClO was prepared from diluting NaClO solution by deionized water, and absorption intensity was determined at 292 nm (ε = 391 M-1 cm-1) to quantitate its content. H2O2 solution was directly acquired by diluting commercial aqueous solution, and the concentration was determined according to the absorption intensity at 240 nm (ε = 43.6 M-1 cm-1). O2•− was produced by dissolving KO2 in DMSO solution and NO was derived from the solution

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of SNAP. Fenton reaction between FeSO4 and H2O2 was used to supply •OH. The

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•OH concentration was equal to the Fe2+ concentration used. 1O2 was prepared from

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the reaction of NaClO with H2O2. ONOO− was generated from the chemical reaction

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between NaNO2 and H2O2, and the concentration was calculated according to the

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2.2 Instruments

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absorption intensity at 302 nm (ε = 1670 M-1 cm-1).

Fluorescence emission and excitation spectra were measured on a Cary Eclipse

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fluorescence spectrophotometer with emission and excitation slit widths both setting at 10 nm. Absorption spectra were carried out on a Shimadzu UV-2550 spectrophotometer. High-resolution mass spectra (HR-MS) were analyzed by an Agilent 6890 mass spectrometer with electron spray ionization/time-of-flight system. 13

C NMR and 1H NMR spectra were determined using a Bruker 500 MHz

spectrometer. Confocal fluorescence and bright field images (512 × 512 pixels) were captured with a TCS-SP8 confocal laser scanning microscope. Cells were imaged using a 63× objective lens, while for zebrafish, an objective lens of 10× was used. The intensities of the obtained fluorescence images were quantified with the help of 6

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ImageJ software. 2.3 Synthesis of hypobromous acid probe (JX-1) Firstly,

4-bromo-1,8-naphthalic

anhydride

(0.50

g,

1.8

mmol)

and

N-tosylethylenediamine (0.41 g, 1.9 mmol) were dissolved in ethanol (100 mL). The mixture was refluxed for 24 h. After completion, the reaction was naturally cooled to room temperature. During this period, precipitates were generated, which were then

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filtered, rinsed, and dried overnight in a desiccator to obtain 0.35 g of compound 1. 1H

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NMR (CDCl3): δ = 8.65 (t, 2H), 8.38 (d, 1H), 8.11 (d, 1H), 7.93 (t, 1H), 7.57 (d, 2H),

-p

6.77 (d, 2H), 4.33 (t, 2H), 3.56 (m, 2H), 1.98 (s, 3H). 13C NMR (CDCl3): δ = 164.08,

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142.70, 137.19, 133.67, 132.30, 131.45, 131.18, 129.20, 128.19, 126.68, 122.63,

na

473.0171; found: 473.0165.

lP

121.73, 42.48, 39.24, 21.16. HRMS (m/z): [M+H]+ calcd. for C21H18BrN2O4S:

Subsequently, to a round bottom flask (100 mL) containing DMF (50 mL), Et3N

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(0.04 g, 0.4 mmol) and compound 1 (0.19 g, 0.4 mmol), CH3SNa (0.03 g, 0.4 mmol) was introduced. The resulted hybrid was vigorously stirred and maintained at room temperature for 12 h. Afterwards, the solvent was evaporated. Then, CH2Cl2 was added to dissolve the crude product, followed by the introduction of H2O. The organic layer was isolated from the H2O layer, which was extracted by an extra amount of CH2Cl2. The CH2Cl2 solutions were combined, dried over anhydrous Na2SO4, and further removed on a rotavapor to give 0.18 g of compound 2 (JX-1) as yellow powder. 1H NMR (CDCl3): δ = 8.58 (t, 2H), 8.44 (d, 1H), 7.82 (t, 1H), 7.56 (d, 2H), 7.50 (d, 1H), 6.74 (d, 2H), 4.32 (t, 2H), 3.55 (m, 2H), 2.77 (s, 3H), 1.94 (s, 3H). 7

13

C

Journal Pre-proof NMR (CDCl3): δ = 164.45, 147.19, 142.60, 131.71, 131.15, 130.00, 129.14, 126.63, 122.61, 120.85, 118.13, 42.72, 39.05, 21.07, 14.86. HRMS (m/z): [M+Na]+ calcd. for C22H20N2NaO4S2: 463.0762; found:463.0757. 2.4 Reaction between JX-1 and HClO NaClO (15 mL, 1.0 mM) was added to a JX-1 (0.08 g, 0.18 mmol) solution in

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DMSO (6 mL), and the resulted solution kept stirring for 10 min. Next, the solvents were removed, and the residue was obtained, and subjected to silica gel column

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chromatography to generate compound 3 as white solid. 1H NMR [(CD3)2SO]: δ =

-p

8.65 (d, 1H), 8.55 (d, 1H), 8.48 (d, 1H), 8.34 (d, 1H), 7.99 (t, 1H), 7.78 (t, 1H), 7.59

re

(d, 2H), 7.25 (d, 2H), 4.13 (t, 2H), 3.11 (m, 2H), 2.92 (s, 3H), 2.26 (s, 3H). 13C NMR

lP

[(CD3)2SO]: δ = 163.48, 150.16, 142.94, 138.10, 131.55, 130.71, 129.98, 128.92,

na

126.81, 124.88, 123.57, 123.18, 43.49, 21.36. HRMS (m/z): [M+Na]+ calcd. for C22H20N2NaO5S2: 479.0711; found 479.0708.

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2.5 Cell culture and cytotoxicity assay HeLa cells were planted in 96-well cell culture plates (1 × 104 cells per well), and cultured in DMEM supplemented with 10% fetal bovine serum, 80 U·mL–1 penicillin, and 80 μg·mL–1 streptomycin in a 95% air/5% CO2 incubator. The culture media were deserted after fostered for 12 h, and the fresh media with varied dosages of the probe (0-50 μM) was introduced into the wells and fostered for 48 h. For each concentration, five parallel experiments were conducted. Subsequently, MTT solution (0.5 mg mL–1) with a volume of 40 μL was injected to each well, allowing the production of formazan crystals for 4 h. Following this, 150 μL of DMSO was injected into the 8

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wells. Absorption intensity (A) of the resultant hybrid was determined. The cellular viability rates were assessed by the following equation: cellular viability (%) = Atest/Acontrol × 100%, where Acontrol and Atest, respectively, denote the absorbance value obtained from the untreated cells and absorbance value recorded in the presence of the synthesized probe.

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2.6 Confocal fluorescence imaging Before bioimaging tests, HeLa cells were detached, replanted on 35 mm confocal

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dishes and adhered for about 12 h. Subsequently, the medium in these wells was

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replaced by new one supplemented with the probe (10 μM) and further fostered for

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0.5 h. Thenceforth, the attached cells were rinsed with phosphate buffer saline (PBS),

lP

and this washing process was conducted for three times. The two-photon fluorescence

na

imaging of the probe loaded cells was acquired in the wavelength range of 470-550 nm under the excitation of 800 nm.

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The subcellular location of the fluorescent probe JX-1 was checked by co-localization bioimaging experiments, where JX-1 labeled HeLa cells were further labeled with ER-Tracker Red (100 nM) for an additional 15 min. After the labeling experiments, the labeled cells were cleaned with PBS before confocal imaging experiments. Using a semiconductor laser at 552 nm as the excitation resource, the one-photon fluorescence image of ER-Tracker was acquired in the 580-650 nm wavelength range, whereas for JX-1, a 800 nm excitation wavelength was employed and the two-photon fluorescence emission was acquired in the 470-550 nm wavelength range. 9

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For fluorescence imaging of exogenous HClO, the HeLa cells were labeled with JX-1 (10 µM) and then stimulated with different amounts of NaClO (0-160 μM). For fluorescence tracking of endogenous HClO, HeLa cells were sequentially stimulated by PMA (1.5 μg/mL) for 3 h, and JX-1 (10 µM) for 0.5 h. For inhibition tests, cells were precultured with ABH (200 μM) for 4 h or NAC (1.0 mM) for 0.5 h, and then

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successively treated with PMA (1.5 μg/mL) for 3 h and JX-1 (10 µM) for 0.5 h. Two-photon fluorescence bioimaging of HClO in zebrafish were conducted in the

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way similar to that of HeLa cells. The 6-day-old zebrafish was maintained at a

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controlled temperature of 27.5 °C. These zebrafish were divided into two groups:

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sleep-rested group and sleep-deprived group. For the sleep-rested group, a 12 : 12

lP

(light : dark) photoperiod was employed, while for the sleep-deprived group,

na

zebrafish were exposed to light for 24 h, according to the protocol described by Luchiari and coworkers [37]. Light with an intensity of approximately 300 lx,

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achieved by a 100 W incandescent bulb, was used in the experiment.

3. Results and discussion

Inspired by the pioneering works reported previously [33, 38, 39], in this study, we developed an ER-targeted two-photon fluorescent probe, JX-1, for HClO detection. The JX-1 probe was synthesized via two steps according to the synthesis routes depicted in Fig. S1. For fabricating the probe, naphthalimide, a classic two-photon fluorophore, was chosen as the fluorophore scaffold, on account of its extraordinary two-photon property [40, 41]. Besides, JX-1 also possesses a S-methyl group, acting 10

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as the recognition unit, which can be readily oxidized by HClO to form -S=O group, thus modulates the push-pull electronic structure of naphthalimide and affects its two-photon fluorescence [33, 42]. Moreover, JX-1 bearing a methyl sulphonamide group, which has a targetable capacity to the sulfonylurea receptors on the ER membrane, endowing the probe with ER-targeting ability [43, 44]. The detailed synthesis procedures are provided in the Experimental Section. The chemical

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structures of the intermediate and JX-1 were fully characterized with 1H NMR,

C

ro

NMR, and HRMS (Fig. S2-S7).

13

-p

After structure confirmation, spectrometric studies of the probe JX-1 toward HClO

re

were then implemented at room temperature in PBS buffer (pH = 7.4, 10 mM) with

lP

1.0% DMSO. As displayed in Fig. 1A, probe JX-1 exhibits one obvious absorption

na

peak at 402 nm, and one shoulder at 274 nm (curve a). However, after being reacted with HClO, the intensity of the absorption at 402 nm significantly decreased, along

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with the increase of the absorption at 274 nm (curve b). In the following fluorescence characterization (Fig. 1B), probe JX-1 showed a strong fluorescence emission at 509 nm (curve a). Using fluorescein in 0.1 M NaOH as the reference, the fluorescence quantum yield (φ) of the probe was determined to be 42.7%. Upon introduction of HClO, the fluorescence emission band of JX-1 at 509 nm presented a dramatic decrease (curve b), suggesting the potential use of JX-1 for HClO detection. To fully study the corresponding sensing mechanism, the produced main product was collected, purified and characterized using 1H NMR, 13C NMR, as well as HRMS (Fig. S8-S10). All these results proved the proposed responding mechanism illustrated in Scheme 1. 11

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Then, the sensing performance of JX-1 for HClO quantification was studied. Titration of the JX-1 solution (10 μM) with HClO (0-180 μM) revealed that the green fluorescence of JX-1 (Fgreen, 470-550 nm) decreased gradually with the increase of HClO concentration (Fig. 2A), as can be clearly distinguished by the naked eye (Fig. 2B). Accordingly, as displayed in Fig. 2C, the fluorescence intensity at 509 nm showed an excellent linear relationship with the amount of HClO in the range of

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1.0-160 μM. Remarkably, the limit of detection (LOD) was calculated to be as low as

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0.12 ± 0.04 μM (according to the formula of 3ζ/k, n = 5), which makes the probe

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feasible for quantitative detection of HClO at trace levels. The LOD value gained in

lP

HClO (Table S1) [10, 33-35, 45-48].

re

this work was comparable or lower than previously reported fluorescent probes for

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Additionally, the response rate of JX-1 to HClO was studied. The observation demonstrated that intensity of the emission at 509 nm reached a plateau within about

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150 s, suggesting a fast recognition process between the S-methyl group and HClO (Fig. S11A). Such a quick response will enable JX-1 to be employed for the real-time tracking of HClO. Besides, the performance of the proposed probe over a pH range of 6.6-8.2, covering the physiological pH range, was estimated. For JX-1 solution, as displayed in Fig. S11B, nearly no variation of the fluorescence signal was found, suggesting good stability of the probe under different pH values. Moreover, the response of the probe to HClO was revealed to be irrespective of pH value from 6.6 to 8.2, suggesting that JX-1 can work well in the physiological pH range without being affected. Furthermore, the photostability of the probe was also tested. After constantly 12

Journal Pre-proof irradiated for 1 h by a HITACHI F4600 fluorescence spectrophotometer (λex = 405 nm), the fluorescence signal at 509 nm showed negligible alterations (< 4.1%), which implies the robust photostability of the probe (Fig. S11C). For further determination of HClO in complex biological system, selectivity tests were conducted. For selectivity experiment, other physiologically related species

of

including other ROS, metal ions, amino acids and anions were investigated. As displayed in Fig. S12 and Fig. S13, no noticeable effects (<4.8%) were found from

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these interferences, in comparison to that for HClO. The achieved data reflect that

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JX-1 can be applied for selective determination of HClO in biological system.

re

The remarkable fluorescence properties of JX-1 for HClO, including high

lP

sensitivity, selectivity and fast response rate, prompted us to further evaluate its utility

na

in the determination of HClO in biological systems. In ahead of this, cellular toxicity of JX-1 to HeLa cells was tested using standard MTT method. The findings suggested

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that cellular viability rate still kept higher than 90% (Fig. S13) even when the cells were cultured in growth medium with a high amount of the present probe (50 μM). Hence, JX-1 is of low cytotoxicity to live cells. As the probe JX-1 was functionalized with methyl sulphonamide moiety, a group possessing ER-targeting capability, the potential of JX-1 to accumulate into ER of live cells was then examined. To achieve this goal, co-localization experiments were conducted through co-labeling HeLa cells with JX-1 (labelling HeLa cells with JX-1 for 0.5 h) and a broadly used ER-specific dye ER-Tracker Red (labelling JX-1 labelled cells with ER-Tracker Red for 15 min). As shown in Fig. 3, the observed 13

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green fluorescence signal from JX-1 (Fig. 3a) displayed a great degree of overlap with the red fluorescence of ER-Tracker (Fig. 3b), as easily observed from the intense yellow signals in Fig. 3c. Additionally, a high Pearson’s coefficient of 0.94 is acquired from the corresponding intensity correlation plots (Fig. 3f). These findings confirm that with the functionalization of methyl sulphonamide group, JX-1, as anticipated,

of

precisely accumulated into ER region. Subsequently, we tried to evaluate the property of probe JX-1 for in situ biosensing

ro

and bioimaging of HClO in ER region of live cells by fluorescent confocal

-p

microscopy. After staining with JX-1, HeLa cells were further treated with changed

re

amounts of HClO (0, 40, 80, 120, and 160 μM). As displayed in Fig. 4a, fluorescence

lP

signal observed from the green channel was relatively strong, revealing a low basal

na

content of HClO in the normal condition. For the cells further treated with HClO, a significantly decreased green fluorescence was observed (Fig. 4b-e). Concomitantly,

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the mean fluorescence intensities also diminished with the increasing of HClO concentration (Fig. 4k). The above observations indicate that the probe JX-1 is competent to detect exogenous HClO. JX-1 was then applied to image fluctuations of endogenously generated HClO. It is known that PMA can generate HClO in living cells [49, 50]. Specifically, PMA stimulates cells to produce H2O2, and then MPO converts H2O2 into HClO. Fig. 5 shows the two-photon fluorescent images of HeLa cells with endogenous HClO. As displayed, JX-1 labeled cells showed bright green fluorescence (Fig. 5a), while the fluorescence of JX-1 in the cells pretreated with PMA was much weaker (Fig. 5b), 14

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suggesting the implication of HClO. To verify whether the fluorescence intensity change was ascribed to the generation of endogenous HClO, ABH, an MPO inhibitor was used [51]. In this bioimaging test, living cells were orderly treated with ABH, PMA, and JX-1. In this case, no apparent variation of the fluorescence intensity was visualized from the green channel (Fig. 5c), irrespective of the presence of PMA, as

of

compared with the control group (Fig. 5a). Furthermore, in another control experiment, live cells were incubated with NAC, an effective ROS scavenger [52],

ro

PMA, and JX-1 successively. We found that with the existence of NAC, stimulation

-p

of HeLa cells with PMA would witness no fluorescence change (Fig. 5d), again

re

validating the fluorescence decrease observed in Fig. 5b was truly due to the

lP

generation of endogenous HClO. Together, our data evidently confirm the suitability

live cells.

na

of JX-1 for two-photon fluorescence imaging the formation of endogenous HClO in

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Having established the desirable capability of JX-1 for biosensing exogenous and endogenous HClO in live HeLa cells, we next interrogated its efficacy for visualization of HClO in zebrafish. As illustrated in Fig. 6, under two-photon excitation, JX-1 stained zebrafish displayed intense green fluorescence (Fig. 6a). However, after the treatment with HClO or PMA, seriously quenched fluorescence signal can be gained from the corresponding green channels (Fig. 6b and 6d). To affirm the fluorescence quenching was attributed to HClO, NAC was introduced. In this context, the fluorescence changes caused by HClO or PMA were efficiently suppressed (Fig. 6c and 6e). These experimental results provided compelling evidence 15

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to support that the probe JX-1 can be applied to determine exogenous HClO, as well as endogenously generated HClO in zebrafish. Subsequently, for the first time, we attempted to explore the contribution of HClO in zebrafish during sleep deprivation. For performing the experiments, the 6-day-old zebrafish was divided into two groups: sleep-rested group (control group) and

of

sleep-deprived group (testing group). As shown, for the control group, a strong green fluorescence can be readily seen under two-photon excitation (Fig. 7a), while for the

ro

testing group, a relatively weak fluorescence was observed (Fig. 7b), suggesting the

-p

involvement of HClO. To ascertain the generation of HClO during sleep deprivation,

re

the sleep-deprived fish was further fed with NAC. In this case, no marked

lP

fluorescence signal change was observed from the zebrafish that was subjected to

na

sleep deprivation for 24 h (Fig. 7c), demonstrating that sleep deprivation can indeed

Conclusion

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trigger the burst of HClO in zebrafish.

In conclusion, a novel naphthalimide-based two-photon fluorescent probe, JX-1, for biosensing of HClO in ER region of living cells was rationally designed. The present fluorescent probe is capable of detecting HClO with rapid response rate, high sensitivity and selectivity. Owing to these prominent sensing performances, the fluorescent probe with low cytotoxicity has been triumphantly utilized for monitoring the generation of exogenous and endogenous HClO in live cells and zebrafish. From the investigation in zebrafish, we have found that sleep deprivation can induce the 16

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generation of HClO in zebrafish for the first time. We believe this probe holds great promise as a useful imaging tool to study the metabolism of HClO during sleep deprivation and its function in biological activities.

Conflicts of interest

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There are no conflicts to declare.

Acknowledgements

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The authors gratefully thank the National Key Research and Development Program of

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China (Grant No. 2018YFB1502900), National Natural Science Foundation of China

lP

(Grant No. 21103024), Zhejiang Provincial Natural Science Foundation of China

na

(Grant No. LY19B060006), and Technology Development Project of Jiaxing

References

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University for financial support.

[1] A. Kumar and A. Singh, Possible nitric oxide modulation in protective effect of (Curcuma longa, Zingiberaceae) against sleep deprivation-induced behavioral alterations and oxidative damage in mice, Phytomedicine, 15 (2008) 577-586. [2] E. Tobaldini, G. Costantino, M. Solbiati, C. Cogliati, T. Kara, L. Nobili and N. Montano, Sleep, sleep deprivation, autonomic nervous system and cardiovascular diseases, Neurosci. Biobehav. Rev., 74 (2017) 321-329. [3] Y. Nir, T. Andrillon, A. Marmelshtein, N. Suthana, C. Cirelli, G. Tononi and I. 17

Journal Pre-proof

Fried, Selective neuronal lapses precede human cognitive lapses following sleep deprivation, Nat. Med., 23 (2017) 1474. [4] J. Noguti, M. L. Andersen, C. Cirelli and D. A. Ribeiro, Oxidative stress, cancer, and sleep deprivation: is there a logical link in this association?, Sleep Breath., 17 (2013) 905-910.

of

[5] G. Villafuerte, A. Miguel-Puga, E. Murillo Rodrguez, S. Machado, E. Manjarrez and O. Arias-Carrion, Sleep deprivation and oxidative stress in animal models: a

ro

systematic review, Oxid. Med. Cell. Longev., 2015 (2015) 15.

-p

[6] X. Chen, F. Wang, J. Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin and J. Yoon,

re

Recent progress in the development of fluorescent, luminescent and colorimetric

na

(2016) 2976-3016.

lP

probes for detection of reactive oxygen and nitrogen species, Chem. Soc. Rev., 45

[7] H. Huang and Y. Tian, A ratiometric fluorescent probe for bioimaging and

Jo ur

biosensing of HBrO in mitochondria upon oxidative stress, Chem. Commun., 54 (2018) 12198-12201.

[8] D. Zhou, H. Huang, Y. Wang, Y. Wang, Z. Hu and X. Li, A yellow-emissive carbon nanodot-based ratiometric fluorescent nanosensor for visualization of exogenous and endogenous hydroxyl radicals in the mitochondria of live cells, J. Mater. Chem. B, 7 (2019) 3737-3744. [9] J. Zhou, L. Li, W. Shi, X. Gao, X. Li and H. Ma, HOCl can appear in the mitochondria of macrophages during bacterial infection as revealed by a sensitive mitochondrial-targeting fluorescent probe, Chem. Sci., 6 (2015) 4884-4888. 18

Journal Pre-proof

[10] Z. Mao, M. Ye, W. Hu, X. Ye, Y. Wang, H. Zhang, C. Li and Z. Liu, Design of a ratiometric two-photon probe for imaging of hypochlorous acid (HClO) in wounded tissues, Chem. Sci., 9 (2018) 6035-6040. [11] M. Ren, Z. Li, J. Nie, L. Wang and W. Lin, A photocaged fluorescent probe for imaging hypochlorous acid in lysosomes, Chem. Commun., 54 (2018) 9238-9241.

of

[12] Z. Lou, P. Li, Q. Pan and K. Han, A reversible fluorescent probe for detecting hypochloric acid in living cells and animals: utilizing a novel strategy for effectively

ro

modulating the fluorescence of selenide and selenoxide, Chem. Commun., 49 (2013)

-p

2445-2447.

re

[13] B. Wang, P. Li, F. Yu, P. Song, X. Sun, S. Yang, Z. Lou and K. Han, A reversible

lP

fluorescence probe based on Se–BODIPY for the redox cycle between HClO

na

oxidative stress and H2S repair in living cells, Chem. Commun., 49 (2013) 1014-1016.

Jo ur

[14] Y. Yue, F. Huo, C. Yin, J. O. Escobedo and R. M. Strongin, Recent progress in chromogenic and fluorogenic chemosensors for hypochlorous acid, Analyst, 141 (2016) 1859-1873.

[15] H. Li and H. Ma, New progress in spectroscopic probes for reactive oxygen species, J. Anal. Test., 2 (2018) 2-19. [16] X. Jiao, Y. Li, J. Niu, X. Xie, X. Wang and B. Tang, Small-molecule fluorescent probes for imaging and detection of reactive oxygen, nitrogen, and sulfur species in biological systems, Anal. Chem., 90 (2018) 533-555. [17] Z. Liu, H. Pei, L. Zhang and Y. Tian, Mitochondria-targeted DNA nanoprobe for 19

Journal Pre-proof real-time imaging and simultaneous quantification of Ca2+ and pH in neurons, ACS Nano, 12 (2018) 12357-12368. [18] H. Zhu, J. Fan, J. Du and X. Peng, Fluorescent probes for sensing and imaging within specific cellular organelles, Acc. Chem. Res., 49 (2016) 2115-2126. [19] Z. Lou, P. Li and K. Han, Redox-responsive fluorescent probes with different

of

design strategies, Acc. Chem. Res., 48 (2015) 1358-1368. [20] F. Yu, P. Li, B. Wang and K. Han, Reversible near-Infrared fluorescent probe

ro

introducing tellurium to mimetic glutathione peroxidase for monitoring the redox

-p

cycles between peroxynitrite and glutathione in vivo, J. Am. Chem. Soc., 135 (2013)

re

7674-7680.

lP

[21] F. Yu, P. Li, G. Li, G. Zhao, T. Chu and K. Han, A near-IR reversible fluorescent

na

probe modulated by selenium for monitoring peroxynitrite and imaging in living cells, J. Am. Chem. Soc., 133 (2011) 11030-11033.

Jo ur

[22] W. Li, B. Fang, M. Jin and Y. Tian, Two-photon ratiometric fluorescence probe with enhanced absorption cross section for imaging and biosensing of zinc ions in hippocampal tissue and zebrafish, Anal. Chem., 89 (2017) 2553-2560. [23] B. Kong, A. Zhu, C. Ding, X. Zhao, B. Li and Y. Tian, Carbon dot-based inorganic-organic nanosystem for two-photon imaging and biosensing of pH variation in living cells and tissues, Adv. Mater., 24 (2012) 5844-5848. [24] Q. Xu, C. H. Heo, G. Kim, H. W. Lee, H. M. Kim and J. Yoon, Development of imidazoline-2-thiones

based

two-photon

fluorescence

probes

for

imaging

hypochlorite generation in a co-culture system, Angew. Chem. Int. Ed., 54 (2015) 20

Journal Pre-proof

4890-4894. [25] Y. Tang, X. Kong, A. Xu, B. Dong and W. Lin, Development of a two-photon fluorescent probe for imaging of endogenous formaldehyde in living tissues, Angew. Chem. Int. Ed., 55 (2016) 3356-3359. [26] H. M. Kim and B. R. Cho, Small-molecule two-photon probes for bioimaging

of

applications, Chem. Rev., 115 (2015) 5014-5055. [27] Z. Mao, W. Feng, Z. Li, L. Zeng, W. Lv and Z. Liu, NIR in, far-red out:

-p

Chem. Sci., 7 (2016) 5230-5235.

ro

developing a two-photon fluorescent probe for tracking nitric oxide in deep tissue,

re

[28] Z. Mao, H. Jiang, Z. Li, C. Zhong, W. Zhang and Z. Liu, An N-nitrosation

lP

reactivity-based two-photon fluorescent probe for the specific in situ detection of

na

nitric oxide, Chem. Sci., 8 (2017) 4533-4538. [29] L. Yuan, L. Wang, B. K. Agrawalla, S.-J. Park, H. Zhu, B. Sivaraman, J. Peng,

Jo ur

Q.-H. Xu and Y.-T. Chang, Development of targetable two-photon fluorescent probes to image hypochlorous acid in mitochondria and lysosome in live cell and inflamed mouse model, J. Am. Chem. Soc., 137 (2015) 5930-5938. [30] X. Jiao, Y. Xiao, Y. Li, M. Liang, X. Xie, X. Wang and B. Tang, Evaluating drug-induced liver injury and its remission via discrimination and imaging of HClO and H2S with a two-photon fluorescent probe, Anal. Chem., 90 (2018) 7510-7516. [31] B. Zhu, P. Li, W. Shu, X. Wang, C. Liu, Y. Wang, Z. Wang, Y. Wang and B. Tang, Highly specific and ultrasensitive two-photon fluorescence imaging of native HOCl in lysosomes and tissues based on thiocarbamate derivatives, Anal. Chem., 88 (2016) 21

Journal Pre-proof

12532-12538. [32] H. Chen, H. Shang, Y. Liu, R. Guo and W. Lin, Development of a unique class of spiro-type two-photon functional fluorescent dyes and their applications for sensing and bioimaging, Adv. Funct. Mater., 26 (2016) 8128-8136. [33] B. Zhang, X. Yang, R. Zhang, Y. Liu, X. Ren, M. Xian, Y. Ye and Y. Zhao,

of

Lysosomal-targeted two-photon fluorescent probe to sense hypochlorous acid in live cells, Anal. Chem., 89 (2017) 10384-10390.

ro

[34] Q. Xu, C. H. Heo, J. A. Kim, H. S. Lee, Y. Hu, D. Kim, K. M. K. Swamy, G. Kim,

-p

S.-J. Nam, H. M. Kim and J. Yoon, A selective imidazoline-2-thione-bearing

re

two-photon fluorescent probe for hypochlorous acid in mitochondria, Anal. Chem., 88

lP

(2016) 6615-6620.

na

[35] Y. L. Pak, S. J. Park, G. Song, Y. Yim, H. Kang, H. M. Kim, J. Bouffard and J. Yoon, Endoplasmic reticulum-targeted ratiometric N-heterocyclic carbene borane

Jo ur

probe for two-photon microscopic imaging of hypochlorous acid, Anal. Chem., 90 (2018) 12937-12943.

[36] N. Naidoo, Cellular stress/the unfolded protein response: relevance to sleep and sleep disorders, Sleep Med. Rev., 13 (2009) 195-204. [37] J. Pinheiro-da-Silva, P. F. Silva, M. B. Nogueira and A. C. Luchiari, Sleep deprivation effects on object discrimination task in zebrafish (Danio rerio), Anim. Cogn., 20 (2017) 159-169. [38] C. Duan, M. Won, P. Verwilst, J. Xu, H. S. Kim, L. Zeng and J. S. Kim, In vivo imaging of endogenously produced HClO in zebrafish and mice using a bright, 22

Journal Pre-proof

photostable ratiometric fluorescent probe, Anal. Chem., 91 (2019) 4172-4178. [39] S.-R. Liu, M. Vedamalai and S.-P. Wu, Hypochlorous acid turn-on boron dipyrromethene probe based on oxidation of methyl phenyl sulfide, Anal. Chim. Acta, 800 (2013) 71-76. [40] H. Xiao, X. Liu, C. Wu, Y. Wu, P. Li, X. Guo and B. Tang, A new endoplasmic

of

reticulum-targeted two-photon fluorescent probe for imaging of superoxide anion in diabetic mice, Biosens. Bioelectron., 91 (2017) 449-455.

ro

[41] M. Yang, J. Fan, J. Zhang, J. Du and X. Peng, Visualization of methylglyoxal in

-p

living cells and diabetic mice model with a 1,8-naphthalimide-based two-photon

re

fluorescent probe, Chem. Sci., 9 (2018) 6758-6764.

lP

[42] M. Ren, K. Zhou, L. He and W. Lin, Mitochondria and lysosome-targetable

(2018) 1716-1733.

na

fluorescent probes for HOCl: recent advances and perspectives, J. Mater. Chem. B, 6

Jo ur

[43] H. Xiao, P. Li, X. Hu, X. Shi, W. Zhang and B. Tang, Simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during apoptosis, Chem. Sci., 7 (2016) 6153-6159. [44] S.-J. Li, D.-Y. Zhou, Y. Li, H.-W. Liu, P. Wu, J. Ou-Yang, W.-L. Jiang and C.-Y. Li, Efficient two-photon fluorescent probe for imaging of nitric oxide during endoplasmic reticulum stress, ACS Sens., 3 (2018) 2311-2319. [45] G. Cheng, J. Fan, W. Sun, J. Cao, C. Hu and X. Peng, A near-infrared fluorescent probe for selective detection of HClO based on Se-sensitized aggregation of heptamethine cyanine dye, Chem. Commun., 50 (2014) 1018-1020. 23

Journal Pre-proof

[46] D. Li, Y. Feng, J. Lin, M. Chen, S. Wang, X. Wang, H. Sheng, Z. Shao, M. Zhu and X. Meng, A mitochondria-targeted two-photon fluorescent probe for highly selective and rapid detection of hypochlorite and its bio-imaging in living cells, Sens. Actuators B: Chem., 222 (2016) 483-491. [47] Y. Feng, S. Li, D. Li, Q. Wang, P. Ning, M. Chen, X. Tian and X. Wang, Rational design of a diaminomaleonitrile-based mitochondria-targeted two-photon fluorescent

ro

Sens. Actuators B: Chem., 254 (2018) 282-290.

of

probe for hypochlorite in vivo: solvent-independent and high selectivity over Cu2+,

-p

[48] X. Wang, X. Wang, Y. Feng, M. Zhu, H. Yin, Q. Guo and X. Meng, A two-photon

re

fluorescent probe for detecting endogenous hypochlorite in living cells, Dalton Trans.,

lP

44 (2015) 6613-6619.

na

[49] H. Li, X. Li, W. Shi, Y. Xu and H. Ma, Rationally designed fluorescence •OH probe with high sensitivity and selectivity for monitoring the generation of •OH in

Jo ur

iron autoxidation without addition of H2O2, Angew. Chem. Int. Ed., 57 (2018) 12830-12834.

[50] R. Zhang, J. Zhao, G. Han, Z. Liu, C. Liu, C. Zhang, B. Liu, C. Jiang, R. Liu, T. Zhao, M.-Y. Han and Z. Zhang, Real-time discrimination and versatile profiling of spontaneous reactive oxygen species in living organisms with a single fluorescent probe, J. Am. Chem. Soc., 138 (2016) 3769-3778. [51] Y. Jiang, G. Zheng, Q. Duan, L. Yang, J. Zhang, H. Zhang, J. He, H. Sun and D. Ho, Ultra-sensitive fluorescent probes for hypochlorite acid detection and exogenous/endogenous imaging of living cells, Chem. Commun., 54 (2018) 24

Journal Pre-proof

7967-7970. [52] L. Liang, C. Liu, X. Jiao, L. Zhao and X. Zeng, A highly selective and sensitive photoinduced electron transfer (PET) based HOCl fluorescent probe in water and its

Jo ur

na

lP

re

-p

ro

of

endogenous imaging in living cells, Chem. Commun., 52 (2016) 7982-7985.

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Declaration of Interest Statement

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There are no conflicts to declare.

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Authorship contribution statement Qineng Xia: Investigation, Data curation, Formal analysis, Writing -original draft. Xiaoyan Wang: Investigation, Data curation. Yanan Liu: Investigation, Data curation. Zhangfeng Shen: Cell culture, Methodology.

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Zhigang Ge: Investigation, Data curation. Hong Huang: Investigation, Conceptualization, Methodology, Writing -review &

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Xi Li: Investigation, Data curation.

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editing.

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Yangang Wang: Conceptualization, Funding acquisition, Project administration,

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Supervision, Methodology, Writing - review & editing.

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Figures:

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Scheme 1 Structure of JX-1 and the corresponding sensing mechanism for HClO.

Fig. 1 Spectral responses of JX-1 to HClO in PBS (pH = 7.4, 10 mM) containing 1.0% DMSO: (A) absorption and (B) fluorescence spectra of JX-1 (10 μM) (a) without or (b) with the existence of HClO (160 μM).

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Fig. 2 (A) Fluorescence spectra of JX-1 (10 μM) in PBS (pH = 7.4, 10 mM)

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containing 1.0% DMSO with various concentrations of HClO (from top to bottom: 0, 1.0, 2.5, 5.0, 10, 20, 40, 60, 80, 100, 120, 140, 160 and 180 μM). (B) The

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fluorescence quenching photographs of JX-1 with different HClO concentrations (from left to right: 0, 25, 50, 75, 100, 120, 140, and 160 μM). λex = 365 nm. (C) Plot of the emission intensity at 509 nm versus the concentration of HClO.

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Fig. 3 Co-localization investigations in HeLa cells that were co-labeled with JX-1 (10 μM) and ER-Tracker Red (100 nM): (a) Two-photon fluorescence image of the green

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channel from the probe (λem = 470-550 nm, λex = 800 nm); (b) one-photon fluorescence image of the red channel from ER-Tracker Red (λem = 580-650 nm, λex =

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552 nm); (c) the merged image of (a) and (b); (d) the bright field image; (e) the merged image of (c) and (d); and (f) the corresponding intensity correlation plot of the green channel with red channel. Scale bar: 10 μm.

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Fig. 4 Two-photon fluorescence imaging of JX-1 labeled HeLa cells fostered in

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media containing various amounts of HClO: (a, f) 0, (b, g) 40, (c, h) 80, (d, i) 120, and

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(e, j) 160 μM. (a-e) Two-photon fluorescence images from the green channel (λem =

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470-550 nm, λex = 800 nm). (f-j) The corresponding bright field images. (k) Mean fluorescence intensities from the green channel vs. different concentrations of HClO.

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The cell images shown are representative. Scale bar: 10 μm.

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Fig. 5 Two-photon fluorescence imaging of HeLa cells labeled with 10 μM JX-1 (λem

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= 470-550 nm, λex = 800 nm): (a) control image; (b) cells pretreated with PMA (1.5

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μg/mL) for 3 h, and then incubated with JX-1; (c) cells pretreated with ABH (200 μM) for 4 h, PMA (1.5 μg/mL) for 3 h, and then incubated with JX-1; (d) cells pretreated

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with NAC (1.0 mM) for 0.5 h, PMA (1.5 μg/mL) for 3 h, and then incubated with JX-1. (e-h) The corresponding bright field images. (i) Mean fluorescence intensities

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of cells in images (a)-(d). The cell images shown are representative. Scale bar: 10 μm.

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Fig. 6 Two-photon fluorescence imaging of HClO in 6-day-old zebrafish (λem =

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470-550 nm, λex = 800 nm): (a) zebrafish fed with JX-1 (10 μM) for 0.5 h; (b) zebrafish fed with NaClO (200 μM) for 0.5 h and JX-1 (10 μM) for 0.5 h; (c)

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zebrafish fed with NAC (1.0 mM) for 0.5 h, NaClO (200 μM) for 0.5 h, and then fed with JX-1 (10 μM) for 0.5 h; (d) zebrafish fed with PMA (1.5 μg/mL) for 3 h and

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JX-1 (10 μM) for 0.5 h; (e) zebrafish fed with NAC (1.0 mM) for 0.5 h, PMA (1.5 μg/mL) for 3 h, and then fed with JX-1 (10 μM) for 0.5 h. (f) Mean fluorescence intensities of zebrafish treated at different conditions shown in (a)-(e). Scale bar: 250 μm.

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Fig. 7 Two-photon fluorescence imaging of HClO in 6-day-old zebrafish (λem = 470-550 nm, λex = 800 nm): (a) sleep-rested zebrafish fed with JX-1 (10 μM) for 0.5 h;

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(b) sleep-deprived zebrafish fed with JX-1 (10 μM) for 0.5 h; (c) sleep-deprived zebrafish fed with NAC (1.0 mM) for 0.5 h and JX-1 (10 μM) for 0.5 h. (d) Mean fluorescence intensities of zebrafish treated at different conditions shown in (a)-(c). The zebrafish was sleep deprived for 24 h. Scale bar: 250 μm.

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Table of contents

Graphical abstract

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An ER targetable two-photon fluorescent probe was synthesized for in situ

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visualization of HClO in living cells and zebrafish.

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Highlights  A novel endoplasmic reticulum-targeted two-photon fluorescent probe for biosensing HClO was developed.  The developed probe is capable of detecting HClO with fast response rate, high selectivity and sensitivity.

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 The developed probe can be applied for monitoring exogenous and endogenous

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HClO.

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