A highly selective and sensitive fluorescent probe for hypochlorous acid and its lysosome-targetable biological applications

A highly selective and sensitive fluorescent probe for hypochlorous acid and its lysosome-targetable biological applications

Talanta 174 (2017) 234–242 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta A highly selective a...

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Talanta 174 (2017) 234–242

Contents lists available at ScienceDirect

Talanta journal homepage: www.elsevier.com/locate/talanta

A highly selective and sensitive fluorescent probe for hypochlorous acid and its lysosome-targetable biological applications Chang Liua,b, Xiaojie Jiaoa, Song Hea, Liancheng Zhaoa,b, Xianshun Zenga,b,

MARK



a Tianjin Key Laboratory for Photoelectric Materials and Devices, and Key Laboratory of Display Materials & Photoelectric Devices, Ministry of Education, School of Materials Science & Engineering, Tianjin University of Technology, Tianjin 300384, China b School of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001, China

A R T I C L E I N F O

A BS T RAC T

Keywords: Fluorescent probe 1,8-Naphthalimide Hypochlorous acid PET Lysosome Imaging

A novel lysosome-targetable fluorescence probe PT-1 based on a photoinduced electron transfer (PET) mechanism has been designed and synthesized. For comparison, the probe PT-2 without morpholine moiety as a lysosome-directing group was also synthesized and investigated. Probes PT-1 and PT-2 exhibited high selectivity, high sensitivity (with the detection limits down to the 10−10 M range) and response in real time (within 10 s) toward HOCl over other reactive oxygen species (ROS). Both PT-1 and PT-2 were cell permeable and enabled them to be used for monitoring of HOCl in living cells. Meanwhile, the probe PT-1 demonstrated an accurately lysosome-targeting ability, and was successfully applied to image of exogenous, endogenous produced HOCl in living cells. The success of subcellular imaging suggested that the probe PT-1 could be used in further applications for the investigation of biological functions and pathological roles of HOCl at organelle levels.

1. Introduction Reactive oxygen species (ROS) are associated with various physiological and pathological processes [1–5]. As a type of ROS, hypochlorous acid (HOCl, pKa 7.53) and hypochlorite (ClO-) are widely employed as strong oxidizing agents in our daily life. In living organisms, hypochlorous acid, is associated with innate host defence for killing a wide range of pathogens [6], which is produced mainly from hydrogen peroxide and chloride ions in a heme enzyme myeloperoxidase (MPO)involved reaction [7–9]. Additionally, aberrant accumulation of HOCl in phagocytes can trigger tissue damage or remodeling, with profound implications for many human diseases including cardiovascular diseases [10], nephropathies [11], neurodegenerative disorders [2,12], inflammatory diseases [13], and certain cancers [9,14]. At the same time, HOCl could cause cell dysfunction at organelle levels, such as mitochondrial permeabilization [15], lysosomal rupture through calcium dependent calpain activation [15,16]. Accordingly, the detection of the distribution of HOCl at the organelle level would be more important for better understanding of its origins, activities, and functions. In recent years, fluorescence imaging technologies have been proven to be the most efficient tools for detecting and imaging trace

amounts of biomolecules in the living cell due to its nature of high sensitivity, high spatiotemporal resolution and nondestructive detection [17,18]. In the past decade, tremendous efforts have been payed for the development of fluorescent probes via distinct strategies for HOCl detection and imaging by taking advantage of the strong oxidization ability of this reactive oxygen species [19–47]. Even though many of these probes have been successfully applied to image intracellular HOCl, only a few of them were able to real-time detect HOCl at subcellular levels [19–22]. To the best of our knowledge, up to now several mitochondria-targeted fluorescent probes have been reported for the detection of intracellular HOCl [48–54], and only a very limited number of lysosome-targeted fluorescent probes have been reported for the detection of intracellular HOCl in lysosomes [55–58]. Lysosomes containing digestive enzymes are the major degradation machinery in cells and recycle damaged organelles as well as digest nucleic acids, polysaccharides, fats and proteins. The HOCl level in lysosomes is tightly related to the redox balance in lysosomes, which is significant to maintain the normal lysosomal function. Abnormal endogenous and exogenous HOCl could induce apoptosis of cultured cells through the rupture of lysosomes [16]. Therefore, it is challenging and highly desired to develop novel fluorescent probes for real-time elucidating the distribution and functions of HOCl in lysosomes.

⁎ Corresponding author at: Tianjin Key Laboratory for Photoelectric Materials and Devices, and Key Laboratory of Display Materials & Photoelectric Devices, Ministry of Education, School of Materials Science & Engineering, Tianjin University of Technology, Tianjin 300384, China. E-mail address: [email protected] (X. Zeng).

http://dx.doi.org/10.1016/j.talanta.2017.06.012 Received 7 March 2017; Received in revised form 26 May 2017; Accepted 2 June 2017 0039-9140/ © 2017 Elsevier B.V. All rights reserved.

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Agilent 6510 Q-TOF LC/MS instrument (Agilent Technologies, Palo Alto, CA) equipped with an electrospray ionization (ESI) source. Elemental analyses were performed on a Vanio-EL elemental analyzer (Analyze-system GmbH, Germany). Melting points were recorded on a Boethius Block apparatus. All absorption spectra were recorded using a Shimadzu UV-2550 UV/Vis spectrophotometer with 1 cm quartz cell. In a similar manner, fluorescence spectra were recorded on a Hitachi F-4600 spectrofluorophotometer with a 1 cm quartz cell. Cells were imaged on a confocal microscope (Olympus FV1000-IX81). All images were analyzed with Olympus FV1000-ASW. The concentration of NaOCl was measured by optical density at 292 nm in 0.01 M NaOH aqueous (ε292 = 350 cm−1 M−1). The concentration of the commercially available stock H2O2 solution was estimated by optical absorbance at 240 nm (ε240 = 43.6 cm−1 M−1). Hydroxyl radical (·OH) was prepared by Fenton reaction on mixing (NH4)2Fe(SO4)2·6H2O with 2.0 equivalents of H2O2 and its concentration was estimated by amount of Fe2+. The concentration of peroxynitrite (ONOO-) was estimated in 0.1 M NaOH aqueous (ε302 = 1670 cm−1 M−1). Singlet oxygen (1O2) was generated by the mixture of MoO42- and H2O2 in the molar ratio of 1: 2. Superoxide (O2-·) was produced from KO2. The saturated nitric oxide (NO) aqueous solution (2 mM, 20 °C) was prepared by bubbling NO gas into deoxygenated deionized water until saturation.

Chart 1. Structures depiction of probes PT-1 and PT-2.

O

O O

N

N

Br

O

O

N

N

2

O O

NH

O

N

PT-1 O

Br

O

S

O

1

N

HN

N

Br

O 3

S

2.2. The synthesis of N-(morpholinoethyl)-4-bromo-1,8naphthalimide 2

O

N

To a 500 mL flask, was added 1 (16.556 g, 60 mmol), N-(2aminoethyl)morpholine (8.066 g, 62 mmol), anhydrous ethanol (350 mL). The reaction mixture was refluxed for 1 h. The reactant was cooled down to room temperature. The precipitates were collected, and washed with ethanol (3 × 50 mL), dried under vacuum to get a gray powder 20 g (86%). The compound was used for the next reaction without further purification [59].

PT-2

Scheme 1. Synthesis routes of PT-1 and PT-2.

Herein, we reported a novel lysosome-targetable probe PT-1 for HOCl (Chart 1), a photostable signal transduction unit 1,8-naphthalimide tethered with morpholine moiety and an electron-rich phenothiazine moiety. The incorporation of a morpholine moiety into the probe is based on its lysosome-directing ability [58]. However, as we demonstrated recently [22], the introduce of phenothiazine moiety into the probe acts not only as a real time responsive unit for HOCl, but also offer a pH-independent electron-donating unit for this PET-based probe for acidic organelles, such as lysosomes. For comparison, the probe PT-2 without morpholine moiety as a lysosome-directing group was also synthesized. Probes PT-1 and PT-2 demonstrated high selectivity, high sensitivity (with the detection limit down to the 10−10 M range) and fast response (within 10 s) towards HOCl. Both PT-1 and PT-2 were cell permeable and enabled it to be used for monitoring of HOCl in living cells. Meanwhile, the probe PT-1 demonstrated a highly lysosome-targeting ability, and was successfully applied to image of exogenous, endogenous produced lysosomal HOCl in living cells. The success of subcellular imaging suggested that the probe PT-1 could be used in further applications for the investigation of biological functions and pathological roles of HOCl at organelle levels.

2.3. The synthesis of PT-1 To a 50 mL flask, was charged 2 (100 mg, 0.26 mmol), N-(3aminopropyl)phenothiazine [60] (132 mg, 0.52 mmol). The reaction mixture was stirred for 6 h at 140 °C under an argon atmosphere. The reaction mixture was purified by silica gel column chromatography (SiO2, CH2Cl2/EtOH, gradient) to obtain a yellow solid PT-1 in 69% yield (100 mg); m.p. 100–102 °C. HRMS: m/z [M + H]+ = 565.2268; Calcd: 565.2280; 1H NMR (CDCl3, 400 MHz, ppm): 8.50 (d, J = 8.0 Hz, 1 H), 8.30 (d, J = 8.0 Hz, 1 H), 7.72 (d, J = 8.0 Hz, 1 H), 7.44 (t, J = 8.0 Hz, 1 H), 7.24 (d, J = 8.0 Hz, 2 H), 7.16 (t, J = 8.0 Hz, 2 H), 6.98 (t, J = 8.0 Hz, 2 H), 6.91 (d, J = 8.0 Hz, 2 H), 6.58 (d, J = 8.0 Hz, 1 H), 5.57 (t, J = 6.0 Hz, 1 H), 4.31 (t, J = 6.0 Hz, 2 H), 4.08 (t, J = 6.0 Hz, 2 H), 3.70 (t, J = 8.0 Hz, 4 H), 3.60-3.56 (m, J = 7.2 Hz, 2 H), 2.69 (t, J = 8.0 Hz, 2 H), 2.61 (s, 4 H), 2.30-2.24 (m, J = 6.0 Hz, 2 H); 13 C NMR (CDCl3, 75 MHz, ppm): 163.47, 148.92, 144.80, 133.98, 130.75, 127.69, 127.38, 126.03, 124.26, 123.00, 122.86, 120.21, 115.91, 104.14, 67.48, 56.84, 54.39, 45.46, 42.10, 37.70, 26.03.

2. Experimental

2.4. The preparation of PT-2

2.1. Materials and equipments

To a 50 mL reactor, was charged 3 [61] (100 mg, 0.3 mmol), N-(3aminopropyl)phenothiazine (155 mg, 0.6 mmol). The reaction mixture was stirred for 6 h at 140 °C under argon atmosphere. The reaction mixture was purified by silica gel column chromatography (SiO2, CH2Cl2/EtOH, gradient) to obtain a yellow solid PT-2 in 67% yield (103 mg); m.p. 86–88 °C. HRMS: m/z [M + H]+ = 508.2053; Calcd: 508.2052; 1H NMR (CDCl3, 400 MHz, ppm): 8.50 (d, J = 8.0 Hz, 1 H), 8.32 (d, J = 8.0 Hz, 1 H), 7.72 (d, J = 8.0 Hz, 1 H), 7.44 (t, J = 7.6 Hz, 1 H),7.24 (d, J = 8.0 Hz, 2 H), 7.16 (t, J = 7.6 Hz, 2 H), 6.98 (t, J = 7.6 Hz, 2 H), 6.91 (d, J = 8.0 Hz, 2 H), 6.58 (d, J = 8.0 Hz, 2 H), 4.14

Starting materials and reagents were purchased from Tokyo Kasei Kogyo (TCI: Tokyo, Japan), AR grade or dry grade solvents were purchased from Alfa-Aesar, and used without further purification. The reactions were carried out in oven-dried glassware with a magnetic stirring. NMR spectra were recorded on a Bruker spectrometer at 400 (1H NMR) MHz and 75 (13C NMR) MHz. Chemical shifts (d values) were reported in ppm down field from internal Me4Si (1H and 13C NMR). High resolution mass spectra (HRMS) were acquired on an 235

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Fig. 1. Fluorescence spectra of PT-1 and PT-2. a) PT-1 (2 µM) and b) PT-2 (2 µM) in the presence of different concentrations of NaOCl in H2O-EtOH (1:1, v/v); inset: a) the fluorescence at 535 nm of PT-1 (2 µM) as a function of the NaOCl concentration; b) the fluorescence at 543 nm of PT-2 (2 µM) as a function of the NaOCl concentration; c) and d) Fluorescence spectra of PT-1 (2 µM) and PT-2 (2 µM) upon the addition of 2.0 equiv. of ClO-,·OH, Ac-, H2O2, TBHP, NO, ONOO-, NO2-, Br-, I-, F-, Cl-, 1O2, SO42-, O2-, CO32-, Ca2+, Mg2+, Cu2+ and Zn2+ in H2O-EtOH (1:1, v/v). λex = 460 nm, slit = 5 nm, 5 nm.

Φ1 = ΦB ×

AbsB × F1 × η12 Abs1 × FB × ηB2

(1)

Where Φ1 is the fluorescence quantum yield, A is the absorbance at the excitation wavelength, F is the area under the corrected emission curve, and η is the refractive index of the solvent used. Subscripts 1 and B refer to the unknown and to the standard, respectively. 2.6. Cell imaging of exogenous HOCl in L929 cells

Scheme 2. Proposed sensing mechanism of the probes towards HOCl.

(t, J = 7.2 Hz, 2 H), 4.07 (t, J = 5.6 Hz, 2 H), 3.58 (t, J = 6.4 Hz, 2 H), 2.30-2.24 (m, J = 6.0 Hz, 2 H), 1.73-1.65 (m, J = 5.6 Hz, 2 H), 1.471.38 (m, J = 7.6 Hz, 2 H), 0.96 (t, J = 8.0 Hz, 3 H); 13C NMR (CDCl3, 75 MHz, ppm): 164.14, 163.59, 148.81, 144.85, 133.94, 130.73, 129.64, 127.72, 127.40, 126.11, 125.86, 124.30, 123.02, 120.25, 115.94, 110.57, 104.18, 45.50, 42.11, 40.64, 31.16, 26.06, 21.31, 14.76.

The first group was incubated with the probe PT-1 or PT-2 (1 μM) for 30 min at 37 °C, washed by PBS buffer (10 mM, pH 7.4). The second group was incubating with the probe PT-1 or PT-2 (1 μM) for 30 min at 37 °C, and then the cell was incubated with NaOCl (2 µM) for 10 min at 37 °C. Fluorescence images were acquired.

2.5. Determination of the fluorescence quantum yield

L929 cells incubating with the probe PT-1 (200 nM) (containing 0.1% DMSO as a cosolvent) and 50 nM Lyso Tracker Red (DND-99) and Mito Tracker Red for 30 min at 37 °C, cells were rinsed with PBS three times and then incubated with NaOCl (400 nM) for 10 min at 37 °C. After being washed with PBS three times, fluorescence images were acquired.

2.7. Colocation experiment in L929 cells

Fluorescence quantum yield (Φ1) was determined by using rhodamine B (Φ1 = 0.71, in ethanol) [62] as the fluorescence standard. The quantum yield was calculated using the following Eq. (1). 236

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150 °C under an argon atmosphere in 67% yield. The new compounds were fully characterized by 1H NMR, 13C NMR and mass spectra. (see ESI). 3.2. Spectroscopic properties of PT-1 and PT-2 toward HOCl

3. Results and discussion

Firstly, we investigated the reactivity of PT-1 and PT-2 toward hypochlorite in H2O-EtOH (1:1, v/v). As indicated in Fig. 1a, the probe PT-1 (2 µM) was nonfluorescent (Φ = 0.0035) prior to its reaction with hypochlorite due to the strong electron-dononating properties of phenothiazine unit which led the fluorescence emission quenching of 1,8-naphthalimide via the photoinduced electron transfer (PET) mechanism. The fluorescence signal appeared immediately and increased significantly at 535 nm with increasing hypochlorite concentration due to the HOCl-promoted oxidation of the phenothiazine unit to its sulfoxide which prevented the PET process from the electron donor phenothiazine to the fluorescence transducer 1,8-naphthalimide (Scheme 2, Fig. S1) [22]. While the ratio of CNaOCl/CPT-1 reached 3: 1, higher concentration of NaOCl did not lead to any further emission changes as shown in the inset of Fig. 1a. Over 160-fold fluorescence enhancement (Φ = 0.57) was observed under saturated conditions. From the sigmoidal curves of the concentration-dependent fluorescence intensities at 535 nm, the fluorescence turn-on constant (Kturnon) values 3.14 ± 0.021 µM (R = 0.999) for PT-1 was obtained (Fig. S2a). Based on the HOCl-dependent changes of fluorescence intensity at 535 nm, the detection limit of PT-1 toward HOCl was estimated to be 0.32 nM (Fig. S3a). After the addition of NaOCl, the probe PT-2 also showed prominent emission enhancement like that of PT-1. Over 34fold enhancement of fluorescence intensity at 543 nm was observed under saturated conditions (Fig. 1b). The fluorescence quantum yield of PT-2 (Φ =0.0089) was increased to 0.32 upon the addition of three equivalents of NaOCl. The Kturn-on values for PT-2 was determined to be 3.14 ± 0.049 µM (R = 0.999) (Fig. S2b). The detection limit of PT2 was estimated to be 0.88 nM (Fig. S3b). Compared to PT-2, the probe PT-1 bore a lysosome-directing morphorline unit (Scheme 2). Although the nitrogen atom within morphorline moiety of PT-1 also acted as electron donor to quench the fluorescence of 1,8-naphthalimide via the PET mechanism, the significant fluorescence enhancement of PT-2 suggested that the phenothiazine unit was an efficient electron-donating system for the fluorescence emission quenching of 1,8-naphthalimide [22]. Although the fluorescence intensity increment of PT-2 was lower than that of PT-1, both probes exhibited high sensitivities toward HOCl. As the pathophysiologically relevant concentrations of HOCl are found in the low to medium micromolar ranges [63], two probes are potentially suitable for medical and biological use. To evaluate the specific nature of PT-1 and PT-2 towards HOCl, we then investigated the influence of other oxidants, cations, and anions. It is interesting to note that, the strong green fluorescence emission only occurred for the addition of HOCl; other representative species such as ·OH, Ac-, H2O2, TBHP, NO, ONOO-, NO2-, Br-, I-, F-, Cl-, 1O2, SO42-, O2-, CO32-, Ca2+, Mg2+, Cu2+ and Zn2+ produced no change in fluorescence intensities (Fig. 1c and d). Thus, the results indicated that PT-1 and PT-2 can be used as high selective probes for ClO-. The fluorescence responses of two probes with ClO- under different pH conditions were examined. PT-1 displayed enough fluorescence turn on response from pH 2–6 and PT-2 showed sufficient fluorescence turn on signal from pH 2–5 (Fig. S4). The results suggested that two probes can be used within an acidic condition.

3.1. The synthesis of PT-1 and PT-2

3.3. Time-dependent responses of PT-1 and PT-2 toward HOCl

Synthetic routes for PT-1 and PT-2 were given in Scheme 1. PT-1 was prepared by the reaction of 2 [59] and N-(3-aminopropyl) phenothiazine [60] at 140–150 °C under an argon atmosphere in 69% yield. PT-2 was obtained by the reaction of N-butyl-4-bromo1,8-naphthalimide [61] and N-(3-aminopropyl)phenothiazine at 140–

Finally, the time-dependent fluorescence response and the photostability of probes PT-1 and PT-2 under excitation at 460 nm were measured in the absence and presence of NaOCl. As shown in Fig. 2, a weak fluorescence signal was observed with the probe alone. Upon addition of NaOCl (4 equiv.), however, the

Fig. 2. Time-dependent fluorescence intensity of PT-1 and PT-2 (2 μM) upon the addition of 4.0 equiv. NaOCl in H2O-EtOH (1:1, v/v). λex = 460 nm, slit = 5 nm, 5 nm.

2.8. Imaging of endogenous HOCl in RAW 264.7 cells Cells were incubated with the probe PT-1 (1 μM) for 30 min at 37 °C, washed by PBS buffer (10 mM, pH 7.4) and subsequently incubated with 1 µg mL−1 LPS (lipopolysaccharides) for 5 h and 2.5 µg mL−1 PMA (phorbol 12-myristate13-acetate) for 30 min. For the control experiments, cells without treated with PMA/LPS were incubated with the probe PT-1 (1 μM) for 30 min under the same conditions. Fluorescence images were acquired. 2.9. Statistical analysis Descriptive statistical analyses were performed using Excel software (Microsoft Office 2002) for calculating the means and the standard error of the mean. Results were expressed as the meanstandard deviation (SD). Using SPSS software for Windows (version 13), the data were evaluated by two-way Analysis of Variance (ANOVA).

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Fig. 3. Fluorescence image of L929 cells incubated with 1 µM of probes PT−1 A) and PT-2 B). a) and b) Fluorescence and brightfield images after staining for 30 min; d) and e) Fluorescence and brightfield images after incubated with NaOCl (2 µM) for 10 min; c) and f) Merged fluorescence and brightfield; For A) images from the band path of 501–540 nm upon excitation at 405 nm; For B) images from the band path of 522–572 nm upon excitation at 405 nm.

fluorescence intensity increased immediately and reached the maximum within 10 s. Then the fluorescence intensity was stable under a continuous wavelength of 460 nm laser excitation from 5 s

to 130 s. The results suggested that probes were photostable. Meanwhile, the fast response to ClO- allowed them to be applied for the real-time detection of HOCl. 238

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Fig. 4. (a1-a3) Fluorescence confocal images of L929 cells costained with PT-1 (200 nM, 30 min), NaOCl (400 nM, 10 min), and then Lyso Tracker Red (50 nM, 20 min); (b1-b3) Fluorescence confocal images of L929 cells costained with PT-1 (200 nM, 30 min), NaOCl (400 nM, 10 min), and then Mito Tracker Red (50 nM, 20 min); (c1-c3) Fluorescence confocal images of L929 cells costained with PT-2 (0.5 µM, 30 min), NaOCl (1 µM, 10 min), and then Lyso Tracker Red (100 nM, 20 min). For a1 and b1, images from the band path of 501– 540 nm upon excitation at 405 nm. For c1, images from the band path of 522–572 nm upon excitation at 405 nm. For a2, b2 and c2, images from the band path of 588–613 nm upon excitation at 559 nm; (a3, b3, c3) The corresponding overlay images; (a4, b4) Intensity profiles of Lyso Tracker Red, Mito Tracker Red and PT-1 within the linear ROIs; (c4) Intensity profiles of Lyso Tracker Red and PT-2 within the linear ROIs (green lines in (c1) and (c2)) across the L929 cell. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the cytoplasm in living L929 cells (Fig. S5). The results indicated that the probes were able to detect HOCl in living cells.

3.4. Fluorescence imaging of exogenous HOCl in living cells The highly desirable HOCl-specific fluorescence properties of PT-1 and PT-2 prompted us to utilize them for the detection of intracellular HOCl. We first explored whether probes could detect HOCl in L929 cells, a cell line from mouse C3H/An connective tissue (Fig. 3A and B). As determined by laser scanning confocal microscopy (Fig. 3A, a-c and Fig. 3B, a-c), when L929 cells were incubated with probes PT-1 (1 µM) and PT-2 (1 µM) at 37 °C for 30 min in the growth medium and then washed three times with DPBS, very weak fluorescence signals of probes were present in cells with an excitation of blue light (405 nm). By contrast, after the addition of NaOCl (2 μM) and then incubation for another 10 min, fluorescence signals in the green channel increased obviously (Fig. 3A, d-f and Fig. 3B, df), indicated that probes were living cell membrane permeable. By comparison with the brightfield images of PT-1 and PT-2, the fluorescence signals distributed at subcellular levels of PT-1 were localized to lysosomes. However, the fluorescence signals of PT-2 were located in cytoplasm, no obvious organelle-riched fluorescence signals could be differenced. Furthermore, co-staining with DAPI (labels the nucleus) revealed that the probe PT-2 localized mainly to

3.5. The ability of localization in lysosomes of the probe PT-1 To assure that the probe PT-1 can accumulate specifically in lysosomes, co-localization experiments were carried out by co-staining L929 cells with Lyso Tracker Red DND-99 (50 nM) (a commercially available lysosomal marker) and the probe PT-1. The fluorescence of DND-99 (Fig. 4a2) from the co-stained cells overlaid well with that of PT1 in the presence of NaOCl (Fig. 4a1), as shown in the merged image (Fig. 4a3). To characterize the degree of overlap between images, a Pearson's colocalization coefficient of 0.91 and an overlap coefficient of 0.92 were obtained from the intensity correlation plots (Fig. S6). At the same time, the change in the intensity profiles of linear regions of interest (ROIs) (PT-1 and DND-99 co-staining) were synchronous, the fluorescence signals of the probe PT-1 overlaid well with the fluorescence of DND-99 (Fig. 4a4). The results indicated that PT-1 was a lysosometargetable probe for HOCl. On the other hand, a negative control experiment was performed by co-staining L929 cells with a mitochondria-localizing fluorescent dye (MitoTracker Red, a known fluorescence 239

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Fig. 5. Confocal fluorescence images of PT-1 in living RAW264.7 cells (a-f). a) Incubated with 1 µM PT-1 for 30 min; d) RAW264.7 cells were pretreated with 1 µg mL−1 LPS for 5 h; 2.5 µg mL−1 PMA for 30 min; and then incubated with 1 µM PT-1 for 30 min; b) and e) birghtfield; c) overlay a) and b); f) overlay d) and e). (λex = 405 nm, λem: 501–540 nm).

Fig. 6. Colocalization of Lyso Tracker Red (DND-99) and PT-1 in RAW264.7 cells upon stimulation by LPS and PMA. a) Fluorescence image from the PT-1 channel (1 µg mL−1 LPS for 5 h, 2.5 µg mL−1 PMA for 30 min, λex = 405 nm, λem: 501–540 nm); b) Fluorescence image from the Lyso Tracker Red (λex = 559 nm, λem: 588–613 nm); c) Merged image of a) and b); d) Merged image of a), b) and brightfield; e) Intensity correlation plot of Lyso Tracker Red and PT-1.

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marker for mitochondria) and PT-1. In the presence of HOCl, the costained cells exhibited significantly different fluorescence regions for both Mito tracker and PT-1, accompanied by a rather poor Pearson's coefficient of 0.35 and an overlap coefficient of 0.36 (Fig. S7A). Meanwhile, completely different changes in the intensity profiles of the linear ROIs were found (Fig. 4b4). At the same time, co-localization experiments of PT-2 with Lyso Tracker Red were also investigated in the study. As shown in Fig. 4c1-c4, no co-localization was essentially observed in above case. These findings further confirmed the high lysosometargeting ability of PT-1 in living cells.

Acknowledgements

3.6. Time-dependent fluorescence image

References

We gratefully acknowledge the Natural Science Foundation of China (NNSFC 21272172), and the Natural Science Foundation of Tianjin (12JCZDJC21000). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.talanta.2017.06.012.

Because HOCl is highly reactive and short-lived in physiological environments, probe that can monitor in real-time of the cellular-HOCl level is vigorously pursued [19–22]. To elucidate the real-time responsive properties of the probe, we then investigated the time-dependent fluorescence response of the probe PT-1 in lysosomes (Fig. S8). When L929 cells were incubated with PT-1 (1 µM) for 30 min in PBS at 37 °C, almost no fluorescence signals were observed. Upon the addition of NaOCl (2 µM), a weak fluorescence signal was observed within 1 min. The fluorescence signals were prominently increased from 1 to 5 min after the addition of NaOCl. The integrated optical densities (IOD) of the imagings with the incubated time indicated that there was a rapid increase of the fluorescence intensities from 1 min to 5 min (Fig. S9). Therefore, the probe PT-1 was very effective for the detection of lysosomal HOCl and should be suitable for practical application in vitro.

[1] [2] [3] [4] [5] [6]

[7] [8] [9] [10] [11] [12] [13] [14] [15]

3.7. Fluorescence imaging of endogenous HOCl in living cell

[16] [17] [18]

For the excellent properties of PT-1 for the detection and imaging of lysosomal HOCl in living cells, we finally investigated whether the probe PT-1 was capable of detecting endogenous HOCl in lysosomes. To stimulate the increased production of endogenous HOCl, macrophages RAW 264.7, a murine live macrophage cell line, were successively pretreated with bacterial cell wall lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). After the LPS/PMA stimulation, significant enhanced fluorescence signals were observed from the lysosomes in RAW 264.7 cells compared with the controls (Fig. 5), indicating the activation of the probe PT-1. To interpret localization of the probe in the RAW 264.7 cells, co-localization experiments were carried out by co-staining the cells with DND-99 (50 nM) and PT-1 (Fig. 6). As can be seen from Fig. 6c and d, the fluorescence signals overlapped very well with the endogenous HOCl-promoted fluorescence signals of PT-1. A Pearson's colocalization coefficient of 0.87 and an overlap coefficient of 0.88 were obtained from the intensity correlation plots (Fig. 6e). The data clearly indicated that the probe PT-1 can efficiently detect endogenous HOCl in lysosomes in the living RAW 264.7 macrophage cell.

[19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]

4. Conclusion

[35] [36] [37]

In summary, a novel lysosome-targetable fluorescence probe PT-1 has been synthesized by the reaction of N-(3-aminopropyl)phenothiazine with N-(morpholinoethyl)-4-bromo-1,8-naphthalimide. For comparison, PT-2 without morpholine moiety has also been synthesized. Based on the PET mechanism, probes PT-1 and PT-2 exhibit an excellent selectivity toward HOCl with a fast and sensitive response. Both PT-1 and PT-2 are cell permeable and enabled it to be used for imaging of HOCl in living cells. At the same time, PT-1 functions as a highly lysosome-targetable fluorescence probe for exogenous, endogenous and real-time imaging of lysosomal HOCl by means of fluorescence microscopy. We anticipate that this organelle-targetable fluorescent probe should prove very useful for investigation of the detailed network of lysosomal HOCl biology in cells.

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