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A Deep-Red AIE Active Fluorophore for Hypochlorite Detection and Bioimaging in Live Cells Lei Wang, Xiuli Chen, Qi Xia, Ruiyuan Liu, and Jinqing Qu Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b01071 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Industrial & Engineering Chemistry Research

A Deep-Red AIE Active Fluorophore for Hypochlorite Detection and Bioimaging in Live Cells Lei Wang a, Xiuli Chen a, Qi Xia c, Ruiyuan Liu b,c* and Jinqing Qu a* a

School of Chemistry and Chemical Engineering, South China University of Technology,

Guangzhou 510640, P.R.China. b

School of Biomedical Engineering, Southern Medical University, Guangzhou 510515, P.R.

China c

School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, P.R.

China. KEYWORDS: deep-red fluorophore; AIE; hypochlorite detection; bioimaging.

ACS Paragon Plus Environment

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ABSTRACT ： Hypochlorite (ClO-) is widely presented in daily life and human body. Appropriate amount of hypochlorite may help body regulate immune system, while excessive hypochlorite can cause a variety of diseases. Herein, a novel fluorescence probe PTZ-BT containing large conjugate plane was designed and synthesized. It exhibited a large Stocks shift of 158 nm, extreme AIE activity and emitted deep-red fluorescence (~642 nm). Moreover， PTZ-BT showed specific turn-off fluorescent responsive and high sensitive detection （detection limit equals to 0.64 µM） toward ClO- in PBS buffer solutions. Job’s plot showed that the binding stoichiometry of PTZ-BT with ClO- was 1:1. It was further demonstrated that sulfur atoms in the phenothiazine groups were oxidized to sulfoxide by ClO-, resulting in fluorescence quenching supported by high-resolution mass spectrometry. In addition, PTZ-BT was used for bioimaging in HepG-2 cells with low cell toxicity and good cell permeability. Furthermore, live cells studies also demonstrated PTZ-BT can detect exogenous ClO- inside live cells.


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INTRODUCTION As a type of reactive oxygen species (ROS), hypochlorite (ClO-) is biologically produced by immune cells through the reaction between hydrogen peroxide (H2O2) and chloride ions catalyzed by myeloperoxidase (MPO).1 Endogenous hypochlorite (ClO-) plays crucial roles in many biological processes, including signal transduction, inflammation, carcinogenesis and neurodegenerative injury.2-6 However, uncontrolled production of ClO- may lead to progression of numerous human diseases.7-10 Thereby, efficient detection toward ClO- is of great scientific importance for life science. Among several detection techniques, fluorescence detection is widely utilized because of its excellent sensitivity, selectivity and low detection limit.11 In recent years, various fluorescent probes have been explored for specific detection toward ClO- and were based on fluorophores, such as coumarin, BODIPY, cyanine, fluorescein and rhodamine.12 Although these fluorescent probes were used for ClO- detection in vitro and in vivo, some of them were still lack of selectively, poor photostability, ultraviolet (UV) light excitation, visible light emission, and their Stokes shifts are small.13-26 Hence, it is necessary to preferentially employ the bright and stable red and/or near-IR (NIR) to efficiently detect ClO-. Up to now, several red and/or near-IR (NIR) fluorescent probes have been applied for ClOdetection.27-30 Most of them suffer from aggregation-induced quenching (ACQ) effect due to the π−π* stacking interactions, are only effective in high proportion of organic solvents or in the solid state, which limit the practical applications in biological systems. Recently, Tang and coworkers have explored an “abnormal” phenomenon of aggregation-induced emission (AIE) that is exactly opposite to the ACQ effect.31-33 Since then, AIE-active fluorescent probes have received intense attention due to the unique optical properties and extensive applications.34-42 Our group has also introduced several AIE fluorogens for various anion and cations detection


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and received extensive progress. Unfortunately, these fluorogens all emit orange fluorescence, which would limit their further utilization in vivo.43, 44 Herein, we design and synthesize a novel Donor-π-Acceptor (D-π-A) based deep-red AIE-active probe, PTZ-BT, for detection hypochlorite (ClO-) in vitro. PTZ-BT exhibites remarkable optical properties, including deep-red emission (642 nm) and large Stokes shift (158 nm). In addition, PTZ-BT displays a highly selective fluorescence “turn-off” effect toward ClO- in PBS buffer solutions with 0.64 µM of detection limit. More importantly, with low cytotoxicity and outstanding biocompatibility, PTZ-BT could detect exogenous ClO- in live cells. EXPERIMENTAL DETAILS Materials: 2-Benzothiazoleacetonitrile, phenothiazine, ammonium acetate and bromoethane were purchased from Adamas. POCl3 was obtained from Aldrich. KO2, sodium hypochlorite (NaClO, 7.5% availablechlorine), Tert-butylhydroperoxide (t-BuOOH, 70%), nipridedihydrate, 3-morpholinosydnonimine hydrochloride were purchased from J&K Chemical. Ethanol was purified by simple distillation prior to use. Water used herein was the twice-distilled water upon being further treated with ion exchange columns and a Milli-Q water purification system. All other chemicals and solvents were commercially available and used without further purifications. Characterization: 1H NMR and

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C NMR spectra were measured on a Bruker Avance III 400

MHz NMR in CDCl3. Infrared (IR) spectroscopy was taken on a Shimadzu FTIR-8100 spectrophotometer. High resolution mass spectra (HR-MS) were performed by a Bruker spectrometer using ESI ionization. UV-vis absorption spectra were carried out on a Shimadzu UV-2450 spectropolarimeter. FL spectra were recorded on a Hitachi F-4500 spectrofluorometer. Elemental analysis was measured on an Eager 300 elemental microanalyzer.


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Quantum yield analysis: Rhodamine B (ΦF = 0.69 in dilute ethanol solution under 365 nm excitation) was chose as a control group, the ΦF of the sample were measured as the reference. Synthesis of PTZ-BT: PTZ-BT was prepared by a knoevenagel condensation reaction between 10-ethyl-phenothiazine-3-carbaldehyde and 2-benzothiazoleacetonitrile, as shown in Scheme 1. 10-ethyl-phenothiazine-3-carbaldehyde (2) was synthesized according to the previous literature.45 Generally, ammonium acetate (0.77 g, 10 mmol), 10-ethyl-phenothiazine-3carbaldehyde (2.56 g, 10 mmol) and 2-benzothiazoleacetonitrile (1.7422 g, 10 mmol) and ethanol (30 mL) were stirred at room temperature overnight. Then the crude product was filtered and recrystallized from ethanol to give the PTZ-BT as a dark red solid (3.01 g, 70% yield). The structure of PTZ-BT was confirmed by NMR and MS, the results of which are presented in Figure S1–S4 (ESI†), respectively, indicating that PTZ-BT was successfully synthesized.1H NMR (400 MHz, CDCl3) δ (ppm): 7.97(d, J = 7.2 Hz, 2H), 7.80(m, 2H), 7.60(d, J = 2.0 Hz, 1H), 7.43(t, J = 7.6 Hz, 1H), 7.32(t, J = 7.6 Hz, 1H), 7.08(t, J = 7.8 Hz, 1H), 7.01(d, J = 7.2 Hz，1H), 6.87(t, J = 7.4 Hz，1H), 6.81(d, J = 8.4 Hz，2H), 3.90(q, 2H), 1.38(t, J = 7.0 Hz， 3H).

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C

NMR (100 MHz, CDCl3) δ (ppm): 163.44, 153.73, 148.31, 145.28, 142.98, 130.48, 129.22, 127.60, 127.48, 126.82, 126.50, 125.66, 123.50, 123.32, 121.61, 115.40, 114.70, 102.06, 42.43, 12.83. IR (cm-1, KBr): 3708, 3062, 2973, 2890, 2212, 1756, 1572, 1464, 1376, 1250, 1179, 996, 812, 774, 723. HR-MS (ESI): C24H17N3S2, m/z, 411.0864, for [M + H]+: 412.0935. Elemental analysis: calcd C, 70.04; H, 4.16; N, 10.21; S, 15.58. Found C, 69.83; H, 3.889; N, 9.38; S, 14.837.


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Scheme 1. Synthesis route of PTZ-BT. Preparation of ROS: The ROS including ClO-, O2•-, HO•, H2O2, 1O2, t-BuOOH, NO• and ONOO- were prepared as follows: ClO- was obtained from sodium hypochlorite and determined the concentration by using an extinction coefficient of 350 M-1.cm-1 (292 nm) at pH 12.0. Superoxide (O2•-) was prepared from KO2. The hydroxyl radical (HO•) was generated from Fenton reaction between H2O2 and Fe (II). Singlet oxygen (1O2) was obtained from the reaction between H2O2 and sodium molybdat solution. t-BuOOH was obtained by diluted in DMSO. Optical properties analysis: A stock solution of PTZ-BT (DMSO, 1mM) in a 10 mL volumetric flask was prepared as the primary solutions. Subsequently, 100 µL of primary PTZ-BT solution was transformed to another fresh volumetric flask and diluted to volume with PBS buffer solutions as the sample solution at the room temperature. To analysis AIE behavior of PTZ-BT, the UV-vis and FL spectra of the mixture solutions with PTZ-BT (10 µM) in DMSO/PBS (from 100: 0 to 1: 99, v/v) were performed at room temperature after incubation for 10 min.


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Cell viabilities analysis: Cell viabilities were measured using MTT assay. HepG-2 cells (1 × 106 cell/mL) were grown in 96-well plates. After maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C for 4 h, PTZ-BT was diluted to a gradient concentration range of 0-9.6 µM in primary cocultured medium and subsequently transplanted to each well after the primary medium has been removed. After cocultured HepG-2 cells with various PTZ-BT concentrations for 24 or 48 h, the absorbance of each well was measured at 490 nm using a BIOTEK ELX80enzyme-linked immunosorbent assay reader. Cellular imaging: HepG-2 cells were seeded in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) at 37 °C in humidified environment of 5% CO2. 6-well plate at 5 × 104 cells per well was used to plated cells and adhered for overnight. PTZ-BT (10 µM) were stained the cells in the medium. Excess PTZ-BT was removed by PBS buffer solutions 3 times after coculturing for 30 min. A DAPI (10 µg /mL) solution was costained the nucleus of the cells for another 30 min to observe the subcellular distributions of the probes. After that, remove the culture media and wash the cells with PBS buffer solutions 3 times. For confocal fluorescence images of exogenous ClO-, HepG-2 cells loaded with PTZ-BT (10 µM) treated with different concentrations of ClO-. The CLSM images were collected using an Olympus FV1000 confocal laser-scanning microscope at blue (450-490 nm) and red channel (570-610 nm). RESULTS AND DISCUSSION Optical properties of D-π π-A based deep-red AIE-active probe: PTZ-BT is comprised of phenothiazine as electron donor (D), benzothiazole core as the electron acceptor (A) and the vinyl group as the π unit. This D-π-A system is designed to obtain the fluorophore with long wavelength absorption. PTZ-BT was synthesized via 10-ethyl-phenothiazine-3-carbaldehyde


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and 2-benzothiazoleacetonitrile with 70% yield (Scheme 1). PTZ-BT exhibits favourable solubility in common organic solvents, such as tetrahydrofuran, chloroform, dichloromethane, acetone, acetonitrile, dimethyl sulfoxide, and dimethylformamide, but is insoluble in water and methanol. As a simpler structure, the α-cyanostilbene is usually employed as the building block to construct an AIE fluorogen.46 Through the condensation reaction between phenothiazine and benzothiazole, PTZ-BT contains the α-cyanostilbene in the molecular skeleton, which should possess the AIE behaviour. As depicted in Figure S5, the absorption maximum displays a small red shift from 474 nm to 484 nm when fw (water volume fraction %) increases from 0 to 99%. The absorption intensity slightly changes when fw is lower than 50%, but it decreases rapidly when fw is over 60%. This phenomenon is caused by the formation of aggregates in high water fractions. The aggregation reduces the number of the molecules available for light absorption. Figure 1a shows the fluorescence spectra of PTZ-BT. The dilute DMSO solution of PTZ-BT emits deep-red fluorescence with an emission maximum at 635 nm and the initial ΦF is 9.7%. While gradually adding water into DMSO (fw is lower than 40%), the fluorescence of PTZ-BT is dramatically quenched and nearly non-emissive when fw = 40%. This is attributed to the increase the solvent polarity, which hence the transformation to the twisted intramolecular charge transfer (TICT) state. With further increase of water fractions, the fluorescence intensity of PTZ-BT is enhanced and the emission maximum presents a small red shift from 635 nm to 642 nm. The fluorescence intensity reaches the maximum value at fw =70%, and then with increasing the water content, the fluorescence intensity begins to decrease when further increase the water content. This is due to the increase of the autofluorescence absorption phenomenon of the large number of probe particles. These results demonstrate that PTZ-BT presents both TICT and AIE


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features (Figure 1b). Taking environment protection and practical use in to account, the rest of characterizations in this paper were carried out in DMSO/PBS (1:99, v/v) and the corresponding

ΦF is 9.2%.

Figure 1. (a) Fluorescence spectra of PTZ-BT (10 µM) in DMSO/PBS mixtures with water fractions (fw); (b) Plot of peak intensity versus water fraction in the DMSO-PBS mixture. Optical response to ROS: The optical response of PTZ-BT toward ROS was evaluated in DMSO ： PBS=1:99 as presented in Figure 2. PTZ-BT still displays bright deep-red fluorescence and slightly changes after addition of 10 equiv. of O2•-, HO•, H2O2, 1O2, t-BuOOH, NO• or ONOO-. However, while adding the same concentration of ClO-, the fluorescence of PTZ-BT at 642 nm decreases 98% and the fluorescence changed from deep red to colorless. These results show specific selective response of PTZ-BT towards ROS. Additionally, PTZ-BT exhibits a large Stokes shift, deep-red emissive and typical AIE behavior, which is more suitable for practical application compared with some of other reported fluorescent probes.


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Figure 2. Fluorescence spectra of PTZ-BT (10 µM) in the presence of different ROS (10 equiv.). Subsequently, the optical spectrum of PTZ-BT toward ClO- titration was obtained in PBS buffer solutions to further investigate the “turn-off” response toward ClO-. As presented in Figure 3a, the maximum absorption peak of PTZ-BT is located at 484 nm. With the addition of ClO-, the absorption peak of PTZ-BT at 484 nm gradually decreases and displays a blue shift to 434 nm with 10 equiv. of ClO-. Furthermore, the appearance of the mixture changed from red to colorless. Additionally, Figure 3b depicts the fluorescence of PTZ-BT shows an interesting “turn-off” response with the addition of ClO- and nearly non-emissive upon addition of 10 equiv. of ClO-.


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Figure 3. (a) UV-vis absorption spectra of PTZ-BT (10 µM) toward various concentration of ClO- in PBS buffer solution (10 mM, pH = 7.4). Inset: the color responses of PTZ-BT toward ClO- under sunlight; (b) Fluorescence spectra of PTZ-BT exposed to various concentration of ClO- in aqueous solution. Inset: the color of fluorescent responses of PTZ-BT toward ClO- under UV lamp (365 nm). The fluorescence intensity of PTZ-BT was dramatically decreased upon the addition of ClO- in the low concentration ranges. Moreover, Figure 4 demonstrates that the fluorescence intensity of PTZ-BT shows a good linear response in the range from 0-10 µM, indicating PTZ-BT is capable in quantitatively evaluating ClO-. The detection limit is 0.64 µM, which suggests a promising ClO- detection of PTZ-BT in practical application. Figure S6 also presents the complex of PTZ-BT with ClO- has a possible bonding ratio of 1:1 according to Job's plot.


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Figure 4. Fluorescence titration curve of PTZ-BT (10 µM) with ClO- in PBS buffer solution (10 mM, pH = 7.4). Inset: the relationship between fluorescence intensity and ClO- concentration. To further study the interference of other ROS for detection of ClO-, PTZ-BT was treated with 40 µM of ClO- in the presence of 100 µM other ROS. Due to the presence of other ROS, Figure 5 shows that there is negative interference in the detection of ClO-. These results demonstrate the specific response of PTZ-BT toward ClO- in PBS buffer solutions.


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Figure 5. Fluorescence intensity at 642 nm of PTZ-BT (10µM) exposed to 4 equiv. of ClO- and 10 equiv. of other anions in PBS buffer solution (10 mM, pH = 7.4). Bonding mode of PTZ-BT toward ClO-: The sensing mechanism of PTZ-BT toward ClO- was subsequently investigated. In order to further study the inner change toward reaction between PTZ-BT and ClO-, we tested the reaction mixture of PTZ-BT and ClO- by HRMS. To our delight, Figure 6 illustrates an increased molecular weight of 16, which is equal to the molecular weight of oxygen.

Figure 6. HR-MS spectrum of PTZ-BT (10 µM) treated with 10 equiv. ClO- for 1 h. To further study the sensing mechanism for PTZ-BT toward ClO-, the DFT calculations were conducted. The molecular orbital density of PTZ-BT and its corresponding sulfoxide product are depicted in Figure S7. For PTZ-BT, the molecular orbital density of HOMO is mainly located in phenothiazine, while that of the LUMO is spread over on the benzothiazole skeleton. This result indicates the intramolecular charge transfer (ICT) processes between the two units. After treated with ClO-, the HOMO and LUMO of the corresponding sulfoxide product aredistributed on the whole molecular structure, which reflect the weak ICT progress in sulfoxide product and reduce the fluorescence intensity. On the other hand, the increased energy gap for sulfoxide


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product (3.145 eV) relative to PTZ-BT (2.833 eV) is also explained for the change of absorption peaks. These results are in accordance with the colorimetric and fluorescent transformation. These results were correlated with those reported in literature.29, 30 Moreover, it revealed that ClO- mediated oxidation occurred and turned the divalent sulphur in PTZ-BT into sulfoxide (Scheme 2).

S S N

ClO-

O S

S N

N CN

PBS buff er solutions

N

CN

Scheme 2. Proposed sensing mechanism of PTZ-BT toward ClO-. Cytotoxicity of PTZ-BT: The cytotoxicity of PTZ-BT was evaluated using MTT assays. Figure 7 depicts the cell viability after incubating HepG-2 cells and PTZ-BT with concentration ranges from 0 to 20 µM for 24 h. Cell viabilities remain above 98% treated with 1.6 µM and above 85% with 20 µM, indicating PTZ-BT exhibits low cytotoxicity and good biocompatibility.


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Figure 7. Cytotoxicity test of HepG-2 cells treated with various concentrations of PTZ-BT after 24 h incubation.

Cellular imaging: We hypothesized that the remarkable properties of PTZ-BT made it possible for ClO- detection in live cells. We selected HepG-2 cells as a model to investigate ClOdetection of PTZ-BT in live cells by CLSM images. After coculturing HepG-2 cells with PTZ-

BT (10 µM) overnight at 37 °C, the commercially nucleus stain DAPI (10 µg mL−1) was chosen to stain the nucleus in HepG-2 cells. To our delight, Figure 8 reveals that PTZ-BT mainly localizes in the cytoplasm region, which indicates good cell membrane permeable. In control groups, HepG-2 cells were firstly pretreated with PTZ-BT and further cocultured with ClO- at the range of 0-100 µM for another 4 h. From CLSM images in Figure 8, bright deep-red fluorescence could be observed from pretreated HepG-2 cells without ClO-. However, deep-red fluorescence gradually disappears after further pretreated HepG-2 cells with ClO-. Moreover, deep-red fluorescence is nearly non-emissive with a higher concentration of ClO-. These results obviously indicate PTZ-BT is capable of detecting exogenous ClO- in live cells.


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Figure 8. Confocal fluorescence imaging of exogenous ClO- in HepG-2 cells. HepG-2 cells were first incubated overnight with PTZ-BT (10 µM) at 37 oC and then incubated with different ClOranges from 0-100 µM for 4 h. Scale bar: 10 µm. CONCLUSIONS


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To conclude, we report the design and synthesis of a novel D-π-A based AIE-active deep-red fluorophore, PTZ-BT. PTZ-BT displays a large Stokes shift (~158 nm), deep-red emission (642 nm) and interesting AIE characteristics. PTZ-BT is able to detect ClO- in PBS buffer solution and shows specific selective with detection limit down to 0.64 µM. Via HRMS experiment, we confirm that the fluorescence response of PTZ-BT toward ClO- is attributed to oxidation of sulphur in phenothiazine. PTZ-BT shows high cellular-internalization efficiency with low cytotoxicity. Furthermore, PTZ-BT could also be used to detect exogenous ClO- inside live cells. ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. The characterization of PTZ-BT; UV-vis absorption spectra of PTZ-BT (10 µM) in DMSO/PBS mixtures with water fractions (fw); Job’s plot of PTZ-BT for ClO- in PBS buffer solutions (10 mM, pH = 7.4) (PDF); and DFT calculation of PTZ-BT and its corresponding sulfoxide product. AUTHOR INFORMATION

Corresponding Author Email: Prof. Ruiyuan Liu, [email protected] Email: Prof. Jinqing Qu, [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (No. 81671749), Natural Science Foundation of Guangdong Province, China (No. 2015A030313205 and No. 2016A030313546), the Science and Technology Program of Guangdong Province, China

(No.

2014B090903010,

2015A010105010,

2015B090925006,

2015B020233019,

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