Novel Cyanostilbene-Based Fluorescent Chemoprobe for Hydroxyl

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A Novel Cyanostilbene-Based Fluorescent Chemoprobe for Hydroxyl Radicals and Its Two-Photon Bioimaging in Living Cells Na Young Lim, Junho Ahn, Miae Won, Wonjin Choi, Jong Seung Kim, and Jong Hwa Jung ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00796 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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ACS Applied Bio Materials

A Novel Cyanostilbene-Based Fluorescent Chemoprobe for Hydroxyl Radicals and Its TwoPhoton Bioimaging in Living Cells Na Young Lim,†,‡ Junho Ahn,†,‡,§ Miae Won,†,∥ Wonjin Choi,‡ Jong Seung Kim∥,* and Jong Hwa Jung‡,*

‡Department of Chemistry and Research Institute of Natural Sciences, Gyeongsang National University, Jinju 52828, Korea

§Composites Research Division, Korea Institute of Materials Science, 797 Changwondaero, Changwon, Gyeongnam 51508, South Korea

∥Department of Chemistry, Korea University, Seoul 02841, Korea

KEYWORDS: hydroxyl radical, fluorescent chemoprobe, cyanostilbene, reactive oxygen species, living cell

ABSTRACT: A novel cyanostilbene derivative as a selective fluorescent chemoprobe for hydroxyl radicals was synthesized. The chemoprobe shows strong green emission in aqueous solution with the addition of hydroxyl radicals. Conversely, negligible emission changes are observed upon

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addition of other reactive oxygen species. The chemoprobe 1 shows high sensitivity, having the low detection limit of ca. 1.0 × 10−7 M. Furthermore, the fluorescent chemoprobe exhibits low cytotoxicity and was effectively applied to bioimaging of hydroxyl radicals by two-photon confocal microscopy in HeLa cells. These results indicate that the new chemoprobe has great potential for bioimaging in vivo and in vitro systems.

INTRODUCTION Reactive oxygen species (ROSs) have attracted notable research attention in the areas of chemistry, biology, and medicine owing to their significant functions in physiological and pathological events1,2, including diabetes, cerebrovascular disease, neurodegeneration, Parkinson’s disease3-7. The most important ROSs are hydrogen peroxide, singlet oxygen, hypochlorous acid, hypochlorite, superoxide anions, peroxynitrite, and hydroxyl radicals. Of these, the hydroxyl radical exhibits high reactivity and causes oxidative damage to numerous biomolecules, including DNA, carbohydrates, lipids, and protein8-11. Accordingly, many monitoring methods for hydroxyl radicals have been developed, including those based on electron paramagnetic resonance spectroscopy, UV-Vis absorption spectroscopy, electrochemical system, and photoluminescence spectroscopy12-18. Of these methodologies, the use of fluorescence chemoprobes has several advantages, including its high selectivity, high sensitivity, and its ability to provide confined site specific information at the target region, allowing real-time sensing in living cells19-23. During the past decade, several techniques involving fluorescent chemoprobes have been studied for the recognition of hydroxyl radicals. Spin-trapping chemoprobes for hydroxyl radicals that shows excellent fluorescence improvement in the presence of free radicals, are the most common. For example, aromatic hydroxylation chemoprobes based on coumarin24,25 and Tb3+ complexes26


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have been reported. Furthermore, rhodamine-based chemoprobes have been used to detect hydroxyl radicals using oxidative hydrogen abstraction27. However, even though these fluorescent chemoprobes exhibit high sensitivity, they suffer from extremely small area observation and are easily influenced by various biological conditions. Thus, the development of fluorescent chemoprobes with high selectivity and sensitivity for ROSs is required. Stilbene derivatives show excellent fluorescent properties, including high photo- and thermostability, excellent fluorescence quantum yields, as well as a controllable emission wavelength28,29. The fluorescence spectra of stilbene derivatives are dependent mainly upon the electron donating and withdrawing groups attached to the stilbene core. Additionally, the 4position of cyanostilbene can be substituted with aromatic groups, allowing it to be excited with weak energy in visible area (400–800 nm) and induce a large Stokes shift30. Furthermore, stilbene derivatives exhibit good biocompatibility and low cytotoxicity31,32, making them very attractive for the recognition of special ROS in living cells as well as tissue. Furthermore, dicyanodistyrylbenzenes are p-conjugated molecules with unique optical properties, such as aggregation-induced emission (AIE) in emission properties and tunable luminescence emission33. Compared to homologous a-cyanostilbenes, emission spectra occur at higher wavelengths due to their longer conjugation length34. We prepared the fluorescent chemoprobe 1 by coupling p-xylene dicyanide and 4propoxybenzaldehyde (Figure 1). The alkylammonium moieties of chemoprobe 1 were attached to increase solubility in water and to increase cell permeability in living cells. The cyanostilbene derivative 1 acts both as the specific detection component for hydroxyl radicals and as a response signal provider because non-emissive 1 specifically reacts with hydroxyl radicals to produce a dianionic species that emits at 500 nm. Additionally, the green emission of the dianionic emitter


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is linearly dependent on hydroxyl radical concentration. Finally, fluorescent chemoprobe 1 was used successfully for bioimaging hydroxyl radicals in live cells.

EXPERIMENTAL SECTION Reagents and Instruments. All reagents were bought from Sigma-Aldrich or Samchun Pure Chemicals Co., Ltd. and used without purifications. A Bruker DRX 300 spectrophotometer was applied to obtain 1H and 13C spectra. Mass spectra were measured using a Xevo TQ-S micro mass spectrometer. An ultraviolet-visible (UV-Vis) absorption spectrophotometer (U-4100) was applied to obtain the solution absorption spectra. Additionally, the emission spectra were recorded on a scinco FS-2 fluorescence spectrometer. Synthesis of Chemoprobe 1. Chemoprobe 1 was prepared using a previously reported method. The detailed information about synthesis and characterization data is included in the supplementary materials. Fluorescence Spectroscopy. In all fluorescence assays, 1-cm length cuvettes were used. All samples were excited at 367 nm, and the fluorescence bands were recorded at 500 nm. All hydroxyl radical detection experiments were performed in triplicate. For the monitoring hydroxyl radicals by chemoprobe 1, 3 mL of phosphate-buffered solution (pH = 7.4) was first added to the cuvette, and hydroxyl radicals were produced via the Fenton reaction using various concentrations of iron perchlorate and hydrogen peroxide (Fe2+/H2O2 = 1:10)35. The solutions were treated with the chemoprobe 1 for 15 min. For the selectivity experiments, hydroxyl radicals were produced by the Fenton reaction (Fe2+/H2O2 = 200 μM, 2000 μM). Tert-butyl hydroperoxide (TBHP) was produced by dilution of commercial TBHP solution (200 μM). Hypochlorite anions (ClO−) were prepared by dilution of NaClO solution (200 μM). Singlet oxygen (1O2) was produced by the reaction of


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H2O2 with NaClO (H2O2/NaClO = 200 μM, 200 μM)36,37. Superoxide anions (O2•−) were produced by dissolved KO2 (200 μM) in DMSO. Nitric oxide (NO) was generated by the dilution of Snitroso-N-acetyl-DL-penicillamine solution (200 μM). Glutathione (GSH) was prepared by dissolved commercial GSH in distilled water. All experiments were performed after treatment with ROSs for 10 min at RT. All fluorescence spectra were measured at the same condition. Cell Culture. HeLa cell lines originated from the human cervical adenocarcinoma cells that were purchased from the KCLP (No.10002). Cells were maintained and grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone) supplemented with 10% heat-inactivated FBS (Hyclone), 1% penicillin-streptomycin (Hyclone) at 37 °C in an incubator with 95% air and 5% CO2. Two-Photon Fluorescence Microscopy. HeLa cells were seeded with 1 × 105 cells onto 35 mm confocal dishes (SPL Life Science) and stabilized for 24 h. When the cells are approximately 80 percent confluency, they were incubated with chemoprobe 1 (30 μM in water) for 2 h at 37 °C. To assess the ROS effect, the cells were treated in the absence or presence of 4-hydroxy-2,2,6,6tetramethylpiperidin-1-oxyl (TEMPOL, 5 mM) for 6 h or lipopolysaccharide (LPS, 1 mg/mL) for 2 h. And then treated with chemoprobe 1 (30 µM) for 2 h at 37 °C. To obtain fluorescence images of the two-photon, the chemoprobe 1-labeled cell images were acquired with a two-photon microscope (Leica TCS SP2) and spectral confocal and possessing a mode-locked Ti:Sapphire laser system (Coherent Chameleon, 90 MHz, 200 fs) with a 100× (NA = 1.30) objective oil lens using a DM IRE2 microscope (Leica) by exciting chemoprobe 1 set at wavelength 750 nm and emission was entered in the range 380–640 nm. . Internal PMTs were utilized to collect the signals as an 8-bit unsigned 512 × 512 pixels at a 400 Hz scan speed.


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Cytotoxicity Assay. HeLa cells were seeded with 2 × 104 cells/well on a 96 well microplate (SPL Life Science) and incubated overnight. Subsequently incubation, the cells were treated with chemoprobe 1 or water as a control for 12 or 24 h. To measure cytotoxicity of the chemoprobe on cells, we used a CytoTox96® Non-Radioactive Cytotoxicity Assay Kit (Promega) according to the manufacturer’s instructions in the presence of the chemoprobes. The absorbance levels were detected at 490 nm by a SpectraMax Gemini EM microplate reader (Molecular Devices). The cell viability assays were performed in triplicate and the cytotoxicity was measured as a percentage calculated in the treated cells relative to that in the control.

RESULT AND DISCUSSION Characterization of Chemoprobe 1. The dicyanostilbene-based fluorogenic chemoprobe was synthesized using three steps (Figure 1). 4-Propoxybenzaldehyde was synthesized by the reaction of 4-hydroxylbenzaldehyde with dibromopropane in acetone in the presence of K2CO3 as a Lewis base. p-xylene dicyanide as the core moiety was introduced by treatment of 4propoxylbenzaldehyde in the presence of sodium methoxide in ethanol. Finally, the desired dicyanostilbene-based chemoprobe 1 was obtained by treatment with trimethylamine in DMF and was analyzed using 1H and 13C NMR and electrospray ionization (ESI) mass spectroscopy.


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Figure 1. Synthetic method of chemoprobe 1. Sensing Ability and Selectivity of Fluorescent Chemoprobe 1 for Hydroxyl Radical in Aqueous Solution. The absorption spectrum of 1 recorded in water (20 μM) at pH 7.4 presents one typical absorption band at 367 nm (Figure S1A), which originates from π-π transitions in the cyanostilbene moiety The fluorescence emission band for 1 (20 μM) under excitation at 365 nm is observed at 500 nm, as shown in Figure S1B, demonstrating that chemoprobe 1 alone exhibits extremely weak fluorescence intensity with excitation at 365 nm. However, chemoprobe 1 exhibits fluorescence spectral changes upon addition of species related to ROSs, including hydroxyl radicals (10 equiv.) and other ROSs (10 equiv. of TBHP, ClO−, O2•−, H2O2, 1O2, NO, and glutathione (GSH)) in water at pH 7.4 (Figure 2). Upon addition of 10 equiv. of hydroxyl radicals,


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a strong green emission at 500 nm is noticed below 1 min, indicating that hydroxyl radicals react with 1 quickly at RT. Additionally, the emission intensity of chemoprobe 1 in the presence of hydroxyl radicals is enhanced 200-fold compared to that in the absence of hydroxyl radicals. Conversely, no significant changes in emission are indicated upon addition of TBHP, ClO−, O2•−, H2O2, 1O2, NO, or GSH (Figure 2). The fluorescence images also demonstrate the significant differences in the response of 1 to hydroxyl radicals and to other ROSs (Figure 2B). Moreover, the fluorescence wavelength changes insignificantly upon changing the pH range from 3.0 to 10.0 (Figure S2), demonstrating that pH has no significant effect on the recognition of hydroxyl radicals by chemoprobe 1. It is slightly influenced in the high pH range, but it has no effect on biological experiments.

Figure 2. (A) Fluorescence spectra of 1 (20 μM) with ROSs (200 μM) in PBS buffer solution at pH 7.4. (B) Plot for fluorescence intensity change of (A) (insert: photographs of (A)). To examine the reactivity and spectral changes of 1 (20 μM) in the presence of hydroxyl radicals, UV-Vis and fluorescence titrations were carried out by adding hydroxyl radicals (0-200 μM) quantitatively in water at room temperature (Figure 3), which was clear solution. The absorbance of 1 at 365 nm decreases below 0.5 equiv. of hydroxyl radicals. The transparent solution upon addition of hydroxyl radicals changed into an opaque solution. The reaction of chemoprobe 1 with


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hydroxyl radicals produced aggregates. As result, new two bands at 430 and 450 nm were due to aggregates. Correspondingly, the fluorescence intensity of 1 at 500 nm, which originates from the cyanostilbene moiety, is dramatically improved by the titration. In addition, an excellent linear relationship between fluorescence intensity and concentration (R2 = 0.99301) is obtained for hydroxyl radical levels below 0.1 equiv. This turn-on mechanism originated from the blocking of the photo-induced electron transfer process38-40.

Figure 3. (A) UV-Vis absorption spectra of chemoprobe 1(20 μM) in presence of various concentration of hydroxyl radicals (0–200 μM) in aqueous solution. (B) Plot for maximum absorbance of 1 against concentrations of hydroxyl radical in aqueous solution. (C) Fluorescence spectra of 1 (20 μM) at different concentrations of hydroxyl radicals (0–200 μM) in aqueous


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solution. (D) Plot for PL intensity of 1 against concentrations of hydroxyl radical in PBS buffer solution (pH = 7.4). The limit of detection (LOD) of 1 for the hydroxyl radical is 1.0 × 10−7 M (Figrue S3), which is better than that of previously reported chemoprobes (7.3 × 10−7 M)41,42. Therefore, chemoprobe 1 shows good selectivity, rapid response, as well as a low LOD for hydroxyl radicals, indicating that it may possibly be used to detect intracellular hydroxyl radicals in biological tissues. Accordingly, to further evaluate the usefulness of 1 as a selective sensing chemoprobe for hydroxyl radicals, the fluorescence spectral changes of 1 with binary ROSs were examined (Figure S4). The other ROSs assessed were TBHP, ClO−, O2•−, H2O2, 1O2, NO, and GSH. The results revealed that chemoprobe 1 shows a strong green emission only with hydroxyl radicals. As anticipated, the fluorescence spectral changes of 1 with hydroxyl radicals was no observed upon treatment with other ROSs, such as TBHP, ClO−, O2•−, H2O2, 1O2, NO, and GSH, indicating that 1 is a promising, selective, turn-on fluorescent chemoprobe for hydroxyl radicals, even when applied in a mixture of ROSs. To demonstrate the reaction mechanism between the chemoprobe 1 and hydroxyl radicals, the 1

H-NMR of 1 before and after treatment with hydroxyl radical was measured. After reaction of 1

with hydroxyl radicals, solid products form with low solubility in water. This main product, i.e., the green emission specie was collected by filtration as a yellow solid, and the product was dried in a high vacuum oven for several hours. The 1H NMR spectra of the solid product are presented in Figure 4. Four proton peaks appear in the aromatic region (Figure 4B), with none in the aliphatic region. These data indicate that the product is a hydroxyl-modified cyanostilbene moiety. These results also indicate that chemoprobe 1 dissociates, generating a green-emissive cyanostilbene moiety upon treatment with hydroxyl radicals.


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Figure 4. 1H NMR (300 MHz) spectra of chemoprobe 1 (A) before and (B) after treatment of hydroxyl radical in DMSO-d6. Bioimaging of Chemoprobe 1 for Hydroxyl Radicals in the Living Cell. Having established the superior sensitivity and specificity of chemoprobe 1 for hydroxyl radicals in living cells, we explored its biological applicability by monitoring intracellular hydroxyl radical levels in HeLa cells. However, before the bioimaging experiments, we assessed the cytotoxicity of the chemoprobe 1. In the presence of chemoprobe 1 at concentrations from 1 to 20 μM, the cell viabilities were estimated to be higher than 92% and 80% after incubation for 12 and 24 h, respectively, as shown in Figure S5, demonstrating the potential of the current chemoprobe for the biosensing applications of hydroxyl radicals.


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Subsequently, chemoprobe 1 was utilized to the bioimaging of endogenous hydroxyl radicals in HeLa cells (Figure 5). In two-photon microscopy, the Goppert-Mayer (GM) values of chemoprobe 1 (10 μM) show a significant increase in the presence of Fenton’s agent (Fe(ClO4)2 and H2O2 = 1:10)27, but chemoprobe 1 alone exhibits very weak intensity (Figure S6). In the absence of chemoprobe 1, there is no background fluorescence. In contrast, the fluorescence is ca. 7.5-foldenhanced in the 380–640 nm emission wavelength region when HeLa cells are treated with chemoprobe 1 (30 μM) for 2 h (Figure S6 and Figure 6B).

Figure 5. Change of intracellular two-photon imaging of endogenous hydroxyl radicals by chemoprobe 1 in HeLa cells (magnification, × 80). (A) Scheme endogenous ∙OH generation by


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chemoprobe 1. (B) HeLa cells were treated with chemoprobe 1 (30 µM) for 2 h. Excitation and emission wavelengths were 750 nm and 380–640 nm, respectively. Scale bar = 20 μm.

Figure 6. Two-photon images of the chemoprobe 1 in HeLa cells (magnification, ×80). (A) Scheme for ROS inhibited by TEMPOL and activates oxidative stress by LPS. (B) Cells were pretreated the TEMPOL (5 mM: ROS scavenger) for 6 h and LPS (1 mg/ml: ROS inducer) for 2 h and then treated with chemoprobe 1 (30 µM) for 2 h. Excitation and emission wavelengths were 750 nm and 380–640 nm, respectively. Scale bar = 20 μm. We then investigated the sensing ability of chemoprobe 1 for hydroxyl radicals generated intracellularly (Figure 6). Thus, HeLa cells were treated with TEMPOL as a radical scavenger 43 for 6 h and LPS (50 μM) as a ROS inducer44 for 2 h, followed by incubation with chemoprobe 1 (30 μM) for 2 h. Two photon imaging was then performed under excitation scanning at λex = 750


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nm after thorough washing with phosphate-buffered saline (PBS) (Figure 6A). Following any treatment of the cell, a weak fluorescence image due to the existence of a small amount of hydroxyl radicals induced by stress is observed (Figure 6B). No significant fluorescence image is observed in the presence of TEMPOL or LPS/TEMPOL, indicating that chemoprobe 1 acts as a selective bioimaging chemoprobe for hydroxyl radicals in the living cell. Conversely, a strong fluorescence image is observed in the LPS-pretreated cells, indicating that the fluorescence change observed is due to generated hydroxyl radicals in the cells. Thus, these initial experiments using Hela cells demonstrate that chemoprobe 1 can be utilized to observe changes in the hydroxyl radical levels in living cells.

CONCLUSIONS In summary, a novel fluorescent cyanostilbene derivative as the sensing chemoprobe for hydroxyl radicals has been synthesized. The cyanostilbene derivative shows a good selectivity, obviously distinguishing hydroxyl radicals from among other ROSs. Furthermore, chemoprobe 1 exhibits excellent sensitivity to hydroxyl radicals and rapid responding time in aqueous solution, showing an LOD of 1.0 × 10-7 M. Primarily, fluorescence chemoprobe 1 exhibits sufficient selectivity for the bioimaging of hydroxyl radicals in living cells. Thus, we believe that this study has not only founded a trustworthy and precise approach to the detection of hydroxyl radicals in living cells but has also established the design methodology for of fluorescence chemoprobes for other ROSs.

ASSOCIATED CONTENT


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Supporting Information. The supporting information is available free of charge on the ACS Publications website at DOI: XX. UV-Vis and fluorescence spectra of chemoprobe 1; plot of fluorescence intensity of chemoprobe 1; limit of detection of chemoprobe 1; cell viability of chemoprobe 1; 1H-NMR spectra and 13CNMR spectra of chemoprobe 1; ESI Mass spectra of chemoprobe 1 (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Na Young Lim: 0000-0001-7233-4083 Junho Ahn: 0000-0002-1903-0248 Miae Won: 0000-0002-1656-6362 Jong Seung Kim: 0000-0003-3477-1172 Jong Hwa Jung: 0000-0002-8936-2272 Author Contributions †These authors contributed equally in this work.

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The authors declare no competing financial interest. ACKNOWLEDGENMENT We thankfully acknowledge the financial support by a grant from NRF (2017R1A4A1014595, 2018R1A2B2003637, 2018R1A3B1052702 and 2017R1D1A1B03030062). Additionally, this work was partially supported by a grant from the Next Generation BioGreen 21 Program (SSAC, grant no. PJ013186052018 for J. H. Jung), Rural development Administration, Korea. REFERENCES (1) D'Autreaux, B.; Toledano, M. B. ROS as Signaling Molecules: Mechanisms That Generate Specificity in ROS Homeostasis. Nat. Rev. Mol. Cell. Biol. 2007, 8, 813–824 (2) Veal, E. A.; Day, A. M.; Morgan, B. A. Hydrogen Peroxide Sensing and Signaling. Mol. Cell. 2007, 26, 1–14. (3) Houstis, N.; Rosen, E. D.; Lander, E.S. Reactive Oxygen Species Have a Causal Role in Multiple Forms of Insulin Resistance. Nature 2006, 440, 944–948. (4) Mattson, M.P. Pathways toward and away from Alzheimer's Disease. Nature 2007, 430, 631639. (5) Diehn, M.; Cho, R. W.; Lobo, N. A.; Kalisky, T.; Dorie, M. J.; Kulp, A. N.; Qian, D.; Lam, J. S.; Ailles, L. E.; Wong, M.; Joshua, B.; Kaplan, M. J.; Wapnir, I.; Dirbas, F. M.; Somlo, G.; Garberoglio, C.; Paz, B.; Shen, J.; Lau, S. K.; Quake, S. R.; Brown, J. M.; Weissman, I. L.; Clarke, M. F. Association of Reactive Oxygen Species Levels and Radioresistance in Cancer Stem Cells. Nature 2009, 458, 780–783.


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Novel Cyanostilbene-Based Fluorescent Chemoprobe for Hydroxyl

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