An Amphiphilic Fluorescent Probe Designed for Extracellular

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An Amphiphilic Fluorescent Probe Designed for Extracellular Visualization of Nitric Oxide Released from Living Cells Hui-Wen Yao, Xiao-Yan Zhu, Xiao-Feng Guo, and Hong Wang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: Nitric oxide (NO) is an intracellular and intercellular messenger involved in numerous physiological and pathophysiological processes. Small-molecule fluorescent probes coupled with fluorescence microscopy provide excellent tools for real-time detection of NO in situ. However, most probes are designed for imaging intracellular NO, which cannot reflect the release behavior of endogenously produced NO. In order to visualize extracellular NO released from living cells, we report herein a particularly designed amphiphilic fluorescent probe, disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4′-difluoro-4-bora3a,4a-diaza-s-indacene (DSDMHDAB), in which hydrophilic groups are introduced to keep the fluorophore and recognition domain outside the cell and a hydrophobic C16 alkyl chain acts as the membrane anchor. Based on this design, NO released out of the cells has been visualized on the outer surface of the plasma membrane. Using RAW 264.7 cells and ECV-304 cells as models, the diffusion of NO across the plasma membrane has been directly observed. The amphiphilic design strategy of fluorescent probes holds great promise for developing fluorescent imaging probes to study the release behaviors of other endogenous gasotransmitters.

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Apparently, these probes cannot be kept outside cells and used for extracellular NO imaging. Even though some previously reported sensors, such as Cu2(FL2A),16 can be used to detect extracellular NO because of their adventitious cellular impermeability, no rationally designed membrane-anchored analogues were yet developed. In order to visualize NO released out of living cells, a new design strategy of the functional fluorescent probes totally different from the previous ones should be proposed. Here, we developed a novel amphiphilic fluorescent probe, disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), which is composed of a highly water-soluble fluorescent sensor and a hydrophobic alkyl tail. High watersolubility of the fluorescent sensor makes it possible to be kept outside the cells and the hydrophobic alkyl tail with structural properties similar to those of phospholipids acts as a membrane anchor. Bright turn-on fluorescence of DSDMHDAB is generated upon the rapid and sensitive reaction with NO, and it maintains the intrinsic virtues of the BODIPY fluorophore, including insensitivity to pH and good photostability. Taking advantage of the autolocalization on the outer surface of the plasma membrane, the probe was successfully

itric oxide (NO) is a small uncharged free radical implicated in numerous physiological and pathophysiological processes and exerts diverse effects both intracellularly and extracellularly.1−5 Because NO is highly diffusive and reactive, extracellular NO released from cells is difficult to trap, quantify, and visualize accurately, which impedes the studies of NO with respect to its release behaviors and intercellular signaling functions. For example, there is a divergence on the basic event whether NO diffuses across the plasma membrane freely or the plasma membrane acts as a barrier for NO.6−9 Of all the existing methods for NO analysis, electrochemical sensors are effective to monitor NO release from cells by sensing NO at the electrode tip.10,11 Recently, we have proposed a robust and facile capillary electrophoresis (CE) strategy to determine extracellular NO released from single cells, which obtained the total amount of NO released to the outside of the cell.12 However, none of these methods can be applied to visualize the panorama of release behavior of NO from cells. The use of fluorescence microscopy in combination with various fluorescent probes can provide an excellent approach for the visualization of target molecules with high temporal and spatial resolution. Many efforts have been made to trap and image NO produced in cells using this approach, and a number of fluorescent probes for NO have been developed, including DAF-2 DA,13 DAR-4 M AM,14 NO550,15 Cu 2 (FL2E), 1 6 Cou-Rho-NO, 1 7 DANPBOs, 1 8 among others.19−35 However, these fluorescent probes are all designed membrane-permeable to allow them to enter cells easily. © 2016 American Chemical Society

Received: April 19, 2016 Accepted: August 21, 2016 Published: August 21, 2016 9014

DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021

Article

Analytical Chemistry

effective way to reach the goal. Such amphiphilic character is expected to drive the probes to accumulate on the outer surface of the plasma membrane without getting into the cell interior or dissociating far away. In our previous work, a completely water-soluble turn-on fluorescent probe for NO, disodium 2,6disulfonate-1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl) difluoroboradiaza-s-indacene (TMDSDAB), has been developed with its saturation concentration in water higher than 130 mg/mL over the pH range of 4−10 at room temperature.12 Owing to the excellent water-solubility, TMDSDAB cannot permeate the cell membrane and is blocked outside the cells. Therefore, we attempted to use TMDSDAB acting as a hydrophilic fluorescence sensor head which remains in the solution. Since straight chains and large head groups favor close and effective packing at the boundary between the cell membrane and bulk solution, hydrophobic alkyl chains which have similarities in structure with phospholipid molecules are selected as a tail to anchor to the cell membrane.37−39 Because increasing the hydrocarbon chain length from C8 to C20 will have little effect on adsorption effectiveness in single straight-chain surfactants,40 the hexadecyl chain is finally adopted considering biological compatibility and hydrophile−lipophile balance. Consequently, the novel amphiphilic fluorescent probe, disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), has been designed and synthesized. Completely different from the existing fluorescent probes with hydrophobic alkyl chains for near-membrane metal ion imaging,41 DSDMHDAB is characteristic with its amphiphilicity and the fluorescence sensor head is kept outside the cell to mirror the situation exactly outside the plasma membrane. Besides amphiphilic feature, the designed probe will inherit the chemical and fluorescence characteristics of TMDSDAB. Realtime monitoring of NO release from cells is illustrated in Scheme 1. Turn-on fluorescence is displayed when NO is released out of the cells and trapped by DSDMHDAB, which accumulates on the outer surface of the plasma membrane.

applied to monitor the extracellular release of NO from RAW 264.7 murine macrophages and human vascular endothelial (ECV-304) cells for the first time.

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EXPERIMENTAL SECTION Apparatus. 1H NMR spectra were recorded on a Varian Mercury VS instrument at 300 MHz and a Bruker instrument at 400 MHz. High-resolution mass spectra were obtained using LTQ Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany). HPLC analysis was performed to examine the purity of DSDMHDAB on a Fortis Xi C18 column (150 × 4.6 mm i.d., 5 μm) using an Agilent 1100 HPLC system with UV− vis detector. Absorption spectra were measured on a UV-3600 UV−vis-NIR spectrophotometer (Shimadzu, Tokyo, Japan). Fluorescence spectra were recorded on a RF5301PC spectrofluorophotometer (Shimadzu, Tokyo, Japan). Cytotoxicity was determined on a Thermo Scientific microplate reader. Fluorescence imaging experiments were performed on a Nikon confocal laser scanning microscope (TE2000, Japan). The objective used for imaging was a 60× oil-immersion objective (Nikon). Fluorescence images (515−545 nm) were obtained by excitation with a laser of 488 nm and analyzed by EZ-C1 software. Chemicals and Reagents. Unless otherwise stated, all chemical materials were of analytical grade, purchased from commercial sources, and used without further purification. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Thermo scientific (Waltham, MA, U.S.A.). Roswell Park Memorial Institute (RPMI) 1640 medium was obtained from Gibco. Fetal bovine serum (FBS) was taken from Tianhang Biological Technology (Zhejiang, China). Penicillin, streptomycin, and trypsin were obtained from Amresco (Solon, Ohio, U.S.A.). LPS and JS-K were purchased from Sigma-Aldrich (St. Louis, Missouri, U.S.A.). Interferon-γ (IFN-γ) was purchased from ProSpec-Tany (Rehovot, Israel). 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Biosharp (Hefei, China). 1,1′-Dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DiI) was purchased from Sigma-Aldrich. Water was purified on a Milli-Q system (Millipore, Bedford, MA, U.S.A.). Phosphate-buffered saline (PBS) solution consisted of 8.00 g/L NaCl, 0.20 g/L KCl, 0.24 g/L KH2PO4, 3.63 g/L Na2HPO4·12H2O, and pH values were adjusted with 1.0 M HCl and 0.1 M NaOH. NO gas was generated by slowly dropping 2 M H2SO4 into saturated NaNO2 solution. The gas was forced through a 30% NaOH solution to remove any acidic components and higher oxides of nitrogen. Before the addition of H2SO4, the entire apparatus was degassed carefully with nitrogen for 30 min.36 Saturated NO solution (at 25 °C, NO ≈ 2.0 mM) and standard solutions were prepared with reference to the literature.22 LPS and IFN-γ were dissolved in sterilized PBS solution for use. DSDMHDAB was synthesized in our laboratory and dissolved in dimethyl sulfoxide to prepare a stock solution of 2.5 mM.

Scheme 1. Schematic Illustration of Monitoring the Extracellular Release of NO with DSDMHDAB

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RESULTS AND DISCUSSION Design and Synthesis of the Amphiphilic Fluorescent Probe DSDMHDAB. To trap and visualize extracellular NO released from cells efficiently, the probes should adsorb at the outer surface of cells and not disperse in the surrounding solution. Naturally, we hypothesized that a novel design of amphiphilic fluorescent probes, like surfactants, would be an

The detailed synthesis route is outlined in Scheme 2. First of all, a reaction between 3,4-dinitrobenzoyl chloride (1) and 2hexadecylpyrrole (2) was carried out. The obtained 2ketopyrrole reacted with 2,4-dimethylpyrrole, which was catalyzed by phosphorus oxychloride, to provide dipyrromethene, followed by treatment with triethylamine and complex9015

DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021

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Analytical Chemistry

Table 1. Spectroscopic Propertiesa of DSDMHDAB and DSDMHDAB-T

Scheme 2. Synthesis of the Probe DSDMHDAB

compound

λabs,max (nm)

εmax (L·mol−1·cm−1)

λem,max (nm)

Φb

DSDMHDAB DSDMHDAB-T

508 512

58300 71100

526 529

0.003 0.183

a All data were obtained in PBS buffer (pH 7.4) with 0.01% Triton X100. bQuantum yields were measured by using fluorescein in 0.1 M NaOH (Φ = 0.92) as a standard.

emit at 526, 529 nm, respectively. As expected, DSDMHDAB luminesces weakly with a very low quantum yield of 0.003. Bright turn-on fluorescence is generated upon the reaction with NO and the quantum yield of DSDMHDAB-T is 0.183. On the contrast, the fluorescence of DSDMHDAB and DSDMHDABT decreases remarkably with the quantum yields dropping to 0 and 0.027, respectively, in aqueous solutions without Triton X100. Because the stability of probes under various pH conditions affects the biological applicability, we evaluated the fluorescence intensity of DSDMHDAB and DSDMHDAB-T under different pH conditions. As shown in Figure 1, the fluorescence of

ation with BF3·OEt2, and the desired asymmetric BODIPY (3) was generated in 28% yield.42,43 Sulfonated product (4) was produced by the reaction of (3) with fresh chlorosulfonic acid in anhydrous CH2Cl2 and treated with NaHCO3. After reduction with hydrazine and catalytic palladium on carbon, DSDMHDAB was obtained with a reasonable yield. On the chromatogram, only one peak of DSDMHDAB was found, which demonstrated the high purity of this probe (Figure S1). Its corresponding triazole derivative with NO, DSDMHDAB-T, was synthesized by excessive introduction of saturated NO solution to DSDMHDAB in the presence of dioxygen under a neutral condition (Scheme 3).13,44 Scheme 3. Reaction of DSDMHDAB with NO To Form DSDMHDAB-T

Figure 1. pH-dependent fluorescence profile of DSDMHDAB (1 μM, ■, λex/λem = 514/526 nm) and corresponding DSDMHDAB-T (1 μM, red ●, λex/λem = 515/529 nm) in 0.1 M phosphate buffers with 0.01% Triton X-100. Slit widths: 1.5 and 3 nm for excitation and emission spectra, respectively.

DSDMHDAB is extremely weak without obvious fluctuation throughout the tested pH from 2 to 12. For DSDMHDAB-T, the formation of triazolate (the deprotonated form of the triazole) of DSDMHDAB-T results in the reduction of fluorescence intensities under alkaline conditions.19 The high fluorescence intensity of DSDMHDAB-T at neutral pH, however, indicates that DSDMHDAB is qualified to image NO under physiological conditions. In the effort to develop efficient fluorescent probes for bioimaging, photostability of fluorescent molecules is a key factor which is closely related to the detection sensitivity and reproducibility. The photostability of DSDMHDAB and DSDMHDAB-T has been assessed under the laser irradiation in 430−473 nm wavelength range for 12 h, which is shown in Figure 2. There is no obvious change in the fluorescence intensity of DSDMHDAB-T within 3 h and 93.7% of the initial intensity is maintained after irradiation for 12 h. Meanwhile, the fluorescence enhancement of DSDMHDAB within 12 h is negligible compared to its corresponding DSDMHDAB-T. The results provide a strong support for DSDMHDAB as a good choice for continuous monitoring of NO over a long period in living systems. Response of DSDMHDAB to Nitric Oxide and Other Reactive Species. To evaluate the performance of

Fluorescence Properties of DSDMHDAB and DSDMHDAB-T. Because of the amphiphilic characteristic, DSDMHDAB may form micelles when its concentration is higher than critical micellar concentration (CMC), and micelle formation may affect its ability to detect NO in buffer and with cells. Thus, the CMC of DSDMHDAB was determined with a capillary electrophoresis method,45,46 which was found to be 1.0 mM in water (Figure S2). Although the salts in buffer solutions can decrease the CMC of DSDMHDAB, the reduction should not be more than 1 order of magnitude.46,47 In all the following experiments, the concentration of DSDMHDAB was as low as 1% of its CMC in water, so the effect of micelle formation can be ruled out. DSDMHDAB or DSDMHDAB-T at low concentration tends to accumulate on the surface of bulk solution to form oriented interfacial monolayer and cannot disperse evenly in it.48 Therefore, Triton X-100 was added to the bulk solution to study the fluorescence properties of DSDMHDAB and DSDMHDAB-T. Table 1 summarizes the fluorescence properties of DSDMHDAB and DSDMHDAB-T under physiological, membrane-like conditions (PBS buffer, pH 7.4, 0.01% Triton X-100). They show a strong absorption at 508, 512 nm and 9016

DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021

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Analytical Chemistry

Figure 4. Time-dependent fluorescence of (A) 10 μM DSDMHDAB in the presence of NO at different concentrations and (B) different concentrations of DSDMHDAB in the presence of 5 μM NO in PBS buffer (pH 7.4, 0.01% Triton X-100) at 37 °C. Slit widths: 1.5 nm for both excitation and emission spectra.

Figure 2. Photostability of DSDMHDAB (1 μM, ■, λex/λem = 514/ 526 nm) and DSDMHDAB-T (1 μM, red ●, λex/λem = 515/529 nm) in PBS buffer (pH 7.4, 0.01% Triton X-100) under the laser irradiation in the 430−473 nm wavelength range. Slit widths: 1.5 and 3 nm for excitation and emission spectra, respectively.

physiological environment, which is greatly beneficial to the fluorescence imaging of extracellular NO released from cells. The high specificity of NO probe is essential for its application to complex biological samples. Therefore, the fluorescence responses of DSDMHDAB toward potentially interfering species including reactive oxygen species (ROS), reactive nitrogen species (RNS), S-nitrosothiols (GSNO and SNAP, reactions were kept in dark), ascorbic acid (AA), dehydroascorbic acid (DHA), and methylglyoxal (MGO) were investigated. Meanwhile, the response of DSDMHDAB to NO was also evaluated in the presence of each of interfering species mentioned above. It can be seen from Figure 5 that the

DSDMHDAB in detecting NO under physiological conditions, the concentration- and time-dependent reactions between DSDMHDAB and NO were carried out in PBS buffer (pH 7.4, 0.01% Triton X-100). DSDMHDAB responds linearly to the concentration of NO and its limit of detection was estimated to be 0.83 nM, as shown in Figure 3A. As the probe

Figure 3. Fluorescence responses of 10 μM DSDMHDAB to (A) different concentrations of NO in PBS buffer with 0.01% Triton X-100 at pH 7.4 (B) 5 μM NO in 0.1 M phosphate buffers with 0.01% Triton X-100 at different pH. The spectra were measured after the incubation of DSDMHDAB with NO for 5 min at 37 °C. Slit widths: 1.5 nm for both excitation and emission. Figure 5. Fluorescence responses of DSDMHDAB (10 μM) to various ROS, RNS, GSNO, SNAP, AA, DHA, and MGO (8 μM for NO; 8 mM for NO3−, HNO, H2O2, ONOO−, AA; 2 mM for NO2−, ClO−, · OH, 1O2, DHA; 1 mM for GSNO and SNAP; 0.2 mM for MGO). Black bars denote the addition of one of these interfering substances or NO to a 10 μM solution of DSDMHDAB. Gray bars denote the addition of NO to the probe solution with one of these interfering substances. All data were acquired in PBS buffer (pH 7.4, 0.01% Triton X-100) at 37 °C (λex/λem = 515/529 nm). Slit widths: 1.5 nm for both excitation and emission spectra.

for NO is significantly dependent on medium pH, we further assessed pH effect on fluorescence responses of DSDMHDAB to NO (Figure 3B). The limits of detection were estimated to be 0.73 nM from pH 3.0 to 6.0, 0.78 nM at pH 7.0, 0.91 nM at pH 8.0, and 1.23 nM at pH 9.0. In addition, linear responsive ranges were 0.5−6.0 μM when pH ranged from 3.0 to 9.0. When Figure 1 is combined with Figure 3B, the similar trends indicate that the pH effect on fluorescence responses of the system to NO is mainly determined by pH effect on DSDMHDAB-T rather than the derivatization procedure. This effect should be attributed to the formation of triazolate of DSDMHDAB-T at higher pH value.19 Furthermore, the time-dependent turn-on fluorescence of DSDMHDAB exhibits a highly uniform pattern with NO at different levels (Figure 4A), and the fluorescence intensities increase dramatically and reach their plateau within 1 min. Compared to the other ophenylenediamine fluorescent probes, for example, DAMBOPH, DAC and DANPBOs which need 30, 15, and 15 min to complete the reaction with NO, respectively, 18,19,49 DSDMHDAB reacts with NO much faster. Further experiments with different concentrations of DSDMHDAB indicate that almost all the NO can be captured with 2 equiv of DSDMHDAB (Figure 4B). These excellent characteristics allow DSDMHDAB to trap NO sensitively and rapidly in a

response signal of DSDMHDAB to interfering substances is minimal, in contrast to the performance in the reaction with NO. Moreover, the reaction of DSDMHDAB with NO will not be interfered by these species. Because the decomposition of Snitrosothiols is a very slow process at physiological pH and temperature, in the absence of light and transition-metal ion catalysis,50 1 mM (100-fold excess) S-nitrosothiols could not interfere with NO detection in our experiments. However, they can release NO in light.51,52 Therefore, we further examined the response of DSDMHDAB to 1 mM S-nitrosothiols in daylight, and a significant turn-on of the probe was observed in 25 min for GSNO and 30 min for SNAP. Potential of DSDMHDAB for NO Imaging in Vitro. In order to trap NO released out of cells, probes designed should self-assemble on the outer surface of plasma membrane 9017

DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021

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Analytical Chemistry uniformly. As the fluorescence of DSDMHDAB is extremely weak, its distribution cannot be observed directly. Therefore, NO was added to the culture medium to light DSDMHDAB after its arrangement on the plasma membrane. RAW 264.7 murine macrophages and human vascular endothelial (ECV304) cells were adopted as the models and both subjected to confocal laser scanning microscopy (CLSM) analysis. Apart from some fluorescence resulting from intercellular tight junction in human vascular endothelial (ECV-304) cells,53 homogeneous and bright fluorescence was predominantly observed on the outer surface of the plasma membrane in both cell lines, but not from their interior (Figure 6). The

Figure 6. Confocal fluorescence imaging of (A) RAW 264.7 murine macrophages and (B) human vascular endothelial (ECV-304) cells loaded with 5 μM DSDMHDAB upon treatment with 20 μM NO. Cells shown are representative images from replicate experiments (n = 5). Scale bar: 20 μm.

Figure 8. (A) Confocal fluorescence images of RAW 264.7 murine macrophages loaded with 5 μM DSDMHDAB upon treatment with NO at different amounts. (B) Average fluorescence intensity of the plasma membrane surface in (A). Each column was obtained from regions of interest (ROIs, n = 6) inside cells. Cells shown are representative images from replicate experiments (n = 5). Scale bar: 20 μm.

results suggest that DSDMHDAB is cell-impermeable and can aggregate on the plasma membrane surface spontaneously and uniformly, which may trap NO released out of cells. The location of DSDMHDAB in the cellular plasma membrane was further confirmed by a fluorescence overlay of green emissive DSDMHDAB-T with red emission from DiI (Figure 7).

Figure 7. Confocal fluorescence images of RAW 264.7 murine macrophages loaded with both 5 μM DiI and 5 μM DSDMHDAB-T: (A) emission from labeling with DiI, (B) emission from labeling with DSDMHDAB-T, (C) overlay of (A) and (B), (D) brightfield image. Cells shown are representative images from replicate experiments (n = 5). Scale bar: 20 μm.

DSDMHDAB in vitro, its ability to monitor extracellular release of NO from living cells was evaluated. At first, the cytotoxicity of DSDMHDAB to living cells was tested using RAW 264.7 murine macrophages. After incubation with DSDMHDAB at different amounts for 24 h, cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.54 It can be seen from Figure 9 that survival rate under treatment with 10 μM and 100 μM DSDMHDAB is 83.7% and 72.1%, respectively, indicating that the probe displays low cytotoxicity to living cells. To visualize the extracellular release of NO from living cells, RAW 264.7 murine macrophages and human vascular endothelial (ECV-304) cells were used for experiments. RAW 264.7 murine macrophages were preincubated with lip-

Moreover, more junctions could be observed in the dark field of Figure 6B than in the bright field, which indicated the high sensitivity of DSDMHDAB after trapping NO. To evaluate the ability of DSDMHDAB adsorbed on cells to reflect the variation of NO amount, NO at different levels was used. As shown in Figure 8, the fluorescence intensity on the plasma membrane surface of RAW 264.7 murine macrophages increases along with NO from 0 to 20 μM. These results indicate that DSDMHDAB can fluorometrically detect NO at the outer surface edge of the plasma membrane with rapid and high response. Fluorescence Monitoring of Nitric Oxide Release from Living Cells. Having characterized the sensing properties of

Figure 9. Survival rate of macrophage cells (RAW 264.7) after treatment with DSDMHDAB at different amounts. 9018

DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021

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Analytical Chemistry opolysaccharide (LPS) and recombinant murine interferon-γ (IFN-γ), which can stimulate the expression of iNOS gene and lead to the production of NO,16 whereas human vascular endothelial (ECV-304) cells were incubated with JS-K (O2(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, a GST-activated NO generator) to simulate the release of NO.55 Upon following incubation with DSDMHDAB, bright and interrupted fluorescence was observed on the plasma membrane surface in both kinds of cells, as shown in Figure 10. Similar phenomenon was also

Figure 11. (A) Confocal fluorescence images of human vascular endothelial (ECV-304) cells loaded with 5 μM DSDMHDAB after treatment with JS-K at different amounts. (B) Average fluorescence intensity of the plasma membrane surface in (A). Each column was obtained from ROIs (n = 6) inside cells. Cells shown are representative images from replicate experiments (n = 5). Scale bar: 20 μm.

Figure 10. (A) Confocal fluorescence images of RAW 264.7 murine macrophages loaded with 5 μM DSDMHDAB after treatment with LPS (0.5 μg/mL) and IFN-γ (0.01 μg/mL) for 12 h. (B) Confocal fluorescence images of human vascular endothelial (ECV-304) cells loaded with 5 μM DSDMHDAB after treatment with 10 μM JS-K. (C) and (D) were average fluorescence intensity of the plasma membrane surface in (A) and (B), respectively. Each column was obtained from ROIs (n = 6) inside cells. Cells shown are representative images from replicate experiments (n = 5). Scale bar: 20 μm.

Figure 12. (A) Confocal fluorescence images of human vascular endothelial (ECV-304) cells loaded with 5 μM DSDMHDAB after treatment with 10 μM JS-K. (B) Images of adding extra 20 μM NO solution to the treated cells (A). (C) DIC images of (A). Cells shown are representative images from replicate experiments (n = 5). Scale bar: 20 μm.

observed in replicate experiments (n = 5). With elevated level of JS-K in ECV-304 cells, the fluorescence was brighter while still intermittent (Figure 11). Because the addition of JS-K might affect the distribution of the probe, we added NO (final concentration of 20 μM) to the same human vascular endothelial (ECV-304) cells which were previously treated with JS-K, and we found the interruption on the plasma membrane disappeared, as shown in Figure 12. There could be the following three explanations that account for such interrupted fluorescence on the plasma membrane surface: (1) distribution of NO produced in cells is uneven; (2) distribution of DSDMHDAB on the plasma membrane is uneven; (3) NO tends to permeate through some active areas on the plasma membrane. At the current time, there are many reports using liposoluble fluorescent probes to observe the generation and distribution of intracellular NO, and the results showed bright and almost uniform fluorescence within cells except in the nucleus, indicating that intracellular produced NO

can diffuse evenly in the cytoplasm.22,30 Therefore, the first explanation is unlikely. Figure 6 indicates that the distribution of DSDMHDAB on the plasma membrane of control cells is uniform. Additionally, treatment of cells with JS-K has almost not altered the uniform attachment of the probe (Figure 12). Thus, the second explanation is also unlikely. Lastly, it was reported that cell membrane acted as a barrier for NO in some publications,8,9 which means NO could not permeate through cell membrane freely. Consequently, we think the third speculation about this phenomenon should be the most plausible one. Based on this speculation, NO might not diffuse across the plasma membrane at the dark regions, and thus, DSDMHDAB is not converted to fluorescent DSDMHDAB-T. 9019

DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021

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Analytical Chemistry Author Contributions

That is, NO tends to permeate through some active areas of the plasma membrane rather than the whole plasma membrane. Furthermore, the fluorescence stain at the inner fluorescent plasma membrane circle of RAW 264.7 cells may be attributed to the phagocytosis nature of macrophages. The phagocytosis function of macrophages can be activated when stimulated by LPS and IFN-γ, and the plasma membrane will invaginate to form a vesicle to help engulf exotic substances accompanied by releasing more NO during phagocytosis.56,57 Thus, the phenomenon we observed is presented. For ECV-304 cells, the endothelial cells which do not have the ability of phagocytosis, no fluorescence can be observed in cell interior. From Figure 10B/Figure 11 and Figure 6B, another interesting phenomenon was observed. In bright fields of Figure 6B and Figure 10B/Figure 11, the morphologies of ECV-304 cells are similar, and some junctions could be observed clearly. However, in the dark fields of these figures, highly fluorescent junctions could only be observed clearly in Figure 6B. In Figure 10B and Figure 11, only some fluorescent parts of junctions are fluorescent. This phenomenon can be attributed to the difference of intercellular NO concentrations and distribution. In all three figures, cells were incubated with DSDMHDAB of the same concentration. Then, in Figure 6B, ECV-304 cells were treated with 20 μM NO in solution out of cells, but in Figure 10B/Figure 11, cells were treated with JS-K, a GST-activated NO generator, not NO. Thus, NO in Figure 10B/Figure 11 was generated in cells, so the amount of intercellular NO present was much less than Figure 6B and distributed unevenly.

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 20835004 and 31170344, Beijing, China).

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CONCLUSION In summary, we have developed a novel amphiphilic fluorescent probe, DSDMHDAB, for visualization of extracellular release of NO. DSDMHDAB can accumulate on cell membranes spontaneously and uniformly with the fluorescence sensor being kept outside the cell, and it can respond to NO released out of cells rapidly and sensitively. The amphiphilic character makes DSDMHDAB completely different from the existing fluorescent probes for imaging and suitable for extracellular visualization of targets. Successful applications of DSDMHDAB in the fluorescence imaging of RAW 264.7 cells and ECV-304 cells demonstrate that DSDMHDAB is quite qualified to visually monitor extracellular NO released from living cells. Furthermore, the observation of interrupted fluorescence at the fluorescent plasma membrane suggests the amphiphilicitybased design strategy for this fluorescent probe is expected to provide new opportunities for the study of other endogenous gasotransmitters.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01532.

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REFERENCES

(1) Moncada, S.; Palmer, R. M.; Higgs, E. A. Pharmacol. Rev. 1991, 43, 109−142. (2) Pfeiffer, S.; Mayer, B.; Hemmens, B. Angew. Chem., Int. Ed. 1999, 38, 1714−1731. (3) Bogdan, C. Nat. Immunol. 2001, 2, 907−916. (4) Fukumura, D.; Kashiwagi, S.; Jain, R. K. Nat. Rev. Cancer 2006, 6, 521−534. (5) Garthwaite, J. Eur. J. Neurosci. 2008, 27, 2783−2802. (6) Goretski, J.; Hollocher, T. C. J. Biol. Chem. 1988, 263, 2316− 2323. (7) Stamler, J. S.; Singel, D. J.; Loscalzo, J. Science 1992, 258, 1898− 1902. (8) Han, T. H.; Qamirani, E.; Nelson, A. G.; Hyduke, D. R.; Chaudhuri, G.; Kuo, L.; Liao, J. C. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 12504−12509. (9) Kleinbongard, P.; Schulz, R.; Rassaf, T.; Lauer, T.; Dejam, A.; Jax, T.; Kumara, I.; Gharini, P.; Kabanova, S.; Ozuyaman, B.; Schnurch, H. G.; Godecke, A.; Weber, A. A.; Robenek, M.; Robenek, H.; Bloch, W.; Rosen, P.; Kelm, M. Blood 2006, 107, 2943−2951. (10) Malinski, T.; Taha, Z. Nature 1992, 358, 676−678. (11) Du, F.; Huang, W.; Shi, Y.; Wang, Z.; Cheng, J. Biosens. Bioelectron. 2008, 24, 415−421. (12) Zhang, Z. X.; Guo, X. F.; Wang, H.; Zhang, H. S. Anal. Chem. 2015, 87, 3989−3995. (13) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446− 2453. (14) Kojima, H.; Hirotani, M.; Nakatsubo, N.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Hirata, Y.; Nagano, T. Anal. Chem. 2001, 73, 1967−1973. (15) Yang, Y.; Seidlits, S. K.; Adams, M. M.; Lynch, V. M.; Schmidt, C. E.; Anslyn, E. V.; Shear, J. B. J. Am. Chem. Soc. 2010, 132, 13114− 13116. (16) McQuade, L. E.; Ma, J.; Lowe, G.; Ghatpande, A.; Gelperin, A.; Lippard, S. J. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 8525−8530. (17) Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Song, J. Chem. Commun. 2011, 47, 9372. (18) Zhang, H. X.; Chen, J. B.; Guo, X. F.; Wang, H.; Zhang, H. S. Anal. Chem. 2014, 86, 3115−3123. (19) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357−3367. (20) Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684−3685. (21) Gabe, Y.; Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. Anal. Bioanal. Chem. 2006, 386, 621−626. (22) Ouyang, J.; Hong, H.; Shen, C.; Zhao, Y.; Ouyang, C.; Dong, L.; Zhu, J.; Guo, Z.; Zeng, K.; Chen, J.; Zhang, C.; Zhang, J. Free Radical Biol. Med. 2008, 45, 1426−1436. (23) Sun, C. D.; Shi, W.; Song, Y. C.; Chen, W.; Ma, H. M. Chem. Commun. 2011, 47, 8638−8640. (24) Yu, H.; Xiao, Y.; Jin, L. J. Am. Chem. Soc. 2012, 134, 17486− 17489. (25) Shiue, T.-W.; Chen, Y.-H.; Wu, C.-M.; Singh, G.; Chen, H.-Y.; Hung, C.-H.; Liaw, W.-F.; Wang, Y.-M. Inorg. Chem. 2012, 51, 5400− 5408.

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DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021

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(57) Stuart, L. M.; Ezekowitz, R. A. B. Immunity 2005, 22, 539−550.

(26) Yu, H. B.; Zhang, X. F.; Xiao, Y.; Zou, W.; Wang, L. P.; Jin, L. J. Anal. Chem. 2013, 85, 7076−7084. (27) Vegesna, G. K.; Sripathi, S. R.; Zhang, J.; Zhu, S.; He, W.; Luo, F. T.; Jahng, W. J.; Frost, M.; Liu, H. ACS Appl. Mater. Interfaces 2013, 5, 4107−4112. (28) Yu, H.; Jin, L.; Dai, Y.; Li, H.; Xiao, Y. New J. Chem. 2013, 37, 1688. (29) Beltran, A.; Burguete, M. I.; Abanades, D. R.; Perez-Sala, D.; Luis, S. V.; Galindo, F. Chem. Commun. 2014, 50, 3579−3581. (30) Dong, X. H.; Heo, C. H.; Chen, S. Y.; Kim, H. M.; Liu, Z. H. Anal. Chem. 2014, 86, 308−311. (31) Lv, X.; Wang, Y.; Zhang, S.; Liu, Y.; Zhang, J.; Guo, W. Chem. Commun. 2014, 50, 7499−7502. (32) Adarsh, N.; Krishnan, M. S.; Ramaiah, D. Anal. Chem. 2014, 86, 9335−9342. (33) Juarez, L. A.; Barba-Bon, A.; Costero, A. M.; Martinez-Manez, R.; Sancenon, F.; Parra, M.; Gavina, P.; Terencio, M. C.; Alcaraz, M. J. Chem. - Eur. J. 2015, 21, 15486−15490. (34) Liu, B.; Hu, X.; Chai, J.; Zhu, J.; Yang, B.; Li, Y. J. Mater. Chem. B 2016, 4, 3358−3364. (35) Miao, J. F.; Huo, Y. Y.; Lv, X.; Li, Z.; Cao, H. L.; Shi, H. P.; Shi, Y. W.; Guo, W. Biomaterials 2016, 78, 11−19. (36) Kanai, A. J.; Strauss, H. C.; Truskey, G. A.; Crews, A. L.; Grunfeld, S.; Malinski, T. Circ. Res. 1995, 77, 284−293. (37) Shynkar, V. V.; Klymchenko, A. S.; Kunzelmann, C.; Duportail, G.; Muller, C. D.; Demchenko, A. P.; Freyssinet, J. M.; Mely, Y. J. Am. Chem. Soc. 2007, 129, 2187−2193. (38) Neef, A. B.; Schultz, C. Angew. Chem., Int. Ed. 2009, 48, 1498− 1500. (39) Shim, S. H.; Xia, C. L.; Zhong, G. S.; Babcock, H. P.; Vaughan, J. C.; Huang, B.; Wang, X.; Xu, C.; Bi, G. Q.; Zhuang, X. W. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 13978−13983. (40) Rosen, M. J. Surfactants and interfacial phenomena, 2nd ed; John Wiley & Sons: Hoboken, NJ, 1989; pp xv, 431 (41) Mohan, P. S.; Lim, C. S.; Tian, Y. S.; Roh, W. Y.; Lee, J. H.; Cho, B. R. Chem. Commun. 2009, 5365−5367. (42) Tahtaoui, C.; Thomas, C.; Rohmer, F.; Klotz, P.; Duportail, G.; Mély, Y.; Bonnet, D.; Hibert, M. J. Org. Chem. 2007, 72, 269−272. (43) Jiao, L.; Yu, C.; Liu, M.; Wu, Y.; Cong, K.; Meng, T.; Wang, Y.; Hao, E. J. Org. Chem. 2010, 75, 6035−6038. (44) Kojima, H.; Sakurai, K.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Akaike, T.; Maeda, H.; Nagano, T. Biol. Pharm. Bull. 1997, 20, 1229−1232. (45) Lin, C.-E. J. Chromatogr. A 2004, 1037, 467−478. (46) Cifuentes, A.; Bernal, J. L.; DiezMasa, J. C. Anal. Chem. 1997, 69, 4271−4274. (47) Fuguet, E.; Ràfols, C.; Rosés, M.; Bosch, E. Anal. Chim. Acta 2005, 548, 95−100. (48) Xu, J.-P.; Ji, J.; Chen, W.-D.; Shen, J.-C. Macromol. Biosci. 2005, 5, 164−171. (49) Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684−3685. (50) Zhang, X. J.; Cardosa, L.; Broderick, M.; Fein, H.; Davies, I. R. Electroanalysis 2000, 12, 425−428. (51) Hunter, R. A.; Schoenfisch, M. H. Anal. Chem. 2015, 87, 3171− 3176. (52) Ismail, A.; d’Orlye, F.; Griveau, S.; Bedioui, F.; Varenne, A.; da Silva, J. A. Electrophoresis 2015, 36, 1982−1988. (53) Hirase, T.; Staddon, J. M.; Saitou, M.; Ando-Akatsuka, Y.; Itoh, M.; Furuse, M.; Fujimoto, K.; Tsukita, S.; Rubin, L. L. J. Cell Sci. 1997, 110, 1603−1613. (54) Plumb, J. A.; Milroy, R.; Kaye, S. B. Cancer Res. 1989, 49, 4435− 4440. (55) Shami, P. J.; Saavedra, J. E.; Wang, L. Y.; Bonifant, C. L.; Diwan, B. A.; Singh, S. V.; Gu, Y.; Fox, S. D.; Buzard, G. S.; Citro, M. L.; Waterhouse, D. J.; Davies, K. M.; Ji, X.; Keefer, L. K. Mol. Cancer Ther. 2003, 2, 409−417. (56) Nakanishi-Matsui, M.; Yano, S.; Matsumoto, N.; Futai, M. Biochem. Biophys. Res. Commun. 2012, 425, 144−149. 9021

DOI: 10.1021/acs.analchem.6b01532 Anal. Chem. 2016, 88, 9014−9021