Design and Synthesis of an Activatable Photoacoustic Probe for

Jun 20, 2019 - Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetrati...
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Article Cite This: Anal. Chem. 2019, 91, 9086−9092

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Design and Synthesis of an Activatable Photoacoustic Probe for Hypochlorous Acid Takayuki Ikeno,† Kenjiro Hanaoka,*,† Shimpei Iwaki,† Takuya Myochin,† Yoshiaki Murayama,∥ Hisashi Ohde,∥ Toru Komatsu,† Tasuku Ueno,† Tetsuo Nagano,‡ and Yasuteru Urano*,†,§,⊥ Graduate School of Pharmaceutical Sciences, ‡Drug Discovery Initiative, and §Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ∥ Olympus Corporation, 2-3 Kuboyamacho, Hachioji-shi, Tokyo 192-8512, Japan ⊥ Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan Downloaded via UNIV OF SOUTHERN INDIANA on July 17, 2019 at 13:06:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (watersoluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.

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molecules or processes that can be imaged in this way is quite limited, and therefore, exogenous contrast agents designed to visualize specific biological molecules or processes are required for further development of PA imaging.11,12 Carbon nanotubes,13 gold nanoparticles,14 polymer nanoparticles,15,16 and organic small molecules17,18 have been used for this purpose, but the signal-to-noise ratio (S/N) of these contrast agents is mostly dependent on their accumulation at the region of interest (ROI). For this reason, activatable PA probes, whose PA signal changes in the presence of a specific target, are attractive candidates as contrast agents12,19 for the sensitive detection and real-time imaging of target biomolecules or biological processes. So far, activatable PA probes for enzymes, 20−22 metal ions, 23−26 reactive oxygen species,16,22,27,28 and pH29 have been reported. Nevertheless, the availability of activatable PA probes is still limited, probably due to the relative difficulty of their molecular design.19 To tackle this problem, we here propose a rational design strategy for the development of activatable PA probes, employing the silicon-rhodamine based near-infrared (NIR) dark quenchers (SiNQs)30 developed by our group for the efficient conversion

urrently, there are many in vivo imaging modalities, such as computerized tomography (CT), magnetic resonance imaging (MRI), positron-emission tomography (PET), ultrasound, etc., that can provide anatomical and physiological information,1 and these techniques are routinely used in clinical medicine. Furthermore, molecular imaging with functionalized imaging agents has also become indispensable in clinical practice and life science research. In this context, photoacoustic (PA) imaging, which combines the high contrast of optical imaging and the deep tissue penetration of ultrasound, has recently attracted great attention.2−4 PA imaging is based on the PA effect, i.e., the generation of acoustic waves by photoexcitation of an optical absorber. When optical absorbers absorb short laser pulses, they release heat, and the subsequent adiabatic expansion generates ultrasound, which can be detected by a transducer as PA signals. PA imaging of endogenous chromophores such as hemoglobin,5,6 lipids,7,8 and melanin9,10 can provide structural and functional information about the interior of the body. For example, since hemoglobin shows an absorption spectral change upon binding of oxygen, it is not only a useful absorber for PA imaging of blood vessels but also provides information about oxygen saturation, which is important for studying tumor angiogenesis.6 Although the PA imaging of such endogenous chromophores is advantageous because no exogenous contrast agent is required, the range of biological © 2019 American Chemical Society

Received: March 27, 2019 Accepted: June 20, 2019 Published: June 20, 2019 9086

DOI: 10.1021/acs.analchem.9b01529 Anal. Chem. 2019, 91, 9086−9092

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where A is the absorbance at the excitation wavelength, n is the refractive index, and D is the area under the fluorescence spectra on an energy scale. Photobleaching Experiment. Samples were exposed for 30 min to light from a xenon lamp (MAX-301; Asahi Spectra), which was passed through a single-band band-pass filter (FF02-655/40-25; Laser 2000 (U.K.) Ltd.), and the timedependent absorption spectral changes of the dyes were measured. Assay of Enzymatic HOCl Generation. Enzymatic assay was performed in PBS (phosphate-buffered saline, pH 7.4). Myeloperoxidase (MPO) from purulent human sputum was from Wako Pure Chemical (no. 531-26181). The MPO inhibitor, 4-aminobenzoic acid hydrazide, was from Tokyo Chemical Industries (no. A1211). UV−vis spectral measurements were conducted as follows. (MPO/H2O2/Cl− system): PA-MMSiNQ (final concentration, 1 μM) was added to PBS (pH 7.4) containing 1 nM MPO at 37 °C, and the measurement of absorption change at 660 nm was started. Five minutes later, H2O2 (final concentration, 5 μM) was added, and the measurement was continued for a further 25 min. (MPO/−/Cl− system): PAMMSiNQ (final concentration, 1 μM) was added to PBS (pH 7.4) containing 1 nM MPO at 37 °C, and the measurement of absorption change at 660 nm was started. Five minutes later, 1.5 μL of PBS (pH 7.4) was added, and the measurement was continued for a further 25 min. (MPO/H2O2/Cl−/inhibitor system): PA-MMSiNQ (final concentration, 1 μM) and 4aminobenzohydrazide (final concentration, 5 μM) were added to PBS (pH 7.4) containing 1 nM MPO at 37 °C, and the measurement of absorption change at 660 nm was started. Five minutes later, H2O2 (final concentration, 5 μM) was added, and the measurement was continued for a further 25 min. PA imaging experiments were conducted as follows. (MPO/ H2O2/Cl− system): PA-MMSiNQ (final concentration, 100 μM) and MPO (final concentration, 100 nM) were dissolved in PBS (pH 7.4). Five minutes later, H2O2 (final concentration, 500 μM) was added to the solution. (MPO/−/Cl− system): PA-MMSiNQ (final concentration, 100 μM) and MPO (final concentration, 100 nM) were dissolved in PBS (pH 7.4). (MPO/H2O2/Cl−/inhibitor system): PA-MMSiNQ (final concentration, 100 μM), MPO (final concentration, 100 nM), and MPO inhibitor (4-aminobenzohydrazide) (final concentration, 50 μM) were dissolved in PBS (pH 7.4). Five minutes later, H2O2 (final concentration, 500 μM) was added to the solution. The silicon tubes were filled with these solutions, and PA imaging was performed. In Vivo PA Imaging of Mice. All procedures were approved by the Animal Care and Use Committee, The University of Tokyo. BALB/cAJc1-nu-nu (male, 6 weeks old) were purchased from CLEA Japan, Inc. Mice were anesthetized with isoflurane using a small animal anesthetizer MK-A110 (MuromachiKikai Co., Ltd., Tokyo, Japan). The PA images were captured with our photoacoustic microscope (Figure S3). Computation Details. Time-dependent density functional theory (TDDFT) calculations were performed at the CAMB3LYP31−35 level as implemented in Gaussian 09.36 The 631+G(d) basis set was used for all atoms. The number of imaginary frequencies was 0 for all structures.

of absorbed light energy to a PA signal in combination with the established spirocyclization strategy for the absorption off/on switching of rhodamines. To validate this approach, we applied it to develop a novel activatable PA probe for hypochlorous acid (HOCl). We confirmed that the probe is effective for three-dimensional imaging of HOCl in mouse subcutis.

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EXPERIMENTAL SECTION General Procedures and Materials. Reagents and solvents were of the best grade available, supplied by Tokyo Chemical Industries, Wako Pure Chemical, Aldrich Chemical Company, Dojindo, Kanto Chemical Co., and Watanabe Chemical Industry, and were used without further purification. Saline was purchased from Otsuka Pharmaceutical Co. Ltd. Purification and Identification of Compounds. Reactions were monitored by means of thin-layer chromatography (TLC) with visual or UV detection (254 nm) of the dye, and by electrospray ionization (ESI) mass spectrometry or ultraperformance liquid chromatography−mass spectrometry (UPLC−MS), as appropriate. UPLC−MS analyses were performed on an ACQUITY UPLC column (ODS, 1.7 μm, 2.1 mm × 50 mm, Waters) using a UPLC−MS system composed of a pump (L12QSM375A, Waters), UV−vis detector (D13UPL585A, Waters), and MS detector (KAB0812, Waters). All compounds were purified by medium pressure liquid chromatography (MPLC) or semipreparative high-performance liquid chromatography (HPLC). MPLC was performed on a Yamazen Smart Flash EPCLC AI-5805 (Tokyo, Japan). HPLC was performed on a reversed-phase column (Inertsil ODS-3 10.0 mm × 250 mm for purification, GL Sciences, Tokyo, Japan) equipped with a pump (PU-2080 or PU-2086, JASCO) and a detector (MD-2015, JASCO), using eluent A [H2O containing 0.1% TFA (v/v)] and eluent B [CH3CN with 20% H2O containing 0.1% TFA (v/v)]. 1H or 13 C NMR spectra were recorded on a JEOL JNM-LA300, JMN-LA400, or JMN-ECZ400S. All chemical shifts (δ) were reported in ppm relative to internal standard tetramethylsilane (δ = 0.0 ppm) or relative to the signals of residual solvent CDCl3 (7.76 ppm for 1H, 77.16 ppm for 13C), CD3OD (3.31 ppm for 1H, 49.00 ppm for 13C), or CD2Cl2 (5.32 ppm for 1H, 53.84 ppm for 13C), and coupling constants are given in hertz. Mass spectra were measured with a JEOL JMS-T100LC AccuToF (ESI). HPLC analyses were performed on a reversed-phase column (Inertsil ODS-3 4.6 mm × 250 mm for analysis, GL Sciences, Tokyo, Japan) equipped with a pump (PU-2080, JASCO) and a detector (MD-4010, JASCO), using eluent A [H2O containing 0.1% TFA (v/v)] and eluent B [CH3CN with 20% H2O containing 0.1% TFA (v/v)]. Measurements of Photophysical Properties. Absorption spectra were obtained with a Shimadzu UV-1650 or UV1850 (Tokyo, Japan). Fluorescence spectroscopic studies were performed with a Hitachi F7100 (Tokyo, Japan). Absolute fluorescence quantum yield was determined with a Hamamatsu Quantaurus QY C11347 (Shizuoka, Japan). For determination of the relative fluorescence quantum yields of wsSiNQ660 and Cy5, 2-Me SiR650 in H2O/EtOH = 1/1 containing 0.05% TFA (Φfl = 0.50) was used as a standard, and the results were calculated according to the following equation (subscript “st” stands for the reference and “x” for the sample):

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RESULTS AND DISCUSSION Evaluation of wsSiNQ660 as a Dye Scaffold for PA Imaging Probes. To establish a design strategy for

Φx /Φst = [A st /Ax ][nx 2 /nst 2][Dx /Dst ] 9087

DOI: 10.1021/acs.analchem.9b01529 Anal. Chem. 2019, 91, 9086−9092

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experimental setting used in this study (excitation at 671 nm, Figure S3). In this experiment, thin silicon tubes (inside diameter, 1 mm) were filled with the three dyes dissolved in H2O/EtOH = 1/1 containing 0.05% trifluoroacetic acid (TFA), and their concentrations were adjusted so as to give the same absorbance at 671 nm in order to compare the PA signal conversion efficiency. We first confirmed that these dyes showed concentration-dependent changes in PA signal intensity (Figure S4), and we then performed the above experiment. The PA signal intensities in Figure S2c were quantified by ImageJ39 (Figure S2d). As shown in Figure S2, parts c and d, 2-Me wsSiNQ660 showed the strongest PA signal among them, as expected. The ratio of PA signal intensities was 2-Me wsSiNQ660/2-Me SiR650/Cy5 = 1:0.22:0.74, which is somewhat different from the ratio expected from the values of 1 − Φfl of the dyes (2-Me wsSiNQ660/2-Me SiR 650/Cy5 = 1:0.5:0.78). As judged from the fluorescence quantum yields of the dyes, the PA signal intensity of 2-Me SiR650 seemed to be relatively weak compared to those of 2-Me wsSiNQ660 and Cy5. This result may indicate that, besides the fluorescence quantum yield, there are other processes that affect the PA signal conversion efficiency, or that the detection frequency of this experiment (5 MHz) may influence the PA signal intensity. The finding that 2-Me wsSiNQ660 showed high PA signal conversion efficiency supports the idea that lowering the fluorescence quantum yield of the dye by introduction of phenyl groups at the N atoms of the xanthene moiety of rhodamine derivatives is a useful design strategy to maximize the light-energy-to-PA-signal conversion efficiency of the dye. It has also been reported that rhodamine derivatives exhibit higher tolerance to photobleaching than cyanine dyes,37 and indeed, we confirmed that 2-Me wsSiNQ660 showed higher photostability than Cy5 (Figure S5). Moreover, many rhodamine-based activatable fluorescent probes have already been developed,38,40−42 and some of them utilize absorption off/on switching.43−45 In particular, the intramolecular spirocyclization of rhodamine dyes has often been utilized for this purpose (Figure S6). These rhodamine dyes take an intramolecular spirocyclic form due to nucleophilic attack of the substituent at the 2′-position of the benzene moiety of the dye on the 9-position of the xanthene moiety. The intramolecular spirocycle shows no absorption in the visible light region, but after reaction with the target molecule, the dye can no longer undergo intramolecular spirocyclization, restoring the absorption and fluorescence of the xanthene moiety. This switching mechanism has been used in various fluorescent probes targeting enzymes,45,46 metal ions,42,47 reactive oxygen species,43,44,48 pH,49,50 and it should also be applicable to 2-Me wsSiNQ660. For these reasons, we thought that wsSiNQs would be a desirable scaffold for activatable PA imaging probes, and we next set out to confirm this idea. Design and Synthesis of PA-MMSiNQ. In developing an activatable PA imaging probe, we decided to focus on hypochlorous acid (HOCl), one of the reactive oxygen species (ROS), as a target biomolecule. HOCl is a strong oxidant that kills a wide range of pathogens in the body and plays an important role in the human immune system.51 Various optical probes for detection of HOCl have been developed,43,44,52,53 and we have previously reported activatable fluorescent probes for HOCl (Figure S6).43,44 Our rhodamine-based selective fluorescent probes for HOCl take an intramolecular spirocyclic form before reaction with HOCl due to the nucleophilic attack

developing activatable PA probes, we focused on our previously reported SiNQs. Silicon-rhodamine derivatives, in which the O atom at the 10-position of the xanthene moiety of rhodamine is replaced with a Si atom,37,38 show a 90 nm redshifted absorption wavelength compared with oxygen-rhodamine dyes (Figure 1a and Figure S2a), and this long

Figure 1. (a and b) Structures and photophysical properties of 2-Me SiR650 (a) and SiNQs (b) (ref 30). (c) Structure of 2-Me wsSiNQ660.

absorption wavelength is considered to be favorable for PA imaging because the NIR light penetrates deeper into tissue, as compared with UV or visible light, mainly due to lower light scattering and relatively weak absorption of endogenous molecules such as hemoglobin in this wavelength region. In addition, the SiNQs show no fluorescence as a result of the introduction of aromatic rings on the N atoms at the 3,6positions of the xanthene moiety (Figure 1b), thereby enabling high conversion efficiency from absorbed light to ultrasound, because the PA signal is derived from nonradiative relaxation of excited dyes. For these reasons, we expected that SiNQs would prove advantageous for PA imaging. To confirm this idea, we newly designed and synthesized 2-Me wsSiNQ660 (Figure 1c and Schemes S1 and S2) and examined its photophysical and PA properties. DFT calculation for 2-Me tetramethylrhodamine (2-Me TMR), 2-Me QSY7, 2-Me SiR650, and 2-Me SiNQ660 at the B3LYP/6-31+G(d) level (Figure S1) indicated that the introduction of benzene rings on the N atoms of the xanthene moiety does not significantly affect the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels of the xanthene moiety, and indeed 2-Me wsSiNQ660 showed NIR light absorption (λabs max = 660 nm) similar to that of 2Me SiR650 (Figure S2, parts a and b). As expected, wsSiNQ660 exhibits no fluorescence (Φfl < 0.001), as shown in Figure S2, parts a and b, and it is also highly water-soluble due to the introduction of two sulfonate groups on the phenyl moieties. Next, we performed PA imaging of 2-Me wsSiNQ660 to examine whether or not it shows stronger PA signal than typical existing NIR fluorescent dyes. For this experiment, we prepared 2-Me SiR650 and Cy5 (Figure 1 and Figure S2a), both of which are NIR fluorescent dyes excitable in the 9088

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addition of HOCl or PBS (pH 7.4) alone (right). Only the PAMMSiNQ solution showed a strong PA signal upon addition of HOCl (S/N > 5) (Figure 2c). In contrast, the PA signal intensity of PA-MMSiNQ before reaction with HOCl was as weak as that of PBS alone. This result demonstrates that PAMMSiNQ emits a strong PA signal only after reaction with HOCl and can work as an activatable PA imaging probe for HOCl. Next, we examined the specificity of PA-MMSiNQ for HOCl versus various other ROS by UV−vis spectroscopy (Figure S8) and PA imaging (Figure 3). For PA imaging,

of mercaptomethyl group at the 2′-position of the benzene moiety on the 9-position of the xanthene moiety. However, once they react with HOCl, they take an open form due to the oxygenation of the S atom of mercaptomethyl group, recovering absorption and fluorescence in the visible light region (Figure S6). Therefore, we designed and synthesized an activatable PA probe for HOCl, PA-MMSiNQ (Figure 2a), by combining this intramolecular spirocyclization strategy for detection of HOCl with the 2-Me wsSiNQ660 scaffold described above.

Figure 3. (a) PA image of 100 μM PA-MMSiNQ after addition of various ROS in PBS (pH 7.4) containing 1% DMSO as a cosolvent. HOCl, 500 μM NaOCl solution; ONOOH, 500 μM sodium peroxynitrite solution; H2O2, 500 μM H2O2 solution; ·OH, 500 μM H2O2 and 500 μM Fe(ClO4)2 (Fenton reaction to generate hydroxyl radical); O2•−, 500 μM KO2 solution; ·NO, 250 μM NOC7 (nitric oxide donor) solution. Excitation wavelength was 671 nm. (b) PA signal intensities of three ROIs (9 × 9 pixels) from each silicon tube in panel a were calculated with ImageJ and averaged. Data are shown as mean ± SD (n = 3 ROIs).

Figure 2. (a) Design of an activatable PA probe for HOCl. Photographs show cuvettes containing the reaction mixture before and after reaction with HOCl. (b) PA image of 100 μM PA-MMSiNQ after (left) and before (middle) reaction with 600 μM HOCl in PBS (pH 7.4) and PBS only (right). The excitation wavelength was 671 nm. (c) PA signal intensities of three ROIs (8 × 8 pixels) from each silicon tube in panel b were calculated with ImageJ and averaged. Data are shown as mean ± SD (n = 3 ROIs). * indicates p < 0.05 (onesided Student t test).

various ROS (HOCl, ONOOH, H2O2, ·OH, O2•−, ·NO) were added to a solution of PA-MMSiNQ in PBS (pH 7.4), and PA imaging was performed in silicon tubes (inside diameter, 1 mm) filled with these solutions. As shown in Figure 3, PAMMSiNQ showed a large increase in PA signal intensity in response to HOCl and also reacted weakly with ONOOH. Thus, PA-MMSiNQ showed high selectivity for HOCl among various ROS. We then examined whether or not PA-MMSiNQ could detect HOCl generated by MPO, which catalyzes the oxidation of Cl− by H2O2 and produces HOCl.54 We first measured the absorption changes of PA-MMSiNQ in the presence of MPO with or without H2O2 (MPO/H2O2/Cl− system) (Figure S9). The absorbance at 660 nm was greatly increased upon addition of MPO with H2O2, and this absorption increase was suppressed by 4-aminobenzoic acid hydrazide, an MPO inhibitor.55 Moreover, no absorption increase was observed upon addition of MPO without H2O2. Thus, PA-MMSiNQ showed a selective absorption change in response to HOCl. In PA imaging, a strong PA signal was also observed from a solution of PA-MMSiNQ in which HOCl was generated by the MPO/H2O2/Cl− system (Figure 4, + H2O2). No PA signal was observed in the absence of H2O2 (Figure 4, − H2O2). Further, addition of the MPO inhibitor to the MPO/H2O2/Cl− system decreased the PA signal intensity (Figure 4, + inhibitor). These

PA-MMSiNQ was synthesized according to Scheme S1, i.e., we first synthesized N-Ph silicon-xanthone by use of the Buchwald−Hartwig reaction to introduce the phenyl groups at the 3,6-positions of the xanthene moiety, and then a lithiated benzene moiety bearing a mercaptomethyl group protected by a tert-butyl group was reacted with N-Ph silicon-xanthone. Finally, deprotection of the tert-butyl and isopropyl groups afforded PA-MMSiNQ. Reactivity of PA-MMSiNQ with HOCl. We evaluated the reactivity of PA-MMSiNQ with HOCl. First, we examined whether or not PA-MMSiNQ could detect HOCl in terms of an absorption spectral change. Although PA-MMSiNQ showed little absorption in the NIR light region before addition of HOCl, a large absorption at 660 nm quickly developed upon addition of HOCl (S/N > 70) (Figure S7). As shown in Figure S7, the absorption spectrum of PA-MMSiNQ after reaction with HOCl was similar to that of 2-Me wsSiNQ660 (Figure S2), indicating that the ring-opening reaction upon oxygenation of the S atom proceeds in a similar manner to that in our previously reported fluorescent probes.43,44 We then examined whether or not PA-MMSiNQ could work as an activatable PA probe for HOCl. Figure 2b shows the PA image of silicon tubes (inside diameter, 1 mm) filled with PA-MMSiNQ solution in PBS (pH 7.4) before (middle) and after (left) 9089

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Figure 4. Reactivity of PA-MMSiNQ with HOCl-generating enzymatic system (MPO/H2O2/Cl− system). (a) PA image of 100 μM MMSiNQ in PBS (pH 7.4) containing 100 nM MPO with (left) or without (middle) 500 μM H2O2 or with 500 μM H2O2 and 50 μM 4-aminobenzoic acid hydrazide (MPO inhibitor) (right). The excitation wavelength was 671 nm. (b) PA signal intensities of three ROIs (11 × 11 pixels) from each silicon tube in panel a were calculated with ImageJ and averaged. Data are shown as mean ± SD (n = 3 ROIs). * indicates p < 0.05 (one-sided Welch’s t test).

results indicate that PA-MMSiNQ is effective for PA imaging of enzymatically generated HOCl. PA Imaging of PA-MMSiNQ in Mouse Subcutis. Finally, we investigated whether PA-MMSiNQ can detect HOCl in mouse subcutis. PA-MMSiNQ in saline was subcutaneously injected into the thigh of a mouse, and then HOCl in saline was injected at the same location (Figure 5a and Figure S10, left circle). Only PA-MMSiNQ in saline (Figure 5a, right circle) and only HOCl in saline (Figure S10, right circle) were also injected at nearby sites as controls. As shown in Figure 5a (and Figure S10), a strong PA signal was observed only from the location at which both PA-MMSiNQ and HOCl were injected. These results demonstrate that PAMMSiNQ can work as an activatable PA probe for HOCl even in subcutis of a mouse. Further, three-dimensional PA images can be constructed without the need for a process such as Zstacking, as used in fluorescence imaging, because the depth of the probe can be determined from the propagation time of ultrasound. Thus, we succeeded in obtaining three-dimensional PA images of PA-MMSiNQ in subcutis of a mouse (Figure 5b and Movie S1).

Figure 5. (a) White-light and PA images of HOCl in mouse subcutis obtained with PA-MMSiNQ. Left circle: 200 μM PA-MMSiNQ in 20 μL of saline containing 2% DMSO as a cosolvent was subcutaneously injected into the thigh of a mouse. Subsequently, 500 μM HOCl in 20 μL of saline was injected at the same location. Right circle: 200 μM PA-MMSiNQ in 20 μL of saline containing 2% DMSO as a cosolvent was subcutaneously injected into the thigh of a mouse. The whitelight image was obtained just after the injection, and the PA image was obtained 40 min after the injection (the delay was due to image integration). The excitation wavelength was 671 nm. (b) PA imaging in the XY-direction (left) and the XZ-direction (right). The XZdirection image was constructed from the orange square area in the XY-direction image. The XZ-direction image was constructed from the PA signal whose waveform was correlated to the waveform from PA-MMSiNQ (correlation coefficient: r ≥ 0.4).

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NIR region is able to monitor tissues quite deep inside the body. Since we have previously developed a series of wsSiNQ780 derivatives as NIR dark quenchers having absorbance around 780 nm,30 the approach described here may also be applicable to design PA imaging probes with longer excitation wavelengths up to 780 nm.

CONCLUSION Our present findings indicate that wsSiNQ660, a NIRabsorbing, nonfluorescent rhodamine dye, is a promising scaffold for activatable PA imaging probes. wsSiNQ660 showed efficient PA signal generation compared with typical NIR fluorescent dyes. Furthermore, established molecular design strategies for activatable fluorescent probes should also be applicable to this nonfluorescent rhodamine dye. Indeed, we developed an activatable PA probe for HOCl by applying the well-known spirocyclization off/on switching strategy to wsSiNQ660 and succeeded in three-dimensional PA imaging of HOCl in mouse subcutis. Fluorescent probes targeting a range of biological phenomena have been developed based on intramolecular spirocyclization off/on switching (Figure S6), and our results suggest that a similar range of activatable PA probes targeting enzymes, metal ions, small biomolecules, pH, and so on could be developed by utilizing the wsSiNQ660 scaffold. Indeed, by applying other established absorption off/ on switching strategies of fluorescent probes to the wsSiNQ660 dye scaffold, it should be possible to develop a broad range of activatable PA probes with high conversion efficiency from light to ultrasound. Notably, excitation in the

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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.9b01529. Experimental procedures and setup, synthesis description, energy levels of HOMO and LUMO orbitals, PA signal intensities of dyes, concentration dependences of PA signal changes, photobleaching experiments, reported rhodamine-based activatable fluorescent probes, absorption spectra, white-light and PA images of PAMMSiNQ in mouse subcutis, pulse energy of PA imaging, Cartesian coordinates and total electron energies, and NMR and mass spectra (PDF) Movie showing the three-dimensional image of PAMMSiNQ (AVI) 9090

DOI: 10.1021/acs.analchem.9b01529 Anal. Chem. 2019, 91, 9086−9092

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Takayuki Ikeno: 0000-0002-5298-8415 Kenjiro Hanaoka: 0000-0003-0797-4038 Toru Komatsu: 0000-0002-9268-6964 Yasuteru Urano: 0000-0002-1220-6327 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by grants by JSPS KAKENHI Grant Nos. JP16H00823, JP16H05099, and JP18H04609 to K.H., JP16H06574 to T.U., and SENTAN, JST to K.H. K.H. was also supported by a grant JSPS Core-to-Core program, A. Advanced Research Networks and a Grant-in-Aid for Scientific Research on Innovative Areas “Singularity Biology (No. 8007)” (JP19H05414 to K.H.) of The Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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DOI: 10.1021/acs.analchem.9b01529 Anal. Chem. 2019, 91, 9086−9092