論文 - 花岡　健二郎

Detection of singularity in immunity and cancer by novel imaging techniques Comment

Nagai Takeharu, Chikuma Shunsuke, Hanaoka Kenjiro

BIOPHYSICS AND PHYSICOBIOLOGY 17   98 - 99 2020年

研究論文（国際会議プロシーディングス）

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Design strategy for germanium-rhodamine based pH-activatable near-infrared fluorescence probes suitable for biological applications

Koide Y., Kojima R., Hanaoka K., Numasawa K., Komatsu T., Nagano T., Kobayashi H., Urano Y.

Communications Chemistry （Communications Chemistry）  2 （ 1 ）  2019年12月

研究論文（学術雑誌）, 査読有り

　概要を見る

Fluorescence probes that can detect pH are useful tools for biological research and clinical diagnosis. Here we report pH-activatable near-infrared fluorescence probes, based on hydroxymethyl germanium-rhodamine (HMGeR), that are suitable for a range of biological applications. The pKa, the ratio of the fluorescent form in an acidic environment, and the absorption/emission wavelengths can all be conveniently optimized. The most promising probe, 2-HM IGeR, offers significant advantages over currently available near-infrared pH probes, notably high quantum efficiency, appropriate pKa value for biological applications, and high photostability. Further, our molecular design strategy allows easy conjugation of the probes to biomolecules without loss of functionality. We illustrate the value of this strategy by developing probe-Herceptin® and probe-avidin conjugates to visualize pH change in cellular vesicles during endocytosis, and to visualize tumors in a mouse model, respectively. We believe 2-HM IGeR is currently among the best-in-class pH-activatable near-infrared probes for biological and medical research.

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Development of ratiometric carbohydrate sensor based on boron dipyrromethene (BODIPY) scaffold

Cheruthu N.M., Komatsu T., Ueno T., Hanaoka K., Urano Y.

Bioorganic and Medicinal Chemistry Letters （Bioorganic and Medicinal Chemistry Letters）  29 （ 22 ） 126684 2019年11月

研究論文（学術雑誌）, 査読有り,  ISSN  0960894X

　概要を見る

We designed a ratiometric carbohydrate sensor consisting of the boron dipyrromethene fluorophore substituted with boronic acid at the 2-position, based upon the strong substituent dependency of the absorbance/fluorescence wavelengths of BODIPY. The substituent is in equilibrium between the boronic acid B(OH)2 and boronate (B(OH)3−) forms, which have different absorbance/fluorescence wavelengths in the visible region. Reaction of the boronic acid moiety with hydroxy groups of carbohydrate affords a cyclic ester and shifts the equilibrium in favor of the boronate (B(OR)3−) form, resulting in a carbohydrate-concentration-dependent change of the fluorescence ratio. Thus, the sensor, BA-BODIPY, can ratiometrically detect carbohydrate at a pH near the pKa of cyclic ester formation.

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Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples

Ichihashi Y., Komatsu T., Kyo E., Matsuzaki H., Hata K., Watanabe T., Ueno T., Hanaoka K., Urano Y.

Analytical Chemistry （Analytical Chemistry）  91 （ 18 ） 11497 - 11501 2019年09月

研究論文（学術雑誌）, 査読有り,  ISSN  00032700

　概要を見る

We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.

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Design and Synthesis of an Activatable Photoacoustic Probe for Hypochlorous Acid

Ikeno T., Hanaoka K., Iwaki S., Myochin T., Murayama Y., Ohde H., Komatsu T., Ueno T., Nagano T., Urano Y.

Analytical Chemistry （Analytical Chemistry）  91 （ 14 ） 9086 - 9092 2019年06月

研究論文（学術雑誌）, 査読有り,  ISSN  00032700

　概要を見る

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 (water-soluble 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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Design of Photostable, Activatable Near-Infrared Photoacoustic Probes Using Tautomeric Benziphthalocyanine as a Platform

Toriumi N., Asano N., Ikeno T., Muranaka A., Hanaoka K., Urano Y., Uchiyama M.

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition）  58 （ 23 ） 7788 - 7791 2019年06月

研究論文（学術雑誌）, 査読有り,  ISSN  14337851

　概要を見る

Near-infrared (NIR) imaging techniques have attracted significant attention for biological and medicinal applications due to the ability of NIR to penetrate deeply into tissues. However, there are very few stable, activatable molecular probes that can utilize NIR light in the wavelength range beyond 800 nm. Herein, we report a new activatable NIR system for photoacoustic imaging based on tautomeric benziphthalocyanines (BPcs). We found that the existence of a free hydroxyl group is crucial for NIR absorption of BPcs. Synthesized water-soluble hydroxy BPcs exhibited high photostability and no fluorescence, which are desirable features for photoacoustic imaging. We synthesized BPcs in which the free hydroxyl group was masked by an esterase-labile or an H2O2-labile group. The photoacoustic signals of these hydroxy-masked BPcs were increased upon NIR excitation at 880 nm in the presence of esterase or H2O2, respectively. These are rare examples of activatable probes utilizing NIR light at around 900 nm.

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Development of a platform for activatable fluorescent substrates of glucose transporters (GLUTs)

Takasugi T., Hanaoka K., Sasaki A., Ikeno T., Komatsu T., Ueno T., Yamada K., Urano Y.

Bioorganic and Medicinal Chemistry （Bioorganic and Medicinal Chemistry）  27 （ 10 ） 2122 - 2126 2019年05月

研究論文（学術雑誌）, 査読有り,  ISSN  09680896

　概要を見る

We have developed a platform for activatable fluorescent substrates of glucose transporters (GLUTs). We firstly conjugated fluorescein to glucosamine via an amide or methylene linker at the C-2 position of D-glucosamine, but the resulting compounds, FLG1 and FLG2, showed no uptake into MIN6 cells. So, we changed the fluorophore moiety to a fluorescein analogue, 2-Me TokyoGreen, which is less negatively charged. TokyoGreen-conjugated glucosamines TGG1 and TGG2 were successfully taken up into cells via GLUT. We further derivatized TGG1 and TGG2, and among the synthesized compounds, 2-Me-4-OMe TGG showed weak fluorescence under the acidic conditions of the extracellular environment inside tumors and in gastric cancers, and strong fluorescence at the intracellular physiological pH, under the control of a photoinduced electron transfer (PeT) process. This fluorogenic platform should be useful for developing a range of activatable fluorescent substrates targeting GLUTs, as well as derivatives that would be fluorescently activated by various intracellular enzymes, such as esterases, β-galactosidase and bioreductases.

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Red Fluorescence Probe Targeted to Dipeptidylpeptidase-IV for Highly Sensitive Detection of Esophageal Cancer

Ogasawara A., Kamiya M., Sakamoto K., Kuriki Y., Fujita K., Komatsu T., Ueno T., Hanaoka K., Onoyama H., Abe H., Tsuji Y., Fujishiro M., Koike K., Fukayama M., Seto Y., Urano Y.

Bioconjugate Chemistry （Bioconjugate Chemistry）  30 （ 4 ） 1055 - 1060 2019年04月

研究論文（学術雑誌）, 査読有り,  ISSN  10431802

　概要を見る

We have developed an activatable red fluorescence probe for dipeptidylpeptidase-IV (DPP-IV) by precisely controlling the photoinduced electron transfer (PeT) process of a red fluorescent scaffold, SiR600. The developed probe exhibited an extremely low background signal and showed significant fluorescence activation upon reaction with DPP-IV, enabling sensitive detection of esophageal cancer in clinical specimens from cancer patients.

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Development of novel dark quenchers and their application to imaging probes

Hanaoka K.

Yakugaku Zasshi （Yakugaku Zasshi）  139 （ 2 ） 277 - 283 2019年

研究論文（学術雑誌）, 査読有り,  ISSN  00316903

　概要を見る

Rhodamine dyes are among the most widely used fluorescent dyes for bioimaging due to their high fluorescence quantum yield and high photostability. Recently, novel far-red to near-infrared (NIR) fluorescent dyes have been developed: Si-rhodamines (SiRs), in which the O atom of conventional rhodamine dyes at the 10 position of the xanthene moiety is replaced with a Si atom. These SiRs are excellent long-wavelength fluorophores for bioimaging, as they retain the advantageous photophysical properties of conventional rhodamine dyes. Further, we focused on the QSY dark quenchers, which contain the rhodamine scaŠold bearing aromatic rings at the N atoms at the 3,6-positions of the xanthene moiety; these show no fluorescence, irrespective of solvent polarity and pH. NIR fluorescent probes based on the F orster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). We successfully developed SiR-based NIR dark quenchers (SiNQs) which show broad absorption covering this region. To demonstrate their usefulness, we designed and synthesized a NIR fluorescence probe for matrix metalloproteinase (MMP) activity using SiNQs. The developed probe was able to detect MMP activity in terms of NIR fluorescence, not only in vitro, but also in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized.

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Development of fluorescent probes for detecting reactive sulfur species and their application to development of inhibitors for 3MST

Echizen H., Hanaoka K.

Folia Pharmacologica Japonica （Folia Pharmacologica Japonica）  154 （ 3 ） 121 - 127 2019年

研究論文（学術雑誌）, 査読有り,  ISSN  00155691

　概要を見る

Hydrogen sulfide (H2S) has been reported to play an important role in biological systems. More recently, sulfane sulfur (sulfur with 0 or −1 charge) molecules have been also reported to be involved in various biological phenomena such as regulation of redox signaling and antioxidant functions. Fluorescent probes are one of the important chemical tools because it is easy to use and enable the real-time detection of the target molecules in living cells and tissues. We have successfully developed a highly selective H2S-detecting fluorescent probe, HSip-1. HSip-1 has been designed on the basis of the facts that the macrocyclic polyamine ligands form a stable complex with Cu2+, and Cu2+ also reacts with H2S and make a stable CuS complex. SSip-1 is a fluorescent probe for detecting sulfane sulfur and this fluorescent probe is designed on the basis of the unique feature of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. SSip-1 is a highly selective fluorescent probe and can detect sulfane sulfur reversibly. Both HSip-1 and SSip-1 were able to be used for the live-cell fluorescence imaging. Further, we applied HSip-1 to the high-throughput screening (HTS) for the inhibitors of 3-mercaptopyruvate sulfurtransferase (3MST), one of the reactive sulfur species (RSS)-generating enzymes. We successfully found new 3MST inhibitors by screening of 174,118 compounds. We expect that these fluorescent probes and inhibitors would be useful to elucidate new functions of RSS and RSS-generating enzymes.

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Rapid detection of metastatic lymph nodes of colorectal cancer with a gamma-glutamyl transpeptidase-activatable fluorescence probe

Kubo H., Hanaoka K., Kuriki Y., Komatsu T., Ueno T., Kojima R., Kamiya M., Murayama Y., Otsuji E., Urano Y.

Scientific Reports （Scientific Reports）  8 （ 1 ） 17781 2018年12月

研究論文（学術雑誌）, 査読有り

　概要を見る

Rapid diagnosis of metastatic lymph nodes (mLNs) of colorectal cancer (CRC) is desirable either intraoperatively or in resected fresh specimens. We have developed a series of activatable fluorescence probes for peptidase activities that are specifically upregulated in various tumors. We aimed to discover a target enzyme for detecting mLNs of CRC. Among our probes, we found that gGlu-HMRG, a gamma-glutamyl transpeptidase (GGT)-activatable fluorescence probe, could detect mLNs. This was unexpected, because we have previously reported that gGlu-HMRG could not detect primary CRC. We confirmed that the GGT activity of mLNs was high, whereas that of non-metastatic lymph nodes and CRC cell lines was low. We investigated the reason why GGT activity was upregulated in mLNs, and found that GGT was induced under conditions of hypoxia or low nutritional status. We utilized this feature to achieve rapid detection of mLNs with gGlu-HMRG. GGT appears to be a promising candidate enzyme for fluorescence imaging of mLNs.

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Red-Shifted Fluorogenic Substrate for Detection of lacZ-Positive Cells in Living Tissue with Single-Cell Resolution

Ito H., Kawamata Y., Kamiya M., Tsuda-Sakurai K., Tanaka S., Ueno T., Komatsu T., Hanaoka K., Okabe S., Miura M., Urano Y.

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition）  57 （ 48 ） 15702 - 15706 2018年11月

研究論文（学術雑誌）, 査読有り,  ISSN  14337851

　概要を見る

The Escherichia coli lacZ gene encoding β-galactosidase is a widely used reporter, but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples. Our recently reported fluorogenic substrate SPiDER-βGal is suitable for this purpose, but its hydrolysis product shows green fluorescence emission, and a red-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers. Herein, we describe the development of a red-shifted fluorogenic substrate for β-galactosidase, SPiDER-Red-βGal, based on a silicon rhodol scaffold and a carboxylic group as the intramolecular nucleophile. LacZ-positive cells were successfully labeled with SPiDER-Red-βGal at single-cell resolution in living samples, which enabled us to visualize different cell types in combination with GFP markers.

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A protein-coupled fluorescent probe for organelle-specific imaging of Na⁺

Taguchi R., Terai T., Ueno T., Komatsu T., Hanaoka K., Urano Y.

Sensors and Actuators, B: Chemical （Sensors and Actuators, B: Chemical）  265   575 - 581 2018年07月

研究論文（学術雑誌）, 査読有り,  ISSN  09254005

　概要を見る

Intracellular sodium concentration plays important roles in many cellular processes, but methods to analyze dynamic change of [Na+] in organelles of interest are still lacking. To address this problem, controlled localization of fluorescent Na+ probes is a key issue. In this work, we developed a protein-coupled Na+-sensitive fluorescent probe, HaloNa-1, which covalently labelled a HaloTag protein expressed at specific organelles in living cells. Fluorescence intensity of HaloNa-1 increased by approximately three-fold when complexed with Na+, and the dissociation constant was within physiological range. Notably, by conjugating a fluorescent protein to the HaloTag protein and using it as internal standard, one can reduce unwanted variation of probe fluorescence due to different cell volume, HaloTag expression level, etc. With this technique, we could observe elevation of intracellular [Na+] after ionophore stimulation with higher reliability than conventional sensors. We believe our probe is potentially useful to understand Na+ dynamics inside cells.

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Development of a Series of Practical Fluorescent Chemical Tools to Measure pH Values in Living Samples

Takahashi S., Kagami Y., Hanaoka K., Terai T., Komatsu T., Ueno T., Uchiyama M., Koyama-Honda I., Mizushima N., Taguchi T., Arai H., Nagano T., Urano Y.

Journal of the American Chemical Society （Journal of the American Chemical Society）  140 （ 18 ） 5925 - 5933 2018年05月

研究論文（学術雑誌）, 査読有り,  ISSN  00027863

　概要を見る

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate pKa for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable pKa and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.

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N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H <inf>2</inf> S and Sulfane Sulfur Production

Ezeriņa D., Takano Y., Hanaoka K., Urano Y., Dick T.P.

Cell Chemical Biology （Cell Chemical Biology）  25 （ 4 ） 447 - 459.e4 2018年04月

研究論文（学術雑誌）, 査読有り,  ISSN  24519456

　概要を見る

The cysteine prodrug N-acetyl cysteine (NAC) is widely used as a pharmacological antioxidant and cytoprotectant. It has been reported to lower endogenous oxidant levels and to protect cells against a wide range of pro-oxidative insults. As NAC itself is a poor scavenger of oxidants, the molecular mechanisms behind the antioxidative effects of NAC have remained uncertain. Here we show that NAC-derived cysteine is desulfurated to generate hydrogen sulfide, which in turn is oxidized to sulfane sulfur species, predominantly within mitochondria. We provide evidence suggesting the possibility that sulfane sulfur species produced by 3-mercaptopyruvate sulfurtransferase and sulfide:quinone oxidoreductase are the actual mediators of the immediate antioxidative and cytoprotective effects provided by NAC. N-Acetyl cysteine (NAC), by itself a poor scavenger of oxidants, is converted inside cells to yield sulfane sulfur species, which are very potent scavengers of oxidants. This conversion may account for many of the antioxidative effects provided by NAC which have hitherto remained unexplained.

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Establishment of Molecular Design Strategy to Obtain Activatable Fluorescent Probes for Carboxypeptidases

Kuriki Y., Kamiya M., Kubo H., Komatsu T., Ueno T., Tachibana R., Hayashi K., Hanaoka K., Yamashita S., Ishizawa T., Kokudo N., Urano Y.

Journal of the American Chemical Society （Journal of the American Chemical Society）  140 （ 5 ） 1767 - 1773 2018年02月

研究論文（学術雑誌）,  ISSN  00027863

　概要を見る

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the "aliphatic carboxamide to aliphatic carboxylate" structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.

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Development of a novel inhibitor for reactive sulfur species-producing enzymes

Echizen H., Hanaoka K.

Seikagaku （Seikagaku）  90 （ 3 ） 388 - 393 2018年

研究論文（学術雑誌）, 査読有り,  ISSN  00371017

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Synthesis of unsymmetrical Si-rhodamine fluorophores and application to a far-red to near-infrared fluorescence probe for hypoxia

Hanaoka K., Kagami Y., Piao W., Myochin T., Numasawa K., Kuriki Y., Ikeno T., Ueno T., Komatsu T., Terai T., Nagano T., Urano Y.

Chemical Communications （Chemical Communications）  54 （ 50 ） 6939 - 6942 2018年

研究論文（学術雑誌）, 査読有り,  ISSN  13597345

　概要を見る

Si-Rhodamines are bright fluorophores with red to near-infrared (NIR) emission, and are widely used for fluorescence imaging of biological phenomena. Here, in order to extend the scope of Si-rhodamine fluorophores, we established a versatile synthesis of unsymmetrical Si-rhodamines. To illustrate its value, we used one of these new fluorophores to synthesize a far-red to NIR fluorescence probe for hypoxia, and showed that it can visualize hepatic ischemia in mice in vivo.

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Molecular imaging of aberrant crypt foci in the human colon targeting glutathione S-transferase P1-1

Muguruma N., Okamoto K., Nakagawa T., Sannomiya K., Fujimoto S., Mitsui Y., Kimura T., Miyamoto H., Higashijima J., Shimada M., Horino Y., Matsumoto S., Hanaoka K., Nagano T., Shibutani M., Takayama T.

Scientific Reports （Scientific Reports）  7 （ 1 ）  2017年12月

研究論文（学術雑誌）

　概要を見る

Aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancers (CRCs), are a good surrogate marker for CRC risk stratification and chemoprevention. However, the conventional ACF detection method with dye-spraying by magnifying colonoscopy is labor- A nd skill-intensive. We sought to identify rat and human ACF using a fluorescent imaging technique that targets a molecule specific for ACF. We found that glutathione S-transferase (GST) P1-1 was overexpressed in ACF tissues in a screening experiment. We then synthesized the fluorogenic probe, DNAT-Me, which is fluorescently quenched but is activated by GSTP1-1. A CRC cell line incubated with DNAT-Me showed strong fluorescence in the cytosol. Fluorescence intensities correlated significantly with GST activities in cancer cell lines. When we sprayed DNAT-Me onto colorectal mucosa excised from azoxymethane-treated rats and surgically resected from CRC patients, ACF with strong fluorescent signals were clearly observed. The ACF number determined by postoperative DNAT-Me imaging was almost identical to that determined by preoperative methylene blue staining. The signal-to-noise ratio for ACF in DNAT-Me images was significantly higher than that in methylene blue staining. Thus, we sensitively visualized ACF on rat and human colorectal mucosa by using a GST-activated fluorogenic probe without dye-spraying and magnifying colonoscopy.

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Development of an azo-based photosensitizer activated under mild hypoxia for photodynamic therapy

Piao W., Hanaoka K., Fujisawa T., Takeuchi S., Komatsu T., Ueno T., Terai T., Tahara T., Nagano T., Urano Y.

Journal of the American Chemical Society （Journal of the American Chemical Society）  139 （ 39 ） 13713 - 13719 2017年10月

研究論文（学術雑誌）, 査読有り,  ISSN  00027863

　概要を見る

Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (1O2), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate 1O2 only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to 1O2 generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of 1O2 to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azobased strategy should be widely applicable to design a range of activatable photosensitizers.

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