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Peptide Receptor-Targeted Fluorescent Probe: Visualization and Discrimination between Chronic and Acute Ulcerative Colitis Meiying Zeng, Andong Shao, Hui Li, Yan Tang, Qiang Li, Zhiqian Guo, Chungen Wu, Yingsheng Cheng, He Tian, and Wei-Hong Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00936 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Peptide Receptor-Targeted Fluorescent Probe: Visualization and Discrimination between Chronic and Acute Ulcerative Colitis Meiying Zeng,†,§ Andong Shao,‡,§ Hui Li,†,§ Yan Tang,† Qiang Li,‡ Zhiqian Guo,‡ Chungen Wu,† Yingsheng Cheng,*,† He Tian,‡ and Wei-Hong Zhu*,‡ †

Department of Radiology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Rd, Shanghai, 200233, China. E-mail: [email protected]. ‡ Key Laboratory for Advanced Materials and Institute of Fine Chemicals, Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China. E-mail: [email protected].

ABSTRACT: The inflammatory activity of ulcerative colitis plays an important role in the medical treatment. However, accurate and real-time monitoring of the colitis activity with noninvasive bioimaging method is still challenging, especially in distinguishing between chronic and acute colitis. As a good receptor, the oligopeptide transporter (PepT1) is over-expressed in colonic epithelial cells of chronic ulcerative colitis, which can deliver the tripeptide KPV (Lys-Pro-Val, the C-terminal sequence of α-MSH) into cytosol in the intestine. Herein, we report a PepT1 peptide receptor-targeted fluorescent probe DCM-KPV, with the strategy of conjugating the KPV into dicyanomethylene-4H-pyran (DCM) chromophore. The diagnostic fluorescent probe bestows a specific receptor-targeted interaction with PepT1 through the KPV moiety, possessing several beneficial characteristics, such as the efficient long emission, low photobleaching, negligible cytotoxicity and high cytocompatibility in living cells. We build the overexpressed PepT1 on the cytomembrane of ulcerative colitis model Caco-2 cell as the efficient receptor to accumulate the targeted tripeptide KPV in the cytoplasm and nucleus. With the co-localization of DCM-KPV and the DNA-specific fluorophore DAPI, the specifically long emission from chromophore DCM and efficient receptor-targeted peptide KPV, the fluorescent probe of DCM-KPV makes a breakthrough to the direct noninvasive observation to the accumulation in colon inflammation regions via intestinal mucosa, even successfully distinguishing the chronic, acute ulcerative colitis and normal groups. Compared with traditional unenhanced magnetic resonance imaging (MRI), and hematoxylin and eosin (H&E) staining, we make full use of exploiting the specific target-receptor interaction between the tripeptide unit KPV and oligopeptide transporter PepT1 for sensing selectivity. The desirable diagnostic ability of DCM-KPV can guarantee the real-time tracking and visualization of intracellular KPV role on ulcerative colitis, which provides an alternative to replace the time-consuming and tissue sampling-invasive H&E staining diagnosis. KEYWORDS: fluorescent probe, receptor, dicyanomethylene-4H-pyran, molecular imaging, inflammation activity INTRODUCTION As an immune-mediate inflammatory bowel disease,1,2 ulcerative colitis is strictly closed with abdominal pain, bloody diarrhea and fatigue,3,4 whose inflammatory activity plays an important role in clinical symptoms and medical treatments.5,6 Although the pathological diagnosis like hematoxylin and eosin (H&E) staining is a conventional method to trap the ulcerative colitis activity, the tissues sampling is invasive, and the staining process is time-consuming and tedious. The efficient and individual therapeutic schedule on basis of inflammatory activity is very indispensable, especially in real-time distinguishing between chronic and acute ulcerative colitis with the clinical noninvasive imaging is still challenging and highly desirable.7-11 The oligopeptide transporter (PepT1) is located at the apical membrane of small intestinal epithelial cells (IECs), capable of mediating the absorption of small peptides from diet. Moreover, PepT1 could be over-expressed in colonic epithelial cells of chronic ulcerative colitis, and is not expressed in normal colonic

epithelial cells.12-14 Therefore, PepT1 is expected as a potential targeted receptor for tracking chronic and acute ulcerative colitis. α-MSH (alpha-melanocyte–stimulating hormone) possesses anti-inflammatory effect on the immune-mediated inflammation,15 such as experimental colitis16 and arthritis.17 Particularly, the tripeptide KPV (Lys-Pro-Val, the C-terminal sequence of α-MSH) has been demonstrated strong anti-inflammatory effects.18 Based on the monitoring of pro-inflammatory cytokines and mediators in vitro, KPV could be transported into cytosol by PepT1 protein,19 which encourages us to explore its potential role in the ulcerative colitis diagnosis as a promising target unit to the receptor PepT1. Owing to the unique role of KPV/PepT1 ligand-receptor system and the overexpression of PepT1 in chronic ulcerative colitis, conjugating KPV with image contrast agents may provide a promising platform for the direct observation to the KPV accumulation in colon inflammation regions. Fluorescent chromophores have been widely utilized as

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biomarkers due to their excellent photostability and photophysical properties,20-30 as well as highly compatible ability with living systems.31-34 Especially, long emission fluorescent probes with deep tissue penetration and low autofluorescence background are preferable for bioimaging in vivo.35-40 The combination of long emission chromophore with tripeptide KPV would be desirable to visualize and real-time track the intracellular KPV role on ulcerative colitis with fluorescence imaging. Herein, we report a peptide receptor-targeted fluorescent probe DCM-KPV for in vivo tracking ulcerative colitis diagnosis (Scheme 1), in which the chromophore of dicyanomethylene-4H-pyran (DCM)41-45 is utilized as a fluorescence reporter, and the tripeptide unit KPV is utilized as the PepT1 receptor target. As expected, probe DCM-KPV demonstrates the outstanding photostability, negligible cytotoxicity and high cytocompatibility in living cells. Indeed the targeting absorption of KPV can accumulate in the cytoplasm and nucleus by mediation of the overexpressed receptor PepT1 on the cytomembrane of Caco-2 cell.46-49 The probe DCM-KPV has achieved the direct observation to the accumulation in colon inflammation regions via intestinal mucosa, and for the first time realized in distinguishing the chronic, acute ulcerative colitis as well as normal groups due to the specific advantages of long emission from chromophore DCM and efficient peptide-target KPV. Scheme 1. Proposed Sensing Mechanism Receptor-Targeted Activation of DCM−KPV.

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emission, large spectral shift, high fluorescent brightness and photostability,41-45 which are critical and required for excellent imaging contrast agents. Owing to its favorable donor-π-acceptor (D-π-A) structure, DCM exhibits a typical intra-molecular charge transfer (ICT) broad absorption band, and the emission wavelength could be changed distinctly via modulation of the electron-donating capability.41 In this regard, we exploit the specific target-receptor interaction between the tripeptide unit KPV and oligopeptide transporter PepT1 for sensing selectivity, designed a peptide receptor-targeted probe DCM-KPV (Scheme 1) by conjugating the tripeptide KPV unit to the DCM moiety. The synthetic route for DCM-KPV is depicted in Scheme 2, and the high resolution mass spectrometry (HRMS) and 1H NMR were performed to fully characterize the fluorescent probe DCM-KPV (Supporting Information). As shown in Figure 1A, compared with the reference intermediate DCM-N (fluorescence quantum yield 4.2%), the probe DCM-KPV demonstrated the favorable long emission at 629 nm and large stokes shift (170 nm), along with 4.9% in fluorescence quantum yield. Furthermore, given the broad the absorption, the fluorescent tail fell into the near-infrared (NIR) region as far as 760 nm, which is highly preferable to the in vivo bioimaging. Scheme 2. Synthetic Route to Receptor-Targeted Fluorescent Probe DCM−KPV.

for

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Reagents and conditions: (i) piperidine, toluene, AcOH, yield 20.0%; (ii) Fmoc-Lys(Fmoc)-Pro-Val-COOH, DCC, HOBt, yield 32.6%; (iii) piperidine, DMF, yield 12.5%.

RESULTS AND DISCUSSION Design of Probe DCM-KPV Containing Peptide Receptor–Target and Fluorescent Chromophore. DCM chromophore has been characterized by controllable

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monitored at 684 nm, and λex = 480 nm), and DCM-KPV (10 µM, monitored at 629 nm, and λex = 480 nm) under sustained illumination.

Low Photobleaching of DCM-KPV with Respect to Commercial Clinical Imaging Agent ICG. Considering bioimaging performance in vivo, the long-time exposure of fluorescence probes to high density light is always inevitable. Hence the photostability with low photobleaching is one of key factors to assess the fluorophore performance for practical applications, which is still a challenge for most commercial available fluorescent materials in bioimaging. A typical example is the cyanine dye ICG (FDA approved commercial clinical imaging agents) which is demonstrated with very poor photostability. Here the photostability of piperazine substituted DCM (DCM-N, its chemical structure shown in Scheme 2) and DCM-KPV were evaluated by time-course fluorescence measurements via uninterrupted continuous exposure to the light irradiation (Hamamatsu, LC8 Lightningcure, 300 W), and ICG was chosen as a standard reference. As shown in Figure 1B, the fluorescence intensity of ICG sharply decreased and hit a minimum level even after 2 min illumination, suggesting that ICG is vulnerable to photobleaching. In contrast, under the same light irradiation condition, the florescence intensity of DCM-N (> 80%) or DCM-KPV (> 60%) was still survived. Specifically, the fluorescence half-life period of DCM-N (420 s) or DCM-KPV (180 s) was exhibited, as 14-fold or 8-fold longer than that of ICG (30 s). As a consequence, both

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Time (min) Figure 1. (A) Absorption spectra (red curve) and photoluminescence spectra (blue curve) of DCM-KPV (10-5 M) in DMSO/H2O mixtures (V : V = 1 : 1), λex = 480 nm. (B) Time-dependent fluorescence intensity of ICG (10 µM, monitored at 812 nm, and λex = 780 nm), DCM (10 µM,

Figure 2. Cell cytotoxicity study of DCM-KPV: In the given concentration range (0, 1, 10 and 30 µM) of DCM-KPV, cell viability (A), cell proliferation rate (B), and cell inhibition rate (C) tests after incubation for 24, 48, and 72 h by MTS assay, respectively, and cell apoptosis rate (D) after incubation for 24 h by TUNEL assay. Note: all these high cell viability, high proliferation rate, low inhibition rate and low apoptosis rate indicate the negligible cytotoxicity of DCM-KPV.



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DCM-N and DCM-KPV display higher photostability than ICG, which was a beneficial factor serving as a promising bioprobe for long-time tracking and bioimaging in vivo. Negligible Cytotoxicity of DCM-KPV to Colon Cells with PepT1 Overexpression. We choose Ulcerative colitis model Caco-2 cells (Human colon adenocarcinoma, ATCC-HTB-37, Manassas, VA, USA) in which the oligopeptide transporter PepT1 was over-expressed.46,47 The cell cytotoxicity of DCM-KPV against Caco-2 cells was firstly assessed by MTS assay. Figure 2 indicates that DCM-KPV shows negligible cytotoxicity towards Caco-2 cells at high concentration and long incubation time. Specifically, after cultivation of Caco-2 cells with DCM-KPV at different concentration in the range of 0, 1, 10 and 30 µM, the cell lines still maintained larger than 95% viability (Figure 2A) along with incubation time (24, 48 and 72 h, respectively). Moreover, the cell viability was not obviously affected even when the concentration of DCM-KPV reached up to 30 µM in the culture medium. In addition, the negligible cytotoxic effects of DCM-KPV were also reflected by the non-significant difference in cell proliferation rate (Figure 2B) and cell growth inhibition rate (Figure 2C) of Caco-2 cells with respect to the control experiments. The cytocompatibility of DCM-KPV was further validated by detecting the apoptotic Caco-2 cells using TUNEL assay after cells were cultured in the given concentration range (0 30 µM) of DCM-KPV for 24 h. As shown in Figure 3A-C, the weak fluorescence signals indicated a tiny number of apoptosis cells. According to the quantitative analysis (Figure 2D), no appreciable apoptosis rate was observed both in control or DCM-KPV treated Caco-2 cells. Taken together, DCM-KPV displays good cytocompatibility towards Caco-2 cells, and can be further used to study its

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fluorescence imaging as the receptor-targeted probe in vitro and in vivo, assuring its safety for cell imaging and tracking in vivo.

Figure 3. Cell apoptosis study by TUNEL staining. Fluorescence images of Caco-2 cells with TUNEL/FITC staining (A-C; 515-565 nm emission, λex = 470 nm) and DAPI staining (D-F; 420-470 nm emission, λex = 405 nm) after incubation in the given concentration range (0, 10, and 30 µM) of DCM-KPV for 24 h. Merged images of PI and TUNEL panels (G-I). Scale bar = 100 µm. Note: no appreciable apoptosis of Caco-2 cells was found both in the DCM-KPV group and in the control group by TUNEL assay.

DCM-KPV Intracellular Transport Tracing through Receptor-Targeted Interaction. To study the intracellular transport of DCM-KPV into living cells by the receptor PepT1 mediation, the cellular uptake of DCM-KPV in PepT1-overexpressed Caco-2 cells48,49 was observed over

Figure 4. CLSM images of Caco-2 cells: (A-C and G-I) control groups, with cells incubated in DAPI (10 µg mL-1) for 50 min; (D-F) colocalization of DCM-KPV and DAPI; (J-L) colocalization of reference DCM-N and DAPI. Fluorescence image collection windows: Blue channel obtained from 420-470 nm emission, λex = 405 nm; Red channel (DCM-KPV) obtained from 620-670 nm emission, λex = 480 nm; Red channel (DCM-N) obtained from 670-690 nm emission, λex = 480 nm. Scale bar = 20 µm. Schematic illustration of the internalization pathway of DCM-KPV (M, the cellular uptake process through receptor targeted internalization) and DCM-N (N, the cellular uptake process through simple diffusion method) into Caco-2 cells. Note: the intracellular fluorescence intensity in image E (DCM-KPV) is highly brighter than that in image K (DCM-N), indicative of the efficient targeting of KPV to the receptor PepT1.48,49 4

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time by confocal laser scanning microscopy (CLSM). Commercial dye DAPI was used for cellular nucleus staining, and the reference compound DCM-N was used as control. As shown in Figure 4, when incubation in Caco-2 cells after 50 min, the fluorescence intensity of probe DCM-KPV and reference DCM-N was significantly different in the given concentration range (1.0 µM). That is, the brighter fluorescence signal of DCM-KPV (Figure 4E) in the cytoplasm and nucleus of Caco-2 cells was detected than that of DCM-N (Figure 4K) at the same condition. Here the significant difference between the cellular uptake of DCM-KPV and DCM-N in Caco-2 cells can be attributed to the PepT1 overexpression on the surface of Caco-2 cells. As illustrated with Figure 4M, the high affinity interaction between KPV and PepT1 facilitates the accumulation of probe DCM-KPV in the cytoplasm and nucleus (the cellular uptake process through receptor targeted internalization). In contrast, the weak brightness in control group of DCM-N is indicative of the cellular uptake process through a simple diffusion method (Figure 4N). In Figure 4E, the red fluorescence was not only detected at the cell membrane, but also accumulated in the cytoplasm and nucleus after 50 min incubation. It is suggestive that, based on the receptor mediation of PepT1 on the cell membrane, the receptor-targeted probes of DCM-KPV could accumulate both in the cytoplasm and in the cell nucleus of Caco-2 cells. Therefore, it is based on the specific target-receptor interaction19 via PepT1-KPV mediated endocytosis process that the peptide receptor-targeted fluorescent probe DCM-KPV provide a potential platform to directly visualize intracellular transport tracing towards Caco-2 cells (Figure 4M). In Situ Tracking and Discrimination of

Receptor-Targeted Activity in Ulcerative Colitis. When building ulcerative colitis model, we randomly divided 24 purchased BALB/c mice (male, 6-8-week-old, Shanghai SLAC Laboratory Animal Company) into three groups. Acute colitis group (n = 8) and chronic colitis group (n = 8) were induced with dextran sodium sulphate (DSS, MP, Biochemicals, 5000). The control group (n = 8) was given to normal drinking water. For fluorescence imaging, DCM-KPV solution was administered to the colon via anus and reserved for 1 h. Then both colitis and normal mice with injecting the same dose of DCM-KPV were euthanized, and distal colons were prepared for the fluorescence imaging. Before utilizing the fluorescence probe of DCM-KPV as contrast agent for colon tissue imaging in mice model, mild cleaning of the colon and air clysis were carried out on the chronic colitis, acute colitis and normal mice. Firstly, we exploited the traditional diagnostic magnetic resonance imaging (MRI) method to monitor the clysis process. The unenhanced MRI indicated that there was no waste residue left in the colon of mice model (Figure 5A-C). Although the distal colon wall of chronic and acute ulcerative colitis model became thickened, it was difficult to distinguish the inflammatory activity with the traditional MRI method. Up to date, H&E staining of local colon tissue is widely used to diagnose the inflammatory activity. As shown in H&E staining photomicrograph Figure 5F, the low intensity area of colon sample in normal mice indicated the intact viable folded mucosa with thin submucosal layer. The bright intensity area of chronic (Figure 5D) and acute colitis sample (Figure 5E) exhibited marked mucosal gland hyperplasia and mucosal necrosis, respectively. As a direct evaluation from the H&E staining histology changes of colon tissues in

Figure 5. T2 weighted MR imaging of the distal colons (A-C): chronic colitis model (A), acute colitis model (B) and control normal model (C). Arrows indicate distal colon pneumatosis, without waste residual. Photomicrographs of corresponding colon tissue slices with H&E staining (D-F): chronic colitis (D) with mucosal gland hyperplasia, acute colitis (E) with mucosa damage, and normal mucosal (F) with intact viable folded mucosa. Arrows indicate distal colon mucosa. Scale bar = 50 µm. Fluorescence image (G) and fluorescence intensity distribution (H) of the distal colon tissues of mice sacrificed at 1 h post-injection with DCM-KPV (2.5 mg kg-1, via anus, each model). Note: in spite of significant histology changes (D-F), the differential diagnosis for the chronic and acute ulcerative colitis is still challenging to MRI (A-C) by the morphological changes, while fluorescence images (G, H) using DCM-KPV probes could visualize the intensity difference directly.



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based on the confocal laser endomicroscopy, for the purpose of detecting the inflammatory activity of ulcerative colitis models in vivo and guiding local anti-inflammatory therapy.

different mice models, these induced ulcerative colitis model is very effective. However, in the H&E staining diagnosis, the sampling with corresponding colon tissue slices is invasive, and the staining process is tedious time-consuming. It is still highly desirable for identifying inflammatory activity in vivo and in direct noninvasive way, which is critical for clinical treatment. As mentioned above, DCM-KPV probes possess better photostability compared with commercial ICG, long emission character, as well as the outstanding cytocompatibility for the negligible cytotoxicity and cell apoptosis. Based on the fluorescence images (Figure 5G), the strong fluorescence can be detected in the distal colon of colitis model with respect to the weak signal in normal mice, which might be possibly attributed to PepT1-KPV mediated interaction during DCM-KPV transportation in colitis colon tissue.12,14 Moreover, the fluorescence signal in the group of chronic colitis mice model exhibited brighter emission than that of the acute colitis group. It is indeed in accordance with the higher overexpression of PepT1 in chronic colitis tissue than that in the acute colitis sample. When obtained by the semi-quantitative analysis data, the corresponding average fluorescence intensity distribution in distal colons (Figure 5H) also showed the distinction of DCM-KPV targeting absorption ability among chronic, acute and control normal groups. As a consequence, the fluorescent probe of DCM-KPV achieves a direct observation to the accumulation in colon inflammation regions via intestinal mucosa, even successfully distinguishing the chronic, acute ulcerative colitis and normal groups (Figure 5G and 5H). Taken together, the peptide receptor-targeted fluorescent probe DCM-KPV can visualize the activity of ulcerative colitis directly and rapidly, which is expected as an integrated gastrointestinal endomicroscopy alternative to replace the extremely time-consuming and tissue sampling-invasive H&E staining diagnosis.50

ASSOCIATED CONTENT More detailed experimental procedures, characterizations, supplementary optical spectra and figures can be found in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *W.-H.Z. fax: (+86) 21-6425-2758; e-mail: [email protected]. cn. *Y. S. C. e-mail: [email protected]

Author Contributions §

M.Y.Z, A.D.S and H. L contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National 973 Program (No. 2013CB733700), NSFC for Creative Research Groups (21421004), Key Project (21636002), and Distinguished Young Scholars (21325625), NSFC/China, the Oriental Scholarship, Science and Technology Commission of Shanghai Municipality (15XD1501400), and Programme of Introducing Talents of Discipline to Universities (B16017).

REFERENCES (1)

(2) (3)

CONCLUSIONS We have successfully developed a fluorescent peptide receptor-targeted probe DCM-KPV, by integrating tripeptide KPV as the PepT1 receptor-target into the fluorescent reporter DCM chromophore. It bestows the efficient long emission, low photobleaching, and outstanding cytocompatibility for the negligible cytotoxicity and cell apoptosis. The cellular uptake process through receptor targeted internalization of KPV can accumulate in the cytoplasm and nucleus by mediation of the overexpressed receptor PepT1 on the cytomembrane of Caco-2 cell. Importantly, DCM-KPV can realize the real-time tracking of the intracellular accumulation of KPV in Caco-2 cells with PepT1 overexpression, and directly observe the accumulation of KPV in colon inflammation regions via intestinal mucosa to efficiently distinguish the chronic, acute and normal activity in ulcerative colitis. In a potential clinical application prospect, to our knowledge, this is the first report on the peptide receptor-targeted fluorescent probe to distinguish the inflammatory activity in the ulcerative colitis models in vivo. As an alternative to replace the extremely time-consuming and tissue sampling-invasive H&E staining diagnosis, the fluorescent peptide receptor-targeted probe DCM-KPV provides a platform to build the novel gastrointestinal fluorescence imaging method

(4) (5)

(6)

(7)

(8) (9)

Ordás, I.; Eckmann, L.; Talamini, M.; Baumgart, D. C.; Sandborn, W. J. Ulcerative Colitis. Lancet 2012, 380, 1606−1619. Abraham, C; Cho, J. H. Inflammatory Bowel Disease. N. Engl. J. Med. 2009, 361, 2066–2078. Danese, S.; Fiocchi, C. Ulcerative Colitis. N. Engl. J. Med. 2011, 365, 1713−1725. Ford, A. C.; Moayyedi, P.; Hanauer, S. B. Ulcerative Colitis. BMJ 2013, 346, f432. Mowat, C.; Cole, A.; Windsor, A.; Ahmad, T.; Arnott, I.; Driscoll, R.; Mitton, S.; Orchard, T.; Rutter, M.; Younge, L.; Lees, C.; Ho, G. T.; Satsangi, J.; Bloom, S. Guidelines for the Management of Inflammatory Bowel Disease in Adults. Gut 2011, 60, 571−607. Nakase, H.; Keum, B.; Ye, B. D.; Park, S. J.; Koo, H. S.; Eun, C. S. Treatment of Inflammatory Bowel Sisease in Asia: the Results of a Multinational Web-Based Survey in the 2(nd) Asian Organization of Crohn's and Colitis (AOCC) Meeting in Seoul. Intest. Res. 2016, 14, 231−239. Socaciu, M.; Ciobanu, L.; Diaconu, B.; Hagiu, C.; Seicean, A.; Badea, R. Non-Invasive Assessment of Inflammation and Treatment Response in Patients with Crohn's Disease and Ulcerative Colitis using Contrast-Enhanced Ultrasonography Quantification. J. Gastrointestin. Liver. Dis. 2015, 24, 457−465. Deepak, P.; Bruining, D. H. Radiographical Evaluation of Ulcerative Colitis. Gastroenterol. Rep. 2014, 2, 169−177. Andersen, K.; Vogt, C.; Blondin, D.; Beck, A.; Heinen, W.; Aurich, V.; Häussinger, D.; Mödder, U.; Cohnen, M. Multi-Detector CT-Colonography in Inflammatory Bowel Disease: Prospective Analysis of CT-Findings to High-Resolution Video Colonoscopy. Eur. J. Radiol. 2006, 58, 140−146.

(10) Bianchi, A.; Bluhmki, T.; Schönberger, T.; Kaaru, E.; Beltzer,


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ACS Applied Materials & Interfaces A.; Raymond, E.; Wunder, A.; Thakker, P.; Stierstorfer, B.; Stiller, D. Noninvasive Longitudinal Study of a Magnetic Resonance Imaging Biomarker for the Quantification of Colon Inflammation in a Mouse Model of Colitis. Inflamm. Bowel. Dis. 2016, 22, 1286−1295.

(24) Bai, H. T.; Chen, H.; Hu, R.; Li, M.; Lv, F. T.; Liu, L. B.; Wang, S. Supramolecular Conjugated Polymer Materials for in Situ Pathogen Detection. ACS Appl. Mater. Interfaces 2016, 8, 31550−31557. (25) Hu, X. L.; Hu, J. M.; Tian, J.; Ge, Z. S.; Zhang, G. Y.; Luo, K. F.; Liu, S. Y. Polyprodrug Amphiphiles: Hierarchical Assemblies for Shape-Regulated Cellular Internalization, Trafficking, and Drug Delivery. J. Am. Chem. Soc. 2013, 135, 17617−17629. (26) Guo, Z. Q.; Park, S.; Yoon, J.; Shin, I. Recent Progress in the Development of Near-Infrared Fluorescent Probes for Bioimaging Applications. Chem. Soc. Rev. 2014, 43, 16−29. (27) Xuan, W. M.; Sheng, C. Q.; Cao, Y. T.; He, W. H.; Wang, W. Fluorescent Probes for the Detection of Hydrogen Sulfide in Biological Systems. Angew. Chem. Int. Ed. 2012, 51, 2282−2284. (28) W. H. Zhu, Visualizing Deeper into the Body with a NIR-II Small-Molecule Fluorophore. Sci. China Chem. 2016, 59, 203−204. (29) Liu, X. G.; Qiao, Q. L.; Tian, W. M.; Liu, W. J.; Chen, J.; Lang, M. J.; Xu, Z. C. Aziridinyl Fluorophores Demonstrate Bright Fluorescence and Superior Photostability by Effectively Inhibiting Twisted Intramolecular Charge Transfer. J. Am. Chem. Soc. 2016, 138, 6960–6963. (30) Tong, H. J.; Zheng, Y. J.; Zhou, L.; Li, X. M.; Qian, R.; Wang, R.; Zhao, J. H.; Lou, K. Y.; Wang, W. Enzymatic Cleavage and Subsequent Facile Intramolecular Transcyclization for in Situ Fluorescence Detection of gamma-Glutamyltranspetidase Activities. Anal. Chem. 2016, 88, 10816–10820 (31) Yoshii, T.; Mizusawa, K.; Takaoka, Y.; Hamachi, I. Intracellular Protein-Responsive Supramolecules: Protein Sensing and In-Cell Construction of Inhibitor Assay System. J. Am. Chem. Soc. 2014, 136, 16635−16642. (32) Kumar, V.; Anslyn, E. V. A Selective Turn-on Fluorescent Sensor for Sulfur Mustard Simulants. J. Am. Chem. Soc. 2013, 135, 6338−6344. (33) Hu, Q. L.; Gao, M.; Feng, G. X.; Liu, B. Mitochondria-Targeted Cancer Therapy Using a Light-up Probe with Aggregation-Induced-Emission Characteristics. Angew. Chem. Int. Ed. 2014, 53, 14225−14229. (34) Duan, X. R.; Liu, L. B.; Feng, F. D.; Wang, S. Cationic Conjugated Polymers for Optical Detection of DNA Methylation, Lesions, and Single Nucleotide Polymorphisms. Acc. Chem. Res. 2010, 43, 260−270. (35) Chan, J.; Dodani, S. C.; Chang, C. J. Reaction-Based Small-Molecule Fluorescent Probes for Chemoselective Bioimaging. Nat. Chem. 2012, 4, 973−984. (36) Liu, Y.; Chen, M.; Cao, T. Y.; Sun, Y.; Li, C. Y.; Liu, Q.; Yang, T. S.; Yao, L. M.; Feng, W.; Li, F. Y. A Cyanine-Modified Nanosystem For in Vivo Upconversion Luminescence Bioimaging of Methylmercury. J. Am. Chem. Soc. 2013, 135, 9869−9876. (37) Atilgan, A.; Ecik, E. T.; Guliyev, R.; Uyar, T. B.; Erbas-Cakmak, S.; Akkaya, E. U. Near-IR-Triggered, Remote-Controlled Release of Metal Ions: A Novel Strategy for Caged Ions. Angew. Chem. Int. Ed. 2014, 53, 10678−10681. (38) Myochin, T.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. Design Strategy for a Near-Infrared Fluorescence Probe for Matrix Metalloproteinase Utilizing Highly Cell Permeable Boron Dipyrromethene. J. Am. Chem. Soc. 2012, 134, 13730−13737. (39) Dong, B. L.; Song, X. Z.; Kong, X. Q.; Wang, C.; Tang, Y. H.; Liu, Y.; Lin, W. Y. Simultaneous Near-Infrared and Two-Photon In Vivo Imaging of H2O2 Using a Ratiometric Fluorescent Probe based on the Unique Oxidative Rearrangement of Oxonium. Adv. Mater. 2016, 28, 8755−8759.

(11) Beltzer, A.; Kaulisch, T.; Bluhmki, T.; Schoenberger, T.; Stierstorfer, B.; Stiller, D. Evaluation of Quantitative Imaging Biomarkers in the DSS Colitis Model. Mol. Imaging. Biol. 2016, 18, 697−704. (12) Abidi, S. A. The Oligopeptide (PepT1) in Human Intestine: Biology and Function. Gastroenterology 1997, 113, 332−340. (13) Ziegler, T. R.; Fernández-Estívariz, C.; Gu, L. H.; Bazargan, N.; Umeakunne, K.; Wallace, T. M.; Diaz, E. E.; Rosado, K. E.; Pascal, R. R.; Galloway, J. R.; Wilcox, J. N.; Leader, L. M. Distribution of the H/Peptide Transporter PepT1 in Human Intestine: Up-Regulated Expression in the Colonic Mucosa of Patients with Short-Bowel Syndrome. Am. J. Clin. Nutr. 2002, 75, 922−930. (14) Merlin, D.; Si-Tahar, M.; Sitaraman, S. V.; Eastburn, K.; Williams, I.; Liu, X.; Hediger, M. A.; Madara, J. L. Colonic Epithelial hPepT1 Expression Occurs in Inflammatory Bowel Disease: Transport of Bacterial Peptides Influence Expression of MHC Class 1 Molecules. Gastroenterology 2001, 120, 1666−1679. (15) Brzoska, T.; Luger, T. A.; Maaser, C.; Abels, C.; Böhm, M. Alpha-Melanocyte-Stimulating Hormone and Related Tripeptides: Biochemistry, Anti-Inflammatory and Protective Effects In Vitro and In Vivo, and Future Perspectives for the Treatment of Immune-Mediated Inflammatory Diseases. Endocr. Rev. 2008, 29, 581−602. (16) Oktar, B. K.; Ercan, F.; Yeğen, B. C.; Alı̇ can, I. The Effect of Alpha-Melanocyte Stimulating Hormone on Colonic Inflammation in the Rat. Peptides 2000, 21, 1271−1277. (17) Gonzalez-Rey, E.; Chorny, A.; O'Valle, F.; Delgado, M. Adrenomedullin Protects from Experimental Arthritis by Down-Regulating Inflammation and Th1 Response and Inducing Regulatory T Cells. Am. J. Pathol. 2007, 170, 263−271. (18) Schaible, E. V.; Steinsträßer, A.; Jahn-Eimermacher, A.; Luh, C.; Sebastiani, A.; Kornes, F.; Pieter, D.; Schäfer, M. K.; Engelhard, K.; Thal, S. C. Single Administration of Tripeptide α-MSH(11-13) Attenuates Brain Damage by Reduced Inflammation and Apoptosis after Experimental Traumatic Brain Injury in Mice. PLoS. One. 2013, 8, e71056. (19) Ingersoll, S. A.; Ayyadurai, S.; Charania, M. A.; Laroui, H.; Yan Y.; Merlin, D. The Role and Pathophysiological Relevance of Membrane Transporter PepT1 in Intestinal Inflammation and Inflammatory Bowel Disease. Am. J. Physiol. Gastrointest. Liver. Physiol. 2012, 302, G484−492. (20) Cheng, Y. Y.; Li, G. C.; Liu, Y.; Shi, Y.; Gao, G.; Wu, D.; Lan, J. B.; You, J. S. Unparalleled Ease of Access to a Library of Biheteroaryl Fluorophores via Oxidative Cross-Coupling Reactions: Discovery of Photostable NIR Probe for Mitochondria. J. Am. Chem. Soc., 2016, 138, 4730−4738. (21) Jana, A.; Bai, L.; Li, X.; Ågren, H.; Zhao, Y. L. Morphology Tuning of Self-Assembled Perylene Monoimide from Nanoparticles to Colloidosomes with Enhanced Excimeric NIR Emission for Bioimaging. ACS. Appl. Mater. Interfaces, 2016, 8, 2336−2347. (22) Zhu, H.; Fan, J. L.; Wang, J. Y.; Mu, H. Y.; Peng, X. J. An "Enhanced PET"-Based Fluorescent Probe with Ultrasensitivity for Imaging Basal and Elesclomol-Induced HClO in Cancer Cells. J. Am. Chem. Soc. 2014, 136, 12820−12823. (23) Lee, M. H.; Park, N.; Yi, C.; Han, J. H.; Hong, J. H.; Kim, K. P.; Kang, D. H.; Sessler, J. L.; Kang, C.; Kim, J. S. Mitochondria-Immobilized pH-Sensitive Off-On Fluorescent Probe. J. Am. Chem. Soc. 2014, 136, 14136−14142.

(40) Cheng, D.; Pan, Y.; Wang, L.; Zeng, Z. B.; Yuan, L.; Zhang, X.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B.; Chang, Y.-T. Selective Visualization of the Endogenous Peroxynitrite in an Inflamed Mouse Model by a Mitochondria-Targetable Two-Photon Ratiometric Fluorescent Probe. J. Am. Chem. Soc. 2017, 139, 285–292.

Page 8 of 9

(45) Shao, A. D.; Xie, Y. S.; Zhu, S. J.; Guo, Z. Q.; Zhu, S. Q.; Guo, J.; Shi, P.; James, T. D.; Tian, H.; Zhu, W. H. Far-Red and Near-IR AIEActive Fluorescent Organic Nanoprobes with Enhanced Tumor Targeting Efficacy: Shape-Specific Effects. Angew. Chem., Int. Ed. 2015, 54, 7275−7280.

(41) Guo, Z. Q.; Zhu, W. H.; Tian, H. Dicyanomethylene-4H-Pyran Chromophores for OLED Emitters, Logic Gates and Optical Chemosensors. Chem. Commun. 2012, 48, 6073−6084.

(46) Geillinger, K. E.; Kipp, A. P.; Schink, K.; Röder, P.V.; Spanier, B.; Daniel, H. Nrf2 Regulates the Expression of the Peptide Transporter PEPT1 in the Human Colon Carcinoma Cell Line Caco-2. Biochim. Biophys. Acta. 2014, 1840, 1747−1754.

(42) Wang, X.; Guo, Z.; Zhu, S.; Liu, Y.; Shi, P.; Tian, H.; Zhu, W. H. Rational Design of Novel Near-Infrared Fluorescent DCM Derivatives and Its Application for Bioimaging. J. Mater. Chem. B. 2016, 4, 4683−4689.

(47) Shimakura, J.; Terada, T.; Katsura, T.; Inui, K. Characterization of the Human Peptide Transporter PEPT1 Promoter: Sp1 Functions as a Basal Transcriptional Regulator of Human PEPT1. Am. J. Physiol. Gastrointest. Liver. Physiol. 2005, 289, G471−477.

(43) Gu, K. Z.; Xu, Y. S.; Li, H.; Guo, Z. Q.; Zhu, S. J.; Zhu, S. Q.; Shi, P.; James, T. D.; Tian, H.; Zhu, W. H. Real-Time Tracking and In Vivo Visualization of β-Galactosidase Activity in Colorectal Tumor with a Ratiometric Near-Infrared Fluorescent Probe. J. Am. Chem. Soc. 2016, 138, 5334−5340.

(48) Wang, W.; Liu, Q.; Wang, C.; Meng, Q.; Kaku, T.; Liu, K.; Effects of JBP485 on the Expression And Function Of PepT1 in Indomethacin-Induced Intestinal Injury in Rats and Damage In Caco-2 Cells. Peptides 2011, 32, 946−955.

(44) Barbon, A.; Bott. E. D.; Brustolon, M.; Fabris, M.; Kahr, B.; Kaminsky, W.; Reid, P. J.; Wong, S. M.; Wustholz, K. L.; Zanré, R. Triplet States of the Nonlinear Optical Chromophore DCM in Single Crystals of Potassium Hydrogen Phthalate and Their Relationship to Single-Molecule Dark States. J. Am. Chem. Soc. 2009, 131, 11548−11557.

(49) Wang, B.; Li, B. Effect of Molecular Weight on the Transepithelial Transport and Peptidase Degradation of Casein-Derived Peptides by Using Caco-2 Cell Model. Food. Chem. 2017, 218, 1−8. (50) Fan, C. H.; Tamiya, E. Translating the Advances of Biosensors from Bench to Bedside. Biotechnol. J. 2016, 11, 727−728.


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Peptide Receptor-Targeted Fluorescent Probe - ACS Publications

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