An azulene dimer as a near-infrared quencher.
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Amino Acid Sequence↗
Biomedical subjects
Ching-Hsuan Tung
Publications and source records attributed to Ching-Hsuan Tung.
A receptor-targeted near-infrared fluorescence probe for in vivo tumor imaging.
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Animals↗
Fluorescence molecular tomography resolves protease activity in vivo.
Systematic efforts are under way to develop novel technologies that would allow molecular sensing in intact organisms in vivo. Using near-infrared fluorescent molecular beacons and inversion techniques that take into account the diffuse nature of photon propagation in tissue, we were able to obtain three-dimensional in vivo images of a protease in orthopic gliomas. We demonstrate that enzyme-activatable fluorochromes can be detected with high positional accuracy in deep tissues, that molecular specificities of different beacons towards enzymes can be resolved and that tomography of beacon activation is linearly related to enzyme concentration. The tomographic imaging method offers a range of new capabilities for studying biological function; for example, identifying molecular-expression patterns by multispectral imaging or continuously monitoring the efficacy of therapeutic drugs.
Endopeptidases↗
In vivo imaging of proteolytic activity in atherosclerosis.
BACKGROUND: Atherosclerotic plaque rupture, the most important cause of acute cardiovascular incidents, has been strongly associated with vascular inflammation. On the basis of the hypothesis that the inflammatory response and proteolysis lead to plaque rupture, we have examined the role of cathepsin B as a model proteolytic enzyme. METHODS AND RESULTS: Using western-type diet-fed apoE and apoE/endothelial NO synthase double knockout mice as models of atherosclerosis, we show (1) that cathepsin B is upregulated in atherosclerotic lesions characterized by high degrees of inflammation compared with normal aorta or silent lesions, (2) that intravenously injectable novel cathepsin B imaging beacons are highly activated within active atherosclerotic lesions and colocalize with cathepsin B immunoreactivity, and (3) that cathepsin B activity in atherosclerotic lesions can be imaged in whole animals by using a novel near-infrared tomographic imaging system. CONCLUSIONS: These studies indicate that cathepsin B, and potentially other proteases, may serve as a biomarker for vulnerable plaques when probed with beacons. The tomographic in vivo imaging method as well as catheter-based optical sensing methods could be readily adapted to screening and potentially to the molecular profiling of a number of proteases in vulnerable plaque in vivo.
Animals↗
A novel near-infrared fluorescence sensor for detection of thrombin activation in blood.
Thrombosis is an important pathophysiologic component of many cardiovascular diseases. Thrombin, a serine protease, plays a central role in thrombosis formation. Detection and imaging of thrombin activity may thus be of considerable biomedical interest. The goal of this study was to design, synthesize, and characterize a novel thrombin-activated near-infrared fluorescence (NIRF) probe. The probe consisted of a thrombin-cleavable peptide spacer (...D-Phe-Pip-Arg...; Pip=pipecolic acid) and contained a terminal fluorescence reporter which was quenched when conjugated to a biocompatible delivery vehicle. A control peptide spacer was synthesized that differed by one amino acid. Following thrombin addition, the probe was activated within minutes. The NIRF signal increased by a factor of 27-fold within 20 minutes, and was inhibited by hirudin, a specific thrombin inhibitor. NIRF optical imaging experiments confirmed rapid activation of the probe in both buffer and human blood. The control probe showed minimal activation in all experiments. In addition to potentially furthering our understanding of thrombin regulation in vivo, the thrombin-activated near-infrared probe may have broad clinical application to the diagnosis of arterial and venous thrombosis.
Enzyme Activation↗
Novel branching membrane translocational peptide as gene delivery vector.
A fragment of HIV-tat protein, RKKRRQRRR, has been shown to have membrane penetration and nuclear localization properties, which are critical attributes of gene therapy agents. In this study, we designed a series of arborizing tat peptides, containing 1-8 tat moieties, and evaluated them as transfection enhancers in a variety of cell lines. We found that all compounds complexed with plasmid DNA, but only the molecule containing 8 tat-peptide chains shows significant transfection capabilities. Using rhodamine labeled plasmid and eight tat-peptide complex, we were also able to demonstrate intracellular delivery of the complex by fluorescence microscopy.
Animals↗
Molecular imaging of MMP expression and therapeutic MMP inhibition.
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Animals↗
Detection of dysplastic intestinal adenomas using enzyme-sensing molecular beacons in mice.
BACKGROUND & AIMS: Proteases play key roles in the pathogenesis of tumor growth and invasion. This study assesses the expression of cathepsin B in dysplastic adenomatous polyps. METHODS: Aged Apc(Min/+) mice served as an experimental model for familial adenomatous polyposis. The 4 experimental groups consisted of (a) animals injected with a novel activatable, cathepsin B sensing near infrared fluorescence (NIRF) imaging probe; (b) animals injected with a nonspecific NIRF; (c) uninjected control animals; and (d) non-APC(Min/+) mice injected with the cathepsin B probe. Lesions were analyzed by immunohistochemistry, Western blotting, reverse transcription-polymerase chain reaction, and optical imaging. RESULTS: Cathepsin B was consistently overexpressed in adenomatous polyps. When mice were injected intravenously with the cathepsin reporter probe, intestinal adenomas became highly fluorescent indicative of high cathepsin B enzyme activity. Even microscopic adenomas were readily detectable by fluorescence, but not light, imaging. The smallest lesion detectable measured 50 microm in diameter. Adenomas in the indocyanine green and/or noninjected group were only barely detectable above the background. CONCLUSIONS: The current experimental study shows that cathepsin B is up-regulated in a mouse model of adenomatous polyposis. Cathepsin B activity can be used as a biomarker to readily identify such lesions, particularly when contrasted against normal adjacent mucosa. This detection technology can be adapted to endoscopy or tomographic optical imaging methods for screening of suspicious lesions and potentially for molecular profiling in vivo.
Adenomatous Polyps↗
Survival of Tropheryma whipplei, the agent of Whipple's disease, requires phagosome acidification.
Tropheryma whipplei was established as the agent of Whipple's disease in 2000, but the mechanisms by which it survives within host cells are still unknown. We show here that T. whipplei survives within HeLa cells by controlling the biogenesis of its phagosome. Indeed, T. whipplei colocalized with lysosome-associated membrane protein 1, a membrane marker of late endosomal and lysosomal compartments, but not with cathepsin D, a lysosomal hydrolase. This defect in phagosome maturation is specific to live organisms, since heat-killed bacilli colocalized with cathepsin D. In addition, T. whipplei survived within HeLa cells by adapting to acidic pH. The vacuoles containing T. whipplei were acidic (pH 4.7 +/- 0.3) and acquired vacuolar ATPase, responsible for the acidic pH of late phagosomes. The treatment of HeLa cells with pH-neutralizing reagents, such as ammonium chloride, N-ethylmaleimide, bafilomycin A1, and chloroquine, increased the intravacuolar pH and promoted the killing of T. whipplei. The ability of T. whipplei to survive in an acidic environment and to interfere with phagosome-lysosome fusion is likely critical for its prolonged persistence in host cells during the course of Whipple's disease. Our results suggest that manipulating the intravacuolar pH may provide a new approach for the treatment of Whipple's disease.
Acids↗
Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes.
PURPOSE: To determine if different expression levels of tumor cathepsin-B activity in well differentiated and undifferentiated breast cancers could be revealed in vivo with optical imaging. MATERIALS AND METHODS: A well differentiated human breast cancer (BT20, n = 8) and a highly invasive metastatic human breast cancer (DU4475, n = 8) were implanted orthotopically in athymic nude mice. Tumor-bearing animals were examined in vivo with near-infrared fluorescence (NIRF) imaging 24 hours after intravenous injection of an enzyme-sensing imaging probe. Immunohistochemistry, Western blotting (on cells and whole tumor samples), and correlative fluorescence microscopy were performed. RESULTS: Both types of breast cancers activated the NIRF probe so that tumors became readily detectable. However, in tumors of equal size, there was a 1.5-fold higher fluorescence signal in the highly invasive breast cancer (861 arbitrary units +/- 88) compared with the well differentiated lesion (566 arbitrary units +/- 36, P <.01). Western blotting confirmed a higher cathepsin-B protein content in the highly invasive breast cancer (DU4475) of about 1.4-fold (whole tumor samples) to 1.7-fold (cells). Immunohistochemistry and fluorescence microscopy findings confirmed the imaging findings. CONCLUSION: Cathepsin-B enzyme activity can be determined in vivo with NIRF optical imaging, while differences in tumoral expression may correlate with tumor aggressiveness.
Adenocarcinoma↗
Feasibility of in vivo multichannel optical imaging of gene expression: experimental study in mice.
PURPOSE: To develop and test a multichannel reflectance imaging system for small animals on the basis of a previously developed single-channel setup. MATERIALS AND METHODS: The imaging system was composed of modular parts, including a light source, excitation filters, emission filters, and a charged-coupled device for recording images. On the basis of generated excitation and absorption spectra of green fluorescent protein (GFP), tricarbocyanine 5.5 (Cy5.5), and indocyanine green (ICG), filters were selected to allow spectral separation and optimize resultant recorded signal. The system was tested by using a combination of the fluorochromes to confirm spectral separation. In vivo tests were performed in nude mice with tumors that expressed cathepsin B, which could be evaluated by using a Cy5.5-based activatable probe and GFP. For each in vivo tumor type and channel, statistical analysis was performed on the basis of signal intensity in the region of interest. RESULTS: The different fluorochromes were readily distinguished with the system; characteristics such as power were determined for all wavelengths. The system demonstrated a linear response for GFP, a monotonic response for Cy5.5 over a range of more than three orders of magnitude of concentration, and a more complex response for ICG. In vivo analysis demonstrated the ability to image GFP expression and cathepsin B expression separately in tumors: As expected, marked differences were observed in GFP-expression imaging between tumor types (1,363 arbitrary units [AU] +/- 236 [SD] vs 110 AU +/- 11 for GFP-positive and GFP-negative tumors, respectively; P <.001), whereas similar cathepsin B expression (1,070 AU +/- 285 vs 1,168 AU +/- 367; P >.5) was observed. Histologic analysis confirmed in vivo findings. CONCLUSION: Imaging multiple gene expressions simultaneously in vivo by using optical imaging is feasible.
Animals↗
Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation.
Recently, near-infrared (NIR) fluorescence light has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than light in the visible spectrum. Compounds exhibiting fluorescent properties in the NIR range are key elements for this upcoming optical imaging technology. In this paper, we report the synthesis of four new, water-soluble NIR cyanine fluorochromes which have superior chemical stability and optical properties. Each fluorochrome was designed with a monoreactive carboxyl group for labeling purposes. When multiple fluorochromes were attached to a single macromolecule, fluorescence quenching was observed. On the basis of this property, a novel autoquenched enzyme sensitive NIR fluorescence probe was prepared.
Amides↗
Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate.
Fluorescence optical imaging technologies are currently being developed to image specific molecular targets in vivo. Detection technologies range from those providing microscopic detail to whole body imaging systems with potential clinical use. A number of target-specific near-infrared imaging probes have recently been developed to image receptors, antigens, and enzymes. The goal of the current study was to evaluate a new near-infrared (NIR) folate receptor (FR)-targeted imaging probe for its ability to improve detection of FR-positive cancers. We hypothesized that modification of folate would retain receptor affinity in vivo, despite the bulkier NIR fluorochrome, NIR2 (em = 682 nm). Cellular uptake of the NIR conjugates was significantly higher in FR-positive nasopharyngeal epidermoid carcinoma, KB cells, compared to FR-negative human fibrosarcoma, HT1080 cells. When tumors were implanted in vivo, equal-sized KB tumors showed a 2.4-fold higher signal intensity compared to HT1080 tumors (24 h). The maximum signal-to-background ratio (3-fold) was observed at 24 h in KB tumor. Injection of the unmodified NIR2 fluorochrome did not result in persistent contrast increases under similar conditions. Furthermore, tumor enhancement with the NIR2-folate probe persisted over 48 h and was inhibitable in vivo by administration of unlabeled folate. These results indicate that folate-modified NIR fluorochrome conjugate can be used for improved detection of FR-positive tumors.
Animals↗
High efficiency synthesis of a bioconjugatable near-infrared fluorochrome.
Near-infrared (NIR) fluorochromes have become important reporter molecules for many biomedical applications, including FACS sorting, confocal microscopy, and more recently in vivo imaging. While the structures of several stable 800 nm fluorochromes have been published, their synthesis is often complex and there are difficulties in rapidly purifying these compounds in large quantities. Here we report on the synthesis of NIR820, ex/em = 790/820, with excellent physicochemical properties. Importantly, NIR820 is conveniently synthesized in a three-step reaction and can be purified by flash column chromatography rather than by HPLC. NIR820 is chemically stable and can be directly coupled to peptides during the solid-phase synthesis. In addition, NIR820 is also suitable for conjugation to proteins and other affinity molecules in aqueous buffer.
Animals↗
Developing a peptide-based near-infrared molecular probe for protease sensing.
Recently near-infrared (NIR) molecular probes have become important reporter molecules for a number of types of in vivo biomedical imaging. A peptide-based NIR fluorescence probe consisting of a NIR fluorescence emitter (Cy5.5), a NIR fluorescence absorber (NIRQ820), and a protease selective peptide sequence was designed to sense protease activity. Using a MMP-7 model, we showed that NIRQ820 efficiently absorbs the emission energy of Cy5.5 resulting in a low initial signal. Upon reacting with its target, MMP-7, the fluorescence signal of the designed probe was increased by 7-fold with a K(cat)/K(m) of 100 000 M(-)(1) s(-)(1). The described synthetic strategy should have wide application for other NIR probe preparations.
Animals↗
Monofunctional near-infrared fluorochromes for imaging applications.
In this report, the development of a new class of monocarboxylate functionalized cyanine derivatives using improved synthetic procedures is detailed. The employed synthetic strategy relies on efficient nucleophilic attack of alkyl-thiols on cyanine dyes bearing chloro-substituted polymethinic linkers. Monocarboxylate derivatized fluorochromes (CyTE dyes) can be prepared in one step in greater than 90% yield without the need for additional purification. Several of the fluorochromes synthesized by this route show no tendency to aggregate in aqueous solution and have excitation and emission maxima greater than 800 nm. The potential utility of the CyTE fluorochromes was demonstrated through direct labeling of phage displaying a vascular cellular adhesion molecule-1 (VCAM-1) targeting peptide. Endothelial cell internalization of the VCAM-1 targeted phage was monitored via near-infrared fluorescence microscopy.
Animals↗
Fluorescence probe with a pH-sensitive trigger.
Acid-catalyzed hydrolysis was used as the mechanism to design a new type of environmentally sensitive fluorescence probe. A mild and selective periodate oxidation of the 2-amino alcohol of serine in the presence of a disulfide bond was developed to prepare dialdehyde peptides. Two identical fluorochrome hydrazide derivatives were then linked to the dialdehyde peptide forming an acid-labile hydrazone linkage. This self-quenched probe is weakly fluorescent at a physiological pH of 7.4 but shows more than 3-fold fluorescence enhancement at pH 4.5.
Amino Acid Sequence↗
An effective method of on-resin disulfide bond formation in peptides.
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Cyclization↗