Shou-Cheng Wu

A N-(2-aminophenyl)-5-(dimethylamino)-1-naphthalenesulfonic amide (Ds-DAB) based fluorescent chemosensor for peroxynitrite.

A dansyl derivative (Ds-DAB) was prepared and used as a fluorescent probe for peroxynitrite (ONOO(-)) detection. The results showed that the addition of peroxynitrite to the aqueous solution of Ds-DAB would result in obvious fluorescence enhancement. This probe is highly specific for peroxynitrite in aqueous solution, avoiding interference from other reactive oxygen species (ROS) and nitrogen species (RNS). The advantages of high selectivity, fast reaction rate, and peroxynitrite bioimaging render Ds-DAB suitable for peroxynitrite detection.

Peptide-based MRI contrast agent and near-infrared fluorescent probe for intratumoral legumain detection

Recent studies suggest that intratumoral legumain promotes tumorigenesis. To monitor legumain activity in tumors, we developed a new MRI contrast agent ([Gd-NBCB-TTDA-Leg(L)]) and a NIR fluorescence probe (CyTE777-Leg(L)-CyTE807). The MRI contrast agent was prepared by introduction of cyclobutyl and benzyl group residues to TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triaza-dodecanedioic acid), followed by the attachment of a legumain-specific substrate peptide (Leg(L)). The NIR fluorescence probe was designed by conjugating two NIR fluorochromes (CyTE777 and CyTE807) with Leg(L). Peptide cleavage of the MRI contrast agent by legumain can increase its hydrophobicity and promote rotational correlation time (τ(R)). Peptide cleavage of the NIR probes by the legumain relieves the self quench of the probe. Peptide cleavage of the MRI contrast agent and the NIR fluorescence probe by legumain were confirmed by T1 relaxometric studies and by fluorescence studies, respectively. In vivo MR images showed that [Gd-NBCB-TTDA-Leg(L)] attained 55.3 fold (254.2% versus 4.6%, at 2.0 h post-injection) higher imaging enhancement, as compared with control contrast agent bearing a noncleaveable peptide ([Gd-NBCB-TTDA-Leg(D)], in the CT-26 (legumain(+)) tumors. Similarly, optical imaging probe CyTE777-Leg(L)-CyTE807 attained 15.2 fold (3.34 × 10(9) photons/min versus 0.22 × 10(9) photons/min, at 24.0 h post-injection) higher imaging enhancement in the CT-26 (legumain(+)) tumors, compared to a NIR control probe (CyTE777-Leg(D)-CyTE807). These data indicate that the [Gd-NBCB-TTDA-Leg(L)] and the CyTE777-Leg(L)-CyTE807 probes may be promising tools to image the legumain-expressing cancers for diagnoses and targeted treatments.

In vitro and in vivo imaging of peroxynitrite by a ratiometric boronate-based fluorescent probe

Peroxynitrite (ONOO-) is an important species involved in many physiopathological processes. Progresses have been made in developing novel fluorescent probes to detect peroxynitrite with relatively high sensitivity and specificity. Herein, we report the synthesis, characterization and biological applications of a new boronate-based fluorescent probe, 4-MB. The studies showed that 4-MB exhibits a dual ratiometric and calorimetric response toward peroxynitrite due to ONOO--triggered oxidative reaction. A possible mechanism of the oxidation reaction was proposed and the reaction product was isolated and characterized using different spectroscopic methods. We have thoroughly demonstrated the utility of 4-MB for intracellular peroxynitrite imaging. Further, we showed that 4-MB can be potentially employed to visualize exogenous and endogenous peroxynitrite in RAW264.7 macrophages, EAhy926 cells, zebrafish and in live tissues from a high-fat diet-induced obese mouse model.

Reaction-based epoxide fluorescent probe for in vivo visualization of hydrogen sulfide.

Hydrogen sulfide (H2S) has emerged as the most important biosynthetic gasotransmitters along with nitric oxide (NO) and carbon monoxide (CO). In this study, we report the design and the synthesis of a new epoxide fluorescent probe 7-glycidyloxy-9-(2-glycidyloxycarbonylphenyl)-2-xanthone (FEPO) for use in in vivo visualization of hydrogen sulfide. The probe employs a fluorescein as a fluorophore, and is equipped with an operating epoxide unit. FEPO functions via epoxide ring opening upon nucleophilic attack of H2S. This ring opening strategy may open a new avenue for the development of various H2S fluorescent sensors. FEPO showed high selectivity and high sensitivity for H2S. FEPOs cytotoxicity was tested using MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide) assay. Furthermore, the use of confocal imaging of H2S and in vivo imaging in live zebra fish demonstrated FEPOs potential biological applications. We anticipate that, owing to their ideal properties, probes of this type will find great uses in exploring the role of H2S in biology.

Bispecific antibody conjugated manganese-based magnetic engineered iron oxide for imaging of HER2/neu- and EGFR-expressing tumors

The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR.

Development of a Mucin4-Targeting SPIO Contrast Agent for Effective Detection of Pancreatic Tumor Cells in Vitro and in Vivo

In search of a unique and reliable contrast agent targeting pancreatic adenocarcinoma, new multifunctional nanoparticles (MnMEIO-silane-NH2-(MUC4)-mPEG NPs) were successfully developed in this study. Mucin4-expression levels were determined through different imaging studies in a panel of pancreatic tumor cells (HPAC, BxPC-3, and Panc-1) both in vitro and in vivo studies. The in vitro T2-weighted MR imaging study in HPAC and Panc-1 tumor cells treated with NPs showed -89.1 ± 5.7% and -0.9 ± 0.2% contrast enhancement, whereas in in vivo study, it is found to be -81.5 ± 4.5% versus -19.6 ± 5.2% (24 h postinjection, 7.0 T), respectively. The T2-weighted MR and optical imaging studies revealed that the novel contrast agent can specifically and effectively target to mucin4-expressing tumors in nude mice. Hence, it is suggested that MnMEIO-silane-NH2-(MUC4)-mPEG NPs are able to provide an efficient and targeted delivery of MUC4 antibodies to mucin4-expressing pancreatic tumors.

Water-Soluble Dinitrosyl Iron Complex (DNIC): a Nitric Oxide Vehicle Triggering Cancer Cell Death via Apoptosis.

Nitric oxide (NO) is an important cellular signaling molecule that modulates various physiological activities. Angiogenesis-promoting activities of NO-donor drugs have been explored in both experimental and clinical studies. In this study, a structurally well characterized and water-soluble neutral {Fe(NO)2}(9) DNIC [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2) was synthesized to serve as a NO-donor species. The antitumor activity of DNIC 2 was determined by MTT assay, confocal imaging, and Annexin-V/PI staining. The IC50 values of DNIC 2 were 18.8, 42.9, and 38.6 μM for PC-3, SKBR-3, and CRL5866 tumor cells, respectively. Moreover, DNIC 2 promoted apoptotic cell death via activation of apoptosis-associated proteins and inhibition of survival associated proteins. In particular, DNIC 2 treatment suppressed PC-3 tumor growth by 2.34- and 19.3-fold at 7 and 21 days, in comparison with the control group. These results indicate that water-soluble DNIC 2 may serve as a promising drug for cancer therapy.