Yun-Ming Wang

Nitric Oxide Turn-on Fluorescent Probe Based on Deamination of Aromatic Primary Monoamines

The stable, water-soluble, and nonfluorescent FA-OMe can sense nitric oxide (NO) and form the intensely fluorescent product dA-FA-OMe via reductive deamination of the aromatic primary amine. The reaction is accompanied by a notable increase of the fluorescent quantum yield from 1.5 to 88.8%. The deamination mechanism of FA-OMe with NO was proposed in this study. The turn-on fluorescence signals were performed by suppression of photoinduced electron transfer (PeT), which was demonstrated by density functional theory (DFT) calculations of the components forming FA-OMe and dA-FA-OMe. Furthermore, FA-OMe showed water solubility and good stability at physiological pHs. Moreover, the selectivity study indicated that FA-OMe had high specificity for NO over other reactive oxygen/nitrogen species. In an endogenously generated NO detection study, increasing the incubation time of FA-OMe with lipopolysaccharide (LPS) pretreated Raw 264.7 murine macrophages could cause an enhanced fluorescence intensity image. In addition, a diffusion/localization cell imaging study showed that FA-OMe could be trapped in Raw 264.7 cells. These cell imaging results demonstrated that FA-OMe could be used as a turn-on fluorescent sensor for the detection of endogenously generated NO.

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.

Sensitivity evaluation of NBD-SCN towards cysteine/homocysteine and its bioimaging applications

A push-pull fluorogenic reagent, NBD-SCN, was applied for specific detection of cysteine (Cys) and homocysteine (Hcy). Replacing thiocyanato group with Cys/Hcy increased the push-pull characteristic of the probe and resulted in emission of fluorescence. The fluorescent response of the probe toward Cys/Hcy was significantly higher than toward glutathione and other amino acids. The probe showed a 470- and 745-fold fluorescence enhancement at 550 nm and detection limit of 2.99 and 1.43 nM for Cys and Hcy, respectively. Time-dependent fluorescence assays showed that the fluorescence intensity reached a plateau within 20s after addition of Cys and within 10 min after addition of Hcy. Furthermore, the fluorescence images of Cys/Hcy in Raw 264.7 cells were obtained after adding this probe to the cells. These results indicate that NBD-SCN not only possesses good selectivity and sensitivity for Cys/Hcy but also can penetrate cells for Cys/Hcy bioimaging.

Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging

A new magnetic resonance imaging (MRI) contrast bearing RGD peptide is reported. In this study, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with various sizes were prepared. Particles sizes between 6 and 13 nm were tuned by varying the stirring rate. Remarkable negative contrast was observed because USPIO nanoparticles (13.1  ±  2.1 nm) generated high transversal relaxivity r₂ (188  ±  3 m m(-1)  s(-1) ) and saturation magnetization (94 emu g(-1) Fe). The USPIO nanoparticles were coated with PDA [2-(pyridyldithio)-ethylamine; PDA nanoparticles] containing functional polymer, which can be readily synthesized by Michael addition. The PDA nanoparticles were conjugated with RGD peptide (RGD nanoparticles) for targeting the specific site. The target specificity and high relaxivity allowed RGD nanoparticles to differentiate the expression level of integrin receptor on several cell lines and tumors (MCF-7, A-549, HT-29 and HT-1080) by in vitro and in vivo MR imaging. Importantly, a remarkable negative contrast (-51.3 ± 6.7%) was observed for in vivo MR imaging of MCF-7 tumor. This result implies that the RGD nanoparticles that greatly enhance the MR imaging are highly sensitive for early stage tumor detection.

Relaxivity studies and X-ray structure of Gd(III)-diethylenetriamine-N,N′,N″-triacetic acid-N,N″-bis(2-methoxyphenethylamide) as a potential contrast agent for magnetic resonance imaging

Abstract A new bis(amide) derivative of diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (H5dtpa), diethylenetriamine-N,N′,N″-triacetic-N,N″-bis(2-methoxyphenethylamide) (H3L), has been synthesized. The crystal structure of gadolinium(III) complex of H3L ([GdL]) has been determined by X-ray crystallography. The coordination sphere of Gd(III) comprises three amine nitrogens, two amide oxygens, three carboxylic acid oxygens, and one water molecule. 17O NMR shifts showed that the [DyL] complex had one inner-sphere water molecule. Relaxivity studies with the gadolinium(III) complex showed that they associate strongly with proteins leading to a significant relaxivity enhancement.

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.

Sensitivity evaluation of rhodamine B hydrazide towards nitric oxide and its application for macrophage cells imaging

A colorless and non-fluorescent rhodamine derivative, rhodamine B hydrazide (RH), is applied to detect nitric oxide and form fluorescent rhodamine B (RB). The reaction mechanism of RH with NO is proposed in this study. The probe shows good stability over a broad pH range (pH>4). Furthermore, fluorescence intensity of RH displays an excellent linearity to the NO concentration and the detection limit is as low as 20 nM. A 1000-fold fluorescence turn-on from a dark background was observed. Moreover, the selectivity study indicated that the fluorescence intensity increasing in the presence of NO was significantly higher than those of other reactive oxygen/nitrogen species. In exogenously generated NO detection study, clear intracellular red fluorescence was observed in the presence of S-nitroso-N-acetyl-D,L-penicillamine (SNAP, a kind of NO releasing agent). In endogenously generated NO detection study, increasing incubation time of RH with lipopolysaccharied (LPS) pre-treated cells could obtain a highly fluorescent cell image. These cell imaging results demonstrated that RH can efficiently penetrate into Raw 264.7 cells and be used for detection of exogenously and endogenously generated nitric oxide.

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.

A water soluble and fast response fluorescent turn-on copper complex probe for H2S detection in zebra fish

According to the displacement method, herein we reported a water soluble copper complex [Cu(MaT-cyclen)2] as a fluorescent probe for the detection of H2S. For this, 1-((1-((10-methylanthracen-9-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,7,10-tetraazacyclododecane (MaT-cyclen) was synthesized first. To improve its solubility in aqueous media, sodium acetate group was introduced into 8-hydroxy-2-quinoline successfully. MaT-cyclen was chelated with Cu(II) to form [Cu(MaT-cyclen)2] complex, which displayed high sensitivity and selectivity for H2S over the other possible competitive substances on the basis of forming CuS. Meanwhile, [Cu(MaT-cyclen)2] displayed rapid response (<1min), well reversibility, lowest detection limit (205nM), and high sensitivity for recognizing H2S in aqueous solution. Furthermore, its potential utility for biological applications was confirmed by fluorescence imaging of H2S in live cells as well as in zebra fish.

Measurement of Poly(ethylene glycol) by Cell-Based Anti-poly(ethylene glycol) ELISA

Poly(ethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/alphaPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The alphaPEG cell-coated plate bound biotinylated PEG(5K) (CH(3)-PEG(5K)-biotin) and CH(3)-PEG(5K)-(131)I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5, 10, or 20 kDa) and a known amount of CH(3)-PEG(5K)-biotin allowed construction of a reproducible competition curve. The alphaPEG cell-based competition ELISA measured small molecules derivatized by PEG(2K), PEG(5K), PEG(10K), PEG(20K), and PEG(5K) at concentrations as low as 58.6, 14.6, 3.7, 3.7, and 14.6 ng/mL, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the alphaPEG cell-based competition ELISA. Finally, we show here that the alphaPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG(5K), comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH(3)-PEG(5K)-(131)I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.