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Biomedical subjects

Wellington Pham

Publications and source records attributed to Wellington Pham.

9 recordsLinked to original sources
PubMed2006

Imaging farnesyl protein transferase using a topologically activated probe.

We report on the development and application of a "smart" activatable peptide-based probe for detection of Ras-related farnesyl protein transferase (FPT). Upon farnesylation by FPT, the probe was brought close to a hydrophobic milieu and as a consequence emitted fluorescent light that could be detected by several media, such as fluorescence microscopy, a plate reader, and an optical imaging system. A FPT activity assay confirmed the specificity of the probe (IC50 = 1.2 muM) for FPT compared to that of the native peptide (IC50 = 0.17 muM). In addition, the probe has remarkable binding constant, Kd = 26 nM. The specificity of enzyme activation was proved in pure enzyme assays as well as in cell-based assays. Furthermore, the fluorescent enhancement of the probe was 30-fold stronger than the control peptide in a live cell assay.

Alkyl and Aryl Transferases↗
PubMed2006

In vivo imaging of tumor response to therapy using a dual-modality imaging strategy.

In vivo assessment of the outcome of cancer therapy is hampered by the paucity of imaging probes that target tumors specifically and noninvasively. The importance of such probes increases with the continuous development of chemotherapeutics and the necessity to evaluate their effectiveness in a clinical setting. We have recently reported on a dual-modality imaging probe specifically targeting the underglycosylated mucin-1 tumor-specific antigen (uMUC-1), which is one of the early hallmarks of tumorigenesis in a wide variety of tumors. This probe consists of crosslinked superparamagnetic iron oxide nanoparticles (CLIO) for MR imaging, modified with Cy5.5 dye (for near infrared optical fluorescence imaging (NIRF)), and has peptides (EPPT), specifically recognizing uMUC-1, attached to the nanoparticle's dextran coat. In the present study, we demonstrated that this probe could not only detect orthotopically implanted preclinical models of adenocarcinomas but could also track tumor response to chemotherapy in vivo in real time. Considering the high cost associated with the development and testing of new cancer therapeutics and the need for accurate, noninvasive assessment of their effectiveness, we believe that the developed probe represents a valuable research tool relevant to clinical discovery.

Adenocarcinoma↗
PubMed2005

Crossing the blood-brain barrier: a potential application of myristoylated polyarginine for in vivo neuroimaging.

As basic neurological research continues to reveal novel targets for therapy, the need to deliver therapeutic agents across the blood-brain barrier (BBB) becomes increasingly important. If developed, delivery modules would bring targeting molecules across the BBB to their respective active sites. In addition, it would be highly advantageous if the bioavailability of these delivered agents could be monitored over time using non-invasive imaging techniques. Here, we describe a versatile delivery module based on a myristoylated polyarginine backbone, which crosses the BBB. Incorporation of the fatty acid group was achieved using a Schotten-Bauman reaction with quantitative yield, and the peptide was further synthesized by conventional solid phase peptide synthesis (SPPS). We report for the first time the in vivo distribution of the delivery module over time into mouse brain using near-infrared (NIR) fluorescence imaging. The fluorescent cargo was detected in vivo from 24-48 h post IV injection and was further characterized in perfused brains. Immunohistochemical staining of excised brain showed that the delivery module primarily accumulated in neurons with occasional localization in astrocytes and endothelial cells. We conclude that this approach can be used for the delivery of imaging probes and potentially targeted therapeutics across the BBB.

Animals↗
PubMed2004

Human myeloperoxidase: a potential target for molecular MR imaging in atherosclerosis.

Plaque rupture in atherosclerotic disease is the major cause of morbidity and correlates well with myeloperoxidase (MPO) secretion by activated neutrophils and macrophages in humans. We hypothesized that paramagnetic electron donor compounds that rapidly oxidize and polymerize in the presence of MPO could be designed to enable imaging of local MPO activity levels in arterial segments at risk. Several potential substrates for MPO were synthesized and tested. One lead compound consisting of a covalent conjugate of GdDOTA and serotonin (3-(2-aminoethyl)-5-hydroxyindole) was efficiently polymerized in the presence of human neutrophil MPO resulting in a 70-100% increase in proton relaxivity. As a result, we were able to demonstrate MPO activity in enzyme solutions and in a model tissue-like system. These studies suggest that activatable paramagnetic MR imaging agents can be used to directly image MPO activity.

Arteriosclerosis↗
PubMedDate not supplied

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↗
PubMedDate not supplied

Synthesis and application of a water-soluble near-infrared dye for cancer detection using optical imaging.

A novel water-soluble 2-[2-(2-chloro-3-{2-[3,3-dimethyl-5-sulfo-1-(4-sulfo-butyl)-3H-indol-2-yl]-vinyl}-cyclohex-2-enylidene)-ethylidene]-3,3-dimethyl-1-(4-sulfo-butyl)-2,3-dihydro-1H-indole-5-carboxylic acid (dye 2) was developed via an asymmetric approach. With an additional sulfonate group, the near-infrared feature of this dye exhibited a 2-fold increase in quantum yield compared to the previous generation. The current synthetic strategy provided a single carboxylic group as a handle for conjugation, thus allowing selectivity for bioconjugation. The stability of this dye was demonstrated by labeling peptides via solid-phase peptide chemistry. The in vivo optical imaging showed potential and broad applications of this dye in developing molecular-based beacons for cancer detection.

Animals↗
PubMedDate not supplied

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↗