ABSTRACT
In vivo blood vessels imaging is crucial to study blood vessels related diseases in real-time. For this purpose, fluorescent based imaging is one of the utmost techniques for imaging a living system. The discovery of a new near-infrared probe (CyA-B2) by screening chemical probe library in our previous report which showed the most specific binding on the blood capillaries of the 3D-tissue models give us interest to study more about the binding site of this probe to the surface of endothelial cells main component cell of blood capillaries. By studying the competition assays of CyA-B2 using several potential surface markers of endothelial cells found through the chemical database (ChEMBL) and manually selected, CD133 gave the lowest IC50 (half maximal inhibitory concentration) value. Hence, CD133 protein which is expressed on the endothelial cell membrane was postulated to be the binding site due to the suppression of CyA-B2 on the blood capillaries by the competition assays. Since, CD133 is also expressed on many types of cancer cells, it would be useful to use CyA-B2 as a bioprobe to monitor or diagnostic tumor growth.
ABSTRACT
Blood vessels are present in all of the organs, reflecting their importance for oxygen and nutrient delivery to the cells. Until now, no organic fluorophore has been reported for the live imaging of endothelium although the layer is the key to blood vessel functions. Here, the discovery of a blood vessel organic probe at near-infrared (NIR) wavelength range (BV-NIR) through an engineered blood capillary-based screening system, which is a more physiological model than a conventional cell culture condition, is reported. This selected Cy5 based probe shows the highest specific adsorption property out of 240 candidates on the endothelium and is equivalent to an anti-CD31 antibody in terms of intensity. The BV-NIR probe indicating strong and stable in vitro, ex vivo, and in vivo imaging of the endothelium even after histological immunostaining processes shows potential as a convenient tool for live imaging as well as for covisualization with a specific antibody.
Subject(s)
Fluorescent Dyes , High-Throughput Screening Assays , Spectroscopy, Near-Infrared/methodsABSTRACT
Conventional microarray analysis usually deals with the monolayer or two-dimensional (2D) assays for the high-throughput screening applications. Even though these cell-based assays are effective for preliminary screening at least to have information on cytotoxicity, they do not adequately re-create the in vivo complexity of three-dimensional (3D) tissues. In this study, 3D-blood capillary models were constructed by using physiological collagen microfibers (CMF), which provide the extracellular matrix in the complex tissue. Micro-droplets of fibrin gels containing CMF, endothelial cells, and fibroblasts were cultured for five days in 48-wells plate to provide a medium-throughput system for screening applications. Blood capillaries networks were formed by optimizing the concentration of CMF used and the number of cells. Finally, this screening method was a powerful assay for the application on the selection of not only a specific chemical probe for blood capillary live-imaging, but also a drug, aptamer, and peptide with potential blood vessel targeting property.
