Cy5.5-Single-chain Cys-tagged vascular endothelial growth factor-121

Optical fluorescence imaging is increasingly used to observe biological functions of specific targets (1, 2) in small animals. However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging in small animals. Vascular endothelial growth factor (VEGF) consists of at least six isoforms with various numbers of amino acids (121, 145, 165, 183, 189, and 206 amino acids) produced through alternative splicing (3). VEGF, VEGF, and VEGF are the forms secreted by most cell types and are active as homodimers linked by disulfide bonds. VEGF does not bind to heparin like the other VEGF species (4). VEGF is a potent angiogenic factor that induces proliferation, sprouting, migration, and tube formation of endothelial cells. There are three high-affinity tyrosine kinase VEGF receptors on endothelial cells (VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; and VEGFR-3, Flt-4). Several types of non-endothelial cells such as hematopoietic stem cells, melanoma cells, monocytes, osteoblasts, and pancreatic β cells also express VEGF receptors (3). VEGF receptors were found to be overexpressed in various tumor cells and tumor-associated endothelial cells (5). Inhibition of VEGF receptor function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (6, 7). Radiolabeled VEGF has been developed as a tracer for imaging solid tumors and angiogenesis in humans (8-10). Cys-tag, a fusion tag comprising 15 amino acids, was developed for site-specific conjugation the free sulfhydryl group of Cys. Backer et al. (11) prepared a Cys-tagged vector of VEGF by cloning two single-chain fragments (amino acid sequence 3–112) of VEGF joining head-to-tail to express as scVEGF, which can be labeled as Cu-1,4,7,10-tetraazacyclododecane--tetraacetic acid (DOTA)-scVEGF (Cu-DOTA-scVEGF), Tc-hydrazinonicotinic acid (HYNIC)-scVEGF (Tc-HYNIC-scVEGF), and Cy5.5-scVEGF for imaging VEGFR expression to study tumor angiogenesis (12). Cy5.5 is a NIR fluorescent dye with an absorbance maximum at 675 nm and an emission maximum at 694 nm with a high extinction coefficient of 250,000 Mcm according to measurement. Cy5.5-scVEGF is being developed for NIR fluorescence imaging of VEGFR-2 in tumor vasculature.