Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets in small animals (1-3). 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 natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive complement to radionuclide imaging in small animals.
Vascular endothelial growth factor (VEGF) consists of at least six isoforms of various numbers of amino acids (121, 145, 165, 183, 189, and 206 amino acids) produced through alternative splicing (4). VEGF121, VEGF165, and VEGF189 are the forms secreted by most cell types and are active as homodimers linked by disulfide bonds. VEGF121 does not bind to heparin like the other VEGF species (5). 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 (VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; and VEGFR-3, Flt-4) on endothelial cells. Several types of non-endothelial cells such as hematopoietic stem cells, melanoma cells, monocytes, osteoblasts, and pancreatic β cells also express VEGF receptors (4).
VEGF receptors were found to be overexpressed in various tumor cells and tumor-associated endothelial cells (6). Inhibition of VEGF receptor function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (7, 8). Radiolabeled VEGF tracers have been developed for imaging solid tumors and angiogenesis in humans (9-11). Wang et al. (12) fused the Avi peptide (14 amino acids) to the C-terminus of VEGF121 to allow site-specific biotinylation of the epsilon amine group of a central lysine residue of Avi. The binotinylated VEGF121-Avi (VEGF121-Avib) was able to form a tight complex with streptavidin-IRDye800 (SA800) as VEGF121-Avib-SA800 for NIR imaging of VEGFR expression in vivo to study tumor angiogenesis.