Indocyanine green derivative 02-Glu-c(RGDyK)

Optical fluorescence imaging is increasingly 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 (650–900 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low background fluorescence, 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 a noninvasive complement to radionuclide imaging in small animals or with probes in close proximity to the target in humans (4). Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment (5-8). It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumor cells and endoscopic imaging of the pancreas and colon. Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (9). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The αβ integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (10-17). Expression of the αβ integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The peptide sequence Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αβ. The αβ antagonists are being studied as anti-tumor and anti-angiogenic agents (12, 16, 18). Various radiolabeled (such as F, Cu, Ga, and Tc) and NIR fluorescence-labeled (such as Cy5.5, Cy7, and Cypate) RGD peptides have been introduced for imaging of tumors and tumor angiogenesis (19). ICG derivative 02 (ICG-Der-02), a hydrophilic dye, contains one carboxyl functional group for covalent conjugation to the amino group of biomolecules. Cao et al. (20) conjugated ICG-Der-02 the α-amino group of Glu residue of Glu-c(RGDyK) (dimer) peptide to form ICG-Der-02-c(RGDyK) ICG-Der-02 was also conjugated to the Ɛ-amino group of the lysine residue of RGD (linear) and c(RGDyK) (monomer) to form ICG-Der-02-RGD and ICG-Der-02-c(RGDyK), respectively. The three ICD-Der-02-labeled conjugates were evaluated for NIR optical imaging in tumor-bearing mice.