IRDye800CW-Cyclic albumin-binding domain (Ac-RLIEDICLPRWGCLWEDDK-NH)

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 (700–1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues. 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, 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 alternative to radionuclide imaging in small animals or with probes in close proximity to the target in humans (4, 5). 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. 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 tumors and endoscopic imaging of the pancreas and colon. The primary function of the lymphatic system is to drain ~10% of the interstitial fluid from small capillaries to lymphatic vessels through lymph nodes and finally to the venous system (6-10). Lymph nodes form a natural filter for the lymphatic drainage and prevent the possible migration of cancer cells from the lymphatic system into the body. Serum proteins and macromolecules can be taken up by the large openings in the lymphatic capillaries. However, an accumulation of protein molecules may impair lymphatic flow and cause lymphedema and tissue edema. For NIR fluorescence imaging, ICG has been used in lymphatic imaging because of its association with serum proteins. However, ICG is degraded in aqueous conditions with a half-life of ~20 h and is sensitive to light (half-life, 2.3 h). IRDye 800CW (IRDye800) is an indocyanine-type NIR fluorophore with peak absorption at 785 nm and peak excitation emission at 803 nm, and it is about four-fold brighter than ICG. Dennis et al. (11) identified the peptide Ac-RLIEDICLPRWGCLWEDD (SA21), which bound with high affinity to human and murine serum albumin. Davies-Venn et al. (12) prepared a cyclic version of SA21 called cyclic albumin-binding domain (Ac-RLIEDICLPRWGCLWEDDK-NH, cABD) and conjugated this with IRDye800 to form IRDye800-cABD for NIR fluorescence lymphatic imaging in mice.