D-Cys-D-Asp-Gly-Tyr(3-NO)-Gly-ProOH-Asn-D-Cys-biotin-streptavidin-Cy5.5

Optical fluorescence imaging is increasingly used to obtain biological functions of specific targets small animals (1, 2). 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 a 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. Integrins are a family of cell surface heterodimeric glycoproteins that mediate diverse biological events involving cell–cell and cell–matrix interactions (3). They consist of an α and a β subunit, and they are important for cell adhesion and signal transduction. The αβ integrin plays an important role in normal lung, kidney, cerebral cortical, and epithelial development (4). On the other hand, it affects tumor growth, tumor invasiveness, and metastasis, as the α integrin is strongly expressed on tumor cells (5, 6). D-Cys-D-Asp-Gly-Leu-Gly-ProOH-Asn-D-Cys (LXY1), a cyclic peptide, was identified to bind to α integrin on human ovarian cancer cells using “one-bead one compound” combinatorial libraries (7, 8). LXY1 was conjugated with Cy5.5 biotin-streptavidin (SA) to study biodistribution of the tracer in tumor-bearing mice. LXY1-biotin-SA-Cy5.5 was found to have a high specific accumulation in α-positive human glioblastoma U-87MG cells in nude mice (9). Another LXY1 analog, D-Cys-D-Asp-Gly-Tyr(3-NO)-Gly-ProOH-Asn-D-Cys (LXY2), was identified to exhibit a higher binding affinity than LXY1 (10). LXY2 was conjugated with Cy5.5 biotin-SA to study biodistribution of the tracer in tumor-bearing mice. LXY2-biotin-SA-Cy5.5 was found to have a high specific accumulation in α-positve human breast adenocarcinoma MDA-MB-231 cells in nude mice. Cy5.5 is a NIR fluorescence dye with an absorbance maximum at 675 nm and an emission maximum at 694 nm with a high extinction coefficient of 250,000 Mcm.