Self-quenched-regioselectively addressable functionalized template-[cyclo-(RGD-d-Phe-Lys)] peptide-Cy5-fluorescence quencher QSY21

Self-quenched-regioselectively addressable functionalized template-[cyclo-(RGD-d-Phe-Lys)] peptide-Cy5-fluorescence quencher QSY21(RAFT-c-(-RGDfK-)-Cy5-SS-Q) is an integrin-targeted molecular imaging agent developed for near-infrared (NIR) fluorescence imaging of tumor vasculature and tumor angiogenesis (1). Cy5 is a fluorophore with a maximal absorption (Abs) wavelength of 649 nm and a maximal emission (Em) wavelength of 670 nm (2). The molar extinction coefficient (є) is 250,000 M−cm−, and the quantum yield (QY) is >0.28. Cellular survival, invasion, and migration control embryonic development, angiogenesis, tumor metastasis, and other physiologic processes (3, 4). Among the molecules that regulate angiogenesis are integrins, which comprise a superfamily of cell adhesion proteins that form heterodimeric receptors for extracellular matrix (ECM) molecules (2, 3). These transmembrane glycoproteins consist of two noncovalently associated subunits, α and β, which are assembled into at least 24 α/β pairs. Several integrins, such as integrin αβ, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin αβ receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin αβ receptor is generally not found in normal tissue, but it is strongly expressed in vessels with increased angiogenesis, such as tumor vasculature. It is significantly unregulated in certain types of tumor cells and in almost all tumor vasculature. Molecular imaging probes carrying the RGD motif that binds to integrin αβ can be used to image tumor vasculature and evaluate angiogenic response to tumor therapy (4-6). Various RGD peptides in both linear and cyclic forms have been developed for binding to integrin αβ (7). Optical imaging utilizes light photons emitted from bioluminescence and fluorescence probes (5). Depth penetration is one major limiting factor in optical imaging. Currently, optical imaging has wide applications in small animal studies but only limited applications in large animal and human studies (8). NIR fluorescence imaging (light range, 650−900 nm) has the advantages of relatively higher tissue penetration and lower autofluorescence from nontarget tissue. NIR fluorescent dyes are conjugated RGD peptides such as Cy5.5-c(RGDyK), Cy5.5-c(RGDfK), and Cyp-RGD, which have been shown to visualize subcutaneously implanted integrin αβ−positive tumors (6, 9-11). Improved biophysical and pharmacodynamic properties of these RGD peptides appear to greatly augment their performance as molecular imaging probes (12). Multivalent presentation appears to be one of the possible approaches that can improve these properties. Boturyn et al. (13) generated a versatile molecular RAFT platform with a cyclic decapeptide [c(-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-)] that forms a ring with two faces. The RAFT platform is used as a suitable scaffold to independently and separately direct the cyclopentapeptide ligands and the reporter groups. The upper face is linked to four copies of the c(RGDfK) peptide for integrin αβ targeting, and the bottom face is linked to Cy5 for NIR imaging. Garanger et al. (14) reported that Cy5-RAFT-c(-RGDfK-) efficiently accumulated into tumors in nude mice. Jin et al. (12) showed that Cy5-RAFT-c(-RGDfK-) could allow optical imaging of subcutaneous and intraperitoneal tumors in nude mice. Based on the concept of “smart-probes” initially described by Weissleder et al. (15), Jin et al. (1) further improved this probe by linking a fluorescence quencher (QSY21) to Cy5 via a disulfide bond (-SS-) to produce RAFT-c(-RGDfK-)-Cy5-SS-Q. This activatable optical probe does not fluoresce unless it is activated by reduction of the disulfide bond during integrin αβ-mediated internalization.