Synthesis, Characterization, and Biological Properties of Cyanine-Labeled Somatostatin Analogues as Receptor-Targeted Fluorescent Probes

Abstract

We present the synthesis and characterization of the somatostatin receptor-specific peptide H(2)N-(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H(2)N-(D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.

Shedding light onto live molecular targets

“…A variety of reporter probes have been used for enhanced detection of early cancers, including somatostatin receptor-targeted probes [32][33][34], folate receptor-targeted agents [35], tumor cell-targeted agents [36][37][38][39], agents that incorporate into areas of calcification, bone formation or both [40], and agents being activated by tumor-associated proteases [41][42][43]. Many of these agents accumulate in (and thus enhance) tumors to a certain degree; however, FRET-based agents can yield particularly high tumor/background signal ratios because of their nondetectability in the native state.…”

Optical Imaging in Cancer Research: Basic Principles, Tumor Detection, and Therapeutic Monitoring

“…Nontargeted optical contrast agents typically accumulate in tumors because of increased tumor vascularization and/or leaky blood vessels [12,99,124,125] . The approved use of the NIR fluorescent dye indocyanine green (ICG) by the US Food and Drug Administration for non- imaging indications has allowed its adoption as a nonspecific and biocompatible optical imaging agent in humans.…”

Tumor Receptor Imaging

“…For small-molecule ligands with a high affinity and a low capacity for receptor binding, such as the ligands used for steroid receptor imaging, radionuclide detection methods are required; the most success has been associated with high-specific-activity PET compounds (Table 1) (33,56). Peptide ligand imaging, such as SSTR imaging, is somewhat more flexible, and some success has been achieved with optical imaging probes for SSTR expression (151)(152)(153)(154) or MRI probes for targets close to the vasculature (155). Studies of optical imaging of SSTRexpressing tumors in animal models have supported the feasibility of optical imaging of peptide ligands.…”