A Target Cell–Specific Activatable Fluorescence Probe for In vivo Molecular Imaging of Cancer Based on a Self-Quenched Avidin-Rhodamine Conjugate

Abstract

A target cell-specific activation strategy for improved molecular imaging of peritoneal implants has been proposed, in which fluorophores are activated only in living targeted cells. A current example of an activatable fluorophore is one that is normally self-quenched by attachment to a peptide backbone but which can be activated by specific proteases that degrade the peptide resulting in ''dequenching.'' In this study, an alternate fluorescence activation strategy is proposed whereby self-quenching avidin-rhodamine X, which has affinity for lectin on cancer cells, is activated after endocytosis and degradation within the lysosome. Using this approach in a mouse model of peritoneal ovarian metastases, we document target-specific molecular imaging of submillimeter cancer nodules with minimal contamination by background signal. Cellular internalization of receptor-ligand pairs with subsequent activation of fluorescence via dequenching provides a generalizable and highly sensitive method of detecting cancer microfoci in vivo and has practical implications for assisting surgical and endoscopic procedures. [Cancer Res 2007;67(6):2791-9]

Activatable aptamer probe for contrast-enhanced in vivo cancer imaging based on cell membrane protein-triggered conformation alteration

“…In the context of molecular imaging, a variety of such probes have been developed (14)(15)(16)(17)(18)(19)(20)(21). Typical mechanisms include enzymatic cleavage of a quencher group from the fluorescence moiety using cancer-related enzymes (15)(16)(17)(18), intracellular degradation of fluorescence-quenched protein against cancer cell receptors (19,20), and surrounding-sensitized fluorescence labels conjugated to macromolecular ligands selectively endocytosed by cancer cells (8).…”

“…Typical mechanisms include enzymatic cleavage of a quencher group from the fluorescence moiety using cancer-related enzymes (15)(16)(17)(18), intracellular degradation of fluorescence-quenched protein against cancer cell receptors (19,20), and surrounding-sensitized fluorescence labels conjugated to macromolecular ligands selectively endocytosed by cancer cells (8). However, these mechanisms cannot be utilized to develop aptamer-based activatable probes.…”

A Self-Quenched Galactosamine-Serum Albumin-RhodamineX Conjugate: A “Smart” Fluorescent Molecular Imaging Probe Synthesized with Clinically Applicable Material for Detecting Peritoneal Ovarian Cancer Metastases

“…Because GmSA synthesis involves the conjugation of 23 Dgalactosamines reacted with carboxyl groups rather than amino groups on an albumin molecule (7), there are multiple binding sites for the D-galactose receptor and the molecule has a favorably high isoelectric point (7,16). Moreover, activation via dequenching of the rhodamineX after cellular internalization provides a generalizable platform for targeted fluorescent probes by exchanging the targeting moiety (5). We used the D-galactose receptor as a target in this study because it is expressed in a wide range of cancers, which metastasize i.p.…”

“…For instance, Weissleder et al have developed a series of agents activated by tumor-associated proteases [e.g., cathepsin D and matrix metalloproteinase-2 (MMP-2)], which function to dequench the complex resulting in a multifold increase in fluorescence (9 -11). As an alternative, a self-quenching avidin-3 rhodamineX probe was synthesized, which binds D-galactose receptor on cancer cells and then is internalized (5). Intracellular endosomal or lysosomal dissociation of the avidin-3 rhodamineX complex leads to ''dequenching'' of the 3 rhodamineX molecules, and hence, activation of the fluorophore occurs but only within live targeted cells and not in surrounding normal cells that lack the D-galactose receptor (5).…”

“…As an alternative, a self-quenching avidin-3 rhodamineX probe was synthesized, which binds D-galactose receptor on cancer cells and then is internalized (5). Intracellular endosomal or lysosomal dissociation of the avidin-3 rhodamineX complex leads to ''dequenching'' of the 3 rhodamineX molecules, and hence, activation of the fluorophore occurs but only within live targeted cells and not in surrounding normal cells that lack the D-galactose receptor (5). Although this approach is highly sensitive for detecting cancer microfoci in vivo, the immunogenicity of avidin has been an obstacle to its translation into clinical studies (13,14).…”