H-Type Dimer Formation of Fluorophores: A Mechanism for Activatable, in Vivo Optical Molecular Imaging

In vivo molecular imaging with target-specific activatable smart probes, which yield fluorescence only at the intended target, enables sensitive and specific cancer detection. Dimerization and fluorescence quenching has been shown to occur in concentrated aqueous solutions of various fluorophores. Here, we hypothesized that fluorophore dimerization and quenching after conjugation to targeting proteins can occur at low concentration. This dimerization can be exploited as a mechanism for fluorescence activation. Rhodamine derivatives were conjugated to avidin and trastuzumab, which target D-galactose receptor and HER2/neu antigen, respectively. After conjugation, a large proportion of R6G and TAMRA formed H-type dinners, even at low concentrations, but could be fully dequenched upon dissociation of the dinners to monomers. To demonstrate the fluorescence activation effect during in vivo fluorescence endoscopic molecular imaging, a highly quenched probe, avidin-TAMRA, or a minimally quenched probe, avidin-Alexa488, was administered into mice with ovarian metastases to the peritoneum. The tumors were clearly visualized with avidin-TAMRA, with low background fluorescence; in contrast, the background fluorescence was high for avidin-Alexa488. Thus, H-dimer formation as a mechanism of fluorescence quenching could be used to develop fluorescence activatable probes for in vivo molecular imaging.