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H-type dimer formation of fluorophores: a mechanism for activatable, in vivo optical molecular imaging
Mikako Ogawa1, Nobuyuki Kosaka, Peter L Choyke
1Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1088, USA.
Abstract:
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 dimers, even at low concentrations, but could be fully dequenched upon dissociation of the dimers 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.
Insights
Fluorophore dimerization enables "smart" probes for sensitive cancer imaging. This mechanism activates fluorescence at the target, improving visualization in vivo.
Area of Science:
- Biomedical Engineering
- Molecular Imaging
- Cancer Diagnostics
Background:
- Activatable "smart" probes offer sensitive and specific cancer detection via targeted fluorescence.
- Fluorophore dimerization and quenching are known in concentrated solutions.
- Hypothesis: Dimerization and quenching occur at low concentrations after protein conjugation.
Purpose of the Study:
- To investigate fluorophore dimerization and quenching at low concentrations after conjugation to targeting proteins.
- To develop fluorescence activatable probes for in vivo molecular imaging.
- To demonstrate fluorescence activation for enhanced cancer visualization.
Main Methods:
- Conjugating Rhodamine derivatives (R6G, TAMRA) to avidin and trastuzumab.
- Assessing H-type dimer formation and fluorescence quenching.
- Administering avidin-TAMRA and avidin-Alexa488 probes in mice with ovarian metastases.
- Performing in vivo fluorescence endoscopic molecular imaging.
Main Results:
- Rhodamine derivatives formed H-type dimers at low concentrations after conjugation.
- Dimerization led to fluorescence quenching, which was reversible upon monomer dissociation.
- Avidin-TAMRA probes clearly visualized tumors with low background fluorescence in mice.
- Avidin-Alexa488 probes resulted in high background fluorescence.
Conclusions:
- H-dimer formation is a viable mechanism for fluorescence quenching in activatable probes.
- This approach enhances in vivo molecular imaging sensitivity and specificity for cancer detection.
- Developed probes show potential for improved endoscopic visualization of peritoneal metastases.
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