Multivalent Fluorinated Nanorings for On-Cell 19 F NMR.

The design of imaging agents with a high fluorine content is necessary for overcoming the challenges of low sensitivity in 19 F magnetic resonance imaging (MRI)-based molecular imaging. Chemically self-assembled nanorings (CSANs) provide a strategy to increase the fluorine content through multivalent display. We previously reported an 19 F NMR-based imaging tracer, in which case a CSAN-compatible epidermal growth factor receptor (EGFR)-targeting protein E 1 -dimeric dihydrofolate (E 1 -DD) was bioconjugated to a highly fluorinated peptide. Despite good 19 F NMR performance in aqueous solutions, a limited signal was observed in cell-based 19 F NMR using this monomeric construct, motivating further design. Here, we design several new E 1 -DD proteins bioconjugated to peptides of different fluorine contents. Flow cytometry analysis was used to assess the effect of variable fluorinated peptide sequences on the cellular binding characteristics. Structure-optimized protein, RTC-3 , displayed an optimal spectral performance with high affinity and specificity for EGFR-overexpressing cells. To further improve the fluorine content, we next engineered monomeric RTC-3 into CSAN, η-RTC-3 . With an approximate eightfold increase in the fluorine content, multivalent η-RTC-3 maintained high cellular specificity and optimal 19 F NMR spectral behavior. Importantly, the first cell-based 19 F NMR spectra of η-RTC-3 were obtained bound to EGFR-expressing A431 cells, showing a significant amplification in the signal. This new design illustrated the potential of multivalent fluorinated CSANs for future 19 F MRI molecular imaging applications.