Multivalent Fluorinated Nanorings for On-Cell ¹⁹F NMR

The design of imaging agents with a high fluorine content is necessary for overcoming the challenges of low sensitivity in F-19 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 F-19 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 F-19 NMR performance in aqueous solutions, a limited signal was observed in cell-based F-19 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, eta-RTC-3. With an approximate eightfold increase in the fluorine content, multivalent eta-RTC-3 maintained high cellular specificity and optimal F-19 NMR spectral behavior. Importantly, the first cell-based F-19 NMR spectra of eta-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 F-19 MRI molecular imaging applications.