Multivalent Fluorinated Nanorings for On-Cell F NMR.

The design of imaging agents with a high fluorine content is necessary for overcoming the challenges of low sensitivity in 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 F NMR-based imaging tracer, in which case a CSAN-compatible epidermal growth factor receptor (EGFR)-targeting protein E-dimeric dihydrofolate (E-DD) was bioconjugated to a highly fluorinated peptide. Despite good F NMR performance in aqueous solutions, a limited signal was observed in cell-based F NMR using this monomeric construct, motivating further design. Here, we design several new E-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, , displayed an optimal spectral performance with high affinity and specificity for EGFR-overexpressing cells. To further improve the fluorine content, we next engineered monomeric into CSAN, . With an approximate eightfold increase in the fluorine content, multivalent maintained high cellular specificity and optimal F NMR spectral behavior. Importantly, the first cell-based F NMR spectra of 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 MRI molecular imaging applications.