P-Sulfonatocalix[4]arene turns peptide aggregates into an efficient cell-penetrating peptide†

A novel cell-penetrating peptide (CPP) called FAM-Y₄R₄, with FAM as a fluorescent probe, was developed. Initially, we aimed to use Y₄ as a supramolecular host for water-insoluble drugs, with R₄ driving the complex into cells. However, an unexpected hurdle was discovered; the peptide self-assembled into amorphous aggregates, rendering it ineffective for our intended purpose. Molecular dynamics simulations revealed that intermolecular cation–π interactions between arginine and tyrosine caused this aggregation. By decorating the R₄ sidechains with p-sulfonatocalix[4]arene (CX4), we successfully dissolved most of the aggregates, significantly improved the peptide's solubility and enhanced the cell uptake with MCF7 and A549 cells via both direct penetration and endocytosis. The binding strength between CX4 and R₄, as well as the interaction between curcumin and tyrosines was quantified. Encouragingly, our results showed that FAM-Y₄R₄, with CX4, effectively delivered curcumin – as a model for poorly water-soluble drugs – into cells which exhibited potent anticancer activity. Using R₄/CX4 instead of the conventional R_7–9 oligoarginine-based CPP simplifies peptide synthesis and offers higher yields. CX4 shows promise for addressing aggregation issues in other peptides that undergo a similar aggregation mechanism.