CPProtectides: Rapid uptake of well-folded β-hairpin peptides with enhanced resistance to intracellular degradation

: Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors or drugs to intact cells. One limitation of CPPs is their rapid degradation by intracellular proteases. β-hairpin "protectides" have previously been demonstrated to be long-lived under cytosolic conditions due to their secondary structure. The goal of this work was to demonstrate that arginine-rich β-hairpin peptides function as both protectides and as CPPs. Peptides exhibiting a β-hairpin motif were found to be rapidly internalized into cells with their uptake efficiency dependent on the number of arginine residues in the sequence. Cellular internalization of the β-hairpin peptides was compared to unstructured, scrambled sequences and to commercially available, arginine-rich CPPs. The unstructured peptides displayed greater uptake kinetics compared to the structured β-hairpin sequences; however, intracellular stability studies revealed that the β-hairpin peptides exhibited superior stability under cytosolic conditions with a 16-fold increase in peptide half-life. This study identifies a new class of long-lived CPPs that can overcome the stability limitations of peptide-based reporters or bioactive delivery mechanisms in intact cells.