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Most cell penetrating peptides (CPPs) are unstructured and susceptible to proteolytic degradation. One alternative is to incorporate D‐chirality amino acids into unstructured CPPs to allow for enhanced uptake and intracellular stability. This work investigates CPP internalization using a series of time, concentration, temperature, and energy dependent studies, resulting in a three‐fold increase in uptake and 50‐fold increase in stability of D‐chirality peptides over L‐chirality counterparts. CPP internalization occurred via a combination of direct penetration and endocytosis, with a percentage of internalized CPP expelling from cells in a time‐dependent manner. Mechanistic studies identified that cells exported the intact internalized D‐chirality CPPs via an exocytosis independent pathway, analogous to a direct penetration method out of the cells. These findings highlight the potential of a D‐chirality CPP as bio‐vector in therapeutic and biosensing applications, but also identify a new expulsion method suggesting a relationship between uptake kinetics, intracellular stability, and export kinetics.

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.