Preparation and cellular uptake of bicyclic-peptide cargo clicked to cell penetrating peptides

Cell penetrating peptides (CPPs) are frequently used to deliver peptide cargo into cells where they can exert their biological activity. Their success, however, can be dependent on the cargo, the cell type, and other variables of peptide application. Here we describe the preparation of a bicyclic peptide inhibitor of the Grb7 cancer target and its conjugation via click chemistry to FITC-labeled derivatives of the classic CPP Penetratin, as well as the equivalent with an additional nuclear localization signal (NLS). We discovered that uptake of the cargo-Penetratin into the breast cancer cell line MDA-MB-231 was slow and led to limited cytosolic distribution. However, the addition of the NLS greatly enhanced the delivery leading to both cytosolic and nuclear distribution.