Effective cell penetration of negatively-charged proline-rich SAP(E) peptides with cysteine mutation

Most of cell penetrating peptides (CPPs) are rich in positively-charged amino acids but the cationic property may provoke possible problems in practical applications. In this study, we carefully substituted the hydrophobic amino acids in the SAP(E) sequence, a rare example of negatively-charged proline-rich CPP, with cysteine for enhancement of cell penetrating activity as well as reversible conjugation of cargo molecules. Most substituents showed almost negligible cell penetrating activity, but a cysteine substituent on the 7th valine (SAP(E)-7C) showed more improved cell penetrating activity than SAP(E). When treated to cells, the negatively-charged SAP(E)-7C exhibited much lower degree of co-localization with acidic endosomes or lysosomes compared to positively-charged TAT. SAP(E)-7C could significantly enhance the PTX efficacy on MDA-MB-231 cells by non-covalent complexation with PTX. As a proof-of-concept for covalent conjugation of cargo drugs, mercaptoethanol, a model drug, was conjugated to the cysteine residue of SAP(E)-7C via a disulfide bond, and the glutathione-dependent release from the conjugate was confirmed. The negatively-charged SAP(E)-7C with a cysteine handle can be a useful molecular module for the development of CPP-based drug delivery carrier.