Novel cleavable cell-penetrating peptide-drug conjugates: synthesis and characterization.

We report the first drug conjugate with a negatively charged amphipathic cell-penetrating peptide. Furthermore, we compare two different doxorubicin cell-penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline-rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N-terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C-13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug-release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF-7 and HT-29 cell lines.