Synthesis of doxorubicin cell‐penetrating peptide bioconjugates
Cell‐penetrating peptides, also known as protein transduction domain (PTD), have attracted interest as carriers for intracellular drug delivery. 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.
Heterobifunctional crosslinkers are extensively applied in drug modifications, and their utilization has proven to be very important for the attachment of diverse carriers, such as monoclonal antibodies, proteins, polymers, and peptides, to doxorubicin. Established conjugation technique is applied at the C‐13 keto group by hydrazones, due to their fast hydrolysis in acidic environment existing in biological compartments like endosomes and lysosomes. However, the insufficient stability of the doxorubicin hydrazone conjugates has been reported even at physiological pH (7.4), leading to the release of the free drug in the bloodstream. In order to overcome these difficulties, we have chosen a crosslinker capable of creating an oxime bond on doxorubicin’s ketone, due to the higher hydrolytic stability of the oxime group. Additionally, thiol‐containing carriers, like albumin proteins, have also been conjugated to anthracyclines utilizing functional groups that are highly specific for sulfhydryl groups, e.g. maleimides and pyridyl disulfides. The application of pyridyl disulfides is advantageous, because a disulfide bond between the linker and the cargo is formed, which can be reduced in the cytoplasm by glutathione to deliver the freight. Furthermore, pyridyl disulfides can serve as a protective group during synthesis to avoid undesired dimerization as well as an activating group for the thiol to facilitate disulfide formation. In contrast, maleimides react with a thiol via Michael addition; thus, a covalent bond is created that cannot be cleaved under physiological conditions. Therefore, we have selected a heterobifunctional crosslinker that contains a protected aminooxy group and pyridyl disulfide.