Pro-apoptotic peptide-polymer conjugates to induce mitochondrial-dependent cell death

The treatment of cancer using pro-apoptotic peptides requires efficient intracellular delivery to trigger mitochondrial disruption. When the widely used non-toxic heptapeptide dimer (D)(KLAKLAK)(2) is coupled with tumor targeting peptides that allow receptor-mediated internalization, it disrupts negatively charged membranes and induces mitochondrial-dependent apoptosis. Here, we describe the design, synthesis, and characterization of novel water-soluble HPMA copolymers bearing a multivalent display of NGR motifs that can target the pro-apoptotic drug (D)(KLAKLAK)(2) to CD13-overexpressing cells. (D)(KLAKLAK)(2) was attached to the polymeric backbone through an acid-sensitive hydrazone linker to facilitate intracellular drug release. The ability of an array of NGR motifs, with different structural conformations, to target several CD13 receptors and thus enhance the binding avidity and the pro-apoptotic activity of (D)(KLAKLAK)(2) in human cell lines was tested. HPMA copolymer conjugates bearing the dimeric (GNGRG)(2) and cyclic (CNGRC) NGR motifs exhibited preferential binding to CD13-overexpressing cells relative to the linear (GNGRG) targeting peptide. The binding of (D)(KLAKLAK)(2) to the polymer facilitated rapid internalization and translocation into endosomal/lysosomal compartments. The polymer-bound (D)(KLAKLAK)(2) markedly increased the cytotoxicity and the pro-apoptotic activity of the drug, relative to free (D)(KLAKLAK)(2) and the targeted pro-apoptotic peptide (GNGRGG-(D)(KLAKLAK)(2)) in CD13(R) cells. This research is the first attempt to apply polymer-conjugates with NGR motifs as targeting ligand to deliver the pro-apoptotic (D)(KLAKLAK)(2) sequence into CD13-overexpressing cells and thereby to promote the death of cancer cells. Copyright (C) 2010 John Wiley & Sons, Ltd.