Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16

Abstract

The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 μM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 μM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 μM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells.

Poly(2‐propylacrylic acid)/poly(lactic‐co‐glycolic acid) blend microparticles as a targeted antigen delivery system to direct either CD4+ or CD8+ T cell activation

“…These CD8 + T cells are capable of specifically recognizing malignant cells or cells infected with pathogens, leading to the activation and proliferation of effector cells which then exert cytotoxic functions to eliminate compromised cells. As such, various mechanism for endosomal membrane disruption have been proposed in the last decade, including strategies utilizing ultrasound, enzymatic degradation, pH, and light . In addition to these efforts, biodegradable polymeric delivery systems have attracted significant attention due to their loading capability, low toxicity, and ease of use.…”

Novel delivery approaches for cancer therapeutics

“…On the other hand, an arginine analogue with 308 base pair (BP308) conjugated to CLB (CLB-BP308) had an IC 50 of 25.5µM against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines compared to CLB alone which had an IC 50 of 73.7µM. A significant improvement in activity of CLB was evidenced when conjugated to either at the N or C-terminal of B16 [41]. A number of small molecular weight peptides have also been applied to deliver targeted MNP (see section: Magnetic nanoparticles) …”

Delivering aminopyridine ligands into cancer cells through conjugation to the cell-penetrating peptide BP16

“…Recently, we described the undecapeptide BP16 as a CPP able to favor the translocation of biologically active compounds across the cell membrane. 30,31 Based on this, herein we designed peptide conjugates resulting from the combination of the Me2 PyTACN or the (S,S′)-BPBP ligands with BP16.…”

“…Previous studies revealed the importance of evaluating the influence of the position of a drug in a peptide sequence on its activity. 30,[47][48][49] Thus, we first designed conjugates PyTACN-BP16 (BP341), BP16-PyTACN (BP342), BPBP-BP16 (BP343) and BP16-BPBP (BP344), incorporating the Me2 PyTACN or the (S,S′)-BPBP ligand at either the N-or the C-terminal end of BP16 (Fig. 1).…”

“…This fact points out that the position of a cargo in a peptide sequence influences the biological activity of the resulting conjugate, constituting a crucial factor in the design of biologically active peptide conjugates. 30,[47][48][49] The higher activity of the BPBP-metal binding peptides compared to the Me2 PyTACN conjugates prompted us to focus next studies on sequences bearing the former ligand. Remarkably, the incorporation of the cathepsin B cleavable sequence Gly-Phe-Leu-Gly in conjugates BP343 and BP344 resulted in metal binding peptides BP347 and BP348, respectively, with an excellent activity (IC 50 = 5.0-11.7 μM).…”