[(WR)8WKβA]-Doxorubicin Conjugate: A Delivery System to Overcome Multi-Drug Resistance against Doxorubicin

Abstract

Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR)8WKβA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR)8WKβA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 μM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 μM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR)8WKβA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 μM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t1/2) for [(WR)8WKβA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it.

Cyclic and Linear Peptides Containing Alternate WW and RR Residues as Molecular Cargo Delivery Tools

“…Our previous studies showed that cyclic CPPs, [WR] 4 and [WR] 5 , with alternation of tryptophan (W) and arginine (R) residues, increased the cellular uptake of different compounds such as phosphopeptides, doxorubicin, ,,, paclitaxel, camptothecin, curcumin, , anti-HIV drugs, , gadolinium nanoparticles, selenium nanoparticles, gold nanoparticles, , and oligonucleotides. , Cyclic CPPs containing alternate W and R residues demonstrated improved cellular uptake delivery when compared to linear CPPs . Moreover, the uptake of both [WR] 4 and [WR] 5 was not dependent on the endocytosis pathway. , …”

“…They can transport small molecules that cannot penetrate through the cell membrane inside the cell. − These peptides use energy-independent and energy-dependent pathways to penetrate the cell membrane . CPPs are used in different ways to mediate cellular cargo delivery, which are covalent conjugating with the cargo − or creating stable noncovalent − complexes. Some of these peptides can penetrate the cell without generating significant cytotoxicity.…”

Amphiphilic Cell-Penetrating Peptides Containing Arginine and Hydrophobic Residues as Protein Delivery Agents

“…However, we noticed cell morphology changed when 10 µM of [WR] 9 was used, as shown in Figure 6 . We have previously shown that [WR] 9 had minimal toxicity at a concentration of 10 µM in SK-OV-3 and MDA-MB-231 after 24 h of incubation [ 52 ]. Herein, as shown in Figure 2 and Figure 3 ( Tables S1 and S2 ), the cell viability was ≥90%.…”

Redox-Responsive Disulfide Cyclic Peptides: A New Strategy for siRNA Delivery

“…This could potentially be due to the mechanism of internalization. We recently reported that internalization of doxorubicin via conjugation of the drug to hybrid [R 5 K]­W 7 A and cyclic [(W R ) 8 WKβA] peptides is endocytosis-independent. While we have not examined the mechanism of internalization of siRNA via H4 and C4, these results might indicate an endocytosis-independent mechanism.…”