Amphiphilic Cyclic Cell-Penetrating Peptides Containing Tryptophan and Arginine as Anticancer Agents and Drug Delivery System

Abstract

Amphiphilic cyclic cell-penetrating peptides composed of an increasing number of alternative tryptophan (W) and arginine (R) were synthesized and evaluated as a molecular transporter and for their ability to deliver doxorubicin (Dox) and large molecular weight molecules, such as siRNA and proteins. We prepared a cyclic peptide containing alternative tryptophan (W) and arginine (R) residues and a lysine containing a free side chain amino group. Cyclic peptide [(WR)8WK βA] was conjugated through the free side chain amino group of β-alanine with Dox via a glutarate linker to afford [(WR)8WK]bA-Dox conjugate. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and 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. Furthermore, [(WR)8WKbA]-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. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. The intracellular release of free Dox was assessed in CCRF-CEM cell line. The experiment exhibited that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h.Cyclic peptide [WR]9 efficiently increased the intracellular delivery of siRNA to triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) by 15-folds and 9-folds, respectively, compared to siRNA alone. Flow cytometry (FACS) and confocal microscopy confirmed the uptake of Alexa-Fluor siRNA (AF-488 siRNA) in the presence of [WR]9 at different concentrations. The calculated binding affinity (BC50) of siRNA to the peptide was 1.9, indicating strong binding for siRNA to the peptide. As a result, the peptide siRNA combination displayed only minimal silencing efficiency for signal transducer and activator of transcription 3 (STAT3). Furthermore, the peptide showed concentration and time-dependent cargo uptake when physically mixed with green fluorescent protein (GFP) and red fluorescent protein (RFP)as model proteins. [WR]9 was also able to internalize therapeutically relevant histone protein at different ratios.