Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity.

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [RK]WA and linear peptide RKWA were conjugated with Dox through a glutarate linker to afford [RK]WA-Dox and RKWA-Dox conjugates to generate Dox derivatives. Alternatively, [RK]WC was conjugated with Dox via a disulfide linker to generate [RK]WC-S-S-Dox conjugate, where S-S is a disulfide bond. Comparative antiproliferative assays between conjugates [RK]WA-Dox, [RK]WC-S-S-Dox, linear RKWA-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [RK]WA-Dox conjugate was 2-fold more efficient than RKWA-Dox, and [RK]WC-S-S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [RK]WA-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 μM after 72 h of incubation, which was comparable to Dox (5 μM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [RK]WA-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5-7%) and heart cells (H9C2, <9%) at concentrations of 5-10 μM (compared to free Dox at 5 μM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [RK]WA-Dox (5 μM) in H9C2 cells. In comparison, Dox (5 μM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [RK]WA-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC of 2.3 and 4.3 μM, respectively) compared to free Dox (LC of 36-41 μM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [RK]WA-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [RK]WA-Dox.