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 [R₅K]W₇A and linear peptide R₅KW₇A were conjugated with Dox through a glutarate linker to afford [R₅K]W₇A-Dox and R₅KW₇A-Dox conjugates to generate Dox derivatives. Alternatively, [R₅K]W₇C was conjugated with Dox via a disulfide linker to generate [R₅K]W₇C–S–S-Dox conjugate, where S–S is a disulfide bond. Comparative antiproliferative assays between conjugates [R₅K]W₇A-Dox, [R₅K]W₇C–S–S-Dox, linear R₅KW₇A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R₅K]W₇A-Dox conjugate was 2-fold more efficient than R₅KW₇A-Dox, and [R₅K]W₇C–S–S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R₅K]W₇A-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 [R₅K]W₇A-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 [R₅K]W₇A-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. [R₅K]W₇A-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 LC₅₀ of 2.3 and 4.3 μM, respectively) compared to free Dox (LC₅₀ of 36–41 μM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R₅K]W₇A-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 [R₅K]W₇A-Dox.

多柔比星 (Dox) 作为有效的化学治疗剂用于治疗乳腺癌、白血病和淋巴瘤。然而，由于固有和获得性耐药性以及潜在致命心脏毒性风险增加 8 倍，Dox 的使用受到限制。混合环状线性肽 [R ₅ K]W ₇ A 和线性肽 R ₅ KW ₇ A 通过戊二酸接头与 Dox 缀合，得到 [R ₅ K]W ₇ A-Dox 和 R ₅ KW ₇ A-Dox 缀合物生成 Dox 衍生物。或者，[R ₅ K]W ₇ C 通过二硫键与 Dox 结合生成 [R ₅ K]W ₇C–S–S-Dox 共轭，其中 S–S 是二硫键。_{[R 5} K]W ₇ A-Dox、[R ₅ K]W ₇ C–S–S-Dox、线性 R ₅ KW ₇ A-Dox、肽的相应物理混合物和 Dox之间的比较抗增殖试验在正常细胞和癌细胞中进行。[R ₅ K]W ₇ A-Dox 偶联物抑制人白血病细胞增殖的效率是 R ₅ KW ₇ A-Dox 和 [R ₅ K]W ₇ C-S-S-Dox 偶联物的 2 倍细胞（CCRF-CEM）。因此，混合循环线性[R ₅ K]W ₇选择 A-Dox 偶联物进行进一步研究，并以 5 的浓度抑制 CCRF-CEM (84%)、卵巢腺癌 (SK-OV-3, 39%) 和胃癌 (AGS, 73%) 的细胞活力孵育 72 小时后的 μM，与 Dox (5 μM) 功效（CCRF-CEM (85%)、SK-OV-3 (33%) 和 AGS (87%)）相当。虽然 [R ₅ K]W ₇ A-Dox 对癌细胞的活力有显着影响，但它对正常肾脏 (LLC-PK1, 5-7%) 和心脏细胞 (H9C2, <9%) 在浓度为 5-10 μM（与 5 μM 的游离 Dox 相比，分别将肾脏和心脏细胞的活力降低 85% 和 44%）。荧光显微镜图像与细胞毒性研究一致，表明循环线性 [R ₅ K]W的摄取量最小_{H9C2 细胞中的7} A-Dox (5 μM)。相比之下，Dox (5 μM) 显示出显着的摄取，降低了细胞活力，并在 24 小时后改变了细胞的形态。[R ₅ K]W ₇ A-Dox 对 Dox 抗性细胞 MDA231R 和 MES-SA/MX2 的活性分别高出 16 倍和 9.5 倍（50% 细胞死亡的致死剂量或 LC ₅₀分别为 2.3 和 4.3 μM ) 与游离 Dox 相比（LC ₅₀分别为 36–41 μM）。这些数据以及从细胞活力测试中获得的结果表明，[R ₅ K]W _{7的效率相当}A-Dox 可在白血病、卵巢癌细胞和胃癌细胞中释放 Dox，显着降低正常肾脏 LLC-PK1 和心脏 H9C2 细胞的毒性，并显着提高抗 Dox 细胞的效率。许多内吞作用抑制剂不影响[R ₅ K]W ₇ A-Dox 的细胞摄取。