The peptide-drug conjugates caused much attention currently. The purpose of present study was to elucidate the possible synergistic effect between ligand peptide and stimuli-responsive linkage in amphiphilic peptide-drug conjugates (APDCs) with different linkers. Especially, the superiority of each strategy as well as the synergistic effect between them was carefully investigated via the parallel comparisons of the three systems throughout of the whole study. Here, we synthesized three APDCs, namely, cRGD-SS-DOX (RSSDOX), cRGD-S-DOX (RSDOX) and cRGD-VC-DOX (RVCDOX), using doxorubicin (DOX) as a model cytotoxic agent, cRGDfC as a homing peptide, and reduction cleavable disulfide (SS), noncleavable single thioether (S) or cathepsin B cleavable valine-citrulline dipeptide (VC) as linker. The APDCs showed high drug loading capacity and they were evaluated in vitro in the integrin αvβ3-overexpressing B16 cells and in vivo in tumor-bearing C57BL/6 mice. Endocytosis mechanism assay demonstrated that three types of APDCs internalized into cells through adynamin and actin depolymerizing-mediated pathway following receptor-mediated endocytosis. Notably, RSDOX or RVCDOX induced stronger antitumor efficacy, which depended on their cellular uptake levels, intracellular trafficking and the colocalization rates with lysosomes. The in vivo efficacy of RSDOX or RVCDOX was 1.4–1.7 fold of free DOX and 1.7–2.0 fold of RSSDOX, respectively. In addition, RSDOX or RVCDOX demonstrated acceptable system, tissue and blood compatibility. The compromised efficacy of RSSDOX might be due to the generation of DOX-SH during degradation of prodrug, but not DOX. Taken together, our studies suggest that certain type of APDCs can significantly decrease the toxicity of free DOX and improve therapy outcome, which provides insight for the design of peptide-drug conjugates integrating ligand peptide and stimuli-responsive linkage.

肽-药物缀合物目前引起了很多关注。本研究的目的是阐明在具有不同接头的两亲性肽-药物缀合物（APDC）中，配体肽与刺激响应性连接之间可能的协同作用。特别是，在整个研究过程中，通过对三个系统的平行比较，仔细研究了每种策略的优势以及它们之间的协同效应。在这里，我们使用阿霉素（DOX）作为模型细胞毒剂，将cRGDfC作为归巢肽和还原型可裂解的二硫键（SS），不可裂解的单个硫醚（S）或组织蛋白酶B的可裂解缬氨酸-瓜氨酸二肽（VC）作为接头。APDCs具有很高的载药量，并在过表达整联蛋白αvβ3的B16细胞中进行了体外评估，并在荷瘤的C57BL / 6小鼠中进行了体内评估。内吞作用机制测定表明，三种类型的APDCs在受体介导的内吞作用后，通过腺苷和肌动蛋白解聚介导的途径内化进入细胞。值得注意的是，RSDOX或RVCDOX诱导了更强的抗肿瘤功效，这取决于它们的细胞摄取水平，细胞内运输和与溶酶体的共定位率。RSDOX或RVCDOX的体内功效分别是游离DOX的1.4–1.7倍和RSSDOX的1.7–2.0倍。此外，RSDOX或RVCDOX表现出可接受的系统，组织和血液相容性。RSSDOX的功效受损可能是由于前药降解过程中产生了DOX-SH，但不是DOX。综上，我们的研究表明，某些类型的APDC可以显着降低游离DOX的毒性并改善治疗效果，这为整合配体肽和刺激反应性连接的肽-药物缀合物的设计提供了见识。