Design of peptide-based targeted delivery vectors with attributes of specificity and selective cellular targeting by fixing their topology and resulting electrostatic fingerprint is the objective of this study. We formulated our peptide design platform by utilizing the possibilities of side-chain induced geometric restrictions in a typical peptide molecule. Conceptually, we locked the conformation of the RGD/NGR motif of tumor homing peptides (THPs) by mutating glycine in these motifs with D-proline and tailed the peptides with a syndiotactic amphipathic segment for cellular penetration. The designed peptides were synthesized, characterized, and tested in vitro on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS) and non-cancer mammary epithelial cells (MCF-10A), by flow cytometry and confocal microscopy. The results showed differential cellular uptake in different cell types, as a result of the distinct electrostatic fingerprint encoded in their design. The uptake of serum pre-treated peptides by cells reveals the retention of peptide activity even after the incubation with serum. In addition, peptide–methotrexate (MTX) conjugates compared to the methotrexate drug showed enhanced apoptotic cell death in MTX-resistant MDA-MB-231 cells, indicating the increase in MTX bioavailability.

中文翻译：

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具有预定义几何锁的基于肽的递送载体

本研究的目标是通过固定拓扑结构和产生的静电指纹来设计具有特异性和选择性细胞靶向属性的基于肽的靶向递送载体。我们通过利用典型肽分子中侧链诱导的几何限制的可能性来制定我们的肽设计平台。从概念上讲，我们通过用D-脯氨酸突变这些基序中的甘氨酸来锁定肿瘤归巢肽 (THP) 的 RGD/NGR 基序的构象，并用间规两亲性片段尾随肽以进行细胞渗透。设计的肽在体外合成、表征和测试通过流式细胞术和共聚焦显微镜检测各种细胞系，包括乳腺癌 (MDA-MB-231)、宫颈癌 (HeLa)、骨肉瘤 (U2-OS) 和非癌乳腺上皮细胞 (MCF-10A)。结果表明，由于设计中编码的不同静电指纹，不同细胞类型中的细胞摄取不同。细胞对血清预处理肽的摄取揭示了即使在与血清一起温育之后肽活性的保留。此外，与甲氨蝶呤药物相比，肽-甲氨蝶呤 (MTX) 缀合物在 MTX 抗性 MDA-MB-231 细胞中显示出增强的凋亡细胞死亡，表明 MTX 生物利用度增加。