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Intelligent “Peptide-Gathering Mechanical Arm” Tames Wild “Trojan-Horse” Peptides for the Controlled Delivery of Cancer Nanotherapeutics
ACS Applied Materials & Interfaces ( IF 9.5 ) Pub Date : 2017-11-21 00:00:00 , DOI: 10.1021/acsami.7b15523
Nian-Qiu Shi 1 , Yan Li , Yong Zhang 2 , Nan Shen , Ling Qi , Shu-Ran Wang , Xian-Rong Qi 3
Affiliation  

Cell-penetrating peptides (CPPs), also called “Trojan-Horse” peptides, have been used for facilitating intracellular delivery of numerous diverse cargoes and even nanocarriers. However, the lack of targeting specificity (“wildness” or nonselectivity) of CPP-nanocarriers remains an intractable challenge for many in vivo applications. In this work, we used an intelligent “peptide-gathering mechanical arm” (Int PMA) to curb CPPs’ wildness and enhance the selectivity of R9-liposome-based cargo delivery for tumor targeting. The peptide NGR, serving as a cell-targeting peptide for anchoring, and peptide PLGLAG, serving as a substrate peptide for deanchoring, were embedded in the Int PMA motif. The Int PMA construct was designed to be sensitive to tumor microenvironmental stimuli, including aminopeptidase N (CD13) and matrix metalloproteinases (MMP-2/9). Moreover, Int PMA could be specifically recognized by tumor tissues via CD13-mediated anchoring and released for cell entry by MMP-2/9-mediated deanchoring. To test the Int PMA design, a series of experiments were conducted in vitro and in vivo. Functional conjugates Int PMA-R9-poly(ethylene glycol) (PEG)2000-distearoylphosphatidyl-ethanolamine (DSPE) and R9-PEG2000-DSPE were synthesized by Michael addition reaction and were characterized by thin-layer chromatography and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The Int PMA-R9-modified doxorubicin-loaded liposomes (Int PMA-R9-Lip-DOX) exhibited a proper particle diameter (approximately 155 nm) with in vitro sustained release characteristics. Cleavage assay showed that Int PMA-R9 peptide molecules could be cleaved by MMP-2/9 for completion of deanchoring. Flow cytometry and confocal microscopy studies indicated that Int PMA-R9-Lip-DOX can respond to both endogenous and exogenous stimuli in the presence/absence of excess MMP-2/9 and MMP-2/9 inhibitor (GM6001) and effectively function under competitive receptor-binding conditions. Moreover, Int PMA-R9-Lip-DOX generated more significant subcellular dispersions that were especially evident within endoplasmic reticulum (ER) and Golgi apparatus. Notably, Int PMA-R9-Lip-DOX could induce enhanced apoptosis, during which caspase 3/7 might be activated. In addition, Int PMA-R9-Lip-DOX displayed enhanced in vitro and in vivo antitumor efficacy versus “wild” R9-Lip-DOX. On the basis of investigations at the molecular level, cellular level, and animals’ level, the control of Int PMA was effective and promoted selective delivery of R9-liposome cargo to the target site and reduced nonspecific uptake. This Int PMA-controlled strategy based on aminopeptidase-guided anchoring and protease-triggered deanchoring effectively curbed the wildness of CPPs and bolstered their effectiveness for in vivo delivery of nanotherapeutics. The specific nanocarrier delivery system used here could be adapted using a variety of intelligent designs based on combinations of multifunctional peptides that would specifically and preferentially bind to tumors versus nontumor tissues for tumor-localized accumulation in vivo. Thus, CPPs have a strong advantage for the development of intelligent nanomedicines for targeted tumor therapy.

中文翻译:

智能的“集肽机械臂”驯服野生的“特洛伊木马”肽以控制癌症纳米治疗剂的递送

细胞穿透肽(CPPs),也称为“特洛伊木马(Trojan-Horse)”肽,已被用于促进细胞内多种多样的货物甚至纳米载体的递送。然而,对于许多体内应用,CPP纳米载体缺乏靶向特异性(“野生性”或非选择性)仍然是棘手的挑战。在这项工作中,我们使用了智能的“肽聚集机械臂”(Int PMA)来抑制CPP的野生性并提高R 9的选择性-基于脂质体的货物递送以靶向肿瘤。Int PMA基序中嵌入了用作锚定细胞靶向肽的肽NGR和用作去锚定的底物肽的PLGLAG肽。Int PMA构建体被设计为对肿瘤微环境刺激敏感,包括氨基肽酶N(CD13)和基质金属蛋白酶(MMP-2 / 9)。此外,Int PMA可以通过CD13介导的锚定被肿瘤组织特异性识别,并通过MMP-2 / 9介导的去锚定释放进入细胞。为了测试Int PMA设计,在体外和体内进行了一系列实验。功能偶联物Int PMA-R 9-聚(乙二醇)(PEG)2000-二硬脂酰磷脂酰乙醇胺(DSPE)和R 9 -PEG 2000通过迈克尔加成反应合成-DSPE,并通过薄层色谱和基质辅助激光解吸电离飞行时间质谱进行表征。Int PMA-R 9修饰的负载阿霉素的脂质体(Int PMA-R 9 -Lip-DOX)表现出合适的粒径(约155 nm),并具有体外持续释放特性。切割试验表明,Int PMA-R 9肽分子可以被MMP-2 / 9切割,从而完成去锚定。流式细胞术和共聚焦显微镜研究表明Int PMA-R 9-Lip-DOX在存在/不存在过量MMP-2 / 9和MMP-2 / 9抑制剂(GM6001)的情况下,可以对内源性和外源性刺激作出反应,并在竞争性受体结合条件下有效发挥作用。此外,Int PMA-R 9 -Lip-DOX产生了更明显的亚细胞分散体,这在内质网(ER)和高尔基体中尤为明显。值得注意的是,Int PMA-R 9 -Lip-DOX可以诱导增强的细胞凋亡,在此期间caspase 3/7可能被激活。此外,与“野生” R 9相比,Int PMA-R 9 -Lip-DOX具有增强的体外和体内抗肿瘤功效。-Lip-DOX。根据分子水平,细胞水平和动物水平的研究,对Int PMA的控制有效并促进了R 9的选择性转运。-脂质体货物到达目标部位并减少非特异性摄取。基于氨基肽酶引导的锚定和蛋白酶触发的去锚定的这种Int PMA控制策略有效地抑制了CPP的野生性,并增强了其在体内递送纳米治疗剂的有效性。基于多功能肽的组合,可以使用多种智能设计来适应在此使用的特定纳米载体递送系统,所述多功能肽的组合将特异性地并优先地与肿瘤而非非肿瘤组织结合,从而在体内进行肿瘤定位的积累。因此,CPP在开发用于靶向肿瘤治疗的智能纳米药物方面具有强大的优势。
更新日期:2017-11-21
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