Cell-free synthesis of connexin 43-integrated exosome-mimetic nanoparticles for siRNA delivery
Cell-free synthesis of connexin 43-integrated exosome-mimetic nanoparticles for siRNA delivery
复制标题
用于 siRNA 递送的连接蛋白 43 整合的外泌体模拟纳米颗粒的无细胞合成
DOI：
10.1016/j.actbio.2019.07.006
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发表时间：
2019-09-15
影响因子：
9.7
通讯作者：
Ding, Pingtian
中科院分区：
文献类型：
--
作者：
Lu, Mei;Zhao, Xiaoyun;Ding, Pingtian
外泌体是天然分泌的纳米囊泡，由于其特定的组成和生物学特性，已成为有前途的治疗纳米递送平台。然而，诸如相当大的复杂性、低分离产率、药物有效载荷和潜在的安全性问题等挑战大大降低了它们的药学可接受性。鉴于纳米生物界面是纳米载体行为和功能的关键因素，用外来体膜的固有标志修饰合成纳米颗粒以产生外来体模拟物可以允许以更安全和更有效的方式递送siRNA。本文中，通过使用无细胞（CF）合成系统，在脂质包被的CS NP（L/CS NP）的存在下，用编码连接蛋白43（Cx43）的质粒构建嵌入的、外泌体模拟的脂质双层包被的壳聚糖纳米颗粒（Cx43/L/CS NP）。在一锅法反应过程中，重新合成的Cx43整合到L/CS纳米粒的脂质双层中是协同发生的，更重要的是，整合的Cx43在转运中具有功能活性。除了比阳离子Lipo 2000显著更低的细胞毒性（<4倍）之外，所获得的Cx43/L/CS-siRNA NP显示出可行的细胞摄取和沉默功效，其显著高于游离siRNA和CS-siRNA NP。通过使用间隙连接（GJ）抑制剂18 β-大黄酸，我们证明了Cx43促进siRNA递送到表达Cx43的U87 MG细胞中。此外，Cx43/L/CS-siRNA NP的细胞进入可能依赖于不同的内吞机制，这取决于受体细胞的类型。然而，与转染试剂Lipo 2000相比，Cx43/L/CS-siRNA NPs仍然表现出远远不够的递送效率。综上所述，我们的研究提供了一个全新的策略，以构建Cx43功能化的，外泌体模拟纳米粒子，这可能会进一步鼓励建立更多的仿生纳米载体具有更高的生物相容性和交付efficiency.Significance声明的主要问题，将RNA干扰（RNAi）治疗从实验室到床边是缺乏安全和有效的交付车辆。鉴于外泌体和合成纳米载体的某些优点和局限性，一种有前途的策略是促进两个领域之间的正反馈，其中外泌体关于有利于细胞质递送的特殊膜组成的优势和合成纳米载体的更好的药学接受性可以结合。在这项研究中，我们报告了构建Cx43整合，外泌体模拟脂质双层包覆纳米粒子，通过使用CF合成技术。所获得的Cx43/L/CS-siRNA NPs的特征在于理想的细胞毒性特征和可行的递送效率。本研究为合成生物相容性更好、更有效的siRNA仿生载体提供了新的思路和思路。(C)2019 Acta Materialia Inc.由爱思唯尔有限公司出版。保留所有权利。
Exosomes are naturally secreted nanovesicles that have emerged as a promising therapeutic nanodelivery platform, due to their specific composition and biological properties. However, challenges like considerable complexity, low isolation yield, drug payload, and potential safety concerns substantially reduce their pharmaceutical acceptability. Given that the nano-bio-interface is a crucial factor for nanocarrier behavior and function, modification of synthetic nanoparticles with the intrinsic hallmarks of exosomes' membrane to create exosome mimetics could allow for siRNA delivery in a safer and more efficient manner. Herein, connexin 43 (Cx43)-embedded, exosome-mimicking lipid bilayers coated chitosan nanoparticles (Cx43/L/CS NPs) were constructed by using cell-free (CF) synthesis systems with plasmids encoding Cx43 in the presence of lipid-coated CS NPs (L/CS NPs). The integration of de novo synthesized Cx43 into the lipid bilayers of L/CS NPs occurred cotranslationally during one-pot reaction and, more importantly, the integrated Cx43 was functionally active in transport. In addition to considerably lower cytotoxicity ( < four-fold) than cationic Lipo 2000, the obtained Cx43/L/CS-siRNA NPs showed feasible cellular uptake and silencing efficacy that was significantly higher than free siRNA and CS-siRNA NPs. By using a gap junction (GJ) inhibitor, 18 beta-glycyrrhetinic acid, we demonstrated that Cx43 facilitated the delivery of siRNA into Cx43-expressing U87 MG cells. Additionally, the cellular entry of Cx43/L/CS-siRNA NPs may rely on different endocytic mechanisms, depending on the types of recipient cells. However, Cx43/L/CS-siRNA NPs still exhibited far from adequate delivery efficiency compared with transfection reagent Lipo 2000. Taken together, our study provides a brand new strategy to construct Cx43-functionalized, exosome-mimetic nanoparticles, which may further encourage the establishment of more biomimetic nanocarriers with higher biocompatibility and delivery efficiency.Significance of statementThe major issue to move RNA interference (RNAi) therapy from bench to bedside is the lack of safe and efficient delivery vehicles. Given the certain advantages and limitations of exosomes and synthetic nanocarriers, a promising strategy is to facilitate positive feedbacks between the two fields, in which the superiority of exosomes regarding special membrane composition beneficial for cytoplasmic delivery and the better pharmaceutical acceptance of synthetic nanocarriers could be combined. In this study, we reported to construct Cx43-integrated, exosome-mimetic lipid bilayers coated nanoparticles by using CF synthesis technique. The obtained Cx43/L/CS-siRNA NPs were characterized by desirable cytotoxicity profile and feasible delivery efficiency. This study provides a new avenue and insights for the synthesis of more biocompatible and effective bio-mimetic siRNA delivery platforms. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.