Graduate School of Chemical Sciences and Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo 060-8628, Japan.
Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo 060-8628, Japan.
ACS Appl Bio Mater. 2021 Feb 15;4(2):1783-1793. doi: 10.1021/acsabm.0c01519. Epub 2021 Jan 12.
Size-controlled lipid nanoparticle (LNP)-based DNA/RNA delivery is a leading technology for gene therapies. For DNA/RNA delivery, typically, a cationic lipid is used to encapsulate DNA/RNA into LNPs. However, the use of the cationic lipid leads to cytotoxicity. In contrast, noncationic NPs, such as exosomes, are ideal nanocarriers for DNA/RNA delivery. However, the development of a simple one-step method for the production of size-controlled noncationic NPs encapsulating DNA/RNA is still challenging because of the lack of electrostatic interactions between the cationic lipid and negatively charged DNA/RNA. Herein, we report a microfluidic-based one-step method for the production of size-controlled noncationic NPs encapsulating small interfering RNA (siRNA). Our microfluidic device, named iLiNP, enables the efficient encapsulation of siRNA, as well as control over the NP size, by varying the flow conditions. We applied this method to produce size-controlled exosome-like NPs. The siRNA-loaded exosome-like NPs did not show in vitro cytotoxicity at a high siRNA dosage. In addition, we investigated the effect of the size of the exosome-like NPs on the target gene silencing and found that the 40-50 nm-sized NPs suppressed target protein expression at a dose of 20 nM siRNA. The iLiNP-based one-step production method for size-controlled noncationic-NP-encapsulated RNA is a promising method for the production of artificial exosomes and functionally modified exosomes for gene and cell therapies.
基于脂质纳米颗粒（LNP）的大小可控 DNA/RNA 递药技术是基因治疗的主要技术。对于 DNA/RNA 递药，通常使用阳离子脂质将 DNA/RNA 包裹到 LNPs 中。然而，阳离子脂质的使用会导致细胞毒性。相比之下，非阳离子 NPs，如外泌体，是 DNA/RNA 递药的理想纳米载体。然而，由于阳离子脂质与带负电荷的 DNA/RNA 之间缺乏静电相互作用，开发一种简单的一步法生产大小可控的非阳离子 NPs 包裹 DNA/RNA 仍然具有挑战性。在此，我们报告了一种基于微流控的一步法生产大小可控的非阳离子 NPs 包裹小干扰 RNA（siRNA）的方法。我们的微流控装置命名为 iLiNP，通过改变流动条件，可以高效地将 siRNA 包裹在 NP 中，并控制 NP 的大小。我们应用该方法生产大小可控的类外泌体 NPs。负载 siRNA 的类外泌体 NPs 在高 siRNA 剂量下没有表现出体外细胞毒性。此外，我们研究了类外泌体 NPs 大小对靶基因沉默的影响，发现 40-50nm 大小的 NPs 在 20nM siRNA 剂量下抑制靶蛋白表达。基于 iLiNP 的一步法生产大小可控的非阳离子-NP-包裹 RNA 的方法是生产用于基因和细胞治疗的人工外泌体和功能修饰外泌体的有前途的方法。
