Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama, 350-0295, Japan.
Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane, Chiba-Ken, 283-8555, Japan.
Sci Rep. 2023 Feb 22;13(1):3102. doi: 10.1038/s41598-023-30180-3.
Small interfering RNAs (siRNAs) knockdown the expression of target genes by causing mRNA degradation and are a promising therapeutic modality. In clinical practice, lipid nanoparticles (LNPs) are used to deliver RNAs, such as siRNA and mRNA, into cells. However, these artificial nanoparticles are toxic and immunogenic. Thus, we focused on extracellular vesicles (EVs), natural drug delivery systems, for the delivery of nucleic acids. EVs deliver RNAs and proteins to specific tissues to regulate various physiological phenomena in vivo. Here, we propose a novel method for the preparation siRNAs encapsulated in EVs using a microfluidic device (MD). MDs can be used to generate nanoparticles, such as LNPs, by controlling flow rate to the device, but the loading of siRNAs into EVs using MDs has not been reported previously. In this study, we demonstrated a method for loading siRNAs into grapefruit-derived EVs (GEVs), which have gained attention in recent years for being plant-derived EVs developed using an MD. GEVs were collected from grapefruit juice using the one-step sucrose cushion method, and then GEVs-siRNA-GEVs were prepared using an MD device. The morphology of GEVs and siRNA-GEVs was observed using a cryogenic transmission electron microscope. Cellular uptake and intracellular trafficking of GEVs or siRNA-GEVs to human keratinocytes were evaluated by microscopy using HaCaT cells. The prepared siRNA-GEVs encapsulated 11% of siRNAs. Moreover, intracellular delivery of siRNA and gene suppression effects in HaCaT cells were achieved using these siRNA-GEVs. Our findings suggested that MDs can be used to prepare siRNA-EV formulations.
小干扰 RNA（siRNA）通过引起 mRNA 降解来降低靶基因的表达，是一种很有前途的治疗方式。在临床实践中，脂质纳米颗粒（LNP）被用于将 RNA（如 siRNA 和 mRNA）递送到细胞中。然而，这些人工纳米颗粒具有毒性和免疫原性。因此，我们专注于细胞外囊泡（EVs），这是一种天然的药物传递系统，用于递送核酸。EVs 将 RNA 和蛋白质递送到特定组织中，以调节体内的各种生理现象。在这里，我们提出了一种使用微流控装置（MD）将 siRNA 封装在 EVs 中的新方法。MD 可用于通过控制流向装置的流速来生成纳米颗粒，如 LNP，但以前没有报道过使用 MD 将 siRNA 加载到 EVs 中。在这项研究中，我们展示了一种将 siRNA 加载到柚子衍生的 EVs（GEVs）中的方法，近年来，由于使用 MD 开发的植物衍生 EVs，GEVs 引起了人们的关注。使用一步蔗糖垫方法从柚子汁中收集 GEVs，然后使用 MD 装置制备 GEVs-siRNA-GEVs。使用低温传输电子显微镜观察 GEVs 和 siRNA-GEVs 的形态。通过使用 HaCaT 细胞进行显微镜观察，评估 GEVs 或 siRNA-GEVs 对人角质形成细胞的细胞摄取和细胞内转运。所制备的 siRNA-GEVs 封装了 11%的 siRNA。此外，使用这些 siRNA-GEVs 在 HaCaT 细胞中实现了 siRNA 的细胞内递送和基因抑制作用。我们的研究结果表明，MD 可用于制备 siRNA-EV 制剂。
