Interdiscilinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark.
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark.
J Am Chem Soc. 2024 May 8;146(18):12410-12422. doi: 10.1021/jacs.3c13123. Epub 2024 Apr 26.
Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.
合成纳米颗粒作为脂质纳米颗粒 (LNPs) 被广泛用作药物递送载体。然而，它们存在一些缺点，包括低生物相容性和不利的免疫反应。天然存在的细胞外囊泡 (EVs) 作为天然的、安全的和多功能的纳米囊泡载体具有潜力。然而，将大生物分子装载到 EVs 中仍然是一个挑战。在这里，我们提出了一种使用 DNA 介导的和编程融合的方法，用于 EVs 和负载信使 RNA (mRNA) 的脂质体之间的受控加载。通过在脂质化生物素-DNA 处理后通过 EV 表面固定化在单个颗粒水平上对融合效率进行实时显微镜检查来进行表征。随后，可以通过采用 DNA 链置换反应收集融合的 EV-脂质体颗粒 (EVL)。将融合反应转移到磁珠上，使我们能够将 EVL 的产量扩大一百万倍。最后，我们证明了与脂质体或 LNPs 相比，使用 EVL 在 HEK293-H 细胞中封装 mCherry mRNA、转染和提高翻译效率。我们设想这是 EV 介导的 RNA 治疗药物递送的重要工具。
