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.

中文翻译：

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通过立足点释放纯化对细胞外囊泡进行可编程 RNA 加载

合成纳米颗粒作为脂质纳米颗粒（LNP）被广泛用作药物递送囊泡。然而，它们有一些缺点，包括生物相容性低和不利的免疫反应。天然存在的细胞外囊泡（EV）具有作为天然、安全和多功能纳米囊泡载体的潜力。然而，电动汽车装载大生物分子仍然是一个挑战。在这里，我们提出了一种利用 DNA 介导和程序化的 EV 与信使 RNA (mRNA) 装载脂质体之间的融合的受控装载方法。通过脂质化生物素-DNA 手柄固定 EV 表面，通过实时显微镜在单颗粒水平上表征融合效率。随后，可以通过 DNA 链置换反应收集融合的 EV-脂质体颗粒 (EVL)。将聚变反应转移到磁珠上使我们能够将 EVL 的生产规模扩大一百万倍。最后，我们在 HEK293-H 细胞中证明了与脂质体或 LNP 相比，使用 EVL 封装 mCherry mRNA、转染和改进的翻译。我们设想这是 EV 介导的 RNA 治疗递送的重要工具。