Based on their identification as physiological nucleic acid carriers in humans and other organisms, extracellular vesicles (EVs) have been explored as therapeutic delivery vehicles for DNA, RNA, and other cargo. However, efficient loading and functional delivery of nucleic acids remain a challenge, largely because of potential sources of degradation and aggregation. Here, we report that protonation of EVs to generate a pH gradient across EV membranes can be utilized to enhance vesicle loading of nucleic acid cargo, specifically microRNA (miRNA), small interfering RNA (siRNA), and single-stranded DNA (ssDNA). The loading process did not impair cellular uptake of EVs, nor did it promote any significant EV-induced toxicity response in mice. Cargo functionality was verified by loading HEK293T EVs with either pro- or anti-inflammatory miRNAs and observing the effective regulation of corresponding cellular cytokine levels. Critically, this loading increase is comparable with what can be accomplished by methods such as sonication and electroporation, and is achievable without the introduction of energy associated with these methods that can potentially damage labile nucleic acid cargo.

中文翻译：

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通过细胞外囊泡的 pH 梯度修饰增强功能性 miRNA 货物的负载。

基于它们作为人类和其他生物体中的生理核酸载体的鉴定，细胞外囊泡 (EV) 已被探索为 DNA、RNA 和其他货物的治疗性递送载体。然而，核酸的有效装载和功能性递送仍然是一个挑战，主要是因为潜在的降解和聚集来源。在这里，我们报告了 EV 的质子化以在 EV 膜上产生 pH 梯度，可用于增强核酸货物的囊泡负载，特别是 microRNA (miRNA)、小干扰 RNA (siRNA) 和单链 DNA (ssDNA)。加载过程不会损害 EV 的细胞摄取，也不会促进小鼠体内任何显着的 EV 诱导的毒性反应。通过在 HEK293T EV 中加载促炎或抗炎 miRNA 并观察相应细胞细胞因子水平的有效调节来验证货物功能。至关重要的是，这种负载增加与超声和电穿孔等方法可以实现的效果相当，并且无需引入与这些方法相关的能量，这些方法可能会损坏不稳定的核酸货物。