Enhanced Loading of Functional miRNA Cargo via pH Gradient Modification of Extracellular Vesicles
Enhanced Loading of Functional miRNA Cargo via pH Gradient Modification of Extracellular Vesicles
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DOI：
10.1016/j.ymthe.2019.12.007
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发表时间：
2020-03-04
影响因子：
12.4
通讯作者：
Jay, Steven M.
中科院分区：
文献类型：
--
作者：
Jeyaram, Anjana;Lamichhane, Tek N.;Jay, Steven M.
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）已经被探索作为DNA、RNA和其他货物的治疗递送载体。然而，核酸的有效装载和功能性递送仍然是一个挑战，主要是因为降解和聚集的潜在来源。在这里，我们报告说，质子化的EV产生跨EV膜的pH梯度，可用于增强囊泡装载的核酸货物，特别是微小RNA（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，也不会促进小鼠中任何显著的EV诱导的毒性反应。通过用促炎或抗炎miRNA加载HEK293T EV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。重要的是，这种负载增加与可以通过诸如超声处理和电穿孔的方法实现的负载增加相当，并且可以在不引入与这些方法相关的能量的情况下实现，所述能量可以潜在地损伤不稳定的核酸货物。
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.