Extracellular vesicles are considered a novel therapeutic tool, due to their ability to transfer their cargoes to target cells. Different strategies to directly load extracellular vesicles with RNA species have been proposed. Electroporation has been used for the loading of non-active vesicles; however, the engineering of vesicles already carrying a therapeutically active cargo is still under investigation. Here, we set up a coincubation method to increase the anti-tumor effect of extracellular vesicles isolated from human liver stem cells (HLSC-EVs). Using the coincubation protocol, vesicles were loaded with the anti-tumor miRNA-145, and their effect was evaluated on renal cancer stem cell invasion. Loaded HLSC-EVs maintained their integrity and miR transfer ability. Loaded miR-145, but not miR-145 alone, was protected by RNAse digestion, possibly due to its binding to RNA-binding proteins on HLSC-EV surface, such as Annexin A2. Moreover, miR-145 coincubated HLSC-EVs were more effective in inhibiting the invasive properties of cancer stem cells, in comparison to naïve vesicles. The protocol reported here exploits a well described property of extracellular vesicles to bind nucleic acids on their surface and protect them from degradation, in order to obtain an effective miRNA loading, thus increasing the activity of therapeutically active naïve extracellular vesicles.

中文翻译：

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共孵育作为miR加载策略以提高干细胞衍生电动汽车的抗肿瘤作用

由于细胞外囊泡将其货物转移至靶细胞的能力，因此被认为是一种新颖的治疗工具。已经提出了将RNA种类直接加载到细胞外囊泡的不同策略。电穿孔已被用于非活性囊泡的装载。然而，已经在研究已经运载了具有治疗活性的货物的囊泡的工程设计。在这里，我们建立了一种共孵育方法，以增强从人肝干细胞（HLSC-EVs）分离的细胞外囊泡的抗肿瘤作用。使用共孵育方案，将抗肿瘤miRNA-145装在囊泡中，并评估其对肾癌干细胞侵袭的作用。装载的HLSC-EV保持其完整性和miR传递能力。装载的miR-145（但不是单独的miR-145）受到RNA酶消化的保护，可能是由于其与HLSC-EV表面的RNA结合蛋白（如膜联蛋白A2）结合所致。此外，与幼稚囊泡相比，miR-145共孵育的HLSC-EV在抑制癌症干细胞的侵袭特性方面更有效。此处报道的方案利用细胞外囊泡的良好描述特性来结合其表面的核酸并保护其免于降解，从而获得有效的miRNA负载量，从而提高了具有治疗活性的幼稚细胞外囊泡的活性。