Brossa Alessia, Tapparo Marta, Fonsato Valentina, Papadimitriou Elli, Delena Michela, Camussi Giovanni, Bussolati Benedetta
Department of Molecular Biotechnology and Health Sciences, University of Torino, 10126 Torino, Italy.
Molecular Biotechnology Center, University of Torino, 10126 Torino, Italy.
Pharmaceutics. 2021 Jan 8;13(1):76. doi: 10.3390/pharmaceutics13010076.
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.
细胞外囊泡因其能够将其携带的物质转移至靶细胞而被视为一种新型治疗工具。人们已提出了多种将RNA种类直接装载到细胞外囊泡中的策略。电穿孔法已用于装载无活性的囊泡；然而，对已携带治疗活性物质的囊泡进行工程改造仍在研究之中。在此，我们建立了一种共孵育方法，以增强从人肝干细胞分离出的细胞外囊泡（HLSC-EVs）的抗肿瘤作用。使用该共孵育方案，将抗肿瘤miRNA-145装载到囊泡中，并评估其对肾癌干细胞侵袭的影响。装载后的HLSC-EVs保持了其完整性和miR转移能力。装载的miR-145（而非单独的miR-145）受到RNA酶消化的保护，这可能是由于其与HLSC-EV表面的RNA结合蛋白（如膜联蛋白A2）结合。此外，与未处理的囊泡相比，miR-145共孵育的HLSC-EVs在抑制癌干细胞侵袭特性方面更有效。本文报道的方案利用了细胞外囊泡的一种已充分描述的特性，即其能够在表面结合核酸并保护其免受降解，从而实现有效的miRNA装载，进而增强未处理的具有治疗活性的细胞外囊泡的活性。
