Molecular Imaging Program at Stanford, Stanford University School of Medicine, Palo Alto, CA, USA.
Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, Palo Alto, CA, USA.
Methods Mol Biol. 2022;2525:227-238. doi: 10.1007/978-1-0716-2473-9_17.
Extracellular vesicles (EVs) derived from various cell lines have been extensively used as natural nanodelivery vehicles for drug, protein, and nucleic acid deliveries in therapeutic applications for cancer. Recently, we developed a microfluidic-based reconstruction strategy as a novel method to generate microRNA-loaded membrane vesicles for cancer therapy in vivo. We used EVs and cell membranes isolated from different source of cells for this reconstruction process. The microfluidic system produced reconstructed vesicles of uniform sizes with high microRNA loading efficiency independent of input membrane sources (EVs or cell membranes). To address the functional integrity of the membrane structure and of proteins in the reconstructed EVs, we introduce a membrane-insertable bioluminescence resonance energy transfer (BRET) sensor system. This sensor, with its membrane-insertable palmitoylation signal peptide sequence derived from a growth-associated protein 43 (GAP43), helps in trafficking the fusion protein to the cell membrane upon its expression in cells and allows for imaging reconstructed membrane vesicles using optical imaging. In this chapter, we detail the stepwise methods used for the engineering of cells using this sensor, isolation of EVs from the engineered cells, preparation of reconstructed EVs by microfluidic processing, and BRET imaging of reconstructed EVs for membrane integrity evaluation.
细胞外囊泡 (EVs) 源自各种细胞系，已被广泛用作药物、蛋白质和核酸递送至癌症治疗应用的天然纳米载体。最近，我们开发了一种基于微流控的重建策略，作为一种新方法来生成用于体内癌症治疗的负载 microRNA 的膜囊泡。我们在这个重建过程中使用了来自不同细胞来源的 EVs 和细胞膜。微流控系统产生了具有均匀大小的重建囊泡，具有高 microRNA 加载效率，与输入膜源（EVs 或细胞膜）无关。为了解决重建 EVs 中膜结构和蛋白质的功能完整性问题，我们引入了一种膜可插入的生物发光共振能量转移 (BRET) 传感器系统。该传感器具有来源于生长相关蛋白 43 (GAP43) 的膜可插入的棕榈酰化信号肽序列，有助于在细胞中表达融合蛋白时将其转运到细胞膜，并允许使用光学成像对重建的膜囊泡进行成像。在本章中，我们详细介绍了使用该传感器对细胞进行工程改造、从工程化细胞中分离 EVs、通过微流处理制备重建 EVs 以及对重建 EVs 进行 BRET 成像以评估膜完整性的分步方法。
