BRET Sensors for Imaging Membrane Integrity of Microfluidically Generated Extracellular Vesicles.

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.