Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

Mitochondrial reactive oxygen species modify extracellular vesicles secretion rate.

Extracellular vesicle (EV) secretion rate is stimulated by hypoxia that causes increased reactive oxygen species (ROS) production by the mitochondrial electron transport chain (ETC) and hypoxia-induced factor (HIF)-1 signaling; however, their contribution to the increased EV secretion rate is unknown. We found that the EV marker secretion rate in our EV reporter cell line CD9truc-EGFP was unaffected by the HIF-1α stabilizer roxadustat; yet, ETC stimulation by dichloroacetic acid (DCA) significantly increased EV secretion. The DCA-induced EV secretion was blocked by the antioxidant TEMPO and rotenone, an inhibitor of the ETC's Complex I. Under hypoxic conditions, the limited oxygen reduction impedes the ETC's Complex III. To mimic this, we inhibited Complex III with antimycin A, which increased ROS-dependent EV secretion. The electron transport between Complex I and III is accomplished by coenzyme Q created by the mevalonate pathway and tyrosine metabolites. Blocking an early step in the mevalonate pathway using pitavastatin augmented the DCA-induced EV secretion, and 4-nitrobenzoate-an inhibitor of the condensation of the mevalonate pathway with tyrosine metabolites-increased ROS-dependent EV secretion. Our findings indicate that hypoxia-mimetics targeting the ETC modify EV secretion and that ROS produced by the ETC is a potent stimulus for EV secretion.