Iron Salts, but Not Iron Oxide Nanoparticles, Drive High-Yield Production of Iron-Engineered Extracellular Vesicles

Efficient production of iron-engineered extracellular vesicles (EVs) is of interest for their use as delivery systems in biotherapy. In this study, we investigated the role of iron in EV biogenesis and evaluated a scalable fluidic strategy for their mass production using turbulence stimulation in bioreactors. Murine mesenchymal stem cells were loaded with iron via ferric quinate or iron oxide nanoparticles (IONPs) and subsequently subjected to turbulence stimulation or serum starvation to induce EV secretion. Using elemental spectroscopy and cryogenic electron microscopy, we found that prolonged incubation with ferric quinate combined with turbulence stimulation resulted in the highest EV yield-60,000 EVs per cell in 3 h-and enabled efficient iron transfer from cells to EVs. In contrast, IONP treatment did not increase EV yield compared to control and showed high variability in iron transfer, as probed by EV magnetophoretic mobility. TEM and XEDS analyses confirmed ferritin-bound iron encapsulation within EVs, whereas IONPs were poorly internalised. These results highlight the promising role of ionic iron in EV formation and content, demonstrating how iron source, concentration, and incubation duration influence both EV biogenesis and iron engineering.