Round window membrane extracellular vesicles facilitate inner ear drug delivery

Over 400 million people are affected by hearing loss, but currently the promising treatments remain limited in part due to challenges of inner ear drug delivery, which is risky, variable, and not efficient. To improve clinical treatments for hearing loss, a safe, reliable, and effective local delivery system is needed to ensure the therapeutic dose reaches the inner ear. Intratympanic (IT) injection is currently the safest method, preserving cochlear integrity, but has low efficiency due to the presence of the round window membrane (RWM), gatekeeper of the inner ear equipped with tight junctions, limiting therapy-related substance passage. Extracellular vesicles (EVs) released by RWM cells have promising properties for drug delivery, as they possess membrane features and intravesicular molecular components that could provide enhanced IT delivery and elicit minimal immune responses. This study explores EV release from RWM cells and characterizes the associated pathways for drug delivery, such as clathrin-mediated endocytosis, retrograde Golgi to ER transport, and gap junction trafficking. We evaluate the uptake and passage efficiency of RWM-derived EVs by porcine RWM cells, comparing them with mesenchymal stem cell (MSC) EVs and liposomes. In addition, we perform in-vivo testing to assess the passage of dexamethasone-loaded EVs following IT delivery in pigs. We also loaded EVs with brain-derived neurotrophic factor (BDNF) and adeno-associated viruses (AAVs) for gene therapy and investigated their uptake ability as well as transduction by human HEK293T and porcine RWM cells.