Round window membrane extracellular vesicles facilitate inner ear drug delivery

Hearing loss affects over 430 million people worldwide, yet effective treatments remain limited, in part due to challenges of inner ear drug delivery, a process that is risky, unreliable, and inefficient. Addressing this challenge calls for delivery approaches that ensure therapies reach the inner ear safely and effectively. Intratympanic (IT) injection, which delivers drugs directly into the middle ear, is currently the safest method for preserving cochlear integrity. However, its efficiency is limited by the round window membrane (RWM), a barrier with tight junctions that restricts therapy-related substance passage into the inner ear. We propose that extracellular vesicles (EVs) released by RWM cells could serve as a novel vehicle for drug delivery, as their membrane features and intravesicular components may facilitate IT transport. Importantly, EVs are also expected to elicit minimal immune responses, addressing a key safety concern for inner ear therapies. We isolated and characterized EVs derived from RWM cells, including sequencing and Ingenuity Pathway Analysis to predict drug delivery pathways and immune-related functions. To establish translational relevance, we investigated their uptake in vitro and assessed passage across the RWM ex vivo and in vivo, demonstrating that dexamethasone-loaded EVs effectively crossed the membrane in a porcine model. We further demonstrated functional delivery potential by showing enhanced cytoplasmic retention of brain-derived neurotrophic factor (BDNF) and improved adeno-associated virus (AAV)-mediated transduction in vitro.