Preliminary Characterization of Extracellular Vesicles From Auditory HEI-OC1 Cells.

Objectives: Isolate, purify, and characterize extracellular vesicles (EVs) obtained from auditory HEI-OCI cells, and evaluate their suitability for intracochlear transport and delivery of pharmacological drugs and/or pro-resolution mediators of acute inflammatory processes. Methods: HEI-OCI EVs were isolated and purified using the exoEasy Maxi Kit, and their size was evaluated by nanoparticle tracking techniques. Bottom-up proteomics of the EVs, either freshly obtained or stored for up to 4 months at -20 degrees C, was performed by LC-ESI-MS/MS. LC-ESI-MS/MS-MRM was used to measure the loading of dexamethasone inside EVs following co-incubation at room temperature for I hour with and without 5 minutes sonication. Results: Routinely, we were able to obtain purified fractions of >2 X 10(9) EVs/mL, with diameters varying between 50 and 800 nm. Bottom-up proteomics showed that among the most abundant EVs proteins, 19.2% were cytoplasmic, 17.2% were membrane localized, 12.3% were cytosolic, and 14.6% were nucleolar. No significant differences between fresh and stored EVs were detected. Importantly, co-incubation of HEI-OCI EVs (1 X 10(8) EVs/nnL) with dexamethasone (10 mM) resulted in the incorporation of 10.1 +/- 1.9 nM dexamethasone per milliliter of EVs suspension. Conclusions: Altogether, the results suggest that EVs from HEI-OCI cells could be advantageously used as biological nanocarriers for the delivery of specific molecules and pharmacological drugs into the inner ear.