Nebulization-Based Pulmonary Delivery of Extracellular Vesicles in Mice

Extracellular vesicles (EVs) are membrane-bound particles involved in intercellular communication and have attracted increasing interest as carriers for therapeutic molecules. Direct pulmonary administration provides an opportunity to deliver EVs to the respiratory system while potentially limiting systemic exposure. Among pulmonary administration approaches, nebulization offers a noninvasive strategy that avoids direct airway manipulation and can facilitate repeated administration. However, aerosolization may alter EV particle characteristics, making it important to evaluate both the effects of nebulization on the EV preparation and subsequent pulmonary delivery. Here, we describe an integrated workflow for nebulization-based pulmonary delivery of EVs in mice. The protocol includes EV preparation and fluorescent labeling, pre- and post-nebulization EV characterization, assembly and operation of the nebulization system, aerosol exposure of mice, and evaluation of pulmonary delivery and PKH26-associated fluorescence in bronchoalveolar lavage fluid (BALF) cells. Post-nebulization EV collection provides an additional quality-control step for assessing particle characteristics after aerosolization. This method provides a practical experimental framework for investigating inhaled EVs and for developing EV-based pulmonary delivery strategies.