To verify dry powder nanoparticle stability and shelf life, lyophilized nanoparticle cargo leakage was tested by an enzyme-linked immunosorbent assay (ELISA), where nanoparticles had less than 2.4% of total pg/mL cargo leakage at day 28 of room-temperature storage (Figures?2B and S3). exosomes (Lung-Exos) ITGA9 as mRNA and protein drug carriers. Compared with standard synthetic nanoparticle liposomes (Lipos), Lung-Exos exhibited superior distribution to the bronchioles and parenchyma and are deliverable to the lungs of rodents and nonhuman primates (NHPs) by dry powder inhalation. In a vaccine application, severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S)?protein encoding mRNA-loaded Lung-Exos (S-Exos) elicited greater immunoglobulin G (IgG) and secretory IgA (SIgA) responses than its loaded liposome (S-Lipo) counterpart. Importantly, S-Exos remained functional at room-temperature storage for one month. Our results suggest that extracellular vesicles can serve as an inhaled mRNA drug-delivery system that is superior to synthetic liposomes. were evaluated through light-sheet fluorescence microscopy (LSFM) (Figure?1F). Healthy mice BAY-678 received a single dose of RFP-Exos or RFP-Lipos via nebulization and were sacrificed after 24 h. LSFM imaging confirmed nanoparticle delivery to the conducting airways and the deep lung, with an accumulation of RFP-Exos in the upper pulmonary regions (Videos S1 and S2). Quantification of nanoparticle delivery to the whole lung demonstrated a 3.7-fold improvement in RFP-Exo retention and uptake compared with RFP-Lipo (Figure?1G). Segmentation of the lung into bronchial and parenchymal regions revealed 2.9- and 3.8-fold improvements in RFP-Exo retention and uptake, respectively, compared with RFP-Lipo (Figure?1H). Flow cytometry analysis in lung parenchymal cells (Figure?1I) and in the murine lung following nebulization (Figure?1J) confirmed greater cellular uptake of RFP-Exos than RFP-Lipos. The drug-loading capabilities of lung-derived exosomes (Lung-Exos and Lipos were expanded by loading GFP-encoding mRNA to evaluate nanoparticle mRNA uptake. Lung parenchymal cells that received GFP-Exos demonstrated more rapid internalization of exosomal mRNA than liposomal mRNA (Figure?S1). These data confirm that our nanoparticle labeling system maintains nanoparticle integrity while delivering functional and translatable cargo after jet nebulization. and analyses suggest superior retention and cellular uptake of exosomes over Lipos in the lung. The native lung signature of lung-derived exosomes may enhance pulmonary bioavailability, resulting in an optimized nanoparticle vesicle for drug delivery for respiratory diseases. Open in a separate window Figure?1 Fabrication and distribution of exosomes and liposomes (A) Schematic showing protein loading into lung-derived exosomes (RFP-Exos) and liposomes (RFP-Lipos), nebulization administration, lung-tissue clearing, and 3D imaging by LSFM. Created with BioRender.com. (B) TEM images of RFP-Exos and RFP-Lipos; scale bar: 50?nm. (C) Immunoblot of RFP in exosome and liposome lysate. (D) Representative immunostaining images of lung parenchymal cells for RFP (red) and DAPI (blue); scale bar: 50?m. (E) Quantification of RFP-Exo and RFP-Lipo pixel intensity normalized to nuclei in lung parenchymal cell images; n?= 6 per group; data are represented as mean? standard deviation. (F) LSFM images of cleared mouse lungs after RFP-Exo and RFP-Lipo nebulization; scale bar: 1,000?m. (G) Quantification of the integrated density of RFP normalized to the whole-lung area; n?= 74 total BAY-678 slices from two biological replicates per group; data are represented as mean? standard deviation. (H) Quantification of the integrated density of RFP normalized to segmented bronchiole and parenchymal regions from whole-lung images; n?= 74 total slices from two biological replicates per group; data are represented as mean? standard deviation. (I and J) Flow cytometry analysis of lung parenchymal cells co-cultured with RFP-Exos or RFP-Lipos (I)?and murine lung cells that received nebulized RFP-Exos or RFP-Lipos (J). Video S1. Biodistribution of nebulized RFP-Exos in mouse BAY-678 lungs: LSFM imaging and 3D rendering and animation by Imaris confirms labeled exosome distribution throughout the lung. Tissue autofluorescence allows for morphological segmentation of bronchioles and parenchyma to quantify exosome distribution. Click here to view.(30M, mp4) Video S2. Biodistribution of nebulized RFP-Lipos in mouse lungs: LSFM imaging and 3D rendering and animation by Imaris confirms labeled liposome distribution throughout the lung. Tissue autofluorescence allows for morphological segmentation of bronchioles and parenchyma to quantify liposome distribution. Click here to view.(38M, mp4) Lung-derived exosomes efficiently penetrate mucus Delivery of inhaled therapeutics must penetrate the lungs protective mucus lining to provide pulmonary bioavailability. Lung-Exos were compared against human embryonic kidney (HEK)-derived exosomes (HEK-Exos) and Lipos to determine if nanoparticle derivation affected mucus BAY-678 penetrance. To test this, we used a model of the human airway at the air-liquid interface (Figure?S2A), with human mucus-secreting bronchial epithelial cells lining the transwell membrane and human lung parenchymal cells lining the well (Figure?S2B). Immunostaining confirmed the mucus lining in the transwell.