@article{moatti_silkstone_martin_abbey_hutson_fitzpatrick_zdanski_cheng_ligler_greenbaum_2023, title={Assessment of drug permeability through an ex vivo porcine round window membrane model}, volume={26}, ISSN={["2589-0042"]}, DOI={10.1016/j.isci.2023.106789}, abstractNote={Delivery of pharmaceutical therapeutics to the inner ear to treat and prevent hearing loss is challenging. Systemic delivery is not effective as only a small fraction of the therapeutic agent reaches the inner ear. Invasive surgeries to inject through the round window membrane (RWM) or cochleostomy may cause damage to the inner ear. An alternative approach is to administer drugs into the middle ear using an intratympanic injection, with the drugs primarily passing through the RWM to the inner ear. However, the RWM is a barrier, only permeable to a small number of molecules. To study and enhance the RWM permeability, we developed an ex vivo porcine RWM model, similar in structure and thickness to the human RWM. The model is viable for days, and drug passage can be measured at multiple time points. This model provides a straightforward approach to developing effective and non-invasive delivery methods to the inner ear.}, number={6}, journal={ISCIENCE}, author={Moatti, Adele and Silkstone, Dylan and Martin, Taylor and Abbey, Keith and Hutson, Kendall A. and Fitzpatrick, Douglas C. and Zdanski, Carlton J. and Cheng, Alan G. and Ligler, Frances S. and Greenbaum, Alon}, year={2023}, month={Jun} }
 @article{popowski_moatti_scull_silkstone_lutz_lópez de juan abad_george_belcher_zhu_mei_et al._2022, title={Inhalable dry powder mRNA vaccines based on extracellular vesicles}, volume={5}, ISSN={2590-2385}, url={http://dx.doi.org/10.1016/j.matt.2022.06.012}, DOI={10.1016/j.matt.2022.06.012}, abstractNote={•Lung extracellular vesicles (Lung-Exos) can package mRNA and protein drugs•Lung-Exos are deliverable through nebulization and dry powder inhalation•Dry powder Lung-Exos are room-temperature stable up to 28 days•Drug-loaded Lung-Exos can serve as an inhalable vaccine to illicit immune responses Research in extracellular vesicles (EVs) is important to the field of translational medicine to develop therapeutics that are limited by poor cellular targeting and efficacy. The biological composition of EVs can be exploited as drug-delivery vehicles that may be engineered for cellular targeting or eliciting specific immune responses through their functions in membrane trafficking and cellular signaling. With the molecular composition of EVs varying depending on their parent-cell origin, the derivation of EVs can further refine nanomedicine by utilizing nanoparticles that are recognized by specific cellular microenvironments. EVs are found in almost all biological fluids, opening the application of EVs as tailored drug-delivery vesicles to a wide range of diseases. Respiratory diseases are a global burden, with millions of deaths attributed to pulmonary illnesses and dysfunctions. Therapeutics have been developed, but they present major limitations regarding pulmonary bioavailability and product stability. To circumvent such limitations, we developed room-temperature-stable inhalable lung-derived extracellular vesicles or exosomes (Lung-Exos) 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. Respiratory diseases are a global burden, with millions of deaths attributed to pulmonary illnesses and dysfunctions. Therapeutics have been developed, but they present major limitations regarding pulmonary bioavailability and product stability. To circumvent such limitations, we developed room-temperature-stable inhalable lung-derived extracellular vesicles or exosomes (Lung-Exos) 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. IntroductionRespiratory diseases are among the leading causes of morbidity and mortality worldwide,1Wisnivesky J. De-Torres J.P. The global burden of pulmonary diseases: most prevalent problems and opportunities for improvement.Ann. Global Health. 2019; 85https://doi.org/10.5334/aogh.2411Crossref Scopus (17) Google Scholar with coronavirus disease 2019 (COVID-19)2Wang C. Horby P.W. Hayden F.G. Gao G.F. A novel coronavirus outbreak of global health concern.Lancet. 2020; 395: 470-473https://doi.org/10.1016/S0140-6736(20)30185-9Abstract Full Text Full Text PDF PubMed Scopus (3967) Google Scholar remaining prevalent in the ongoing pandemic. A wide range of therapeutics have been developed and repurposed to treat respiratory diseases, including small-molecule drugs,3Li D. 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Med. 2020; 9: 786-798https://doi.org/10.1002/sctm.19-0167Crossref PubMed Scopus (5) Google Scholar in rodent models of idiopathic pulmonary fibrosis (IPF), and their safety and efficacy are being tested in a human clinical trial (HALT-IPF, ClinicialTrials.gov: NCT04262167). LSCs and their secreted exosomes (lung-derived extracellular vesicles [Lung-Exos]) have regenerative abilities in IPF models13Dinh P.-U.C. Paudel D. Brochu H. Popowski K.D. Gracieux M.C. Cores J. Huang K. Hensley M.T. Harrell E. Vandergriff A.C. et al.Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis.Nat. Commun. 2020; 11: 1064https://doi.org/10.1038/s41467-020-14344-7Crossref PubMed Scopus (127) Google Scholar and protective abilities against COVID-19 as decoys.32Li Z. Wang Z. Dinh P.-U.C. Zhu D. Popowski K.D. Lutz H. Hu S. Lewis M.G. Cook A. Andersen H. et al.Cell-mimicking nanodecoys neutralize SARS-CoV-2 and mitigate lung injury in a non-human primate model of COVID-19.Nat. Nanotechnol. 2021; 16: 942-951https://doi.org/10.1038/s41565-021-00923-2Crossref PubMed Scopus (43) Google Scholar In both disease models, Lung-Exos maintained therapeutic efficacy through jet-nebulization administration, demonstrating the ability of Lung-Exos to function as an inhalable drug-delivery and vaccine vehicle. Additionally, exosomes can be synthetically supplemented to enhance cellular targeting and therapeutic efficacy.33Hutcheson J.D. Aikawa E. Extracellular vesicles in cardiovascular homeostasis and disease.Curr. Opin. Cardiol. 2018; 33: 290-297https://doi.org/10.1097/HCO.0000000000000510Crossref PubMed Scopus (29) Google Scholar, 34Li S.p. Lin Z.x. Jiang X. Yu X.y. Yu X. Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools.Acta Pharmacol. 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Paudel D. Brochu H. Popowski K.D. Gracieux M.C. Cores J. Huang K. Hensley M.T. Harrell E. Vandergriff A.C. et al.Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis.Nat. Commun. 2020; 11: 1064https://doi.org/10.1038/s41467-020-14344-7Crossref PubMed Scopus (127) Google Scholar,37Zhang D. Lee H. Wang X. Rai A. Groot M. Jin Y. Exosome-Mediated small RNA delivery: a novel therapeutic approach for inflammatory lung responses.Mol. Ther. 2018; 26: 2119-2130https://doi.org/10.1016/j.ymthe.2018.06.007Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar the distribution and retention of exosome particles in the lung have yet to be determined. Drug effectiveness depends on proper deposition of particles within the respirable fraction, requiring optimized nanoparticle formulation. In this study, we sought to elucidate the biodistribution of lung-derived exosomes upon nebulization, baselined to liposomes (Lipos) as a commercial standard. Furthermore, to provide a room-temperature-stable product, we formulated exosomes as a lyophilized dry powder to investigate their stability and inhaled biodistribution in the lung of both the mouse and African green monkey (AGM). The parent-cell signature of Lung-Exos may suggest that they are naturally optimized for the distribution and retention within the lung, which may allow them to bypass pulmonary clearance more efficiently than Lipos or exosomes derived from other cell types. Through this enhanced pulmonary bioavailability, we hypothesize that lung-derived exosomes elicit greater therapeutic responses for pulmonary diseases and serve as a customizable drug-delivery vehicle for room-temperature-stable inhaled mRNA therapeutics.ResultsExosome distribution in the bronchioles and parenchyma are superior to that of synthetic nanoparticlesRed fluorescent protein (RFP)-labeled lung-derived exosomes (RFP-Exos) and Lipos (RFP-Lipos) were fabricated to generate trackable nanoparticles for biodistribution analysis in the murine lung after inhalation treatment through three-dimensional (3D) imaging (Figure 1A ). The nanoparticles were characterized by transmission electron microscopy (TEM), confirming that the isolation of exosomes and Lipos did not disrupt vesicular membrane integrity (Figure 1B). RFP loading was verified by immunoblotting (Figure 1C). When co-cultured with lung parenchymal cells, RFP-Exo had a 6.7-fold increase in cellular uptake and RFP protein expression compared with cells cultured with RFP-Lipo (Figures 1D and 1E). Next, the biodistribution of nanoparticles in vivo were evaluated through light-sheet fluorescence microscopy (LSFM) (Figure 1F). Healthy mice 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. In vitro and in vivo 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.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyZDE5ZTI0ODk4NDRkYTFhYjJlNGUzMWEwMGE4OTEyYyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjY5OTY2NjUwfQ.ZP_2__aVFlfG_3XKl06osGFI0GeEXb27ElTxZ-KGGM8IcSO676pqZKF6h65jz0cnnYuojIwvoPOnatMAd9qqR65EtMzNhGLYU-TdT2hEgjFIUL9AatnXvIyrTXEJt4Orap9iWn8eBBxr63jGbLdLvzqnA2A05saBmcwbPhbN3bVzqdWYJQCLOpzv6z8Yi-0i3xtJfwEmLHgdsvSLgQt8vnGKzY4IUA9jBXCdfho1Cp5PVg3eCXd0p9bQo-Df_q9s-Ghg1957V4C1OlRDoKdNdXo62FKjOcdLZPrCnVgzDNqmk_IPHTSkzr3rdFCG41UJVBXHBYkJMpXnhOvsDyhoXw Download .mp4 (30.4 MB) Help with .mp4 files Video S1. Biodistribution of nebulized RFP-Exos in mouse lungsLSFM 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.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiNTYzZTFmZDNiYjJiOTQzMWQyMDAwODQzYzA2MDcxNCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjY5OTY2NjUwfQ.rrwycH66nt21uB8nXcVXQaa02xcRy_2vJlbQsIigymt2QlJ24qCloQxenvyl4rqzmA9WIpCs6ZdvZ5yRY5FWlu-j2SfO4Sl8lcL2Xz3jrbu8PU-Ge_avejxOWdeaYEEEGIbmAWmXlVHWH2p6_LlFpfPB-j4E-KTapjf5CHzT4rGRom3mXroj6OiJkAkRl3ylnmxTdNaQDUbtDr2dJvIYzLCX_8TUNx9a7L4Rz-65ZWZD_ZXLADu_bgTFdnNOGodGmEY-9NZhPBqraOljjTPxzbe4TJJOttzGsmAySGpVDMWGFGcStxkuif_tgwujLF5wcLU1o-KQtk8eosKrcuRuxA Download .mp4 (38.39 MB) Help with .mp4 files Video S2. Biodistribution of nebulized RFP-Lipos in mouse lungsLSFM 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.Lung-derived exosomes efficiently penetrate mucusDelivery of inhaled therapeutics must penetrate the lung’s 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 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 membrane and delivery of DiD-labeled nanoparticles (Figure S2C). Quantification of nanoparticle penetrance into the wells revealed the greatest uptake of Lung-Exos (Figure S2D), with the highest percentage of cellular uptake by lung parenchymal cells (Figure S2E) by 24 h. Likewise, Lung-Exos had the least entrapment by the mucus-lined membrane (Figure S2F) and the lowest percentage of cellular uptake by bronchial epithelial cells (Figure S2G). These data confirm mucus penetrance of the nanoparticles and suggest that Lung-Exos can most efficiently evade mucoadhesion, overcoming the lung’s natural defense mechanism and allowing for greater parenchymal bioavailability.Lung-derived exosomes are room-temperature stable and distributable in dry powder formulation in the murine lungRoom-temperature formulation of therapeutics circumvents major limitations in traditional IM vaccine delivery: deep-freezing storage, healthcare professional administration, and reduced patient compliance. Therefore, we reformulated our liquid nanoparticle suspensions into dry, lyophilized powder for dry powder inhalation (DPI) administration. We verified the efficacy and stability of room-temperature lyophilized Lung-Exos up to 28 days in the murine lung (Figure 2A ). 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). Next, the morphology of nanoparticles was evaluated across their fresh and lyophilized formulations, as well as lyophilized powder reconstituted in water (reconstituted), to mimic rehydration of dry powder by saliva and mucus. TEM (Figures 2E and S4) and atomic force microscopy (AFM) verified that reformulation and rehydration did not affect nanoparticle membrane integrity (Figures 2C and S5) but did affect size distributions through clumping (Figure S6–S8). Lyophilization increased nanoparticle height and diameter (Figure 2D) but remained as small respiratory droplets upon reconstitution. Across all formulations, the nanoparticle diameters are approximately 10-fold larger, which may be explained by tip dilation38Wong C. West P.E. Olson K.S. Mecartney M.L. Starostina N. Tip dilation and AFM capabilities in the characterization of nanoparticles.JOM. 2007; 59: 12-16https://doi.org/10.1007/s11837-007-0003-xCrossref Scopus (40) Google Scholar that reports larger lateral dimensions than 2D analysis such as through TEM.39Eaton P. Quaresma P. Soares C. Neves C. de Almeida M.P. Pereira E. West P. A direct comparison of experimental methods to measure dimensions of synthetic nanoparticles.Ultramicroscopy. 2017; 182: 179-190https://doi.org/10.1016/j.ultramic.2017.07.001Crossref PubMed Scopus (164) Google Scholar Cross-section measurement curves demonstrate a restoration of membrane “smoothness” in reconstituted nanoparticles, mimicking fresh formulation (Figure S9). Next, we delivered the lyophilized Lung-Exos via DPI, where ex vivo images (Figure 2F) of mouse lungs who received fresh (fresh lyophilized) and 28-day-old (28-day lyophilized) dry powder Lung-Exos had no significant difference in exosomal mRNA and protein distribution (Figure 2G). mRNA activity showed greater variability at it 28-day-old state, but protein activity remained more stable.Figure 2Stability and distribution of lung-derived exosomes in dry powder formulation in the murine lungShow full caption(A) Schematic of mRNA and protein-loaded lung-derived exosome lyophilization, encapsulation, rodent DPI administration, and ex vivo histology. Created with BioRender.com.(B) Heatmaps of RFP leakage from Lung-Exos, HEK-Exos, and Lipos detected by ELISA; n = 2 per group.(C) Representative AFM height (I), amplitude (II), and phase (III) images of Lung-Exos; scale bar: 50 nm.(D) Quantification of the height and diameter of Lung-Exos, HEK-Exos, and Lipos from AFM images; n = 9 per group; data are represented as mean ± standard deviation.(E) TEM images of Lung-Exos at frozen (Frozen) or room (Lyophilized) temperatures; scale bar: 50 nm.(F) Ex vivo images of mouse lungs that received fresh lyophilized (0 days) and 28-day-old lyophilized Lung-Exos via dry powder inhalation after 24 h.(G) Quantification of the integrated density of GFP and RFP fluorescence in ex vivo mouse lungs 24 h after fresh (Fresh-Lyos) and 28-day-old (28-Day Lyos) dry powder inhalation; n = 3 per group; data are represented as mean ± standard deviation.(H) Quantification of the integrated density of GFP and RFP fluorescence in ex vivo mouse lungs 24 h after nebulization and fresh (Fresh-Lyos) dry powder inhalation; n = 3 per group; data are represented as mean ± standard deviation.(I) Quantification of the integrated density of GFP and RFP fluorescence in ex vivo mouse lungs 24 h after nebulization and 28-day-old (28-Day Lyos) dry powder inhalation; n = 3 per group; data are represented as mean ± standard deviation.View Large}, number={9}, journal={Matter}, publisher={Elsevier BV}, author={Popowski, Kristen D. and Moatti, Adele and Scull, Grant and Silkstone, Dylan and Lutz, Halle and López de Juan Abad, Blanca and George, Arianna and Belcher, Elizabeth and Zhu, Dashuai and Mei, Xuan and et al.}, year={2022}, month={Sep}, pages={2960–2974} }