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Abstract Introduction The inhalation of aerosolized therapeutics is a promising strategy for the targeted delivery of drugs and biologics to the lungs. We have previously demonstrated the efficacy and feasibility of nebulized exosomes using a platelet-derived exosome product (PEP: Rion, Inc.), which significantly attenuated emphysema in a murine model (PMID: 38325750). However, the PEP aerosol’s physical properties—such as size, tissue deposition, anatomic location, and stability remain incompletely characterized. To address this, we utilized a cascade impactor to define aerosol particle size and deposition patterns and employed immunofluorescence staining to assess the regional and anatomic localization of nebulized PEP within the murine lung. Methods PEP particle size and tissue deposition were determined by passing the aerosol through a Marple cascade impactor (CI, DSI), which was connected to an inhalation tower (DSI). PEP aerosols were generated by a vibrating mesh nebulizer (Aerogen, Cat No AG-AL1100) at a controlled velocity and humidity and delivered to the CI at a flow rate of 2L/min. Aerosols were separated by size and allowed to deposit at the corresponding stages as they passed through the pinholes of the stage, by design. The stages were weighed after nebulization, and the percentage of aerosol deposition was calculated to simulate the human respiratory tree, according to the manufacturer’s specifications. In parallel, fluorescence DiR-labeled PEP was nebulized to spontaneously breathing mice, and confocal microscopy was used to determine intrapulmonary distribution. Results A total of 2.25 mL of PEP reconstituted in 0.9% normal saline (11.4 mg/mL final concentration) was nebulized for 35 minutes at a flow rate of 2 L/min, yielding 3.5 mg of total aerosol deposition, which is 13.6% of the total PEP mass (25.65mg) being nebulized, across the CI stages. Of this, 74% of the aerosolized particles with a particle size ≤ 1µm and deposited in Stages 7, 8 & F, corresponding to the deep regions of the lungs, specifically regions beyond the alveolar ducts and within the alveoli. In vivo 3D confocal imaging corroborated these findings, revealing abundant DiR-labeled PEP deep into the alveolar space within the peripheral regions of all lung lobes. Conclusion The study result demonstrates the feasibility and capability of delivering aerosolized PEP deeply into the alveolar regions of the lung at the given pressure, humidity, flow rate and nebulizer settings. This study establishes a methodological framework for characterizing and optimizing aerosolized exosome therapeutics for precise, region-specific delivery within the respiratory tract. This abstract is funded by: Department of Defense, Mayo Clinic Funding

Abstract Rationale Alpha1-antitrypsin (AAT; gene name SERPINA1) is a critical antiprotease that protects the lung from neutrophil elastase-mediated injury. AAT deficiency predisposes to emphysema, which is further exacerbated by cigarette smoke (CS) exposure. We previously reported (PMID: 38325750) that a nebulized human platelet-derived exosome product (PEP) attenuated CS-induced emphysema in C57BL/6 mice. Given the presence of AAT protein in these platelet-derived exosomes (identified by proteomics), we hypothesized that PEP nebulization would significantly attenuate both spontaneous and CS-induced emphysema in SERPINA1 knockout (KO) mice. Methods Transgenic SERPINA1 KO C57BL/6 mice (both sexes) exposed to CS (10 cigarettes/day and 5 days/week) for 2 months were then randomized to receive nebulized PEP or 0.9% saline (placebo) twice weekly for 4 additional weeks, with continued CS exposure. PEP was reconstituted in 0.9% saline (22.7mg/mL; ∼3 × 10¹0 vesicles/mL) and administered via pressure- and humidity-controlled nose-only inhalation tower (2.25 mL of PEP or saline over 35 minutes). Lung static compliance (Cst), cutaneous O2 saturation, mean linear intercepts (Lm), and plethysmographic parameters were measured during and after the study. Results Histological analysis of the lung tissue sections revealed that PEP significantly attenuated the CS-induced increase in alveolar size, as determined by measurement of the mean linear intercepts (Lm, p < 0.001). Lung static compliance (Cst), which was increased in CS-exposed mice, was also significantly decreased in the PEP-treated group (p < 0.001) compared to placebo in the male and female combined group. Functional respiratory parameters further supported the protective effect of PEP in this AAT-deficient model. In female mice, PEP nebulization significantly improved several CS-impaired pulmonary function parameters, including minute volume (MV, p = 0.024), the flow at the point 50% of tidal volume is expired (EF50, p = 0.021), Peak Inspiratory Flow (PIF, p = 0.009), Peak Expiratory Flow (PEF, p = 0.018), and O2 saturation (p = 0.035). However, these improvements were not observed in male mice. Conclusions These findings extend prior observations and underscore the therapeutic potential of nebulized AAT-enriched PEP directly to the alveolar region, effectively attenuating injury and improving pulmonary function in AAT deficiency-related emphysema. This abstract is funded by: Department of Defense, Mayo Clinic Funding, The Young and Middle-aged Medical High-end Talents in Chongqing Program

Abstract Introduction We previously demonstrated that nebulized platelet-derived exosome product (PEP) alleviates cigarette smoke (CS)-induced murine emphysema. To elucidate the underlying molecular mechanisms, we conducted single-nucleus RNA sequencing of lung tissue, which initially revealed a shift in the cell source for TNF signaling (a critical regulator of inflammation in COPD lungs) from innate lymphoid cells (ILC) to alveolar macrophages (aMac) after chronic CS exposure. In this study, we conducted a more in-depth analysis of cellular communication to determine the mechanism by which PEP modulates CS-induced TNF signaling in the lung microenvironment. Methods Single-cell transcriptomics and cell-cell communication analyses were performed between CS-exposed placebo control lungs vs. sham control lungs and CS-exposed PEP-treated vs. placebo control lungs using R packages, including Seurat, CellChat, Patchwork, Future, and NMF. A comparative analysis of ligand-receptor interactions across these datasets was conducted using CellChat to infer intercellular signaling networks. Results Single-nuclear RNA-seq analysis revealed that the Thy1-ITGAX_ITGB2 signaling pathway was significantly upregulated by CS, acting as the adhesive and activating bridge that facilitates the switch in TNF production from ILC to aMac. Thy1 is a GPI-anchored ligand expressed on ILCs, and ITGAX_ITGB2 is its cognate receptor on aMacs. Concurrently, Ptprc-Mrc1 signaling, an inhibitory pathway in ILC-aMac crosstalk, was downregulated, contributing to sustained aMac activation by CS. Furthermore, analysis of aMac-aMac interactions revealed upregulated GRN/Sort1 signaling, which promotes the internalization of progranulin, an anti-inflammatory glycoprotein that competes for TNF/TNFR signaling. Ptprc-Mrc1 signaling was also downregulated within the aMac autocrine/paracrine network, further perpetuating a pro-inflammatory state. In contrast, treatment with PEP downregulated Thy1-ITGAX_ITGB2 signaling and upregulated Lamc1-Cd44 signaling, a hallmark of aMac deactivation, thereby inducing a phenotypic switch in inflammatory macrophages toward a reparative M2 state, promoting the resolution of CS-induced chronic inflammation. Conclusions These data suggest that the transition of TNF production from ILCs to aMacs under chronic CS stress is driven by increased Thy1-ITGAX_ITGB2 interactions and decreased Ptprc-Mrc1 signaling between ILCs and aMacs crosstalk within the local tissue. Enhancing the crosstalk between Lamc1-Cd44 and Ptprc-Mrc1 may suppress TNF production and attenuate CS induced aMac activation. Targeting these pathways, as demonstrated by nebulized PEP treatment, holds therapeutic potential to deactivate inflamed aMacs and promote homeostasis in the inflamed lung. This abstract is funded by: Department of Defense, Mayo Clinic Funding