Khan, I

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 Background Sputum color is a visual marker of airway inflammation and disease activity in bronchiectasis, ranging from clear or light gray (mucoid) to yellow-green (purulent), the latter largely reflecting neutrophil myeloperoxidase release. This study evaluated associations between sputum color and common clinical markers of disease activity to determine which parameters differed most between color categories. Methods Forty spontaneous sputum samples were analyzed from 80 consecutive adults with non-cystic fibrosis bronchiectasis (median age 70 years [IQR 12]; 74% female) enrolled between January 2024 and April 2025 in an observational study. Sputum color was graded using the Murray Sputum Color Chart and classified as mucoid (color #1) or purulent (colors #2-4). Clinical parameters, including blood cell counts, C-reactive protein (CRP), fractional exhaled nitric oxide (FeNO), and results of bacterial and acid-fast bacillus cultures (induced or bronchoscopic), were obtained from medical records if available within one month of enrollment. Categorical variables were analyzed using chi-square or Fisher’s exact tests and continuous variables with the Mann-Whitney U test. A secondary analysis compared patients who could versus could not produce spontaneous sputum. Results Of 40 samples, 20 (50%) were purulent. Blood neutrophil counts (median 4.5 vs. 3.9 x109/L), neutrophil-to-lymphocyte ratios (2.5 vs. 2.6), and eosinophil counts (1.7 vs. 1.7 x109/L) did not differ significantly between groups (n = 26). FeNO was normal in all 10 patients with available data and was not analyzed further. CRP was higher among patients with purulent sputum (4.6 vs. <3 mg/L; n = 20; p = 0.044). Bacterial growth was more frequent in purulent than mucoid samples (73% vs. 31%; n = 31; p = 0.032). Isolates in the purulent group included Klebsiella, Pseudomonas, Staphylococcus, and Streptococcus (n = 2 each), and single isolates of Acetobacter, Acinetobacter, Chryseobacterium, Delftia, Enterobacter, Escherichia, and Moraxella. In the mucoid group, Pseudomonas, Staphylococcus (n = 2 each), and Stenotrophomonas (n = 1) were identified. Mycobacterial growth did not differ significantly (20% vs. 12.5%; n = 31; p = 0.65). Patients unable to produce spontaneous sputum had a higher rate of mycobacterial isolation on induced/bronchoscopic samples (43% vs. 16%; n = 54; p = 0.027), though other parameters were similar. Over 90% of measurements were obtained within three days of enrollment, and freezing did not appear to alter sputum color. Conclusions Even within a small cohort, purulent sputum was associated with higher CRP and greater bacterial growth, supporting sputum color as a rapid, inexpensive marker of disease activity in bronchiectasis. Further research should clarify whether sputum color primarily reflects bacterial colonization, airway inflammation, or both. This abstract is funded by: None