Supramolecular Organization Predicts Protein Nanoparticle Delivery to Neutrophils for Acute Lung Inflammation Diagnosis and Treatment

Jacob W. Myerson; Priyal N. Patel; Nahal Habibi; Landis R. Walsh; Yi-Wei Lee; David C. Luther; Laura T. Ferguson; Michael H. Zaleski; Marco E. Zamora; Oscar A. Marcos-Contreras; Patrick M. Glassman; Ian Johnston; Elizabeth D. Hood; Tea Shuvaeva; Jason V. Gregory; Raisa Y. Kiseleva; Jia Nong; Kathryn M. Rubey; Colin F. Greineder; Samir Mitragotri; George S. Worthen; Vincent M. Rotello; Joerg Lahann; Vladimir R. Muzykantov; Jacob S. Brenner

Single cell suspensions were prepared from lungs for flow cytometric analysis of cell type composition of the lungs and/or nanoparticle distribution among different cell types in the lungs. C57BL/6 male mice were anesthetized with ketamine/xylazine (10 mg/kg ketamine, 100 mg/kg xylazine, intramuscular administration) prior to installation of tracheal catheter secured by suture. After sacrifice by terminal exsanguination via the vena cava, lungs were perfused by right ventricle injection of ~10 mL of cold PBS. The lungs were then infused via the tracheal catheter with 1 mL of a digestive enzyme solution consisting of 5 U/mL dispase, 2.5 mg/mL collagenase type I, and 1 mg/mL of DNAse I in cold PBS. Immediately after infusion, the trachea was sutured shut while removing the tracheal catheter. The lungs with intact trachea were removed via thoracotomy and kept on ice prior to manual disaggregation. Disaggregated lung tissue was aspirated in 2 mL of digestive enzyme solution and incubated at 37°C for 45 minutes, with vortexing every 10 minutes. After addition of 1 mL of fetal calf serum, tissue suspensions were strained through 100 µm filters and centrifuged at 400xg for 5 minutes. After removal of supernatant, the pelleted material was resuspended in 10 mL of cold ACK lysing buffer. The resulting suspensions were strained through 40 µm filter and incubated for 10 minutes on ice. The suspensions were centrifuged at 400xg for 5 minutes and the resulting pellets were rinsed in 10 mL of FACS buffer (2% fetal calf . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.037564 doi: bioRxiv preprint serum and 1 mM EDTA in PBS). After centrifugation at 400xg for 5 minutes, the rinsed cell pellets were resuspended in 2% PFA in 1 mL FACS buffer for 10 minutes incubation. The fixed cell suspensions were centrifuged at 400xg for 5 minutes and resuspended in 1 mL of FACS buffer.