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

For experiments with cell suspensions derived from human lungs (chosen for research use according to the above standards), single cell suspensions were generously provided by the laboratory of Edward E. Morrisey at the University of Pennsylvania. Aliquots of 600,000 cells were pelleted at 400xg for 5 minutes and resuspended in 100 µL PBS containing different quantities of lysozyme-dextran nanogels synthesized with FITC-labeled dextran. Cells and nanogels were incubated at room temperature for 30 minutes before two-fold pelleting at 400xg with 1 mL PBS washes. Cells were re-suspended in 200 µL FACS buffer for staining with APCconjugated anti-human CD45, applied by 20-minute incubation with a 1:500 dilution of the antibody stock. Cells were pelleted at 400xg for 5 minutes and resuspended in 200 µL PBS for immediate analysis with flow cytometry (BD Accuri). Negative/positive nanogel or anti-CD45 signal was established by comparison to unstained cells. Singlestained controls indicated no spectral overlap between FITC-nanogel fluorescence and anti-CD45 APC fluorescence.