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

Similar staining and analysis protocols enabled identification of nanoparticle distribution among different cell types in the lungs. To enable fluorescent tracing, lysozyme-dextran nanogels contained FITC-dextran, DBCO-IgG liposomes contained green fluorescent Top Fluor PC lipid, and crosslinked albumin nanoparticles were labeled with NHS ester Alexa Fluor 488. Alexa Fluor 488 labeling of albumin nanoparticles was accomplished by incubation of the NHS ester fluorophore with nanoparticles at 1:25 mass:mass fluorophore:nanoparticle ratio for two hours on ice. Excess fluorophore was removed from nanoparticles by 3-fold centrifugation at 16000xg for 15 minutes followed by washing with PBS. Nanoparticles were administered at 2.5 mg/kg via jugular vein injection and circulated for 30 minutes, prior to preparation of single cell suspensions from lungs as above. Fixed single cell suspensions were stained with APC-conjugated anti-Ly6G or PerCP-conjugated anti-CD45 as above. Additional suspensions were stained with 1:150 dilution of APC-conjugated anti-CD31, in lieu of anti-Ly6G, to identify endothelial cells. Association of nanoparticles with cell types was identified by coincidence of green fluorescent signal with anti-CD45, anti-Ly6G, or anti-CD31 signal.