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 conjugation to liposomes, rat IgG was modified with dibenzylcyclooctyne-PEG4-NHS ester (DBCO, Jena Bioscience). IgG solutions (PBS) were adjusted to pH 8.3 with 1 M NaHCO3 buffer and reacted with DBCO for 1 hour at room temperature at molar ratios of 2.5:1, 5:1, 10:1, or 20:1 DBCO:IgG. Unreacted DBCO was removed after reaction via centrifugal filtration against 10 kDa cutoff filters (Amicon . DBCOmodified IgG was incubated with azide liposomes at 200 IgG per liposome overnight at room temperature. Unreacted antibody was removed via size exclusion chromatography, and purified liposomes were concentrated to original volume against centrifugal filters (Amicon). Maleimide liposomes were also prepared via lipid film hydration. 66 Lipid films comprised 54% DPPC, 40% cholesterol, and 6% MPB-PE (1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide]), with lipids prepared, dried, resuspended, and extruded as described above for azide liposomes.