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

Protein, horse spleen ferritin nanocages (Sigma), or adeno-associated virus (empty capsids, serotype 8) were prepared in PBS at concentrations between 1 and 2 mg/mL in volumes between 100 and 200 µL. Films of oxidizing agent were prepared in borosilicate tubes by drying 300 µL of 0.5 mg/mL Iodogen (Perkin-Elmer, chloroform solution) under nitrogen gas. Alternatively, Iodobeads (Perkin-Elmer) were added to borosilicate tubes (one per reaction). Protein solutions were added to coated or beadcontaining tubes, before addition of Na 125/131 I at 25 µCi per 100 µg of protein. Protein was incubated with radioiodine at room temperature for 5 minutes under parafilm in a ventilated hood. Iodide-protein reacottions were terminated by purifying protein solutions through a 7 kDa cutoff gel column (Zeba). Additional passages through gel filtration columns or against centrifugal filters (Amicon, 10 kDa cutoff) were employed to remove free iodine, assuring that >95% of radioactivity was associated with protein.