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

Flow cytometry showed greater concentrations of neutrophils in LPS-injured lungs, compared to naïve lungs ( Figure 1C , counts above horizontal threshold indicate positive staining for neutrophils, Figure 1D , rightmost peak indicates positive staining for neutrophils). Comparison of Ly6G stain to total CD45-positive cells indicated 53.5% of leukocytes in the lungs were Ly6G-positive after LPS injury, compared to 5.6% in the naïve control ( Figure 1D , center panel). Comparison of Ly6G stain to IV CD45 stain indicated that the majority of neutrophils were intravascular, in both naïve and LPSinjured mice. In naïve mice, 85.3% of neutrophils were intravascular and in LPS-injured mice, 96.2% of neutrophils were intravascular ( Figure 1D , right panel). The presence of large populations of intravascular neutrophils following inflammatory injury is consistent with previously published observations. 6, 10, 14, 18, 33 Histological analysis confirmed results obtained with flow cytometry and radiolabeled anti-Ly6G biodistributions. Staining of lung sections indicated increased concentration of neutrophils in the lungs following IV LPS injury ( Figure 1E , left panels). Co-registration of neutrophil staining with tissue autofluorescence (indicating tissue architecture) broadly supported the finding that pulmonary neutrophils reside in the vasculature ( Figure 1E , right panels).