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

A mouse was anesthetized with ketamine/xylazine five hours after intravenous administration of 2 mg/kg B4 LPS. A jugular vein catheter was fixed in place for injection of lysozyme-dextran nanogels, anti-CD45 antibody, and fluorescent dextran during imaging. In preparation for exposure of the lungs, a patch of skin on the back of the mouse, around the juncture between the ribcage and the diaphragm, was denuded. While the mouse was maintained on mechanical ventilation, an incision at the juncture between the ribs and the diaphragm, towards the posterior, exposed a portion of the lungs. A coverslip affixed to a rubber o-ring was sealed to the incision by vacuum. The exposed portion of the mouse lung was placed in focus under the objective by locating autofluorescence signal in the "FITC" channel. With 100 ms exposure, fluorescent images from channels corresponding to violet, green, near red, and far red fluorescence were sequentially acquired. A mixture of rhodamine-dextran nanogels (2.5 mg/kg), Brilliant Violet-conjugated anti-CD45 antibody (0.8 mg/kg), and Alexa Fluor 647 labeled 70 kDa dextran (40 mg/kg) for vascular contrast was administered via jugular vein catheter and images were recorded for 30 minutes. Images were recorded in SlideBook software and opened in ImageJ (FIJI distribution) for composition in movies with coregistration of the four fluorescent channels.