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

Lysozyme-dextran nanogels were prepared for chelation to 111 In by conjugation to S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA, Macrocyclics). Nanogels were moved to metal free pH 8.3 1 M NaHCO3 buffer by three-fold centrifugation (16000xg for 15 minutes) and pellet washing with metal free buffer. p-SCN-Bn-DOTA was added to nanogels at 1:25 mass:mass ratio, prior to reaction for 30 minutes at room temperature. Free p-SCN-Bn-DOTA was removed by three-fold centrifugal filtration against 10 kDa cutoff centrifugal filters, with resuspension of nanogels in metal-free pH 4 citrate buffer after each centrifugation. DOTA-conjugated nanogels or DTPA-containing liposomes in pH 4 citrate buffer were combined with 111 InCl3 for one-hour chelation at 37°C. Nanoparticle/ 111 InCl3 mixtures were treated with free DTPA (1 mM final concentration) to remove 111 In not incorporated in nanoparticles. Efficiency of 111 In incorporation in nanoparticles was assessed by thin film chromatography (aluminum/silica strips, Sigma) with 10 µM EDTA mobile phase. Chromatography strips were divided between origin and mobile front and the two portions of the strip were analyzed in a gamma counter to assess nanoparticleassociated (origin) vs. free (mobile front) 111 In. Free 111 In was separated from nanoparticles by centrifugal filtration and nanoparticles were resuspended in PBS (liposomes) or saline (nanogels). For SPECT/CT imaging experiments (see SPECT/CT Imaging methods below) with nanogels, 80 µCi of 111 In-labeled nanogels, used within one day 111 In labeling as described above, were administered to each mouse. For tracing 111 In-labeled liposomes in biodistribution studies, liposomes were labeled with 50 µCi 111 In per µmol of lipid.