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

Previous work has indicated that NPs based on denatured albumin accumulate in neutrophils in inflamed lungs and at sites of acute vascular injury, whereas NPs coated with native albumin do not. 26, 27 We have characterized lysozyme-dextran nanogels and crosslinked human albumin NPs with circular dichroism (CD) spectroscopy to compare secondary structure of proteins in the NPs to secondary structure of the native component proteins (Supplementary Figure 7A-B) . Identical CD spectra were recorded for LDNGs vs. lysozyme and for albumin NPs vs. human albumin. Deconvolution of the CD spectra via neural network algorithm trained against a library of CD spectra for known structures verified that secondary structure composition of lysozyme and albumin was unchanged by incorporation of the proteins in the NPs. 40 Free protein and protein NPs were also probed with 8-anilino-1naphthalenesulfonic acid (ANSA), previously established as a tool for determining the extent to which hydrophobic domains are exposed on proteins. 41 Consistent with known structures of the two proteins, ANSA staining indicated few available hydrophobic domains on lysozyme and substantial hydrophobic exposure on albumin (Supplementary Figure 7C -D, blue curves). LDNGs had increased hydrophobic accessibility vs. native lysozyme whereas albumin NPs had reduced hydrophobic accessibility compared with native albumin. Therefore, our data indicate that lysozyme and albumin are not denatured in LDNGs and albumin NPs, but the NPs composed of the two proteins present a balance of hydrophobic and hydrophilic surfaces differing from the native proteins.