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

We traced recently-developed poly(glutamate) tagged green fluorescent protein (E-GFP) NPs, representing a third class of protein NP based on electrostatic interactions between proteins and carrier polymer or metallic particles. 43 Negatively-. CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.037564 doi: bioRxiv preprint charged E-GFP was paired to arginine-presenting gold nanoparticles (89.0±1.6 nm diameter, PDI 0.14±0.04) or to poly(oxanorborneneimide) (PONI) functionalized with guanidino and tyrosyl side chains (158.9±6.2 nm diameter, PDI 0.17±0.03) (Supplementary Figure 1D) . For biodistribution experiments with PONI/E-GFP hybrid NPs, tyrosine-bearing PONI was labeled with 131 I and E-GFP was labeled with 125 I, allowing simultaneous tracing of each component of the hybrid NPs. The two E-GFP NPs, with structure based on charge interactions, had specificity for IV LPS-injured lungs. Comparing uptake in LPS-injured lungs to naïve lungs, we observe an LPS:naïve ratio of 2.37 for PONI/E-GFP NPs as traced by the PONI component, 2.57 for PONI/E-GFP NPs as traced by the E-GFP component, and 2.79 for Au/E-GFP NPs ( Figure 3C , Supplementary Figure 10 ). PONI/E-GFP particles, specifically, accumulated in LPSinjured lungs at 26.77% initial dose as measured by PONI tracing and 27.24% initial dose as measured by GFP tracing, indicating effective co-delivery in the inflamed organ. Acute inflammatory injury therefore resulted in a two-to three-fold increase in pulmonary uptake of NPs constructed via electrostatic protein interactions. Nanoparticles Based on Symmetric Protein Organization Adeno-associated virus (AAV), adenovirus, and horse spleen ferritin nanocages were employed as examples of protein-based NPs with highly symmetrical structure (See Supplementary Figure 1D for DLS confirmation of structure). [44] [45] [46] For each of these highly ordered protein NPs, IV LPS injury had no significant effect on biodistribution and levels of uptake in the injured lungs were minimal ( Figure 3D Table 5 ). Therefore, highly ordered protein NPs traced in our studies did not have tropism for the lungs after acute inflammatory injury.