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

Neutrophils are "first responder" cells in acute inflammation, rapidly adhering and activating in large numbers in inflamed vessels and forming populations of "marginated" neutrophils along the vascular lumen. [10] [11] [12] [13] [14] [15] [16] Neutrophils can be activated by a variety of initiating factors, including pathogen-and damage-associated molecular patterns such as bacterial lipopolysaccharides (LPS). 17, 18 After acute inflammatory insults, neutrophils marginate in most organs, but by far most avidly in the lung capillaries. 6, 14, 15, 19, 20 Neutrophils are therefore key cell types in most forms of ARDS. In ARDS, marginated neutrophils can secrete tissue-damaging substances (proteases, reactive oxygen species) and extravasate into the alveoli, leading to disruption of the endothelial barrier and accumulation of neutrophils and edematous fluid in the air space of the lungs ( Figure 1A ). 6, 15, 16, [19] [20] [21] Targeted nanoparticle delivery to marginated neutrophils could provide an ARDS treatment with minimal side effects, but specific delivery to marginated neutrophils remains an open challenge. Antibodies against markers such as Ly6G have achieved targeting to neutrophils in mice, but also deplete populations of circulating neutrophils. [22] [23] [24] [25] Additionally, while Ly6G readily marks neutrophils in mice, there is no analogous specific and ubiquitous marker on human neutrophils. 22 Therefore, antibody targeting strategies have not been widely adopted for targeted drug delivery to these cells. 24 As another route to neutrophil targeting, two previous studies noted that activated neutrophils take up denatured and agglutinated bovine albumin, concluding that denatured protein was critical in neutrophil-particle interactions. 26, 27 Nanoparticle structural properties such as shape, size, and deformability can define unique targeting behaviors. [28] [29] [30] [31] [32] Here, we screened a diverse panel of nanoparticles to determine the nanostructural properties that predict uptake in pulmonary marginated neutrophils during acute inflammation. As a high-throughput animal model for ARDS, we administered LPS to mice, causing a massive increase in pulmonary marginated neutrophils. We show that two initial leads in our screen, lysozyme-dextran nanogels (LDNGs) and crosslinked albumin nanoparticles (ANPs), selectively home to marginated neutrophils in inflamed lungs, but not naïve lungs. In our subsequent screen of over 20 diverse nanoparticles, we find that 13 protein nanoparticles, all defined by agglutination of protein in amorphous nanostructures (nanoparticles with agglutinated proteins, NAPs), but not by denatured protein, have specificity for LPS-inflamed lungs. In contrast to NAPs, we demonstrate that three symmetric protein nanostructures (viruses/nanocages) have biodistributions unaffected by LPS injury. We show that polystyrene nanoparticles and five liposome formulations do not accumulate in injured lungs, indicating that nanostructures that are not based on protein are not intrinsically drawn to marginated neutrophils in acute inflammation. We then engineered liposomes (the most clinically relevant nanoparticle drug carriers) as NAPs, through conjugation to protein modified with hydrophobic cyclooctynes, encouraging protein agglutination on the liposome surface by hydrophobic interactions. We thus show that supramolecular organization of proteins, rather than chemical composition, best predicts uptake in mar