617 - Neutrophils retain bacterial phagocytic capacity after interaction with nanoparticles with agglutinated protein (NAP)

Poster Number: 617
Publication Number: 617.200

Kathryn Rubey, Children's Hospital of Philadelphia, Philadelphia, PA, United States; Jichuan Wu, Perelman School of Medicine at the University of Pennsylvania, Mount Laurel, NJ, United States; Jacob W. Myerson, University of Pennsylvania, Philadelphia, PA, United States; Jacob Brenner, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; G. Scott Worthen, Childrens Hospital of Philadelphia, Philadelphia, PA, United States

Background: Using an in vivo model of systemic inflammation, as well as in vitro modeling, we have previously shown that nanoparticles with agglutinated protein (NAPs) are taken up by both murine and human neutrophils (Nature Nanotechnology accepted). Uptake of NAPs by neutrophils is enhanced by complement protein C3b coating the NAPs. We aim to improve treatment of pneumonia by delivering antibiotic-loaded NAPs to lung neutrophils responsible for phagocytosing and killing the offending bacteria. Whether presence of NAPs changes the ability of neutrophils to phagocytose and kill bacteria is unknown. In this study, we examine uptake and localization of bacteria within NAP-containing neutrophils.

Objective: We hypothesize that neutrophil phagocytosis of NAPs and bacterial bioparticles is enhanced by serum opsonization, and that bacterial phagocytosis is preserved after neutrophil interaction with NAPs.

Design/Methods: Murine neutrophils were enriched by magnetic cell sorting of bone marrow. For complement opsonization, FITC-nanogels were exposed to mouse serum. Purified neutrophils were incubated with serum-opsonized nanogels. Neutrophils were then incubated with pHrodo red E. coli bioparticles. Bacterial uptake was quantified via flow cytometry, and cell localization was analyzed via confocal microscopy.

Results: Our results show that neutrophils are still able to phagocytose after interacting with nanoparticles. The mean fluorescence, surrogate measure of particle uptake, showed no correlation between FITC and pHrodo red fluorescence, suggesting that there is not decreased bacterial phagocytosis as more nanoparticles are phagocytosed. On microscopic evaluation, there is partial co-localization of nanoparticles with bacteria within the neutrophil.Conclusion(s): We have previously shown that nanogels are taken up by neutrophils in a complement-enhanced interaction, and inflammation causes concentration within the lungs. These in vitro results presented here, suggest that neutrophils that home to the lungs in pneumonia will be able to phagocytose both nanoparticles and pathogenic bacteria, and will colocalize them within the cell, key steps to being able to improve bacterial killing by the neutrophil. Our next steps will be to continue this work with antibiotic-loaded nanoparticles, and then translating into an in vivo model.