Researcher(s)
- Vilina Akala, Biomedical Engineering, University of Delaware
Faculty Mentor(s)
- Catherine Fromen, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Neutrophils have garnered attention as a method for combating various pathologies due to their varied functionality and designation as a phagocyte. However, neutrophil-based therapeutics face roadblocks that limit their adoption, including low ex vivo viability, phenotype maintenance, and quantity for a therapeutically relevant dose. The Fromen lab has characterized a nanoparticle (NP) therapeutic with the capacity to prolong phagocyte lifespan while minimizing phenotypical changes. We hypothesize that this survival response is conserved across phagocytes and can facilitate a non-activating stimulus to promote neutrophil survival in vitro. A histopage-based density gradient was used to isolate neutrophils from c57B6 mice bone marrow and confirmed using flow cytometry and antibody stains (CD11b+, CD45+, MHCii–, Ly6G+ and CD182). Flow cytometry analysis after isolation showed that there were significantly lower monocyte counts than neutrophils. Furthermore, there was no significant difference between naive and mature neutrophil count, confirmed with low and high CD182 expression respectively. After isolation, neutrophils were dosed with positive (AEM) and negative (CEA) PEGDA nanoparticles to observe if NPs were phagocytosed and a corresponding lifespan increase as hypothesized. The results showed that neutrophils phagocytosed both NPs, with naive neutrophils having the highest uptake of CEA NPs. Neutrophil count was analyzed after, which showed a significant decrease in count for both particle dosing conditions. To determine if the loss was due to NP phagocytosis, cell death over time, or neutrophil activation, neutrophils were then dosed with varying CEA concentrations (10, 20, 40, 50 ng/mL) and LPS as an inflammatory control. An antibody stain of CD63 was applied to analyze if degranulation occurs. Results showed that cell death was not related to higher dosage of NP’s and that CD63 expression was insignificantly different from the LPS condition, showing no degranulation. Future work would include analyzing additional types of neutrophil activation and extracellular vesicle secretion.