(266g) Modulation of Neutrophil Extracellular Trap Formation through Polymer Coating of Metal Oxide Nanoparticles

Given that 1 in 8 women develop breast cancer throughout their lifetime, the disease is a substantial public health burden. Patient morbidity and mortality occurs primarily due to metastatic spread, but is compounded by thrombosis, or the formation of blood clots. Recently, neutrophils activated by the inflammatory tumor microenvironment have been observed to release web-like DNA fibers known as neutrophil extracellular traps (NETs). NETs have been linked to several negative downstream effects including thrombosis, damage to vessel endothelial cells, and capture of circulating tumor cells to promote metastasis. Due to the pathogenic role of NETs in breast cancer, it is critical to ensure that contrast agents used in diagnostic imaging do not elicit this response. Breast MRI is often used as a supplement to mammography for high-risk women, as it detects breast cancers that may be missed by mammography and provides superior soft-tissue contrast; however, the standard MRI contrast agents used in the clinic (e.g. gadolinium chelates) cause high false-positive rates, as high as 25%. Thus, alternative contrast agents to improve diagnostic accuracy are being developed, including metal oxide nanoparticles such as iron oxide (Fe₃O₄) and manganese oxide (MnO). Prior studies have demonstrated that certain nanoparticle formulations may promote NETosis, which could encourage cancer progression. The goal of this study was to evaluate the effect of altered polymer coating of MnO nanoparticles on NET formation.

MnO nanocrystals were synthesized using thermal decomposition of Mn (II) acetylacetonate in dibenzyl ether and oleylamine at 280°C. Nanocrystal size and chemistry were evaluated with TEM, XRD, and FTIR. Hydrophobic MnO nanocrystals were encapsulated with biodegradable poly(lactic-co-glycolic acid) (PLGA) and increasing amounts of polyethylene glycol (PEG) including 0%, 2.5%, 5%, and 10%. Hydrodynamic particle size was evaluated by DLS and confirmed to be ~180 nm. Neutrophils were isolated from the bone marrow of healthy female BALB/c mice femurs via density gradient. Neutrophils were stained with CellTracker Deep Red, plated, and stimulated for 3 hours with either media only (negative control), phorbol-12-myristate-13-acetate (PMA) (positive control), unencapsulated MnO nanoparticles, or PLGA MnO nanoparticles with 0-10% attached PEG. DNA was stained with Hoechst and plates were imaged with an inverted confocal fluorescence microscope to evaluate total NET formation. Nikon General Analysis software was used to quantify the number of neutrophils and NETs per field of view.

The greatest NET formation resulted from PMA stimulated neutrophils with an average of 35.83 NETs per 100 neutrophils. Unstimulated neutrophils caused the second-highest amount of NET formation at 19.28 NETs per 100 neutrophils. Bare MnO nanocrystals and PLGA MnO nanoparticles without PEG attached resulted in similar NETosis to unstimulated controls, whereas particles coated with a low percentage of PEG, provoked less NETosis than did stimulated neutrophils; 2.5% PEG PLGA nanoparticles had the smallest response of 11.06 NETs per 100 neutrophils. These results indicated that polymer-coated MnO nanoparticles do not enhance NETosis, supporting their safety as future breast cancer MRI contrast agents. Future studies will elucidate the mechanism responsible for the observed reduced NETotic response with nanoparticle PEG coating including assessment of nanoparticle stability, particle uptake into neutrophils, neutrophil activation, and formation of reactive oxygen species as a result of nanoparticle exposure.