(264e) Neutrophil?Particle Interactions in Blood Circulation Drive Particle Clearance and Alter Neutrophil Responses in Acute Inflammation

Introduction: Particulate drug carriers for targeted drug delivery have been widely studied for their potential to enhance the therapeutic impact of treatments for a variety of diseases, including cancer, cardiovascular disease, and inflammation. However, the interactions between these drug carriers and circulating leukocytes, particularly neutrophils, remain relatively explored. Such interactions are crucial, as they may impact the ultimate therapeutic efficacy of particulate drug carriers, and may have a therapeutic impact themselves. In this work, we sought to determine what impact, if any, injectable particulate drug carriers have on neutrophil adhesion and transmigration in an inflammatory environment in vivo.

Materials and Methods: The impact of injected particulate drug carriers on neutrophil adhesion and migration was evaluated using two different in vivo models: a mesentery inflammation model, and an acute lung injury model. For the mesentery inflammation model, female C57BL/6 mice (Jackson) between 3 and 4 weeks in age were used. Neutrophil rolling and adhesion in mesenteric veins were visualized using a 25× oil objective on an inverted fluorescence microscope (Zeiss Axio Observer Z1Marianas microscope) using Slidebook 6 software. Mice were anesthetized, and a tail vein catheter was placed for delivery of reagents. Mice were placed on a custom-made microscopic heated stage at 37 °C, and the mesentery was exteriorized to a glass coverslip through a midline incision. Rhodamine 6G (Rh6G, Sigma, 100 μL of 0.1 mg/mL in PBS) or antiLy6G (Biolegend) was injected IV, and a local injury was induced by topical application of TNF-α (Fitzgerald, 10 μL of 200 μg/mL in PBS). FITC-labeled particles suspended in PBS were injected via IV catheter 3 min following topical TNF-α application and imaged for another 7 min. Analysis was performed using Slidebook 6 and ImageJ by a blinded investigator. Images were recorded continuously in green, red, and bright field channels every 100 ms. Vessels were chosen in each mouse based on size and vessel exposure, with the average diameter of veins ranging from 90 to 190 μm, with an average of 130 μm. The total number of NΦs and particles were counted per frame to obtain average NΦ counts per 3 s of capture in 125 μm length of mesentery vessel, corresponding to 30 frames at 100 ms/ frame.

For the acute lung injury model, male C57BL/6 and BALB/c mice (Jackson) were anesthetized using isofluorane, and then LPS (20 μg of LPS at 0.4 mg/mL) was delivered to the lungs through an orotracheal instillation in a 50 μL volume in PBS. One hour after instillation, mice were injected with a suspension of particles in PBS through the tail vein. Mice received 2 μm particles or 0.5 μm particles in 200 μL injection volume, corresponding to ∼0.6 mg/mouse, ∼30 mg/kg. Two or 3 h after LPS instillation, mice were euthanized, and blood was collected via cardiac puncture in acid citrate dextrose (ACD) and perfused with PBS. BALF was collected by inserting a cannula in an incision in the trachea and flushing the lungs with three sequential 1 mL washes of PBS. BALF cells were obtained by centrifugation, separating BALF cells from the supernatant. Following the lavage, organs were harvested and imaged using an Odyssey CLx infrared imaging system (LI-COR), and whole organ scans performed. Following NIR scan, lung, liver, and spleen were homogenized into single-cell suspensions or frozen for histological examination. Frozen samples were embedded in OCT (Fisher) in disposable cassettes and flash frozen with an isopentane slurry; additional liver samples were fixed in 4% paraformaldehyde (PFA) or flash frozen in liquid nitrogen and stored for histology or protein analysis.

Results and Discussion: Circulating blood neutrophils in vivo were found to be capable of rapidly binding and sequestering injected carboxylate-modified particles of both 2 and 0.5 μm diameter within the bloodstream. These neutrophil−particle associations within the vasculature were found to suppress neutrophil interactions with an inflamed mesentery vascular wall and hindered neutrophil adhesion. Furthermore, in a model of acute lung injury, intravenously administered drug-free particles reduced normal neutrophil accumulation in the airways of C57BL/6 mice between 52% and 60% versus particle-free mice and between 93% and 98% in BALB/c mice. This suppressed neutrophil migration resulted from particle-induced neutrophil diversion to the liver.

Conclusions: These data indicate a considerable acute interaction between injected particles and circulating neutrophils that can drive variations in neutrophil function during inflammation and implicate neutrophil involvement in the clearance process of intravenously injected particle therapeutics. Such an understanding will be critical toward both enhancing designs of drug delivery carriers and developing effective therapeutic interventions in diseases where neutrophils have been implicated.