Cargo-free particles divert neutrophil-platelet aggregates to reduce thromboinflammation

Introduction:: Platelets are essential in maintaining hemostasis to prevent extreme blood loss and permanent injuries. Platelets' close interaction with the inflamed endothelium and their contribution to intravascular thrombi formation (or blood clot) can often exacerbate thromboinflammatory pathologies such as ischemic stroke, deep vein thrombosis, and myocardial infarction. Leukocytes, particularly neutrophils, also contribute to thrombus development by forming aggregates with platelets. During thromboinflammation, neutrophils-platelet aggregates are formed via the interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can together bind and accumulate at the injured endothelium. Particulate drug carriers have been applied to redirect and prevent leukocyte adhesion from areas of injury or inflammation. However, the downstream effect of these particulate carriers on neutrophil-platelet and platelet accumulation in thromboinflammatory conditions has yet to be explored. This work investigates the ability of cargo-free polymeric carriers to divert platelets away from inflamed vasculature both in vitro and in vivo.

Materials and Methods:: Human umbilical vein endothelial cells (HUVEC)s are isolated from umbilical cords and grown at 37℃ and 5% CO₂ until confluent. For in vitro blood flow assays, HUVECs were seeded onto gelatin-coated glass coverslips and activated with IL-1β for 4 hours to upregulate the expression of leukocyte adhesion molecules. HUVECs were further damaged via histamine stimulation and a scalpel score to facilitate platelet adhesion to injured areas. Whole blood from healthy donors was treated with adenosine diphosphate (ADP) for 1 hour before flow assays to activate platelets. After HUVEC and platelet activation, whole blood containing polystyrene untargeted or targeted polymeric particles was perfused over HUVEC/coverslip in a parallel plate flow chamber for 5 mins under laminar flow at 1000s^-1. After blood perfusion, HUVEC-bound leukocytes and bound platelets were recorded to examine the effect of particles on activated leukocyte and platelet adhesion under conditions of thromboinflammation.

Additionally, we utilized a murine model of thromboinflammation to study activated neutrophil and platelet adhesion after particle treatment in vivo. C57BL/6 mice were administered an intraperitoneal lipopolysaccharide (LPS) injection to induce systemic inflammation. Mice were treated with untargeted or targeted polystyrene particles either during LPS administration (Preventative group) or 2 hours after LPS injection (Intervention group). Mice are anesthetized at 3 hours post LPS exposure, and 2-4 independent mesentery vessels were visualized via intravital fluorescence microscopy. We recorded adherent and non-adherent Ly6G+ neutrophils, platelets, and fluorescent particles within blood vessels to elucidate the impact of particles on neutrophil and platelet adhesion.


Results, Conclusions, and Discussions:: We find that cargo-free particles, in a particle concentration and size manner, modulate platelet binding to inflamed endothelial by interfering with neutrophil and neutrophil-platelet aggregates adhesion. Notably, we find that untargeted and targeted micro-sized polymeric particles outperformed nano-sized carriers at decreasing platelet adhesion to endothelial cells in vitro in blood flow assays. Similarly, untargeted and targeted polystyrene microparticles successfully prevented platelet and neutrophil adhesion to mesentery vessels of particle-treated mice. Overall, our results showcase that platelet binding can be modulated mainly by controlling neutrophil-platelet aggregates' adhesion to areas of thromboinflammation via particle-neutrophil interactions.

Conclusion: The presence of neutrophil-platelet aggregates is prominent in several inflammatory conditions and often indicates the severity of diseases, including chronic cardiovascular disorders, COVID-19, and sepsis. Thus, this data represents initial work in creating new, drug-free, anti-platelet therapeutics, particularly for conditions of thromboinflammation.


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