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

The combination of inflammation and thrombosis is a hallmark of many cardiovascular diseases. Under such conditions, platelets are recruited to an area of inflammation by forming platelet-leukocyte aggregates via interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can bind to the endothelium. While particulate drug carriers have been utilized to passively redirect leukocytes from areas of inflammation, the downstream impact of these carriers on platelet accumulation in thromboinflammatory conditions has yet to be studied. Here, we explore the ability of polymeric particles to divert platelets away from inflamed blood vessels both in vitro and in vivo. We find that untargeted and targeted micron-sized polymeric particles can successfully reduce platelet adhesion to an inflamed endothelial monolayer in vitro in blood flow systems and in vivo in a lipopolysaccharide-induced, systemic inflammation murine model. Our data represent initial work in developing cargo-free, anti-platelet therapeutics specifically for conditions of thromboinflammation.

Polysalicylic Acid Polymer Microparticle Decoys Therapeutically Treat Acute Respiratory Distress Syndrome.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain problematic due to high mortality rates and lack of effective treatments. Neutrophilic injury contributes to mortality in ALI/ARDS. Here, technology for rapid ARDS intervention is developed and evaluated, where intravenous salicylic acid-based polymer microparticles, i.e., Poly-Aspirin (Poly-A), interfere with neutrophils in blood, reducing lung neutrophil infiltration and injury in vivo in mouse models of ALI/ARDS. Importantly, Poly-A particles reduce multiple inflammatory cytokines in the airway and bacterial load in the bloodstream in a live bacteria lung infection model of ARDS, drastically improving survival. It is observed that phagocytosis of the Poly-A microparticles, with salicylic acid in the polymer backbone, alters the neutrophil surface expression of adhesion molecules, potentially contributing to their added therapeutic benefits. Given the proven safety profile of the microparticle degradation products-salicylic acid and adipic acid-it is anticipated that the Poly-A particles represent a therapeutic strategy in ARDS with a rare opportunity for rapid clinical translation.

Deformable microparticles for shuttling nanoparticles to the vascular wall.

Vascular-targeted drug carriers must localize to the wall (i.e., marginate) and adhere to a diseased endothelium to achieve clinical utility. The particle size has been reported as a critical physical property prescribing particle margination in vitro and in vivo blood flows. Different transport process steps yield conflicting requirements-microparticles are optimal for margination, but nanoparticles are better for intracellular or tissue delivery. Here, we evaluate deformable hydrogel microparticles as carriers for transporting nanoparticles to a diseased vascular wall. Depending on microparticle modulus, nanoparticle-loaded poly(ethylene glycol)-based hydrogel microparticles delivered significantly more 50-nm nanoparticles to the vessel wall than freely injected nanoparticles alone, resulting in >3000% delivery increase. This work demonstrates the benefit of optimizing microparticles' efficient margination to enhance nanocarriers' transport to the vascular wall.

Increased Adhesive Potential of Antiphospholipid Syndrome Neutrophils Mediated by ß2 Integrin Mac-1.

OBJECTIVE: While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received less attention. This study was undertaken to investigate the extent to which antiphospholipid syndrome (APS) neutrophils adhere to resting endothelial cells under physiologic flow conditions and the surface molecules required for that adhesion. METHODS: Patients with primary APS (n = 43), patients with a history of venous thrombosis but negative test results for antiphospholipid antibodies (n = 11), and healthy controls (n = 38) were studied. Cells were introduced into a flow chamber and perfused across resting human umbilical vein endothelial cells (HUVECs). Surface adhesion molecules were quantified by flow cytometry. Neutrophil extracellular trap release (NETosis) was assessed in neutrophil-HUVEC cocultures. RESULTS: Upon perfusion of anticoagulated blood through the flow chamber, APS neutrophils demonstrated increased adhesion as compared to control neutrophils under conditions representative of either venous (n = 8; P < 0.05) or arterial (n = 15; P < 0.0001) flow. At the same time, APS neutrophils were characterized by up-regulation of CD64, CEACAM1, ß2 -glycoprotein I, and activated Mac-1 on their surface (n = 12-18; P < 0.05 for all markers). Exposing control neutrophils to APS plasma or APS IgG resulted in increased neutrophil adhesion (n = 10-11; P < 0.0001) and surface marker up-regulation as compared to controls. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils in the flow-chamber assay (P < 0.01). The same monoclonal antibody reduced NETosis in neutrophil-HUVEC cocultures (P < 0.01). CONCLUSION: APS neutrophils demonstrate increased adhesive potential, which is dependent upon the activated form of Mac-1. In patients, this could lower the threshold for neutrophil-endothelium interactions, NETosis, and possibly thrombotic events.

Secretory expression and characterization of two hemicellulases, xylanase, and ß-xylosidase, isolated from Bacillus subtilis M015.

Microbial hydrolysis of lignocellulosic biomass is becoming increasingly important for the production of renewable biofuels to address global energy concerns. Hemicellulose is the second most abundant lignocellulosic biopolymer consisting of mostly xylan and other polysaccharides. A variety of enzymes is involved in complete hydrolysis of xylan into its constituent sugars for subsequent biofuel fermentation. Two enzymes, endo-ß-xylanase and ß-xylosidase, are particularly important in hydrolyzing the xylan backbone into xylooligosaccharides and individual xylose units. In this study, we describe the cloning, expression, and characterization of xylanase and ß-xylosidase isolated from Bacillus subtilis M015 in Escherichia coli. The genes were identified to encode a 213 amino acid protein for xylanase (glycoside hydrolase (GH) family 11) and a 533 amino acid protein for ß-xylosidase (GH family 43). Recombinant enzymes were produced by periplasmic-leaky E. coli JE5505 and therefore secreted into the supernatant during growth. Temperature and pH optima were determined to be 50 °C and 5.5-6 for xylanase and 35 °C and 7.0-7.5 for ß-xylosidase using beech wood xylan and p-nitrophenyl-ß-D-xylopyranoside as the substrates, respectively. We have also investigated the synergy of two enzymes on xylan hydrolysis and observed 90 % increase in total sugar release (composed of xylose, xylobiose, xylotriose, and xylotetraose) for xylanase/ß-xylosidase combination as opposed to xylanase alone.