Trauma-targeted delivery of tranexamic acid improves hemostasis and survival in rat liver hemorrhage model.

BACKGROUND: Trauma-associated hemorrhage and coagulopathy remain leading causes of mortality. Such coagulopathy often leads to a hyperfibrinolytic phenotype where hemostatic clots become unstable because of upregulated tissue plasminogen activator (tPA) activity. Tranexamic acid (TXA), a synthetic inhibitor of tPA, has emerged as a promising drug to mitigate fibrinolysis. TXA is US Food and Drug Administration-approved for treating heavy menstrual and postpartum bleeding, and has shown promise in trauma treatment. However, emerging reports also implicate TXA for off-target systemic coagulopathy, thromboembolic complications, and neuropathy. OBJECTIVE: We hypothesized that targeted delivery of TXA to traumatic injury site can enable its clot-stabilizing action site-selectively, to improve hemostasis and survival while avoiding off-target effects. To test this, we used liposomes as a model delivery vehicle, decorated their surface with a fibrinogen-mimetic peptide for anchorage to active platelets within trauma-associated clots, and encapsulated TXA within them. METHODS: The TXA-loaded trauma-targeted nanovesicles (T-tNVs) were evaluated in vitro in rat blood, and then in vivo in a liver trauma model in rats. TXA-loaded control (untargeted) nanovesicles (TNVs), free TXA, or saline were studied as comparison groups. RESULTS: Our studies show that in vitro, the T-tNVs could resist lysis in tPA-spiked rat blood. In vivo, T-tNVs maintained systemic safety, significantly reduced blood loss and improved survival in the rat liver hemorrhage model. Postmortem evaluation of excised tissue from euthanized rats confirmed systemic safety and trauma-targeted activity of the T-tNVs. CONCLUSION: Overall, the studies establish the potential of targeted TXA delivery for safe injury site-selective enhancement and stabilization of hemostatic clots to improve survival in trauma.

Engineering Intravenously Administered Nanoparticles to Reduce Infusion Reaction and Stop Bleeding in a Large Animal Model of Trauma.

Bleeding from traumatic injury is the leading cause of death for young people across the world, but interventions are lacking. While many agents have shown promise in small animal models, translating the work to large animal models has been exceptionally difficult in great part because of infusion-associated complement activation to nanomaterials that leads to cardiopulmonary complications. Unfortunately, this reaction is seen in at least 10% of the population. We developed intravenously infusible hemostatic nanoparticles that were effective in stopping bleeding and improving survival in rodent models of trauma. To translate this work, we developed a porcine liver injury model. Infusion of the first generation of hemostatic nanoparticles and controls 5 min after injury led to massive vasodilation and exsanguination even at extremely low doses. In naïve animals, the physiological changes were consistent with a complement-associated infusion reaction. By tailoring the zeta potential, we were able to engineer a second generation of hemostatic nanoparticles and controls that did not exhibit the complement response at low and moderate doses but did at the highest doses. These second-generation nanoparticles led to cessation of bleeding within 10 min of administration even though some signs of vasodilation were still seen. While the complement response is still a challenge, this work is extremely encouraging in that it demonstrates that when the infusion-associated complement response is managed, hemostatic nanoparticles are capable of rapidly stopping bleeding in a large animal model of trauma.

Intravenous administration of synthetic platelets (SynthoPlate) in a mouse liver injury model of uncontrolled hemorrhage improves hemostasis.

BACKGROUND: Clinical resuscitative treatment of traumatic hemorrhage involves transfusion of RBC, platelets and plasma in controlled ratios. However, use of such blood components, especially platelets, present many challenges including availability, portability, contamination risks, and short shelf-life, which limit the use of platelet transfusions outside of large trauma centers such as remote civilian hospitals and austere prehospital settings. This has prompted significant research in platelet substitutes that may resolve the above issues while providing platelet-mimetic hemostatic action. In this framework, we have developed a synthetic platelet surrogate, SynthoPlate, by integrative decoration of platelet function mimetic peptides on a biocompatible lipid nanovesicle platform. We have previously demonstrated hemostatic capability of SynthoPlate in correcting tail-bleeding time in thrombocytopenic mice. Building on this, we hypothesized that SynthoPlate transfusion would decrease bleeding in a murine model of acute hemorrhagic shock. METHODS: A validated model of uncontrolled intraperitoneal hemorrhage, via liver laceration was used to induce hemorrhagic shock in mice. SynthoPlate, control (unmodified) particles, and normal saline were administered as pretreatment and recue infusions to mice undergoing liver laceration and evaluated for hemostatic benefit by determining differences in blood loss and monitoring real-time hemodynamic data. RESULTS: Pretreatment SynthoPlate transfusion resulted in significant reduction of blood loss following hemorrhage, compared with control particles or normal saline treatment (0.86 ± 0.16 g control particles [CP] vs. 0.84 ± 0.13 g normal saline [NS] vs. 0.68 ± 0.09 g SynthoPlate, p < 0.005). SynthoPlate transfused mice demonstrated improved hemodynamics taking significantly longer to develop post-injury hypotension (168.3 ± 106.6 seconds CP vs. 137 ± 58 seconds NS vs. 546.7 ± 329.8 seconds SynthoPlate, p < 0.05). SynthoPlate infusion following liver laceration, that is, rescue transfusion, also resulted in a significant decrease in blood loss (0.89 ± 0.17 g CP vs. 0.92 ± 0.19 g NS vs. 0.69 ± 0.18 g SynthoPlate, p < 0.05). CONCLUSION: Transfusion of SynthoPlate particles reduces blood loss in a murine model of liver injury, and SynthoPlates may represent a viable transfusion product for the mitigation of blood loss in acute, severe hemorrhagic shock.

Intravenous synthetic platelet (SynthoPlate) nanoconstructs reduce bleeding and improve 'golden hour' survival in a porcine model of traumatic arterial hemorrhage.

Traumatic non-compressible hemorrhage is a leading cause of civilian and military mortality and its treatment requires massive transfusion of blood components, especially platelets. However, in austere civilian and battlefield locations, access to platelets is highly challenging due to limited supply and portability, high risk of bacterial contamination and short shelf-life. To resolve this, we have developed an I.V.-administrable 'synthetic platelet' nanoconstruct (SynthoPlate), that can mimic and amplify body's natural hemostatic mechanisms specifically at the bleeding site while maintaining systemic safety. Previously we have reported the detailed biochemical and hemostatic characterization of SynthoPlate in a non-trauma tail-bleeding model in mice. Building on this, here we sought to evaluate the hemostatic ability of SynthoPlate in emergency administration within the 'golden hour' following traumatic hemorrhagic injury in the femoral artery, in a pig model. We first characterized the storage stability and post-sterilization biofunctionality of SynthoPlate in vitro. The nanoconstructs were then I.V.-administered to pigs and their systemic safety and biodistribution were characterized. Subsequently we demonstrated that, following femoral artery injury, bolus administration of SynthoPlate could reduce blood loss, stabilize blood pressure and significantly improve survival. Our results indicate substantial promise of SynthoPlate as a viable platelet surrogate for emergency management of traumatic bleeding.

Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding.

Bleeding complications arising from trauma, surgery, and as congenital, disease-associated, or drug-induced blood disorders can cause significant morbidities and mortalities in civilian and military populations. Therefore, stoppage of bleeding (hemostasis) is of paramount clinical significance in prophylactic, surgical, and emergency scenarios. For externally accessible injuries, a variety of natural and synthetic biomaterials have undergone robust research, leading to hemostatic technologies including glues, bandages, tamponades, tourniquets, dressings, and procoagulant powders. In contrast, treatment of internal noncompressible hemorrhage still heavily depends on transfusion of whole blood or blood's hemostatic components (platelets, fibrinogen, and coagulation factors). Transfusion of platelets poses significant challenges of limited availability, high cost, contamination risks, short shelf-life, low portability, performance variability, and immunological side effects, while use of fibrinogen or coagulation factors provides only partial mechanisms for hemostasis. With such considerations, significant interdisciplinary research endeavors have been focused on developing materials and technologies that can be manufactured conveniently, sterilized to minimize contamination and enhance shelf-life, and administered intravenously to mimic, leverage, and amplify physiological hemostatic mechanisms. Here, a comprehensive review regarding the various topical, intracavitary, and intravenous hemostatic technologies in terms of materials, mechanisms, and state-of-art is provided, and challenges and opportunities to help advancement of the field are discussed.

Platelet microparticle-inspired clot-responsive nanomedicine for targeted fibrinolysis.

Intravascular administration of plasminogen activators is a clinically important thrombolytic strategy to treat occlusive vascular conditions. A major issue with this strategy is the systemic off-target drug action, which affects hemostatic capabilities and causes substantial hemorrhagic risks. This issue can be potentially resolved by designing technologies that allow thrombus-targeted delivery and site-specific action of thrombolytic drugs. To this end, leveraging a liposomal platform, we have developed platelet microparticle (PMP)-inspired nanovesicles (PMINs), that can protect encapsulated thrombolytic drugs in circulation to prevent off-target uptake and action, anchor actively onto thrombus via PMP-relevant molecular mechanisms and allow drug release via thrombus-relevant enzymatic trigger. Specifically, the PMINs can anchor onto thrombus via heteromultivalent ligand-mediated binding to active platelet integrin GPIIb-IIIa and P-selectin, and release the thrombolytic payload due to vesicle destabilization triggered by clot-relevant enzyme phospholipase-A2. Here we report on the evaluation of clot-targeting efficacy, lipase-triggered drug release and resultant thrombolytic capability of the PMINs in vitro, and subsequently demonstrate that intravenous delivery of thrombolytic-loaded PMINs can render targeted fibrinolysis without affecting systemic hemostasis, in vivo, in a carotid artery thrombosis model in mice. Our studies establish significant promise of the PMIN technology for safe and site-targeted nanomedicine therapies in the vascular compartment.

MCP-1 downregulates MMP-9 export via vesicular redistribution to lysosomes in rat portal fibroblasts.

Portal fibroblasts (PF) are one of the two primary cell types contributing to the myofibroblast population of the liver and are thus essential to the pathogenesis of liver fibrosis. Monocyte chemoattractant protein-1 (MCP-1) is a known profibrogenic chemokine that may be of particular importance in biliary fibrosis. We examined the effect of MCP-1 on release of matrix metalloproteinase-9 (MMP-9) by rat PF. We found that MCP-1 blocks PF release of MMP-9 in a posttranslational fashion. We employed an optical and electron microscopic approach to determine the mechanism of this downregulation. Our data demonstrated that, in the presence of MCP-1, MMP-9-containing vesicles were shunted to a lysosome-like compartment. This is the first report of a secretory protein to be so regulated in fibrogenic cells.

CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway.

Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension.

Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis.

Liver myofibroblasts derived from hepatic stellate cells (HSC) are critical mediators of liver fibrosis. Release of tissue inhibitor of metalloproteinase-1 (TIMP-1) advances liver fibrosis by blocking fibrinolysis. The mechanisms responsible for the posttranslational regulation of TIMP-1 by myofibroblastic HSC are unknown. Here, we demonstrate that TIMP-1 release by HSC is regulated in a posttranslational fashion via calcium-sensitive vesicular exocytosis. To our knowledge, this is the first article to directly examine vesicular trafficking in myofibroblastic HSC, potentially providing a new target to treat and or prevent liver fibrosis.

Biodegradation of polyester polyurethane by endophytic fungi.

Bioremediation is an important approach to waste reduction that relies on biological processes to break down a variety of pollutants. This is made possible by the vast metabolic diversity of the microbial world. To explore this diversity for the breakdown of plastic, we screened several dozen endophytic fungi for their ability to degrade the synthetic polymer polyester polyurethane (PUR). Several organisms demonstrated the ability to efficiently degrade PUR in both solid and liquid suspensions. Particularly robust activity was observed among several isolates in the genus Pestalotiopsis, although it was not a universal feature of this genus. Two Pestalotiopsis microspora isolates were uniquely able to grow on PUR as the sole carbon source under both aerobic and anaerobic conditions. Molecular characterization of this activity suggests that a serine hydrolase is responsible for degradation of PUR. The broad distribution of activity observed and the unprecedented case of anaerobic growth using PUR as the sole carbon source suggest that endophytes are a promising source of biodiversity from which to screen for metabolic properties useful for bioremediation.