Clot-targeted nanogels for anticoagulant and fibrinolytic dual-delivery to treat disseminated intravascular coagulation

Introduction:: Disseminated Intravascular Coagulation (DIC) is a dangerous consumptive coagulopathy that disrupts hemostatic balance by excessive thrombin generation. Increased thrombin levels cause fibrin clot and microthrombi formation throughout the entire body. Concurrently, excessive microthrombi formation consumes critical clotting factors which paradoxically leads to high risk for excessive bleeding along with the prothrombotic concerns of blood vessel blockage. These dual dangers, combined with lack of a direct treatment for DIC, account for the high mortality rate. Attempts at mitigating DIC symptoms include systemic delivery of anticoagulants and clotting factors; however, evidence supporting the efficacy of these treatments is poor. A clot-targeted therapy that lyses clots and downregulates thrombin activity could cease the prothrombotic state, release critical clotting factors, and restore hemostatic balance. To that end, our group has developed fibrin-targeting nanogels to deliver both anticoagulant and fibrinolytic agents. Tissue plasminogen activator (tPA), a fibrinolytic drug, activates the fibrinolytic pathway and lyses clots. The anticoagulant, Antithrombin III (ATIII), inhibits thrombin and prevents further clotting. Here we (1) examine the ability of fibrin-specific nanogels (FSNs) to deliver tPA and ATIII and (2) evaluate their efficacy in restoring hemostatic balance in two DIC rodent models representing the excessive clotting and bleeding state of DIC. We hypothesize both ATIII-FSNs and tPA-ATIII-FSNs will target clots and decrease microthrombi; however, the addition of tPA in tPA-ATIII-FSNs will help promote clot lysis and restore critical clotting factor concentration. Subsequently, the dissolution of microthrombi will increase platelet and fibrin accumulation at the wound site decreasing total blood loss.


Materials and Methods:: Core-shell poly(N-isopropylacrylamide) (pNIPAM) nanogels were synthesized through two polymerization reactions with N, N′-Methylenebis (acrylamide) (BIS) and acrylic acid and then conjugated to anti-fibrin fragment E antibody via EDC/NHS coupling. Next, nanogels were loaded with ATIII and/or tPA through a rehydration technique and lyophilized. To investigate nanogel impact on clotting dynamics in vivo, a lipopolysaccharide (LPS)-induced DIC rodent model was optimized. After sufficient DIC induction, ATIII-FSNs and tPA-ATIII-FSNs were administered via tail vein and allowed to circulate. Blood was then collected via cardiac puncture and organs were harvested. Platelet count, fibrinogen concentration, and d-dimer concentration were quantified. To investigate nanogel impact on an excessive bleeding state of DIC, a second set of experiments was conducted where LPS-induced DIC rodents received liver lacerations. Blood loss was measured overtime followed by cardiac puncture and organ harvest. Platelet, fibrinogen, and d-dimer concentrations were quantified once again. Additionally, an activated partial thromboplastin time (aPTT) assay was used to evaluate ex vivo clotting parameters. Finally, fibrin and FSN accumulation within organs and at the laceration site were evaluated with both Martius Scarlet Blue (MSB) staining and immunohistochemistry (IHC).


Results, Conclusions, and Discussions:: Results: In DIC rodents without injury, a 10 mg/kg ATIII-FSN and tPA-ATIII-FSN (Figure 1A) dosage decreased fibrin accumulation in organs. Additionally, in DIC rodents with an injury, both ATIII-FSNs and tPA-ATIII-FSNs reduced total blood loss (Figure 1B) and increased fibrin localization at the liver laceration site (Figure 1C). Individually, tPA-ATIII-FSNs restored platelet and d-dimer concentrations in DIC rodents without a liver laceration injury (Figure 2A), while ATIII-FSNs increased platelet and fibrinogen concentrations and restored plasma’s ex vivo clotting ability in DIC rodent models coupled with a liver laceration injury (Figure 2B).

Conclusions: Both single-loaded ATIII-FSNs and dual-loaded tPA-ATIII-FSNs are effective at targeting fibrin clots and releasing ATIII and tPA in vivo. ATIII-FSNs inhibit overactive thrombin and reduce the number of clots formed in a DIC rodent model allowing fibrin to localize at the wound site and stop bleeding. tPA-ATIII-FSNs promote clot lysis and increase the number of available platelets, so when faced with injury, platelets can localize to the wound site and stop bleeding. Further evaluation of clotting factor concentration and blood loss is ongoing for ATIII-FSN and tPA-ATIII-FSN treated animals.


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