Magnetically powered microwheel thrombolysis of occlusive thrombi in zebrafish.

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here, we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-functionalized µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-functionalized µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models.

The lack of innovation in von Willebrand disease (VWD) originates from many factors including the complexity and heterogeneity of the disease but also from a lack of recognition of the impact of the bleeding symptoms experienced by patients with VWD. Recently, a few research initiatives aiming to move past replacement therapies using plasma-derived or recombinant von Willebrand factor (VWF) concentrates have started to emerge. Here, we report an original approach using synthetic platelet (SP) nanoparticles for the treatment of VWD type 2B (VWD-2B) and severe VWD (type 3 VWD). SP are liposomal nanoparticles decorated with peptides enabling them to concomitantly bind to collagen, VWF, and activated platelets. In vitro, using various microfluidic assays, we show the efficacy of SPs to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from mice with VWD-2B and deficient VWF (VWF-KO, ie, type 3 VWD). In vivo, using a tail-clip assay, SP treatment reduced blood loss by 35% in mice with VWD-2B and 68% in mice with VWF-KO. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen-binding peptide, although not sufficient by itself, was crucial for SP efficacy in VWD-2B; whereas all 3 peptides appeared necessary for mice with VWF-KO. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of mice with VWF-KO led to a strong clot, similar to those obtained in wild-type mice. Altogether, our results show that SP could represent an attractive therapeutic alternative for VWD, especially considering their long half-life and stability.

ac/dc Magnetic Fields for Enhanced Translation of Colloidal Microwheels.

Microscale devices must overcome fluid reversibility to propel themselves in environments where viscous forces dominate. One approach, used by colloidal microwheels (µwheels) consisting of superparamagnetic particles assembled and powered by rotating ac magnetic fields, is to employ a nearby surface to provide friction. Here, we used total internal reflection microscopy to show that individual 8.3 µm particles roll inefficiently with significant slip because of a particle-surface fluid gap of 20-80 nm. We determined that both gap width and slip increase with the increasing particle rotation rate when the load force is provided by gravity alone, thus providing an upper bound on translational velocity. By imposing an additional load force with a dc magnetic field gradient superimposed on the ac field, we were able to decrease the gap width and thereby enhance translation velocities. For example, an additional load force of 0.2 Fg provided by a dc field gradient increased the translational velocity from 40 to 80 µm/s for a 40 Hz rotation rate. The translation velocity increases with the decreasing gap width whether the gap is varied by dc field gradient-induced load forces or by reducing the Debye length with salt. These results present a strategy to accelerate surface-enabled rolling of microscale particles and open the possibility of high-speed µwheel rolling independent of the gravitational field.

Enhanced Fibrinolysis with Magnetically Powered Colloidal Microwheels.

Thrombi that occlude blood vessels can be resolved with fibrinolytic agents that degrade fibrin, the polymer that forms between and around platelets to provide mechanical stability. Fibrinolysis rates however are often constrained by transport-limited delivery to and penetration of fibrinolytics into the thrombus. Here, these limitations are overcome with colloidal microwheel (µwheel) assemblies functionalized with the fibrinolytic tissue-type plasminogen activator (tPA) that assemble, rotate, translate, and eventually disassemble via applied magnetic fields. These microwheels lead to rapid fibrinolysis by delivering a high local concentration of tPA to induce surface lysis and, by taking advantage of corkscrew motion, mechanically penetrating into fibrin gels and platelet-rich thrombi to initiate bulk degradation. Fibrinolysis of plasma-derived fibrin gels by tPA-microwheels is fivefold faster than with 1 µg mL-1 tPA. µWheels following corkscrew trajectories can also penetrate through 100 µm sized platelet-rich thrombi formed in a microfluidic model of hemostasis in ≈5 min. This unique combination of surface and bulk dissolution mechanisms with mechanical action yields a targeted fibrinolysis strategy that could be significantly faster than approaches relying on diffusion alone, making it well-suited for occlusions in small or penetrating vessels not accessible to catheter-based removal.

Platelet-inspired synthetic nanoparticles improve hemostasis and hemodynamics in a rabbit model of abdominal hemorrhage.

BACKGROUND: Early platelet transfusion is associated with reduced mortality in traumatic hemorrhage. However, platelet usage is severely limited because of the challenges of donor availability, platelet portability, and storage. Here, we report on a bioinspired synthetic platelet (SP) nanoconstruct that utilizes liposome surface-decoration with peptides that mimic injury site-specific platelet adhesion to von Willebrand Factor and collagen, and fibrinogen-mediated platelet aggregation. Synthetic platelet has previously shown promising hemostatic outcomes in vitro and in vivo. Here, we evaluated hemostasis and hemodynamic effects of SP in a rabbit model of abdominal hemorrhage. METHODS: Twenty-three adult male New Zealand white rabbits (2.5-3.5 kg) were treated with either buffer, control particles (CPs), or SP. Under general anesthesia with invasive monitoring, rabbits underwent laparotomy with combined splenic and hepatic injury. Hemodynamics were monitored for 30 minutes and blood loss was quantified. Blood counts, aggregometry, catecholamine and platelet factor 4 (PF4) assays were performed at multiple timepoints. Analysis used analysis of variance and post hoc Tukey testing with α = 0.05. RESULTS: Rabbits in the SP (n = 7) group had significantly lower weight-normalized blood loss compared with both buffer (n = 8) and CP (n = 8) animals (21.1 vs. 33.2 vs. 40.4 g/kg, p < 0.001). Synthetic platelet-treated animals had higher systolic blood pressure area under curve compared with buffer- and CP-treated animals (1567 vs. 1281 vs. 1109 mm Hg*min, p = 0.006), although post hoc differences were only significant for the SP/CP comparison ( p = 0.005). Platelet counts, catecholamine levels, PF4, and aggregometry were similar between groups. CONCLUSION: Synthetic platelet treatment significantly reduced blood loss and improved hemodynamics in a rabbit abdominal hemorrhage model. Synthetic platelet has potential as an intravenous hemostatic platelet surrogate with donor-independent availability and scalable manufacture.

A Multichannel Portable Platform With Embedded Thermal Management for Miniaturized Dielectric Blood Coagulometry.

This article presents a standalone, multichannel, miniaturized impedance analyzer (MIA) system for dielectric blood coagulometry measurements with a microfluidic sensor termed ClotChip. The system incorporates a front-end interface board for 4-channel impedance measurements at an excitation frequency of 1 MHz, an integrated resistive heater formed by a pair of printed-circuit board (PCB) traces to keep the blood sample near a physiologic temperature of 37 °C, a software-defined instrument module for signal generation and data acquisition, and a Raspberry Pi-based embedded computer with 7-inch touchscreen display for signal processing and user interface. When measuring fixed test impedances across all four channels, the MIA system exhibits an excellent agreement with a benchtop impedance analyzer, with rms errors of ≤0.30% over a capacitance range of 47-330 pF and ≤0.35% over a conductance range of 2.13-10 mS. Using in vitro-modified human whole blood samples, the two ClotChip output parameters, namely, the time to reach a permittivity peak (Tpeak) and maximum change in permittivity after the peak (Δϵr,max) are assessed by the MIA system and benchmarked against the corresponding parameters of a rotational thromboelastometry (ROTEM) assay. Tpeak exhibits a very strong positive correlation (r = 0.98, p < 10-6, n = 20) with the ROTEM clotting time (CT) parameter, while Δϵr,max exhibits a very strong positive correlation (r = 0.92, p < 10-6, n = 20) with the ROTEM maximum clot firmness (MCF) parameter. This work shows the potential of the MIA system as a standalone, multichannel, portable platform for comprehensive assessment of hemostasis at the point-of-care/point-of-injury (POC/POI).

Magnetically Powered Microwheel Thrombolysis of Occlusive Thrombi in Zebrafish.

Tissue plasminogen activator (tPA) is the only FDA approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Providing a living microfluidic analog, transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Breaking the fibrinolytic speed limit with microwheel co-delivery of tissue plasminogen activator and plasminogen.

BACKGROUND: To reestablish blood flow in vessels occluded by clots, tissue plasminogen activator (tPA) can be used; however, its efficacy is limited by transport to and into a clot and by the depletion of its substrate, plasminogen. OBJECTIVES: To overcome these rate limitations, a platform was designed to co-deliver tPA and plasminogen based on microwheels (µwheels), wheel-like assemblies of superparamagnetic colloidal beads that roll along surfaces at high speeds. METHODS: The biochemical speed limit was determined by measuring fibrinolysis of plasma clots at varying concentrations of tPA (10-800 nM) and plasminogen (1-6 µM). Biotinylated magnetic mesoporous silica nanoparticles were synthesized and bound to streptavidin-coated superparamagnetic beads to make studded beads. Studded beads were loaded with plasminogen and tPA was immobilized on their surface. Plasminogen release and tPA activity were measured on the studded beads. Studded beads were assembled into µwheels with rotating magnetic fields and fibrinolysis of plasma clots was measured in a microfluidic device. RESULTS: The biochemical speed limit for plasma clots was ~15 µm/min. Plasminogen-loaded, tPA-immobilized µwheels lyse plasma clots at rates comparableto the biochemical speed limit. With the addition of a corkscrew motion, µwheels penetrate clots, thereby exceeding the biochemical speed limit (~20 µm/min) and achieving lysis rates 40-fold higher than 50 nM tPA. CONCLUSIONS: Co-delivery of an immobilized enzyme and its substrate via a microbot capable of mechanical work has the potential to target and rapidly lyse clots that are inaccessible by mechanical thrombectomy devices or recalcitrant to systemic tPA delivery.

Assessment of fibrinolytic status in whole blood using a dielectric coagulometry microsensor.

Rapid assessment of the fibrinolytic status in whole blood at the point-of-care/point-of-injury (POC/POI) is clinically important to guide timely management of uncontrolled bleeding in patients suffering from hyperfibrinolysis after a traumatic injury. In this work, we present a three-dimensional, parallel-plate, capacitive sensor - termed ClotChip - that measures the temporal variation in the real part of blood dielectric permittivity at 1 MHz as the sample undergoes coagulation within a microfluidic channel with <10 µL of total volume. The ClotChip sensor features two distinct readout parameters, namely, lysis time (LT) and maximum lysis rate (MLR) that are shown to be sensitive to the fibrinolytic status in whole blood. Specifically, LT identifies the time that it takes from the onset of coagulation for the fibrin clot to mostly dissolve in the blood sample during fibrinolysis, whereas MLR captures the rate of fibrin clot lysis. Our findings are validated through correlative measurements with a rotational thromboelastometry (ROTEM) assay of clot viscoelasticity, qualitative/quantitative assessments of clot stability, and scanning electron microscope imaging of clot ultrastructural changes, all in a tissue plasminogen activator (tPA)-induced fibrinolytic environment. Moreover, we demonstrate the ClotChip sensor ability to detect the hemostatic rescue that occurs when the tPA-induced upregulated fibrinolysis is inhibited by addition of tranexamic acid (TXA) - a potent antifibrinolytic drug. This work demonstrates the potential of ClotChip as a diagnostic platform for rapid POC/POI assessment of fibrinolysis-related hemostatic abnormalities in whole blood to guide therapy.

Platelet-Inspired Intravenous Nanomedicine for Injury-Targeted Direct Delivery of Thrombin to Augment Hemostasis in Coagulopathies.

Severe hemorrhage associated with trauma, surgery, and congenital or drug-induced coagulopathies can be life-threatening and requires rapid hemostatic management via topical, intracavitary, or intravenous routes. For injuries that are not easily accessible externally, intravenous hemostatic approaches are needed. The clinical gold standard for this is transfusion of blood products, but due to donor dependence, specialized storage requirements, high risk of contamination, and short shelf life, blood product use faces significant challenges. Consequently, recent research efforts are being focused on designing biosynthetic intravenous hemostats, using intravenous nanoparticles and polymer systems. Here we report on the design and evaluation of thrombin-loaded injury-site-targeted lipid nanoparticles (t-TLNPs) that can specifically localize at an injury site via platelet-mimetic anchorage to the von Willebrand factor (vWF) and collagen and directly release thrombin via diffusion and phospholipase-triggered particle destabilization, which can locally augment fibrin generation from fibrinogen for hemostatic action. We evaluated t-TLNPs in vitro in human blood and plasma, where hemostatic defects were created by platelet depletion and anticoagulation. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM)-based studies of clot viscoelasticity, and BioFlux-based real-time imaging of fibrin generation under simulated vascular flow conditions confirmed that t-TLNPs can restore fibrin in hemostatic dysfunction settings. Finally, the in vivo feasibility of t-TLNPs was tested by prophylactic administration in a tail-clip model and emergency administration in a liver-laceration model in mice with induced hemostatic defects. Treatment with t-TLNPs was able to significantly reduce bleeding in both models. Our studies demonstrate an intravenous nanomedicine approach for injury-site-targeted direct delivery of thrombin to augment hemostasis.

Engineered microparticles and nanoparticles for fibrinolysis.

Fibrinolytic agents including plasmin and plasminogen activators improve outcomes in acute ischemic stroke and thrombosis by recanalizing occluded vessels. In the decades since their introduction into clinical practice, several limitations of have been identified in terms of both efficacy and bleeding risk associated with these agents. Engineered nanoparticles and microparticles address some of these limitations by improving circulation time, reducing inhibition and degradation in circulation, accelerating recanalization, improving targeting to thrombotic occlusions, and reducing off-target effects; however, many particle-based approaches have only been used in preclinical studies to date. This review covers four advances in coupling fibrinolytic agents with engineered particles: (a) modifications of plasminogen activators with macromolecules, (b) encapsulation of plasminogen activators and plasmin in polymer and liposomal particles, (c) triggered release of encapsulated fibrinolytic agents and mechanical disruption of clots with ultrasound, and (d) enhancing targeting with magnetic particles and magnetic fields. Technical challenges for the translation of these approaches to the clinic are discussed.