(476e) Enhanced Fibrinolysis Using Magnetically Powered Colloidal Microwheels

Background: The mechanical stability of blood clots is derived from the biopolymer fibrin. Blood clots can be biochemically decomposed via systemic or local delivery of tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Such drugs activate plasminogen to plasmin, which binds to and lyses fibrin. Fibrinolytic treatment is limited by two processes: the transport of plasminogen activator (PA) to a clot, and the dissolution of the clot itself. Delivery of fibrinolytics depends on method of administration. Blood flow is minimal in vessels that are occluded, and intravenously administered PA must diffuse to clot interface. Both the rate of diffusion and the rate of clot dissolution can be increased by using a higher systemic concentration of PA, but high concentrations of PA can induce hemorrhaging in the patient. In large arteries, catheterization can be used to deliver a high local concentration of PA while keeping systemic concentration relatively low; however, catheters provide no solution to thrombi that occur in smaller arteries.

Clot dissolution rates depend on several factors, including the pressure gradient across the thrombus, the composition of the thrombus, and PA concentration; however, lysis rates are typically limited by transport of PA to and throughout the thrombus. Microparticles functionalized with tPA offer an alternative method for delivery of PA to a thrombus that can generate high localized concentrations and accelerate transport of PA to and through a thrombus. Such a method overcomes the transport restrictions associated with PA delivery and accelerates fibrin degradation. Previously, we have demonstrated assembly of microparticles into “microwheels" which translate under a magnetic field with rapid velocities. Such microwheels are well-suited to carry PA to a thrombus in an occluded blood vessel.

Methods: One micrometer superparamagnetic beads subjected to a 9 mT rotating magnetic field (f= 100 Hz) assemble spontaneously into microwheels because of net induced dipole interactions. In this work, the magnetic field is generated externally by five solenoids. A sinusoidal function of alternating current is passed through two pairs of coils on the x-y plane. A π/2 phase lag between the coils on the x-axis and those on the y-axis cause the field to rotate at the center of the coils. A fifth solenoid positioned on the z-axis cants the magnetic field such that it rotates at an angle with respect to the horizontal. Microwheels align with the angle of the field and roll because of wet friction between the wheel and the surface. The direction of microwheel translation is readily controllable in real time, or can be pre-programmed to direct the wheels along user-defined paths.

Fibrin gels were formed inside a microfluidic channel from recalcified normal pooled plasma (NPP) or platelet rich plasma (PRP) (20 mM CaCl₂) with 4.5 nM thrombin. Fibrinolysis rates achieved by 1.5*10^6 tPA-beads/μL, corresponding to an effective tPA concentration of 3.6 ug/mL, were compared to lysis rates induced by 1 and 10 μg/mL soluble tPA in a microfluidic device. The beads were guided by the magnetic field to translate with two different modalities: direct and corkscrew motions. Under the direct modality, wheels translate unidirectionally towards the fibrin front. In contrast, the corkscrew modality causes wheels to approach the fibrin front in spiral pattern with a directional bias.

Results:We observe fibrinolysis rates of 0.81 ± 0.25 and 5.0 ± 1.5 um/min for 1 ug/mL and 10 ug/mL soluble tPA, respectively. Lysis rates achieved by functionalized microwheels driven by direct and corkscrew modalities were 4.8 ± 0.3 and 9.6 ± 1.5 um/min. This data supports the approach of a magnetic field-powered microwheel as a viable drug delivery mechanism for tPA. Similar lysis rates for microwheel experiments were observed in clots formed from both NPP and PRP, indicating that microwheels can bust fibrin networks despite the presence of platelets. It should be noted that natural tPA inhibitors such as plasminogen activator inhibitor one (PAI-1) are present in physiologic concentrations since the experiments are performed in plasma.

Both the unidirectional and corkscrew translation modalities offer improved performance over soluble tPA. Translational velocities of beads approaching the fibrin front were 4.5 ± 3.8 um/s and 2.1 ± 1.8 um/s for unidirectional and corkscrew motion. Since these velocities are high relative to fibrinolysis rates, microwheels accumulated at the gel interface, resulting in a local concentration of tPA of up to 50-fold higher than the initial bulk concentration. Moreover, field-driven microwheels penetrated the fibrin gel, transporting drug within the fibrin network itself. The direct motion modality drove small wheels consisting of 1-4 microparticles approximately 100-200 um into the gel, whereas the corkscrew modality drove much larger microwheels (consisting of >4 colloids) from 20-60 um into the fibrin network. Roughly twice as many beads penetrated the gel under the corkscrew modality than the unidirectional one, corresponding to an increase in lysis velocity. The combination of biochemical and mechanical action contributed to enhanced fibrinolysis.

Conclusions: Fibrinolysis using soluble tPA is a transport-limited process that relies heavily on diffusion. Colloidal microparticles functionalized with tPA offer a lysis strategy that overcome the transport limitations inherent in soluble tPA fibrinolysis. Colloids are dispersed into a microfluidic system, and assembled into microwheels via subsequent application of a magnetic field. The low-density functionalized microwheels are driven to the fibrin interface and rapidly accumulate near the gel surface. Uniquely, microwheels driven by the magnetic field also penetrate into the fibrin gel, transporting tPA within the fibrin network and enhancing dissolution rates. Microwheels have potential as a drug delivery method against thrombi occurring in small vessels where catheterization and systemic soluble PA delivery are ineffective.