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.

Blood flow and mass transfer regulation of coagulation.

Blood flow regulates coagulation and fibrin formation by controlling the transport, or mass transfer, of zymogens, co-factors, enzymes, and inhibitors to, from, and within a growing thrombus. The rate of mass transfer of these solutes relative to their consumption or production by coagulation reactions determines, in part, the rate of thrombin generation, fibrin deposition, and thrombi growth. Experimental studies on the influence of blood flow on specific coagulation reactions are reviewed here, along with a theoretical framework that predicts how flow influences surface-bound coagulation binding and enzymatic reactions. These flow-mediated transport mechanisms are also used to interpret the role of binding site densities and injury size on initiating coagulation and fibrin deposition. The importance of transport of coagulation proteins within the interstitial spaces of thrombi is shown to influence thrombi architecture, growth, and arrest.

On-chip recalcification of citrated whole blood using a microfluidic herringbone mixer.

In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.

Elastic behavior and platelet retraction in low- and high-density fibrin gels.

Fibrin is a biopolymer that gives thrombi the mechanical strength to withstand the forces imparted on them by blood flow. Importantly, fibrin is highly extensible, but strain hardens at low deformation rates. The density of fibrin in clots, especially arterial clots, is higher than that in gels made at plasma concentrations of fibrinogen (3-10 mg/mL), where most rheology studies have been conducted. Our objective in this study was to measure and characterize the elastic regimes of low (3-10 mg/mL) and high (30-100 mg/mL) density fibrin gels using shear and extensional rheology. Confocal microscopy of the gels shows that fiber density increases with fibrinogen concentration. At low strains, fibrin gels act as thermal networks independent of fibrinogen concentration. Within the low-strain regime, one can predict the mesh size of fibrin gels by the elastic modulus using semiflexible polymer theory. Significantly, this provides a link between gel mechanics and interstitial fluid flow. At moderate strains, we find that low-density fibrin gels act as nonaffine mechanical networks and transition to affine mechanical networks with increasing strains within the moderate regime, whereas high-density fibrin gels only act as affine mechanical networks. At high strains, the backbone of individual fibrin fibers stretches for all fibrin gels. Platelets can retract low-density gels by >80% of their initial volumes, but retraction is attenuated in high-density fibrin gels and with decreasing platelet density. Taken together, these results show that the nature of fibrin deformation is a strong function of fibrin fiber density, which has ramifications for the growth, embolization, and lysis of thrombi.

A combined microfluidic-microstencil method for patterning biomolecules and cells.

Despite the myriad of soft lithography based micropatterning methods available to researchers, it is still challenging to define small features (10-100 µm) that are spaced far apart (1-10 mm). In this report, we describe a combined microfluidic-microstencil patterning method that can produce multifunctional substrates of small features, O(10 µm), with a large pitch, O(1 mm). In that, we fabricate microstencils using an UV curable polyurethane (Norland Optical Adhesive 81) with dense arrays of 10-100 µm holes. Overlaying arrays of microfluidic channels over these microstencils allow for the control of the spacing between features and the ability to pattern multiple substrates. We show that this method is capable of patterning soluble proteins, fibrillar insoluble collagen, liposomes, cells, and nanoparticles. We demonstrate the utility of the method by measuring platelet adhesion under flow to three adhesive proteins (insoluble fibrillar collagen, laminin, and reconstituted acid solubilized collagen fibers) in a single assay.

TRAIL-coated leukocytes that kill cancer cells in the circulation.

Metastasis through the bloodstream contributes to poor prognosis in many types of cancer. Mounting evidence implicates selectin-based adhesive interactions between cancer cells and the blood vessel wall as facilitating this process, in a manner similar to leukocyte trafficking during inflammation. Here, we describe a unique approach to target and kill colon and prostate cancer cells in the blood that causes circulating leukocytes to present the cancer-specific TNF-related apoptosis inducing ligand (TRAIL) on their surface along with E-selectin adhesion receptor. This approach, demonstrated in vitro with human blood and also in mice, mimics the cytotoxic activity of natural killer cells and increases the surface area available for delivery of the receptor-mediated signal. The resulting "unnatural killer cells" hold promise as an effective means to neutralize circulating tumor cells that enter blood with the potential to form new metastases.

Inducing apoptosis in rolling cancer cells: a combined therapy with aspirin and immobilized TRAIL and E-selectin.

Though metastasis is considered an inefficient process, over 90% of cancer related deaths are attributed to the formation of secondary tumors. Thus, eliminating circulating cancer cells could lead to improved patient survival. This study was aimed at exploiting the interactions of cancer cells with selectins under flow to selectively kill captured colon cancer cells. Microtubes functionalized with E-selectin and TRAIL were perfused with colon cancer cell line Colo205 either treated with 1 mM aspirin or untreated for 1 or 2 h. Cells were collected from the microtube and analyzed by flow cytometry. Aspirin treatment alone killed only 3% cells in culture. A 95% difference in the number of cells killed between control and TRAIL + ES surfaces was seen when aspirin treated cells were perfused over the functionalized surface for 2 h. We have demonstrated a novel biomimetic method to capture and neutralize cancer cells in flow, thus reducing the chances for the formation of secondary tumors.

Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration.

Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability--a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or small interfering RNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.

Knockdown of fucosyltransferase III disrupts the adhesion of circulating cancer cells to E-selectin without affecting hematopoietic cell adhesion.

Adhesive interactions between selectins and their ligands play an essential role during cancer extravasation. Fucosylation of these proteins by fucosyltransferases, or FUTs, is critical for their functions. Using quantitative RT-PCR, we demonstrated that FUT4 and FUT7 are the predominant FUTs expressed in hematopoietic cell line, while FUT3 is heavily expressed by multiple cancer cell lines including the prostate cancer cell line MDA PCa2b. Knockdown of FUT3 expression in MDA PCa2b cells by small interference RNA (siRNA) significantly reduced FUT3 expression. Cell-surface sialyl Lewis antigens were largely abolished. Cell adhesion and cell rolling on the blood vessel wall were simulated by perfusing cancer cells through microtubes coated with recombinant human E-selectin. At physiological levels of wall shear stress, the number of flowing cancer cells recruited to the microtube surface was dramatically reduced by FUT3 knockdown. Higher rolling velocity was also observed, which is consistent with reduced E-selectin binding activity. Interestingly, FUT3 siRNA treatment also significantly reduced the cell growth rate. Combined with the novel siRNA delivery platform recently developed in our laboratory, FUT3 siRNA could be a promising conjunctive therapy aiming at reducing the metastatic virulence of circulating epithelial cancer cells.

Delivery of apoptotic signal to rolling cancer cells: a novel biomimetic technique using immobilized TRAIL and E-selectin.

The survival rate for patients with metastases versus localized cancer is dramatically reduced, with most deaths being associated with the formation of secondary tumors. Circulating cancer cells interact with the endothelial lining of the vasculature via a series of adhesive interactions that facilitate tethering and firm adhesion of cancer cells in the initial steps of metastasis. TNF-related apoptosis-inducing ligand (TRAIL) holds promise as a tumor-specific cancer therapeutic, by inducing a death signal by apoptosis via the caspase pathway. In this study, we exploit this phenomenon to deliver a receptor-mediated apoptosis signal to leukemic cells adhesively rolling along a TRAIL and selectin-bearing surface. Results show that cancer cells exhibit selectin-mediated rolling in capillary flow chambers, and that the rolling velocities can be controlled by varying the selectin and selectin surface density and the applied shear stress. It was determined that a 1 h rolling exposure to a functionalized TRAIL and E-selectin surface was sufficient to kill 30% of captured cells compared to static conditions in which 4 h exposure was necessary to kill 30% of the cells. Thus, we conclude that rolling delivery is more effective than static exposure to a TRAIL immobilized surface. We have also verified that there is no significant effect of TRAIL on hematopoietic stem cells and other normal blood cells. This represents the first demonstration of a novel biomimetic method to capture metastatic cells from circulation and deliver an apoptotic signal.