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.

Reversible Microwheel Translation Induced by Polymer Depletion.

For in vivo applications, microbots (µbots) must move, which is a need that has led to designs, such as helical swimmers, that translate through the bulk fluid. We have previously demonstrated that, upon application of a rotating magnetic field, colloidal particles in aqueous systems can be reversibly assembled from superparamagnetic particles into µbots that translate along surfaces using wet friction. Here, we show that high-molecular-weight polymers of a size that approaches the length scale of the gap between the µbot and surface can be excluded, impacting µbot transport. Using xanthan gum as a convenient high-molecular-weight model, we determine that polymer depletion imparts only a weak effect on colloid-surface interactions but has a significant influence on local viscosity, which is an effect great enough to induce a reversal in the µbot translation direction.

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.

Magnetically Powered Chitosan Milliwheels for Rapid Translation, Barrier Function Rescue, and Delivery of Therapeutic Proteins to the Inflamed Gut Epithelium.

Inflammatory bowel disease (IBD) is mediated by an overexpression of tumor necrosis factor-α (TNF) by mononuclear cells in the intestinal mucosa. Intravenous delivery of neutralizing anti-TNF antibodies can cause systemic immunosuppression, and up to one-third of people are non-responsive to treatment. Oral delivery of anti-TNF could reduce adverse effects; however, it is hampered by antibody degradation in the harsh gut environment during transit and poor bioavailability. To overcome these shortcomings, we demonstrate magnetically powered hydrogel particles that roll along mucosal surfaces, provide protection from degradation, and sustain the local release of anti-TNF. Iron oxide particles are embedded into a cross-linked chitosan hydrogel and sieved to produce 100-200 µm particles called milliwheels (m-wheels). Once loaded with anti-TNF, these m-wheels release 10 to 80% of their payload over 1 week at a rate that depends on the cross-linking density and pH. A rotating magnetic field induces a torque on the m-wheels that results in rolling velocities greater than 500 µm/s on glass and mucus-secreting cells. The permeability of the TNF-challenged gut epithelial cell monolayers was rescued in the presence of anti-TNF carrying m-wheels, which both neutralized the TNF and created an impermeable patch over leaky cell junctions. With the ability to translate over mucosal surfaces at high speed, provide sustained release directly to the inflamed epithelium, and provide barrier rescue, m-wheels demonstrate a potential strategy to deliver therapeutic proteins for the treatment of IBD.

Rolling of soft microbots with tunable traction.

Microbot (µbot)-based targeted drug delivery has attracted increasing attention due to its potential for avoiding side effects associated with systemic delivery. To date, most µbots are rigid. When rolling on surfaces, they exhibit substantial slip due to the liquid lubrication layer. Here, we introduce magnetically controlled soft rollers based on Pickering emulsions that, because of their intrinsic deformability, fundamentally change the nature of the lubrication layer and roll like deflated tires. With a large contact area between µbot and wall, soft µbots exhibit tractions higher than their rigid counterparts, results that we support with both theory and simulation. Upon changing the external field, surface particles can be reconfigured, strongly influencing both the translation speed and traction. These µbots can also be destabilized upon pH changes and used to deliver their contents to a desired location, overcoming the limitations of low translation efficiency and drug loading capacity associated with rigid structures.

Coupling magnetic torque and force for colloidal microbot assembly and manipulation.

For targeted transport in the body, biomedical microbots (µbots) must move effectively in three-dimensional (3D) microenvironments. Swimming µbots translate via asymmetric or screw-like motions while rolling ones use friction with available surfaces to generate propulsive forces. We have previously shown that planar rotating magnetic fields assemble µm-scale superparamagnetic beads into circular µbots that roll along surfaces. In this, gravity is required to pull µbots near the surface; however, this is not necessarily practical in complex geometries. Here we show that rotating magnetic fields, in tandem with directional magnetic gradient forces, can be used to roll µbots on surfaces regardless of orientation. Simplifying implementation, we use a spinning permanent magnet to generate differing ratios of rotating and gradient fields, optimizing control for different environments. This use of a single magnetic actuator sidesteps the need for complex electromagnet or tandem field setups, removes requisite gravitational load forces, and enables µbot targeting in complex 3D biomimetic microenvironments.

Chain Assembly Kinetics from Magnetic Colloidal Spheres.

Magnetic colloidal chains are a microrobotic system with promising applications due to their versatility, biocompatibility, and ease of manipulation under magnetic fields. Their synthesis involves kinetic pathways that control chain quality, length, and flexibility, a process performed by first aligning superparamagnetic particles under a one-dimensional magnetic field and then chemically linking them using a four-armed maleimide-functionalized poly(ethylene glycol). Here, we systematically vary the concentration of the poly(ethylene glycol) linkers, the reaction temperature, and the magnetic field strength to study their impact on the physical properties of synthesized chains, including the chain length distribution, reaction temperature, and bending modulus. We find that this chain fabrication process resembles step-growth polymerization and can be accurately described by the Flory-Schulz model. Under optimized experimental conditions, we have successfully synthesized long flexible colloidal chains with a bending modulus, which is 4 orders of magnitude smaller than previous studies. Such flexible and long chains can be folded entirely into concentric rings and helices with multiple turns, demonstrating the potential for investigating the actuation, assembly, and folding behaviors of these colloidal polymer analogues.

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.

Delivery and actuation of aerosolized microbots.

For disease of the lung, the physical key to effective inhalation-based therapy is size; too large (10's of µm) and the particles or droplets do not remain suspended in air to reach deep within the lungs, too small (subµm) and they are simply exhaled without deposition. µBots within this ideal low-µm size range however are challenging to fabricate and would lead to devices that lack the speed and power necessary for performing work throughout the pulmonary network. To uncouple size from structure and function, here we demonstrate an approach where individual building blocks are aerosolized and subsequently assembled in situ into µbots capable of translation, drug delivery, and mechanical work deep within lung mimics. With this strategy, a variety of pulmonary diseases previously difficult to treat may now be receptive to µbot-based therapies.

Reconfigurable microbots folded from simple colloidal chains.

To overcome the reversible nature of low-Reynolds-number flow, a variety of biomimetic microrobotic propulsion schemes and devices capable of rapid transport have been developed. However, these approaches have been typically optimized for a specific function or environment and do not have the flexibility that many real organisms exhibit to thrive in complex microenvironments. Here, inspired by adaptable microbes and using a combination of experiment and simulation, we demonstrate that one-dimensional colloidal chains can fold into geometrically complex morphologies, including helices, plectonemes, lassos, and coils, and translate via multiple mechanisms that can be varied with applied magnetic field. With chains of multiblock asymmetry, the propulsion mode can be switched from bulk to surface-enabled, mimicking the swimming of microorganisms such as flagella-rotating bacteria and tail-whipping sperm and the surface-enabled motion of arching and stretching inchworms and sidewinding snakes. We also demonstrate that reconfigurability enables navigation through three-dimensional and narrow channels simulating capillary blood vessels. Our results show that flexible microdevices based on simple chains can transform both shape and motility under varying magnetic fields, a capability we expect will be particularly beneficial in complex in vivo microenvironments.

Microwheels on Microroads: Enhanced Translation on Topographic Surfaces.

Microbot locomotion is challenging because of the reversible nature of microscale fluid flow, a limitation that can be overcome by breaking flowfield symmetry with a nearby surface. We have used this strategy with rotating wheel-shaped microbots, µwheels, that roll on surfaces leading to enhanced propulsion and fast translation speeds. Despite this, studies to date on flat surfaces show that µwheels roll inefficiently with significant slip. Taking inspiration from the mathematics of roads and wheels, here we demonstrate that µwheel velocities can be significantly enhanced by changing microroad topography. In this, we observe that periodic bumps in the road can be used to enhance the traction between µwheels and nearby walls. While continuous µwheel rotation with slip is observed on flat surfaces, a combination of rotation with slip and non-slip flip occurs when µwheels roll upon surfaces with periodic features, resulting in up to four-fold enhancement in translation velocity. The surprisingly fast rolling speed of µwheels on bumpy roads can be attributed to the hydrodynamic coupling between µwheels and road surface features, allowing non-slip rotation of entire wheels along one of their stationary edges. This road/wheel coupling can also be used to enhance µwheel sorting and separation where the gravitational potential energy barrier induced by topographic surfaces can lead to motion in only one direction and to different rolling speeds between isomeric wheels, allowing one to separate them not based on size but on symmetry.

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.

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.

Magnetic Microlassos for Reversible Cargo Capture, Transport, and Release.

Microbot propulsion has seen increasing interest in recent years as artificial methods that overcome the well-established reversible and challenging nature of microscale fluid mechanics. While controlled movement is an important feature of microbot action, many envisioned applications also involve cargo transport where microbots must be able to load and unload contents on command while tolerating complex solution chemistry. Here we introduce a physical method that uses flexible and linked superparamagnetic colloidal chains, which can form closed rings or "lassos" in the presence of a planar rotating magnetic field. By adding an additional AC magnetic field along the direction perpendicular to the substrate, we can orient the lasso at a tilted camber angle. We show that these magnetic lassos can roll at substantial velocities, with precise spatial control by manipulating both field strength and phase lag. Moreover, the lasso can curl around and capture cargo tightly and transport it based on a wheel-type mechanism. At the targeted destination, cargo is easily released upon field removal and the lasso can be readily reused. Since the entire process is physically controlled with no chemistry for attachment or disengagement involved, our system can potentially be used for transporting diverse types of cargo under different solution conditions.

Non reciprocal skewed rolling of a colloidal wheel due to induced chirality.

We use a rotating magnetic field to assemble an oblate cluster of paramagnetic colloidal particles. If the field is rotating about a horizontal axis, the cluster acts as a colloidal wheel rolling across the supporting glass surface. The motion is reversible upon switching the direction of rotation. Surprisingly, the reversibility is lost if the axis of field rotation is tilted with respect to the surface. The wheel then rolls in a direction that is not perpendicular to the field rotation axis. We explain the skewed rotation with an interplay between a magnetic driving torque, magnetic anisotropy and an anisotropy in the hydrodynamic mobility tensor in the vicinity of a surface. The opposing forward and backward drive induce opposite chirality in the degrees of freedom of the mechanically achiral colloidal wheel.

High-throughput linear optical stretcher for mechanical characterization of blood cells.

This study describes a linear optical stretcher as a high-throughput mechanical property cytometer. Custom, inexpensive, and scalable optics image a linear diode bar source into a microfluidic channel, where cells are hydrodynamically focused into the optical stretcher. Upon entering the stretching region, antipodal optical forces generated by the refraction of tightly focused laser light at the cell membrane deform each cell in flow. Each cell relaxes as it flows out of the trap and is compared to the stretched state to determine deformation. The deformation response of untreated red blood cells and neutrophils were compared to chemically treated cells. Statistically significant differences were observed between normal, diamide-treated, and glutaraldehyde-treated red blood cells, as well as between normal and cytochalasin D-treated neutrophils. Based on the behavior of the pure, untreated populations of red cells and neutrophils, a mixed population of these cells was tested and the discrete populations were identified by deformability. © 2015 International Society for Advancement of Cytometry.

Viscoelasticity as a biomarker for high-throughput flow cytometry.

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.

Measuring cell mechanics by optical alignment compression cytometry.

To address the need for a high throughput, non-destructive technique for measuring individual cell mechanical properties, we have developed optical alignment compression (OAC) cytometry. OAC combines hydrodynamic drag in an extensional flow microfluidic device with optical forces created with an inexpensive diode laser to induce measurable deformations between compressed cells. In this, a low-intensity linear optical trap aligns incoming cells with the flow stagnation point allowing hydrodynamic drag to induce deformation during cell-cell interaction. With this novel approach, we measure cell mechanical properties with a throughput that improves significantly on current non-destructive individual cell testing methods.

Erythrocyte deformation in high-throughput optical stretchers.

Optical stretchers can be used to quantify elastic and homeostatic properties of cells. Because they can apply forces to cells without requiring direct contact, they may noninvasively measure mechanical properties related to cell and membrane health. Present-day optical stretchers are, however, limited to measurements on individual stationary cells, limiting throughput. To overcome this limitation and allow study of variations in cell populations, we recently developed and tested a microfluidic chamber that measures optical stretching parameters for erythrocytes under dynamic flowing conditions. The method uses a single linear diode laser bar and permitted measurements at low flow rates and higher throughput. Here, we numerically investigate the feasibility of further increasing the measurement rates of the optical stretcher in parameter domains where hydrodynamic and optical forces are of comparable magnitude. To do this we couple a recently implemented dynamic optical ray-tracing technique with a fluid-structure interaction solver to simulate the deformation of osmotically swollen erythrocytes in fluid flow of variable rate. Our results demonstrate that a detectable steady-state stretch is induced at nominal optical powers and flow rates. In addition, we find that flow rates can be increased significantly with no major effect on net cell stretch showing the feasibility of application of this technique at greatly increased throughputs.