Dissertation

Development of Single-Molecule-Based Materials With Structures and Functions Responsive to Shear for Targeted Therapeutic Delivery

Ismail, Esraa

2025

Overview

This dissertation presents the development and characterization of shear-responsive platforms for targeted therapeutic delivery, inspired by the mechanosensitive properties of von Willebrand Factor (VWF), a key blood-clotting protein. By leveraging biomolecular insights into VWF unraveling under flow, a Single-Molecule-Based Material with Structures and Functions Responsive to Shear (SMORES) platform was engineered to address critical challenges in vascular disorders and enhance hemostatic regulation.The initial studies investigated how flow dynamics influence VWF conformation, particularly the effect of pulse frequency on VWF extension, to elucidate the disease mechanism underlying acquired von Willebrand syndrome (AVWS), a bleeding disorder observed in patients supported by continuous-flow ventricular assist devices (CF-VADs). CF-VADs, implantable mechanical pumps designed to maintain systemic circulation in patients with advanced heart failure, are associated with an elevated risk of non-surgical bleeding. This complication has been attributed to the loss of physiological pulsatility, which destabilizes VWF structure, promoting its unraveling and enzymatic degradation, thereby impairing clotting function. Although artificial pulsatility has been incorporated into some device designs, the specific impact of pulse frequency on VWF conformational stability remains poorly defined. To address this gap, in vitro microfluidic models were developed to study endothelial membrane-bound VWF and single-tethered immobilized VWF within devices connected to programmable fluidic pumps, enabling precise generation of continuous and pulsatile flow waveforms replicating physiological shear forces. These platforms facilitated real-time visualization of VWF conformational dynamics under distinct flow conditions. By comparing continuous flow to pulsatile profiles, this work aims to uncover mechanistic insights that inform the optimization of flow modulation protocols, including the identification of pulse frequencies that preserve VWF integrity and mitigate bleeding complications.Building on mechanistic insights into how flow modulates the structure and function of VWF, the SMORES platform was developed to enable shear-triggered molecular release. A well-established aptamer, selected for its high affinity to the VWF A1 domain, was incorporated into a construct designed for the controlled release of VWF A1—the platelet-binding domain—under pathological shear conditions indicative of vascular injury. This strategy aims to initiate clot formation at sites of vascular damage, particularly in patients with acquired von Willebrand syndrome (AVWS), where VWF is either dysfunctional or deficient. Each SMORES construct employs an aptamer molecule as the flow transducer and a microparticle to sense and amplify the hydrodynamic force. Within the construct, the aptamer, ARC1172, undergoes conformational changes beyond a shear stress threshold, mimicking the shear-responsive behavior of VWF. This conformational alteration modulates the bioavailability of its target, the VWF A1 domain, ultimately releasing it at elevated shear. Single-molecule optical tweezer experiments were used to characterize the force-responsive behavior of the aptamer, while flow-based microfluidic assays confirmed shear-dependent release. Functional assays demonstrated that the released A1 domain retained its biological activity. Building on aptamer biomechanics, this platform presents a new strategy for engineering shear-responsive biomaterials based on single-molecule designs.Building on the modularity of the SMORES platform, the shear-responsive system was adapted for the targeted delivery of tissue plasminogen activator (TPA), a clot-dissolving enzyme used in thrombolytic therapy. An anti-PLAT (plasminogen activator) aptamer with high affinity for TPA was incorporated into the construct, which also employed a shear-amplifying microparticle to facilitate force transmission. Upon exposure to pathological shear stresses, indicative of occluded vessels, conformational changes in the aptamer modulate the availability of TPA, enabling its release at the clot site. To enhance selective targeting, the construct was functionalized with monoclonal anti-αIIbβ3 antibodies, allowing specific binding to activated platelets within thrombi. Shear-triggered release of TPA was demonstrated using microfluidic flow models, and a clot dissolution assay was developed to further confirm the biological activity of the released payload. By combining shear-responsive release with platelet-specific targeting, this platform offers a promising strategy for localized thrombolysis with reduced systemic exposure, addressing key limitations of current thrombolytic therapies.Altogether, this work establishes SMORES as a promising strategy for shear-responsive, targeted therapeutic delivery in vascular diseases, and advances the development of physiologically relevant in vitro models for neurodegenerative disease research.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Bioengineering / Biomedical engineering
• ISBN: 9798291578278