Shear stress-activated synthetic cells for targeted drug release in stenotic blood vessels

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PROJECT SUMMARY Narrowing of critical blood vessels due to thrombosis or embolism is one of the most prevalent heart valve diseases and the leading cause of death with aging. Their hemodynamic environment significantly changes as a result, with an increase in shear stress up to >1000 dyne/cm2 in highly constricted vessels compared to 1–70 dyne/cm2 in normal vessels. Since the increased shear stress activates platelets, the vessels become even more narrow at the stenotic site from the platelet aggregation, leading to a life-threatening stroke or long-term disability. The current treatment for obstructed vessels is to administer thrombolytic or anticoagulant drugs, but it entails high bleeding risk as active drugs are distributed throughout the body. Thus, to overcome current limitations, the goal of this proposal is to develop a synthetic cell system that only releases drugs in constricted vessels where it exhibits abnormally high shear stress. A synthetic cell is a bilayer membrane structure (e.g., vesicle) that includes various biomolecules to carry out cell-like behaviors. They are gaining attention in the drug delivery field as they can present sense-responsive behavior towards the surrounding environment when engineered with membrane proteins. To develop a shear stress-responsive synthetic cell that can be used for targeted drug delivery in stenotic blood vessels, we will use the most well-studied bacterial mechanosensitive channels, the mechanosensitive channel of large conductance (MscL). MscL is a non-selective channel that opens upon an increase in membrane tension. Our lab is the first group, to our knowledge, to develop synthetic cells using MscL and successfully demonstrate their function under hypo-osmotic condition. Recent theoretical studies have shown that the MscL reconstituted in vesicles can also be activated by shear stress when flowing through a narrowing constriction channel. Our hypothesis is that MscL incorporated in vesicles will be opened under shear stress by vesicle-shape deformation-driven membrane stretch and release the loaded drugs. We will investigate MscL activity under shear stress using constricted microfluidic channels in Aim 1. Contributing factors, such as vesicle size and lipid compositions, will be tuned to understand their effects on MscL response. In Aim 2, we will examine the potential value of the system in vitro. We will introduce thrombolytic drug-loaded synthetic cells into microfluidic channels that are constricted with experimentally induced fibrin emboli and monitor the dissolution of the clots. Successful completion of this work will result in the development of shear stress-responsive synthetic cells that can locally release thrombolytic or anticoagulant drugs in constricted or stenotic vessels. This work will further expand the application boundary of the synthetic cell field by utilizing mechanical stimulus-responsive synthetic cells as drug carriers. Additionally, successful activation of MscL under shear stress will provide a deeper understanding of MscL and demonstrates this channel's effectiveness in the drug delivery field as a drug- releasing valve in more diverse contexts.