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

项目总结 血栓形成或栓塞导致的重要血管狭窄是最常见的心脏瓣膜之一。 随着年龄的增长，疾病和死亡的主要原因。他们的血液动力学环境随着 结果，与1-70相比，高度狭窄的血管的剪应力增加了高达1000达因/平方厘米。 正常血管达因/厘米~2。由于增加的切应力激活了血小板，血管变得更多 狭窄处狭窄，远离血小板聚集，导致危及生命的中风或长期残疾。 目前对阻塞血管的治疗是使用溶栓或抗凝药物，但这需要 由于活性药物分布在全身，出血风险很高。因此，为了克服目前的限制， 这项提议的目标是开发一种合成细胞系统，它只在狭窄的血管中释放药物，在 它表现出异常高的剪应力。合成细胞是双层膜结构(例如，囊泡) 包括执行细胞样行为的各种生物分子。他们正在药物输送领域获得关注。 因为当它们被设计成与周围环境一起时，它们可以呈现对周围环境的感觉反应行为 膜蛋白。开发一种可用于靶向药物的剪应力响应型合成细胞 在狭窄的血管中输送，我们将使用研究最充分的细菌机械敏感通道， 大电导机械敏感通道(MSCL)。MSCL是一种非选择性通道，在 膜张力增加。据我们所知，我们的实验室是第一个使用骨髓间充质干细胞开发合成细胞的小组 并成功地演示了它们在低渗透条件下的功能。最近的理论研究已经 研究表明，在囊泡中重组的MSCL在流经 缩小狭窄航道。我们的假设是，整合在囊泡中的MSCL将在剪切下打开。 应激由囊泡状变形驱动，膜拉伸释放所载药物。我们会调查的 利用狭窄的微流体通道在切应力下的MSCL活性在AIM 1.促成因素，例如 囊泡大小和脂质成分，将被调整，以了解它们对MSCL反应的影响。在目标2中，我们将 在体外检测该系统的潜在价值。我们将把溶栓药物负载的合成细胞引入 用实验诱导的纤维蛋白栓子收缩并监测溶解的微流控通道 血块的含量。这项工作的成功完成将导致剪切应力响应型复合材料的开发 能在狭窄或狭窄的血管中局部释放溶栓或抗凝药物的细胞。这项工作将 利用机械刺激响应进一步拓展合成细胞领域的应用边界 人工合成细胞作为药物载体。此外，MSCL在剪切力下的成功激活将提供 加深对MSCL的了解，并展示该渠道作为一种药物在药物输送领域的有效性- 在更多的环境中释放阀。

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.