Probing the Bioeffects of Cavitation at Single-Cell Level

描述(申请人提供)：由于其非侵入性和非电离性质，治疗性超声波和聚焦冲击波已在临床上广泛用于癌症的治疗、靶向药物输送(如开放血脑屏障)、结石粉碎、软组织分离、骨不连骨折的愈合以及疼痛治疗。虽然在所有这些应用中，空化通常被认为是产生理想治疗效果以及在某些情况下产生不良反应的关键机制，但气泡(S)-组织相互作用的动态过程，特别是气泡(S)-细胞相互作用的动态过程，在很大程度上是未知的。这主要是由于缺乏可行的实验系统，可以用来研究这种具有足够的空间和时间分辨率的动态相互作用。为了克服这一主要挑战，我们建议开发一种新的实验系统和相关技术，以在单细胞水平上研究空化气泡产生的生物效应。该项目将由一个在治疗性超声和空化(中实验室)和细胞力学(Guilak实验室)方面拥有公认专业知识的多学科研究团队进行。提出了两个具体目标：1)开发基于微流体的系统，该系统允许精确控制在每个通道中生长的单个细胞的位置和方向，以及它们与激光和超声产生的空化气泡的空间对准；2)研究生物效应(细胞裂解， 使用与治疗性超声波和冲击波治疗相关的代表性细胞系(即HeLa细胞、MDCK上皮细胞和软骨细胞)，研究气泡(S)-细胞相互作用所产生的细胞膜穿孔、钙离子内流、细胞骨架重排、损伤修复、存活和增殖)以及机械变形。随着对气泡(S)-细胞相互作用以及由此在不同细胞类型中引起的机械和生物后果的更好的了解，我们希望获得未来可能用于改进治疗性超声波和冲击波设备的设计以及安全和更有效的医疗应用的治疗方案。此外，从该项目中获得的新知识可能被用于开发新的超声波技术，该技术可以在精确的机械载荷条件下刺激干细胞，以影响其在组织工程和再生医学应用中的分化、生长和表型表达。R03应用的目的是证明在单细胞水平上构建这样一个新的实验系统和开发相关技术以评估空化气泡产生的生物效应的可行性，同时将在后续的RO1应用中开发这一新系统的综合利用。

DESCRIPTION (provided by applicant): Because of their non-invasiveness and non-ionization nature, therapeutic ultrasound and focused shock waves have been used extensively in clinic for the treatment of cancers, targeted drug delivery (such as opening of blood-brain-barriers), disintegration of concretions, fractionation of soft tissues, and healing of non-union fractures, a well as pain therapy. Although in all these applications cavitation has often been implicated as a key mechanism whereby the desirable therapeutic effects and, in some cases, the undesirable adverse effects are produced, the dynamic processes of bubble(s)-tissue interaction and, in particular, bubble(s)-cell interaction, are largely unknown. This is primarily due to the lack of viable experimental systems that can be used to investigate such dynamic interactions with sufficient spatial and temporal resolutions. To overcome this primary challenge, we propose to develop a new experimental system and associated technologies to investigate the bioeffects produced by cavitation bubbles at the single cell level. This project will be carried out by a multidisciplinary research team with recognized expertise in therapeutic ultrasound and cavitation (Zhong Lab) and cell mechanics (Guilak Lab). Two specific aims are proposed: 1) development of a microfluidic based system that allows for precise control of the location and orientation of individual cells grown in each channel, as well as their spatial alignment with laser- and ultrasound-generated cavitation bubbles, 2) investigation of the bioeffects (cell lysis, membrane poration, Ca2+ influx, cytoskeleton re-arrangement, injury repair, viability, and proliferation) and mechanical deformation of the cell produced by the bubble(s)-cell interaction using representative cell lines (i.e., HeLa cells, MDCK epithelial cells, and chondrocytes) relevant to therapeutic ultrasound and shock wave therapy. With a better understanding of the bubble(s)-cell interaction and the resultant mechanical and biological consequences elicited in different cell types, we hope to gain insights that may be used in the future to improve the design of therapeutic ultrasound and shock wave devices, as well as treatment protocols for safe and more effective medical applications. Furthermore, the new knowledge acquired from this project may be used to develop novel ultrasonic techniques that can be applied to stimulate stem cells under precise mechanical loading conditions to impact their differentiation, growth and phenotypic expression with tissue engineering and regenerative medicine applications. The goal of this R03 application is to demonstrate the feasibility in constructing such a novel experimental system and developing associated technologies for assessing the bioeffects produced by cavitation bubbles at the single-cell level, while the comprehensive utilization of this novel system will be exploited in a follow-up RO1 application.