Probing the Bioeffects of Cavitation at Single-Cell Level

描述（由申请人提供）：由于其非侵入性和非电离性质，治疗性超声和聚焦冲击波已在临床上广泛用于治疗癌症、靶向药物递送（如打开血脑屏障）、崩解结石、分离软组织和愈合骨折不愈合以及疼痛治疗。尽管在所有这些应用中，空化通常被认为是产生期望的治疗效果和在某些情况下产生不期望的副作用的关键机制，但是气泡-组织相互作用，特别是气泡-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，可以用来调查这种动态的相互作用，具有足够的空间和时间分辨率。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，以研究空化气泡在单细胞水平上产生的生物效应。该项目将由一个多学科研究团队进行，该团队在治疗性超声和空化（Zhong Lab）和细胞力学（Guilak Lab）方面具有公认的专业知识。提出了两个具体目标：1）开发基于微流体的系统，其允许精确控制在每个通道中生长的单个细胞的位置和取向，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， 膜穿孔、Ca 2+内流、细胞骨架重排、损伤修复、存活力和增殖）和由气泡-细胞相互作用产生的细胞的机械变形（使用代表性细胞系（即，HeLa细胞、MDCK上皮细胞和软骨细胞）。随着对气泡-细胞相互作用以及不同细胞类型引起的机械和生物学后果的更好理解，我们希望获得未来可能用于改善治疗性超声和冲击波设备设计的见解，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用于开发新型超声技术，该技术可用于在精确的机械负载条件下刺激干细胞，以影响其分化，生长和表型表达，并具有组织工程和再生医学应用。R 03申请的目的是证明构建这种新型实验系统和开发相关技术的可行性，以评估单细胞水平空化气泡产生的生物效应，而这种新型系统的综合利用将在后续的RO 1申请中进行开发。

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.