Mechanisms of Ultrasound-Mediated Bioeffects

描述（由申请人提供）：细胞内递送药物、蛋白质和基因到活细胞是药物和基因递送中最大的挑战之一。以往的研究表明，在适当的条件下将细胞暴露在超声波中，可以驱动分子进入细胞，非侵入性地增加药物的药理作用，并在体外和动物体内提高多种不同细胞类型的基因表达。这些影响发生在不同的超声条件下，从那些用于临床超声成像或加热。在这种超声波新应用的背景下，化合物进入细胞的机制尚不清楚，作为一个常见的副作用，一些细胞可能在这个过程中被杀死。我们之前在这一领域的工作强调了将分子传递到活细胞中的声学和物理条件。因此，本提案的具体目标通过确定(1)分子被吸收到暴露于超声波的细胞中的机制和(2)细胞在暴露于超声波时死亡的机制来解决细胞反应的生物学和生物物理机制。实现这些目标将对合理设计方案、配方和设备的长期目标取得重大进展，这些方案、配方和设备可以在保持高细胞活力的同时实现高水平的细胞内递送。具体来说，Aim 1研究的指导假设是，分子摄取是通过1微米大小的膜破坏扩散发生的，通过细胞内囊泡的主动修补，在一生中大约1分钟的时间内重新密封。实验将确定膜破坏的存在、大小和寿命，以及破坏被主动或被动重新密封的机制。通过膜破坏的运输将被数学建模，主动运输的作用也将被检查。Aim 2的研究基于这样的假设：细胞死亡可以在几秒的时间尺度上发生，并表现出细胞凋亡、坏死和细胞凋亡的特征。实验将确定细胞凋亡的核、线粒体、超微结构和酶的特征，并将其与坏死和凋亡的特征进行对比。细胞死亡的动力学是特别感兴趣的，因为初步结果表明，细胞在暴露于超声波后几秒钟内表现出晚期凋亡的特征，这与其他凋亡机制通常观察到的数小时动力学形成对比。研究将采用多种形式的电子和共聚焦显微镜、流式细胞术和数学分析作为其核心工具，部分通过与已知细胞膜破坏机制（包括电穿孔和机械细胞损伤）的比较来指导。在最后一年，机制的发现将用于设计和测试超声方案，优化分子摄取和细胞活力。

DESCRIPTION (provided by applicant): Intracellular delivery of drugs, proteins and genes into viable cells in one of the greatest challenges in drug and gene delivery. Previous studies show that exposure of cells to ultrasound under appropriate conditions drives molecules into cells to non-invasively increase pharmacological effect of drugs and expression of genes in a variety of different cell types in vitro and in animals in vivo. These effects occur under different ultrasound conditions from those employed in clinical ultrasonic imaging or heating. In the context of this new use of ultrasound, the mechanisms are not known by which compounds can be driven into cells and, as a common side effect, some cells can be killed in the process. Our previous work in this area has emphasized the acoustic and physical conditions that deliver molecules into viable cells. For this reason, the Specific Aims of this proposal address biological and biophysical mechanisms of the cell's response by determining (1) the mechanism(s) by which molecules are taken up into cells exposed to ultrasound and (2) the mechanism(s) by which ceils die when exposed to ultrasound. Carrying out these Aims will make significant advances toward the long term goal of rationally designing protocols, formulations and devices that achieve high levels of intracellular delivery while maintaining high cell viability. Specifically, Aim 1 studies are guided by the hypothesis that molecular uptake occurs by diffusion through membrane disruptions on the order of 1 micron in size that reseal over a lifetime on the order of 1 minute by active patching using intracellular vesicles. Experiments will determine the existence, size and lifetime of membrane disruptions, as well as the mechanism by which disruptions are actively or passively resealed. Transport through membrane disruptions will be modeled mathematically and the role of active transport will be examined too. Aim 2 studies are guided by the hypothesis that cell death can occur on a timescale of seconds and exhibits characteristic features of apoptosis, necrosis and paraptosis. Experiments will identify characteristic nuclear, mitochondrial, ultrastructural and enzymatic features of apoptosis and contrast them with features of necrosis and paraptosis. The kinetics of cell death is of special interest, since preliminary results suggest that cells exhibit characteristics of late stage apoptosis within seconds after exposure to ultrasound, which contrasts with kinetics typically of hours observed by other apoptosis mechanisms. Studies will employ multiple forms of electron and confocal microscopy, flow cytometry and mathematical analysis as their core tools guided in part by comparison with known mechanisms of cell membrane disruption, including electroporation and mechanical cell wounding. During the final year, mechanistic findings will be used to design and test ultrasound protocols that optimize molecular uptake and cell viability.