Mechanisms of Ultrasound-Mediated Bioeffects

描述(申请人提供)：药物、蛋白质和基因在细胞内输送到活细胞，这是药物和基因输送中最大的挑战之一。以往的研究表明，在适当的条件下对细胞进行超声波辐照，可以在体外和动物体内无创地增加药物的药理作用和多种不同细胞类型的基因表达，促使分子进入细胞内。这些效应发生在不同于临床超声成像或加热的超声条件下。在超声波的这种新用途的背景下，机制尚不清楚，哪些化合物可以被驱动到细胞中，作为一个常见的副作用，一些细胞可以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输送到活细胞的声学和物理条件。为此，这项建议的具体目标是通过确定(1)分子被吸收到暴露于超声波的细胞中的机制(S)和(2)细胞在暴露于超声波时死亡的机制(S)来解决细胞反应的生物学和生物物理机制。实现这些目标将朝着合理设计方案、配方和设备的长期目标取得重大进展，这些方案、配方和设备在保持高细胞活力的同时实现高水平的细胞内递送。具体地说，目标1的研究遵循这样的假设，即分子摄取是通过大小约为1微米的膜破裂扩散发生的，这些破裂通过使用细胞内小泡的主动修补在生命周期约1分钟内重新密封。实验将确定膜破裂的存在、大小和寿命，以及破坏被主动或被动重新密封的机制。通过膜破坏的传输将被数学建模，主动传输的作用也将被研究。目的2研究假设细胞死亡可以发生在秒的时间尺度上，并表现出细胞凋亡、坏死和细胞凋亡的特征。实验将确定凋亡的核、线粒体、超微结构和酶的特征，并将它们与坏死和凋亡的特征进行对比。细胞死亡的动力学特别令人感兴趣，因为初步结果表明，细胞在暴露于超声波后的几秒钟内表现出晚期凋亡的特征，这与其他凋亡机制观察到的典型的数小时动力学形成鲜明对比。研究将使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，部分指导方法是与已知的细胞膜破坏机制进行比较，包括电穿孔和细胞机械损伤。在最后一年，机械发现将被用于设计和测试超声方案，以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.