Mechanisms of ultrasound mediated intracellular drug and gene delivery

描述(申请人提供)：这项研究的长期目标是开发强大和可靠的超声策略，用于生物医学应用，如靶向癌症治疗、分子成像和基因治疗，用于理想的药物(例如，药物、基因、成像标记)的细胞内传递。人们普遍认为，需要开发方法来实现药物的靶向递送，并改进基于基因的多种疾病治疗的基因递送方法。由于超声照射是安全和非侵入性的，并且允许暂时和空间上的靶向应用，超声介导的传递有可能为体内临床应用提供一种有利的策略，以克服与电穿孔和病毒转染等方法相关的安全问题、可能的突变和免疫反应的限制。 已有研究表明，超声波可增强细胞内对化疗药物、遗传物质和荧光葡聚糖分子的摄取，而这些物质通常不能透过完整的细胞膜。假说是超声波作用，在此过程中，由于超声波照射，细胞膜上形成了毛孔，允许细胞外分子和物质在重新密封之前进入细胞。然而，尽管该领域最近取得了一些进展，但其机制仍不完全清楚，在提高传递效率和细胞存活率方面仍存在许多问题和挑战。 为了实现我们开发最终临床应用的最佳超声传递策略的目标，本研究基于大量的统计事件，通过在单细胞水平和细胞水平上研究超声修饰的动态过程来研究超声修饰的机制。具体目标是： 1.从单细胞水平研究声学修饰的过程和机制。为了系统地研究和表征超声操作，我们将开发和建立一种集成的方法，包括膜片钳技术的新应用，动态荧光成像，以及用于超声后分析的各种检测方法。我们计划 A)定量描述声学参数(频率、强度、暴露方案)和微泡造影剂在细胞水平上影响声波修复的关键方面(气孔形成、持续时间和再密封)； B)研究Ca~(2+)对超声修复动力学(尤其是膜再封闭)和超声后细胞存活的影响； 2.发展超声技术，以达到最佳的细胞内给药效果。我们将使用两个模型系统来研究和优化各种相关剂型(如DNA、荧光探针、抗癌药物和纳米颗粒)中的超声细胞内递送药物：结肠癌细胞系和成人心肌细胞。我们会 A)建立传递结果(细胞内摄取和细胞存活)与超声操作条件(超声参数、Optison浓度和钙)的相关性； B)通过控制声学手术条件来实现最佳的分娩效果。

DESCRIPTION (provided by applicant): The long term goal of this research is to develop robust and reliable ultrasound strategy for intracellular delivery of desirable agents (e.g. drugs, genes, imaging markers) for biomedical applications such as targeted cancer treatment, molecular imaging, and gene therapy. There exists a widely recognized need to develop methods to achieve targeted delivery of drugs and to improve the methods of gene delivery for gene-based therapy of numerous diseases. As ultrasound exposure is safe and non-invasive, and allows targeted application both temporarily and spatially, ultrasound mediated delivery has the potential to provide an advantageous strategy especially for in vivo clinical applications to overcome the limitations of safety concerns, possibly mutagenesis and immune responses associated with methods such as electroporation and viral transfection. It has been demonstrated that ultrasound application results in enhanced intracellular uptake of chemotherapeutic compounds, genetic materials, and fluorescent dextran molecules, which are normally not permeable through intact cell membrane. The hypothesis is that sonoporation, during which pores form in the cell membrane as the result of ultrasound exposure, allowing entry of extracellular molecules and substances into the cell before resealing. However, despite of the recent progress made in the field, the mechanisms of sonoporation are not completely understood and many problems and challenges remain to improve delivery efficiency and cell survival rate. To achieve our goal of developing optimal ultrasound mediated delivery strategy for ultimate clinical applications, this research focuses on investigating the mechanisms of sonoporation by studying the dynamic processes of sonoporation at both the single cell level and the cellular level based on a large number of statistical events. The specific aims are: 1. To study the sonoporation process and mechanism at the single cell level. To systematically investigate and characterize sonoporation, we will develop and establish an integrated approach including novel application of patch clamp technique, dynamic fluorescent imaging, in addition to various assay methods for post-ultrasound analysis. We plan to a) characterize quantitatively the key aspects of sonoporation (pore formation, duration, and resealing) at the cellular level affected by acoustic parameters (frequency, intensity, exposure protocol) and microbubble contrast agents; b) investigate the effects of Ca2+ on sonoporation dynamics (especially membrane resealing) and post ultrasound cell survival; 2. To develop ultrasound method to achieve optimal intracellular delivery outcome. We will investigate and optimize ultrasound intracellular delivery of agents in various relevant formulations of interest (e.g. DNA, fluorescent probes, cancer drugs, and nano-particles) using two model systems: a colonic cancer cell line and adult cardiac myocytes. We will a) establish correlation of delivery outcome (intracellular uptake and cell survival) with sonoporation conditions (ultrasound parameters, Optison concentration, and Calcium); b) achieve optimal delivery outcome through controlling the sonoporation conditions.