Mechanisms of ultrasound mediated intracellular drug and gene delivery

描述（由申请人提供）：本研究的长期目标是开发稳健可靠的超声策略，用于生物医学应用（如靶向癌症治疗、分子成像和基因治疗）中所需药物（如药物、基因、成像标记物）的细胞内递送。人们普遍认为需要开发实现药物靶向递送的方法，并改进用于许多疾病的基于基因的治疗的基因递送方法。由于超声暴露是安全和非侵入性的，并且允许暂时和空间的靶向应用，因此超声介导的递送具有提供有利策略的潜力，特别是对于体内临床应用，以克服与诸如电穿孔和病毒转染的方法相关的安全性问题、可能的诱变和免疫应答的限制。 已经证明，超声应用导致化疗化合物、遗传物质和荧光葡聚糖分子的细胞内摄取增强，所述分子通常不能透过完整的细胞膜。假设是声孔作用，在此期间，由于超声暴露，细胞膜中形成孔，允许细胞外分子和物质在重新密封之前进入细胞。然而，尽管最近在该领域取得了进展，但声致孔的机制尚未完全理解，并且在提高递送效率和细胞存活率方面仍然存在许多问题和挑战。 为了实现我们的目标，开发最佳的超声介导的传输策略，最终的临床应用，本研究的重点是通过研究声孔效应的动态过程中，在单细胞水平和细胞水平上的基础上，大量的统计事件的调查声孔的机制。具体目标是： 1.在单细胞水平上研究声致穿孔的过程和机理。为了系统地研究和表征声孔效应，我们将开发和建立一种综合方法，包括膜片钳技术的新应用，动态荧光成像，以及各种超声后分析的测定方法。我们计划 a）在细胞水平上定量表征受声学参数（频率、强度、暴露方案）和微泡造影剂影响的声致穿孔的关键方面（孔形成、持续时间和重新密封）; B）研究Ca 2+对声孔动力学（特别是膜再密封）和超声后细胞存活的影响; 2.开发超声方法以获得最佳的细胞内递送结果。我们将使用两个模型系统研究和优化各种相关制剂（例如DNA，荧光探针，抗癌药物和纳米颗粒）中的药物的超声细胞内递送：结肠癌细胞系和成人心肌细胞。我们将 a）建立递送结果（细胞内摄取和细胞存活）与声穿孔条件（超声参数、Optison浓度和钙）的相关性; B）通过控制声致穿孔条件实现最佳递送结果。

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.