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强大而可靠的超声策略，用于细胞内递送所需的药物（例如药物，基因，成像标记物），用于生物医学应用，如靶向癌症治疗，分子成像和基因治疗。人们普遍认识到，需要开发方法来实现药物的靶向递送，并改进基因递送方法，以用于基于基因的多种疾病治疗。由于超声暴露是安全且无创的，并且允许暂时和空间上的靶向应用，超声介导的递送有可能提供一种有利的策略，特别是在体内临床应用中，以克服安全性问题的限制，可能与电穿孔和病毒转染等方法相关的诱变和免疫反应。

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.