Novel Micro/nanofluidic Electroporation Devices for DNA&Oligonucleotide Delivery

描述(申请人提供)：电穿孔是一种广泛使用的将核酸和其他分子探针引入细胞或组织的转基因方法。最近的进展使该方法适用于广泛的细胞和生物分子，用于体外转基因测试，以及体外甚至体内临床试验。目前全球电穿孔市场价值约为2500万美元，在学术界、政府以及制药和生物技术公司的刺激下，预计未来五年将以两位数的增长迅速增长。目前商业化的电穿孔设备和试剂盒(特别是Amaxa Nucleofector技术)方便、易用，当需要非病毒基因转移时，对许多难以转化的细胞系是有利的。然而，一个主要的缺点是，每个新的细胞系统(如实验室特定的克隆或患者的原代细胞)通常需要耗时且昂贵的反复试验过程来确定适当的电穿孔条件和/或溶液组合物，以实现所需的转染率和足够高的细胞存活率。每个电池系统必须使用微调的高电压和不均匀的电场单独进行优化。如果电穿孔能够在温和、均匀的电场中进行，并且对各种细胞系和原代细胞有效，这将是非常有价值的。以此方式，可以消除或高度简化为每个小区系统确定新设置的需要。在临床应用中，需要大量的细胞(如&gt；109细胞)具有较高的转染率和细胞存活率。这在当前商业电穿孔设备中很难实现，其中推荐的细胞数量约为105 106。我们计划设计、建造和表征一种间歇式纳米颗粒夹层电穿孔(NSE)器件和一种连续型收敛流电穿孔(CFE)器件。这两个新的设备将被用来研究选定的寡核苷酸和用于癌症和干细胞应用的基因的体外转染效率和摄取。他们的性能将与最好的商业电穿孔系统进行比较。为了更好地体外和体外转导治疗材料，将开发几种新型的电穿孔设备。在这项研究中，将研究寡核苷酸和质粒DNA在癌细胞和干细胞中的摄取。所提出的装置具有将寡核苷酸和质粒DNA导入大量细胞用于临床应用的巨大潜力。

DESCRIPTION (provided by applicant): Electroporation is a widely used transfection method to introduce nuclear acids and other molecular probes into cells or tissues. Recent progress has made this method applicable to a wide range of cells and biomolecules, for in vitro transfection testing and for ex vivo and even in vivo clinical trials. The current worldwide market for electroporation is about $25 million and is expected to grow rapidly with double-digit gains over the next five years, stimulated by academia, government and pharmaceutical and biotech companies. The current commercial electroporation devices and kits (Amaxa Nucleofector technology in particular) are convenient, easy to use, and beneficial for many hard-to-transfect cell lines when non-viral gene transfer is required. However, a major drawback is that each new cell system (such as lab-specific clones or a patient's primary cells) often requires a time consuming and expensive trial-and-error search process to identify proper electroporation conditions and/or solution composition that will achieve the desired transfection level with high enough cell viability. Each cell system must be optimized individually using a finely-tuned, high electric voltage and non-uniform electric field. It would be very valuable if electroporation could be carried out in a mild, uniform electric field that is effective with various cell lines and primary cells. In this way, the need to determine new settings for each cell system can be eliminated or highly simplified. For clinical applications, there is a need to transfect a large number of cells (e.g. >109 cells) with a high transfection efficiency and cell viability. This is difficult to achieve in the current commercial electroporation device where the recommended cell population is around 105 106. We plan to design, construct and characterize a batch-type Nanonozzle Sandwich Electroporation (NSE) device and a continuous-type Converging Flow Electroporation (CFE) device. These two novel devices will then be used to investigate in vitro transfection efficiency and uptake of selected oligonucleotides and genes for cancer and stem cell applications. Their performance will be compared with the best commercial electroporation systems. Several novel electroporation devices will be developed for better in vitro and ex vivo transfection of therapeutic materials. In this study, the uptake of oligonucleotides and plasmid DNA into cancer cells and stem cells will be investigated. The proposed devices have great potential to transfect oligonucleotides and plasmid DNA into a large number of cells for clinical applications.