Novel Micro/nanofluidic Electroporation Devices for DNA&Oligonucleotide Delivery

新型 DNA 微/纳流体电穿孔装置

• 批准号: 7363207
• 负责人: Ly James Lee
• 金额: $ 17.71万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7363207
• 关键词: Academia; Acceleration; Acids; Area; Biological Models; Cell Count; Cell Line; Cell Survival; Cell membrane; Cells; Clinical; Clinical Trials; Condition; Cultured Cells; DNA; Devices; Digit structure; Electroporation; Encapsulated; Evaluation; Gene Delivery; Gene Transfer; Genes; Government; Growth Factor; In Vitro; Localized; Marketing; Melanoma Cell; Membrane; Methods; MicroRNAs; Microcapsules drug delivery system; Microfluidics; Molecular Probes; Motion; Mus; Nuclear; Nucleic Acids; Oligonucleotides; Operative Surgical Procedures; Patients; Performance; Persons; Pharmacologic Substance; Pharyngeal structure; Physiologic pulse; Polymers; Population; Process; Provider; Pulse taking; Purpose; Range; Reporter Genes; Research; Research Proposals; Small Interfering RNA; Solutions; Stem cells; System; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Transfection; Viral; Work; base; blastomere structure; cancer cell; cancer stem cell; clinical application; design and construction; desire; electric field; embryonic stem cell; experience; in vivo; knock-down; leukemia; nanofluidic; novel; plasmid DNA; tool; transcription factor; two-dimensional; uptake; voltage

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.

描述（由申请人提供）：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染方法。最近的进展使这种方法适用于广泛的细胞和生物分子，用于体外转染测试以及离体甚至体内临床试验。目前全球电穿孔市场规模约为 2500 万美元，预计在学术界、政府以及制药和生物技术公司的刺激下，未来五年将以两位数的增幅快速增长。目前的商用电穿孔设备和试剂盒（尤其是 Amaxa Nucleofector 技术）方便、易于使用，并且在需要非病毒基因转移时对许多难以转染的细胞系有益。然而，一个主要缺点是每个新的细胞系统（例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试错搜索过程来确定适当的电穿孔条件和/或溶液成分，这将导致达到所需的转染水平和足够高的细胞活力。每个电池系统必须使用微调的高电压和非均匀电场单独优化。如果电穿孔能够在对各种细胞系和原代细胞有效的温和、均匀的电场中进行，那将是非常有价值的。通过这种方式，可以消除或高度简化为每个小区系统确定新设置的需要。对于临床应用，需要以高转染效率和细胞活力转染大量细胞（例如>109个细胞）。这在目前的商业电穿孔装置中很难实现，推荐的细胞数约为 105 106。我们计划设计、构建和表征间歇式纳米喷嘴夹心电穿孔（NSE）装置和连续式合流式电穿孔（CFE）装置。 ） 设备。然后，这两种新型设备将用于研究体外转染效率以及癌症和干细胞应用中选定寡核苷酸和基因的摄取。他们的性能将与最好的商业电穿孔系统进行比较。将开发几种新型电穿孔装置，以更好地体外和离体转染治疗材料。在本研究中，将研究癌细胞和干细胞对寡核苷酸和质粒 DNA 的摄取。所提出的装置具有将寡核苷酸和质粒DNA转染到大量细胞中用于临床应用的巨大潜力。