Acoustic-transfection using high frequency ultrasound for intracellular delivery of macromolecules into targeted single cells

使用高频超声进行声转染，将大分子细胞内递送到目标单细胞中

• 批准号: 9889137
• 负责人: Sangpil Yoon
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889137
• 关键词: Acoustics; Address; Area; Award; Biology; Biomedical Engineering; Biosensor; CRISPR/Cas technology; California; Cell Communication; Cell membrane; Cell physiology; Cells; Characteristics; Clinic; Contrast Media; Data; Development; Dextrans; Disease; Dyes; Electroporation; Endothelial Growth Factors; Engineering; Ensure; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Frequencies; Future; Gene Expression; Generations; Genes; Goals; Health; Human; Immune; Label; Laboratory Research; Lipid Bilayers; Location; Longitudinal Studies; Medical; Mentors; Methods; Molecular; Molecular and Cellular Biology; Monitor; Pathway interactions; Patients; Peptides; Phenotype; Phosphotransferases; Physiologic pulse; Plasma Cells; Play; Population; Process; Qi; Radiation; Recombinant Proteins; Regenerative Medicine; Reporting; Research; Research Personnel; Research Proposals; Resolution; Role; Signal Pathway; Signal Transduction; Specificity; System; Techniques; Testing; Therapeutic; Time; Training; Transfection; Ultrasonic Transducer; Ultrasonography; United States National Institutes of Health; Universities; Viral Vector; Work; base; career development; cellular engineering; cytotoxicity; daughter cell; experimental study; human embryonic stem cell; improved; individualized medicine; induced pluripotent stem cell; innovation; macromolecule; molecular imaging; nanoparticle; plasmid DNA; pluripotency; real-time images; screening; spatiotemporal; stem cells; treatment planning; voltage

Project Summary / Abstract Dr. Sangpil Yoon is a postdoctoral scholar at the Department of Biomedical Engineering at the University of Southern California (USC). He has developed an innovative technique for the intracellular delivery of macromolecules using high frequency ultrasound. Dr. Yoon is a perfect candidate for NIH Pathway to Independence Award (K99/R00) and he will smoothly transit from a mentored trainee to an independent investigator by completing research goals and career development training plans, proposed in this award proposal. Dr. Yoon has three mentors to provide their expertise and constructive and critical advice during his award period. Dr. Yoon will have trainings in ultrasound and medical ultrasonic transducer development from Dr. Kirk Shung, USC, molecular and cellular biology from Dr. Yingxiao Wang, University of California, San Diego (UCSD), and stem cell and regenerative medicine from Dr. Qi-Long Ying, USC. Transfection methods currently available in research laboratories and clinics are based on non-targeted and random process such as viral-vectors and electroporation. I have developed an innovative transfection method by focusing acoustic energy within very confined area of less than 10 μm using a very high frequency ultrasonic transducer with a center frequency of over 150 MHz, entitled acoustic-transfection. The key innovation is that the acoustic-transfection has the capabilities of single-cell level targeting and controlling the size of delivered macromolecules with low cytotoxicity. The hypothesis is that the developed acoustic- transfection can deliver various kinds of macromolecules into different cells with the peculiar capabilities that distinguish from other transfection methods. Understanding of signaling pathways and the activation of important molecular events during intracellular and intercellular interactions are important because these are a basic building block to identify cell phenotypes and some molecular events are precedent for certain disease. Based on this understanding, engineering cell fate and cell functions using efficient gene editing with CRISPR/Cas9 and the generation of iPSCs by delivering recombinant proteins for regenerative medicine will further advance human health. Therefore, the primary goal of this proposal is to deliver desired molecules using acoustic-transfection into designated cells to visualize cell-to-cell interactions and the signaling of important molecular events, to induce gene expressions, and to demonstrate the delivery of recombinant proteins labeled with fluorescence dyes. To test hypothesis and to achieve the goal of this proposal, three specific aims were developed. 1) I will further optimize acoustic- transfection using macromolecules with different sizes. 2) I will deliver FRET biosensors into neighboring or single cells to visualize molecular events under stimulation. 3) pCas9_GFP will be delivered into hESCs and GFP expression will be observed. Recombinant proteins conjugated with Alexa 488 will be continuously delivered into hNSCs using acoustic-transfection for 8 days to confirm short-term delivery efficacy and long- term endogenous pluripotency. The proposed acoustic-transfection will advance transfection methods with the safe delivery of versatile molecules into cells to better understand important molecular events in cells and develop improved therapeutic strategies to cure diseases.

项目摘要 /摘要 Sangpil Yoon博士是大学生物医学工程系的博士后科学 南加州（南加州大学）。他开发了一种创新技术，用于细胞内交付 大分子使用高频超声。 Yoon博士是NIH途径的理想候选人 独立奖（K99/R00），他将顺利地从受过指导的实习生到独立 通过完成研究目标和职业发展培训计划，调查员提出了该奖项 提议。 Yoon博士有三位导师在他的期间提供他们的专业知识，建设性和重要建议 奖励期。 Yoon博士将在超声和医疗超声传感器开发中进行培训 Kirk Shung博士，USC，来自加利福尼亚大学SAN的Yingxiao Wang博士的分子和细胞生物学 迭戈（UCSD），以及加州大学Qi-Long Ying博士的干细胞和再生医学。 当前在研究实验室和诊所中可用的转染方法基于非目标和 随机过程，例如病毒向量和电穿孔。我开发了一种创新的翻译方法 通过使用非常高的频率在小于10μm的非常狭窄的区域内将声能聚焦 超声传感器的中心频率超过150 MHz，标题为原声转染。钥匙 创新的是，声学转染具有单细胞水平定位和控制的功能 假设是发达的声学 转染可以通过具有特殊能力的各种大分子将各种大分子传递到不同的细胞中 与其他翻译方法不同。 在细胞内和 细胞间相互作用很重要，因为这些是识别细胞表型和 某些分子事件是某些疾病的先例。基于这种理解，工程细胞的命运 通过使用CRISPR/CAS9进行有效的基因编辑以及IPSC的生成，并通过传递来产生细胞功能 再生医学的重组蛋白将进一步改善人类健康。因此，主要目标 该建议是使用声学转染到指定细胞中传递所需的分子，以可视化 细胞对细胞相互作用以及重要分子事件的信号传导，诱导基因表达和对 证明用荧光染料标记的重组蛋白的递送。检验假设和 1）我将进一步优化声学 - 使用具有不同尺寸的大分子转染。 2）我将将FRET生物传感器传递到附近或 单个细胞可视化刺激下的分子事件。 3）PCAS9_GFP将被输送到hESC和 将观察到GFP表达。与Alexa 488结合的重组蛋白将连续 使用声学转染8天，以确认短期递送效率和长期 术语内源性多能。提出的声学转染将通过 安全递送多功能分子到细胞中，以更好地了解细胞中的重要分子事件 制定改进的治疗策略来治愈疾病。