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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

基本信息

批准号：
10733852
负责人：
Sangpil Yoon
金额：
$ 12.74万
依托单位：
UNIVERSITY OF OKLAHOMA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10733852
关键词：
Acoustic transfection using frequency ultrasound

项目摘要

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.

项目总结/摘要博士Sangyun Yoon是密歇根大学生物医学工程系的博士后学者，南加州（USC）。他开发了一种创新的技术，使用高频超声波的高分子。Yoon博士是NIH Pathway的完美候选人，独立奖（K99/R 00），他将顺利地从一个有指导的实习生过渡到一个独立的研究员通过完成研究目标和职业发展培训计划，在这个奖项提出提议Yoon博士有三位导师，在他的课程中提供他们的专业知识和建设性和关键性的建议。奖励期。Yoon博士将接受超声和医疗超声换能器开发方面的培训，博士Kirk Shung，南加州大学，分子和细胞生物学，来自加州大学旧金山分校王颖晓博士迭戈（加州大学圣地亚哥分校），和干细胞和再生医学博士Qi-Long Ying，南加州大学。目前在研究实验室和诊所中可用的转染方法是基于非靶向的和非靶向的。随机过程，如病毒载体和电穿孔。我发明了一种新的转染方法通过使用非常高的频率将声能聚焦在小于10 μm的非常有限的区域内，中心频率超过150 MHz的超声换能器，称为声转染。关键本发明的创新之处在于，声转染具有单细胞水平靶向和控制细胞增殖的能力，递送的大分子的尺寸具有低细胞毒性。假设是发达的声学- 转染可以将各种大分子递送到具有特殊能力的不同细胞中，区别于其他转染方法。理解细胞内和细胞外信号传导途径和重要分子事件的激活，细胞间相互作用是重要的，因为它们是鉴定细胞表型的基本构件，某些分子事件是某些疾病的前兆。基于这种认识，工程细胞命运利用CRISPR/Cas9的高效基因编辑和iPSC的产生，用于再生医学的重组蛋白将进一步促进人类健康。因此，首要目标该建议的一个重要方面是使用声学转染将所需分子递送到指定细胞中，细胞间相互作用和重要分子事件的信号传导，诱导基因表达，证明了用荧光染料标记的重组蛋白的递送。为了检验假设，为了实现这一目标，制定了三个具体目标。1)我将进一步优化声学- 使用不同大小的大分子进行转染。2)我将把FRET生物传感器送到邻近的单个细胞来可视化刺激下的分子事件。3)pCas9_GFP将被递送到hESC中，将观察GFP表达。与Alexa 488缀合的重组蛋白将被连续地纯化。使用声学转染将其递送到hNSC中8天，以确认短期递送功效和长期递送功效。术语内源性多能性。所提出的声转染将推进转染方法，将多功能分子安全递送到细胞中，以更好地了解细胞中的重要分子事件，开发更好的治疗策略来治愈疾病。