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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：
10116417
负责人：
Sangpil Yoon
金额：
$ 12.16万
依托单位：
UNIVERSITY OF NOTRE DAME
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 2022-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10116417
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

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.

项目摘要/摘要本文作者是加州大学生物医学工程系博士后南加州(南加州大学)。他开发了一种创新的技术，用于细胞内传递使用高频超声波的大分子。Yoon博士是NIH途径的完美候选人独立奖(K99/R00)，他将从一个有指导的学员平稳过渡到一个独立的人调查员通过完成研究目标和职业发展培训计划，在本奖项中提出求婚。尹博士有三位导师提供他们的专业知识以及建设性和批判性的建议获奖期。Yoon博士将接受超声波和医用超声换能器开发方面的培训加州大学旧金山分校分子和细胞生物学博士Kirk Shung博士 Diego博士(UCSD)和南加州大学应启龙博士的干细胞和再生医学。目前研究实验室和诊所可用的转染法是基于非靶向和随机过程，如病毒载体和电穿孔。我开发了一种创新的转基因方法通过使用极高频率将声波能量聚焦在小于10μm的非常有限的区域内中心频率超过150 MHz的超声换能器，称为声学转染法。钥匙创新之处在于，声波转染法具有单细胞水平的靶向和控制输送的大分子大小具有低细胞毒性。假设是发展出来的声学- 转基因可以将各种大分子输送到不同的细胞中，具有独特的能力区别于其他的转染法。了解细胞内和细胞内重要分子事件的信号通路和激活细胞间的相互作用很重要，因为它们是识别细胞表型和某些分子事件是某些疾病的先例。基于这一认识，工程细胞命运和细胞功能，使用CRISPR/Cas9进行高效的基因编辑，并通过提供用于再生医学的重组蛋白将进一步促进人类健康。因此，首要目标是这一提议的目的是将所需的分子通过声学导入指定的细胞进行可视化细胞间的相互作用和重要分子事件的信号传递，以诱导基因表达，并展示用荧光染料标记的重组蛋白的传递。来检验假说和为了实现这一建议的目标，制定了三个具体目标。1)我将进一步优化音响- 用不同大小的大分子进行转染法。2)我将把FRET生物传感器送到邻居或单个细胞在刺激下可视化分子事件。3)pCas9_GFP将被输送到hESCs和观察GFP的表达情况。与Alexa 488结合的重组蛋白将连续用声学转染法将其导入hNSCs，为期8天，以确认短期给药效果和长期给药效果。术语内源性多能性。建议的声学转染法将促进基因转染法安全地将多功能分子输送到细胞内，以更好地了解细胞和制定改进的治疗策略来治愈疾病。