Microfluidics-Integrated Photothermal Nanoblade for High-Throughput Large Cargo D

描述（由申请人提供）：生物材料转移用于所有生物领域，以操纵细胞功能，并在生理和病理环境中解剖分子和细胞机制。需要新的、可行的技术来克服所有当前递送方法固有的尺寸限制，以允许转移大型货物，例如活病原体、整个染色体，以及（最终）用于新时代治疗的生物工程替代部件，例如转基因线粒体。正如《自然方法》的高级编辑Nicole Rusk最近所说的，“令人惊讶的是，没有一种方法可以有效地将不必要的大分子带入活细胞（《自然方法》8:44,2011）。”本提案的重点是我们最近开发的货物转移技术，称为光热纳米叶片，将整个人类染色体输送到人类多能干细胞（hPSCs）中，以检查染色体重编程。这些转移的染色体将在hPSC细胞核内使用标准和尖端的分子技术进行表观遗传重编程分析，并得到强大的机构生物信息学基础设施的支持。该提案还旨在开发一种微流体集成光热纳米刀片平台，用于下一代高通量和几乎同时将大型货物在几秒钟内运送到10,000多个细胞中。实现这些目标将为生物学研究提供定制的工程hPSCs，对联邦注册的H1和H9人类胚胎干细胞（hESCs）的染色体重编程能力进行评估，以及一个先进但易于使用的平台，以克服潜在的生物学障碍，如果它们存在，如果染色体整合是低频事件，则由于通量不足而限制成功，目前尚不清楚。

DESCRIPTION (provided by applicant): Biomaterial transfer is used in all biological fields to manipulate cell function and dissect molecular and cellular mechanisms in physiologic and pathologic settings. New, enabling technologies are required to overcome size limitations inherent in all current delivery methods to allow the transfer of large cargo, such as live pathogens, whole chromosomes, and (eventually) bioengineered replacement components, such as genetically modified mitochondria, for new-age therapies. As Nicole Rusk, senior editor Nature Methods, recently opined "surprisingly, no methods for efficiently bringing impermeant large molecules into living cells exist (Nat Methods 8:44, 2011). This proposal is focused on our recently developed cargo transfer technology, which is called the photothermal nanoblade, to deliver whole human chromosomes into human pluripotent stem cells (hPSCs) in order to examine chromosome reprogramming. These transferred chromosomes will be analyzed for epigenetic reprogramming within the hPSC nucleus using standard and cutting-edge molecular techniques and is supported by a strong institutional bioinformatics infrastructure. This proposal also aims to develop a microfluidics-integrated photothermal nanoblade platform for next-generation high- throughput and near simultaneous delivery of large cargo into more than 10,000 cells in seconds. Achieving these goals will provide custom engineered hPSCs for biological investigations, an assessment of the chromosome reprogramming capability of federally-registered H1 and H9 human embryonic stem cells (hESCs), and an advanced yet simple to use platform to overcome potential biological obstacles that, if they exist, would limit success due to an insufficient throughput, should chromosome integration be a low frequency event, which is currently not known. PUBLIC HEALTH RELEVANCE: Biomaterial transfer is used in all biological fields to manipulate cell function and dissect molecular and cellular mechanisms in physiologic and pathologic settings. New, enabling technologies are required to overcome size limitations inherent in all current delivery methods to allow the transfer of large cargo for new-age therapies. Our R21 proposal is to use a novel photothermal nanoblade system (Wu, et al., in press, Analytical Chemistry, 2011- see paper in appendix) to deliver whole human chromosomes into pluripotent human stem cells, a currently impossible task, in order to examine whole chromosome epigenetic reprogramming.