High-Precision Non-Contact Plasmon-Induced Intracellular Delivery

摘要 最近出现了强大的基因组编辑方法(CRISPR相关的靶向核酸酶技术) 它们在干细胞方面的应用导致了在研究和开发干细胞方面的革命性突破 下一代基因编辑疗法。为了便于转导，基因编辑材料需要 以快速、高效和安全的方式进入细胞。尽管一些生理和生化指标 细胞内给药的方法现在在实验室环境中被常规使用，效率，吞吐量， 和毒性限制了他们在临床上的实施，将各种大小的货物普遍运送到所有类型的 细胞。 为了直接应对上述挑战，本提案旨在开发一种高精度 利用高度局部化和强化的等离子体诱导的细胞内递送方案 等离子体纳米管附近的电磁场。目标是开发一种集成的 平台，重点是干细胞基因编辑，用于细胞内传递的通用方法和 所有种类的货物和细胞类型的高效率和生存能力的表征从单细胞到 数百万个细胞。将开发两个原型平台：i)用于非接触的等离子体纳米管 结合扫描离子电导显微镜的细胞内递送；以及ii)并行化 通过惯性微流体的等离子体纳米管柱。这两个目标的成功实现将导致 实现了无需物理穿透细胞膜的普遍细胞内给药， 单货物，单电池精度。此外，近场光学传感将被引入到开发的 站台。它将为非侵入性、连续、无标记的单分子生物传感提供一条直接途径 敏感度。因此，不仅将询问等离子体诱导递送的基本机制， 但它也将为进一步改进已开发的平台提供现场反馈。

ABSTRACT The recent emergence of robust genome editing methods (CRISPR associated targeted nuclease technologies) and their applications to stem cells has led to revolutionary breakthrough in the research and development of next-generation gene-editing therapies. In order to facilitate the transfection, gene-editing materials need to be delivered into cells in a rapid, efficient, and safe manner. Although some of the physical and biochemical methods for intracellular delivery are now used routinely in laboratory settings, issues with efficiency, throughput, and toxicity have limited their clinical implementations for universal delivering all-sizes of cargos into all-types of cells. To directly address the aforementioned challenges, this proposal aims to develop a high-precision plasmon-induced intracellular delivery scheme which utilizes the highly localized and intensified electromagnetic field in the close proximity of the plasmonic nanopipettes. The goal is to develop an integrated platform, with an emphasis on stem cell gene-editing, for universal approach of intracellular delivery and characterization for all varieties of cargo and cell types with high-efficiency and -viability from single-cell to millions of cells. Two prototype platforms will be developed: i) plasmonic nanopipettes for non-contact intracellular delivery through combining with scanning ion conductance microscopy; and ii) Parallelization of plasmonic nanopipettes via inertial microfluidics. Successfully completion of these two aims would lead to realization of universal intracellular delivery without physically penetrating the cell membranes, with single-cargo, single-cell precision. Furthermore, near-field optical sensing will be introduced into the developed platforms. It will provide a direct route to non-invasive, continuous, label-free biosensing with single-molecule sensitivity. Therefore, not only the fundamental mechanism of the plasmon-induced delivery will be interrogated, but it will also provide an in-situ feedback to further improvement of the developed platforms.