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.
(Relevance to Public Health) The recent emergence of gene-editing therapies has been revolutionary for next-generation treatment strategies for diseases such as cancer, anemia, and cystic fibrosis. The proposed work will pave the way for universal, high-precision, intercellular delivery of gene-editing materials, ushering forward technological advances broadly for a variety of health-related disciplines, including cell-based therapy, diagnostics, and stem-cell-based regenerative medicine.
