UC Riverside

Physical methods of intracellular delivery based on microfluidic approaches have emerged as a promising alternative to conventional delivery and cellular engineering techniques with many possible applications, particularly in the gene therapy sphere. No matter the physical poration method, there are tradeoffs between delivery efficiency and cell viability, and these differ depending on the manner of membrane poration. For several physical delivery methods, the process is difficult to control and random, which can limit their effectiveness and utility. Herein, we propose a novel silicon-based ultrahigh throughput deterministic mechanoporation (DMP) device platform for nanomechanical gene delivery into cells to address the current gap in knowledge and performance in current gene delivery techniques. This platform functions through microfluidic flow control, inducing the impingement of a single cell upon a solid needle resulting in a membrane pore after the cells are released, but on a massively-parallelized scale. This temporary pore allows diffusion of genetic cargo into the cell before membrane repair.In these studies, the performance of DMP delivery of model genetic cargos into human T cells was investigated, as well as how experimental design and analysis influences these types of experimental results. Next, characterization of how T cells interact with and are morphologically impacted by the DMP device was performed along with reassessment of device performance with small molecule cargo delivery. Finally, a preliminary investigation of how larger, complex gene editing constructs can be delivered to cells through conventional nonviral techniques was completed with a novel genetic construct. Through future design and operational parameter optimization, DMP aims to serve as a critical enabler for gene editing applications, given their dependence on successful delivery of these large genetic cargos and the survivability of edited cells for expansion and eventual treatment of patients with gene therapies.