Abstract
Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment by engineering patients' T cells to specifically target cancer cells. Traditional CAR-T cell manufacturing methods use viral transduction to integrate CAR genes into T cells, but this can cause severe side effects and immune reactions and is costly. To overcome these challenges, non-viral methods, such as plasmid DNA (pDNA) transfection, are being explored. Here, a high-throughput intracellular delivery platform that integrates microfluidic mechanoporation with lipid nanoparticle (LNP)-based delivery, LNP + Squeeze, is introduced. This system enhances pDNA transfection efficiency in T cells while maintaining cell viability compared to other non-viral transfection methods like electroporation. This platform successfully engineers CAR-T cells using primary human T cells with a high transfection efficiency and demonstrates potent cytotoxicity against melanoma cells. This approach offers a promising, cost-effective, and scalable alternative to viral methods, potentially improving the accessibility and efficacy of CAR-T cell therapies.