Enhancing non-viral gene delivery to human T cells through tuning nanoparticles physicochemical features, modulation cellular physiology, and refining transfection strategies

Genetically engineered immune cells hold great promise for treating immune-related diseases, but their development is hindered by technical challenges, primarily related to nucleic acid delivery. Polyethylenimine (PEI) is a cost-effective transfection agent, yet it requires significant optimization for effective T cell transfection. In this study, we comprehensively fine-tuned the characteristics of PEI/DNA nanoparticles, culture conditions, cellular physiology, and transfection protocols to enhance gene delivery into T cells. Gel retardation and dynamic light scattering (DLS) analyses confirmed that PEI effectively bound to DNA, forming size- and charge-adjustable particles based on the N/P ratio, which remained stable in RPMI 1640 medium for 3 days at 25°C. At an N/P ratio of 8.0, these nanoparticles achieved an optimal transfection rate, which improved further with adjustments in DNA dosage and complex volume. Additionally, increasing the cell seeding density and adding complete media shortly after transfection significantly boosted PEI-mediated gene delivery. Notably, reversing the transfection in vials resulted in a 20-fold increase in cellular uptake and transfection efficiency compared to the conventional direct transfection method in culture plates. Finally, modifying cellular physiology with hypotonic extracellular media at pH 9.0 dramatically enhanced transfection rates while maintaining minimal cytotoxicity. These findings could reduce the cost and complexity of preparing engineered T cells, potentially accelerating the development of immune cell therapies for human diseases.