T Cell Phenotype Influences Acoustofluidic Delivery of Biomolecules.

OBJECTIVE: Ultrasound-mediated sonoporation has been explored for intracellular delivery in T cells, yet most studies rely on immortalized cell lines, limiting translational relevance. This study aimed to directly compare acoustofluidic molecular delivery efficiency and intracellular distribution in Jurkat T cells and primary human T cells. METHODS: Ultrasound-mediated molecular delivery was assessed using cationic microbubbles in a 3D-printed acoustofluidic device. Ultrasound-mediated delivery efficiency of 150 kDa fluorescein-labeled dextran and green fluorescent protein-expressing plasmid (2.9 MDa) were quantified using flow cytometry. Confocal microscopy was employed to evaluate intracellular distribution. RESULTS: Sonoporation enhanced dextran uptake in both cell types while maintaining high viability; however, Jurkat T cells exhibited significantly greater uptake than primary human T cells under matched conditions. Confocal imaging revealed broad intracellular distribution of dextran in both cell types, with comparable fluorescence intensity in cytoplasmic and nuclear regions. Within primary human T cells, activated cells demonstrated greater dextran uptake than non-activated cells, while no significant differences were observed between CD4⁺ and CD8⁺ subsets. In contrast to dextran delivery, plasmid delivery resulted in modest, non-significant increases in gene expression in both Jurkat and primary T cells. CONCLUSION: Acoustofluidic treatment enhanced intracellular delivery in both Jurkat and primary human T cells, while revealing important cell-type-dependent differences in delivery outcomes. These results reveal key differences in molecular delivery between immortalized T cell lines and primary human T cells, which provide important insights for the development of ultrasound-based delivery approaches to advance clinical T cell engineering technologies.