Application of rapidly prepared nonactivated high-efficiency chimeric antigen receptor T cells in the treatment of bladder cancer

Chimeric antigen receptor (CAR) T-cell therapy is clinically limited by lentiviral vector dependence, ex vivo activation/expansion requirements, complex processes, high costs, and impaired antitumor functions. For advanced cancer, the long manufacturing cycle of traditional CAR-T cells further compromises the treatment timeliness, creating an urgent need for optimized preparation strategies. A gene delivery system was constructed using microtubule-associated sequence and nuclear localization signal peptide–modified poly(β-amino ester) nanoparticles, combined with microfluidic technology. Nonactivated primary human T cells were transfected without ex vivo activation. The transfection efficiency, gene expression persistence, and in vitro cytotoxicity against the B7-H3-positive bladder cancer cell line T24 were evaluated. The system achieved gene delivery to nonactivated T cells within 2 to 6 hours, with a transfection efficiency that was 7-fold higher than that of unmodified nanoparticles. Target genes were stably expressed for over 5 days. The resulting CAR-T cells exhibited potent, specific cytotoxicity against T24 cells, avoiding viral vector risks and reducing manufacturing costs. This strategy innovates CAR-T-cell preparation by targeting nonactivated T cells and overcoming traditional bottlenecks. This provides a simpler, faster, cost-effective, and safe approach for adoptive cellular immunotherapy with significant translational value for bladder cancer and other solid tumors.