Engineering Efficient CAR‐T Cells via Electroactive Nanoinjection

Chimeric antigen receptor (CAR)‐T cell therapy has emerged as a promising cell‐based immunotherapy approach for treating blood disorders and cancers, but genetically engineering CAR‐T cells is challenging due to primary T cells’ sensitivity to conventional gene delivery approaches. The current viral‐based method can typically involve significant operating costs and biosafety hurdles, while bulk electroporation (BEP) can lead to poor cell viability and functionality. Here, a non‐viral electroactive nanoinjection (ENI) platform is developed to efficiently negotiate the plasma membrane of primary human T cells via vertically configured electroactive nanotubes, enabling efficient delivery (68.7%) and expression (43.3%) of CAR genes in the T cells, with minimal cellular perturbation (>90% cell viability). Compared to conventional BEP, the ENI platform achieves an almost threefold higher CAR transfection efficiency, indicated by the significantly higher reporter GFP expression (43.3% compared to 16.3%). By co‐culturing with target lymphoma Raji cells, the ENI‐transfected CAR‐T cells’ ability to effectively suppress lymphoma cell growth (86.9% cytotoxicity) is proved. Taken together, the results demonstrate the platform's remarkable capacity to generate functional and effective anti‐lymphoma CAR‐T cells. Given the growing potential of cell‐based immunotherapies, such a platform holds great promise for ex vivo cell engineering, especially in CAR‐T cell therapy. An electroactive nanoinjection (ENI) platform consisting of arrays of vertically configured conductive nanotubes, which mediate the loading and efficient nanoinjection of CAR construct into primary human T cells, with negligible cellular damage, is developed. The engineered ENI platform provides a non‐viral, low‐voltage (10 V), scalable, and reusable intracellular delivery system that can generate functional and effective anti‐lymphoma CAR‐T cells.