Engineering Efficient Car-T Cells via Electroactive Nanoinjection.

Chimeric antigen receptor (CAR)-T 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 can lead to poor cell viability and functionality. Here, we develop a non-viral electroactive nanoinjection (ENI) platform 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 bulk electroporation (BEP), the ENI platform achieves an almost 3-fold 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, we prove the ENI-transfected CAR-T cells' ability to effectively suppress lymphoma cell growth (86.9% cytotoxicity). 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, we anticipate that a non-viral and efficient nanoinjection platform like the one described here will offer a promising avenue for ex vivo cell engineering, particularly in the context of CAR-T cell therapy. This article is protected by copyright. All rights reserved.