Engineered nano–bio cellular interfaces driven by vertical nanostructured materials are set to spur transformative progress in modulating cellular processes and interrogations. In particular, the intracellular delivery—a core concept in fundamental and translational biomedical research—holds great promise for developing novel cell therapies based on gene modification. This study demonstrates the development of a mechanotransfection platform comprising vertically aligned silicon nanotube (VA‐SiNT) arrays for ex vivo gene editing. The internal hollow structure of SiNTs allows effective loading of various biomolecule cargoes; and SiNTs mediate delivery of those cargoes into GPE86 mouse embryonic fibroblasts without compromising their viability. Focused ion beam scanning electron microscopy (FIB‐SEM) and confocal microscopy results demonstrate localized membrane invaginations and accumulation of caveolin‐1 at the cell–NT interface, suggesting the presence of endocytic pits. Small‐molecule inhibition of endocytosis suggests that active endocytic process plays a role in the intracellular delivery of cargo from SiNTs. SiNT‐mediated siRNA intracellular delivery shows the capacity to reduce expression levels of F‐actin binding protein (Triobp) and alter the cellular morphology of GPE86. Finally, the successful delivery of Cas9 ribonucleoprotein (RNP) to specifically target mouse Hprt gene is achieved. This NT‐enhanced molecular delivery platform has strong potential to support gene editing technologies.
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
Nanofabrication technologies have been recently applied to the development of engineered nano–bio interfaces for manipulating complex cellular processes. In particular, vertically configurated nanostructures such as nanoneedles (NNs) have been adopted for a variety of biological applications such as mechanotransduction, biosensing, and intracellular delivery. Despite their success in delivering a diverse range of biomolecules into cells, the mechanisms for NN-mediated cargo transport remain to be elucidated. Recent studies have suggested that cytoskeletal elements are involved in generating a tight and functional cell–NN interface that can influence cargo delivery. In this study, by inhibiting actin dynamics using two drugs—cytochalasin D (Cyto D) and jasplakinolide (Jas), we demonstrate that the actin cytoskeleton plays an important role in mRNA delivery mediated by silicon nanotubes (SiNTs). Specifically, actin inhibition 12 h before SiNT-cellular interfacing (pre-interface treatment) significantly dampens mRNA delivery (with efficiencies dropping to 17.2% for Cyto D and 33.1% for Jas) into mouse fibroblast GPE86 cells, compared to that of untreated controls (86.9%). However, actin inhibition initiated 2 h after the establishment of GPE86 cell–SiNT interface (post-interface treatment), has negligible impact on mRNA transfection, maintaining > 80% efficiency for both Cyto D and Jas treatment groups. The results contribute to understanding potential mechanisms involved in NN-mediated intracellular delivery, providing insights into strategic design of cell–nano interfacing under temporal control for improved effectiveness.
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