在人类细胞中目击,细胞内通路和治疗货物运输,包括基因编辑工具(例如,CRISPR-Cas9和转座子),核酸(例如,DNA,mRNA和siRNA),肽,和蛋白质(例如,酶和抗体),被严格限制以确保健康的细胞功能和行为。该原理在用于离体免疫疗法的嵌合抗原受体(CAR)-T细胞的递送机制中示例。特别是,CAR-T细胞的临床成功通过治愈以前无法治愈的血癌建立了新的治疗标准.该方法涉及交付,通常通过使用电穿孔(EP)和慢病毒,治疗性CAR基因进入患者自己的T细胞,然后将其设计为表达靶向和对抗血液癌症的CAR。但关键的困难在于对这些细胞进行基因操纵,而不会造成不可逆转的损害或功能丧失,同时将制造的复杂性降至最低。安全问题,和成本,并确保最终CAR-T细胞产品的功效。纳米注射-使用纳米针(NN)进行细胞内递送的过程-是一种新兴的物理递送途径,可有效地协商许多细胞类型的质膜,包括原代人类T细胞。它以最小的扰动发生,侵入性,和毒性,在高空间和时间分辨率下具有高效率和吞吐量。纳米注射有望大大改善广泛的治疗性货物的递送,而对这些货物几乎没有或没有损害。纳米注射平台允许这些货物根据需要在细胞内空间中发挥作用。纳米注射平台的适应性现在在免疫调节方面带来了主要优势,机械传导,细胞状态采样(纳米活检),受控的细胞内询问,以及该帐户的主要焦点-细胞内递送及其在体外细胞工程中的应用。机械纳米注射通常对细胞膜施加直接的机械力,提供了一条直接的途径来改善NN的膜扰动以及随后将遗传货物运输到目标细胞类型(粘附或悬浮细胞)中。相比之下,通过将NN与电场耦合来控制电活性纳米注射,这是在纳米级激活电穿孔(EP)的新途径,可以显着降低施加给细胞的电压,从而最大程度地减少EP对细胞和货物的损伤。并克服了传统散装EP的许多局限性。纳米注射超越了单纯的技术;它是一种离体细胞工程的方法,提供了赋予细胞新的潜力,强大的功能,例如为未来的CAR-T细胞技术产生嵌合抗原受体(CAR)-T细胞。我们首先讨论神经网络器件的制造(第2节),然后深入研究纳米注射介导的细胞工程(第3节),纳米注射机制和接口方法(第4节),以及使用纳米注射产生功能性CAR-T细胞的新兴应用(第5节)。
ConspectusIn human cells, intracellular access and therapeutic cargo transport, including gene-editing tools (e.g., CRISPR-Cas9 and transposons), nucleic acids (e.g., DNA, mRNA, and siRNA), peptides, and proteins (e.g., enzymes and antibodies), are tightly constrained to ensure healthy cell function and behavior. This principle is exemplified in the delivery mechanisms of chimeric antigen receptor (CAR)-T cells for ex-vivo immunotherapy. In particular, the clinical success of CAR-T cells has established a new standard of care by curing previously incurable blood cancers. The approach involves the delivery, typically via the use of electroporation (EP) and lentivirus, of therapeutic CAR genes into a patient\'s own T cells, which are then engineered to express CARs that target and combat their blood cancer. But the key difficulty lies in genetically manipulating these cells without causing irreversible damage or loss of function─all the while minimizing complexities of manufacturing, safety concerns, and costs, and ensuring the efficacy of the final CAR-T cell product.Nanoinjection─the process of intracellular delivery using nanoneedles (NNs)─is an emerging physical delivery route that efficiently negotiates the plasma membrane of many cell types, including primary human T cells. It occurs with minimal perturbation, invasiveness, and toxicity, with high efficiency and throughput at high spatial and temporal resolutions. Nanoinjection promises greatly improved delivery of a broad range of therapeutic cargos with little or no damage to those cargos. A nanoinjection platform allows these cargos to function in the intracellular space as desired. The adaptability of nanoinjection platforms is now bringing major advantages in immunomodulation, mechanotransduction, sampling of cell states (nanobiopsy), controlled intracellular interrogation, and the primary focus of this account─intracellular delivery and its applications in ex vivo cell engineering.Mechanical nanoinjection typically exerts direct mechanical force on the cell membrane, offering a straightforward route to improve membrane perturbation by the NNs and subsequent transport of genetic cargo into targeted cell type (adherent or suspension cells). By contrast, electroactive nanoinjection is controlled by coupling NNs with an electric field─a new route for activating electroporation (EP) at the nanoscale─allowing a dramatic reduction of the applied voltage to a cell and so minimizing post-EP damage to cells and cargo, and overcoming many of the limitations of conventional bulk EP. Nanoinjection transcends mere technique; it is an approach to cell engineering ex vivo, offering the potential to endow cells with new, powerful features such as generating chimeric antigen receptor (CAR)-T cells for future CAR-T cell technologies.We first discuss the manufacturing of NN devices (Section 2), then delve into nanoinjection-mediated cell engineering (Section 3), nanoinjection mechanisms and interfacing methodologies (Section 4), and emerging applications in using nanoinjection to create functional CAR-T cells (Section 5).