Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.
Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, 151 Wellington Road, Clayton, VIC, 3168, Australia.
J Nanobiotechnology. 2023 Aug 17;21(1):273. doi: 10.1186/s12951-023-02056-1.
Nanoinjection-the process of intracellular delivery using vertically configured nanostructures-is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell's intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation.
Through both theoretical simulations and experiments, we demonstrate the capability of the ENI platform to locally perforate GPE-86 mouse fibroblast cells and efficiently inject a diverse range of membrane-impermeable biomolecules with efficacy of 62.5% (antibody), 55.5% (mRNA), and 51.8% (plasmid DNA), with minimal impact on cells' viability post nanoscale-EP (> 90%). We also show gene silencing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of 41.3%.
We anticipate that our non-viral and low-voltage ENI platform is set to offer a new safe path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene silencing.
纳米注射——使用垂直构型纳米结构进行细胞内递药的过程——是一种物理途径，可有效地穿透质膜，对细胞的干扰和毒性最小。纳米注射作为一种物理膜破坏介导的方法，克服了与传统载体介导方法相关的挑战，如安全性问题（与病毒载体相关）、遗传毒性、有限的包装能力、内体逃逸水平低以及对细胞和货物类型的通用性差。然而，尽管在各种细胞操作中实施了纳米注射工具及其辅助类似物，但仍然存在很大的挑战，即如何在不破坏细胞复杂结构的情况下，更精确地利用这些平台进入细胞内部。在这里，我们提出了一种基于垂直构型导电纳米管（NTs）的非病毒、低电压、可重复使用的电活性纳米注射（ENI）平台，该平台允许靶向生物分子货物快速流入细胞内环境，并成功实现基因沉默。导电 NTs 与细胞膜紧密界面处的电场定位极大地降低了将货物递送到细胞内所需的电压，从千伏（用于体电穿孔）降低到仅≤10 V；这增强了对膜破坏的精细控制，并减轻了传统电穿孔中细胞死亡率高的问题。
通过理论模拟和实验，我们证明了 ENI 平台能够局部穿孔 GPE-86 小鼠成纤维细胞，并有效地注射各种具有不同通透性的生物分子，其效率分别为 62.5%（抗体）、55.5%（mRNA）和 51.8%（质粒 DNA），对纳米级电穿孔后细胞活力的影响最小（>90%）。我们还通过递送靶向 TRIOBP 的 siRNA 显示了基因沉默，从而实现了 41.3%的基因敲低效率。
我们预计，我们的非病毒和低电压 ENI 平台将为细胞内递药提供一条新的安全途径，具有更广泛的货物和细胞类型选择，并将为先进的体外细胞工程和基因沉默开辟机会。
