Enhanced intracellular delivery via coordinated acoustically driven shear mechanoporation and electrophoretic insertion.
Enhanced intracellular delivery via coordinated acoustically driven shear mechanoporation and electrophoretic insertion.
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DOI：
10.1038/s41598-018-22042-0
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发表时间：
2018-02-27
影响因子：
4.6
通讯作者：
Fedorov AG
中科院分区：
文献类型：
--
作者：
Meacham JM;Durvasula K;Degertekin FL;Fedorov AG
将大的且结构复杂的靶分子递送到细胞中对于细胞修饰和分子治疗的新兴领域至关重要。用于递送蛋白质的流行体内（病毒）和体外（脂质体）基因转移方法的不足以及合成纳米材料的日益多样化，鼓励了替代物理方法的开发。经由剪切机械穿孔的基于损伤/扩散的递送的功效在很大程度上对细胞类型和靶分子不敏感;然而，增强的灵活性通常伴随着降低的基因转移功效。我们详细介绍了一种方法，以提高转染效率，通过协调的细胞膜的机械破坏和电泳插入的DNA到细胞内部。一系列微机械喷嘴聚焦超声压力波，创造一个高剪切环境，促进传输细胞膜中瞬时孔隙的形成。声剪切穿孔（ASP）允许以大于75%的效率将小到大的非基因大分子被动地细胞质递送到建立的和原代细胞中。电泳作用的加入使得靶DNA分子的主动转运能够显著增加被动机械穿孔/扩散递送的转染效率而不影响活力。这种两阶段穿孔/插入方法保留了基于剪切的递送的令人信服的灵活性，但显著增强了质粒DNA的主动运输和转染的能力。
Delivery of large and structurally complex target molecules into cells is vital to the emerging areas of cellular modification and molecular therapy. Inadequacy of prevailing in vivo (viral) and in vitro (liposomal) gene transfer methods for delivery of proteins and a growing diversity of synthetic nanomaterials has encouraged development of alternative physical approaches. Efficacy of injury/diffusion-based delivery via shear mechanoporation is largely insensitive to cell type and target molecule; however, enhanced flexibility is typically accompanied by reduced gene transfer effectiveness. We detail a method to improve transfection efficiency through coordinated mechanical disruption of the cell membrane and electrophoretic insertion of DNA to the cell interior. An array of micromachined nozzles focuses ultrasonic pressure waves, creating a high-shear environment that promotes transient pore formation in membranes of transmitted cells. Acoustic Shear Poration (ASP) allows passive cytoplasmic delivery of small to large nongene macromolecules into established and primary cells at greater than 75% efficiency. Addition of an electrophoretic action enables active transport of target DNA molecules to substantially augment transfection efficiency of passive mechanoporation/diffusive delivery without affecting viability. This two-stage poration/insertion method preserves the compelling flexibility of shear-based delivery, yet substantially enhances capabilities for active transport and transfection of plasmid DNA.
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