California NanoSystems Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States.
Department of Chemistry and Biochemistry , University of California, Los Angeles , Los Angeles , California 90095 , United States.
ACS Nano. 2018 May 22;12(5):4503-4511. doi: 10.1021/acsnano.8b00763. Epub 2018 Mar 14.
An efficient nonviral platform for high-throughput and subcellular precision targeted intracellular delivery of nucleic acids in cell culture based on magnetic nanospears is reported. These magnetic nanospears are made of Au/Ni/Si (∼5 μm in length with tip diameters <50 nm) and fabricated by nanosphere lithography and metal deposition. A magnet is used to direct the mechanical motion of a single nanospear, enabling precise control of position and three-dimensional rotation. These nanospears were further functionalized with enhanced green fluorescent protein (eGFP)-expression plasmids via a layer-by-layer approach before release from the underlying silicon substrate. Plasmid functionalized nanospears are guided magnetically to approach target adherent U87 glioblastoma cells, penetrating the cell membrane to enable intracellular delivery of the plasmid cargo. After 24 h, the target cell expresses green fluorescence indicating successful transfection. This nanospear-mediated transfection is readily scalable for the simultaneous manipulation of multiple cells using a rotating magnet. Cell viability >90% and transfection rates >80% were achieved, which exceed conventional nonviral intracellular methods. This approach is compatible with good manufacturing practices, circumventing barriers to the translation and clinical deployment of emerging cellular therapies.
一种高效的非病毒平台，用于在细胞培养中高通量和亚细胞精确定位靶向细胞内递核酸，基于磁性纳米矛。这些磁性纳米矛由 Au/Ni/Si 制成（长度约为 5 μm，尖端直径<50 nm），通过纳米球光刻和金属沉积制造。磁体用于引导单个纳米矛的机械运动，从而能够精确控制位置和三维旋转。这些纳米矛通过层层法进一步用增强型绿色荧光蛋白（eGFP）表达质粒功能化，然后从下面的硅衬底上释放。功能化的纳米矛通过磁力引导接近靶标贴壁 U87 神经胶质瘤细胞，穿透细胞膜以实现质粒货物的细胞内递。24 h 后，目标细胞发出绿色荧光，表明转染成功。这种纳米矛介导的转染很容易通过旋转磁体同时操纵多个细胞进行扩展。实现了>90%的细胞存活率和>80%的转染率，超过了传统的非病毒细胞内方法。这种方法与良好的生产规范兼容，避免了新兴细胞疗法转化和临床应用的障碍。
