High-Throughput and Efficient Intracellular Delivery Method via a Vibration-Assisted Nanoneedle/Microfluidic Composite System

纳米针 转染 纳米技术 细胞内 基因传递 微流控 材料科学 细胞生物学 细胞培养 生物 遗传学 纳米结构
作者
Xuan Li,Yuan Ma,Yu Xue,Xuanhe Zhang,Linwen Lv,Qianghua Quan,Yiqing Chen,Guoxu Yu,Zhenwei Liang,Xinping Zhang,Ding Weng,Lei Chen,Kui Chen,Xin Han,Jiadao Wang
出处
期刊:ACS Nano [American Chemical Society]
卷期号:17 (3): 2101-2113 被引量:17
标识
DOI:10.1021/acsnano.2c07852
摘要

Intracellular delivery and genetic modification have brought a significant revolutionary to tumor immunotherapy, yet existing methods are still limited by low delivery efficiency, poor throughput, excessive cell damage, or unsuitability for suspension immune cells, specifically the natural killer cell, which is highly resistant to transfection. Here, we proposed a vibration-assisted nanoneedle/microfluidic composite system that uses large-area nanoneedles to rapidly puncture and detach the fast-moving suspension cells in the microchannel under vibration to achieve continuous high-throughput intracellular delivery. The nanoneedle arrays fabricated based on the large-area self-assembly technique and microchannels can maximize the delivery efficiency. Cas9 ribonucleoprotein complexes (Cas9/RNPs) can be delivered directly into cells due to the sufficient cellular membrane nanoperforation size; for difficult-to-transfect immune cells, the delivery efficiency can be up to 98%, while the cell viability remains at about 80%. Moreover, the throughput is demonstrated to maintain a mL/min level, which is significantly higher than that of conventional delivery techniques. Further, we prepared CD96 knockout NK-92 cells via this platform, and the gene-edited NK-92 cells possessed higher immunity by reversing exhaustion. The high-throughput, high-efficiency, and low-damage performance of our intracellular delivery strategy has great potential for cellular immunotherapy in clinical applications.
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