Department of Mechanical and Aerospace Engineering , University of California, Los Angeles , Los Angeles , California 90095 , United States.
Department of Pathology and Laboratory Medicine , University of California, Los Angeles , Los Angeles , California 90095 , United States.
ACS Nano. 2019 Sep 24;13(9):10835-10844. doi: 10.1021/acsnano.9b06025. Epub 2019 Sep 10.
Efficient intracellular delivery of biomolecules into cells that grow in suspension is of great interest for biomedical research, such as for applications in cancer immunotherapy. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to deliver materials efficiently into suspension cells. Here, we demonstrate a high-efficiency photothermal delivery approach for suspension cells using sharp nanoscale metal-coated tips positioned at the edge of microwells, which provide controllable membrane disruption for each cell in an array. Self-aligned microfabrication generates a uniform microwell array with three-dimensional nanoscale metallic sharp tip structures. Suspension cells self-position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme β-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.
高效地将生物分子递送到悬浮生长的细胞内对于生物医学研究非常重要,例如在癌症免疫治疗中的应用。尽管已经付出了巨大的努力,但现有的传递平台仍然难以有效地将材料递送到悬浮细胞中。在这里,我们展示了一种使用置于微井边缘的尖锐纳米尺度金属涂层尖端的高效光热传递方法,该方法可对每个微井中的细胞进行可控的膜破坏。自对准微加工生成了具有三维纳米尺度金属尖锐尖端结构的均匀微井阵列。悬浮细胞通过重力在每个微井内自定位,与八个尖锐尖端直接接触,其中激光诱导空化气泡在细胞膜中产生瞬时孔,以促进细胞外货物的胞内递送。我们在该平台上测试了一系列不同大小的货物,使用悬浮的 Ramos B 细胞,Calcein green(0.6 kDa)的效率超过 84%,FITC-葡聚糖(2000 kDa)的效率超过 45%,细胞活力保持在>96%,每分钟可传递超过 100000 个细胞。β-内酰胺酶(29 kDa)被递送到 Ramos B 细胞中并保留其生物活性,而绿色荧光蛋白表达质粒被递送到 Ramos B 细胞中,转染效率超过 58%,细胞活力达到>89%。