Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):16486-91. doi: 10.1073/pnas.1208198109. Epub 2012 Sep 24.
This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1.
本文提出了独特的方法，以实现对超声介导的细胞膜破坏（即超声穿孔）的单细胞水平的控制和定量。将靶向质膜的微泡超声激发可产生具有高重复性的空间和时间可控的膜破坏。通过全细胞膜片钳记录结合荧光显微镜，我们获得了单细胞超声穿孔的时程测量，并定量了孔的大小和再封闭率。我们测量了细胞内 RNA/DNA 的扩散系数，方法是将嵌入荧光染料通过超声穿孔诱导的运输进入和穿过单个细胞。通过选择性激发微泡，我们实现了具有亚细胞精度和钙信号的时空控制递药，靶向细胞。最后，我们利用超声穿孔将膜非渗透性多药耐药蛋白 1（MRP1）底物钙黄绿素递送至过度表达 MRP1 的 HEK-MRP1 细胞，并通过 MRP1 监测钙黄绿素的外排。这种方法使得能够测量单个细胞中的外排率，并直接将其与不表达 MRP1 的亲本对照细胞的外排率进行比较。
