Angle Matthew R, Wang Andrew, Thomas Aman, Schaefer Andreas T, Melosh Nicholas A
Department of Materials Science and Engineering, Stanford University, Stanford, California.
Department of Materials Science and Engineering, Stanford University, Stanford, California.
Biophys J. 2014 Nov 4;107(9):2091-100. doi: 10.1016/j.bpj.2014.09.023.
Nanoscale devices have been proposed as tools for measuring and controlling intracellular activity by providing electrical and/or chemical access to the cytosol. Unfortunately, nanostructures with diameters of 50-500 nm do not readily penetrate the cell membrane, and rationally optimizing nanoprobes for cell penetration requires real-time characterization methods that are capable of following the process of membrane penetration with nanometer resolution. Although extensive work has examined the rupture of supported synthetic lipid bilayers, little is known about the applicability of these model systems to living cell membranes with complex lipid compositions, cytoskeletal attachment, and membrane proteins. Here, we describe atomic force microscopy (AFM) membrane penetration experiments in two parallel systems: live HEK293 cells and stacks of synthetic lipid bilayers. By using the same probes in both systems, we were able to clearly identify membrane penetration in synthetic bilayers and compare these events with putative membrane penetration events in cells. We examined membrane penetration forces for three tip geometries and 18 chemical modifications of the probe surface, and in all cases the median forces required to penetrate cellular and synthetic lipid bilayers with nanoprobes were greater than 1 nN. The penetration force was sensitive to the probe's sharpness, but not its surface chemistry, and the force did not depend on cell surface or cytoskeletal properties, with cells and lipid stacks yielding similar forces. This systematic assessment of penetration under various mechanical and chemical conditions provides insights into nanoprobe-cell interactions and informs the design of future intracellular nanoprobes.
纳米级器件已被提议作为通过提供对细胞质的电和/或化学通路来测量和控制细胞内活动的工具。不幸的是，直径为50 - 500纳米的纳米结构不易穿透细胞膜，合理优化用于细胞穿透的纳米探针需要能够以纳米分辨率跟踪膜穿透过程的实时表征方法。尽管已有大量工作研究了支撑的合成脂质双层的破裂，但对于这些模型系统在具有复杂脂质组成、细胞骨架附着和膜蛋白的活细胞膜中的适用性知之甚少。在此，我们描述了在两个平行系统中的原子力显微镜（AFM）膜穿透实验：活的HEK293细胞和合成脂质双层堆叠。通过在两个系统中使用相同的探针，我们能够清楚地识别合成双层中的膜穿透，并将这些事件与细胞中假定的膜穿透事件进行比较。我们研究了三种针尖几何形状和18种探针表面化学修饰的膜穿透力，在所有情况下，用纳米探针穿透细胞和合成脂质双层所需的中值力均大于1纳牛。穿透力对探针的尖锐度敏感，但对其表面化学不敏感，并且该力不依赖于细胞表面或细胞骨架特性，细胞和脂质堆叠产生的力相似。这种在各种机械和化学条件下对穿透的系统评估为纳米探针 - 细胞相互作用提供了见解，并为未来细胞内纳米探针的设计提供了参考。
