Nanostraw-Assisted Cellular Injection of Fluorescent Nanodiamonds via Direct Membrane Opening
Nanostraw-Assisted Cellular Injection of Fluorescent Nanodiamonds via Direct Membrane Opening
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DOI：
10.1002/smll.202006421
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发表时间：
2021-01-27
期刊：
影响因子：
13.3
通讯作者：
Prinz, Christelle N.
中科院分区：
文献类型：
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作者：
Hebisch, Elke;Hjort, Martin;Prinz, Christelle N.
由于其稳定的荧光、生物相容性和易于功能化，荧光纳米金刚石 (FND) 是用于长期细胞标记和跟踪的有前途的材料。然而，由于内化效率低和内体截留，将它们转运到细胞质仍然是一个重大挑战。在这里，纳米管与低压电穿孔脉冲相结合，用于实现 FND 直接递送至细胞质。与在含有 FND 的培养基中孵育细胞相比，纳米管递送可有效、快速地将 FND 转运到细胞中。此外，虽然通过孵育传递的所有内化 FND 最终都在溶酶体中，但纳米管注射的 FND 的大部分在胞质溶胶中，这为使用 FND 作为细胞探针打开了大门。此外，为了回答纳米生物学领域长期存在的有关中空纳米结构上细胞膜状态的问题，采用活细胞受激发射损耗（STED）显微镜直接对纳米管上膜的状态进行成像。延时 STED 图像显示，在施加温和的电脉冲后，纳米管顶部的细胞膜完全打开，这支持了许多 FND 直接递送至细胞质的假设，避免了内吞作用和溶酶体截留。
Due to their stable fluorescence, biocompatibility, and amenability to functionalization, fluorescent nanodiamonds (FND) are promising materials for long term cell labeling and tracking. However, transporting them to the cytosol remains a major challenge, due to low internalization efficiencies and endosomal entrapment. Here, nanostraws in combination with low voltage electroporation pulses are used to achieve direct delivery of FND to the cytosol. The nanostraw delivery leads to efficient and rapid FND transport into cells compared to when incubating cells in a FND-containing medium. Moreover, whereas all internalized FND delivered by incubation end up in lysosomes, a significantly larger proportion of nanostraw-injected FND are in the cytosol, which opens up for using FND as cellular probes. Furthermore, in order to answer the long-standing question in the field of nano-biology regarding the state of the cell membrane on hollow nanostructures, live cell stimulated emission depletion (STED) microscopy is performed to image directly the state of the membrane on nanostraws. The time-lapse STED images reveal that the cell membrane opens entirely on top of nanostraws upon application of gentle electrical pulses, which supports the hypothesis that many FND are delivered directly to the cytosol, avoiding endocytosis and lysosomal entrapment.