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Quantification of nanowire penetration into living cellsAlexander M Xu et al. Nat Commun. 2014.
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AbstractHigh-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.
Conflict of interest statementCompeting financial interests: The authors declare no competing financial interest.
FiguresFigure 1. An ionic delivery assay designed…
Figure 1. An ionic delivery assay designed to probe nanostraw cell penetration
Nanostraw membranes are…
Figure 1. An ionic delivery assay designed to probe nanostraw cell penetrationNanostraw membranes are integrated into a microfluidic device (a) For each cell plated in the device, there are many nanostraw interfaces that may either be non-penetrant or penetrant (b). When the nanostraws penetrate into the cell membrane, the Co2+ ions in the solution below are able to directly enter the cell via passive diffusion and quench GFP fluorescence (c, scale bar, 20 µm).
Figure 2. Quenching spots indicate sites of…
Figure 2. Quenching spots indicate sites of nanostraw penetration
During Co 2+ delivery, quenching spots…
Figure 2. Quenching spots indicate sites of nanostraw penetrationDuring Co2+ delivery, quenching spots grow in size as Co2+ accumulates above a penetrating nanostraw. Spots were counted (a, n = 252, scale bar, 25 µm) to determine the number of spots per cell (b), the density of spots per cell (c) and the dependence of spot density on cell area (d). The largest cells generally had more spots (red) than intermediate sized cells (blue) and small cells (green), but the actual density of spots was largely independent of cell area (c,d). The number of spots and their size increased (e–h, scale bar, 40 µm) as spots accumulated enough Co2+ to be observed.
Figure 3. Alternating intracellular reagent delivery
Solutions…
Figure 3. Alternating intracellular reagent delivery
Solutions of Co 2+ or an ethylenediaminetetraacetic acid (EDTA)…
Figure 3. Alternating intracellular reagent deliverySolutions of Co2+ or an ethylenediaminetetraacetic acid (EDTA) chelator were alternately pumped into the device for ~5 min (a) beginning with the prequenched state (b, scale bar, 25 µm). Quenching spots formed during the first quenched state (c), and were mostly recovered during the first recovery (d). During the second recovery (e), most quenching spots were directly associated with a spot from the first recovery (red arrows). After later recovery (f) and quench cycles (g), the recovery steps become insufficient to fully restore fluorescence. During later quench cycles, neighbouring spots grew large enough to combine with each other, demonstrating that a single pool of cytosolic GFP was quenched (blue arrows).
Figure 4. Kinetics of penetration determined by…
Figure 4. Kinetics of penetration determined by ionic delivery
The Co 2+ penetration assay was…
Figure 4. Kinetics of penetration determined by ionic deliveryThe Co2+ penetration assay was applied to cells immediately as they were plated on nanostraws to determine penetration kinetics. First cells were added to a device (a, scale bar, 10 µm) and tracked as they contacted the nanostraws (b). Eventually a pulse of Co2+ was added to the device, resulting in quenching spots forming within the cells (c). Over 30 min, cells became more likely to be penetrated at least once (d).
Figure 5. Cell adhesion correlated with penetration
Figure 5. Cell adhesion correlated with penetration
Biomolecular adhesion markers indicate that cells interact strongly…
Figure 5. Cell adhesion correlated with penetrationBiomolecular adhesion markers indicate that cells interact strongly with nanostraws. During initial cell contact after 30 min, enhanced cell adhesion was associated with cell spreading and actin and paxillin colocalization (a, scale bar, 10 µm). These features were lost as adhesion was decreased. The mature state after 24 h of adhesion showed stress fibre formation as well as actin and paxillin colocalization (b). Without nanostraws, some actin stress fibres appeared but no paxillin features were observed (c). The mature state without nanostraws featured paxillin puncta attached to stress fibres, but no colocalization (d).
Cited byShakoor A, Gao W, Zhao L, Jiang Z, Sun D. Shakoor A, et al. Microsyst Nanoeng. 2022 Apr 29;8:47. doi: 10.1038/s41378-022-00376-0. eCollection 2022. Microsyst Nanoeng. 2022. PMID: 35502330 Free PMC article. Review.
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