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Molecular machines open cell membranes

Abstract

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes1. These include using electric2 and magnetic3 fields, temperature4, ultrasound5 or light6 to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications7,8,9. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation10.

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Figure 1: Molecular motors for disruption of lipid bilayers through molecular mechanical action and control molecules.
Figure 2: NIH 3T3 cells in the presence of the fluorescent molecular machines 1 and 2.
Figure 3: The effects of nanomachines 3 and 4 and control molecule 5 on PC-3 cells upon UV activation.
Figure 4: Whole-cell patch-clamp studies of the dynamic effects of UV-induced molecular mechanical action of 3 on HEK293 cells.

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Acknowledgements

G.W. acknowledges North Carolina State University start-up funds and the FRPD Award. R.P. acknowledges support from the Royal Society and BSI Durham. J.M.T. acknowledges support from the National Science Foundation (CHE-1007483). Imaging for this project was supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from NIH (DK56338 and CA125123), CPRIT (RP150578), the Dan L. Duncan Comprehensive Cancer Center and the John S. Dunn Gulf Coast Consortium for Chemical Genomics. We thank R. S. Gunasekera for his assistance with imaging and A. Marti for the use of his high-performance liquid chromatography (HPLC) system for the separation of 710. W. Sikkema prepared Fig. 1a.

Author information

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Authors

Contributions

The idea to use the nanomachines to open membranes was suggested by J.M.T., then further discussed with G.W. and R.P. V.G.-L. carried out the synthesis of 113 and 16 and L.G.N. carried out the synthesis of 14, both under the supervision of J.M.T. F.C. performed the experiments on lipid vesicles under the supervision of G.W. R.P. carried out all of the experiments on live cells. A.A. and V.G.-L. carried out the HPLC separation of 710. A.B.K. and G.W. developed the theory. G.D. performed the patch-clamp work under the supervision of J.T.R.; G.D. and J.T.R. also prepared the text for that section. V.G.-L., G.W., R.P. and J.M.T. wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Jacob T. Robinson, Gufeng Wang, Robert Pal or James M. Tour.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks R. D. Astumian, S. Cockroft and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Discussion, Supplementary Figures 1-26, Supplementary Tables 1-2, a Supplementary Video figure and additional references (PDF 7096 kb)

Reporting Summary (PDF 69 kb)

Supplementary Data

This file contains data for Supplementary Figures 3 and 5-12. (ZIP 119 kb)

When 1 was introduced to cells, it localized on the cell surface in pit-like localization patterns

Red-blue-green (RGB) fluorescent and transmission overlay video for NIH 3T3 cells bearing Nanomachine 1 (red, Cloading 500 nM/1 h, λex 633 nm, λem 650-700 nm, 1 mW) and LysoTrackerGreen (green, Cloading 200 nM/5 min, λex 488 nm, λem 500-530 nm, 0.2 mW) depicting natural homeostatic intracellular lysosomal movement. In the absence of UV activation the clusters of nanomachine 1 remains stationary and cell-membrane-bound with subsequent cell starvation and shriveling that leads to apoptotic cell death. (AVI 2407 kb)

When 1 was introduced to cells, it localized on the cell surface.

RGB fluorescent and transmission overlay video for NIH 3T3 cells bearing nanomachine 1 (red, Cloading 500 nM/1 h, λex 633 nm, λem 650-700 nm, 1 mW) and LysoTrackerGreen (green, Cloading 200 nM/5 min, λex 488 nm, λem 500-530 nm, 0.2 mW) showing that after cells died via natural starvation induced apoptosis in the absence of UV activation (confirmed by the absence of both blebbing and lysosomal rupture associated with necrosis), natural homeostatic lysosomal movement can be seen but more importantly the membrane bound nanomachine 1 clusters are inactivated and did not cross the cell membrane and cannot be detected in the cytosol. (AVI 636 kb)

NIH 3T3 cells in the presence of the nanomachines 1 with UV activation.

Upon UV-induced motor activation for 150 s (355 nm), 1, introduced by Method A, was found to cross the cell membrane, and it was internalized into cells in a time-dependent manner, displaying fast accelerated intracellular motion. Fluorescent and transmission overlay video for unwashed NIH 3T3 cells incubated with nanomachine 1 (red, Cloading 500 nM/1, λex 633 nm, λem 650-700 nm, 1 mW) depicting the uptake via membrane penetration of the cell surface formed 1 clusters in the presence of UV activation. (AVI 222 kb)

NIH 3T3 cells in the presence of the nanomachines 1 with UV activation.

RGB fluorescent and transmission overlay video for NIH 3T3 cells introduced to nanomachine 1 (red, Cloading 500 nM, λex 633 nm, λem 640-700 nm, 1 mW) depicting UV-induced necrosis (no acceleration compared to control experiment) characterized by mitochondrial autofluorescence disappearance due to mitochondrial fragmentation. Final stages of necrosis are characterized by blebbing observable at later time-points. That is also accompanied with rapid formation and movement of cytosol internalized 1 clusters which, upon UV activation, burst and contribute to detectable evenly distributed cytosolic cy5 fluorescence. (AVI 7222 kb)

NIH 3T3 cells in the presence of the nanomachines 1 with UV activation.

The same as Supplementary Video 4, but zoomed into the highlighted area on the Video 4 image surrounded by the dashed line. (AVI 2565 kb)

Natural homeostatic cellular organelle movement or 1 in NIH 3T3 cells in the absence of UV-nanomechanical activation.

Fluorescent and transmission overlay video for NIH 3T3 cells in situ loaded nanomachine 1 (red, Cloading 500 nM, λex 633 nm, λem 650-700 nm, 1 mW) depicting the uptake and subsequent clusters formation of 1 in the absence of UV activation followed by at 460 s continuous UV activation (λactivation 355 nm, 12 mW). (AVI 1364 kb)

Small aggregates of 1 inside the cytoplasm prior to UV activation.

Fluorescent and transmission overlay video for washed NIH 3T3 cells incubated with 1 (red, Cloading 500 nM/1, λex 633 nm, λem 650-700 nm, 1 mW) depicting the accumulated 1 clusters being quasi-stationery in the absence of UV activation. (AVI 681 kb)

Small aggregates of 1 inside the cytoplasm upon UV activation.

Fluorescent and transmission overlay video for washed NIH 3T3 cells incubated with 1 (red, Cloading 500 nM/1, λex 633 nm, λem 650-700 nm, 1 mW, λactivation 355 nm, 12 mW) depicting the erratic intracellular movement of the accumulated 1 clusters in the presence of UV activation. This is imaged as shown in Supplementary Video 7 but Supplementary Video 7 had no UV activation. As a further explanation, using nanomachine 1 according to Method B and no UV activation, once the cell membrane adhesion of the small aggregates is observed microscopically, allowing parent cell natural proliferation and division, well-defined and detectable cell membrane-bound clusters of 1 are still present, with detectable cy5 fluorescence in the surrounding culture media. Similar observations were made with nanomachine 2 with traceable BODIPY fluorescence in the daughter cells with predominantly mitochondrial localization with some detectable amount revealed with co-staining in the lysosomes and cytosol, further confirming non-toxicity. In the absence of UV activation, and disruptive cell membrane opening during natural cellular homeostatic division, no traceable amount 2 is detected in the surrounding cell culture media. Both verification experiments have been carried out by employing Method B (2 h nanomachine incubation followed by wash and 48 h subsequent incubation without nanomachines). (AVI 299 kb)

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García-López, V., Chen, F., Nilewski, L. et al. Molecular machines open cell membranes. Nature 548, 567–572 (2017). https://doi.org/10.1038/nature23657

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