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Generation of phospholipid vesicle-nanotube networks and transport of molecules therein

Abstract

We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires 90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60–120 min, depending on the transport regime.

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Figure 1: Schematic overview over the network generation procedure.
Figure 2: Optical differential interference contrast microscopy setup for nanotube-vesicle network fabrication.
Figure 3: Capillary needle and carbon fiber microelectrode preparation.
Figure 4: Electrophoresis electrode assemblies, each consisting of a 1-mm (OD) hydrophobized capillary needle with cross-linked polyacrylamide gel plug and internal Ag/AgCl electrode.
Figure 5: Lipid dehydration/hydration on no. 1 microscope cover slips and subsequent transfer.
Figure 6: Rehydration/swelling of lipid preparations.
Figure 7: Initial stage of network fabrication.
Figure 8: Stepwise fabrication of a linear network from a single GUV/MLV.
Figure 9: Stepwise fabrication of a branched network from a single GUV/MLV.
Figure 10: Confocal microscopy images showing diffusive transport of internalized 20-nm fluorescent latex nanoparticles between container vesicles through a nanotube.
Figure 11: Marangoni transport along a lipid nanotube in a two-vesicle network.
Figure 12: Electrophoretic transport of carboxylated nanoparticles along a lipid nanotube in a two-vesicle network.

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Acknowledgements

We thank all previous research group members and associates, in particular, former graduate students M. Karlsson, R. Karlsson, A. Karlsson, M. Davidson, K. Sott, M. Tokarz, J. Hurtig, M. Markström, B. Bauer and T. Lobovkina, who have contributed greatly to the development of these protocols during the time they completed their individual projects. We also thank P. Dommersnes for advice and discussion. Our research was and is supported by the Royal Swedish Academy of Sciences, the Swedish Research council (VR), the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation and the European Research Council.

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A.J. performed the experimental procedures for network generation, Marangoni transport and took part in manuscript preparation. N.S. and H.Z. performed experimental procedures for diffusive transport. B.O. carried out lipid preparation. B.H. performed electrophoretic transport and took part in the manuscript preparation. O.O. designed the original concept and took part in the manuscript preparation.

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Correspondence to Owe Orwar.

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Jesorka, A., Stepanyants, N., Zhang, H. et al. Generation of phospholipid vesicle-nanotube networks and transport of molecules therein. Nat Protoc 6, 791–805 (2011). https://doi.org/10.1038/nprot.2011.321

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