Liposome-based molecular robots that molecular systems are integrated into a giant liposome have been proposed; they are expected to be applied in the fields of medicine, environmental science, food science, and energy science. However, the performance of these molecular robotic components, including intelligence, sensors, and actuators, still hinders their practical use. In particular, the actuators used in the molecular robots, such as molecular motors, do not provide sufficient performance to move the giant liposomes. Hence, we propose an osmotic-engine-driven liposome and demonstrate the migration of liposomes in a microfluidic channel by applying a salt concentration difference between the front and rear of the liposome. Although the migration mechanism is simple and has the potential to provide sufficient mobility performance, control techniques for the movement speed and on/off switching are not established. Herein, we describe a speed control method of osmotic-engine-driven liposomes using pore-forming membrane proteins. In this study, we evaluated the effect of reconstituted α-hemolysin (αHL) nanopores on the water permeability through lipid bilayers. Thereafter, we demonstrated the change in displacement speeds of liposomes with and without nanopores. We expect the speed control method using nanopores to be applied to the liposome-based molecular robots.

1. [1] S. Murata, A. Konagaya, S. Kobayashi, H. Saito, and M. Hagiya, “Molecular Robotics: A New Paradigm for Artifacts,” New Gener. Comput., Vol.31, No.1, pp. 27-45, 2013. https://doi.org/10.1007/s00354-012-0121-z
2. [2] M. Hagiya, A. Konagaya, S. Kobayashi, H. Saito, and S. Murata, “Molecular Robots with Sensors and Intelligence,” Acc. Chem. Res., Vol.47, No.6, pp. 1681-1690, 2014. https://doi.org/10.1021/ar400318d
3. [3] R. Kawano, “Synthetic Ion Channels and DNA Logic Gates as Components of Molecular Robots,” ChemPhysChem, Vol.19, No.4, pp. 359-366, 2018. https://doi.org/10.1002/cphc.201700982
4. [4] K. Shoji and R. Kawano, “Recent Advances in Liposome-Based Molecular Robots,” Micromachines, Vol.11, No.9, Article No.788, 2020. https://doi.org/10.3390/mi11090788
5. [5] S. Murata, T. Toyota, S. M. Nomura, T. Nakakuki, and A. Kuzuya, “Molecular Cybernetics: Challenges Toward Cellular Chemical Artificial Intelligence,” Advanced Functional Materials, Vol.32, No.37, Article No.2270207, 2022. https://doi.org/10.1002/adfm.202201866
6. [6] K. Akashi, H. Miyata, H. Itoh, and K. Kinosita, Jr., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophysical J., Vol.71, No.6, pp. 3242-3250, 1996. https://doi.org/10.1016/S0006-3495(96)79517-6
7. [7] A. Moscho, O. Orwar, D. T. Chiu, B. P. Modi, and R. N. Zare, “Rapid preparation of giant unilamellar vesicles,” Proc. of the National Academy of Sciences, Vol.93, No.21, pp. 11443-11447, 1996. https://doi.org/10.1073/pnas.93.21.11443
8. [8] C. Kurokawa et al., “DNA cytoskeleton for stabilizing artificial cells,” Proc. of the National Academy of Sciences, Vol.114, No.28, pp. 7228-7233, 2017. https://doi.org/10.1073/pnas.1702208114
9. [9] T. Tohgasaki et al., “A Photocaged DNA Nanocapsule for Controlled Unlocking and Opening inside the Cell,” Bioconjugate Chem., Vol.30, No.7, pp. 1860-1863, 2019. https://doi.org/10.1021/acs.bioconjchem.9b00040
10. [10] M. Hagiya et al., “On DNA-Based Gellular Automata,” O. H. Ibarra, L. Kari, and S. Kopecki (Eds.), “Lecture Notes in Computer Science: 13th Int. Conf. on Unconventional Computation and Natural Computation,” pp. 177-189, Springer, 2014. https://doi.org/10.1007/978-3-319-08123-6_15
11. [11] K. Abe, I. Kawamata, S. M. Nomura, and S. Murata, “Programmable reactions and diffusion using DNA for pattern formation in hydrogel medium,” Mol. Syst. Des. Eng., Vol.4, No.3, pp. 639-643, 2019. https://doi.org/10.1039/C9ME00004F
12. [12] H. Bermudez, A. K. Brannan, D. A. Hammer, F. S. Bates, and D. E. Discher, “Molecular Weight Dependence of Polymersome Membrane Structure, Elasticity, and Stability,” Macromolecules, Vol.35, No.21, pp. 8203-8208, 2002. https://doi.org/10.1021/ma020669l
13. [13] M. Honda, K. Takiguchi, S. Ishikawa, and H. Hotani, “Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking,” J. of Molecular Biology, Vol.287, No.2, pp. 293-300, 1999. https://doi.org/10.1006/jmbi.1999.2592
14. [14] H. Hotani, F. Nomura, and Y. Suzuki, “Giant liposomes: From membrane dynamics to cell morphogenesis,” Current Opinion in Colloid & Interface Science, Vol.4, No.5, pp. 358-368, 1999. https://doi.org/10.1016/S1359-0294(99)90021-3
15. [15] M. Hayashi, M. Nishiyama, Y. Kazayama, T. Toyota, Y. Harada, and K. Takiguchi, “Reversible Morphological Control of Tubulin-Encapsulating Giant Liposomes by Hydrostatic Pressure,” Langmuir, Vol.32, No.15, pp. 3794-3802, 2016. https://doi.org/10.1021/acs.langmuir.6b00799
16. [16] S. Tanaka, K. Takiguchi, and M. Hayashi, “Repetitive stretching of giant liposomes utilizing the nematic alignment of confined actin,” Commun. Phys., Vol.1, Article No.18, 2018. https://doi.org/10.1038/s42005-018-0019-2
17. [17] Y. Sato, Y. Hiratsuka, I. Kawamata, S. Murata, and S. M. Nomura, “Micrometer-sized molecular robot changes its shape in response to signal molecules,” Science Robotics, Vol.2, No.4, Article No.eaal3735, 2017. https://doi.org/10.1126/scirobotics.aal3735
18. [18] M. Nakajima et al., “Quantitative Evaluation of Injected Molecules into Phospholipid-Coated Microdroplets for In situ Biological Reactions,” J. Robot. Mechatron., Vol.22, No.5, pp. 651-658, 2010. https://doi.org/10.20965/jrm.2010.p0651
19. [19] K. Shoji and R. Kawano, “Osmotic-engine-driven liposomes in microfluidic channels,” Lab on a Chip, Vol.19, No.20, pp. 3472-3480, 2019. https://doi.org/10.1039/C9LC00788A
20. [20] K. M. Stroka et al., “Water Permeation Drives Tumor Cell Migration in Confined Microenvironments,” Cell, Vol.157, No.3, pp. 611-623, 2014. https://doi.org/10.1016/j.cell.2014.02.052
21. [21] K. Funakoshi, H. Suzuki, and S. Takeuchi, “Lipid Bilayer Formation by Contacting Monolayers in a Microfluidic Device for Membrane Protein Analysis,” Anal. Chem., Vol.78, No.24, pp. 8169-8174, 2006. https://doi.org/10.1021/ac0613479
22. [22] R. Kawano et al., “Automated Parallel Recordings of Topologically Identified Single Ion Channels,” Sci. Rep., Vol.3, Article No.1995, 2013. https://doi.org/10.1038/srep01995
23. [23] K. Shoji, R. Kawano, and R. J. White, “Recessed Ag/AgCl Microelectrode-Supported Lipid Bilayer for Nanopore Sensing,” Anal. Chem., Vol.92, No.15, pp. 10856-10862, 2020. https://doi.org/10.1021/acs.analchem.0c02720
24. [24] S. Pautot, B. J. Frisken, and D. A. Weitz, “Production of Unilamellar Vesicles Using an Inverted Emulsion,” Langmuir, Vol.19, No.7, pp. 2870-2879, 2003. https://doi.org/10.1021/la026100v
25. [25] N. N. Deng, M. Yelleswarapu, and W. T. S. Huck, “Monodisperse Uni- and Multicompartment Liposomes,” J. Am. Chem. Soc., Vol.138, No.24, pp. 7584-7591, 2016. https://doi.org/10.1021/jacs.6b02107
26. [26] F. A. Edwards, A. J. Gibb, and D. Colquhoun, “ATP receptor-mediated synaptic currents in the central nervous system,” Nature, Vol.359, No.6391, pp. 144-147, 1992. https://doi.org/10.1038/359144a0
27. [27] P. M. Arnott and S. Howorka, “A Temperature-Gated Nanovalve Self-Assembled from DNA to Control Molecular Transport Across Membranes,” ACS Nano, Vol.13, No.3, pp. 3334-3340, 2019. https://doi.org/10.1021/acsnaNo.8b09200
28. [28] J. R. Burns, A. Seifert, N. Fertig, and S. Howorka, “A biomimetic DNA-based channel for the ligand-controlled transport of charged molecular cargo across a biological membrane,” Nature Nanotech., Vol.11, No.2, pp. 152-156, 2016. https://doi.org/10.1038/nnaNo.2015.279
29. [29] C. Lanphere, P. M. Arnott, S. F. Jones, K. Korlova, and S. Howorka, “A Biomimetic DNA-Based Membrane Gate for Protein-Controlled Transport of Cytotoxic Drugs,” Angewandte Chemie Int. Edition, Vol.60, No.4, pp. 1903-1908, 2021. https://doi.org/10.1002/anie.202011583
30. [30] D. Offenbartl-Stiegert, A. Rottensteiner, A. Dorey, and S. Howorka, “A Light-Triggered Synthetic Nanopore for Controlling Molecular Transport Across Biological Membranes,” Angewandte Chemie Int. Edition, Vol.61, No.52, Article No.e202210886, 2022. https://doi.org/10.1002/anie.202210886