Presentation Information
[P01-039]Development of Gel-in-Giant Unilamellar Vesicle System as Artificial Cell Model
○Wancheng ZHANG1 (1. Institute of Science Tokyo (Japan))
Keywords:
Giant unilamellar vesicles,hydrogel,artificial cell,stability
•Wancheng Zhang1, Aileen Cooney2, Kazutoshi Masuda3, Lorenzo Di Michelle4, Yuval Elani2, Miho Yanagisawa3, Tomoaki Matsuura1,˜
1 Earth-Life Science Institute, Institute of Science Tokyo
2 Department of Chemical Engineering, Imperial College London
3 Department of Basic Science, The University of Tokyo
4 Department of Chemical Engineering and Biotechnology, University of Cambridge
[Purpose]
Giant unilamellar vesicles (GUVs) are liposomes with a diameter of 1-100 μm and mainly composed of a bilayer of phospholipids. This bilayer system is similar with the biological membranes and able to compartmentalize an enclosed inner space and outer environment. Henceforth, they have been widely utilized as models for artificial cells. However, GUVs exhibits obvious limitations for its compositional simplicity and low stability under complex conditions such as detergents, mechanical stimuli and so on.
In this study, we present a gel-in-GUV system (GiG), in which a polyethylene glycol (PEG) hydrogel is encapsulated as artificial inner cellular environment and covalently linked to the membrane. The stability and physicochemical properties of formed GiG were extensively investigated to validate its capability as artificial cells.
[Method]
The GiG was prepared by encapsulating 4-arm PEG with thiol terminal group into GUVs by phase transfer method, along with horseradish peroxidase and glycyl-L-tyrosine. Maleimide lipids were added into the membrane during the preparation to enable covalent binding of the membrane and the gel. After incubation overnight, the gelation of the inner PEG core was realized.
[Results]
The resistance against detergent Triton X-100 was tested through flow cytometry after mixing the vesicles with different amount of Triton X-100. After the treatment, the amount of residual fluorescent lipid membrane of the GiG with membrane/gel bridging were higher than those without bridging and normal GUVs and increased with the amount of maleimide lipids, proving an enhanced resistance against detergents.
The GiGs with membrane/gel bridging presented an elevated young’s modulus measured by micropipette aspiration. An increase in the membrane integrity was also observed during the aspiration, as the encapsulated calcein was well constraint but that in GiGs without membrane/gel bridging leaked.
Besides, the GiGs showed increased stability during long-time storage at room temperature. Also, they preserved the ability to incorporate membrane proteins, exemplified by α-hemolysin (AHL), as encapsulated small-molecular calcein was able to leak out through AHL nanopores after incubation of GiGs and AHL monomers.
[Conclusion]
The GiG system using a mild enzymatic crosslinking reaction was constructed. The hydrogel core and its bridging with lipid membrane improved its stability against outer factors, while maintaining benign ability to incorporate membrane proteins. This system offers valuable insights for the design of more robust and complex artificial cell models.
1 Earth-Life Science Institute, Institute of Science Tokyo
2 Department of Chemical Engineering, Imperial College London
3 Department of Basic Science, The University of Tokyo
4 Department of Chemical Engineering and Biotechnology, University of Cambridge
[Purpose]
Giant unilamellar vesicles (GUVs) are liposomes with a diameter of 1-100 μm and mainly composed of a bilayer of phospholipids. This bilayer system is similar with the biological membranes and able to compartmentalize an enclosed inner space and outer environment. Henceforth, they have been widely utilized as models for artificial cells. However, GUVs exhibits obvious limitations for its compositional simplicity and low stability under complex conditions such as detergents, mechanical stimuli and so on.
In this study, we present a gel-in-GUV system (GiG), in which a polyethylene glycol (PEG) hydrogel is encapsulated as artificial inner cellular environment and covalently linked to the membrane. The stability and physicochemical properties of formed GiG were extensively investigated to validate its capability as artificial cells.
[Method]
The GiG was prepared by encapsulating 4-arm PEG with thiol terminal group into GUVs by phase transfer method, along with horseradish peroxidase and glycyl-L-tyrosine. Maleimide lipids were added into the membrane during the preparation to enable covalent binding of the membrane and the gel. After incubation overnight, the gelation of the inner PEG core was realized.
[Results]
The resistance against detergent Triton X-100 was tested through flow cytometry after mixing the vesicles with different amount of Triton X-100. After the treatment, the amount of residual fluorescent lipid membrane of the GiG with membrane/gel bridging were higher than those without bridging and normal GUVs and increased with the amount of maleimide lipids, proving an enhanced resistance against detergents.
The GiGs with membrane/gel bridging presented an elevated young’s modulus measured by micropipette aspiration. An increase in the membrane integrity was also observed during the aspiration, as the encapsulated calcein was well constraint but that in GiGs without membrane/gel bridging leaked.
Besides, the GiGs showed increased stability during long-time storage at room temperature. Also, they preserved the ability to incorporate membrane proteins, exemplified by α-hemolysin (AHL), as encapsulated small-molecular calcein was able to leak out through AHL nanopores after incubation of GiGs and AHL monomers.
[Conclusion]
The GiG system using a mild enzymatic crosslinking reaction was constructed. The hydrogel core and its bridging with lipid membrane improved its stability against outer factors, while maintaining benign ability to incorporate membrane proteins. This system offers valuable insights for the design of more robust and complex artificial cell models.
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