Mardešić, I.; Boban, Z.; Raguz, M. Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content. Membranes2024, 14, 79.
Mardešić, I.; Boban, Z.; Raguz, M. Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content. Membranes 2024, 14, 79.
Mardešić, I.; Boban, Z.; Raguz, M. Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content. Membranes2024, 14, 79.
Mardešić, I.; Boban, Z.; Raguz, M. Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content. Membranes 2024, 14, 79.
Abstract
Giant unilamellar vesicles (GUVs) are membrane model used to study membrane properties. Electroformation is one of the methods used to produce GUVs. During electroformation protocol dry lipid film is formed. Drying of the lipid film induce Cholesterol (Chol) demixing artifact, in which Chol forms anhydrous crystals, where these crystals does not participate in formation of vesicles. This leads to a lower Chol concentration in the vesicle bilayers compared to the Chol concentration in the initial lipid solution. To address this problem, we propose novel electroformation protocol that includes rapid solvent exchange, plasma cleaning and spin-coating methods to produce GUVs. We tested the protocol focusing on vesicles with high Chol content using different spin-coating durations and vesicle types deposition. Additionally, we compared the novel protocol using completely dry lipid film. The optimal spin-coating duration for vesicles made out of phosphatidylcholine/Chol mixture was 30 s. Mulitlamellar vesicles (MLVs), large unilamellar vesicles (LUVs) obtained by extrusion of MLVs through 100 nm membrane pores and LUVs obtained by extrusion of previously obtained LUVs through 50 nm membrane pores were deposited on electrode for 1.5/1 Chol/phosphatidylcholine lipid mixture and the results were compared. Electroformation using all three deposited vesicle types gave a high GUVs yield, but deposition of LUVs obtained by extrusion of MLVs through 100 nm membrane pores gave the most reproducible results. Using deposition of these LUVs, we produced GUVs with high yield for 6 different Chol concentrations (from 0 % to 71.4%). Using protocol that includes dry lipid film GUVs yield was lower and Chol demixing artifact was induced, proving that the lipid film should never undergo drying when Chol content is high.
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