Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content
<p>Adapted electroformation protocol. Lipids dissolved in organic solvent (pink) are mixed with an aqueous solution (light blue). MLVs are formed using the RSE method. MLV solution is extruded through a filter with membrane pores in order to obtain LUVs. LUV solution is spin-coated on a plasma threated electrode, where these vesicle rupture and form a damp lipid film. An electrode with damp lipid film is used to build an electroformation chamber, where lipid bilayers detach and form GUVs under the influence of osmotic pressure and an alternating electric field.</p> "> Figure 2
<p>Electroformation of GUVs for deposition of different vesicle types. (<b>a</b>) Fluorescence microscopic images of GUVs formed when different vesicle types were deposited. MLV represents the deposition of MLVs on the electrode, LUV 100 represents the deposition of LUVs formed by extruding MLVs through 100 nm membrane pores, and LUV 50 represents the deposition of LUVs formed by extruding LUV 100 vesicles through 50 nm membrane pores. The scale bar represents 50 µm. (<b>b</b>) Comparison of average diameters and standard deviations of GUVs for preparations from three different vesicle types. The averages and standard deviations were calculated by averaging the mean diameters from three independent samples for each condition. (<b>c</b>) Size distribution densities of GUVs for deposition of different vesicle types for each sample. Each distribution density represents one independent sample (100 vesicles).</p> "> Figure 3
<p>(<b>a</b>) GUVs mean diameters for different Chol/POPC mixing ratios. Points and bars represent mean values and their standard errors. (<b>b</b>) Size distribution densities of GUVs for different Chol/POPC mixing ratios. All experiments were performed using the deposition of LUV 100 vesicles spin-coated onto electrode for 30 s.</p> "> Figure 4
<p>(<b>a</b>) Schematic depiction of differences obtained using our novel protocol for three different experiments. In the first experiment, damp lipid film was formed by depositing LUV 100 vesicles on an electrode and spinning it for 30 s at 600 rpm angular velocity (Damp 30 s). In the second experiment, Damp 30 s lipid film was held under vacuum for 30 min (Dry 30 s). In the third experiment, instead of spinning for 30 s, the electrode was spun for 240 s, and afterwards held under vacuum for 30 min (Dry 240 s). (<b>b</b>) Success of electroformation using Damp 30 s, Dry 30 s, and Dry 240 s protocols for obtaining lipid films, depending on different Chol/POPC mixing ratios. Success is based on the population homogeneity, yield, and number of defects. It is displayed through circle fullness, where a fuller circle indicates greater success. Empty circles (white circle) denote that no GUVs were formed or that their number was negligible; quarter circles represent low, half and three-quarter circles indicate medium, and full circles (gray circle) indicate high success. (<b>c</b>) Fluorescence microscopy images of GUVs for the different Chol/POPC mixing ratios using three different approaches for obtaining lipid films. The scale bar represents 50 µm.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Preparation of Multilamellar Vesicles Using the Rapid Solvent Exchange Method
2.3. Preparation of Large Unilamellar Vesicles
2.4. Preparation of the Damp Lipid Film
2.5. Electroformation Protocol
2.6. Dynamic Light Scattering
2.7. Fluorescence Imaging and Data Analysis
3. Results and Discussion
3.1. Optimization of the Protocol
3.1.1. The Spin-Coating Duration for Formation of Damp Lipid Film
3.1.2. Comparison of MLVs and LUVs Deposition on the Electrode
3.2. The Effect of Chol Content
3.3. Comparison with Samples Obtained from Completely Dry Lipid Films
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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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. https://doi.org/10.3390/membranes14040079
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(4):79. https://doi.org/10.3390/membranes14040079
Chicago/Turabian StyleMardešić, Ivan, Zvonimir Boban, and Marija Raguz. 2024. "Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content" Membranes 14, no. 4: 79. https://doi.org/10.3390/membranes14040079
APA StyleMardešić, I., Boban, Z., & Raguz, M. (2024). Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content. Membranes, 14(4), 79. https://doi.org/10.3390/membranes14040079