Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures

Abstract

Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency–voltage combinations in the range of 2–6 V and 10–100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion

“…Comparing this to the corresponding result for the POPC/Chol mixture of 42 ± 19 m, we can see that the vesicles were smaller after inclusion of SM ( p = 2) ( Figure 6 ). This is consistent with our previous research, showing that inclusion of SM in the lipid mixture makes it harder to produce GUVs with large average size and yield [ 21 , 22 ]. The average size of the SM-containing GUV population is also on par with the best results obtained using the conventional lipid film deposition methods [ 21 ].…”

“…This is consistent with our previous research, showing that inclusion of SM in the lipid mixture makes it harder to produce GUVs with large average size and yield [ 21 , 22 ]. The average size of the SM-containing GUV population is also on par with the best results obtained using the conventional lipid film deposition methods [ 21 ].…”

“…Compared to previous research from our group which dealt with optimization of GUV electroformation from a dry lipid film [ 21 , 22 ], the sizes and quality of vesicles determined using fluorescence microscopy images were similar or better for both durations of spin-coating, confirming the benefits of our protocol in that regard as well ( Figure 5 b).…”

“…In order to assure good contact between the conductor wires and the electrodes, the outer edges of the electrodes were covered with copper tape. Based on experience from our previous electroformation studies [ 21 , 22 ], the electrical parameters were set to 2 V and 10 Hz. After 2 h, the voltage was turned off and the chamber was kept in the incubator for another hour.…”

“…The uniformity of the lipid film and method reproducibility were validated by ellipsometry and atomic force microscopy. The method has been accepted by several groups using a wide range of lipid compositions to produce GUVs [ 17 , 21 , 22 , 23 , 24 ].…”