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

Abstract

Giant unilamellar vesicles (GUVs) are artificial membrane models which are of special interest to researchers because of their similarity in size to eukaryotic cells. The most commonly used method for GUVs production is electroformation. However, the traditional electroformation protocol involves a step in which the organic solvent is completely evaporated, leaving behind a dry lipid film. This leads to artifactual demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the formation of the lipid bilayer, resulting in a decrease of Chol concentration in the bilayer compared to the initial lipid solution. We propose a novel electroformation protocol which addresses this issue by combining the rapid solvent exchange, plasma cleaning and spin-coating techniques to produce GUVs from damp lipid films in a fast and reproducible manner. We have tested the protocol efficiency using 1/1 phosphatidylcholine/Chol and 1/1/1 phosphatidylcholine/sphingomyelin/Chol lipid mixtures and managed to produce a GUV population of an average diameter around 40 µm, with many GUVs being larger than 100 µm. Additionally, compared to protocols that include the dry film step, the sizes and quality of vesicles determined from fluorescence microscopy images were similar or better, confirming the benefits of our protocol in that regard as well.

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content

“…Subsequently, the chamber was connected to a pulse generator (UTG9005C, UNI-T, Dongguan City, China or PSG 9080, Joy-IT, Neukirchen-Vluyn, Germany) and placed in an incubator set to a temperature of 60 • C. Copper tape was applied to the outer edges of the electrode to enhance wire-electrode contact. Consistent with previous experiments [17,18,25], a voltage of 2 V and a frequency of 10 Hz were maintained. After 2 h, the pulse generator was turned off, and the chamber remained in the incubator for an additional hour.…”

“…A shorter duration of spin-coating resulted in an uneven film thickness or a film that was too wet, leading to the low reproducibility of the experiment and high GUV heterogeneity within the successful samples. As shown in our previous study, for a spin-coating duration of 30 s, the lipid film remained damp, with high GUV electroformation yield [25]. At a duration of 30 s, water-dissolved lipid droplets can still be seen on the electrode, which assures us that the lipid film is still damp.…”

“…In our previous study, we adapted the traditional electroformation protocol to incorporate all of the aforementioned improvements, allowing us to bypass the dry lipid film phase and produce a uniform damp lipid film (Figure 1) [25]. Inspired by the vesicle fusion method for the preparation of supported lipid bilayers [4,26,27], the adapted protocol uses spin-coating to deposit an aqueous solution of LUVs onto a plasma-treated electrode.…”

“…The efficiency of the protocol was tested using 1/1 Chol/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1/1/1 Chol/POPC/sphingomyelin lipid mixtures [25]. This ternary mixture was selected because these three lipid types are highly prevalent in biological membranes.…”

“…Compared to the protocol involving a drying step, the new protocol resulted in a similar or better GUV yield, while exhibiting the potential to significantly reduce the Chol demixing artifact [25]. In order to further increase the reproducibility and yield of the obtained samples, this study will focus on the further optimization of the new protocol.…”