Facile generation of giant unilamellar vesicles using polyacrylamide gels

Abstract

Giant unilamellar vesicles (GUVs) are model cell-sized systems that have broad applications including drug delivery, analysis of membrane biophysics, and synthetic reconstitution of cellular machineries. Although numerous methods for the generation of free-floating GUVs have been established over the past few decades, only a fraction have successfully produced uniform vesicle populations both from charged lipids and in buffers of physiological ionic strength. In the method described here, we generate large numbers of free-floating GUVs through the rehydration of lipid films deposited on soft polyacrylamide (PAA) gels. We show that this technique produces high GUV concentrations for a range of lipid types, including charged ones, independently of the ionic strength of the buffer used. We demonstrate that the gentle hydration of PAA gels results in predominantly unilamellar vesicles, which is in contrast to comparable methods analyzed in this work. Unilamellarity is a defining feature of GUVs and the generation of uniform populations is key for many downstream applications. The PAA method is widely applicable and can be easily implemented with commonly utilized laboratory reagents, making it an appealing platform for the study of membrane biophysics.

Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels

“…The distribution of GUV diameters ranges from ~1 μm to ~40 μm. In our measurements, the mean diameter for various PAA stiffness and porosity conditions was on the order of 10 μm(Parigoris et al, 2020).…”

“…Polyacrylamide formulations were adapted from those used in Wen et al, 2014. To start, we recommend using formulation 1 or 5. b. We have found that the variations in PAA stiffness and pore size do not significantly affect the concentration and size distribution of the free-floating GUVs that are generated (Parigoris et al, 2020 2. Loosen the lid on the conical tube and place it in the desiccator/vacuum.…”

Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

“…Here, using polymer assisted swelling 48–51 and electroformation 52 methods, we compare the produced GUVs on vesicle properties important for investigating MPs in GUVs as outlined above. We compare the ability of these formation methods to produce a high number of vesicles with diameters between 5 – 20 µm under physiologically relevant conditions and characterize whether immobilization using the biotin-streptavidin system was affected by the presence of salt.…”

Nanoscale curvature promotes high yield spontaneous formation of cell-mimetic giant vesicles

“…Surface-assisted assembly, a growing collection [22][23][24][25][26][27][28][29][30] of techniques in which solvent-free lipid films are templated onto surfaces and then hydrated in aqueous solutions, is a highly promising route to obtain giant vesicles 1,2 . In the earliest discovered technique 22,23 , contemporarily known as 'gentle hydration' 1,2 , dry lipid films are templated onto impermeable glass and Teflon surfaces and then hydrated in quiescent solutions.…”

“…Electroformation modifies the procedure by applying an electric field normal to lipid films templated on conductive surfaces 24,25 . Other recent modifications include gel-assisted hydration, where lipid films are templated onto soluble hydrogels supported on glass surfaces [26][27][28] and PAPYRUS, Paper-Abetted amPhiphile hYdRation in aqUeous Solutions, where lipid films are templated onto insoluble fibers of cellulose filter paper 29 and fabrics 30 composed of natural, semi-synthetic, and synthetic fibers.…”