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.
Highlights
Giant unilamellar vesicles (GUVs) are a model system widely used in the fields of membrane biophysics[23,24], in vitro reconstitution of cellular machineries[25,26], and drug delivery[6,8], among others
We have developed a simple and robust method for the reproducible production of large populations of free-floating GUVs in buffers of physiological ionic strength based on the gentle hydration of lipid layers deposited on polyacrylamide (PAA) gels (Fig. 1)
We compared the concentration and mean diameter of the GUVs obtained on PAA gels and using other methods for bulk GUV preparation, namely agarose hydration[19] and gentle hydration on glass[27,28]
Summary
Coverslips were placed in a 6-well plate and 20 μL of lipid solution was pipetted onto the gel. A glass Drigalski spatula was used to evenly spread the solution across the PAA gel and the solvent was evaporated in a vacuum chamber for 15 minutes. The problem of gel incorporation into the formed vesicles was mitigated, these techniques are complex and require chemicals that are uncommon in most laboratories, hindering their broad adoption. We report a gentle hydration method for the generation of large populations of cell-sized free-floating GUVs from hybrid films of dried polyacrylamide (PAA). As with the agarose gel method, gentle hydration of the hybrid PAA-lipid gels allows for the generation of GUVs using common chemicals present in the majority of laboratories
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
