Gel-Assisted Formation of Giant Unilamellar Vesicles

Weinberger, Andreas; Tsai, Feng-Ching; Koenderink, Gijsje H.; Schmidt, Thaís F.; Itri, Rosângela; Meier, Wolfgang; Schmatko, Tatiana; Schröder, A.; Marques, Carlos M.

doi:10.1016/j.bpj.2013.05.024

Cited by 312 publications

(351 citation statements)

References 52 publications

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“…42 If the GUVs of Xad-PC-Xad are prepared at temperatures above the corresponding T m of the lipids, only rapid cooling (≥4°C/ min) leads to faceted vesicles (see Figure 5A). When the GUVs have been cooled down more slowly (≤0.5−1°C/min), they were spherical with no apparent facets (see Figure 5B).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Bilayer Properties of 1,3-Diamidophospholipids

et al. 2015

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A series of 1,3-diamido phosphocholines was synthesized, and their potential to form stable bilayers was investigated. Large and giant unilamellar vesicles produced from these new lipids form a wide variety of faceted liposomes. Factors such as cooling rates and the careful choice of the liposome preparation method influence the formation of facets. Interdigitation was hypothesized as a main factor for the stabilization of facets and effectively monitored by small-angle X-ray scattering measurements.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Bilayer Properties of 1,3-Diamidophospholipids

et al. 2015

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“…In their work, Horger et al (22) suggest that films of crosslinked polyacrylamide gels could be used instead of agarose gels. Very recently, Weinberger et al (29) have replaced agarose for PVA, a less water-soluble polymer, to produce GUVs. It was shown by fluorescence imaging that there is no indication of residual polymer, either encapsulated or bound to the membranes (fluorescence imaging does not fully exclude the possibility of remaining polymers present at the membrane and in the vesicles).…”

Section: Discussionmentioning

confidence: 99%

“…Given the significance of the method, we considered it important to evaluate how the residual polymer affects the overall membrane mechanics rather than simply the lipid diffusion. A subsequent work replaced agarose with the chemically cross-linked polyvinyl alcohol (PVA) to circumvent polymer encapsulation (29). However, the difficulty in detaching the formed GUVs from the PVA film is still an issue.…”

Section: Introductionmentioning

confidence: 99%

Giant Unilamellar Vesicles Formed by Hybrid Films of Agarose and Lipids Display Altered Mechanical Properties

Lira

Dimova

Riske

2014

Biophysical Journal

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Giant unilamellar vesicles (GUVs) are presumably the current most popular biomimetic membrane model. Preparation of GUVs in physiological conditions using the classical electroformation method is challenging. To circumvent these difficulties, a new method was recently reported, by which GUVs spontaneously swell from hybrid films of agarose and lipids. However, agarose is left encapsulated in the vesicles in different amounts. In this work, we thoroughly characterize the mechanical properties of these agarose-GUVs in response to electric pulses, which induce vesicle deformation and can lead to membrane poration. We show that the relaxation dynamics of deformed vesicles, both in the presence and absence of poration, is significantly slowed down for agarose-GUVs when compared to agarose-free GUVs. In the presence of poration, agarose polymers prevent complete pore closure and lead to high membrane permeability. A fraction of the vesicles were found to encapsulate agarose in the form of a gel-like meshwork. These vesicles rupture and open up after electroporation and the meshwork is expelled through a macropore. When the agarose-GUVs are heated above the melting temperature of agarose for 2 h before use, vesicle response is (partially) recovered due to substantial release of encapsulated agarose during temperature treatment. Our findings reveal potential artifactual behavior of agarose-GUVs in processes involving morphological changes in the membrane as well as poration.

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“…GUVs were prepared following published gel-assisted swelling (48) or electroformation (49) protocols using buffers containing 20 mM Tris·HCl (pH 7.5) and 100 mM salt (KCl, NaCl, or CholCl as specified). Solvent effects were studied with GUVs prepared using an inverse emulsion protocol (50), and GUVs containing beta-casein (≥98%; Sigma) were formed by including …”

Section: Methodsmentioning

confidence: 99%

Whole-GUV patch-clamping

Garten

Mosgaard

Bornschlögl

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/ quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape. giant unilamellar vesicle | patch clamp | electrophysiology | biomimetic system | lipid-glass interaction

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Gel-Assisted Formation of Giant Unilamellar Vesicles

Cited by 312 publications

References 52 publications

Bilayer Properties of 1,3-Diamidophospholipids

Bilayer Properties of 1,3-Diamidophospholipids

Giant Unilamellar Vesicles Formed by Hybrid Films of Agarose and Lipids Display Altered Mechanical Properties

Whole-GUV patch-clamping

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