Production of Isolated Giant Unilamellar Vesicles under High Salt Concentrations

Stein, Hannah; Spindler, Susann; Bonakdar, Navid; Chun, Wang; Sandoghdar, Vahid

doi:10.3389/fphys.2017.00063

Cited by 134 publications

(148 citation statements)

References 90 publications

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“…However, low GUV yields in physiological buffer solutions, being heterogeneous in GUV's size and lipid composition, strong limitations to small amounts of charged lipids (<10%) and low efficiency biomolecule encapsulation remain the major drawbacks of these methods towards realizing a synthetic cell. [15][16][17] Other methods like microfluidic jetting 18 or emulsion transfer 19 overcome these limitations to some extent, but compromise the GUV yield. Recently, microfluidic technologies for the highthroughput production of monodisperse GUVs and vesosomes have been developed.…”

Section: Introductionmentioning

confidence: 99%

Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems

et al. 2018

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In this manuscript, we introduce a simple, off-the-shelf approach for the on-demand creation of giant unilamellar vesicles (GUVs) or multicompartment synthetic cell model systems in a high-throughput manner. To achieve this, we use microfluidics to encapsulate small unilamellar vesicles in block-copolymer surfactant-stabilized water-in-oil droplets. By tuning the charge of the inner droplet interface, adsorption of lipids can be either inhibited, leading to multicompartment systems, or induced, leading to the formation of droplet-stabilized GUVs. To control the charge density, we formed droplets using different molar ratios of an uncharged PEG-based fluorosurfactant and a negatively-charged PFPE carboxylic acid fluorosurfactant (Krytox). We systematically studied the transition from a multicompartment system to 3D-supported lipid bilayers as a function of lipid charge and Krytox concentration using confocal fluorescence microscopy, cryo-scanning electron microscopy and interfacial tension measurements. Moreover, we demonstrate a simple method to release GUVs from the surfactant shell and the oil phase into a physiological buffer -providing a remarkably high-yield approach for GUV formation. This widely applicable microfluidics-based technology will increase the scope of GUVs as adaptable cell-like compartments in bottom-up synthetic biology applications and beyond.

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Section: Introductionmentioning

confidence: 99%

Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems

et al. 2018

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“…Electroformation devices. A) Reproduced under the terms and conditions of the Creative Commons Attribution License (CC BY) . Copyright 2017, The Authors; B) Reproduced under the terms and conditions of the Creative Commons Attribution 4.0 International License .…”

Section: Methodsmentioning

confidence: 99%

Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies

2019

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Self‐assembly of lipids or polymeric amphiphiles into vesicular structures has been achieved by various methods since the first generation of liposomes in the 1960s. Vesicles can be obtained with diameters from the nanometer to the micrometer regime. From the perspective of cell mimicking, vesicles with diameters of several micrometers are most relevant. These vesicles are called giant unilamellar vesicles (GUVs). Commonly used methods to form GUVs are solvent‐displacement techniques, especially since the development of microfluidics. These methodologies however, trap undesirable organic solvents in their membrane as well as other potentially undesired additives (surfactants, polyelectrolytes, polymers, etc.). In contrast to those strategies, summarized herein are solvent‐free approaches as suitable clean alternatives. The vesicles are formed from a dry thin layer of the lipid or amphiphilic polymers and are hydrated in aqueous media using the entropically favored self‐assembly of amphiphiles into GUVs. The rearrangement of the amphiphilic films into vesicular structures is usually aided by shear forces such as an alternative current (electroformation) or the swelling of water‐soluble polymeric supports (gel‐assisted hydration).

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“…Approaches based on vesicle swelling on polymer films [23,24] were not employed because they result in polymer residues encapsulated in the vesicles and maybe even in the membrane [25,26], which is more pronounced at high temperature. Methods based on transfer of lipids from an oil phase to an aqueous one [27,28] were also avoided because of the inherent risk of remaining oil residues in the bilayer, which may influence the membrane phase behavior.…”

Section: Vesicle Stability Is Reduced Upon Fast Cooling After Preparamentioning

confidence: 99%

Micron-sized domains in quasi single-component giant vesicles

Knorr

Steinkühler

Dimova

2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Giant unilamellar vesicles (GUVs), are a convenient tool to study membrane-bound processes using optical microscopy. An increasing number of studies highlights the potential of these model membranes when addressing questions in membrane biophysics and cell-biology. Among them, phase transitions and domain formation, dynamics and stability in raft-like mixtures are probably some of the most intensively investigated. In doing so, many research teams rely on standard protocols for GUV preparation and handling involving the use of sugar solutions. Here, we demonstrate that following such a standard approach can lead to the abnormal formation of micron-sized domains in GUVs grown from only a single phospholipid. The membrane heterogeneity is visualized by means of a small fraction (0.1 mol%) of a fluorescent lipid dye. For dipalmitoylphosphatidylcholine GUVs, different types of membrane heterogeneities were detected. First, the unexpected formation of micron-sized dye-depleted domains was observed upon cooling. These domains nucleated about 10 K above the lipid main phase transition temperature, T. In addition, upon further cooling of the GUVs down to the immediate vicinity of T, stripe-like dye-enriched structures around the domains are detected. The micron-sized domains in quasi single-component GUVs were observed also when using two other lipids. Whereas the stripe structures are related to the phase transition of the lipid, the dye-excluding domains seem to be caused by traces of impurities present in the glucose. Supplementing glucose solutions with nm-sized liposomes at millimolar lipid concentration suppresses the formation of the micron-sized domains, presumably by providing competitive binding of the impurities to the liposome membrane in excess. It is likely that such traces of impurities can significantly alter lipid phase diagrams and cause differences among reported ones.

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Production of Isolated Giant Unilamellar Vesicles under High Salt Concentrations

Cited by 134 publications

References 90 publications

Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems

Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems

Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies

Micron-sized domains in quasi single-component giant vesicles

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