Size Of Giant Unilamellar Vesicles Research Articles

Phase separation on giant liposomes prepared by gel-assisted swelling.

Giant unilamellar vesicles (GUVs) are often used as models to study lipid membrane structure and function. The most common method for GUV formation is electroformation, though this technique has its own inherent drawbacks such as low yield in ionic solutions or in the presence of charged lipids. In recent years, the method of gel-assisted swelling has been explored as an alternative for electroformation and has been reported to overcome some of the limitations of this technique. This study aims to assess the ability of gel-assisted swelling to form GUVs of ternary lipid compositions that are known to undergo phase separation. To this end, we utilize different ratios of a 3-component mixture of DOPC/DPPC/Cholesterol to form vesicles capable of separating into phases of liquid-ordered (Lo) and liquid-disordered (Ld). This lipid composition was chosen as previous studies have shown that at room temperature, many of its possible compositions exhibit distinct phase separation. We apply confocal fluorescence microscopy to evaluate the resultant GUVs, in terms of yield, size, and phase behavior. We compare the results of this method to that of electroformation with a focus on GUV size and quantity, as well as formation of lipid domains, their number and area fraction on GUVs. Ultimately, the results show that agarose gel-assisted swelling can parallel electroformation in forming phase-separating GUVs over a series of ternary lipid compositions.

Effects of sugar concentration on the electroporation, size distribution and average size of charged giant unilamellar vesicles.

We investigated the effects of sugar concentration on the electroporation, size distribution and average size of giant unilamellar vesicles (GUVs). GUVs were prepared from 40mol% of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 60mol% of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. Pulsed electric field was applied to the 40%DOPG/60%DOPC-GUVs and it induced lateral electric tension (σc) in the membranes of vesicles. The σc-induced probability of rupture (Ppore) and the rate constant of rupture (kp) of GUVs under the sugar concentration, c = 40, 100 and 300mM, were determined. Both the Ppore and kp increased with the increase of σc, but higher tension was required to generate the same values of Ppore and kp with increasing c. We also investigated average sizes of GUVs from the size distribution of vesicles under various sugar concentrations. With the increase of c, the peak of the size distribution histograms shifted to the region of smaller vesicles. The average size decreased 1.6-fold when c increased from 10 to 300mM. These investigations help to understand various biomedical, biophysical, and biochemical processes in vesicles and cells. Electroporation, size distribution and average size of charged GUVs were investigated under various sugar concentrations. The sugar concentration influences the electroporation of vesicles and the average size of GUVs.

Monodisperse Liposome Preparation from W/O/W Droplets by Removal of the Oil Layer

Giant unilamellar vesicles (GUVs) or giant liposomes have been used as a cell-size bioreactor to recapitulate the physical/chemical characteristics of biological cells. However, conventional preparation methods for GUVs lack precise control of the size of GUVs. Recently, several groups developed a preparation method for monodisperse GUVs using microfluidics, in which water-in-oil-in-water (W/O/W) droplets with a thin oil layer were generated and transformed into GUVs. However, the formation of the thin oil shell requires precise control of flow rates, which is often difficult and unstable. In this study, we investigated the design of a two-step flow-focusing microfluidic channel to generate stable W/O/W droplets, from which a significant portion of the oil layer was spontaneously removed by the fluidic shear. As a result, the oil layer of the W/O/W droplets was thinned down, which could promote spontaneous dewetting of the oil layer to form a lipid bilayer.

Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.

A new purification technique to obtain specific size distribution of giant lipid vesicles using dual filtration.

A new purification technique is developed for obtaining distribution of giant unilamellar vesicles (GUVs) within a specific range of sizes using dual filtration. The GUVs were prepared using well known natural swelling method. For filtration, different combinations of polycarbonate membranes were implemented in filter holders. In our experiment, the combinations of membranes were selected with corresponding pore sizes–(i) 12 and 10 μm, (ii) 12 and 8 μm, and (iii) 10 and 8 μm. By these filtration arrangements, obtained GUVs size distribution were in the ranges of 6−26 μm, 5–38 μm and 5–30 μm, respectively. In comparison, the size distribution range was much higher for single filtration technique, for example, 6−59 μm GUVs found for a membrane with 12 μm pores. Using this technique, the water-soluble fluorescent probe, calcein, can be removed from the suspension of GUVs successfully. The size distributions were analyzed with lognormal distribution. The skewness became smaller (narrow size distribution) when a dual filtration was used instead of single filtration. The mode of the size distribution obtained in dual filtration was also smaller to that of single filtration. By continuing this process of purification for a second time, the GUVs size distribution became even narrower. After using an extra filtration with dual filtration, two different size distributions of GUVs were obtained at a time. This experimental observation suggests that different size specific distributions of GUVs can be obtained easily, even if GUVs are prepared by different other methods.

Confinement Geometry Tunes Fascin-Actin Bundle Structures and Consequently the Shape of a Lipid Bilayer Vesicle.

Depending on the physical and biochemical properties of actin-binding proteins, actin networks form different types of membrane protrusions at the cell periphery. Actin crosslinkers, which facilitate the interaction of actin filaments with one another, are pivotal in determining the mechanical properties and protrusive behavior of actin networks. Short crosslinkers such as fascin bundle F-actin to form rigid spiky filopodial protrusions. By encapsulation of fascin and actin in giant unilamellar vesicles (GUVs), we show that fascin-actin bundles cause various GUV shape changes by forming bundle networks or straight single bundles depending on GUV size and fascin concentration. We also show that the presence of a long crosslinker, α-actinin, impacts fascin-induced GUV shape changes and significantly impairs the formation of filopodia-like protrusions. Actin bundle-induced GUV shape changes are confirmed by light-induced disassembly of actin bundles leading to the reversal of GUV shape. Our study contributes to advancing the design of shape-changing minimal cells for better characterization of the interaction between lipid bilayer membranes and actin cytoskeleton.

Effect of Electrical Parameters and Cholesterol Concentration on Giant Unilamellar Vesicles Electroformation.

Giant unilamellar vesicles (GUVs) are used extensively as models that mimic cell membranes. The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. Thus, a methodological paper pertaining to preparations of model lipid bilayer membranes with high Chol content would significantly help the study of properties of these membranes. Lipid solutions containing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and Chol were fluorescently labeled with phospholipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC18(3)) and spin-coated to produce thin lipid films. GUVs were formed from these films using the electroformation method and the results were obtained using fluorescent microscopy. Electroformation outcomes were examined for different electrical parameters and different Chol concentrations. A wide range of field frequency-field strength (ff-fs) combinations was explored: 10-10,000 Hz and 0.625-9.375 V/mm peak-to-peak. Optimal values for GUVs preparation were found to be 10-100 Hz and 1.25-6.25 V/mm, with largest vesicles occurring for 10 Hz and 3.75 V/mm. Chol:POPC mixing ratios (expressed as a molar ratio) ranged from 0 to 3.5. We show that increasing the Chol concentration decreases the GUVs size, but this effect can be reduced by choosing the appropriate ff-fs combination.

Electrostatic interaction effects on the size distribution of self-assembled giant unilamellar vesicles.

The influence of electrostatic conditions (salt concentration of the solution and vesicle surface charge density) on the size distribution of self-assembled giant unilamellar vesicles (GUVs) is considered. The membranes of GUVs are synthesized by a mixture of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine in a physiological buffer using the natural swelling method. The experimental results are presented in the form of a set of histograms. The log-normal distribution is used for statistical treatment of results. It is obtained that the decrease of salt concentration and the increase of vesicle surface charge density of the membranes increase the average size of the GUV population. To explain the experimental results, a theory using the Helmholtz free energy of the system describing the GUV vesiculation is developed. The size distribution histograms and average size of GUVs under various conditions are fitted with the proposed theory. It is shown that the variation of the bending modulus due to changing of electrostatic parameters of the system is the main factor causing a change in the average size of GUVs.

Study of molecular transport through a single nanopore in the membrane of a giant unilamellar vesicle using COMSOL simulation.

The antimicrobial peptide (AMP) magainin 2 induces nanopores in the lipid membranes of giant unilamellar vesicles (GUVs), as observed by the leakage of water-soluble fluorescent probes from the inside to the outside of GUVs through the pores. However, molecular transport through a single nanopore has not been investigated in detail yet and is studied in the present work by simulation. A single pore was designed in the membrane of a GUV using computer-aided design software. Molecular transport, from the outside to the inside of GUV through the nanopore, of various fluorescent probes such as calcein, Texas-Red Dextran 3000 (TRD-3k), TRD-10k and TRD-40k was then simulated. The effect of variation in GUV size (diameter) was also investigated. A single exponential growth function was fitted to the time course of the fluorescence intensity inside the GUV and the corresponding rate constant of molecular transport was calculated, which decreases with an increase in the size of fluorescent probe and also with an increase in the size of GUV. The rate constant found by simulation agrees reasonably well with reported experimental results for inside-to-outside probe leakage. Based on Fick's law of diffusion an analytical treatment is developed for the rate constant of molecular transport that supports the simulation results. These investigations contribute to a better understanding of the mechanism of pore formation using various membrane-active agents in the lipid membranes of vesicles and the biomembranes of cells.

Prediction of the size of electroformed giant unilamellar vesicle using response surface methodology

The production of giant unilamellar vesicles (GUVs) with specific size and structure has been a challenge on the design of quantitative biological assays in cell-mimetic micro-compartments. In this study, the effect of electroformation parameters (electric potential, frequency, and temperature) on the size of GUVs was investigated. Using response surface methodology based on Box-Behnken design, GUVs from neutral, positive and negative charges were formulated. The average diameter of GUVs was determined for each formulation. The acquired data of these GUVs were successfully fitted with quadratic regression models. These models were applied to visualize the parameters for ideal GUVs with wanted diameters by the obtained phase diagrams. These results show that response surface methodology can be used to estimate the electroformation parameters for specifically sized GUVs.

The role of gel-phase domains in electroporation of vesicles

Transient permeabilisation of the cell membrane is a critical step to introduce drugs or DNA into living cells, yet challenging for both biological research and therapeutic applications. To achieve this, electroporation (or electropermeabilisation) has become a widely used method due to its simplicity to deliver almost any biomolecule to any cell type. Although this method demonstrates promise in the field of drug/gene delivery, the underlying physical mechanisms of the response of the heterogeneous cell membrane to strong electric pulses is still unknown. In this study, we have investigated the role of gel-phase lipids in the electroporation of binary giant unilamellar vesicles (GUVs), composed from DPPC (gel-phase) and DPhPC (fluid-phase) lipids (molar ratio 8:2 and 2:8). We have observed that the exposure to electric pulses leads to expel of fluid-phase lipids and concomitant decrease in GUV size, whereas the gel-phase domains become buckled. Based on experiments on pure fluid-phase and gel-phase GUVs, we have found that fluid-phase lipids can be expelled by electrical forces and the highly viscous gel-phase lipids cannot. Moreover, our analyses suggest that pore formation occurs primarily in fluid-phase domains and that the pore size is similar in all GUVs containing fluid-phase lipids, irrespective of the gel-phase percentage.

Microfluidic Fabrication of Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation

Conventional methods for vesicle production lack the uniformity in size and composition required for many scientific and technological applications. To overcome these limitations, we present a microfluidic approach for the production of giant unilamellar vesicles (GUVs) using monodisperse water-in-oil-in-water (W/O/W) double emulsion drops as templates. The middle oil phase is a mixture of the lipids that ultimately form the GUV membranes, dissolved in a mixture of a highly-volatile good solvent and a less volatile poor solvent. The shell formed by the middle phase around the aqueous double emulsion core is less than a micron thick, much smaller than in typical double emulsion templates; this enables the fabrication of GUVs that contain minimal residual solvent within their membranes. The evaporation of the good solvent forces the bad solvent to dewet from the double emulsion drops, forming GUVs that stably encapsulate the material within their cores. By carefully controlling the lipid composition of the middle phase, we show that lipid microdomains form during the dewetting process. Our work thus demonstrates a straightforward approach to GUV fabrication that enables control over the GUV size, lipid composition, and the formation of microdomains within the GUV membrane.

Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation

A microfluidic approach is reported for the high-throughput, continuous production of giant unilamellar vesicles (GUVs) using water-in-oil-in-water double emulsion drops as templates. Importantly, these emulsion drops have ultrathin shells; this minimizes the amount of residual solvent that remains trapped within the GUV membrane, overcoming a major limitation of typical microfluidic approaches for GUV fabrication. This approach enables the formation of microdomains, characterized by different lipid compositions and structures within the GUV membranes. This work therefore demonstrates a straightforward and versatile approach to GUV fabrication with precise control over the GUV size, lipid composition and the formation of microdomains within the GUV membrane.

Cell-Free Protein Synthesis inside Giant Unilamellar Vesicles Analyzed by Flow Cytometry

Lipid vesicles have been used as model cell systems, in which an in-vitro transcription-translation system (IVTT) is encapsulated to carry out intravesicular protein synthesis. Despite a large number of previous studies, a quantitative understanding of how protein synthesis inside the vesicles is affected by the lipid membrane remains elusive. This is mainly because of the heterogeneity in structural properties of the lipid vesicles used in the experiments. We investigated the effects of the phospholipid membrane on green fluorescent protein (GFP) synthesis occurring inside cell-sized giant unilamellar vesicles (GUV), which have a defined quantity of lipids relative to the reaction volume. We first developed a method to distinguish GUV from multilamellar vesicles using flow cytometry (FCM). Using this method, we investigated the time course of GFP synthesis using one of the IVTT, the PURE system, and found that phospholipid in the form of GUV has little effect on GFP synthesis based on three lines of investigation. (1) GFP synthesis inside the GUV was not dependent on the size of GUV (2) or on the fraction of cholesterol or anionic phospholipid constituting the GUV, and (3) GFP synthesis proceeded similarly in GUV and in the test tube. The present results suggest that GUV provides an ideal reaction environment that does not affect the internal biochemical reaction. On the other hand, we also found that internal GFP synthesis is strongly dependent on the chemical composition of the outer solution.

Measuring Passive Transport Using Confocal Microscopy of Giant Lipid Vesicles

The ability of a molecule to pass through the plasma membrane without the aid of any active cellular mechanisms is central to that molecule's pharmaceutical characteristics. Existing techniques for measuring this passive transport capacity are hampered by the presence of an unstirred layer (USL) which dominates transport considerations across the bilayer. We are developing assays based on confocal microscopy of giant unilamellar vesicles (GUVs) that allow for the detailed investigation of passive transport processes and mechanisms. At the size of GUVs (generally less than 100 μm), the effect of the USL on membrane transport processes is minimized, giving more accurate values of membrane permeability.We have constructed several series of drug-like fluorescent molecules by covalently modifying the dye 4-nitrobenzo-2-oxa-1, 3-diazole (NBD). A series of molecules of increasing hydrophilicity was constructed from polyethylene glycol (PEG) having 4, 8, and 12 repeating units. Alkane chains with 3, 5, and 7 carbons were used as the hydrophobic representatives. Transport of both series of modified NBD molecules was observed by tracking NBD fluorescence as the molecules passed through the GUV membrane. Weak acyclic acid with 2, 4, and 6 carbons were also examined, using a pH-sensitive dye to track their transport.An analytical theoretic passive transport model was devised, original data was regressed to the model, and permeability was calculated for each dataset. Finite element modeling (FEM) was used to simulate the experiments. The simulation supported the experimental results well.

SNARE-Mediated Adhesion Kinetics in Giant Membrane Systems

Giant membrane in vitro systems are powerful and relatively unexplored model systems to probe mechanisms of SNARE-mediated adhesion and fusion. We present results of a joint experimental-modeling study of SNARE-mediated adhesion kinetics of giant unilamellar vesicles (GUVs) with supported bilayers (SBLs). The large size of GUVs allows control of physical parameters such as tension and direct observation of adhesion kinetics unavailable in small unilamellar vesicle (SUV) systems. In the gravity-imposed contact zone between v-SNARE GUVs and t-SNARE SBLs, reflection interference contrast microscopy (RICM) revealed initial membrane separation ∼50 nm, consistent with Helfrich theory predicting intermembrane repulsion from thermal undulations. Tight adhesion in several discrete patches within the contact zone then develops over ∼10 min, with total patch area growing linearly in time. We mathematically modeled the adhesion kinetics, which comprises two stages. (a) Patch nucleation kinetics. Nucleation is limited by the mean membrane separation which exceeds the reach of cognate SNAREs in apposing membranes. Complexation requires fluctuations to bring cognate SNAREs into proximity and the nucleation rate depends on tension and SNARE density. (b) Patch growth. Once nucleated a patch encourages further complexation because the membrane separation is within SNARE reach. We find two classes of patch growth kinetics. (i) Mobile SNARE complexes. Complex osmotic pressure then drives patch growth and patch area grows quadratically in time. (ii) Immobile SNARE complexes. Patches of tightly clustered SNARE complexes are predicted: patch growth, limited by SNARE diffusion, is linear in time. The experimental data are consistent with model (ii). The SNARE complex density in the patch inferred from the areal growth data suggests that complexes were shoulder-to-shoulder as expected in clusters generated by SNARE-SNARE interactions.