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ArticleMarch 16, 2017

Electroformation of Giant Unilamellar Vesicles on Stainless Steel Electrodes
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Cite this: ACS Omega 2017, 2, 3, 994–1002

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https://doi.org/10.1021/acsomega.6b00395

Published March 16, 2017

Abstract

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Giant unilamellar vesicles (GUVs) are well-established model systems for studying membrane structure and dynamics. Electroformation, also referred to as electroswelling, is one of the most prevalent methods for producing GUVs, as it enables modulation of the lipid hydration process to form relatively monodisperse, defect-free vesicles. Currently, however, it is expensive and time-consuming compared with other methods. In this study, we demonstrate that 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine GUVs can be prepared readily at a fraction of the cost on stainless steel electrodes, such as commercially available syringe needles, without any evidence of lipid oxidation or hydrolysis.

Subjects

Introduction

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Giant unilamellar vesicles (GUVs), often referred to as giant liposomes, are fluid-filled membranes that offer a useful basic model of a biological cell. GUVs have been widely used as models to study the fluid–fluid and gel–fluid phase coexistence of membrane lipids, (1-6) membrane transport phenomena, (7-12) the behavior of native membranes, (13-16) the structure of early cells and protocells, (17) and more recently, cells’ biological activity. (10) In its simplest form, a GUV consists of a single amphiphilic lipid bilayer that encloses an aqueous solution.

Among the multitude of production methods developed over several decades, (18-20) lipid-film hydration, (21) electroformation, (22) lipid emulsification, (23, 24) and microfluidic-based methods, such as fluid jetting (25) and hydrodynamic flow focusing, (26) are the most widely used. While the literature on vesicle formation is rich with established formation protocols, each technique has its own inherent drawbacks and trade-offs according to the application. The factors affecting the choice of a specific protocol are technical expertise, the need for specialist equipment, and the degree to which vesicle characteristics need to be tuned. Of the methods mentioned above, electroformation is one of the most widespread. (18)

Figure 1. Schematic of vesicle electroformation from a dry lipid bilayer deposited on a substrate. Vesicles are formed upon hydration and the application of an alternating electric field, Ẽ (not to scale).

Electroformation of GUVs was pioneered by Angelova and Dimitrov in 1986. (22) It involves modulating the spontaneous swelling of lipids within an aqueous solution using an externally applied electric field (Figure 1). Typically, a solution of lipids dissolved in an organic solvent is deposited on two electrodes of indium tin oxide (ITO)-coated glass or platinum. Following solvent evaporation, the electrodes are placed in contact with an aqueous solution. Subsequently, an alternating potential difference is applied across the electrodes, stimulating the swelling process of the hydrated lipid layer. The main advantages of electroformation are that it requires comparatively little technical expertise to implement and that it yields spherical, relatively monodisperse and unilamellar lipid vesicles. Set against this, however, is the cost of the electrodes, which limits the scalability of the technique.

In this article, we present and validate a simple electrode modification in the standard electroformation protocol that reduces the cost and time of chamber preparation while improving scalability. 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) was chosen as a representative unsaturated phospholipid, which is more prone to oxidation than saturated phospholipids, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), to prove that stainless steel does not affect its structural integrity.

Stainless steel electrodes, such as injection needles, provide a significantly lower-cost alternative to platinum and ITO electrodes, which are the established electrode materials reported in the electroformation literature. Other studies have also reported electroformation of vesicles on interdigitated and nonconductive substrates, (27) albeit with limited adoption rates.

Stainless steel electrodes provide numerous advantages. First, the rigidity of stainless steel compared to that of platinum reduces the risk of bending, improving control over the electrode-separation distance, thus leading to a more uniform electric field. In addition, disposable needles do not require cleaning in an ultrasound bath, (28) are readily available, and do not require the modification of current chambers.

The wide cost disparity between stainless steel and platinum enables two secondary modifications to the electroformation protocol: the use of longer electrodes and chamber parallelization. (29) These lead, in turn, to an increase in the overall vesicle production, concurrent electroformation, and multiparametric testing at high throughput.

While a direct comparison between electroformation methods is complex, Table 1 provides an overview of the merits and drawbacks of different electrode materials used in the electroformation of vesicles. To populate the table, the electrode surface area was fixed across all methods. The cost comparison was carried out on the face value of the electrodes, without taking into account the cost of the purpose-built polyoxymethylene electroformation chamber. A full cost breakdown of the electroformation chamber is provided in Table S1. It is noteworthy that, if the stainless steel electrodes were disposed of after every electroformation experiment, it would take approximately 292 experiments to match the cost of the platinum electrodes—ignoring overheads, such as the time and cost of cleaning.

Table 1. Comparison of Costs, Advantages, and Disadvantages of Using Stainless Steel Electrodes and Platinum Electrodes during Electroformation^a

	cost^b	disposable	scalable	machinable	cleaning required	consistent electrode separation
platinum	∼£584	expensive	expensive	yes	yes	difficult
stainless steel	∼£2.00	yes	yes	yes	only if reused	yes
ITO-coated glass	∼£27.00	yes	yes	no	yes	yes

A detailed cost breakdown of the electroformation chamber is provided in the Supporting Information.

Calculated for five pairs of 40 mm cylindrical electrodes with a diameter of 0.8 mm or an ITO surface of equivalent area (10 cm²).

In the experiments reported here, these features are exploited using a purpose-built electroformation device, which consists of a polyoxymethylene base containing five electroformation chambers and a lid to hold the electrodes in position.

To validate the proposed technique, vesicles were characterized in terms of their size distribution, lipid packing and unilamellarity of their membranes. In addition, the constituents of the aqueous electroformation solution were analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES) to test whether any ions are released from the electrodes during electroformation. Lipid degradation was assessed by NMR.

Results and Discussion

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GUV Production and Imaging

Electroformation visualization chambers have been used to monitor and verify the effective production of vesicles. (30) To assess the formation of vesicles on stainless steel electrodes, swelling was visualized in situ (Figure 2a) using a second specially designed electroformation chamber with optical access using the protocol described in the Materials and Methods section. Vesicle formation was observed across most of the electrode surface, even before the application of the electric field (Figure 2a). Upon switching on the signal generator, the swelling was accelerated, and the vesicles reached a moderate size (see video in the Supporting Information). Swelling and detachment of DOPC vesicles from an electrode were also observed (Figure 2b,c).

Figure 2. Vesicles swelling from electroformation electrodes: (a) hydrated lipid layer commencing the swelling process, (b) DiI-labeled single-vesicle swelling while attached to the electroformation electrodes, and (c) vesicles forming, swelling, and detaching along the electrode.

Following the visualization, vesicles were produced inside the device depicted in Figure 2a using the electroformation protocol described in the Materials and Methods section. The electrode material did not produce significant differences in the size distributions of the vesicles (Figure 3) which were within the ranges reported in the literature (10–30 μm for DOPC vesicles). (27, 31, 32)

Figure 3. (a) Size distribution of DOPC vesicles as a function of electrode material. The white bars represent vesicles formed using platinum wires as electrodes, whereas the gray bars represent vesicles formed using stainless steel electrodes. (b) Comparison of average vesicle diameters (±standard deviation) of platinum and stainless steel electrodes. Three independent experiments were conducted, and over 1500 vesicles were analyzed for each electrode material.

The yield of a single electroformation chamber was estimated by suspending 100 μL of a GUV-rich solution in phosphate-buffered saline (PBS) and counting the number of vesicles at the bottom of a visualization plate after sedimentation. The vesicles were counted using a purpose-built algorithm for both electrode materials. On average, electroformation of the stainless steel electrode yielded 2256 vesicles (22 560 vesicles/mL) against the 3114 (31 140 vesicles/mL) yielded by the platinum wire electrodes. The difference, however, was not statistically significant.

It should be noted, however, that a comparison between GUV studies is problematic because of the differences in experimental parameters, such as osmotic pressure, electrode size and separation, electroformation solution, lipid species, electric field strength, imaging plane, and vesicle manipulation technique.

Lipid Order and Size of Vesicles Composed of Binary Lipid Mixtures

To test the parallelization potential, vesicles with different compositions were produced simultaneously by varying the DOPC/cholesterol (DOPC/Chol) molar ratio in the multichamber device (Figure 4).

Figure 4. Simultaneous electroformation of vesicles with dissimilar membrane properties. (a) Generalized polarization (GP) of the vesicles as a function of initial lipid-film formulation. (b) Representative GP false-colored images of four vesicles with different GP values.

The results depicted in Figure 5 were obtained by performing four concurrent electroformation experiments, coating the wires in each chamber with a different lipid composition. Figure 5a displays the average GPs and the standard deviations of the vesicles for each initial lipid composition. The packing of the membrane increases with increasing cholesterol volume fraction, as cholesterol intercalates between the DOPC lipids. (33-35) The electroformation cycle lasted 2.5 h, required limited setup time, and was performed using four pairs of new injection needles (SS304), at a total electrode cost of approximately £2.00. The equivalent cost of Pt wire electrodes would have been approximately £584 and would have required electrode cleaning and straightening.

Figure 5. (a) Vesicle diameters as a function of lipid composition (b) Lipid packing of DOPC vesicles as a function of electrode material.

The consistency in vesicle diameter was determined as a function of lipid composition. The vesicle size was not affected significantly by the molar fraction of cholesterol compared with the 1:0 DOPC/Chol composition (Figure 6a). The electrode material did not have an effect on the lipid packing of GUV electrodes (Figure 6b). A higher osmolarity of the extravesicular environment yields vesicles with a higher membrane order and thus a higher degree of lipid packing. (36)

Figure 6. ¹H NMR spectra of lipids extracted from electroformed GUVs (top trace) and large multilamellar vesicles (LMVs) (bottom trace). The residual solvent peaks of chloroform (7.26 ppm) and methanol (3.49 ppm) were removed for clarity. The inset shows a magnified view of the olefinic and head group protons.

Leaching of Metals from the Electrodes

Leaching of transition metals from the electrodes can be a concern in the electroformation of GUVs. Metal ions can bind to membranes (37) and alter their bilayer structure, (38) phase behavior, (39) and stability against vesicle fusion. (40) Unwanted chemical reactions can also be catalyzed by trace metals: chromium, iron, and nickel can initiate and, in some cases, propagate lipid peroxidation. (41)

The possibility of leaching of metals from stainless steel electrodes under electroformation conditions was assessed by comparing the concentrations of their constituents (Fe, Cr, Ni, and Mn) in GUV samples (Table 2) prepared using stainless steel and platinum electrodes. No increase in metal concentration compared to that of platinum electrodes was detected by ICP-OES. While no evidence of ion leaching was detected when the electroformation was carried out in 200 mM sucrose, this may not be the case in conductive electroformation solutions. On the contrary, platinum electrodes are routinely used to electroform vesicles in solutions of physiological ionic strength. A similar argument holds true for temperature, as the electroformation of vesicles with higher transition temperatures requires a heated chamber.

Table 2. Concentrations of Alloy Constituents Inside the Electroformation Chamber^a

element	% fraction in SS304	concentration with Pt electrodes (ppm)	concentration with SS304 electrodes (ppm)
iron	65–71	<0.1	<0.1
chromium	18–20	<0.1	<0.1
nickel	8–12	<0.1	<0.1
manganese	2	<0.1	<0.1

The concentrations of both platinum and stainless steel electrodes are shown.

Lipid Oxidation and Hydrolysis

The products of lipid oxidation and hydrolysis, which can be generated at the electrode during the electroformation process, (42) are also known to affect the physical properties of phospholipid membranes. Even small amounts of the degradation products can cause structural and dynamic changes, such as the formation of lipid rafts (42) or changes in permeability (43) and mechanical stability. (44)¹H NMR and ³¹P NMR spectroscopies are among the fastest and most informative analytical methods that have been used to characterize the chemical structure of phospholipids extracted from biological membranes and their degradation products. (45-48) The structural integrity of electroformed GUVs was assessed by comparing the NMR spectra of the combined lipid extracts from nine electroformed GUV samples with a control obtained from LMVs prepared by lipid hydration. The ¹H NMR spectra of lipids extracted from electroformed GUVs and LMVs are compared in Figure 6. The spectra were identical and consistent with pure DOPC. No degradation products were detected within a limit of detection of 1 mol %. Although trace impurities can be seen in the electroformed sample, the same impurities are found in the control. Their concentration remains constant when the concentration of DOPC in the control sample is increased from 0.34 to 3.4 mM, suggesting that they could be due to trace contamination rather than lipid degradation.

The absence of hydrolysis products was confirmed by the ³¹P NMR spectrum, which shows only one signal in both samples. All isomers of lyso-PC would resonate downfield, where no signal was detected.

Unilamellarity of Electroformed Vesicles

Vesicle unilamellarity was tested using a fluorescence quenching assay on the basis of the measurement of the ratio of inner to outer layers of fluorescently labeled GUVs. (49, 50) DOPC vesicles symmetrically labeled with the fluorescent lipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE), were electroformed, and a time course of their fluorescence was measured in a plate reader. Sodium dithionite, a membrane-impermeable quencher, was added to the sample to quench the fluorophores on the outer layer of the vesicles. The vesicles were then lysed by addition of Triton X-100, to expose all lipids to the quencher. The ratio of the emission drop caused by the addition of detergent (I_interior) to the total emission drop (I_total = I_interior + I_exterior) is used as a measure of the fraction of leaflets that are not exposed to the solvent (unilamellarity index) (Figure 7). We measured a ratio of 51 ± 2.8%, which is consistent with unilamellarity. The measurements for all three samples are provided in the Supporting Information.

Figure 7. Fluorescence intensity measurements of DOPC/NBD-PE vesicles. The fluorescence of intact vesicles is represented from time = 0 to arrow 1. At arrow 1, the quencher is added to the suspension, and the intensity is recorded until a stable value is reached. The emission drop is a measure of the number of fluorophores in outer leaflets. At arrow 2, Triton X-100 is added to lyse the vesicles, and fluorescence is recorded until a new stable value is reached. The corresponding emission drop is a measure of the number of fluorophores in inner leaflets. After each addition, the plate was removed from the plate reader and shaken for several minutes (the time axis is correspondingly cut for clarity).

Conclusions

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In summary, in this article, giant vesicle electroformation using an alternative electrode material was proposed and validated. We demonstrate that electroswelling of unilamellar vesicles occurs efficiently on stainless steel electrodes without any significant difference in vesicle size distribution, lipid degradation, or leaching of metals in the electroformation chamber from those of platinum electrodes. By using readily available stainless steel electrodes, we show that rapid, low-cost, and scalable electroformation can be achieved. We believe this platform will find application in biophysical investigations of the membrane, particularly in studies where multiplexing is crucial, such as large-scale screening of bioactive compounds.

Materials and Methods

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Electroformation Chamber

The electroformation chamber (Figure 8a) is composed of two parts: the electroformation base and the chamber lid. The electroformation base (150 mm × 40 mm × 50 mm) was designed to accommodate five equidistant electroformation chambers with a length of 50 mm and a diameter of 10 mm. The extremities of the chambers were tapered to increase vesicle concentration and facilitate the collection of the produced GUVs. The electroformation lid (150 mm × 40 mm × 5 mm) consists of five pairs of through holes (⌀ 1 mm), each aligned with an electroformation chamber, that are used to host the electrodes. On the side of the lid, 3 mm brass screws were positioned in correspondence to each electroformation chamber, to hold the electrodes in place once fully screwed. A copper bar (165 mm × 10 mm) ensures an electrical connection between corresponding electrodes. The chamber base and the lid were held together with four 4 mm screws at each corner of the device. The cost of the electroformation device with five electroformation chambers was calculated to be ∼£14.00. The full cost breakdown is provided in the Supporting Information.

Figure 8. Vesicle electroformation devices for high-yield production and formation visualization. (a) Scalable device with five electroformation chambers and needles held by brass screws. (b) Monitoring device with six chambers, brass screws, and stainless steel sheets for electrical connection between common electrodes.

A similar device (Figure 8b) was designed with the aim of visualizing the vesicle electroswelling process. The device was designed to be dimensionally compatible with a microscope stage and the working distance of a 40× objective. In this device, the electroformation electrodes were placed horizontally to allow imaging of the entire electrode surface area. A 170 μm thick glass slide was coupled with the bottom of the chamber with silicone gel. The electrodes were connected using two sheets of stainless steel, to provide a common electrical connection.

The electroformation chamber was cut from a solid piece of polyoxymethylene purchased from RS Components, U.K., brass screws were also supplied by RS Components, and 0.8 mm 21G 50 mm needles were purchased from Becton, Dickinson and Company. DOPC was sourced from Avanti Polar Lipids, Inc.. Cholesterol, sucrose, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. NBD-PE was purchased from ThermoFisher, U.K. All reagents were used as received without further purification. The electrodes were connected to an Agilent 33220A signal generator set to high-impedance load. GUVs were imaged in a μ-Slide eight-well multiwell plate purchased from ibidi GmbH, Germany. Ultrapure water (MilliQ) from a Millipore filtration system (resistivity, >18.2 MΩ cm) was used throughout all experiments.

Electroformation Protocol

The electroformation protocol consisted in preparing solutions of DOPC (1 mg/mL in chloroform) and cholesterol (10 mg/mL in chloroform) at varying molar ratios (1:0–1:3). The electrodes were rinsed in toluene and wiped using Kimwipes (Kimberly-Clark Professional) to remove any traces of silicon lubricant. The desired solution was then pipetted vertically onto the electrodes (50 μL per electrode pair) using a glass syringe, carefully coating the entire surface. After a drying phase of approximately 1 h, the electroformation chamber was filled with a sucrose solution (∼3 mL, 200 mM sucrose in deionized (DI) water at room temperature), the electrodes were submerged, and the signal generator was connected via the brass screws on the sides of the chamber.

A 5 Vpp, 10 Hz sinusoidal excitation was applied for 2 h to induce repetitive stress on the hydrated lipid bilayer, leading to vesicle swelling. The effective voltage on the electroformation chamber was recorded using a high-voltage probe and oscilloscope as 4.87 Vpp, indicating a 0.13 V voltage difference due to an unmatched load of the signal generator. Subsequently, the frequency of the excitation was lowered to 5 Hz for 30 min to facilitate vesicle detachment. The electroformation parameters used in the protocol are summarized in Table 3. At the end of the electroformation process, the lid of the chamber was removed and the vesicles were slowly pipetted into a visualization well using a 100–1000 μL pipette tip. To minimize the shear stress at the orifice, the end of the pipette tip was cut by 2 mm using a pair of scissors. The same protocol was used for both SS304 and platinum electrodes.

Table 3. Electroformation Signal Generator Parameters for GUV Formation

	parameters
electroformation phase	voltage (V)	frequency (Hz)	time (min)
I	5	10	120
II	5	5	30

GUV Detection and Sizing

The GUVs were imaged by phase-contrast microscopy using a Nikon ECLIPSE Ti inverted microscope (Nikon Corporation, Japan), and the images were then processed using a purpose-built MATLAB routine. The algorithm detects the GUVs on the basis of the imfindcircles built-in MATLAB function, calculates their diameter, and plots a vesicle-size histogram for all of the vesicles detected.

To quantify the concentration of DOPC GUVs, a finite volume (100 μL) of the electroformation solution was placed in an 8-well imaging dish (ibidi, U.K.) with 400 μL of PBS. The dish was coated with BSA by letting a 1 mg/mL BSA solution in DI water stand for 2 h. The BSA solution was then rinsed gently with DI water. After allowing the GUVs to sediment for an additional 2 h, the glass surface of the dish was imaged to cover the full area of the dish. A bounding grid that consisted of 39 × 45 tiles with 10% overlap was used for stitching.

Lipid Order Measurements

The lateral organization of the membrane lipids, also referred to as lipid packing, was quantified using a microscope-based spectral imaging technique. (33, 51) For this purpose, an aliquot (100 μL) of GUV-rich solution was placed in an imaging well. The vesicles were then fluorescently labeled with a final concentration of 400 nM c-Laurdan, an environment-sensitive molecular probe. The solution was then diluted with 100 μL of PBS. In the case of the experiment relating lipid packing to electrode material, 400 μL of PBS was added to the solution.

The emission spectrum of c-Laurdan shifts as a function of the dipolar water relaxation and thus the level of hydration within its surrounding microenvironment, indicating lipid packing in membranes. GP (52) was employed as a relative measure of lipid packing on a scale of −1 to 1, where −1 represents the least-packed membrane and 1 represents the most-packed membrane. Equation 1 describes the GP relative to the intensities at two specific wavelengths: 440 (I₄₄₀) and 490 nm (I₄₉₀).

(1)Spectral imaging was performed using a Zeiss LSM780 confocal microscope equipped with a 32-channel GaAsP detector array. The vesicles were excited at 405 nm, and the spectral intensity of the signal was recorded in the 415–691 nm range.

ICP-OES Experiments

The concentration of metal ions in the electroformed samples was measured by ICP-OES with a Varian Vista-MPX spectrometer, for both electrode materials.

Lipid Extraction and NMR Experiments

The samples for NMR analysis were prepared from nine simultaneous electroformation experiments. The content of each electroformation chamber (3 mL) was diluted with an equal volume of PBS in a 50 mL Falcon tube, which was then left to stand overnight at room temperature. The GUV-enriched layers (500 μL each) were then withdrawn from the bottom of each Falcon tube, combined, and freeze-dried. The lipids were then extracted from the dried GUV samples by the Bligh and Dyer method. (53) Briefly, the sample was dispersed in water (0.8 mL), and then methanol (2 mL) and chloroform (1 mL) were added with mixing after each addition. The sample was vortexed at 2500 rpm for 15 s and allowed to stand for 30 min. Chloroform (1 mL) and water (1 mL) were added, and the sample was centrifuged at 1000g for 5 min to achieve a complete phase separation. The bottom organic layer was withdrawn using a glass syringe, dried under a nitrogen stream, and redissolved in CDCl₃ (600 μL) for analysis.

LMV control samples with different lipid concentrations were prepared using the lipid hydration method. A solution of DOPC in chloroform was added to a glass vial and dried under a nitrogen stream. The lipid film was then hydrated by adding the same sucrose solution used for electroformation and vortexed for 2 min at 2500 rpm. After adding an equal volume of PBS, the sample was left to stand at room temperature for 18 h. The lipids were then extracted with the same protocol used for the electroformed sample.

¹H NMR spectra were acquired on a Bruker Ascend 400 spectrometer (at 400 MHz for ¹H and at 162 MHz for ³¹P) with 30° pulses (Bruker zg30 sequence) and a 3 s relaxation delay on a spectral width of 8000 Hz, whereas ³¹P NMR spectra were acquired with proton decoupling and a 3 s relaxation delay on a spectral width of 64 103 Hz. The spectra were apodized by multiplication with an exponential decay equivalent to 0.5 Hz line broadening and a Gaussian function equivalent to 1 Hz line broadening. ¹H NMR spectra were referenced to residual nondeuterated chloroform, whereas ³¹P NMR spectra were externally referenced to triphenylphosphine oxide.

Vesicle Unilamellarity

Three independent suspensions of fluorescently labeled vesicles were prepared using the electroformation protocol outlined previously, by depositing a mixture of DOPC and NBD-PE (1 mol %) in chloroform on the electrodes. The GUV suspension (2.7 mL) was then mixed with 300 μL of a HEPES buffer at pH 7 (0.1 M HEPES and 1 M NaCl) to obtain final concentrations of 10 mM HEPES and 100 mM NaCl. Three wells of a 96-well plate were filled with 200 μL of the buffered GUV suspension. Stable values of fluorescence intensity were measured at 520 nm on a FLUOstar Omega plate reader (BMG Labtech) before and after adding 4 μL of a freshly prepared solution of 1 M Na₂S₂O₄ in 1 M Tris buffer at pH 10, with excitation at 485 nm. Subsequently, 20 μL of a 10% v/v solution of Triton X-100 was added to the solution to cause vesicle lysis and expose all lipid structures to the quencher. A final stable value of fluorescence was recorded after the addition of the detergent. The unilamellarity index, defined as the fraction of leaflets that are not exposed to the solvent, was calculated as the ratio of the emission drop caused by the addition of detergent (I_interior) to the total emission drop (I_total = I_interior + I_exterior).

Statistical Analyses

Statistical testing was performed using one-way analysis of variance for multiple comparison analyses, whereas a Student’s t-test was employed for direct comparison between two data sets. All data were expressed in terms of mean ± standard deviation, and the number of independent replicates was expressed in the figure captions. The following conventions for statistical significance are used throughout the paper: ns, p > 0.05; *, p ≤ 0.05, **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.6b00395.

Cost breakdown of the electroformation chamber, limit of detection in NMR experiments, and unilamellarity experimental data; photograph of electroformation chamber (PDF)
Video of the formation of vesicles on an SS304 electrode upon the application of an external electric field at t = 3 s (scale bar = 20 μm) (AVI)

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Author Information

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Corresponding Author
- Eleanor Stride - Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford OX3 7DQ, U.K.; http://orcid.org/0000-0003-3371-5929; Email: [email protected]
Authors
- Valerio Pereno - Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford OX3 7DQ, U.K.
- Dario Carugo - Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford OX3 7DQ, U.K.; Mechatronics and Bioengineering Science Research Groups, Faculty of Engineering and the Environment, University of Southampton, SO17 1BJ Southampton, U.K.
- Luca Bau - Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford OX3 7DQ, U.K.
- Erdinc Sezgin - Weatherall Institute of Molecular Medicine, MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, U.K.
- Jorge Bernardino de la Serna - Rutherford Appleton Laboratory, Central Laser Facility, Science and Technology Facilities Council, Research Complex at Harwell, Harwell-Oxford, Didcot OX11 0FA, U.K.; http://orcid.org/0000-0002-1396-3338
- Christian Eggeling - Weatherall Institute of Molecular Medicine, MRC Human Immunology Unit, University of Oxford, Oxford OX3 9DS, U.K.
Author Contributions
The article was written through contributions of all authors. All authors have given approval to the final version of the article.
Funding
This work was supported by EPSRC Grant EP/I021795/1. E. Sezgin and C. Eggeling thank Wolfson Foundation, the Medical Research Council (MRC, grant number MC_UU_12010/unit programmes G0902418 and MC_UU_12025), MRC/BBSRC/ESPRC (grant number MR/K01577X/1), and the Wellcome Trust (grant ref 104924/14/Z/14). E. Sezgin was supported by EMBO Long-Term and Marie Curie Intra-European Fellowships (MEMBRANE DYNAMICS). J. Bernardino de la Serna acknowledges support from the Marie Curie Career Integration Grant.
Notes
The authors declare no competing financial interest.
Raw data are available through the University of Oxford ORA data repository doi: 10.5287/bodleian:9ed9ON5wZ.

Acknowledgment

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The authors extend their gratitude to James Fisk and David Salisbury for the fabrication of the electroformation chambers, Graham Brown for helpful discussions on microscopic techniques, and Fabio Bianchi for the helpful technical discussions.

Abbreviations

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GUV	giant unilamellar vesicle
LMV	large multilamellar vesicle
E	electric field
DOPC	1,2-dioleoyl-sn-glycero-3-phosphatidylcholine
DSPC	1,2-distearoyl-sn-glycero-3-phosphocholine
DPPC	1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DMPC	1,2-dimyristoyl-sn-glycero-3-phosphocholine
ITO	indium tin oxide
¹H NMR	proton nuclear magnetic resonance
ICP-OES	inductively coupled plasma optical emission spectrometry
PBS	phosphate-buffered saline
Vpp	peak-to-peak voltage

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Bagatolli, Luis A.; Gratton, Enrico
Biophysical Journal (2000), 79 (1), 434-447CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixts. were studied at different temps., by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temps. corresponding to the fluid phase and the fluid-solid phase coexistence. In addn., we studied the solid-solid temp. regime for the DMPC/DSPC and DMPE/DMPC mixts. From the Laurdan intensity images the generalized polarization function (GP) was calcd. at different temps. to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temps. corresponding to the fluid region for all of the lipid mixts. At temps. corresponding to phase coexistence we obsd. concurrent fluid and solid domains in the GUVs independent of the lipid mixt. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temp. The migration of the solid domains decreased dramatically at temps. close to the solid-fluid→solid phase transition. For the DLPC-contg. mixts., the solid domains showed line, quasi-circular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixt. increases. In addn., for the satd. PC-contg. mixts., we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixt. components. Specifically, at the phase coexistence temp. region the difference in the GP values, assocd. with the fluid and solid domains, increases as the difference in the chain length of the binary mixt. component increases. This last finding suggests that in the solid-phase domains, the local concn. of the low melting temp. phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temp. regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concn. of the low melting temp. phospholipid component in the solid domains is negligible. This last observation extends to the satd. PE/PC mixts. at the phase coexistence temp. range. For the DMPC/DSPC we found that the non-fluorescent solid regions gradually disappear in the solid temp. regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixts., where the solid domains remain on the GUV surface at temps. corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixts.
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Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Lipid Rafts Reconstituted in Model Membranes Biophys. J. 2001, 80, 1417– 1428 DOI: 10.1016/S0006-3495(01)76114-0
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Lipid rafts reconstituted in model membranes
Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E.
Biophysical Journal (2001), 80 (3), 1417-1428CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from (1) equimolar mixts. of phospholipid-cholesterol-sphingomyelin or (2) natural lipids extd. from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diam. were obsd. by fluorescence microscopy at room temp. (24°) whereas non-raft mixts. (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extn., which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixts., in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examd. by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixt. or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.
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Korlach, J.; Schwille, P.; Webb, W. W.; Feigenson, G. W. Characterization of Lipid Bilayer Phases by Confocal Microscopy and Fluorescence Correlation Spectroscopy Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8461– 8466 DOI: 10.1073/pnas.96.15.8461
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Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy
Korlach, Jonas; Schwille, Petra; Webb, Watt W.; Feigenson, Gerald W.
Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (15), 8461-8466CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chem. well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1,1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C20) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC). Phase sepn. was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphol. of coexisting phase domains on the surface of these vesicles with full two-dimensional resoln. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coeff., DT, of the DiI-C20 was measured by FCS. DT values at ∼25° ranged from ∼3×10-8 cm2/s in the fluid phase, to ∼2×10-9 cm2/s in high-cholesterol-content phases, to ∼2×10-10 cm2/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of DT in a region of two-phase coexistence on a single vesicle.
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Feigenson, G. W.; Buboltz, J. T. Ternary Phase Diagram of Dipalmitoyl-PC/Dilauroyl-PC/Cholesterol: Nanoscopic Domain Formation Driven by Cholesterol Biophys. J. 2001, 80, 2775– 2788 DOI: 10.1016/S0006-3495(01)76245-5
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Ternary phase diagram of dipalmitoyl-PC/dilauroyl-PC/cholesterol: nanoscopic domain formation driven by cholesterol
Feigenson, Gerald W.; Buboltz, Jeffrey T.
Biophysical Journal (2001), 80 (6), 2775-2788CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
A ternary phase diagram is proposed for the hydrated lamellar lipid mixt. dipalmitoylphosphatidylcholine/dilauroylphosphatidylcholine/cholesterol (DPPC/DLPC/cholesterol) at room temp. The entire compn. space has been thoroughly mapped by complementary exptl. techniques, revealing interesting phase behavior that has not been previously described. Confocal fluorescence microscopy shows a regime of coexisting DPPC-rich ordered and DLPC-rich fluid lamellar phases, having an upper boundary at apparently const. cholesterol mole fraction χchol ∼ 0.16. Fluorescence resonance energy transfer expts. confirm the identification and extent of this two-phase regime and, furthermore, reveal a 1-phase regime between χchol ∼ 0.16 and 0.25, consisting of ordered and fluid nanoscopic domains. Dipyrene-PC excimer/monomer measurements confirm the new regime between χchol ∼ 0.16 and 0.25 and also show that rigidly ordered phases seem to disappear around χchol ∼ 0.25. This study should be considered as a step toward a more complete understanding of lateral heterogeneity within biomembranes. Cholesterol may play a role in domain sepn. on the nanometer scale.
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Veatch, S. L.; Keller, S. L. Separation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol Biophys. J. 2003, 85, 3074– 3083 DOI: 10.1016/S0006-3495(03)74726-2
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Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol
Veatch, Sarah L.; Keller, Sarah L.
Biophysical Journal (2003), 85 (5), 3074-3083CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
The authors use fluorescence microscopy to directly observe liq. phases in giant unilamellar vesicles. The authors find that a long list of ternary mixts. of high melting temp. (satd.) lipids, low melting temp. (usually unsatd.) lipids, and cholesterol produce liq. domains. For one model mixt. in particular, DPPC/DOPC/Chol, the authors have mapped phase boundaries for the full ternary system. For this mixt. the authors observe two coexisting liq. phases over a wide range of lipid compn. and temp., with one phase rich in the unsatd. lipid and the other rich in the satd. lipid and cholesterol. The authors find a simple relationship between chain melting temp. and miscibility transition temp. that holds for both phosphatidylcholine and sphingomyelin lipids. The authors exptl. cross miscibility boundaries both by changing temp. and by the depletion of cholesterol with β-cyclodextrin. Liq. domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, the authors have not obsd. macroscopic sepn. of liq. phases in only binary lipid mixts.
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Sun, T.-L.; Sun, Y.; Lee, C.-C.; Huang, H. W. Membrane Permeability of Hydrocarbon-Cross-Linked Peptides Biophys. J. 2013, 104, 1923– 1932 DOI: 10.1016/j.bpj.2013.03.039
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Membrane Permeability of Hydrocarbon-Cross-Linked Peptides
Sun, Tzu-Lin; Sun, Yen; Lee, Chang-Chun; Huang, Huey W.
Biophysical Journal (2013), 104 (9), 1923-1932CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
Schafmeister, Po, and Verdine (another study) introduced a method using a hydrocarbon linker (staple) to stabilize a peptide in a helical configuration. One intended goal of this scheme is to facilitate the delivery of peptide drugs into target cells. Here, we investigate whether stapled peptides are intrinsically membrane permeable, by performing a case study on a stapled 12-mer peptide named NYAD-1. We found that the native peptide CAI (an HIV-1 inhibitor) does not bind to lipid bilayers, however NYAD-1 indeed permeates through lipid bilayers even at low soln. concns. To understand the reason for the membrane permeability, we investigated the phys. properties of NYAD-1 as a function of bound peptide/lipid molar ratio P/L. We found that NYAD-1 spontaneously binds to a lipid bilayer. At low P/L, the peptide primarily binds on the polar-apolar interface with its helical axis parallel to the bilayer, which has the effect of stretching the membrane area and thinning the membrane. The membrane thinning reaches its max. at P/L ∼1/15-1/12 in DOPC bilayers. Addnl. bound peptides have little thinning effect and their helical axes are normal to the plane of bilayers. Thus, the stapled peptide has a membrane interaction behavior similar to helical antimicrobial peptides, such as magainin and melittin. We emphasize that not all peptides that bind to lipid bilayers in the α-helical form behave this way.
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Nishimura, K.; Matsuura, T.; Sunami, T.; Fujii, S.; Nishimura, K.; Suzuki, H.; Yomo, T. Identification of Giant Unilamellar Vesicles with Permeability to Small Charged Molecules RSC Adv. 2014, 4, 35224 DOI: 10.1039/C4RA05332J
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Identification of giant unilamellar vesicles with permeability to small charged molecules
Nishimura, Koji; Matsuura, Tomoaki; Sunami, Takeshi; Fujii, Satoshi; Nishimura, Kazuya; Suzuki, Hiroaki; Yomo, Tetsuya
RSC Advances (2014), 4 (66), 35224-35232CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
The permeability of giant unilamellar vesicle (GUV) membranes to various solutes was investigated at the single-vesicle level. Membrane permeability has primarily been studied by obtaining the av. of vesicle ensembles using small or large unilamellar vesicles (SUVs and LUVs, resp.) <1 μm in diam. The av. properties obsd. for biol. mols. or systems may not necessarily represent those of individual vesicles. In addn., although the GUV (>1 μm) is considered to be a primitive cell model, its membrane permeability has rarely been investigated. We investigated the permeation of various mols., including amino acids and mononucleotides, through more than 20 000 GUV membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using flow cytometry. We obsd. a general trend of lower membrane permeability for polar or charged mols. than for nonpolar mols., which is consistent with previous studies. However, we found that the lower permeation of charged mols. resulted from the presence of at least two distinct GUV populations; the larger population consisted of impermeable GUVs, and the smaller population consisted of those with a high permeability. The presence of phospholipid vesicles highly permeable to charged and small mols. (<3 nm) was found through the single vesicular measurement. The obsd. permeability of the GUVs may have played an essential role in the self-reprodn. and evolution of primitive cells.
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Staykova, M.; Lipowsky, R.; Dimova, R. Membrane Flow Patterns in Multicomponent Giant Vesicles Induced by Alternating Electric Fields Soft Matter 2008, 4, 2168 DOI: 10.1039/b811876k
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Fenz, S. F.; Sengupta, K. Giant Vesicles as Cell Models Integr. Biol. 2012, 4, 982 DOI: 10.1039/c2ib00188h
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Giant vesicles as cell models
Fenz, Susanne F.; Sengupta, Kheya
Integrative Biology (2012), 4 (9), 982-995CODEN: IBNIFL; ISSN:1757-9694. (Royal Society of Chemistry)
A review. Tremendous progress has been made in recent years in understanding the working of the living cell, including its micro-anatomy, signalling networks, and regulation of genes. However, an understanding of cellular phenomena using fundamental laws starting from first principles is still very far away. Part of the reason is that a cell is an active and exquisitely complex system where every part is linked to the other. Thus, it is difficult or even impossible to design expts. that selectively and exclusively probe a chosen aspect of the cell. Various kinds of idealised systems and cell models have been used to circumvent this problem. An important example is a giant unilamellar vesicle (GUV, also called giant liposome), which provides a cell-sized confined vol. to study biochem. reactions as well as self-assembly processes that occur on the membrane. The GUV membrane can be designed suitably to present selected, correctly-oriented cell-membrane proteins, whose mobility is confined to two dimensions. Here, we present recent advances in GUV design and the use of GUVs as cell models that enable quant. testing leading to insight into the working of real cells. We briefly recapitulate important classical concepts in membrane biophysics emphasizing the advantages and limitations of GUVs. We then present results obtained over the last decades using GUVs, choosing the formation of membrane domains and cell adhesion as examples for in-depth treatment. Insight into cell adhesion obtained using micro-interferometry is treated in detail. We conclude by summarising the open questions and possible future directions.
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Nappini, S.; Al Kayal, T.; Berti, D.; Nordèn, B.; Baglioni, P. Magnetically Triggered Release From Giant Unilamellar Vesicles: Visualization By Means Of Confocal Microscopy J. Phys. Chem. Lett. 2011, 2, 713– 718 DOI: 10.1021/jz2000936
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Magnetically Triggered Release From Giant Unilamellar Vesicles: Visualization By Means Of Confocal Microscopy
Nappini, Silvia; Al Kayal, Tamer; Berti, Debora; Norden, Bengt; Baglioni, Piero
Journal of Physical Chemistry Letters (2011), 2 (7), 713-718CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
Magnetically triggered release from magnetic giant unilamellar vesicles (GUVs) loaded with Alexa fluorescent dye was studied by means of confocal laser scanning microscopy (CLSM) under a low-frequency alternating magnetic field (LF-AMF). Core/shell cobalt ferrite nanoparticles coated with rhodamine B isothiocyanate (MP@SiO2(RITC)) were prepd. and adsorbed on the GUV membrane. The MP@SiO2(RITC) location and distribution on giant lipid vesicles were detd. by 3D-CLSM projections, and their effect on the release properties and GUV permeability under a LF-AMF was investigated by CLSM time-resolved expts. We show that the mechanism of release of the fluorescent dye during the LF-AMF exposure is induced by magnetic nanoparticle energy and mech. vibration, which promote the perturbation of the GUV membrane without its collapse.
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Dubavik, A.; Sezgin, E.; Lesnyak, V.; Gaponik, N.; Schwille, P.; Eychmüller, A. Penetration of Amphiphilic Quantum Dots through Model and Cellular Plasma Membranes ACS Nano 2012, 6, 2150– 2156 DOI: 10.1021/nn204930y
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Bernardino de la Serna, J.; Perez-Gil, J.; Simonsen, A. C.; Bagatolli, L. A. Cholesterol Rules J. Biol. Chem. 2004, 279, 40715– 40722 DOI: 10.1074/jbc.M404648200
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Bernardino de la Serna, J.; Hansen, S.; Berzina, Z.; Simonsen, A. C.; Hannibal-Bach, H. K.; Knudsen, J.; Ejsing, C. S.; Bagatolli, L. A. Compositional and Structural Characterization of Monolayers and Bilayers Composed of Native Pulmonary Surfactant from Wild Type Mice Biochim. Biophys. Acta, Biomembr. 2013, 1828, 2450– 2459 DOI: 10.1016/j.bbamem.2013.07.008
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Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice
Bernardino de la Serna, Jorge; Hansen, Soren; Berzina, Zane; Simonsen, Adam C.; Hannibal-Bach, Hans K.; Knudsen, Jens; Ejsing, Christer S.; Bagatolli, Luis A.
Biochimica et Biophysica Acta, Biomembranes (2013), 1828 (11), 2450-2459CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
This work comprises a structural and dynamic study of monolayers and bilayers composed of native pulmonary surfactant from mice. Spatially resolved information was obtained using fluorescence (confocal, wide field and two photon excitation) and at. force microscopy methods. Lipid mass spectrometry expts. were also performed in order to obtain relevant information on the lipid compn. of this material. Bilayers composed of mouse pulmonary surfactant showed coexistence of distinct domains at room temp., with morphologies and lateral packing resembling the coexistence of liq. ordered (lo)/liq. disordered (ld)-like phases reported previously in porcine lung surfactant. Interestingly, the molar ratio of satd. (mostly DPPC)/non-satd. phospholipid species and cholesterol measured in the innate material corresponds with that of a DOPC/DPPC/cholesterol mixt. showing lo/ld phase coexistence at a similar temp. This suggests that at quasi-equil. conditions, key lipid classes in this complex biol. material are still able to produce the same scaffold obsd. in relevant but simpler model lipid mixts. Also, robust structural and dynamic similarities between mono- and bi-layers composed of mouse pulmonary surfactant were obsd. when the monolayers reach a surface pressure of 30 mN/m. This value is in line with theor. predicted and recently measured surface pressures, where the monolayer-bilayer equivalence occurs in samples composed of single phospholipids. Finally, squeezed out material attached to pulmonary surfactant monolayers was obsd. at surface pressures near the beginning of the monolayer reversible exclusion plateau (∼ 40 mN/m). Under these conditions this material adopts elongated tubular shapes and displays ordered lateral packing as indicated by spatially resolved LAURDAN GP measurements.
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Heinrich, M. C.; Capraro, B. R.; Tian, A.; Isas, J. M.; Langen, R.; Baumgart, T. Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains J. Phys. Chem. Lett. 2010, 1, 3401– 3406 DOI: 10.1021/jz101403q
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Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains
Heinrich, Michael C.; Capraro, Benjamin R.; Tian, Aiwei; Isas, Jose M.; Langen, Ralf; Baumgart, Tobias
Journal of Physical Chemistry Letters (2010), 1 (23), 3401-3406CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
Biol. membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-assocd. proteins. The membrane-binding N-terminal amphipathic helix-contg. Bin/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quant. exptl. characterization. We have measured tube-pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipet-aspirated giant vesicles. We obsd. that fluorescently labeled DA-N-BAR showed significantly higher protein d. on tubes compared to the connected low-curvature vesicle membrane. Furthermore, we found the equil. tube-pulling force to be systematically dependent on the aq. soln. concn. of DA-N-BAR, thereby providing the first quant. assessment of spontaneous curvature generation. At sufficiently high protein concns., pulled tubes required no external force to maintain mech. equil., in agreement with the qual. spontaneous tubulation previously reported for amphiphysin.
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López-Montero, I.; Rodríguez-García, R.; Monroy, F. Artificial Spectrin Shells Reconstituted on Giant Vesicles J. Phys. Chem. Lett. 2012, 3, 1583– 1588 DOI: 10.1021/jz300377q
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Artificial Spectrin Shells Reconstituted on Giant Vesicles
Lopez-Montero, Ivan; Rodriguez-Garcia, Ruddi; Monroy, Francisco
Journal of Physical Chemistry Letters (2012), 3 (12), 1583-1588CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
In the exptl. approach to a synthetic minimal cell, the membrane compartment is a main component. Lipid vesicles represent the natural host for the artificial reconstruction of a cytomimetic membrane skeleton able to support mech. function. Using the membrane component of human erythroid cells, we have reconstructed a membrane shell composed of a spectrin skeleton and fed by ATP. The structural and mech. anal. reveals this spectrin skeleton as topol. network supporting mech. rigidity. Such an artificial shell would define a membrane compartment mech. stable under physiol. conditions.
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Caschera, F.; Bernardino de la Serna, J.; Löffler, P. M. G.; Rasmussen, T. E.; Hanczyc, M. M.; Bagatolli, L. A.; Monnard, P.-A. Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles Langmuir 2011, 27, 14078 DOI: 10.1021/la203057b
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Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles
Caschera, F.; de la Serna, J. Bernardino; Loffler, P. M. G.; Rasmussen, T. E.; Hanczyc, M. M.; Bagatolli, L. A.; Monnard, P.-A.
Langmuir (2011), 27 (23), 14078-14090CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
The self-assembly of cationic and anionic amphiphile mixts. into vesicles in aq. media was studied using two different systems: (i) decanoic acid and trimethyldecylammonium bromide and (ii) hexadecanedioic acid (a simple bola-amphiphile) and trimethyldecylammonium bromide. The resulting vesicles with varying amphiphile ratios were characterized using parameters such as the crit. vesicle concn., pH sensitivity, and encapsulation efficiency. We also produced and obsd. giant vesicles from these mixts. using the electroformation method and confocal microscopy. The mixed catanionic vesicles were shown to be more stable than those formed by pure fatty acids. Those contg. bola-amphiphile even showed the encapsulation of a small hydrophilic solute (8-hydroxypyrene-1,3,6-trisulfonic-acid), suggesting a denser packing of the amphiphiles. Compression and kinetics anal. of monolayers composed of these amphiphiles mixts. at the air/water interface suggests that the stabilization of the structures can be attributed to two main interactions between headgroups, predominantly the formation of hydrogen bonds between protonated and deprotonated acids and the addnl. electrostatic interactions between ammonium and acid headgroups.
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Walde, P.; Cosentino, K.; Engel, H.; Stano, P. Giant Vesicles: Preparations and Applications ChemBioChem 2010, 11, 848– 865 DOI: 10.1002/cbic.201000010
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Giant Vesicles: preparations and Applications
Walde, Peter; Cosentino, Katia; Engel, Helen; Stano, Pasquale
ChemBioChem (2010), 11 (7), 848-865CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
A review. There is considerable interest in prepg. cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biol. cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chem. compds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible prepn. of giant vesicles are available. The key concepts of currently known methods for prepg. giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
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Méléard, P.; Bagatolli, L. A.; Pott, T. Giant Unilamellar Vesicle Electroformation: From Lipid Mixtures to Native Membranes under Physiological Conditions Methods Enzymol. 2009, 465, 161– 176 DOI: 10.1016/S0076-6879(09)65009-6
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Dimova, R.; Aranda, S.; Bezlyepkina, N.; Nikolov, V.; Riske, K. A.; Lipowsky, R. A Practical Guide to Giant Vesicles. Probing the Membrane Nanoregime via Optical Microscopy J. Phys.: Condens. Matter 2006, 18, S1151– 76 DOI: 10.1088/0953-8984/18/28/S04
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A practical guide to giant vesicles. Probing the membrane nanoregime via optical microscopy
Dimova, Rumiana; Aranda, Said; Bezlyepkina, Natalya; Nikolov, Vesselin; Riske, Karin A.; Lipowsky, Reinhard
Journal of Physics: Condensed Matter (2006), 18 (28), S1151-S1176CODEN: JCOMEL; ISSN:0953-8984. (Institute of Physics Publishing)
A review. Research on giant vesicles is becoming increasingly popular. Giant vesicles provide model biomembrane systems for systematic measurements of mech. and rheol. properties of bilayers as a function of membrane compn. and temp., as well as hydrodynamic interactions. Membrane response to external factors (for example elec. fields, ions and amphiphilic mols.) can be directly visualized under the microscope. In this paper we review our current understanding of lipid bilayers as obtained from studies on giant unilamellar vesicles. Because research on giant vesicles increasingly attracts the interest of scientists from various backgrounds, we also try to provide a concise introduction for newcomers in the field. Finally, we summarize some recent developments on curvature effects induced by polymers, domain formation in membranes and shape transitions induced by elec. fields.
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Reeves, J. P.; Dowben, R. M. Formation and Properties of Thin-Walled Phospholipid Vesicles J. Cell. Physiol. 1969, 73, 49– 60 DOI: 10.1002/jcp.1040730108
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Formation and properties of thin-walled phospholipid vesicles
Reeves, John P.; Dowben, Robert M.
Journal of Cellular Physiology (1969), 73 (1), 49-60CODEN: JCLLAX; ISSN:0021-9541.
Large nos. of thin-walled vesicles, 0.5-10 μ in diam., can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distd. H2O or in an aq. nonelectrolyte soln. Electron micrographs of phospholipid analyses indicated that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium to make them a useful system for studying permeability. The osmolarity of the soln. in the interior of the vesicles can be estd. by immersion refractometry and the osmolarity of the internal aq. phase is linearly related to that of the external medium.
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Angelova, M. I.; Dimitrov, D. S. Liposome Electroformation Faraday Discuss. Chem. Soc. 1986, 81, 303 DOI: 10.1039/dc9868100303
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Liposome electroformation
Angelova, M.; Dimitrov, D.
Faraday Discussions of the Chemical Society (1986), 81 (1), 303-11CODEN: FDCSB7; ISSN:0301-7249.
Liposome formation and lipid swelling on Pt electrodes in distd. water and water solns. in d.c. elec. fields were investigated for different amts. of a neg. charged lipid (mixt. from 71% phosphatidylcholines, 21.5% phosphatidylethanolamines and 7.5% phosphatidylserines), and a neutral lipid (dimyristoylphosphatidylcholine, DMPC). Neg. charged lipids do not form liposomes without fields when the thickness of the dried lipid layer is ≤90 bilayers. The rate and extent of swelling of layers thicker than 90 bilayers is largest on the cathode, smaller without fields and smallest on the anode. The theory, based on the assumption that osmotic and electrostatic forces drive lipid swelling and liposome formation. is in semi-quant. agreement with the exptl. data; in particular, it gives the obsd. linear dependence of the rate of swelling on the inverse lipid layer thickness. To induce liposome formation for layers thinner than 90 bilayers it was necessary to apply a neg. potential which is proportional to the logarithm of the inverse layer thickness. The characteristic crit. potential is proportional to RTk/F; R being the gas const., Tk the abs. temp., and F the Faraday const. This indicates that redistribution of counterions may be the cause which increases the repulsive electrostatic intermembrane forces to overcome van der Waals attraction. For thicknesses <10 bilayers, formation of very thin-walled liposomes of narrow size distribution and mean diam. of ∼30 μm was obsd. These liposomes grow in size before detachment, and a formula for the kinetics of growth was derived, which is in very good agreement with the exptl. data. The effects of d.c. field on DMPC swelling are smaller and lead to formation of liposome-like structures of different appearance. Bilayer sepn. and bending are prerequisites for liposome formation from hydrating lipids. Therefore, a possible mol. mechanism is that membranes should be destabilized to bend and fuse to form liposomes. This requires the right proportion between structured regions, in the form of bilayers, and defects and (or) nonbilayer structures, and in many cases external constraints, in particular, elec. fields.
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Pautot, S.; Frisken, B. J.; Weitz, D. A. Production of Unilamellar Vesicles Using an Inverted Emulsion Langmuir 2003, 19, 2870– 2879 DOI: 10.1021/la026100v
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Production of Unilamellar Vesicles Using an Inverted Emulsion
Pautot, Sophie; Frisken, Barbara J.; Weitz, D. A.
Langmuir (2003), 19 (7), 2870-2879CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
We investigate a method for the controlled assembly of unilamellar vesicles consisting of bilayers assembled one leaflet at a time. We use water-in-oil emulsions stabilized by the material for the inner leaflet and produce vesicles by passing the water droplets through a second oil-water interface, where they become coated with the outer leaflet. We have used this technique to form vesicles from lipids, mixed lipid and surfactant systems, and diblock copolymers. The stability of lipid-stabilized emulsions limits the range of sizes that can be produced and the vesicle yield; nevertheless, there are several advantages with this emulsion-based technique: It is possible to make unilamellar vesicles with sizes ranging from 100 nm to 1 μm. Moreover, the process allows for efficient encapsulation and ensures that the contents of the vesicles remain isolated from the continuous aq. phase. To illustrate possible applications of this technique, we demonstrate the use of vesicles as microreactors where we polymerize actin through the addn. of magnesium and show that the polymn. kinetics are unaffected by the encapsulation.
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Yamada, A.; Le Berre, M.; Yoshikawa, K.; Baigl, D. Spontaneous Generation of Giant Liposomes from an Oil/water Interface ChemBioChem 2007, 8, 2215– 2218 DOI: 10.1002/cbic.200700473
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Stachowiak, J. C.; Richmond, D. L.; Li, T. H.; Liu, A. P.; Parekh, S. H.; Fletcher, D. A. Unilamellar Vesicle Formation and Encapsulation by Microfluidic Jetting Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4697– 4702 DOI: 10.1073/pnas.0710875105
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Unilamellar vesicle formation and encapsulation by microfluidic jetting
Stachowiak, Jeanne C.; Richmond, David L.; Li, Thomas H.; Liu, Allen P.; Parekh, Sapun H.; Fletcher, Daniel A.
Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (12), 4697-4702CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Compartmentalization of biomols. within lipid membranes is a fundamental requirement of living systems and an essential feature of many pharmaceutical therapies. However, applications of membrane-enclosed solns. of proteins, DNA, and other biol. active compds. have been limited by the difficulty of forming unilamellar vesicles with controlled contents in a repeatable manner. Here, we demonstrate a method for simultaneously creating and loading giant unilamellar vesicles (GUVs) using a pulsed microfluidic jet. Akin to blowing a bubble, the microfluidic jet deforms a planar lipid bilayer into a vesicle that is filled with soln. from the jet and separates from the planar bilayer. In contrast with existing techniques, our method rapidly generates multiple monodisperse, unilamellar vesicles contg. solns. of unrestricted compn. and mol. wt. Using the microfluidic jetting technique, we demonstrate repeatable encapsulation of 500-nm particles into GUVs and show that functional pore proteins can be incorporated into the vesicle membrane to mediate transport. The ability of microfluidic jetting to controllably encapsulate solns. inside of GUVs creates new opportunities for the study and use of compartmentalized biomol. systems in science, industry, and medicine.
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Arriaga, L. R.; Datta, S. S.; Kim, S.-H.; Amstad, E.; Kodger, T. E.; Monroy, F.; Weitz, D. A. Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation Small 2014, 10, 950– 956 DOI: 10.1002/smll.201301904
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Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation
Arriaga, Laura R.; Datta, Sujit S.; Kim, Shin-Hyun; Amstad, Esther; Kodger, Thomas E.; Monroy, Francisco; Weitz, David A.
Small (2014), 10 (5), 950-956CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
A microfluidic approach is reported for the high-throughput, continuous prodn. of giant unilamellar vesicles (GUVs) using water-in-oil-in-water double emulsion drops as templates. These emulsion drops have ultrathin shells; this minimizes the amt. 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 compns. and structures within the GUV membranes.
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Bi, H.; Yang, B.; Wang, L.; Cao, W.; Han, X. Electroformation of Giant Unilamellar Vesicles Using Interdigitated ITO Electrodes J. Mater. Chem. A 2013, 1, 7125 DOI: 10.1039/c3ta10323d
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Electroformation of giant unilamellar vesicles using interdigitated ITO electrodes
Bi, Hongmei; Yang, Bin; Wang, Lei; Cao, Wenwu; Han, Xiaojun
Journal of Materials Chemistry A: Materials for Energy and Sustainability (2013), 1 (24), 7125-7130CODEN: JMCAET; ISSN:2050-7496. (Royal Society of Chemistry)
A coplanar interdigitated electrode was used as a new electrode system to form giant unilamellar vesicles (GUVs). The formation of GUVs using interdigitated electrodes was studied in detail with respect to various parameters, including soln. height, electrode width, the amplitude and frequency of AC fields, and temp. Interdigitated electrodes with smaller widths generated bigger GUVs under the same conditions. According to both exptl. and 3D field simulation results, a soln. height >600 μm has no influence on the GUV formation. GUVs were obtained within a wide range of frequency from 1 Hz to 104 Hz and field amplitude from 1 V to 10 V. The diams. of the GUVs decreased with increasing frequency at a const. amplitude, and increased with increasing amplitude from 1 V to 5 V and then decreased from 5 V to 10 V at 10 Hz. A phase diagram based on varying the AC frequency and amplitude was obtained exptl., which can be used to predict the electroformation of GUVs.
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Aimon, S.; Manzi, J.; Schmidt, D.; Poveda Larrosa, J. A.; Bassereau, P.; Toombes, G. E. S. Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles PLoS One 2011, 6e25529 DOI: 10.1371/journal.pone.0025529
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Functional reconstitution of a voltage-gated potassium channel in Giant Unilamellar Vesicles
Aimon, Sophie; Manzi, John; Schmidt, Daniel; Larrosa, Jose Antonio Poveda; Bassereau, Patricia; Toombes, Gilman E. S.
PLoS One (2011), 6 (10), e25529CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid compn. and phys. state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in exptl. settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane compn., tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compns. and buffers contg. low (5 mM) or near-physiol. (100 mM) salt concns. Protein incorporation into GUVs was characterized with quant. confocal microscopy, and the protein d. of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs contg. functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.
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Fidorra, M.; Duelund, L.; Leidy, C.; Simonsen, A. C.; Bagatolli, L. A. Absence of Fluid-Ordered/Fluid-Disordered Phase Coexistence in Ceramide/POPC Mixtures Containing Cholesterol Biophys. J. 2006, 90, 4437– 4451 DOI: 10.1529/biophysj.105.077107
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Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol
Fidorra, M.; Duelund, L.; Leidy, C.; Simonsen, A. C.; Bagatolli, L. A.
Biophysical Journal (2006), 90 (12), 4437-4451CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
The effect of temp. on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), with and without addn. of cholesterol, were studied using differential scanning calorimetry, Fourier transformed IR spectroscopy, at. force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temp. regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was obsd. for different POPC/ceramide mixts. (up to ∼25 mol % ceramide). This obsd. phase coexistence scenario is in contrast to that reported previously for this mixt., where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addn. of cholesterol to the POPC/ceramide mixts. shows a gradual transition from a gel/fluid to gel/liq.-ordered phase coexistence scenario as indicated by the different exptl. techniques used in our expts. This last result suggests the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixts. studied in contrast to that obsd. at similar molar concns. with other ceramide-base-contg. lipid mixts. (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Addnl., we observe a crit. cholesterol concn. in the ternary mixts. that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.
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Montes, L.-R.; Alonso, A.; Goñi, F. M.; Bagatolli, L. A. Giant Unilamellar Vesicles Electroformed from Native Membranes and Organic Lipid Mixtures under Physiological Conditions Biophys. J. 2007, 93, 3548– 3554 DOI: 10.1529/biophysj.107.116228
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Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions
Montes, L.-Ruth; Alonso, Alicia; Goni, Felix M.; Bagatolli, Luis A.
Biophysical Journal (2007), 93 (10), 3548-3554CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biol. membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biol. membranes:. (1), The conventional prepn. of GUVs currently requires very low salt concn., thus precluding experimentation under physiol. conditions, and. (2), The model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepd. using a new protocol based on the electroformation method either from native membranes or org. lipid mixts. at physiol. ionic strength. Addnl., for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low- or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mech. properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amt. of salt present in the soln.
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Lecuyer, S.; Ristenpart, W. D.; Vincent, O.; Stone, H. A. Electrohydrodynamic Size Stratification and Flow Separation of Giant Vesicles Appl. Phys. Lett. 2008, 92104105 DOI: 10.1063/1.2894182
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Electrohydrodynamic size stratification and flow separation of giant vesicles
Lecuyer, S.; Ristenpart, W. D.; Vincent, O.; Stone, H. A.
Applied Physics Letters (2008), 92 (10), 104105/1-104105/3CODEN: APPLAB; ISSN:0003-6951. (American Institute of Physics)
We demonstrate an electrohydrodynamic (EHD) technique for sepg. giant unilamellar vesicles by size in polydisperse suspensions. An oscillatory elec. field (∼30 Hz) generates EHD flow around each vesicle close to an electrode. Nearby vesicles are entrained in the flow and the vesicles move toward one another. Upon aggregation, smaller vesicles are pulled underneath the larger vesicles, which ultimately lifts them off of the electrode. A brief spike in the elec. field then serves to irreversibly adhere the bottom layer of smaller vesicles to the electrode, and the large vesicles are subsequently removed by flow. We demonstrate that a single application of this technique can remove more than 90% of the smallest vesicles (diam. <20 μm) from a suspension of electroformed giant lipid vesicles. (c) 2008 American Institute of Physics.
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Herold, C.; Chwastek, G.; Schwille, P.; Petrov, E. P. Efficient Electroformation of Supergiant Unilamellar Vesicles Containing Cationic Lipids on ITO-Coated Electrodes Langmuir 2012, 28, 5518– 5521 DOI: 10.1021/la3005807
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Efficient Electroformation of Supergiant Unilamellar Vesicles Containing Cationic Lipids on ITO-Coated Electrodes
Herold, Christoph; Chwastek, Grzegorz; Schwille, Petra; Petrov, Eugene P.
Langmuir (2012), 28 (13), 5518-5521CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromols. and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a std. approach to GUV prodn. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topog. on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diam. > 100 μm) unilamellar vesicles contg. cationic lipids.
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Sezgin, E.; Sadowski, T.; Simons, K. Measuring Lipid Packing of Model and Cellular Membranes with Environment Sensitive Probes Langmuir 2014, 30, 8160– 8166 DOI: 10.1021/la501226v
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Measuring Lipid Packing of Model and Cellular Membranes with Environment Sensitive Probes
Sezgin, Erdinc; Sadowski, Tomasz; Simons, Kai
Langmuir (2014), 30 (27), 8160-8166CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
The extent of lipid packing is one of the key physicochem. features of biol. membranes and is involved in many membrane processes. Polarity sensitive fluorescent probes are commonly used tools to measure membrane lipid packing in both artificial and biol. membranes. The authors have systematically compared eight different probes to measure membrane lipid ordering. The authors studied how these probes behave in small unilamellar liposomes, phase-sepd. giant unilamellar vesicles, cell-derived giant plasma membrane vesicles, and live cells. The authors have tested the order sensitivity of a variety of measurable parameters, including generalized polarization, peak shift, or intensity shift. The authors also studied internalization and photostability of the probes to assess probe potential for time-lapse live cell imaging. These results provide a catalog of properties to facilitate the choice of probe according to need.
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Silvius, J. R. Role of Cholesterol in Lipid Raft Formation: Lessons from Lipid Model Systems Biochim. Biophys. Acta, Biomembr. 2003, 1610, 174– 183 DOI: 10.1016/S0005-2736(03)00016-6
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Role of cholesterol in lipid raft formation: lessons from lipid model systems
Silvius, John R.
Biochimica et Biophysica Acta, Biomembranes (2003), 1610 (2), 174-183CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
A review. Biochem. and cell-biol. expts. have identified cholesterol as an important component of lipid rafts' and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in detg. raft stability and organization. Studies using cholesterol-contg. bilayers as model systems have provided important insights into the roles that cholesterol plays in detg. lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, assocg. particularly strongly with satd., high-melting phospho- and sphingolipids and particularly weakly with highly unsatd. lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liq.-ordered) domains in model membranes that mimic the lipid compns. of natural membranes incorporating raft domains.
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Veatch, S. L.; Keller, S. L. Organization in Lipid Membranes Containing Cholesterol Phys. Rev. Lett. 2002, 89268101 DOI: 10.1103/PhysRevLett.89.268101
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Organization in Lipid Membranes Containing Cholesterol
Veatch, Sarah L.; Keller, Sarah L.
Physical Review Letters (2002), 89 (26), 268101/1-268101/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
A fundamental attribute of raft formation in cell membranes is lateral sepn. of lipids into coexisting liq. phases. Using fluorescence microscopy, we observe spontaneous lateral sepn. in free-floating giant unilamellar vesicles. We record coexisting liq. domains over a range of compn. and temp. significantly wider than previously reported. Furthermore, we establish correlations between miscibility in bilayers and in monolayers. For example, the same lipid mixts. that produce liq. domains in bilayer membranes produce two upper miscibility crit. points in the phase diagrams of monolayers.
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Söderlund, T.; Alakoskela, J.-M. I.; Pakkanen, A. L.; Kinnunen, P. K. J. Comparison of the Effects of Surface Tension and Osmotic Pressure on the Interfacial Hydration of a Fluid Phospholipid Bilayer Biophys. J. 2003, 85, 2333– 2341 DOI: 10.1016/S0006-3495(03)74657-8
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Comparison of the effects of surface tension and osmotic pressure on the interfacial hydration of a fluid phospholipid bilayer
Soderlund Tim; Alakoskela Juha-Matti I; Pakkanen Antti L; Kinnunen Paavo K J
Biophysical journal (2003), 85 (4), 2333-41 ISSN:0006-3495.
The effects of three so-called kosmotropic solutes, namely, betaine, sucrose, and choline chloride on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine large unilamellar vesicles, were studied by measuring the generalized polarization (GP) for the fluorescence emission of the membrane partitioning probe Laurdan. The latter has been shown to be sensitive to the depth of water penetration into phospholipid bilayers. At equal osmotic pressures the three solutes produced different increments in GP, with a qualitative positive correlation. However, the increments in GP correlated also quantitatively with the increase of air-water surface tension caused by the three kosmotropes. Our findings suggest surface tension to determine the impact of these solutes on the lateral packing of the lipid bilayer. Based on the changes in area/lipid at different surface tensions, the equilibrium lateral pressure for a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer at 25 degrees C was estimated to be approximately 34 mN/m.
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Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P. Adsorption of Divalent Cations to a Variety of Phosphatidylcholine Bilayers Biochemistry 1981, 20, 1771– 1777 DOI: 10.1021/bi00510a010
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Adsorption of divalent cations to a variety of phosphatidylcholine bilayers
Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P.
Biochemistry (1981), 20 (7), 1771-7CODEN: BICHAW; ISSN:0006-2960.
The degree of binding of divalent cations to several kinds of phosphatidylcholine (PC) bilayers was detd. by measuring the electrostatic interbilayer repuslive force that results when multilamellar lattices are exposed to alk. earth chloride solns. Divalent cations bind to dipalmitoylphosphatidylcholine in the sequence Ca2+ ≃ Cd2+ ≃ Mn2+ > Co2+ ≃ Mg2+ > Ba2+. Among the different synthetic lipids, preference for Ca2+ is in the sequence dioleoylphosphatidylcholine < dilauroylphosphatidylcholine < dimyristoylphosphatidylcholine ≃ dipalmitoylphosphatidylcholine ≃ distearoylphosphatidylcholine. The d. of bound charge is proportional to the d. of polar groups on the bilayer surface. PCs with mixed hydrocarbon chains, e.g. egg PC or 1:1 mixts. of synthetic PCs, form 2 distinct lamellar phase in CaCl2 solns. In all cases the electrostatic force between bilayers decays exponentially with their sepn. but more slowly than expected from ionic double-layer theory. The elec. fields from opposing surfaces evidently perturb the zwitterionic charge-binding polar groups and continuously modify their ion-binding affinities as the bilayers approach.
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Suwalsky, M.; Castro, R.; Villena, F.; Sotomayor, C. P. Cr(III) Exerts Stronger Structural Effects than Cr(VI) on the Human Erythrocyte Membrane and Molecular Models J. Inorg. Biochem. 2008, 102, 842– 849 DOI: 10.1016/j.jinorgbio.2007.11.020
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Binder, H.; Zschörnig, O. The Effect of Metal Cations on the Phase Behavior and Hydration Characteristics of Phospholipid Membranes Chem. Phys. Lipids 2002, 115, 39– 61 DOI: 10.1016/S0009-3084(02)00005-1
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The effect of metal cations on the phase behavior and hydration characteristics of phospholipid membranes
Binder, Hans; Zschornig, Olaf
Chemistry and Physics of Lipids (2002), 115 (1-2), 39-61CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Science Ltd.)
To characterize the specificity of ion binding to phospholipids in terms of headgroup structure, hydration and lyotropic phase behavior we studied 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of relative humidity (RH) at 25° in the presence and absence of Li+, Na+, K+, Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Zn2+ and Cu2+ ions by means of IR (IR) spectroscopy. All divalent cations and Li+ shift the gel-to-liq. cryst. phase transition towards bigger RH values indicating stabilization of the gel state. The obsd. shift correlates in a linearly fashion with the electrostatic solvation free energy for most of the ions in water that in turn, is inversely related to the ionic radius. This interesting result was interpreted in terms of the excess chem. potential of mixing of hydrated ions and lipids. Calcium, zinc and partially lithium, cause a pos. deviation from the linear relationship. IR spectral anal. shows that the carbonyl groups become more accessible to the water in the presence of Mg2+, Ca2+, Sr2+ and Ba2+ probably because of their involvement into the hydration shell of the ions. In contrast, Be2+, Zn2+ and Cu2+ dehydrate the carbonyl groups at small and medium RH. The ability of the lipid to take up water is distinctly reduced in the presence of Zn2+ and, partially, of Cu2+ meaning that the headgroups have become less hydrophilic. The binding mode of Be2+ to lipid headgroups involves hydrolyzed water. Polarized IR spectra show that complex formation of the phosphate groups with divalent ions gives rise to conformational changes and immobilization of the headgroups. The results are discussed in terms of the lyotropic Hofmeister series and of fusogenic activity of the ionic species.
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Ohki, S.; Düzgünes, N. Divalent Cation-Induced Interaction of Phospholipid Vesicle and Monolayer Membranes Biochim. Biophys. Acta, Biomembr. 1979, 552, 438– 449 DOI: 10.1016/0005-2736(79)90188-3
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Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes
Ohki, Shinpei; Duzgunes, Nejat
Biochimica et Biophysica Acta, Biomembranes (1979), 552 (3), 438-49CODEN: BBBMBS; ISSN:0005-2736.
The effects of phospholipid vesicles and divalent cations in the subphase soln. on the surface tension of phospholipid monolayer membranes were studied to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. Various concns. of unilamellar phospholipid vesicles and divalent cations were introduced into the subphase soln. of the monolayers, and the changes of surface tension of monolayers were measured with respect to divalent ion concns. and time. With a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1:1), there were crit. concns. of divalent cations to produce a large redn. in surface tension of the monolayer. The concns. for Mg2+, Sr2+, Ca2+, Ba2+, and Mn2+ are given. For a phosphatidylcholine monolayer and phosphatidylcholine vesicles, no change in monolayer surface tension occurred at ≤25 mM of divalent cation. With a phosphatidylserine monolayer and phosphatidylcholine vesicles, the order of divalent cations to effect the large redn. of surface tension was Mn2+ > Ca2+ > Mg2+. The threshold concns. depended on vesicle concns. as well as the area/mol. of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1:1) vesicles, the surface tension decreased to a value close to the equil. pressure of the monolayers within 0.5 h at concns. of Mn2+ and Ca2+ above the crit. concns. This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes.
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Stohs, S. J.; Bagchi, D. Oxidative Mechanisms in the Toxicity of Metal Ions Free Radicals Biol. Med. 1995, 18, 321– 336 DOI: 10.1016/0891-5849(94)00159-H
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Oxidative mechanisms in the toxicity of metal ions
Stohs S J; Bagchi D
Free radical biology & medicine (1995), 18 (2), 321-36 ISSN:0891-5849.
The role of reactive oxygen species, with the subsequent oxidative deterioration of biological macromolecules in the toxicities associated with transition metal ions, is reviewed. Recent studies have shown that metals, including iron, copper, chromium, and vanadium undergo redox cycling, while cadmium, mercury, and nickel, as well as lead, deplete glutathione and protein-bound sulfhydryl groups, resulting in the production of reactive oxygen species as superoxide ion, hydrogen peroxide, and hydroxyl radical. As a consequence, enhanced lipid peroxidation. DNA damage, and altered calcium and sulfhydryl homeostasis occur. Fenton-like reactions may be commonly associated with most membranous fractions including mitochondria, microsomes, and peroxisomes. Phagocytic cells may be another important source of reactive oxygen species in response to metal ions. Furthermore, various studies have suggested that the ability to generate reactive oxygen species by redox cycling quinones and related compounds may require metal ions. Recent studies have suggested that metal ions may enhance the production of tumor necrosis factor alpha (TNF alpha) and activate protein kinase C, as well as induce the production of stress proteins. Thus, some mechanisms associated with the toxicities of metal ions are very similar to the effects produced by many organic xenobiotics. Specific differences in the toxicities of metal ions may be related to differences in solubilities, absorbability, transport, chemical reactivity, and the complexes that are formed within the body. This review summarizes current studies that have been conducted with transition metal ions as well as lead, regarding the production of reactive oxygen species and oxidative tissue damage.
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Ayuyan, A. G.; Cohen, F. S. Lipid Peroxides Promote Large Rafts: Effects of Excitation of Probes in Fluorescence Microscopy and Electrochemical Reactions during Vesicle Formation Biophys. J. 2006, 91, 2172– 2183 DOI: 10.1529/biophysj.106.087387
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Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation
Ayuyan, Artem G.; Cohen, Fredric S.
Biophysical Journal (2006), 91 (6), 2172-2183CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
Raft formation and enlargement was investigated in liposomes and supported bilayers prepd. from sphingomyelin (SM), cholesterol, and unsatd. phospholipids; NBD-DPPE and rhodamine-(DOPE) were employed as fluorescent probes. Rafts were created by lowering temp. Maintaining 20 mol % SM, fluorescence microscopy showed that, in the absence of photooxidn., large rafts did not form in giant unilamellar vesicles (GUVs) contg. 20 or more mol. % cholesterol. But if photooxidn. was allowed to proceed, large rafts were readily obsd. In population, cuvette expts., small rafts formed without photooxidn. at high cholesterol concns. Thus, photooxidn. was the cause of raft enlargement during microscopy expts. Because photooxidn. results in peroxidn. at lipid double bonds, photosensitization expts. were performed to explicitly produce peroxides of SM and an unsatd. phospholipid. GUVs of high cholesterol content contg. the breakdown products of SM-peroxide, but not phospholipid-peroxide, resulted in large rafts after lowering temp. In addn., GUV prodn. by electroswelling can result in peroxides that cause large raft formation. The use of titanium electrodes eliminates this problem. In conclusion, lipid peroxides and their breakdown products are the cause of large raft formation in GUVs contg. biol. levels of cholesterol. It is crit. that expts. investigating rafts in bilayer membranes avoid the prodn. of peroxides.
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Mills, J. K.; Needham, D. Lysolipid Incorporation in Dipalmitoylphosphatidylcholine Bilayer Membranes Enhances the Ion Permeability and Drug Release Rates at the Membrane Phase Transition Biochim. Biophys. Acta, Biomembr. 2005, 1716, 77– 96 DOI: 10.1016/j.bbamem.2005.08.007
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Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition
Mills, Jeffrey K.; Needham, David
Biochimica et Biophysica Acta, Biomembranes (2005), 1716 (2), 77-96CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
The enhanced permeability of lipid bilayer membranes at their gel-to-liq. phase transition has been explained using a "bilayer lipid heterogeneity" model, postulating leaky interfacial regions between still solid and melting liq. phases. The addn. of lysolipid to dipalmitoylphosphatidylcholine bilayers dramatically enhances the amt. of, and speed at which, encapsulated markers or drugs are released at this, already leaky, phase transition through these interfacial regions. To characterize and attempt to det. the mechanism behind lysolipid-generated permeability enhancement, dithionite permeability and doxorubicin release were measured for lysolipid and non-lysolipid, contg. membranes. Rapid release of contents from lysolipid-contg. membranes appears to occur through lysolipid-stabilized pores rather than a simple enhancement due to increased drug soly. in the bilayer. A dramatic enhancement in the permeability rate const. begins about two degrees below the calorimetric peak of the thermal transition, and extends several degrees past it. The max. permeability rate const. coincides exactly with this calorimetric peak. Although some lysolipid desorption from liq. state membranes cannot be dismissed, dialyzation above T m and mass spectrometry anal. indicate lysolipid must, and can, remain in the membrane for the permeability enhancement, presumably as lysolipid stabilized pores in the grain boundary regions of the partially melted solid phase.
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Fuller, N.; Rand, R. P. The Influence of Lysolipids on the Spontaneous Curvature and Bending Elasticity of Phospholipid Membranes Biophys. J. 2001, 81, 243– 254 DOI: 10.1016/S0006-3495(01)75695-0
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The influence of lysolipids on the spontaneous curvature and bending elasticity of phospholipid membranes
Fuller, N.; Rand, R. P.
Biophysical Journal (2001), 81 (1), 243-254CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
The effects of lysolipids on phospholipid layer curvature and bending elasticity were examd. using x-ray diffraction and the osmotic stress method. Lysolipids with two different head groups, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and differing hydrocarbon chains were mixed with the hexagonal-forming lipid, dioleoylphosphatidylethanolamine (DOPE). With up to 30 mol% lysolipid in DOPE, the mixt. maintains the inverted hexagonal (HII) phase in excess water, where increasing levels of lysolipid result in a systematic increase in the HII lattice dimension. Anal. of the structural changes imposed by lysolipids show that, opposite to DOPE itself, which has an spontaneous radius of curvature (RO) of -30 Å, PC lysolipids add high pos. curvature, with RD = +38 to +60 Å, depending on chain length. LysoPEs, in contrast, add very small curvatures. When both polar group and hydrocarbon chains of the added lysolipid mismatch those of DOPE, the structural effects are qual. different from otherwise. Such mismatched lysolipids "reshape" the effective combination mol. into a longer and more cylindrical configuration compared to those lysolipids with either matching polar group or hydrocarbon chain.
45
Sparling, M. L.; Zidovetzki, R.; Muller, L.; Chan, S. I. Analysis of Membrane Lipids by 500 MHz 1H NMR Anal. Biochem. 1989, 178, 67– 76 DOI: 10.1016/0003-2697(89)90358-8
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Analysis of membrane lipids by 500 MHz proton NMR
Sparling, Mary Lee; Zidovetzki, Raphael; Muller, Luciano; Chan, Sunney I.
Analytical Biochemistry (1989), 178 (1), 67-76CODEN: ANBCA2; ISSN:0003-2697.
A nondestructive method was developed for rapid anal. of lipid content of membrane exts. based on high field proton NMR spectroscopy. Lipid extn. is done by stepwise sonication of purified membranes into chloroform:methanol:water mixts., and 1H spectra are recorded at 35° on final prepns. consisting of ≥1 mg dried lipid solubilized in 2:1 CD3OD:CDCl3. Spectral peaks of lipid mixts. are assigned to lipid classes using a data base of std. lipid characteristic resonances derived from purified single membrane lipids and known mixts. of them. Peak intensities of characteristic peaks yield ratios of various lipids such as cholesterol:phospholipid and phosphatidylcholine:phosphatidylethanolamine, degree of unsatn., av. acyl chain length, total glycerol lipid content, and presence or absence of particular lipids, such as glycolipids or lysolipids. This procedure of membrane lipid anal. was verified using known mixts. of purified std. lipids.
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Nieva-Echevarría, B.; Goicoechea, E.; Manzanos, M. J.; Guillén, M. D. Usefulness of 1H NMR in Assessing the Extent of Lipid Digestion Food Chem. 2015, 179, 182– 190 DOI: 10.1016/j.foodchem.2015.01.104
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Usefulness of 1H NMR in assessing the extent of lipid digestion
Nieva-Echevarria, Barbara; Goicoechea, Encarnacion; Manzanos, Maria J.; Guillen, Maria D.
Food Chemistry (2015), 179 (), 182-190CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
Proton NMR (1H NMR) is proved to be, for the first time, a very useful technique in monitoring the extent of lipid hydrolysis in digestion processes. Sunflower oil and minced fish flesh, as model foods, were subjected to different in vitro digestion expts. and the lipolysis levels reached were evaluated using 1H NMR spectral data. Simple observation of the spectra gives very valuable information about the extent of the lipolysis and enables a rapid discrimination among samples having different hydrolysis degree. Equations were developed to quantify all the lipolytic products, and either referred to acyl groups plus fatty acids, or to glyceryl structures. The main hydrolysis products were 1,2-diglycerides, 2-monoglycerides, glycerol and fatty acids, although small proportions of 1,3-diglycerides and of 1-monoglycerides were also found. With this methodol., detn. of the extent of lipid digestion in its different definitions can be made. It has been shown that these definitions are not equiv., which is evidence for the need for a consensus in this regard.
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Kriat, M.; Vion-Dury, J.; Confort-Gouny, S.; Favre, R.; Viout, P.; Sciaky, M.; Sari, H.; Cozzone, P. J. Analysis of Plasma Lipids by NMR Spectroscopy: Application to Modifications Induced by Malignant Tumors J. Lipid Res. 1993, 34, 1009– 1019
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MacKenzie, A.; Vyssotski, M.; Nekrasov, E. Quantitative Analysis of Dairy Phospholipids by 31P NMR J. Am. Oil Chem. Soc. 2009, 86, 757– 763 DOI: 10.1007/s11746-009-1403-6
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Quantitative Analysis of Dairy Phospholipids by 31P NMR
MacKenzie, Andrew; Vyssotski, Mikhail; Nekrasov, Eduard
Journal of the American Oil Chemists' Society (2009), 86 (8), 757-763CODEN: JAOCA7; ISSN:0003-021X. (Springer)
31P NMR anal. of samples prepd. in a sodium cholate detergent system was assessed as a method for the quant. measurement of dairy phospholipids. Major phospholipid (PL) classes measured included: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM) and dihydrosphingomyelin (DHSM). The 31P NMR method was validated by comparison with a quant. two-dimensional thin-layer chromatog. (2D-TLC) technique. The 2D-TLC system was more sensitive, able to detect some minor compds. not obsd. by 31P NMR. However, 31P NMR is more suited to routine anal., with sample anal. taking 36 min. The method was also more versatile and sample anal. was possible on high phospholipid contg. materials without prior lipid extn. (e.g. buttermilk protein conc., beta serum liq.).
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McIntyre, J. C.; Sleight, R. G. Fluorescence Assay for Phospholipid Membrane Asymmetry Biochemistry 1991, 30, 11819– 11827 DOI: 10.1021/bi00115a012
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Fluorescence assay for phospholipid membrane asymmetry
McIntyre, Jonathan C.; Sleight, Richard G.
Biochemistry (1991), 30 (51), 11819-27CODEN: BICHAW; ISSN:0006-2960.
Highly fluorescent nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogs are widely used to examine lipid transport and membrane structure. A method was developed for chem. modifying NBD-labeled lipids in both artificial and biol. membranes by treating fluorescently, labeled membranes with dithionite (S2O42). When small unilamellar vesicles contg. NBD-labeled phospholipids were treated with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicle bilayer was reduced. Seven different NBD-lipid analogs, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent amino-2,1,3-benzooxadiazol-4-yl-lipid derivs. To assess the feasibility of using this reagent in biol. systems, N-(7-nitro-2,1,3-benzoxa)oxadiazol-4-yl)dioleoylphosphatidylethanolamine was inserted into the outer leaflet of the plasma membrane of CHO-K1 cells. Subsequent incubation of these cells with a nontoxic concn. of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. Apparently dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivs. Redn. of NBD-lipids with dithionite can be used to prep. asym. labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochem. investigations, including the measurement of phospholipid translocase activity.
50
Gruber, H. J.; Schindler, H. External Surface and Lamellarity of Lipid Vesicles: A Practice-Oriented Set of Assay Methods Biochim. Biophys. Acta, Biomembr. 1994, 1189, 212– 224 DOI: 10.1016/0005-2736(94)90068-X
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External surface and lamellarity of lipid vesicles: a practice-oriented set of assay methods
Gruber, Hermann J.; Schindler, Hansgeorg
Biochimica et Biophysica Acta, Biomembranes (1994), 1189 (2), 212-24CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
Three methods are presented for the detn. of external surface of large lipid vesicles of different lamellarity with 2% abs. accuracy. These methods (referred to as EPR, NBD and TNBS assays) use different marker lipids which provide signals (ESR, fluorescence of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) residues, and UV absorption increase of 2,4,6-trinitrobenzenesulfonic acid after reaction with amino lipids, resp.). The signals change upon addn. of different membrane-impermeant reagents due to reaction with marker lipids at the external vesicle surface. They were applied to the same vesicle samples, including unilamellar and multilamellar vesicles, both at two different lipid compns. External surface data matched for the three assays within 2%, but only after appropriate redesign or adaptation of so far published procedures. Main improvements related to slow influx of reagents (TNBS and NBD assays) or to redistribution of marker lipids (EPR assay), obscuring detn. of outer vesicle surface from fast reaction between reagent and readily accessible marker lipids. Furthermore, suitable strategies were found to obtain accurate 100% values (reaction of all marker lipids present), required to relate external vesicle surface to total surface. This included corrections for light scattering (NBD assay) and for turbidity (TNBS assay). These three methods appear to close a gap in the methodol. to det. external surface of vesicles for typical practical needs. In particular, the reliability range of the NBD assay could be extended to marker lipid densities as low as 1 marker lipid per 3000 lipids.
51
Sezgin, E.; Waithe, D.; Bernardino de la Serna, J.; Eggeling, C. Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing ChemPhysChem 2015, 16, 1387– 1394 DOI: 10.1002/cphc.201402794
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51
Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing
Sezgin, Erdinc; Waithe, Dominic; Bernardino de la Serna, Jorge; Eggeling, Christian
ChemPhysChem (2015), 16 (7), 1387-1394CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
Physicochem. properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the mol. order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the mol. order of the immediate membrane environment. Here, we report on an improved detn. of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved anal., which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image-with high accuracy-minute differences in the lipid packing of model and cellular membranes.
52
Parasassi, T.; De Stasio, G.; Ravagnan, G.; Rusch, R. M.; Gratton, E. Quantitation of Lipid Phases in Phospholipid Vesicles by the Generalized Polarization of Laurdan Fluorescence Biophys. J. 1991, 60, 179– 189 DOI: 10.1016/S0006-3495(91)82041-0
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Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence
Parasassi, Tiziana; De Stasio, Gelsomina; Ravagnan, Giampietro; Rusch, Ruth M.; Gratton, Enrico
Biophysical Journal (1991), 60 (1), 179-89CODEN: BIOJAU; ISSN:0006-3495.
The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the phys. state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan mol. Expts. performed by using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the mol. is not responsible for the obsd. effects. Also, pH titrn. in the range pH 4-10 shows that the spectral variations are independent of the charge of the polar head. A 2-state model of dipolar relaxation is used to qual. explain the behavior of Laurdan. Apparently, the presence of water mols. in the phospholipid matrix is responsible for the spectral properties of Laurdan in the gel phase. In the liq. cryst. phase there is a relaxation process that is attributed to water mols. that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained by using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addn. rule.
53
Bligh, E. G.; Dyer, W. J. A Rapid Method of Total Lipid Extraction and Purification Can. J. Biochem. Physiol. 1959, 37, 911 DOI: 10.1139/o59-099
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A rapid method of total lipide extraction and purification
Bligh, E. G.; Dyer, W. J.
Canadian Journal of Biochemistry and Physiology (1959), 37 (), 911-17CODEN: CJBPAZ; ISSN:0576-5544.
The wet tissue is homogenized with a mixt. of CHCl3 and MeOH to form a miscible system with the H2O in the tissue. Diln. with CHCl3 and H2O seps. the homogenate into 2 layers, the CHCl3 layer contg. all the lipides and the methanolic layer contg. all the non-lipides. A purified lipide ext. is obtained merely by isolating the CHCl3 layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.

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https://doi.org/10.1021/acsomega.6b00395

Published March 16, 2017

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Abstract
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Figure 1
Figure 1. Schematic of vesicle electroformation from a dry lipid bilayer deposited on a substrate. Vesicles are formed upon hydration and the application of an alternating electric field, Ẽ (not to scale).
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Figure 2
Figure 2. Vesicles swelling from electroformation electrodes: (a) hydrated lipid layer commencing the swelling process, (b) DiI-labeled single-vesicle swelling while attached to the electroformation electrodes, and (c) vesicles forming, swelling, and detaching along the electrode.
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Figure 3
Figure 3. (a) Size distribution of DOPC vesicles as a function of electrode material. The white bars represent vesicles formed using platinum wires as electrodes, whereas the gray bars represent vesicles formed using stainless steel electrodes. (b) Comparison of average vesicle diameters (±standard deviation) of platinum and stainless steel electrodes. Three independent experiments were conducted, and over 1500 vesicles were analyzed for each electrode material.
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Figure 4
Figure 4. Simultaneous electroformation of vesicles with dissimilar membrane properties. (a) Generalized polarization (GP) of the vesicles as a function of initial lipid-film formulation. (b) Representative GP false-colored images of four vesicles with different GP values.
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Figure 5
Figure 5. (a) Vesicle diameters as a function of lipid composition (b) Lipid packing of DOPC vesicles as a function of electrode material.
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Figure 6
Figure 6. ¹H NMR spectra of lipids extracted from electroformed GUVs (top trace) and large multilamellar vesicles (LMVs) (bottom trace). The residual solvent peaks of chloroform (7.26 ppm) and methanol (3.49 ppm) were removed for clarity. The inset shows a magnified view of the olefinic and head group protons.
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Figure 7
Figure 7. Fluorescence intensity measurements of DOPC/NBD-PE vesicles. The fluorescence of intact vesicles is represented from time = 0 to arrow 1. At arrow 1, the quencher is added to the suspension, and the intensity is recorded until a stable value is reached. The emission drop is a measure of the number of fluorophores in outer leaflets. At arrow 2, Triton X-100 is added to lyse the vesicles, and fluorescence is recorded until a new stable value is reached. The corresponding emission drop is a measure of the number of fluorophores in inner leaflets. After each addition, the plate was removed from the plate reader and shaken for several minutes (the time axis is correspondingly cut for clarity).
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Figure 8
Figure 8. Vesicle electroformation devices for high-yield production and formation visualization. (a) Scalable device with five electroformation chambers and needles held by brass screws. (b) Monitoring device with six chambers, brass screws, and stainless steel sheets for electrical connection between common electrodes.
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References
This article references 53 other publications.
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  Lingwood, D.; Simons, K. Lipid Rafts as a Membrane-Organizing Principle Science 2010, 327, 46– 50 DOI: 10.1126/science.1174621
  1
  Lipid Rafts As a Membrane-Organizing Principle
  Lingwood, Daniel; Simons, Kai
  Science (Washington, DC, United States) (2010), 327 (5961), 46-50CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  A review. Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liq.-liq. immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. We present the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.
2. 2
  Bagatolli, L. A.; Gratton, E. A Correlation between Lipid Domain Shape and Binary Phospholipid Mixture Composition in Free Standing Bilayers: A Two-Photon Fluorescence Microscopy Study Biophys. J. 2000, 79, 434– 447 DOI: 10.1016/S0006-3495(00)76305-3
  2
  A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: a two-photon fluorescence microscopy study
  Bagatolli, Luis A.; Gratton, Enrico
  Biophysical Journal (2000), 79 (1), 434-447CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixts. were studied at different temps., by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temps. corresponding to the fluid phase and the fluid-solid phase coexistence. In addn., we studied the solid-solid temp. regime for the DMPC/DSPC and DMPE/DMPC mixts. From the Laurdan intensity images the generalized polarization function (GP) was calcd. at different temps. to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temps. corresponding to the fluid region for all of the lipid mixts. At temps. corresponding to phase coexistence we obsd. concurrent fluid and solid domains in the GUVs independent of the lipid mixt. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temp. The migration of the solid domains decreased dramatically at temps. close to the solid-fluid→solid phase transition. For the DLPC-contg. mixts., the solid domains showed line, quasi-circular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixt. increases. In addn., for the satd. PC-contg. mixts., we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixt. components. Specifically, at the phase coexistence temp. region the difference in the GP values, assocd. with the fluid and solid domains, increases as the difference in the chain length of the binary mixt. component increases. This last finding suggests that in the solid-phase domains, the local concn. of the low melting temp. phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temp. regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concn. of the low melting temp. phospholipid component in the solid domains is negligible. This last observation extends to the satd. PE/PC mixts. at the phase coexistence temp. range. For the DMPC/DSPC we found that the non-fluorescent solid regions gradually disappear in the solid temp. regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixts., where the solid domains remain on the GUV surface at temps. corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixts.
3. 3
  Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Lipid Rafts Reconstituted in Model Membranes Biophys. J. 2001, 80, 1417– 1428 DOI: 10.1016/S0006-3495(01)76114-0
  3
  Lipid rafts reconstituted in model membranes
  Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E.
  Biophysical Journal (2001), 80 (3), 1417-1428CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from (1) equimolar mixts. of phospholipid-cholesterol-sphingomyelin or (2) natural lipids extd. from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diam. were obsd. by fluorescence microscopy at room temp. (24°) whereas non-raft mixts. (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extn., which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixts., in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examd. by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixt. or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.
4. 4
  Korlach, J.; Schwille, P.; Webb, W. W.; Feigenson, G. W. Characterization of Lipid Bilayer Phases by Confocal Microscopy and Fluorescence Correlation Spectroscopy Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8461– 8466 DOI: 10.1073/pnas.96.15.8461
  4
  Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy
  Korlach, Jonas; Schwille, Petra; Webb, Watt W.; Feigenson, Gerald W.
  Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (15), 8461-8466CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chem. well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1,1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C20) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC). Phase sepn. was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphol. of coexisting phase domains on the surface of these vesicles with full two-dimensional resoln. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coeff., DT, of the DiI-C20 was measured by FCS. DT values at ∼25° ranged from ∼3×10-8 cm2/s in the fluid phase, to ∼2×10-9 cm2/s in high-cholesterol-content phases, to ∼2×10-10 cm2/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of DT in a region of two-phase coexistence on a single vesicle.
5. 5
  Feigenson, G. W.; Buboltz, J. T. Ternary Phase Diagram of Dipalmitoyl-PC/Dilauroyl-PC/Cholesterol: Nanoscopic Domain Formation Driven by Cholesterol Biophys. J. 2001, 80, 2775– 2788 DOI: 10.1016/S0006-3495(01)76245-5
  5
  Ternary phase diagram of dipalmitoyl-PC/dilauroyl-PC/cholesterol: nanoscopic domain formation driven by cholesterol
  Feigenson, Gerald W.; Buboltz, Jeffrey T.
  Biophysical Journal (2001), 80 (6), 2775-2788CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  A ternary phase diagram is proposed for the hydrated lamellar lipid mixt. dipalmitoylphosphatidylcholine/dilauroylphosphatidylcholine/cholesterol (DPPC/DLPC/cholesterol) at room temp. The entire compn. space has been thoroughly mapped by complementary exptl. techniques, revealing interesting phase behavior that has not been previously described. Confocal fluorescence microscopy shows a regime of coexisting DPPC-rich ordered and DLPC-rich fluid lamellar phases, having an upper boundary at apparently const. cholesterol mole fraction χchol ∼ 0.16. Fluorescence resonance energy transfer expts. confirm the identification and extent of this two-phase regime and, furthermore, reveal a 1-phase regime between χchol ∼ 0.16 and 0.25, consisting of ordered and fluid nanoscopic domains. Dipyrene-PC excimer/monomer measurements confirm the new regime between χchol ∼ 0.16 and 0.25 and also show that rigidly ordered phases seem to disappear around χchol ∼ 0.25. This study should be considered as a step toward a more complete understanding of lateral heterogeneity within biomembranes. Cholesterol may play a role in domain sepn. on the nanometer scale.
6. 6
  Veatch, S. L.; Keller, S. L. Separation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol Biophys. J. 2003, 85, 3074– 3083 DOI: 10.1016/S0006-3495(03)74726-2
  6
  Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol
  Veatch, Sarah L.; Keller, Sarah L.
  Biophysical Journal (2003), 85 (5), 3074-3083CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  The authors use fluorescence microscopy to directly observe liq. phases in giant unilamellar vesicles. The authors find that a long list of ternary mixts. of high melting temp. (satd.) lipids, low melting temp. (usually unsatd.) lipids, and cholesterol produce liq. domains. For one model mixt. in particular, DPPC/DOPC/Chol, the authors have mapped phase boundaries for the full ternary system. For this mixt. the authors observe two coexisting liq. phases over a wide range of lipid compn. and temp., with one phase rich in the unsatd. lipid and the other rich in the satd. lipid and cholesterol. The authors find a simple relationship between chain melting temp. and miscibility transition temp. that holds for both phosphatidylcholine and sphingomyelin lipids. The authors exptl. cross miscibility boundaries both by changing temp. and by the depletion of cholesterol with β-cyclodextrin. Liq. domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, the authors have not obsd. macroscopic sepn. of liq. phases in only binary lipid mixts.
7. 7
  Sun, T.-L.; Sun, Y.; Lee, C.-C.; Huang, H. W. Membrane Permeability of Hydrocarbon-Cross-Linked Peptides Biophys. J. 2013, 104, 1923– 1932 DOI: 10.1016/j.bpj.2013.03.039
  7
  Membrane Permeability of Hydrocarbon-Cross-Linked Peptides
  Sun, Tzu-Lin; Sun, Yen; Lee, Chang-Chun; Huang, Huey W.
  Biophysical Journal (2013), 104 (9), 1923-1932CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
  Schafmeister, Po, and Verdine (another study) introduced a method using a hydrocarbon linker (staple) to stabilize a peptide in a helical configuration. One intended goal of this scheme is to facilitate the delivery of peptide drugs into target cells. Here, we investigate whether stapled peptides are intrinsically membrane permeable, by performing a case study on a stapled 12-mer peptide named NYAD-1. We found that the native peptide CAI (an HIV-1 inhibitor) does not bind to lipid bilayers, however NYAD-1 indeed permeates through lipid bilayers even at low soln. concns. To understand the reason for the membrane permeability, we investigated the phys. properties of NYAD-1 as a function of bound peptide/lipid molar ratio P/L. We found that NYAD-1 spontaneously binds to a lipid bilayer. At low P/L, the peptide primarily binds on the polar-apolar interface with its helical axis parallel to the bilayer, which has the effect of stretching the membrane area and thinning the membrane. The membrane thinning reaches its max. at P/L ∼1/15-1/12 in DOPC bilayers. Addnl. bound peptides have little thinning effect and their helical axes are normal to the plane of bilayers. Thus, the stapled peptide has a membrane interaction behavior similar to helical antimicrobial peptides, such as magainin and melittin. We emphasize that not all peptides that bind to lipid bilayers in the α-helical form behave this way.
8. 8
  Nishimura, K.; Matsuura, T.; Sunami, T.; Fujii, S.; Nishimura, K.; Suzuki, H.; Yomo, T. Identification of Giant Unilamellar Vesicles with Permeability to Small Charged Molecules RSC Adv. 2014, 4, 35224 DOI: 10.1039/C4RA05332J
  8
  Identification of giant unilamellar vesicles with permeability to small charged molecules
  Nishimura, Koji; Matsuura, Tomoaki; Sunami, Takeshi; Fujii, Satoshi; Nishimura, Kazuya; Suzuki, Hiroaki; Yomo, Tetsuya
  RSC Advances (2014), 4 (66), 35224-35232CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
  The permeability of giant unilamellar vesicle (GUV) membranes to various solutes was investigated at the single-vesicle level. Membrane permeability has primarily been studied by obtaining the av. of vesicle ensembles using small or large unilamellar vesicles (SUVs and LUVs, resp.) <1 μm in diam. The av. properties obsd. for biol. mols. or systems may not necessarily represent those of individual vesicles. In addn., although the GUV (>1 μm) is considered to be a primitive cell model, its membrane permeability has rarely been investigated. We investigated the permeation of various mols., including amino acids and mononucleotides, through more than 20 000 GUV membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using flow cytometry. We obsd. a general trend of lower membrane permeability for polar or charged mols. than for nonpolar mols., which is consistent with previous studies. However, we found that the lower permeation of charged mols. resulted from the presence of at least two distinct GUV populations; the larger population consisted of impermeable GUVs, and the smaller population consisted of those with a high permeability. The presence of phospholipid vesicles highly permeable to charged and small mols. (<3 nm) was found through the single vesicular measurement. The obsd. permeability of the GUVs may have played an essential role in the self-reprodn. and evolution of primitive cells.
9. 9
  Staykova, M.; Lipowsky, R.; Dimova, R. Membrane Flow Patterns in Multicomponent Giant Vesicles Induced by Alternating Electric Fields Soft Matter 2008, 4, 2168 DOI: 10.1039/b811876k
  There is no corresponding record for this reference.
10. 10
  Fenz, S. F.; Sengupta, K. Giant Vesicles as Cell Models Integr. Biol. 2012, 4, 982 DOI: 10.1039/c2ib00188h
  10
  Giant vesicles as cell models
  Fenz, Susanne F.; Sengupta, Kheya
  Integrative Biology (2012), 4 (9), 982-995CODEN: IBNIFL; ISSN:1757-9694. (Royal Society of Chemistry)
  A review. Tremendous progress has been made in recent years in understanding the working of the living cell, including its micro-anatomy, signalling networks, and regulation of genes. However, an understanding of cellular phenomena using fundamental laws starting from first principles is still very far away. Part of the reason is that a cell is an active and exquisitely complex system where every part is linked to the other. Thus, it is difficult or even impossible to design expts. that selectively and exclusively probe a chosen aspect of the cell. Various kinds of idealised systems and cell models have been used to circumvent this problem. An important example is a giant unilamellar vesicle (GUV, also called giant liposome), which provides a cell-sized confined vol. to study biochem. reactions as well as self-assembly processes that occur on the membrane. The GUV membrane can be designed suitably to present selected, correctly-oriented cell-membrane proteins, whose mobility is confined to two dimensions. Here, we present recent advances in GUV design and the use of GUVs as cell models that enable quant. testing leading to insight into the working of real cells. We briefly recapitulate important classical concepts in membrane biophysics emphasizing the advantages and limitations of GUVs. We then present results obtained over the last decades using GUVs, choosing the formation of membrane domains and cell adhesion as examples for in-depth treatment. Insight into cell adhesion obtained using micro-interferometry is treated in detail. We conclude by summarising the open questions and possible future directions.
11. 11
  Nappini, S.; Al Kayal, T.; Berti, D.; Nordèn, B.; Baglioni, P. Magnetically Triggered Release From Giant Unilamellar Vesicles: Visualization By Means Of Confocal Microscopy J. Phys. Chem. Lett. 2011, 2, 713– 718 DOI: 10.1021/jz2000936
  11
  Magnetically Triggered Release From Giant Unilamellar Vesicles: Visualization By Means Of Confocal Microscopy
  Nappini, Silvia; Al Kayal, Tamer; Berti, Debora; Norden, Bengt; Baglioni, Piero
  Journal of Physical Chemistry Letters (2011), 2 (7), 713-718CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
  Magnetically triggered release from magnetic giant unilamellar vesicles (GUVs) loaded with Alexa fluorescent dye was studied by means of confocal laser scanning microscopy (CLSM) under a low-frequency alternating magnetic field (LF-AMF). Core/shell cobalt ferrite nanoparticles coated with rhodamine B isothiocyanate (MP@SiO2(RITC)) were prepd. and adsorbed on the GUV membrane. The MP@SiO2(RITC) location and distribution on giant lipid vesicles were detd. by 3D-CLSM projections, and their effect on the release properties and GUV permeability under a LF-AMF was investigated by CLSM time-resolved expts. We show that the mechanism of release of the fluorescent dye during the LF-AMF exposure is induced by magnetic nanoparticle energy and mech. vibration, which promote the perturbation of the GUV membrane without its collapse.
12. 12
  Dubavik, A.; Sezgin, E.; Lesnyak, V.; Gaponik, N.; Schwille, P.; Eychmüller, A. Penetration of Amphiphilic Quantum Dots through Model and Cellular Plasma Membranes ACS Nano 2012, 6, 2150– 2156 DOI: 10.1021/nn204930y
  There is no corresponding record for this reference.
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  Bernardino de la Serna, J.; Perez-Gil, J.; Simonsen, A. C.; Bagatolli, L. A. Cholesterol Rules J. Biol. Chem. 2004, 279, 40715– 40722 DOI: 10.1074/jbc.M404648200
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14. 14
  Bernardino de la Serna, J.; Hansen, S.; Berzina, Z.; Simonsen, A. C.; Hannibal-Bach, H. K.; Knudsen, J.; Ejsing, C. S.; Bagatolli, L. A. Compositional and Structural Characterization of Monolayers and Bilayers Composed of Native Pulmonary Surfactant from Wild Type Mice Biochim. Biophys. Acta, Biomembr. 2013, 1828, 2450– 2459 DOI: 10.1016/j.bbamem.2013.07.008
  14
  Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice
  Bernardino de la Serna, Jorge; Hansen, Soren; Berzina, Zane; Simonsen, Adam C.; Hannibal-Bach, Hans K.; Knudsen, Jens; Ejsing, Christer S.; Bagatolli, Luis A.
  Biochimica et Biophysica Acta, Biomembranes (2013), 1828 (11), 2450-2459CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
  This work comprises a structural and dynamic study of monolayers and bilayers composed of native pulmonary surfactant from mice. Spatially resolved information was obtained using fluorescence (confocal, wide field and two photon excitation) and at. force microscopy methods. Lipid mass spectrometry expts. were also performed in order to obtain relevant information on the lipid compn. of this material. Bilayers composed of mouse pulmonary surfactant showed coexistence of distinct domains at room temp., with morphologies and lateral packing resembling the coexistence of liq. ordered (lo)/liq. disordered (ld)-like phases reported previously in porcine lung surfactant. Interestingly, the molar ratio of satd. (mostly DPPC)/non-satd. phospholipid species and cholesterol measured in the innate material corresponds with that of a DOPC/DPPC/cholesterol mixt. showing lo/ld phase coexistence at a similar temp. This suggests that at quasi-equil. conditions, key lipid classes in this complex biol. material are still able to produce the same scaffold obsd. in relevant but simpler model lipid mixts. Also, robust structural and dynamic similarities between mono- and bi-layers composed of mouse pulmonary surfactant were obsd. when the monolayers reach a surface pressure of 30 mN/m. This value is in line with theor. predicted and recently measured surface pressures, where the monolayer-bilayer equivalence occurs in samples composed of single phospholipids. Finally, squeezed out material attached to pulmonary surfactant monolayers was obsd. at surface pressures near the beginning of the monolayer reversible exclusion plateau (∼ 40 mN/m). Under these conditions this material adopts elongated tubular shapes and displays ordered lateral packing as indicated by spatially resolved LAURDAN GP measurements.
15. 15
  Heinrich, M. C.; Capraro, B. R.; Tian, A.; Isas, J. M.; Langen, R.; Baumgart, T. Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains J. Phys. Chem. Lett. 2010, 1, 3401– 3406 DOI: 10.1021/jz101403q
  15
  Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains
  Heinrich, Michael C.; Capraro, Benjamin R.; Tian, Aiwei; Isas, Jose M.; Langen, Ralf; Baumgart, Tobias
  Journal of Physical Chemistry Letters (2010), 1 (23), 3401-3406CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
  Biol. membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-assocd. proteins. The membrane-binding N-terminal amphipathic helix-contg. Bin/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quant. exptl. characterization. We have measured tube-pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipet-aspirated giant vesicles. We obsd. that fluorescently labeled DA-N-BAR showed significantly higher protein d. on tubes compared to the connected low-curvature vesicle membrane. Furthermore, we found the equil. tube-pulling force to be systematically dependent on the aq. soln. concn. of DA-N-BAR, thereby providing the first quant. assessment of spontaneous curvature generation. At sufficiently high protein concns., pulled tubes required no external force to maintain mech. equil., in agreement with the qual. spontaneous tubulation previously reported for amphiphysin.
16. 16
  López-Montero, I.; Rodríguez-García, R.; Monroy, F. Artificial Spectrin Shells Reconstituted on Giant Vesicles J. Phys. Chem. Lett. 2012, 3, 1583– 1588 DOI: 10.1021/jz300377q
  16
  Artificial Spectrin Shells Reconstituted on Giant Vesicles
  Lopez-Montero, Ivan; Rodriguez-Garcia, Ruddi; Monroy, Francisco
  Journal of Physical Chemistry Letters (2012), 3 (12), 1583-1588CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
  In the exptl. approach to a synthetic minimal cell, the membrane compartment is a main component. Lipid vesicles represent the natural host for the artificial reconstruction of a cytomimetic membrane skeleton able to support mech. function. Using the membrane component of human erythroid cells, we have reconstructed a membrane shell composed of a spectrin skeleton and fed by ATP. The structural and mech. anal. reveals this spectrin skeleton as topol. network supporting mech. rigidity. Such an artificial shell would define a membrane compartment mech. stable under physiol. conditions.
17. 17
  Caschera, F.; Bernardino de la Serna, J.; Löffler, P. M. G.; Rasmussen, T. E.; Hanczyc, M. M.; Bagatolli, L. A.; Monnard, P.-A. Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles Langmuir 2011, 27, 14078 DOI: 10.1021/la203057b
  17
  Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles
  Caschera, F.; de la Serna, J. Bernardino; Loffler, P. M. G.; Rasmussen, T. E.; Hanczyc, M. M.; Bagatolli, L. A.; Monnard, P.-A.
  Langmuir (2011), 27 (23), 14078-14090CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
  The self-assembly of cationic and anionic amphiphile mixts. into vesicles in aq. media was studied using two different systems: (i) decanoic acid and trimethyldecylammonium bromide and (ii) hexadecanedioic acid (a simple bola-amphiphile) and trimethyldecylammonium bromide. The resulting vesicles with varying amphiphile ratios were characterized using parameters such as the crit. vesicle concn., pH sensitivity, and encapsulation efficiency. We also produced and obsd. giant vesicles from these mixts. using the electroformation method and confocal microscopy. The mixed catanionic vesicles were shown to be more stable than those formed by pure fatty acids. Those contg. bola-amphiphile even showed the encapsulation of a small hydrophilic solute (8-hydroxypyrene-1,3,6-trisulfonic-acid), suggesting a denser packing of the amphiphiles. Compression and kinetics anal. of monolayers composed of these amphiphiles mixts. at the air/water interface suggests that the stabilization of the structures can be attributed to two main interactions between headgroups, predominantly the formation of hydrogen bonds between protonated and deprotonated acids and the addnl. electrostatic interactions between ammonium and acid headgroups.
18. 18
  Walde, P.; Cosentino, K.; Engel, H.; Stano, P. Giant Vesicles: Preparations and Applications ChemBioChem 2010, 11, 848– 865 DOI: 10.1002/cbic.201000010
  18
  Giant Vesicles: preparations and Applications
  Walde, Peter; Cosentino, Katia; Engel, Helen; Stano, Pasquale
  ChemBioChem (2010), 11 (7), 848-865CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
  A review. There is considerable interest in prepg. cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biol. cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chem. compds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible prepn. of giant vesicles are available. The key concepts of currently known methods for prepg. giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
19. 19
  Méléard, P.; Bagatolli, L. A.; Pott, T. Giant Unilamellar Vesicle Electroformation: From Lipid Mixtures to Native Membranes under Physiological Conditions Methods Enzymol. 2009, 465, 161– 176 DOI: 10.1016/S0076-6879(09)65009-6
  There is no corresponding record for this reference.
20. 20
  Dimova, R.; Aranda, S.; Bezlyepkina, N.; Nikolov, V.; Riske, K. A.; Lipowsky, R. A Practical Guide to Giant Vesicles. Probing the Membrane Nanoregime via Optical Microscopy J. Phys.: Condens. Matter 2006, 18, S1151– 76 DOI: 10.1088/0953-8984/18/28/S04
  20
  A practical guide to giant vesicles. Probing the membrane nanoregime via optical microscopy
  Dimova, Rumiana; Aranda, Said; Bezlyepkina, Natalya; Nikolov, Vesselin; Riske, Karin A.; Lipowsky, Reinhard
  Journal of Physics: Condensed Matter (2006), 18 (28), S1151-S1176CODEN: JCOMEL; ISSN:0953-8984. (Institute of Physics Publishing)
  A review. Research on giant vesicles is becoming increasingly popular. Giant vesicles provide model biomembrane systems for systematic measurements of mech. and rheol. properties of bilayers as a function of membrane compn. and temp., as well as hydrodynamic interactions. Membrane response to external factors (for example elec. fields, ions and amphiphilic mols.) can be directly visualized under the microscope. In this paper we review our current understanding of lipid bilayers as obtained from studies on giant unilamellar vesicles. Because research on giant vesicles increasingly attracts the interest of scientists from various backgrounds, we also try to provide a concise introduction for newcomers in the field. Finally, we summarize some recent developments on curvature effects induced by polymers, domain formation in membranes and shape transitions induced by elec. fields.
21. 21
  Reeves, J. P.; Dowben, R. M. Formation and Properties of Thin-Walled Phospholipid Vesicles J. Cell. Physiol. 1969, 73, 49– 60 DOI: 10.1002/jcp.1040730108
  21
  Formation and properties of thin-walled phospholipid vesicles
  Reeves, John P.; Dowben, Robert M.
  Journal of Cellular Physiology (1969), 73 (1), 49-60CODEN: JCLLAX; ISSN:0021-9541.
  Large nos. of thin-walled vesicles, 0.5-10 μ in diam., can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distd. H2O or in an aq. nonelectrolyte soln. Electron micrographs of phospholipid analyses indicated that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium to make them a useful system for studying permeability. The osmolarity of the soln. in the interior of the vesicles can be estd. by immersion refractometry and the osmolarity of the internal aq. phase is linearly related to that of the external medium.
22. 22
  Angelova, M. I.; Dimitrov, D. S. Liposome Electroformation Faraday Discuss. Chem. Soc. 1986, 81, 303 DOI: 10.1039/dc9868100303
  22
  Liposome electroformation
  Angelova, M.; Dimitrov, D.
  Faraday Discussions of the Chemical Society (1986), 81 (1), 303-11CODEN: FDCSB7; ISSN:0301-7249.
  Liposome formation and lipid swelling on Pt electrodes in distd. water and water solns. in d.c. elec. fields were investigated for different amts. of a neg. charged lipid (mixt. from 71% phosphatidylcholines, 21.5% phosphatidylethanolamines and 7.5% phosphatidylserines), and a neutral lipid (dimyristoylphosphatidylcholine, DMPC). Neg. charged lipids do not form liposomes without fields when the thickness of the dried lipid layer is ≤90 bilayers. The rate and extent of swelling of layers thicker than 90 bilayers is largest on the cathode, smaller without fields and smallest on the anode. The theory, based on the assumption that osmotic and electrostatic forces drive lipid swelling and liposome formation. is in semi-quant. agreement with the exptl. data; in particular, it gives the obsd. linear dependence of the rate of swelling on the inverse lipid layer thickness. To induce liposome formation for layers thinner than 90 bilayers it was necessary to apply a neg. potential which is proportional to the logarithm of the inverse layer thickness. The characteristic crit. potential is proportional to RTk/F; R being the gas const., Tk the abs. temp., and F the Faraday const. This indicates that redistribution of counterions may be the cause which increases the repulsive electrostatic intermembrane forces to overcome van der Waals attraction. For thicknesses <10 bilayers, formation of very thin-walled liposomes of narrow size distribution and mean diam. of ∼30 μm was obsd. These liposomes grow in size before detachment, and a formula for the kinetics of growth was derived, which is in very good agreement with the exptl. data. The effects of d.c. field on DMPC swelling are smaller and lead to formation of liposome-like structures of different appearance. Bilayer sepn. and bending are prerequisites for liposome formation from hydrating lipids. Therefore, a possible mol. mechanism is that membranes should be destabilized to bend and fuse to form liposomes. This requires the right proportion between structured regions, in the form of bilayers, and defects and (or) nonbilayer structures, and in many cases external constraints, in particular, elec. fields.
23. 23
  Pautot, S.; Frisken, B. J.; Weitz, D. A. Production of Unilamellar Vesicles Using an Inverted Emulsion Langmuir 2003, 19, 2870– 2879 DOI: 10.1021/la026100v
  23
  Production of Unilamellar Vesicles Using an Inverted Emulsion
  Pautot, Sophie; Frisken, Barbara J.; Weitz, D. A.
  Langmuir (2003), 19 (7), 2870-2879CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
  We investigate a method for the controlled assembly of unilamellar vesicles consisting of bilayers assembled one leaflet at a time. We use water-in-oil emulsions stabilized by the material for the inner leaflet and produce vesicles by passing the water droplets through a second oil-water interface, where they become coated with the outer leaflet. We have used this technique to form vesicles from lipids, mixed lipid and surfactant systems, and diblock copolymers. The stability of lipid-stabilized emulsions limits the range of sizes that can be produced and the vesicle yield; nevertheless, there are several advantages with this emulsion-based technique: It is possible to make unilamellar vesicles with sizes ranging from 100 nm to 1 μm. Moreover, the process allows for efficient encapsulation and ensures that the contents of the vesicles remain isolated from the continuous aq. phase. To illustrate possible applications of this technique, we demonstrate the use of vesicles as microreactors where we polymerize actin through the addn. of magnesium and show that the polymn. kinetics are unaffected by the encapsulation.
24. 24
  Yamada, A.; Le Berre, M.; Yoshikawa, K.; Baigl, D. Spontaneous Generation of Giant Liposomes from an Oil/water Interface ChemBioChem 2007, 8, 2215– 2218 DOI: 10.1002/cbic.200700473
  There is no corresponding record for this reference.
25. 25
  Stachowiak, J. C.; Richmond, D. L.; Li, T. H.; Liu, A. P.; Parekh, S. H.; Fletcher, D. A. Unilamellar Vesicle Formation and Encapsulation by Microfluidic Jetting Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4697– 4702 DOI: 10.1073/pnas.0710875105
  25
  Unilamellar vesicle formation and encapsulation by microfluidic jetting
  Stachowiak, Jeanne C.; Richmond, David L.; Li, Thomas H.; Liu, Allen P.; Parekh, Sapun H.; Fletcher, Daniel A.
  Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (12), 4697-4702CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Compartmentalization of biomols. within lipid membranes is a fundamental requirement of living systems and an essential feature of many pharmaceutical therapies. However, applications of membrane-enclosed solns. of proteins, DNA, and other biol. active compds. have been limited by the difficulty of forming unilamellar vesicles with controlled contents in a repeatable manner. Here, we demonstrate a method for simultaneously creating and loading giant unilamellar vesicles (GUVs) using a pulsed microfluidic jet. Akin to blowing a bubble, the microfluidic jet deforms a planar lipid bilayer into a vesicle that is filled with soln. from the jet and separates from the planar bilayer. In contrast with existing techniques, our method rapidly generates multiple monodisperse, unilamellar vesicles contg. solns. of unrestricted compn. and mol. wt. Using the microfluidic jetting technique, we demonstrate repeatable encapsulation of 500-nm particles into GUVs and show that functional pore proteins can be incorporated into the vesicle membrane to mediate transport. The ability of microfluidic jetting to controllably encapsulate solns. inside of GUVs creates new opportunities for the study and use of compartmentalized biomol. systems in science, industry, and medicine.
26. 26
  Arriaga, L. R.; Datta, S. S.; Kim, S.-H.; Amstad, E.; Kodger, T. E.; Monroy, F.; Weitz, D. A. Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation Small 2014, 10, 950– 956 DOI: 10.1002/smll.201301904
  26
  Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation
  Arriaga, Laura R.; Datta, Sujit S.; Kim, Shin-Hyun; Amstad, Esther; Kodger, Thomas E.; Monroy, Francisco; Weitz, David A.
  Small (2014), 10 (5), 950-956CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
  A microfluidic approach is reported for the high-throughput, continuous prodn. of giant unilamellar vesicles (GUVs) using water-in-oil-in-water double emulsion drops as templates. These emulsion drops have ultrathin shells; this minimizes the amt. 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 compns. and structures within the GUV membranes.
27. 27
  Bi, H.; Yang, B.; Wang, L.; Cao, W.; Han, X. Electroformation of Giant Unilamellar Vesicles Using Interdigitated ITO Electrodes J. Mater. Chem. A 2013, 1, 7125 DOI: 10.1039/c3ta10323d
  27
  Electroformation of giant unilamellar vesicles using interdigitated ITO electrodes
  Bi, Hongmei; Yang, Bin; Wang, Lei; Cao, Wenwu; Han, Xiaojun
  Journal of Materials Chemistry A: Materials for Energy and Sustainability (2013), 1 (24), 7125-7130CODEN: JMCAET; ISSN:2050-7496. (Royal Society of Chemistry)
  A coplanar interdigitated electrode was used as a new electrode system to form giant unilamellar vesicles (GUVs). The formation of GUVs using interdigitated electrodes was studied in detail with respect to various parameters, including soln. height, electrode width, the amplitude and frequency of AC fields, and temp. Interdigitated electrodes with smaller widths generated bigger GUVs under the same conditions. According to both exptl. and 3D field simulation results, a soln. height >600 μm has no influence on the GUV formation. GUVs were obtained within a wide range of frequency from 1 Hz to 104 Hz and field amplitude from 1 V to 10 V. The diams. of the GUVs decreased with increasing frequency at a const. amplitude, and increased with increasing amplitude from 1 V to 5 V and then decreased from 5 V to 10 V at 10 Hz. A phase diagram based on varying the AC frequency and amplitude was obtained exptl., which can be used to predict the electroformation of GUVs.
28. 28
  Aimon, S.; Manzi, J.; Schmidt, D.; Poveda Larrosa, J. A.; Bassereau, P.; Toombes, G. E. S. Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles PLoS One 2011, 6e25529 DOI: 10.1371/journal.pone.0025529
  28
  Functional reconstitution of a voltage-gated potassium channel in Giant Unilamellar Vesicles
  Aimon, Sophie; Manzi, John; Schmidt, Daniel; Larrosa, Jose Antonio Poveda; Bassereau, Patricia; Toombes, Gilman E. S.
  PLoS One (2011), 6 (10), e25529CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
  Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid compn. and phys. state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in exptl. settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane compn., tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compns. and buffers contg. low (5 mM) or near-physiol. (100 mM) salt concns. Protein incorporation into GUVs was characterized with quant. confocal microscopy, and the protein d. of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs contg. functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.
29. 29
  Fidorra, M.; Duelund, L.; Leidy, C.; Simonsen, A. C.; Bagatolli, L. A. Absence of Fluid-Ordered/Fluid-Disordered Phase Coexistence in Ceramide/POPC Mixtures Containing Cholesterol Biophys. J. 2006, 90, 4437– 4451 DOI: 10.1529/biophysj.105.077107
  29
  Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol
  Fidorra, M.; Duelund, L.; Leidy, C.; Simonsen, A. C.; Bagatolli, L. A.
  Biophysical Journal (2006), 90 (12), 4437-4451CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  The effect of temp. on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), with and without addn. of cholesterol, were studied using differential scanning calorimetry, Fourier transformed IR spectroscopy, at. force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temp. regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was obsd. for different POPC/ceramide mixts. (up to ∼25 mol % ceramide). This obsd. phase coexistence scenario is in contrast to that reported previously for this mixt., where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addn. of cholesterol to the POPC/ceramide mixts. shows a gradual transition from a gel/fluid to gel/liq.-ordered phase coexistence scenario as indicated by the different exptl. techniques used in our expts. This last result suggests the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixts. studied in contrast to that obsd. at similar molar concns. with other ceramide-base-contg. lipid mixts. (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Addnl., we observe a crit. cholesterol concn. in the ternary mixts. that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.
30. 30
  Montes, L.-R.; Alonso, A.; Goñi, F. M.; Bagatolli, L. A. Giant Unilamellar Vesicles Electroformed from Native Membranes and Organic Lipid Mixtures under Physiological Conditions Biophys. J. 2007, 93, 3548– 3554 DOI: 10.1529/biophysj.107.116228
  30
  Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions
  Montes, L.-Ruth; Alonso, Alicia; Goni, Felix M.; Bagatolli, Luis A.
  Biophysical Journal (2007), 93 (10), 3548-3554CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biol. membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biol. membranes:. (1), The conventional prepn. of GUVs currently requires very low salt concn., thus precluding experimentation under physiol. conditions, and. (2), The model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepd. using a new protocol based on the electroformation method either from native membranes or org. lipid mixts. at physiol. ionic strength. Addnl., for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low- or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mech. properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amt. of salt present in the soln.
31. 31
  Lecuyer, S.; Ristenpart, W. D.; Vincent, O.; Stone, H. A. Electrohydrodynamic Size Stratification and Flow Separation of Giant Vesicles Appl. Phys. Lett. 2008, 92104105 DOI: 10.1063/1.2894182
  31
  Electrohydrodynamic size stratification and flow separation of giant vesicles
  Lecuyer, S.; Ristenpart, W. D.; Vincent, O.; Stone, H. A.
  Applied Physics Letters (2008), 92 (10), 104105/1-104105/3CODEN: APPLAB; ISSN:0003-6951. (American Institute of Physics)
  We demonstrate an electrohydrodynamic (EHD) technique for sepg. giant unilamellar vesicles by size in polydisperse suspensions. An oscillatory elec. field (∼30 Hz) generates EHD flow around each vesicle close to an electrode. Nearby vesicles are entrained in the flow and the vesicles move toward one another. Upon aggregation, smaller vesicles are pulled underneath the larger vesicles, which ultimately lifts them off of the electrode. A brief spike in the elec. field then serves to irreversibly adhere the bottom layer of smaller vesicles to the electrode, and the large vesicles are subsequently removed by flow. We demonstrate that a single application of this technique can remove more than 90% of the smallest vesicles (diam. <20 μm) from a suspension of electroformed giant lipid vesicles. (c) 2008 American Institute of Physics.
32. 32
  Herold, C.; Chwastek, G.; Schwille, P.; Petrov, E. P. Efficient Electroformation of Supergiant Unilamellar Vesicles Containing Cationic Lipids on ITO-Coated Electrodes Langmuir 2012, 28, 5518– 5521 DOI: 10.1021/la3005807
  32
  Efficient Electroformation of Supergiant Unilamellar Vesicles Containing Cationic Lipids on ITO-Coated Electrodes
  Herold, Christoph; Chwastek, Grzegorz; Schwille, Petra; Petrov, Eugene P.
  Langmuir (2012), 28 (13), 5518-5521CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
  Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromols. and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a std. approach to GUV prodn. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topog. on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diam. > 100 μm) unilamellar vesicles contg. cationic lipids.
33. 33
  Sezgin, E.; Sadowski, T.; Simons, K. Measuring Lipid Packing of Model and Cellular Membranes with Environment Sensitive Probes Langmuir 2014, 30, 8160– 8166 DOI: 10.1021/la501226v
  33
  Measuring Lipid Packing of Model and Cellular Membranes with Environment Sensitive Probes
  Sezgin, Erdinc; Sadowski, Tomasz; Simons, Kai
  Langmuir (2014), 30 (27), 8160-8166CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
  The extent of lipid packing is one of the key physicochem. features of biol. membranes and is involved in many membrane processes. Polarity sensitive fluorescent probes are commonly used tools to measure membrane lipid packing in both artificial and biol. membranes. The authors have systematically compared eight different probes to measure membrane lipid ordering. The authors studied how these probes behave in small unilamellar liposomes, phase-sepd. giant unilamellar vesicles, cell-derived giant plasma membrane vesicles, and live cells. The authors have tested the order sensitivity of a variety of measurable parameters, including generalized polarization, peak shift, or intensity shift. The authors also studied internalization and photostability of the probes to assess probe potential for time-lapse live cell imaging. These results provide a catalog of properties to facilitate the choice of probe according to need.
34. 34
  Silvius, J. R. Role of Cholesterol in Lipid Raft Formation: Lessons from Lipid Model Systems Biochim. Biophys. Acta, Biomembr. 2003, 1610, 174– 183 DOI: 10.1016/S0005-2736(03)00016-6
  34
  Role of cholesterol in lipid raft formation: lessons from lipid model systems
  Silvius, John R.
  Biochimica et Biophysica Acta, Biomembranes (2003), 1610 (2), 174-183CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
  A review. Biochem. and cell-biol. expts. have identified cholesterol as an important component of lipid rafts' and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in detg. raft stability and organization. Studies using cholesterol-contg. bilayers as model systems have provided important insights into the roles that cholesterol plays in detg. lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, assocg. particularly strongly with satd., high-melting phospho- and sphingolipids and particularly weakly with highly unsatd. lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liq.-ordered) domains in model membranes that mimic the lipid compns. of natural membranes incorporating raft domains.
35. 35
  Veatch, S. L.; Keller, S. L. Organization in Lipid Membranes Containing Cholesterol Phys. Rev. Lett. 2002, 89268101 DOI: 10.1103/PhysRevLett.89.268101
  35
  Organization in Lipid Membranes Containing Cholesterol
  Veatch, Sarah L.; Keller, Sarah L.
  Physical Review Letters (2002), 89 (26), 268101/1-268101/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
  A fundamental attribute of raft formation in cell membranes is lateral sepn. of lipids into coexisting liq. phases. Using fluorescence microscopy, we observe spontaneous lateral sepn. in free-floating giant unilamellar vesicles. We record coexisting liq. domains over a range of compn. and temp. significantly wider than previously reported. Furthermore, we establish correlations between miscibility in bilayers and in monolayers. For example, the same lipid mixts. that produce liq. domains in bilayer membranes produce two upper miscibility crit. points in the phase diagrams of monolayers.
36. 36
  Söderlund, T.; Alakoskela, J.-M. I.; Pakkanen, A. L.; Kinnunen, P. K. J. Comparison of the Effects of Surface Tension and Osmotic Pressure on the Interfacial Hydration of a Fluid Phospholipid Bilayer Biophys. J. 2003, 85, 2333– 2341 DOI: 10.1016/S0006-3495(03)74657-8
  36
  Comparison of the effects of surface tension and osmotic pressure on the interfacial hydration of a fluid phospholipid bilayer
  Soderlund Tim; Alakoskela Juha-Matti I; Pakkanen Antti L; Kinnunen Paavo K J
  Biophysical journal (2003), 85 (4), 2333-41 ISSN:0006-3495.
  The effects of three so-called kosmotropic solutes, namely, betaine, sucrose, and choline chloride on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine large unilamellar vesicles, were studied by measuring the generalized polarization (GP) for the fluorescence emission of the membrane partitioning probe Laurdan. The latter has been shown to be sensitive to the depth of water penetration into phospholipid bilayers. At equal osmotic pressures the three solutes produced different increments in GP, with a qualitative positive correlation. However, the increments in GP correlated also quantitatively with the increase of air-water surface tension caused by the three kosmotropes. Our findings suggest surface tension to determine the impact of these solutes on the lateral packing of the lipid bilayer. Based on the changes in area/lipid at different surface tensions, the equilibrium lateral pressure for a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer at 25 degrees C was estimated to be approximately 34 mN/m.
37. 37
  Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P. Adsorption of Divalent Cations to a Variety of Phosphatidylcholine Bilayers Biochemistry 1981, 20, 1771– 1777 DOI: 10.1021/bi00510a010
  37
  Adsorption of divalent cations to a variety of phosphatidylcholine bilayers
  Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P.
  Biochemistry (1981), 20 (7), 1771-7CODEN: BICHAW; ISSN:0006-2960.
  The degree of binding of divalent cations to several kinds of phosphatidylcholine (PC) bilayers was detd. by measuring the electrostatic interbilayer repuslive force that results when multilamellar lattices are exposed to alk. earth chloride solns. Divalent cations bind to dipalmitoylphosphatidylcholine in the sequence Ca2+ ≃ Cd2+ ≃ Mn2+ > Co2+ ≃ Mg2+ > Ba2+. Among the different synthetic lipids, preference for Ca2+ is in the sequence dioleoylphosphatidylcholine < dilauroylphosphatidylcholine < dimyristoylphosphatidylcholine ≃ dipalmitoylphosphatidylcholine ≃ distearoylphosphatidylcholine. The d. of bound charge is proportional to the d. of polar groups on the bilayer surface. PCs with mixed hydrocarbon chains, e.g. egg PC or 1:1 mixts. of synthetic PCs, form 2 distinct lamellar phase in CaCl2 solns. In all cases the electrostatic force between bilayers decays exponentially with their sepn. but more slowly than expected from ionic double-layer theory. The elec. fields from opposing surfaces evidently perturb the zwitterionic charge-binding polar groups and continuously modify their ion-binding affinities as the bilayers approach.
38. 38
  Suwalsky, M.; Castro, R.; Villena, F.; Sotomayor, C. P. Cr(III) Exerts Stronger Structural Effects than Cr(VI) on the Human Erythrocyte Membrane and Molecular Models J. Inorg. Biochem. 2008, 102, 842– 849 DOI: 10.1016/j.jinorgbio.2007.11.020
  There is no corresponding record for this reference.
39. 39
  Binder, H.; Zschörnig, O. The Effect of Metal Cations on the Phase Behavior and Hydration Characteristics of Phospholipid Membranes Chem. Phys. Lipids 2002, 115, 39– 61 DOI: 10.1016/S0009-3084(02)00005-1
  39
  The effect of metal cations on the phase behavior and hydration characteristics of phospholipid membranes
  Binder, Hans; Zschornig, Olaf
  Chemistry and Physics of Lipids (2002), 115 (1-2), 39-61CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Science Ltd.)
  To characterize the specificity of ion binding to phospholipids in terms of headgroup structure, hydration and lyotropic phase behavior we studied 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of relative humidity (RH) at 25° in the presence and absence of Li+, Na+, K+, Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Zn2+ and Cu2+ ions by means of IR (IR) spectroscopy. All divalent cations and Li+ shift the gel-to-liq. cryst. phase transition towards bigger RH values indicating stabilization of the gel state. The obsd. shift correlates in a linearly fashion with the electrostatic solvation free energy for most of the ions in water that in turn, is inversely related to the ionic radius. This interesting result was interpreted in terms of the excess chem. potential of mixing of hydrated ions and lipids. Calcium, zinc and partially lithium, cause a pos. deviation from the linear relationship. IR spectral anal. shows that the carbonyl groups become more accessible to the water in the presence of Mg2+, Ca2+, Sr2+ and Ba2+ probably because of their involvement into the hydration shell of the ions. In contrast, Be2+, Zn2+ and Cu2+ dehydrate the carbonyl groups at small and medium RH. The ability of the lipid to take up water is distinctly reduced in the presence of Zn2+ and, partially, of Cu2+ meaning that the headgroups have become less hydrophilic. The binding mode of Be2+ to lipid headgroups involves hydrolyzed water. Polarized IR spectra show that complex formation of the phosphate groups with divalent ions gives rise to conformational changes and immobilization of the headgroups. The results are discussed in terms of the lyotropic Hofmeister series and of fusogenic activity of the ionic species.
40. 40
  Ohki, S.; Düzgünes, N. Divalent Cation-Induced Interaction of Phospholipid Vesicle and Monolayer Membranes Biochim. Biophys. Acta, Biomembr. 1979, 552, 438– 449 DOI: 10.1016/0005-2736(79)90188-3
  40
  Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes
  Ohki, Shinpei; Duzgunes, Nejat
  Biochimica et Biophysica Acta, Biomembranes (1979), 552 (3), 438-49CODEN: BBBMBS; ISSN:0005-2736.
  The effects of phospholipid vesicles and divalent cations in the subphase soln. on the surface tension of phospholipid monolayer membranes were studied to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. Various concns. of unilamellar phospholipid vesicles and divalent cations were introduced into the subphase soln. of the monolayers, and the changes of surface tension of monolayers were measured with respect to divalent ion concns. and time. With a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1:1), there were crit. concns. of divalent cations to produce a large redn. in surface tension of the monolayer. The concns. for Mg2+, Sr2+, Ca2+, Ba2+, and Mn2+ are given. For a phosphatidylcholine monolayer and phosphatidylcholine vesicles, no change in monolayer surface tension occurred at ≤25 mM of divalent cation. With a phosphatidylserine monolayer and phosphatidylcholine vesicles, the order of divalent cations to effect the large redn. of surface tension was Mn2+ > Ca2+ > Mg2+. The threshold concns. depended on vesicle concns. as well as the area/mol. of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1:1) vesicles, the surface tension decreased to a value close to the equil. pressure of the monolayers within 0.5 h at concns. of Mn2+ and Ca2+ above the crit. concns. This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes.
41. 41
  Stohs, S. J.; Bagchi, D. Oxidative Mechanisms in the Toxicity of Metal Ions Free Radicals Biol. Med. 1995, 18, 321– 336 DOI: 10.1016/0891-5849(94)00159-H
  41
  Oxidative mechanisms in the toxicity of metal ions
  Stohs S J; Bagchi D
  Free radical biology & medicine (1995), 18 (2), 321-36 ISSN:0891-5849.
  The role of reactive oxygen species, with the subsequent oxidative deterioration of biological macromolecules in the toxicities associated with transition metal ions, is reviewed. Recent studies have shown that metals, including iron, copper, chromium, and vanadium undergo redox cycling, while cadmium, mercury, and nickel, as well as lead, deplete glutathione and protein-bound sulfhydryl groups, resulting in the production of reactive oxygen species as superoxide ion, hydrogen peroxide, and hydroxyl radical. As a consequence, enhanced lipid peroxidation. DNA damage, and altered calcium and sulfhydryl homeostasis occur. Fenton-like reactions may be commonly associated with most membranous fractions including mitochondria, microsomes, and peroxisomes. Phagocytic cells may be another important source of reactive oxygen species in response to metal ions. Furthermore, various studies have suggested that the ability to generate reactive oxygen species by redox cycling quinones and related compounds may require metal ions. Recent studies have suggested that metal ions may enhance the production of tumor necrosis factor alpha (TNF alpha) and activate protein kinase C, as well as induce the production of stress proteins. Thus, some mechanisms associated with the toxicities of metal ions are very similar to the effects produced by many organic xenobiotics. Specific differences in the toxicities of metal ions may be related to differences in solubilities, absorbability, transport, chemical reactivity, and the complexes that are formed within the body. This review summarizes current studies that have been conducted with transition metal ions as well as lead, regarding the production of reactive oxygen species and oxidative tissue damage.
42. 42
  Ayuyan, A. G.; Cohen, F. S. Lipid Peroxides Promote Large Rafts: Effects of Excitation of Probes in Fluorescence Microscopy and Electrochemical Reactions during Vesicle Formation Biophys. J. 2006, 91, 2172– 2183 DOI: 10.1529/biophysj.106.087387
  42
  Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation
  Ayuyan, Artem G.; Cohen, Fredric S.
  Biophysical Journal (2006), 91 (6), 2172-2183CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  Raft formation and enlargement was investigated in liposomes and supported bilayers prepd. from sphingomyelin (SM), cholesterol, and unsatd. phospholipids; NBD-DPPE and rhodamine-(DOPE) were employed as fluorescent probes. Rafts were created by lowering temp. Maintaining 20 mol % SM, fluorescence microscopy showed that, in the absence of photooxidn., large rafts did not form in giant unilamellar vesicles (GUVs) contg. 20 or more mol. % cholesterol. But if photooxidn. was allowed to proceed, large rafts were readily obsd. In population, cuvette expts., small rafts formed without photooxidn. at high cholesterol concns. Thus, photooxidn. was the cause of raft enlargement during microscopy expts. Because photooxidn. results in peroxidn. at lipid double bonds, photosensitization expts. were performed to explicitly produce peroxides of SM and an unsatd. phospholipid. GUVs of high cholesterol content contg. the breakdown products of SM-peroxide, but not phospholipid-peroxide, resulted in large rafts after lowering temp. In addn., GUV prodn. by electroswelling can result in peroxides that cause large raft formation. The use of titanium electrodes eliminates this problem. In conclusion, lipid peroxides and their breakdown products are the cause of large raft formation in GUVs contg. biol. levels of cholesterol. It is crit. that expts. investigating rafts in bilayer membranes avoid the prodn. of peroxides.
43. 43
  Mills, J. K.; Needham, D. Lysolipid Incorporation in Dipalmitoylphosphatidylcholine Bilayer Membranes Enhances the Ion Permeability and Drug Release Rates at the Membrane Phase Transition Biochim. Biophys. Acta, Biomembr. 2005, 1716, 77– 96 DOI: 10.1016/j.bbamem.2005.08.007
  43
  Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition
  Mills, Jeffrey K.; Needham, David
  Biochimica et Biophysica Acta, Biomembranes (2005), 1716 (2), 77-96CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
  The enhanced permeability of lipid bilayer membranes at their gel-to-liq. phase transition has been explained using a "bilayer lipid heterogeneity" model, postulating leaky interfacial regions between still solid and melting liq. phases. The addn. of lysolipid to dipalmitoylphosphatidylcholine bilayers dramatically enhances the amt. of, and speed at which, encapsulated markers or drugs are released at this, already leaky, phase transition through these interfacial regions. To characterize and attempt to det. the mechanism behind lysolipid-generated permeability enhancement, dithionite permeability and doxorubicin release were measured for lysolipid and non-lysolipid, contg. membranes. Rapid release of contents from lysolipid-contg. membranes appears to occur through lysolipid-stabilized pores rather than a simple enhancement due to increased drug soly. in the bilayer. A dramatic enhancement in the permeability rate const. begins about two degrees below the calorimetric peak of the thermal transition, and extends several degrees past it. The max. permeability rate const. coincides exactly with this calorimetric peak. Although some lysolipid desorption from liq. state membranes cannot be dismissed, dialyzation above T m and mass spectrometry anal. indicate lysolipid must, and can, remain in the membrane for the permeability enhancement, presumably as lysolipid stabilized pores in the grain boundary regions of the partially melted solid phase.
44. 44
  Fuller, N.; Rand, R. P. The Influence of Lysolipids on the Spontaneous Curvature and Bending Elasticity of Phospholipid Membranes Biophys. J. 2001, 81, 243– 254 DOI: 10.1016/S0006-3495(01)75695-0
  44
  The influence of lysolipids on the spontaneous curvature and bending elasticity of phospholipid membranes
  Fuller, N.; Rand, R. P.
  Biophysical Journal (2001), 81 (1), 243-254CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  The effects of lysolipids on phospholipid layer curvature and bending elasticity were examd. using x-ray diffraction and the osmotic stress method. Lysolipids with two different head groups, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and differing hydrocarbon chains were mixed with the hexagonal-forming lipid, dioleoylphosphatidylethanolamine (DOPE). With up to 30 mol% lysolipid in DOPE, the mixt. maintains the inverted hexagonal (HII) phase in excess water, where increasing levels of lysolipid result in a systematic increase in the HII lattice dimension. Anal. of the structural changes imposed by lysolipids show that, opposite to DOPE itself, which has an spontaneous radius of curvature (RO) of -30 Å, PC lysolipids add high pos. curvature, with RD = +38 to +60 Å, depending on chain length. LysoPEs, in contrast, add very small curvatures. When both polar group and hydrocarbon chains of the added lysolipid mismatch those of DOPE, the structural effects are qual. different from otherwise. Such mismatched lysolipids "reshape" the effective combination mol. into a longer and more cylindrical configuration compared to those lysolipids with either matching polar group or hydrocarbon chain.
45. 45
  Sparling, M. L.; Zidovetzki, R.; Muller, L.; Chan, S. I. Analysis of Membrane Lipids by 500 MHz 1H NMR Anal. Biochem. 1989, 178, 67– 76 DOI: 10.1016/0003-2697(89)90358-8
  45
  Analysis of membrane lipids by 500 MHz proton NMR
  Sparling, Mary Lee; Zidovetzki, Raphael; Muller, Luciano; Chan, Sunney I.
  Analytical Biochemistry (1989), 178 (1), 67-76CODEN: ANBCA2; ISSN:0003-2697.
  A nondestructive method was developed for rapid anal. of lipid content of membrane exts. based on high field proton NMR spectroscopy. Lipid extn. is done by stepwise sonication of purified membranes into chloroform:methanol:water mixts., and 1H spectra are recorded at 35° on final prepns. consisting of ≥1 mg dried lipid solubilized in 2:1 CD3OD:CDCl3. Spectral peaks of lipid mixts. are assigned to lipid classes using a data base of std. lipid characteristic resonances derived from purified single membrane lipids and known mixts. of them. Peak intensities of characteristic peaks yield ratios of various lipids such as cholesterol:phospholipid and phosphatidylcholine:phosphatidylethanolamine, degree of unsatn., av. acyl chain length, total glycerol lipid content, and presence or absence of particular lipids, such as glycolipids or lysolipids. This procedure of membrane lipid anal. was verified using known mixts. of purified std. lipids.
46. 46
  Nieva-Echevarría, B.; Goicoechea, E.; Manzanos, M. J.; Guillén, M. D. Usefulness of 1H NMR in Assessing the Extent of Lipid Digestion Food Chem. 2015, 179, 182– 190 DOI: 10.1016/j.foodchem.2015.01.104
  46
  Usefulness of 1H NMR in assessing the extent of lipid digestion
  Nieva-Echevarria, Barbara; Goicoechea, Encarnacion; Manzanos, Maria J.; Guillen, Maria D.
  Food Chemistry (2015), 179 (), 182-190CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
  Proton NMR (1H NMR) is proved to be, for the first time, a very useful technique in monitoring the extent of lipid hydrolysis in digestion processes. Sunflower oil and minced fish flesh, as model foods, were subjected to different in vitro digestion expts. and the lipolysis levels reached were evaluated using 1H NMR spectral data. Simple observation of the spectra gives very valuable information about the extent of the lipolysis and enables a rapid discrimination among samples having different hydrolysis degree. Equations were developed to quantify all the lipolytic products, and either referred to acyl groups plus fatty acids, or to glyceryl structures. The main hydrolysis products were 1,2-diglycerides, 2-monoglycerides, glycerol and fatty acids, although small proportions of 1,3-diglycerides and of 1-monoglycerides were also found. With this methodol., detn. of the extent of lipid digestion in its different definitions can be made. It has been shown that these definitions are not equiv., which is evidence for the need for a consensus in this regard.
47. 47
  Kriat, M.; Vion-Dury, J.; Confort-Gouny, S.; Favre, R.; Viout, P.; Sciaky, M.; Sari, H.; Cozzone, P. J. Analysis of Plasma Lipids by NMR Spectroscopy: Application to Modifications Induced by Malignant Tumors J. Lipid Res. 1993, 34, 1009– 1019
  There is no corresponding record for this reference.
48. 48
  MacKenzie, A.; Vyssotski, M.; Nekrasov, E. Quantitative Analysis of Dairy Phospholipids by 31P NMR J. Am. Oil Chem. Soc. 2009, 86, 757– 763 DOI: 10.1007/s11746-009-1403-6
  48
  Quantitative Analysis of Dairy Phospholipids by 31P NMR
  MacKenzie, Andrew; Vyssotski, Mikhail; Nekrasov, Eduard
  Journal of the American Oil Chemists' Society (2009), 86 (8), 757-763CODEN: JAOCA7; ISSN:0003-021X. (Springer)
  31P NMR anal. of samples prepd. in a sodium cholate detergent system was assessed as a method for the quant. measurement of dairy phospholipids. Major phospholipid (PL) classes measured included: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM) and dihydrosphingomyelin (DHSM). The 31P NMR method was validated by comparison with a quant. two-dimensional thin-layer chromatog. (2D-TLC) technique. The 2D-TLC system was more sensitive, able to detect some minor compds. not obsd. by 31P NMR. However, 31P NMR is more suited to routine anal., with sample anal. taking 36 min. The method was also more versatile and sample anal. was possible on high phospholipid contg. materials without prior lipid extn. (e.g. buttermilk protein conc., beta serum liq.).
49. 49
  McIntyre, J. C.; Sleight, R. G. Fluorescence Assay for Phospholipid Membrane Asymmetry Biochemistry 1991, 30, 11819– 11827 DOI: 10.1021/bi00115a012
  49
  Fluorescence assay for phospholipid membrane asymmetry
  McIntyre, Jonathan C.; Sleight, Richard G.
  Biochemistry (1991), 30 (51), 11819-27CODEN: BICHAW; ISSN:0006-2960.
  Highly fluorescent nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogs are widely used to examine lipid transport and membrane structure. A method was developed for chem. modifying NBD-labeled lipids in both artificial and biol. membranes by treating fluorescently, labeled membranes with dithionite (S2O42). When small unilamellar vesicles contg. NBD-labeled phospholipids were treated with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicle bilayer was reduced. Seven different NBD-lipid analogs, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent amino-2,1,3-benzooxadiazol-4-yl-lipid derivs. To assess the feasibility of using this reagent in biol. systems, N-(7-nitro-2,1,3-benzoxa)oxadiazol-4-yl)dioleoylphosphatidylethanolamine was inserted into the outer leaflet of the plasma membrane of CHO-K1 cells. Subsequent incubation of these cells with a nontoxic concn. of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. Apparently dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivs. Redn. of NBD-lipids with dithionite can be used to prep. asym. labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochem. investigations, including the measurement of phospholipid translocase activity.
50. 50
  Gruber, H. J.; Schindler, H. External Surface and Lamellarity of Lipid Vesicles: A Practice-Oriented Set of Assay Methods Biochim. Biophys. Acta, Biomembr. 1994, 1189, 212– 224 DOI: 10.1016/0005-2736(94)90068-X
  50
  External surface and lamellarity of lipid vesicles: a practice-oriented set of assay methods
  Gruber, Hermann J.; Schindler, Hansgeorg
  Biochimica et Biophysica Acta, Biomembranes (1994), 1189 (2), 212-24CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
  Three methods are presented for the detn. of external surface of large lipid vesicles of different lamellarity with 2% abs. accuracy. These methods (referred to as EPR, NBD and TNBS assays) use different marker lipids which provide signals (ESR, fluorescence of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) residues, and UV absorption increase of 2,4,6-trinitrobenzenesulfonic acid after reaction with amino lipids, resp.). The signals change upon addn. of different membrane-impermeant reagents due to reaction with marker lipids at the external vesicle surface. They were applied to the same vesicle samples, including unilamellar and multilamellar vesicles, both at two different lipid compns. External surface data matched for the three assays within 2%, but only after appropriate redesign or adaptation of so far published procedures. Main improvements related to slow influx of reagents (TNBS and NBD assays) or to redistribution of marker lipids (EPR assay), obscuring detn. of outer vesicle surface from fast reaction between reagent and readily accessible marker lipids. Furthermore, suitable strategies were found to obtain accurate 100% values (reaction of all marker lipids present), required to relate external vesicle surface to total surface. This included corrections for light scattering (NBD assay) and for turbidity (TNBS assay). These three methods appear to close a gap in the methodol. to det. external surface of vesicles for typical practical needs. In particular, the reliability range of the NBD assay could be extended to marker lipid densities as low as 1 marker lipid per 3000 lipids.
51. 51
  Sezgin, E.; Waithe, D.; Bernardino de la Serna, J.; Eggeling, C. Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing ChemPhysChem 2015, 16, 1387– 1394 DOI: 10.1002/cphc.201402794
  51
  Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing
  Sezgin, Erdinc; Waithe, Dominic; Bernardino de la Serna, Jorge; Eggeling, Christian
  ChemPhysChem (2015), 16 (7), 1387-1394CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
  Physicochem. properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the mol. order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the mol. order of the immediate membrane environment. Here, we report on an improved detn. of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved anal., which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image-with high accuracy-minute differences in the lipid packing of model and cellular membranes.
52. 52
  Parasassi, T.; De Stasio, G.; Ravagnan, G.; Rusch, R. M.; Gratton, E. Quantitation of Lipid Phases in Phospholipid Vesicles by the Generalized Polarization of Laurdan Fluorescence Biophys. J. 1991, 60, 179– 189 DOI: 10.1016/S0006-3495(91)82041-0
  52
  Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence
  Parasassi, Tiziana; De Stasio, Gelsomina; Ravagnan, Giampietro; Rusch, Ruth M.; Gratton, Enrico
  Biophysical Journal (1991), 60 (1), 179-89CODEN: BIOJAU; ISSN:0006-3495.
  The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the phys. state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan mol. Expts. performed by using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the mol. is not responsible for the obsd. effects. Also, pH titrn. in the range pH 4-10 shows that the spectral variations are independent of the charge of the polar head. A 2-state model of dipolar relaxation is used to qual. explain the behavior of Laurdan. Apparently, the presence of water mols. in the phospholipid matrix is responsible for the spectral properties of Laurdan in the gel phase. In the liq. cryst. phase there is a relaxation process that is attributed to water mols. that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained by using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addn. rule.
53. 53
  Bligh, E. G.; Dyer, W. J. A Rapid Method of Total Lipid Extraction and Purification Can. J. Biochem. Physiol. 1959, 37, 911 DOI: 10.1139/o59-099
  53
  A rapid method of total lipide extraction and purification
  Bligh, E. G.; Dyer, W. J.
  Canadian Journal of Biochemistry and Physiology (1959), 37 (), 911-17CODEN: CJBPAZ; ISSN:0576-5544.
  The wet tissue is homogenized with a mixt. of CHCl3 and MeOH to form a miscible system with the H2O in the tissue. Diln. with CHCl3 and H2O seps. the homogenate into 2 layers, the CHCl3 layer contg. all the lipides and the methanolic layer contg. all the non-lipides. A purified lipide ext. is obtained merely by isolating the CHCl3 layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.6b00395.
- Cost breakdown of the electroformation chamber, limit of detection in NMR experiments, and unilamellarity experimental data; photograph of electroformation chamber (PDF)
- Video of the formation of vesicles on an SS304 electrode upon the application of an external electric field at t = 3 s (scale bar = 20 μm) (AVI)
- ao6b00395_si_002.pdf (199.45 kb)
- ao6b00395_si_003.avi (3.62 MB)
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Electroformation of Giant Unilamellar Vesicles on Stainless Steel ElectrodesClick to copy article linkArticle link copied!

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Abstract

Introduction

Figure 1

Results and Discussion

GUV Production and Imaging

Figure 2

Figure 3

Lipid Order and Size of Vesicles Composed of Binary Lipid Mixtures

Figure 4

Figure 5

Figure 6

Leaching of Metals from the Electrodes

Lipid Oxidation and Hydrolysis

Unilamellarity of Electroformed Vesicles

Figure 7

Conclusions

Materials and Methods

Electroformation Chamber

Figure 8

Electroformation Protocol

GUV Detection and Sizing

Lipid Order Measurements

ICP-OES Experiments

Lipid Extraction and NMR Experiments

Vesicle Unilamellarity

Statistical Analyses

Supporting Information

Terms & Conditions

Author Information

Acknowledgment

Abbreviations

References

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Abstract

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Figure 8

References

Supporting Information

Supporting Information

Terms & Conditions

Electroformation of Giant Unilamellar Vesicles on Stainless Steel Electrodes
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