Electric Destabilization of Supramolecular Lipid Vesicles Subjected to

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Electric Destabilization of Supramolecular Lipid Vesicles Subjected to Fast Electric Pulses Chloé Mauroy,†,‡ Isabelle Rico-Lattes,‡ Justin Teissié,*,†,§ and Marie-Pierre Rols*,† †

Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, UMR 5089 and Université Paul Sabatier, 205 route de Narbonne, BP 64182, 31077 Toulouse, France ‡ Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique, UMR 5623 CNRS and Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France § Emeritus Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, UMR 5089 and Université Paul Sabatier, 205 route de Narbonne, BP 64182, 31077 Toulouse, France S Supporting Information *

ABSTRACT: Biological membranes are weakly permeable to hydrophilic molecules and ions and electric pulses can induce their transient permeabilization, but this process is not well characterized. We directly assay the electropermeabilization process, on the minimum model of lipid vesicles, by using a highly sensitive fluorescence method based on manganese ion transport. The approach gives access, at the single-lipid self-assembly level, to the transmembrane potential needed to detect divalent ion permeabilization on supramolecular giant unilamellar lipid vesicles. The critical values are strongly dependent on the lipid composition and are observed to vary from 10 to 150 mV. These values appear to be much lower than those previously reported in the literature for cells and vesicles. The detection method appears to be a decisive parameter as it is controlled by the transport of the reporter dye. We also provide evidence that the electropermeabilization process is a transient transition of the lipid selforganization due to the loss of assembly cohesion induced by bioelectrochemical perturbations of the zwitterionic interface with the solution.

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INTRODUCTION Cell membranes act as a barrier that prevents free exchanges of ions and other hydrophilic species between cells and the external medium. Within a membrane the lipids are collectively organized. The cohesion of this supramolecular self-assembly comes from a balance between attractive and repulsive lowenergy forces such as van der Waals and electrostatic interactions.1 Transitions of their organization can be disturbed by the temperature, the pressure, or the application of an external electric field.2 Therefore, membrane permeability can be enhanced by electric field pulses.3,4 The application of short DC electric field pulses to the model and natural cell membranes causes their destabilization, resulting in a sharp increase in their permeability to charged species.4,5 The membrane of a spherical vesicle in a homogeneous electric field deforms the field lines, resulting in an accumulation of electric charge on the membrane surface. The vesicle acts as a © XXXX American Chemical Society

spherical capacitor. As a result, a transmembrane potential is created. Its expression, which is valid for DC pulses,6 is given by the Schwan equation7 ΔΨ = 1.5rE cos θ(1 − e−t/ τc)

(1)

with r being the radius of the vesicle, E being the electric field magnitude, θ being the angle between the direction of the electric field and the normal to the surface of the vesicle at the point considered on the membrane, and τc being the time constant of the capacitor charging. Most experiments have shown that membrane permeabilization occurs only when ΔΨ reaches a critical threshold.8 Therefore, the application of electrical pulses beyond a critical field strength value on a lipid Received: August 21, 2015 Revised: October 21, 2015

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DOI: 10.1021/acs.langmuir.5b03090 Langmuir XXXX, XXX, XXX−XXX

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organic fluorescent dyes. Very tiny defects can support transmembrane ionic exchange across lipid assemblies.22,23 These are the early events in electropermeabilization. Permeabilization for larger molecules require more defects (or larger defects) that are obtained with a larger field, with the same critical TMP. Then, we measure the critical transmembrane potential with this highly sensitive detection method at different lipid compositions. The results show that the phase states of the lipid compositions play a decisive role in the threshold of the transmembrane potential. Finally, we proposed a model revisiting the mechanism of membrane permeabilization.

vesicle can lead to its transient permeabilization.9 This phenomenon is a physical process that allows the entry of molecules of different sizes. Permeabilization is a local loss of membrane cohesion. The critical field (i.e., transmembrane potential) is directly linked to the structural forces supporting membrane cohesion. Short electric pulses (in the μs time range) can induce the permeabilization of lipid vesicles as well as cell membranes, leading to the transport of small molecules by electrophoretic drift during the pulse and by diffusion along the resealing events.3 Despite numerous studies,3−10 the mechanisms of electropermeabilization are still poorly understood.10,11 It is already known that the application of an electric field pulse to a lipid vesicle induces a mechanical effect on the membrane.12 The Maxwell stress caused by the field can deform the vesicle and/or change the volume of the vesicle. A spherical vesicle can be deformed into an ellipsoidal or cylindrical one.13−17 Moreover, it has been shown by different groups on different systems (cells, black lipid membranes, and unilamellar and multilamellar vesicles) and with different techniques (sucrose leakage, calcein transport, trypan blue uptake, and current measurements) that the electropermeabilizing transmembrane potential threshold is detected from 0.2 to 1 V.9,16−19 Open questions are that, on one hand, this threshold could be dependent on the sensibility of the detection as transport is detected20 (an operative criterion not really relevant to the physical chemistry of the autoassembly). On the other hand, the composition of the membrane could control this threshold21 (a membrane structural property). These questions are our concerns in the present study. In the present work, we sensitively determined this threshold for transport with a new fluorescence method using the binding competition between manganese and calcium ions on an ionsensitive fluorescent probe, calcium green 2, trapped within the vesicle. We developed a direct sensitive method on single lipid vesicles. Giant unilamellar vesicles (GUV) were chosen as a model system. Indeed, their composition can be varied from one lipid to a mixture of different classes of lipids. The size of these models is large, and they can be observed directly under a fluorescence videomicroscope as a single vesicle. We show the response of a lipid self-assembly submitted to an electric field pulse. The experimental evaluation of the field threshold value and resulting critical transmembrane potential (TMP) depends, on one hand, on the technique used to detect it (transport and sensitivity of the assay, i.e., the amount of substances that should cross the membrane to produce a detectable change in signal) and, on the other hand, on the system composition that is considered. The critical TMP did not depend on the size of the vesicle in the range from 20 to 50 μm but on the field magnitude used to induce it as predicted in eq 1. This last conclusion is supported by our observations that the electric field required to permeabilize small vesicles is stronger than the one for larger vesicles, as predicted with the Schwan equation. We show that the detection method is a very important parameter for the precise determination of the critical transmembrane potential. GUV-trapped calcium green means that fluorescence is sensitive to changes in the concentration of intravesicular divalent ions by a few nM. The use of competiting ion penetration as probes for lipid bilayer permeabilization indeed allows for very highly sensitive detection. We demonstrate in this paper that their penetration is detected with a lower destabilization field (i.e., voltage) threshold than the one needed for larger molecules such as

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EXPERIMENTAL SECTION

Observation and Pulsation of GUVs. Giant unilamellar vesicles composed of egg-PC, POPC, DOPC, DMPC, and DPPC with or without cholesterol (Supporting Information S1) were produced by the electroformation method (Supporting Information S2) . GUVs were visualized using an inverted Leica DM IRB fluorescence microscope (Wetzlar, Germany) equipped with a 40× objective. Images were taken with a Quantem 512SC digital camera (Roper Scientific, Germany) mounted on the microscope and connected to a computer (Supporting Information S3). The Metavue software allowed for taking images of the vesicles. Images were acquired with an exposure time of 25 ms, 1 s before the application of the pulse train and less than 5 s after it. Sample illumination was achieved with a Leica HBO 100 W mercury lamp and a filter set (Leica filter cube L4) at an excitation of 488 nm or by phase contrast with a halogen lamp. The observation chamber consisted of a glass slide and a coverslip with a pair of parallel electrodes spaced 5 mm apart (Supporting Information S3) .25 A 0.5 μL portion of the vesicle solution was placed inside the chamber and was then diluted with 50 μL of the pulsing buffer (pH 7.35) composed of 260 mM glucose, 1 mM NaCl, 1 mM HEPES, and 2 mM MnCl2. Less than 5 min after the insertion of GUVs into the chamber, the vesicles sedimented to the bottom of the chamber due to gravity. The chamber was connected to a high-voltage unipolar S20 βtech generator (L’Union, France) which generated square-wave dc pulses. The vesicles were pulsed with 10 pulses of 100 μs duration at 1 Hz (the 1 s delay was proven to be long enough for the vesicle to reseal) at room temperature (22 °C) and with increasing intensity. The shape of the pulses was checked online with the touch screen. Measurements were performed on a new sample under each condition to submit each vesicle to only a single electric pulse train. Detection Method. CaGr2 (from Molecular Probes/Invitrogen) is a fluorophore that becomes highly fluorescent when complexed with Ca2+ ions (Kd ≈ 550 nM) (Supporting Information S4). CaGr2 was added to the electroformation medium. The conditions under which the electroformation of GUVs was performed resulted in fluorescent vesicles as a result of the use of a glass support that spontaneously released calcium ions. Since the affinity between CaGr2 and Ca2+ is strong, all of the CaGr2 inside the GUVs was already complexed and saturated. This saturation was experimentally confirmed; adding an excess (100 μM) of Ca2+ ions outside GUVs and permeabilizing the membrane did not make them more fluorescent compared to the fluorescence due only to the electroformation. The electropermeabilization threshold was determined by the entry of manganese ions into vesicles when pulsing in a pulsing buffer containing 2 mM manganese. Mn2+ ions are also complexed with CaGr2. As the dissociation constant of manganese ions with CaGr2is lower than that for calcium, this leads to the dissociation of the CaGr2−Ca2+ complexes and the formation of CaGr2−Mn2+ complexes which are less fluorescent than CaGr2−Ca2+ complexes (from the Molecular Probes/Invitrogen Web site), inducing a decrease in vesicle fluorescence. Therefore, the electropermeabilization threshold was determined from the detection of a decrease in fluorescence in lipid vesicles as a result of pulse delivery. This was detected on GUVs formed with lipids of different compositions. B


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Langmuir Data Processing. The sequence of data acquisition was as follows. An image was taken before the pulses for each vesicle and then just after (i.e., within 5 s) the end of the 10 pulses. We noted I0, the mean fluorescence intensity of a given vesicle present on the image before the train of electric pulses, and I, the mean fluorescence intensity of the same vesicle after a given train of electric pulses. By comparison of these two images (before and after the pulse), we could evaluate the change in fluorescence intensity. As the vesicles did not move during the delivery of the pulse train, a mathematical subtraction of the fluorescence levels in the same region of interest (ROI) covering the vesicle was done using the Metavue software. This operation allowed for detecting an increase or decrease in the fluorescence intensity after the application of the pulses. These differential images were then processed with the ImageJ software on a desk computer. As long as permeabilization had not occurred, the differential images were black. When permeabilization occurred, inducing Mn2+ transport, a decrease in vesicle fluorescence intensity resulted in the presence of lighted pixels on the differential images. A medium filter of four pixels was applied. The light profile plot across each vesicle was applied to evaluate the homogeneity of dye distribution, and the mean fluorescence intensity was measured in the region of interest covering the vesicle for quantitative analysis. Synchronized Flashed-Phase Contrast Setup. To study GUV geometrical characteristics during the electrical pulses, vesicles were visualized by videomicroscopy (exposure time, 10 μs) during the last 10 μs of the electric pulse. For this purpose, a synchronized flashed phase contrast setup was used. A light flash of 10 μs allows detection under phase contrast. It was triggered 10 μs before the end of the first electric pulse (Figure 1).

Figure 2. Field-pulse-induced Mn2+ transport. Top row: Evolution of the fluorescence intensity differences due to the entry of Mn2+ in GUV selected with a diameter of 40 μm. Left image: Before the pulse. Middle image: After the pulse. Right image: Differential image. Bottom row: Relative fluorescence change as a function of the induced transmembrane potential. ΔI = I0 − I, with I0 being the intensity corresponding to the ROI before the electric pulse and I being the intensity after a given electric pulse. ΔΨ is the transmembrane potential calculated from eq 1.

was dependent on the size of the lipid assembly. Using Schwan’s equation, we calculated the critical associated transmembrane potential for each vesicle as proposed by Neumann.3 For a pulse duration of 100 μs, a value close to the capacitive loading time constant, τc, is ⎡ 1 1 ⎤ + tc = RCm⎢ ⎥ 2λex ⎦ ⎣ λ in

(2)

τc is long in a poorly conductive buffer because the specific membrane capacitance Cm is estimated to be 1 μF·cm−2.6 The conductivities of the media are 17.1 μS/cm for the internal medium, λin, and 343.0 μS/cm for the external one, λext. Given the size of the vesicle radius, R, the loading time constant is 60 μs for a vesicle with a 10 μm radius. The expression for the transmembrane potential is given by the Schwan equation at the end of the 100 μs pulse. The mean of the critical transmembrane potentials for the vesicles gave ΔΨc = 6 ± 1 mV at the end of the 100 μs pulse for egg-PC assemblies (n = 18). Efflux of CaGr2 Molecules. Classically, electropermeabilization induces the leakage of cytoplasmic compounds. In the present system, we checked the occurrence of leakage of CaGr2. Images were taken before the application of any electric field pulse and after the application of 10 pulses. The ROI before the pulses was subtracted from the one after the pulses. ROI sizes were selected to cover the surroundings of each GUV. The resulting image was analyzed to determine the difference in fluorescence intensity (Figure 3). A decrease in fluorescence inside the GUVs, concomitant with an increase in fluorescence outside the GUVs, would show an efflux of CaGr2 from the vesicles (top row in Figure 3). This change in fluorescence intensity was observed only above the critical electric field needed to obtain lipid bilayer permeabilization to such an extent that CaGr2 efflux was detected. The critical electric field for vesicles with diameters ranging from 20 to 50 μm was

Figure 1. Sequence of pulses in the flashed-phase contrast illumination setup. The 100 μs signal (in white) is the voltage delivered by the pulse generator to apply a field to the GUV suspension. The 10 μs signals (in gray) are those of the generator controlling the electroluminescent diode and the camera acquisition that is used to visualize the effects of the electric pulses on the vesicles. The light is on for 10 μs before the pulse or during the last 10 μs of the electric pulse. The scale for the voltage is taken to be arbitrary to display the two signals on the same graph.

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RESULTS Influx of Manganese Ions. To detect membrane permeabilization for ions, the influx of manganese ions that quenches the CaGr2−Ca2+ complex was used, as described in the methods section (Supporting Information S4). We observed a decrease in the fluorescence of the GUVs composed of egg-PC lipids after applying electric field pulses to the vesicle (Figure 2) (Supporting Information S5). For each single GUV, if the intensity of the electric field was below a critical level, then no fluorescence change was detected. The drastic decrease in fluorescence resulted from a critical transition within a narrow field window (top row in Figure 2). This fluorescence decrease was the signature of the entry of Mn2+. The critical electric field was determined for 18 vesicles with diameters ranging from 20 to 50 μm (Figure 2). The critical field intensity C


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The vesicles in the gel phase, namely, the vesicles composed of DMPC and DPPC, showed a larger ΔΨ, about 21 mV (Table 1) (n = 20). Finally, for the vesicles in the ld/lo phase (POPC with 30% cholesterol), ΔΨ was determined to be 158 ± 25 mV (Table 1) (n = 20). Whatever their composition, the measured critical transmembrane potential determined using vesicles 20 to 50 μm in diameter did not depend on the size of the vesicles (data not shown). The above results show that this critical transmembrane potential does not depend on the length of the carbon chain of the lipids for the same phase state. This threshold appears to depend only on the phase state. Transient Changes in Vesicle Surface and Volume. The changes in vesicle surface and volume (Supporting Information S6) during the application of the pulse were also investigated. A synchronized flashed phase contrast setup was used to measure the surface and volume of the vesicle during the electric pulse. Swelling was observed (Figure 4A,B). The diameter of a single POPC GUV was directly determined during the last 10 μs of one pulse and compared to that measured on the image taken before the application of the electric pulse. As the vesicles remained spherical during the pulse, the surface and volume were estimated from the radius to the powers of 2 and 3, respectively, and compared to the initial measurements. This experiment was done on 22 vesicles with initial diameters ranging from 20 to 50 μm. Linear regression was done on the surface and volume values. We obtained a membrane surface increase of 12 ± 2% (Figure 4D) and a volume increase of 20 ± 1% (Figure 4E) at the end of the pulse. We detected an isotropic stretching/swelling of the vesicle. We checked the reversibility of these increases in surface area and volume by measuring the surface area and volume about 10 s after the application of the pulse. It has already been shown that the resealing of GUVs is effective within 200 ms.25,28 The image taken a few seconds after the application of the electric pulse train allowed us to determine, as described before, the surface and volume of the vesicles after the resealing of the vesicles. These results showed that the GUVs reverted back to their initial state with no change in size when the field was turned off (Figure 4A,C). The field-induced swelling was therefore fully reversible under the present pulsing conditions (0.1 ms duration).

Figure 3. Evolution of the fluorescence intensity differences due to the leakage of CaGr2 from one GUV with a diameter of 21 μm. Top row: Fluorescent images (left) before the pulse, (middle) after the pulse, and (right) the differential image. Lower graph: Change in ΔI/I0 versus ΔΨ. ΔI = I0 − I, with I0 being the intensity corresponding to the picture before the electric pulse and I being the intensity after a given electric pulse. ΔΨ is the transmembrane potential calculated from eq 1.

determined by increasing the electric field from 2 to 20 V/cm (in steps of 2 V/cm). The mean of the critical associated transmembrane potential was calculated by the Schwan equation at the end of the 100 μs pulse and gave ΔΨc = 187 ± 24 mV (n = 16). Effect of Lipid Composition on the Permeabilization Threshold. The method using Mn2+ ion penetration as the permeabilization assay was further used to determine the permeabilizing field thresholds of vesicles with different lipid compositions and phase states. Liquid phases were obtained with the following lipids and lipid mixtures: egg-PC, egg-PC with 5% cholesterol, egg-PC with 30% cholesterol, DOPC, POPC, and POPC with 5% cholesterol. Gel phases were obtained with DMPC and DPPC. Finally, an ld/lo lipid mixture was obtained by mixing POPC with 30% cholesterol. All phospholipids were carrying the same zwitterionic polar heads. All of these vesicles contained CaGr2 already complexed with calcium ions and were fluorescent when excited at 488 nm. The diameters of the GUVs ranged from 20 to 50 μm. Ten electric field pulses of 100 μs duration, at a frequency of 1 Hz and with increasing intensity, were applied to about 20 vesicles of each composition. Room temperature was controlled at 17 °C to maintain the desired phase state. The vesicles, prepared with lipids in the fluid phase, showed a critical transmembrane potential equal to 6 ± 2 mV (Table 1) (n = 20).

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DISCUSSION Under the electropermeabilization hypothesis,3,4 the above results show that the transmembrane voltage threshold needed to induce membrane electropermeability depends (i) on the sensitivity of the detection method (Mn2+ inflow versus CaGr2 outflow), (ii) on the size of the permeant species (Mn2+ versus CaGr2), and (iii) on the lipid composition of the vesicles.

Table 1. Critical Permeabilizing Transmembrane Potential Determined by the Method Using Manganese Ion Inflow for Different System Compositionsa GUVs composition

egg-PC

ΔΨmoy (mV)

6±1

egg-PC + 5% chol egg-PC + 30% chol 5.0 ± 0.3

4.8 ± 0.4

DOPC

POPC

6.1 ± 0.4

6.0 ± 0.3

POPC + 5% chol POPC + 30% chol 5.3 ± 0.3

158 ± 26

DMPC

DPPC

22 ± 4

20 ± 3

a The Schwan equation was used to obtain these voltages at the end of each pulse from the intensity of the critical field. The systems composed of egg-PC, egg-PC + 5% cholesterol, egg-PC + 30% cholesterol, DOPC, POPC, and POPC + 5% cholesterol are in the fluid phase. The system composed of POPC + 30% cholesterol is in the ld/lo phase. The systems composed of DMPC and DPPC are in the gel phase. N = 20, with sizes ranging from 20 and 50 μm. The errors represent standard errors. Temperature is controlled to 17 °C.

D


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Figure 4. Determination of POPC vesicle geometrical change during and after the application of 10 electric pulses (E = 20 V/cm and T = 100 μS). (A) Picture of a vesicle just before the electric pulse train. (B) Picture of the same vesicle during a pulse. (C) Picture of the same vesicle just after the electric pulses train. (D) Plot of the surface during a pulse as a function of the surface before the application of the train of pulses. (E) Plot of the volume during pulses as a function of the volume after the application of the train of pulses.

However, it does not depend on the size of the vesicles, which should be considered to be large under our present conditions and free of curvature constraints. The results show that there was a difference in the values for the critical transmembrane potential depending on which probe we used to measure it. When the electropermeabilization was induced, the exchange of molecules across the lipid membrane was due to the creation of membrane defects. In the case of the leak of CaGr2, the probability to obtain large defects needed to be increased to have enough CaGr2 molecules leaving the GUV for fluorescence detection. The higher critical transmembrane potential may reflect the larger amount of energy needed to get such large defects. But the population density of the membrane defects could play a critical role in terms of detecting electropermeabilization. In comparison, manganese ions, which were smaller, did not need a high probability of defects to be detected inside the GUV (Supporting Information S7). A lower electric field intensity was needed to observe the entry of manganese than to observe the exit of CaGr2. Increasing field intensity induced a larger number of defects on the vesicle, i.e., a higher intravesicular permeability, due to the increase in the size of the vesicle cap affected by the field Ap:10

Furthermore, the control of structural defects induced by the electric field pulse is dependent on the membrane organizational forces, namely, the lipid composition. Indeed, lipids in the fluid phase are less organized than in the gel phase. When applying an electropermeabilizing electric field pulse to a lipid vesicle, the system is destabilized and defects are created in the membrane. The above results show that creating defects in the fluid phase requires a lower critical electropermeabilizing transmembrane electric field than in the gel phase. Our data showed that the addition of 30% cholesterol to the POPC GUVs strongly increased the transmembrane permeabilization threshold. Indeed, the cholesterol induces a better cohesion of the lipid assembly and a greater stability of the membrane. This lipid composition makes the membrane more resistant to the creation of defects. It has already been shown that the addition of cholesterol above 10% (mol/mol) in homogeneous lipid membranes strongly increases the membrane permeabilization threshold by condensing the fluid lipid bilayers.31 This may explain how cholesterol, an important component of the cell membrane, can affect the electropermeabilization process in cells by condensing the membrane, inducing a greater cohesion of the lipid assembly. From all of the results presented above, we revisit in this paper the mechanism of membrane permeabilization. Lipid loss, detected by a loss of surface area and associated with strong membrane destabilization due to a strong electric field pulse and a long pulse duration (5 ms), has already been reported.25,28 In our experiments, using a pulse duration of 100 μs, this phenomenon was not observed. No changes in the vesicle surface were detected after the electric pulse. However, transient changes in volume (20%) and surface area (12%) without deformation were detected during the electric pulse in our experiments. A transient isotropic swelling is therefore present during the pulse delivery. Regarding this transient surface increase, Cruzeiro-Hansson et al. gave theoretical evidence that ion permeation during the phase transition was similar to that due to the application of an electric field on the membrane system.32,33 Some other groups have shown that, during a phase transition, ion transport occurs.34−36 Papahadjopoulos et al.37 have shown experimentally that phase transitions of phospholipid vesicles were

⎛ Ep ⎞ ⎟ Ap = A⎜1 − ⎝ E⎠

A is the vesicle surface, and Ep is the critical permeabilization field strength. As predicted by Fick’s law, the transport is more effective even with a low critical transmembrane potential when the field strength is increased (a larger surface on the vesicle supports the transport). As a conclusion, the population of membrane structural defects and, as a consequence, the size of the hydrophilic species that can be detected after passing across the membrane depend on the applied electric field but not on the critical transmembrane potential. The detection of permeabilization is then dependent on the size of the hydrophilic species used for this detection due to their permeability coefficient (higher for smaller species). These results are further supported by previous results of different groups using cells.26,30 E


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As a conclusion, we have shown here that the electropermeabilization threshold definition is complex. It depends on the cohesion, namely, the stability, of the lipid self-assembly we observe. In this paper, we have shown that the lipid composition of this system gives significantly different values for the electropermeabilization threshold. More precisely, it is the phase state and the quantity of cholesterol that support variations in these values. This is reminiscent of a recent investigation of Dimova on DPPC GUVs, where permeabilization was detected only under drastic field conditions for lipids in the gel state,46 and of the results obtained by pulsing vesicles held on a micropipette tip.47 The electropermeabilization process in lipid assemblies was described as a reversible transition of the membrane organization. Indeed an open question is to know if the permeabilization is due to the induced transmembrane potential or to the fieldinduced stretching of the GUV. More than 20 years ago, it was observed that shear stress was associated with ion transport (Ca2+) across lipid bilayers linearly with the applied shear rate. The fluid force application was proposed to directly influence the molecular area of the bilayer and increase the number of bilayer packing defects through which passive Ca2+ transport was supposed to occur.48 The transport of Ca2+ was dependent on the lipid composition in a rather similar way to that which we observed in the present study.49 In another similar study, where Brij was added to the lipid, exchanges of aqueous solution between the internal and external vesicle pools of Mg2+ using a spectrophotometric approach were again observed. Lipid perturbation relaxed once the shear stress was stopped.50 It was further reported that such a stress could bring about lipid vesicle fusion as it was reported to be a consequence of electric pulse delivery.51,52 The flexibility of large lipid vesicles makes them very sensitive to the deformation due to the electrical stress. With such systems, permeabilization is mechanical and bioelectrochemical. The consequence is that either they are poor models for the investigation of cell electropermeabilization or the stretching is a key factor in the process. But one should take into account that cells are more resistant to stretching due to their cytoskeleton. From a more experimental point of view, the threshold value depends on the detection method used. The size of the probe we used had an effect on the evaluation of the critical transmembrane voltage. CaGr2 was a probe with a size comparable to those of other organic dyes used classically. In this case, we found a critical transmembrane potential similar to those previously published.9 In the 1980s, Neumann predicted that there was no thermodynamic permeabilization threshold.3 When an electric field is applied to a lipid vesicle, membrane permeabilization occurs. The population of the defects is larger when increasing the electric parameters (intensity and duration of the pulse). In this paper, we experimentally validated this theory, proving that it is the size of the molecule to be detected that determines the permeabilization threshold (as the size controls the transport) and that there is no absolute threshold. Finally, we gave evidence that the electropermeabilization process is a reversible transition of the membrane organization due to changes in the balance of forces controlling the assembly cohesion.53,54

associated with the permeabilization (leak) of the lipid bilayers.38 The molecular areas of lipids in different phase states have already been measured: ADPPC (50 °C) = 63.0 Å2,39 ADPPC (19 °C) = 45.8 Å2,40 ADMPC (30 °C) = 60.6 Å2,41 ADMPC (10 °C) = 47.2 Å2,42 APOPC (25 °C) = 64 Å2,43 and APOPC (−13 °C) = 59 Å2.43 The gel−fluid phase transition induces, on one hand, an increase in the membrane surface area and, on the other hand, changes in the distance between the negatively charged phosphate and positively charged ammonium groups. Furthermore, the orientation of the headgroup dipoles also changes.39 For DPPC and DMPC, we can estimate an increase in molecular area of about 25%, and for POPC, the increase would be about 8%. In our experiments, we found an increase in vesicle surface area of 12% during the electric pulse (Supporting Information S9). The increase in membrane surface area due to the phase transition is associated with permeabilization. This increase in surface area during the phase transition is similar to what was found in our electric pulse experiments. Destabilization inducing permeabilization and an increase in the membrane surface area are closely linked. Mismatch defects between lipid domains in two different states are present along the phase transition. A membrane is a stabilized self-assembly of lipids which can be subjected to transitions in its organization, inducing an increase in surface area concomitant with a transient state of permeabilization either by a change in the temperature or by the application of an external electric field.2,44 It has been theoretically proposed that the electropermeabilized state of a membrane occurs in a two-step process.45 A proposal for the mechanism of membrane permeabilization is presented in Figure 5. At the onset of the electric pulse, the

Figure 5. Scheme of the hypothesis for the mechanism of the entry of manganese ions. (a) Increase in the surface area (μs range) with the entry of the external buffer. (b) Increase in the volume of the vesicle until the concentration equilibrium between the external and internal media is reached. (c) Permeabilization at constant volume with an exchange of external and internal buffers. (d) Resealing of the vesicle, regaining its initial volume.

vesicle would be subjected to an increase in surface area associated with permeabilization (in the μs range) with the entry of the external buffer. During this step, no deformation of the vesicle was detected during the application of the electric pulse (Supporting Information S8). The volume of the vesicle would increase until the concentration equilibrium between external and internal media is reached (if the pulse duration is long enough). At this time, an exchange of external and internal media occurs at a constant volume during the electric pulse and before resealing. The resealing induces a decrease in the membrane area and volume as the electric stress is not applied, which is linked to a leak in the internal buffer. F


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03090. Material, preparation of giant unilamellar vesicles, observation chamber for giant unilamellar vesicles, photobleaching of calcium green 2, control of the nonspontaneous permeabilization of the vesicles, determination of the vesicle area and volume, sizes of CaGr2 and manganese ions, transient vesicle deformations, relative contributions of electrophoretic drift and concentration dependent diffusion in Mg transfer (PDF) Observation chamber for giant unilamellar vesicles (PDF) Observation of CaGr2 photobleaching as a function of time (PDF) Phase contrast intensity profile of a vesicle (PDF) Determination of POPC vesicle deformations under electric field pulse application (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Marie-Claire Blache and Florent Bissières for their very useful help with the deformation visualization setup. C.M. was supported by a fellowship from the MRES. We acknowledge financial support from the Association Française Contre les Myopathies (to M.-P.R.), ANR contracts Cemirbio (to J.T.) and CMIDT (to M.-P.R.), the Direction Générale de l’Armement (to J.T.), the Ministère des Affaires Etrangères et Européennes (to M.-P.R.), the EU-funded FP7 Oncomir (to J.T.), and the Région Midi-Pyrénées (grant 11052700 to J.T.). Research was conducted in the scope of the EBAM European Associated Laboratory (LEA) and resulted from the networking efforts of COST Action TD1104 (http://www.electroporation. net).

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REFERENCES

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Electric Destabilization of Supramolecular Lipid Vesicles Subjected to

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