Efficient Electroformation of Supergiant Unilamellar Vesicles

Letter pubs.acs.org/Langmuir

Efficient Electroformation of Supergiant Unilamellar Vesicles Containing Cationic Lipids on ITO-Coated Electrodes Christoph Herold, Grzegorz Chwastek, Petra Schwille, and Eugene P. Petrov* Biophysics, BIOTEC, Technische Universität Dresden, Tatzberg 47/49, 01307 Dresden, Germany S Supporting Information *

ABSTRACT: Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromolecules and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a standard approach to GUV production. 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 topography on the nanoscale. We find that mild annealing of the ITOcoated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diameter > 100 μm) unilamellar vesicles containing cationic lipids.

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INTRODUCTION During the last decades, giant unilamellar vesicles (GUVs) have become an extremely versatile model system which can be successfully used in a wide range of applications.1,2 Establishment of the GUV as a standard model membrane system has become possible, not in the least part, owing to the electroformation method invented by Angelova and co-workers in the end of the 1980s to the beginning of 1990s.3,4 This approach allows one to produce GUVs with sizes ranging from a few to a few tens of micrometers in radius and larger, depending on experimental conditions. A number of variations of the electroformation techniques have been introduced since, which allow for use of various electroformation substrates, as well as wide ranges of membrane and surrounding media compositions.5 GUVs can serve as a perfect model system mimicking a freestanding lipid bilayer. Especially useful in this respect are vesicles containing charged (anionic or cationic) lipids, which allow for investigation of charge-induced interactions between freestanding lipid membranes and colloidal particles or (bio)macromolecules. In this case, interaction forces between the membrane and a colloidal particle or macromolecule can be reduced to electrostatics,6−13 which makes it possible to study charge-related effects under the conditions of a minimum experimental system. Typically, experiments of this type are based on optical (fluorescence) microscopy with the help of which one can observe attachment/detachment of particles to the membrane, as well as Brownian motion and/or conformational dynamics of membrane-attached particles. GUVs with a diameter of 100 μm or larger, which will be referred to in what follows as supergiant unilamellar vesicles (SGUVs), are particularly handy in these experiments. Indeed, if microscopy observations are carried out on the upper pole of the vesicle whose lower part is attached to a coverslip, the combination of the large vesicle size with a typical focal depth of a wide-field optical microscope (∼1 μm) allows one to image more than 300 μm2 of essentially flat freestanding membrane. © 2012 American Chemical Society

While GUVs consisting of zwitterionic or/and anionic lipids can be easily electroformed virtually without any restrictions on the GUV size, production of cationic GUVs with diameters exceeding 2040 μm using the standard electroformation procedure appears to be notoriously difficult for the unknown reason, a conclusion based on both previous reports6,7 and our own experience discussed in the present communication. At the same time, the microfluidics-based approach to GUV formation (see ref 14 and refs therein) which was recently introduced as an alternative to electroformation, still has to be demonstrated to produce stable cationic lipid vesicles. Thus, at the moment, electroformation still remains the method of choice to produce GUVs containing positively charged lipids, and therefore, determining the conditions under which this technique provides best possible results is of considerable practical importance. In the present report, we describe an efficient and reliable approach to electroformation of supergiant unilamellar vesicles from lipid mixtures containing cationic lipid using indium tin oxide (ITO) coated coverslips.

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EXPERIMENTAL SECTION

Materials and Chemicals. Glass coverslips (diameter 25 mm, #1.5, Menzel Gläser, Braunschweig, Germany) were coated with a 100 nm thick ITO layer (In2O3/SnO2 90%/10%) by reactive magnetron sputtering at GeSiM (Grosserkmannsdorf, Germany). 1,2-Dioleoyl-snglycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Fluorescent lipid analogue 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) was obtained from Invitrogen (Darmstadt, Germany). Chloroform (analytical grade, 99+%) for lipid solutions and acetone (analytical grade, > 99.5%) for cleaning of the coverslips Received: February 8, 2012 Revised: March 16, 2012 Published: March 16, 2012 5518

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Figure 1. Fluorescence microscopy images of cationic GUVs (DOPC/DOTAP 95:5) grown on ITO-coated coverslips and distributions of vesicle diameters demonstrating the effects of coverslip aging expressed in the number of times N the coverslips were previously used and coverslip recovery after annealing: N = 0 (as received from the producer) (a, e); N = 2 (b, f); N = 4 (c, g); N = 5, but annealed in air for 20 min at 150 °C prior to electroformation (d, h). Note: only vesicles with diameter larger than 15 μm were included in the statistics. Statistics was collected on 20 (e) or 10 (f−h) randomly chosen regions of interest, each with the area of 0.13 mm2. The number of vesicles included in the statistics: 914 (e); 236 (f); 50 (g); 211 (h). Membrane fluorescent labeling: 0.1 mol % DiD. Scale bar: 50 μm. were purchased at Sigma-Aldrich (Germany). Ethanol was purchased from Merck (Darmstadt, Germany). Deionized water (ELGA, resistivity > 18 MΩ·cm) was used in electroformation experiments. Phosphate buffered saline (PBS) was used for atomic force microscopy imaging of ITO-coated coverslips. Vesicle Electroformation. For electroformation of vesicles, lipid solutions in chloroform with a total lipid concentration of 10 mg/mL were prepared. DOPC was used to form zwitterionic vesicles. In experiments on electroformation of cationic vesicles, a lipid mixture consisting of 95 mol % DOPC and 5 mol % DOTAP was used. Anionic GUVs were formed using 95 mol % DOPC and 5 mol % DOPS. Fluorescence microscopy observations were facilitated by adding the fluorescent membrane label DiD at a concentration of 0.1 mol % to all lipid mixtures. At these concentrations of the fluorescent label, its effect on the mechanical properties of the membrane is known to be negligible.15 Generally, a large amount of GUVs with diameter > 100 μm was obtained under appropriate conditions (see below), if lipid films of approximately 10-bilayer thickness had been deposited onto an ITO-coated surface of a glass slide, as discussed elsewhere.3,16 To this end, 0.7 μL of the lipid solution in chloroform was spread in a snakelike pattern without overlap on a 1.5 × 1.5 cm2 area using a 5 μL Hamilton syringe. After the lipid film deposited on the ITO-coated glass was dried under vacuum for 30 min, the electroformation chamber was assembled. The chamber consisted of two ITO-coated coverslips with conductively attached copper tape contacts (SPI, West Chester, PA) facing each other with their ITO-coated surfaces and separated by a 3 mm thick silicon rubber ring sealing the chamber. The assembled vesicle electroformation chamber was slowly filled with 300 μL of deionized water. After that, a sinusoidal ac electric field of 10 Hz and 1.2 V (rms) was applied for 120 min to form GUVs. Fluorescence Microscopy. Fluorescence microscopy observations on the electroformed vesicles were carried out on a setup built around a Zeiss Axiovert 200 microscope (Zeiss, Germany). The 647 nm line of an Innova 70C Spectrum argon/krypton laser (Coherent, Germany) was used to excite the DiD membrane label. Images were acquired through a 20× N-Achroplan water immersion objective, NA 0.5 (Zeiss, Germany) and appropriate emission filters using an Andor iXon+ EMCCD camera (Andor Technology, Belfast, U.K.). Confocal fluorescence microscopy observations were carried out on an LSM 510 laser scanning microscope (Zeiss, Germany) equipped with a CApochromat 40× NA 1.2 W water-immersion objective (Zeiss,

Germany) using excitation at 633 nm in combination with an appropriate emission filter for detection; the pinhole was set to 100 μm. Handling of ITO-Coated Coverslips: Standard Cleaning Procedure. In order to reuse ITO glass electrodes after electroformation, the chamber was disassembled, and the copper tape contacts were removed from the ITO glass coverslips using acetone to dissolve the conductive glue. The ITO surface was cleaned by swabbing with 80:20 (v/v) ethanoldeionized water mixture, followed by swabbing with acetone and again with ethanol; finally, it was rinsed with deionized water and dried under a flow of nitrogen. Handling of ITO-Coated Coverslips: Mild Annealing in Air. ITO-coated coverslips cleaned with the standard cleaning procedure were annealed immediately before starting the preparations for the electroformation procedure. For annealing, ITO-coated coverslips were placed with the ITO-coated surface facing air on a heating plate with the temperature set to 150 °C. After 20 min, the coverslips were removed from the heater and left to cool down to the room temperature, after which the lipid film was deposited on one of the coverslips, and the normal electroformation procedure was carried out as described above. Atomic Force Microscopy. Atomic force microscopy (AFM) measurements were carried out using a NanoWizard I system (JPK Instruments, Berlin, Germany). In all cases ITO-coated coverslips were imaged in 1× PBS pH 7.4 at room temperature (21 °C). AFM was operated in contact mode using noncoated silicon triangular cantilevers (CSC21, MikroMasch, Estonia) with a typical spring constant of 2 N/m. Each ITO-coated coverslip was scanned at three different spots of a size of 3 × 3 μm2 at a resolution of 512 × 512 pixels. To additionally characterize the image topography, surface height histograms were computed for each of the AFM height images.

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RESULTS AND DISCUSSION In our experiments on cationic vesicle electroformation using DOPC/DOTAP lipid mixtures,11 we have found that the standard electroformation procedure4 based on the use of ITOcoated coverslips does not consistently lead to formation of cationic GUVs in general and SGUVs in particular. We found that sometimes electroformation produced a number of GUVs with diameters in the range of 100−200 μm. In some experiments, however, electroformation resulted in samples 5519

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consisting of substantially smaller GUVs, with diameters mostly below 20 μm, frequently surrounded with tubular vesicular structures. The latter samples are, obviously, far from optimal for carrying out experiments involving interactions between colloidal particles and lipid membranes. By relating the successful and failed attempts to produce SGUVs from cationic lipid mixtures with all experimental parameters, we managed to correlate the electroformation success rate with aging of ITO-coated coverslips. We found that the total number of times N the coverslips had previously been used to prepare vesicles is a convenient and reliable measure of their aging. While the use of a new (N = 0) set of ITO-coated coverslips consistently led to efficient formation of SGUVs (Figure 1a,e), the maximum achievable size of vesicles substantially decreased already when the coverslips were used for the third time (N = 2) (Figure 1b,f), and finally the vesicle quality became absolutely unacceptable already for N > 3 (Figure 1c,g). This is an unexpected and remarkable finding, especially taking into account the extreme reliability of the electroformation technique when it is used to produce GUVs and SGUVs consisting of zwitterionic (DOPC) or anionic (e.g., DOPC/DOPS mixtures) lipids, irrespective of whether new or used ITO-coated coverslips were employed (see the Supporting Information). Using AFM, we found that the aging effect observed in the form of the progressively deteriorating quality of electroformed cationic vesicles is accompanied by morphological changes of the surface of ITO-coated coverslips. We found that the AFM height images of new, as received from the producer, coverslips show small (50−100 nm in diameter, ca. 2 nm deep) pores with a typical surface density of 20−40 μm−2 (Figure 2a,b). Interestingly, after repeated use of the coverslip for vesicle electroformation, these pores virtually completely disappear for N > 3 (Figure 2c,d). By serendipity, we found that the properties of ITO-coated coverslips aged during production of lipid vesicles could be completely restored if, prior to electroformation, ITO-coated coverslips are annealed in air for ca. 20 min at 150 °C. This is illustrated in Figure 1d,h. Remarkably, annealing-induced recovery of the properties of coverslips was reflected in the ITO-coated coverslip surface topography and accompanied by reappearance of pores with the same parameters, as for the new coverslips (Figure 2e,f). Moreover, we observed that typically the quality of cationic vesicles obtained using coverslips recovered by annealing was even improved compared to samples produced using new coverslips (cf. the vesicle size distributions in Figures 1e and 1h). Cationic SGUVs obtained using this procedure are very stable and allow for repeated measurements on the same sample over a period of several days.11 A representative three-dimensional image of a cationic SGUV with a diameter of ∼200 μm is shown in Figure 3. The vesicle has a hemispherical dome shape, and its upper pole is about 100 μm away from the coverslip, which allows one to treat this part of the vesicle as a freestanding membrane. The combination of the large vesicle diameter with a typical depth of focus of a wide-field fluorescence microscope (∼1 μm) provides an observation area of ∼600 μm2 of an essentially flat freestanding membrane. The use of such a vesicle would allow for easy observation of interaction of polymer molecules or colloidal particles with the membrane and tracking their Brownian motion and/or conformational dynamics. Because

Figure 2. Surface topography of ITO-coated coverslips determined by AFM (AFM height images) demonstrating the effects of ITO surface aging expressed in terms of the number of times N the coverslip was used in electroformation and recovery after annealing: N = 0 (as received from the producer) (a, b); N = 4 (c, d); N = 5, but annealed in air for 20 min at 150 °C prior to AFM measurements (e, f). Scale bar: 1 μm.

Figure 3. Three-dimensional image of a supergiant unilamellar vesicle (DOPC/DOTAP 95:5) formed on the surface of an ITO-coated coverslip using electroformation. The 3D image is reconstructed from a set (Z-stack) of laser scanning microscopy images.

of its dome shape, the vesicle strongly adheres to the coverslip and is therefore immobile, which is an important condition carrying out accurate single-particle tracking experiments on membrane-bound particles. We should emphasize that the lipid ratios reported here are related to the original lipid mixture which was deposited on ITO-coated coverslips. It is presently beyond our technical capabilities to check whether the composition of electroformed vesicles is identical to that of the original lipid mixture. What is, however, clear from our previous experiments,11 is that the concentration of the cationic lipid in electroformed vesicles is at least correlated with that in the original lipid mixture. The experiments presented in this Letter were carried out using the DOPC/DOTAP 95:5 lipid mixture, but the same 5520

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(3) Angelova, M. I.; Dimitrov, D. S. Liposome electroformation. Faraday Discuss. 1986, 81, 303−311. (4) Angelova, M. I.; Soléau, S.; Méléard, P.; Faucon, J. F.; Bothorel, P. Preparation of giant vesicles by external AC electric fields. Kinetics and applications. Prog. Colloid Polym. Sci. 1992, 89, 127−131. (5) Méléard, P.; Bagatolli, L.; Pott, T. Giant unilamellar vesicle electroformation: From lipid mixtures to native membranes under physiological conditions. Methods Enzymol. 2009, 465, 161−176. (6) Angelova, M. I.; Tsoneva, I. Interaction of DNA with giant liposomes. Chem. Phys. Lipids 1999, 101, 123−137. (7) Hristova, N. I.; Angelova, M. I.; Tsoneva, I. An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles. Bioelectrochem. Bioenerg. 2002, 58, 65−73. (8) Koltover, I.; Rädler, J. O.; Safinya, C. R. Membrane mediated attraction and ordered aggregation of colloidal particles. Phys. Rev. Lett. 1999, 82, 1991−1994. (9) Dimova, R.; Pouligny, B.; Dietrich, C. Pretransitional effects in DMPC-vesicle membranes: optical dynamometry study. Biophys. J. 2000, 79, 340−356. (10) Fery, A.; Moyab, S.; Puech, P.-H.; Brochard-Wyart, F.; Mohwald, H. Interaction of polyelectrolyte coated beads with phospholipid vesicles. C. R. Phys. 2003, 4, 259−264. (11) Herold, C.; Schwille, P.; Petrov, E. P. DNA condensation at freestanding cationic lipid bilayers. Phys. Rev. Lett. 2010, 101, 148102. (12) Quemeneur, F.; Rinaudo, M.; Maret, G.; Pépin-Donat, B. Decoration of lipid vesicles by polyelectrolytes: mechanism and structure. Soft Matter 2010, 6, 4471−4481. (13) Laurencin, M.; Georgelin, T.; Malezieux, B.; Siaugue, J.-M.; Ménager, C. Interactions between giant unilamellar vesicles and charged core-shell magnetic nanoparticles. Langmuir 2010, 26, 16025− 16030. (14) Richmond, D. L.; Schmid, E. M.; Martens, S.; Stachowiak, J. C.; Liskac, N.; Fletcher, D. A. Forming giant vesicles with controlled membrane composition, asymmetry, and contents. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 9431−9436. (15) Bouvrais, H.; Pott, T; Bagatolli, L. A.; Ipsen, J. H.; Méléard, P. Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers. Biochim. Biophys. Acta 2010, 1798, 1333− 1337. (16) Estes, D. J.; Mayer, M. Electroformation of giant liposomes from spin-coated films of lipids. Colloids Surf., B 2005, 42, 115−123. (17) Mason, M. G.; Hung, L. S.; Tang, C. W.; Lee, S. T.; Wong, K. W.; Wang, M. Characterization of treated indium−tin−oxide surfaces used in electroluminescent devices. J. Appl. Phys. 1999, 86, 1688− 1692. (18) Sugiyama, K.; Ishii, H.; Ouchi, Y.; Seki, K. Dependence of indium−tin−oxide work function on surface cleaning methods as studied by ultraviolet and X-ray photoemission spectroscopies. J. Appl. Phys. 2000, 87, 295−298.

effect is observed also for DOPC/DOTAP mixtures with the cationic DOTAP content from 2 to 10 mol % in the lipid mixture. At DOTAP concentrations of 1 mol % and below, SGUVs can be easily formed using the standard coverslip cleaning procedure. For DOTAP concentrations of 15 mol % and higher, we found that electroformation of GUVs with diameters larger than 20 μm is inhibited. We also explored the behavior of a lipid mixture containing another cationic lipid, EDOPC, and found that essentially the same phenomenology is observed for DOPC/EDOPC lipid mixtures. One can speculate that there might be a direct connection between the quality of electroformed cationic vesicles and the surface properties of the ITO layer reflected in its surface topography. Indeed, it has been shown previously17,18 that the ITO surface is prone to develop a carbon-rich contamination layer. Oxidative surface treatment removes the contamination and substantially increases the work function of the ITO surface. Therefore, we suggest that annealing in air can play a role of a mild oxidative surface treatment, thereby restoring the properties of the ITO layer. Nevertheless, the exact mechanisms of both the effect of aging of the ITO surface on the growth of cationic lipid vesicles and its reversal by annealing in air still remain open questions, which are outside the scope of the present communication.

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CONCLUSIONS To summarize, in this report, we describe a new unexpected effect in vesicle electroformation consisting in degradation of quality of cationic (but not zwitterionic or anionic) vesicles with aging of ITO-coated coverslips used for vesicle production. We find that the effects of aging can be completely reverted by mild annealing of coverslips in air, which results in reliable electroformation of cationic SGUVs. We thus describe a route for efficient and reproducible electroformation of cationic SGUVs on ITO-coated electrodes. Our findings provide a solid experimental basis for the use of cationic SGUVs as a freestanding membrane model in studies of interactions between lipid membranes and colloidal particles and (bio)macromolecules.

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ASSOCIATED CONTENT

S Supporting Information *

Results of additional experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the Deutsche Forschungsgemeinschaft via Research Unit FOR 877.

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REFERENCES

(1) Schwille, P.; Diez, S. Synthetic biology of minimal systems. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 223−242. (2) Walde, P.; Cosentino, K.; Engel, H.; Stano, P. Giant vesicles: Preparations and applications. ChemBioChem 2010, 11, 848−865. 5521

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