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Giant Liposomes in Studies on Membrane Domain Formation
[2]
giant liposomes in studies on membrane domain formation
[2]
15
Giant Liposomes in Studies on Membrane Domain Formation By Juha M. Holopainen, Miglena Angelova and Paavo K. J. Kinnunen
Introduction
There has been a renewal of interest in the organization of biomembranes. Accordingly, it has become well established that biomembranes are laterally highly heterogeneous, being organized into microdomains with specific lipid as well as protein compositions.1 This ordering is due to mechanisms operating at thermal equilibrium as well as those involving an energy flux, that is, dissipative processes.2 Several molecular mechanisms of the former category have been worked out and involve both lipid–protein as well as lipid–lipid interactions, with key contribution by the physicochemical properties of lipids.3–5 Most of these studies have used liposomal model membranes together with various spectroscopic techniques. However, the diameters of liposomes obtained by methods such as extrusion are limited to ~100–200 nm, thus imposing a serious drawback when pursuing structures on the length scales relevant to plasma membranes, for example. This limitation was overcome by the introduction of so-called giant unilamellar vesicles, GUVs (see Luisi and Walde6), with diameters up to hundreds of microns. These model membranes have remained relatively little exploited. Yet, significant new information has been obtained about the physical properties of bilayers and shape transformations of vesicles, and in this context the pioneering studies by the groups of Evans,7–9 Sackmann,10,11 Needham,12–14 and Bothorel15 should be mentioned. 1
P. K. J. Kinnunen, Chem. Phys. Lipids 57, 375 (1991). P. K. J. Kinnunen, Cell Physiol. Biochem. 10, 243 (2000). 3 O. G. Mouritsen and P. K. J. Kinnunen, in ‘‘Biological Membranes’’ (K. Merz, Jr. and B. Roux, eds.), p. 463. Birkha¨user, Boston, 1996. 4 J. Y. A. Lehtonen and P. K. J. Kinnunen, Biophys. J. 68, 1888 (1995). 5 J. M. Holopainen, J. Lemmich, F. Richter, O. G. Mouritsen, G. Rapp, and P. K. J. Kinnunen, Biophys. J. 78, 2459 (2000). 6 P. L. Luisi and P. Walde, eds., ‘‘Giant Vesicles.’’ John Wiley & Sons, New York, 2000. 7 R. Kwok and E. Evans, Biophys. J. 35, 637 (1981). 8 D. Needham, T. J. McIntosh, and E. Evans, Biochemistry 27, 4668 (1988). 9 D. Needham and E. Evans, Biochemistry 27, 8261 (1988). 10 J. Ka¨s and E. Sackmann, Biophys. J. 60, 825 (1991). 11 H. G. Do¨bereiner, J. Ka¨s, D. Noppl, I. Sprenger, and E. Sackmann, Biophys. J. 65, 1396 (1993). 2
METHODS IN ENZYMOLOGY, VOL. 367
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
16
methods of liposome preparation
[2]
Importantly, GUVs can be directly observed by light microscopy. Moreover, they can also be subjected to micromanipulation techniques, including microinjection. Studies are now being published describing their use for the observation of membrane microdomains.16,17 The inclusion of integral membrane proteins into GUVs has been described.18 Clearly, giant liposomes hold great potential and will certainly be helpful in elucidating biomembrane properties and functions in a well-defined model system. Several techniques have been described for the formation of GUVs. In early studies GUVs were made by first depositing the desired lipids in an organic solvent on a Teflon disk, the surface of which had been slightly roughened by sandpaper.9 The disks were subsequently hydrated with water vapor and then immersed into an aqueous buffer. The yields are, however, rather modest and the procedure is time consuming. These problems are alleviated by the use of an ac electric field facilitating formation of the GUVs.19 We describe this technique in detail in this chapter, together with its use to observe microdomain formation by fluorescence microscopy.
Giant Unilamellar Vesicle Electroformation Chamber
The electroformation of GUVs is best performed with a specialized chamber. One possible setup is illustrated in Fig. 1. The chamber consists of a circular cavity with a diameter of 26.5 mm, drilled in a metal plate, and with an opening with a diameter of 19 mm in its bottom. Into this chamber a cuvette (diameter, 26 mm) of optical quality quartz glass is fitted. Attached to the Plexiglas holder are two platinum electrodes (diameter, 0.8 mm), which can be removed for lipid deposition and cleaning. The distance between the parallel electrodes axes in the chamber is 4 mm.
12
D. Needham and R. M. Hochmuth, Biophys. J. 55, 1001 (1989). D. Needham and R. S. Nunn, Biophys. J. 58, 997 (1990). 14 D. V. Zhelev and D. Needham, Biochim. Biophys. Acta 1147, 89 (1993). 15 P. Meleard, C. Gerbeaud, T. Pott, L. Fernandez-Puente, I. Bivas, M. D. Mitov, J. Dufourcq, and P. Bothorel, Biophys. J. 72, 2616 (1997). 16 L. A. Bagatolli and E. Gratton, Biophys. J. 77, 2090 (1999). 17 J. Korlach, P. Schwille, W. W. Webb, and G. W. Feigenson, Proc. Natl. Acad Sci. USA 96, 8461 (1999). 18 N. Kahya, E. I. Pecheur, W. P. de Boeij, D. A. Wiersma, and D. Hoekstra, Biophys. J. 81, 1464 (2001). 19 M. I. Angelova and D. S. Dimitrov, Faraday Discuss. Chem. Soc. 81, 303 (1986). 13
[2]
giant liposomes in studies on membrane domain formation
17
Fig. 1. A schematic diagram of one type of electroformation chamber.
Deposition of Lipids
The desired lipids are dissolved and mixed in an organic solvent at a concentration of 0.3 to 1.6 mg=ml. Of this solution, 1 to 3 l is deposited with a microsyringe [Hamilton (Reno, NV) or an equivalent] in ~1-l aliquots onto the platinum electrodes under a stream of nitrogen, in a manner allowing immediate evaporation of most of the solvent. Subsequently, the electrodes are maintained under reduced pressure for 2 to 24 h, so as to ensure complete removal of solvent residues. The electrodes are then fixed into the chamber inside the quartz cuvette. The formation of sphingomyelin=1 stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) (3:1, molar ratio) GUVs is described in detail in this chapter. For observation of the vesicles as well as to allow monitoring of the progression of enzymatic hydrolysis of sphingomyelin by sphingomyelinase, a fluorescent tracer, BODIPY-labeled sphingomyelin (mole fraction X ¼ 0.05; Molecular Probes, Eugene, OR), is also included. The chamber with the electrodes is then placed on the stage of an inverted fluorescence microscope, equipped with long or extralong working distance objectives and resting on a vibration isolation table (Melles Griot, Carlsbad, CA). Before hydration of the lipid an ac field (0.2-0.4 V, 4-10 Hz) is applied, using a voltage generator (CFG250; Tektronix, Beaverton, OH), and the chamber is then filled with buffer (1.3 ml of 0.5 mM HEPES, pH 7.4). This field is maintained for 1 min, after which the voltage is increased to 1.2-1.3 V and the frequency is adjusted to 4 Hz. The GUVs start to form on the electrodes and become visible by phase-contrast or fluorescence microscopy in about 0.5 h (Fig. 2). Initially, the formed GUVs are small and through subsequent fusions they grow in diameter. For easier
18
methods of liposome preparation
[2]
Fig. 2. Still fluorescence images of giant unilamellar vesicles composed of SOPC, N-palmitoyl–sphingomyelin, and BODIPY–sphingomyelin (0.75:0.20:0.05 molar ratio, respectively) after electroformation.
[2]
giant liposomes in studies on membrane domain formation
19
observation as well as micromanipulation GUVs attached to the electrode surface can be used. Alternatively, the GUV on the electrode may be allowed to become spherical before pulling it away from the electrode with a holding micropipette.20 The number of bilayers in the GUV can be deduced when fluorescent tracer is present. Accordingly, the emission intensity of the liposome membrane is recorded with a charge-coupled device (CCD) camera and should increase as multiples. The bilayers with minimum values should represent unilamellar vesicles.21 An alternative method is based on determining the elastic properties of the bilayer.22 Microinjection
One of the fascinating possibilities of GUVs is to subject them to local perturbation by specific lipid-modifying enzymes23–26 or other membraneactive substances, such as DNAs27 and antimicrobial peptides.28 This is best achieved by microinjection. Micropipettes are pulled from a borosilicate capillary (outer diameter, 1.2 mm), using a programmable puller (Sutter P-87; Sutter Instruments, Novato, CA). Tip diameters are determined by measuring the threshold pressure required to obtain air bubble flow through the micropipette tip immersed into ethanol.29 The micropipette is attached to a micromanipulator (MX831 with MC2000 controller; SD Instruments, Grants Pass, OR) and further connected by plastic tubing to a microinjector (PLI-100; Medical Systems, Greenvale, NY). Before loading the pipette with the enzyme solution the latter must be filtered in order to remove particles and aggregates, which could cause clogging of the micropipette tip. For this purpose the solution is passed through a 0.2-m pore size filter (World Precision Instruments, Sarasota, FL). An aliquot (~100 l) of the filtered enzyme solution is applied onto a clean microscope slide and the micropipette is immersed with the manipulator into the solution. The micropipette is filled by applying 20
F. M. Menger and J. S. Keiper, Curr. Opin. Chem. Biol. 2, 726 (1998). K. Akashi, H. Miyata, H. Itoh, and K. Kinosita, Jr., Biophys. J. 71, 3242 (1996). 22 M. I. Angelova, S. Soleau, P. Meleard, J. F. Faucon, and P. Bothorel, Prog. Colloid Polym. Sci. 89, 127 (1992). 23 R. Wick, M. I. Angelova, P. Walde, and P. L. Luisi, Chem. Biol. 3, 105 (1996). 24 V. Dorovska-Taran, R. Wick, and P. Walde, Anal. Biochem. 240, 37 (1996). 25 J. M. Holopainen, O. Penate Medina, A. J. Metso, and P. K. J. Kinnunen, J. Biol. Chem. 275, 16484 (2000). 26 J. M. Holopainen, M. I. Angelova, and P. K. J. Kinnunen, Biophys. J. 78, 830 (2000). 27 M. I. Angelova and I. Tsoneva, Chem. Phys. Lipids 101, 123 (1999). 28 H. Zhao, J. P. Mattila, J. M. Holopainen, and P. K. J. Kinnunen, Biophys. J. 81, 2979 (2001). 29 M. Schnorf, I. Potrykus, and G. Neuhaus, Exp. Cell Res. 210, 260 (1994). 21
20
methods of liposome preparation
[2]
Fig. 3. A still fluorescence microscopy image of a micropipette close to a giant unilamellar vesicle composed of SOPC, N-palmitoyl–sphingomyelin, and BODIPY–sphingomyelin (0.75:0.20:0.05 molar ratio, respectively).
reduced pressure via the injector. Subsequently, the pipette is brought into the liposome electroformation chamber. Importantly, the view is easily calibrated by using proper multiples of the known step length of the micromanipulator or an object-micrometer. The pipette tip is then adjusted to the vicinity of the GUV surface (Fig. 3), for controlled delivery of the enzyme solution by the microinjector. In the shown study, the applied sphingomyelinase converts the sphingomyelin in the outer surface into ceramide, by hydrolytic cleavage of the phosphocholine head group. Unlike sphingomyelin, which is perfectly miscible with phosphatidylcholine, the produced ceramide readily segregates (within ~10 s) into brightly fluorescent microdomains (Fig. 4A). The latter is driven by intramolecular hydrogen bonding.30 Subsequently, the domains invaginate and form ‘‘endocytotic-like’’ vesicles into the inner space of the GUV (Fig. 4B). One of the interesting possibilities available when using GUVs is microinjection into vesicle inner space. This requires a thin, long micropipette tip (inner=outer diameter, 0.1=0.2 m), and the use of a vibration isolation table. As expected, a number of vesicles burst in the process of inserting 30
I. Pascher, Biochim. Biophys. Acta 455, 433 (1976).
[2]
giant liposomes in studies on membrane domain formation
21
Fig. 4. Sphingomyelinase (Bacillus cereus) was applied in the vicinity of the outer membrane of a GUV composed of SOPC, N-palmitoyl–sphingomyelin, and BODIPY– sphingomyelin (0.75 : 0.20 : 0.05 molar ratio, respectively). In a few tenths of seconds brightly fluorescent spots appeared in the membrane (A). On further incubation these spots were invaginated as smaller vesicles into the interior of the GUV (B). Note that only a small portion of the GUV is shown. Scale bar in (B) corresponds to 100 m.
the micropipette through the bilayer. However, the success rate is reasonable. A GUV with a micropipette tip inside of it is shown in Fig. 5. Aspects to Be Aware of
It should be emphasized that although some theoretical possibilities have been proposed,31 the exact mechanisms of liposome electroformation are still not understood entirely. Each electroformation case, that is, any particular lipid composition and buffer, needs to be considered individually and takes a few trials before setting an efficient protocol. This is not a complicated process, because the operating person can observe the vesicle formation directly and control the processes. The vesicles obtained by electroformation are spherical and when attached to the electrodes lack thermal undulations. Accordingly, the bilayer is under small yet finite tension. The impact of the latter on the bilayer properties is not known yet. If well-isolated and relaxed GUVs are demanded, the suspension should be taken (gently) out of the preparation chamber and transferred into another working chamber. However, once the conditions have been worked out the electroformation method is highly reproducible and fast. The choice of lipids warrants consideration. As 31
M. I. Angelova and D. S. Dimitrov, Prog. Colloid Polymer Sci. 76, 59 (1988).
22
methods of liposome preparation
[2]
Fig. 5. A still fluorescence image of a GUV composed of SOPC, N-palmitoyl– sphingomyelin, and BODIPY–sphingomyelin (0.75:0.20:0.05 molar ratio, respectively) showing a micropipette embedded in the interior of the vesicle.
expected on the basis of augmented van der Waals interactions,4 more stable GUVs are formed when using lipids with longer saturated acyl chains. For instance, SOPC is preferred over 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC). When fluorescence microscopy or
[3]
preparing small and large unilamellar liposomes
23
spectroscopy studies on GUVs are to be performed, the choice of fluorescent moiety is important. Obviously, the quantum yield of the fluorophore should be high and it should be stable against photobleaching. In addition, a small fluorescent moiety is preferable in order to reduce steric perturbation of the lipid packing in the GUV membrane. The most critical factors in the efficient formation of GUVs are the thickness of the initial dried lipid film, and the applied voltage and frequency. Likewise, the duration of the exposure to the ac field varies and must be found experimentally. Some useful values can be found in Fischer et al.32 and can be used as guidelines when initiating studies with a new system. 32
A. Fischer, P. L. Luisi, T. Oberholzer, and P. Walde, in ‘‘Giant Vesicles’’ (P. L. Luisi and P. Walde, eds.), p. 37. John Wiley & Sons, New York, 1999.
[3]
Preparation and Quantitation of Small Unilamellar Liposomes and Large Unilamellar Reverse-Phase Evaporation Liposomes By Nejat Du¨zgu¨nes,
Introduction
Since the publication of the first article on the preparation and characterization of multilamellar liposomes,1 numerous methods have been developed to generate liposomes of different size and characteristics. Here we describe the preparation of two types of liposomes: (1) small unilamellar liposomes prepared by bath sonication, (2) large unilamellar liposomes prepared by reverse-phase evaporation. Preparation of Small Unilamellar Liposomes
Small unilamellar liposomes were developed by Papahadjopoulos and Miller2 and characterized thoroughly by Huang3 and Suurkuusk et al.4 For a typical preparation, for example, to encapsulate a fluorescent 1
A. D. Bangham, M. M. Standish, and J. C. Watkins, J. Mol. Biol. 13, 238 (1965). D. Papahadjopoulos and N. Miller, Biochim. Biophys. Acta 135, 624 (1967). 3 C. Huang, Biochemistry 8, 344 (1969). 4 J. Suurkuusk, B. R. Lentz, Y. Barenholz, R. L. Biltonen, and T. E. Thompson, Biochemistry 15, 1393 (1976). 2
METHODS IN ENZYMOLOGY, VOL. 367
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
giant liposomes in studies on membrane domain formation
[2]
15
Giant Liposomes in Studies on Membrane Domain Formation By Juha M. Holopainen, Miglena Angelova and Paavo K. J. Kinnunen
Introduction
There has been a renewal of interest in the organization of biomembranes. Accordingly, it has become well established that biomembranes are laterally highly heterogeneous, being organized into microdomains with specific lipid as well as protein compositions.1 This ordering is due to mechanisms operating at thermal equilibrium as well as those involving an energy flux, that is, dissipative processes.2 Several molecular mechanisms of the former category have been worked out and involve both lipid–protein as well as lipid–lipid interactions, with key contribution by the physicochemical properties of lipids.3–5 Most of these studies have used liposomal model membranes together with various spectroscopic techniques. However, the diameters of liposomes obtained by methods such as extrusion are limited to ~100–200 nm, thus imposing a serious drawback when pursuing structures on the length scales relevant to plasma membranes, for example. This limitation was overcome by the introduction of so-called giant unilamellar vesicles, GUVs (see Luisi and Walde6), with diameters up to hundreds of microns. These model membranes have remained relatively little exploited. Yet, significant new information has been obtained about the physical properties of bilayers and shape transformations of vesicles, and in this context the pioneering studies by the groups of Evans,7–9 Sackmann,10,11 Needham,12–14 and Bothorel15 should be mentioned. 1
P. K. J. Kinnunen, Chem. Phys. Lipids 57, 375 (1991). P. K. J. Kinnunen, Cell Physiol. Biochem. 10, 243 (2000). 3 O. G. Mouritsen and P. K. J. Kinnunen, in ‘‘Biological Membranes’’ (K. Merz, Jr. and B. Roux, eds.), p. 463. Birkha¨user, Boston, 1996. 4 J. Y. A. Lehtonen and P. K. J. Kinnunen, Biophys. J. 68, 1888 (1995). 5 J. M. Holopainen, J. Lemmich, F. Richter, O. G. Mouritsen, G. Rapp, and P. K. J. Kinnunen, Biophys. J. 78, 2459 (2000). 6 P. L. Luisi and P. Walde, eds., ‘‘Giant Vesicles.’’ John Wiley & Sons, New York, 2000. 7 R. Kwok and E. Evans, Biophys. J. 35, 637 (1981). 8 D. Needham, T. J. McIntosh, and E. Evans, Biochemistry 27, 4668 (1988). 9 D. Needham and E. Evans, Biochemistry 27, 8261 (1988). 10 J. Ka¨s and E. Sackmann, Biophys. J. 60, 825 (1991). 11 H. G. Do¨bereiner, J. Ka¨s, D. Noppl, I. Sprenger, and E. Sackmann, Biophys. J. 65, 1396 (1993). 2
METHODS IN ENZYMOLOGY, VOL. 367
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
16
methods of liposome preparation
[2]
Importantly, GUVs can be directly observed by light microscopy. Moreover, they can also be subjected to micromanipulation techniques, including microinjection. Studies are now being published describing their use for the observation of membrane microdomains.16,17 The inclusion of integral membrane proteins into GUVs has been described.18 Clearly, giant liposomes hold great potential and will certainly be helpful in elucidating biomembrane properties and functions in a well-defined model system. Several techniques have been described for the formation of GUVs. In early studies GUVs were made by first depositing the desired lipids in an organic solvent on a Teflon disk, the surface of which had been slightly roughened by sandpaper.9 The disks were subsequently hydrated with water vapor and then immersed into an aqueous buffer. The yields are, however, rather modest and the procedure is time consuming. These problems are alleviated by the use of an ac electric field facilitating formation of the GUVs.19 We describe this technique in detail in this chapter, together with its use to observe microdomain formation by fluorescence microscopy.
Giant Unilamellar Vesicle Electroformation Chamber
The electroformation of GUVs is best performed with a specialized chamber. One possible setup is illustrated in Fig. 1. The chamber consists of a circular cavity with a diameter of 26.5 mm, drilled in a metal plate, and with an opening with a diameter of 19 mm in its bottom. Into this chamber a cuvette (diameter, 26 mm) of optical quality quartz glass is fitted. Attached to the Plexiglas holder are two platinum electrodes (diameter, 0.8 mm), which can be removed for lipid deposition and cleaning. The distance between the parallel electrodes axes in the chamber is 4 mm.
12
D. Needham and R. M. Hochmuth, Biophys. J. 55, 1001 (1989). D. Needham and R. S. Nunn, Biophys. J. 58, 997 (1990). 14 D. V. Zhelev and D. Needham, Biochim. Biophys. Acta 1147, 89 (1993). 15 P. Meleard, C. Gerbeaud, T. Pott, L. Fernandez-Puente, I. Bivas, M. D. Mitov, J. Dufourcq, and P. Bothorel, Biophys. J. 72, 2616 (1997). 16 L. A. Bagatolli and E. Gratton, Biophys. J. 77, 2090 (1999). 17 J. Korlach, P. Schwille, W. W. Webb, and G. W. Feigenson, Proc. Natl. Acad Sci. USA 96, 8461 (1999). 18 N. Kahya, E. I. Pecheur, W. P. de Boeij, D. A. Wiersma, and D. Hoekstra, Biophys. J. 81, 1464 (2001). 19 M. I. Angelova and D. S. Dimitrov, Faraday Discuss. Chem. Soc. 81, 303 (1986). 13
[2]
giant liposomes in studies on membrane domain formation
17
Fig. 1. A schematic diagram of one type of electroformation chamber.
Deposition of Lipids
The desired lipids are dissolved and mixed in an organic solvent at a concentration of 0.3 to 1.6 mg=ml. Of this solution, 1 to 3 l is deposited with a microsyringe [Hamilton (Reno, NV) or an equivalent] in ~1-l aliquots onto the platinum electrodes under a stream of nitrogen, in a manner allowing immediate evaporation of most of the solvent. Subsequently, the electrodes are maintained under reduced pressure for 2 to 24 h, so as to ensure complete removal of solvent residues. The electrodes are then fixed into the chamber inside the quartz cuvette. The formation of sphingomyelin=1 stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) (3:1, molar ratio) GUVs is described in detail in this chapter. For observation of the vesicles as well as to allow monitoring of the progression of enzymatic hydrolysis of sphingomyelin by sphingomyelinase, a fluorescent tracer, BODIPY-labeled sphingomyelin (mole fraction X ¼ 0.05; Molecular Probes, Eugene, OR), is also included. The chamber with the electrodes is then placed on the stage of an inverted fluorescence microscope, equipped with long or extralong working distance objectives and resting on a vibration isolation table (Melles Griot, Carlsbad, CA). Before hydration of the lipid an ac field (0.2-0.4 V, 4-10 Hz) is applied, using a voltage generator (CFG250; Tektronix, Beaverton, OH), and the chamber is then filled with buffer (1.3 ml of 0.5 mM HEPES, pH 7.4). This field is maintained for 1 min, after which the voltage is increased to 1.2-1.3 V and the frequency is adjusted to 4 Hz. The GUVs start to form on the electrodes and become visible by phase-contrast or fluorescence microscopy in about 0.5 h (Fig. 2). Initially, the formed GUVs are small and through subsequent fusions they grow in diameter. For easier
18
methods of liposome preparation
[2]
Fig. 2. Still fluorescence images of giant unilamellar vesicles composed of SOPC, N-palmitoyl–sphingomyelin, and BODIPY–sphingomyelin (0.75:0.20:0.05 molar ratio, respectively) after electroformation.
[2]
giant liposomes in studies on membrane domain formation
19
observation as well as micromanipulation GUVs attached to the electrode surface can be used. Alternatively, the GUV on the electrode may be allowed to become spherical before pulling it away from the electrode with a holding micropipette.20 The number of bilayers in the GUV can be deduced when fluorescent tracer is present. Accordingly, the emission intensity of the liposome membrane is recorded with a charge-coupled device (CCD) camera and should increase as multiples. The bilayers with minimum values should represent unilamellar vesicles.21 An alternative method is based on determining the elastic properties of the bilayer.22 Microinjection
One of the fascinating possibilities of GUVs is to subject them to local perturbation by specific lipid-modifying enzymes23–26 or other membraneactive substances, such as DNAs27 and antimicrobial peptides.28 This is best achieved by microinjection. Micropipettes are pulled from a borosilicate capillary (outer diameter, 1.2 mm), using a programmable puller (Sutter P-87; Sutter Instruments, Novato, CA). Tip diameters are determined by measuring the threshold pressure required to obtain air bubble flow through the micropipette tip immersed into ethanol.29 The micropipette is attached to a micromanipulator (MX831 with MC2000 controller; SD Instruments, Grants Pass, OR) and further connected by plastic tubing to a microinjector (PLI-100; Medical Systems, Greenvale, NY). Before loading the pipette with the enzyme solution the latter must be filtered in order to remove particles and aggregates, which could cause clogging of the micropipette tip. For this purpose the solution is passed through a 0.2-m pore size filter (World Precision Instruments, Sarasota, FL). An aliquot (~100 l) of the filtered enzyme solution is applied onto a clean microscope slide and the micropipette is immersed with the manipulator into the solution. The micropipette is filled by applying 20
F. M. Menger and J. S. Keiper, Curr. Opin. Chem. Biol. 2, 726 (1998). K. Akashi, H. Miyata, H. Itoh, and K. Kinosita, Jr., Biophys. J. 71, 3242 (1996). 22 M. I. Angelova, S. Soleau, P. Meleard, J. F. Faucon, and P. Bothorel, Prog. Colloid Polym. Sci. 89, 127 (1992). 23 R. Wick, M. I. Angelova, P. Walde, and P. L. Luisi, Chem. Biol. 3, 105 (1996). 24 V. Dorovska-Taran, R. Wick, and P. Walde, Anal. Biochem. 240, 37 (1996). 25 J. M. Holopainen, O. Penate Medina, A. J. Metso, and P. K. J. Kinnunen, J. Biol. Chem. 275, 16484 (2000). 26 J. M. Holopainen, M. I. Angelova, and P. K. J. Kinnunen, Biophys. J. 78, 830 (2000). 27 M. I. Angelova and I. Tsoneva, Chem. Phys. Lipids 101, 123 (1999). 28 H. Zhao, J. P. Mattila, J. M. Holopainen, and P. K. J. Kinnunen, Biophys. J. 81, 2979 (2001). 29 M. Schnorf, I. Potrykus, and G. Neuhaus, Exp. Cell Res. 210, 260 (1994). 21
20
methods of liposome preparation
[2]
Fig. 3. A still fluorescence microscopy image of a micropipette close to a giant unilamellar vesicle composed of SOPC, N-palmitoyl–sphingomyelin, and BODIPY–sphingomyelin (0.75:0.20:0.05 molar ratio, respectively).
reduced pressure via the injector. Subsequently, the pipette is brought into the liposome electroformation chamber. Importantly, the view is easily calibrated by using proper multiples of the known step length of the micromanipulator or an object-micrometer. The pipette tip is then adjusted to the vicinity of the GUV surface (Fig. 3), for controlled delivery of the enzyme solution by the microinjector. In the shown study, the applied sphingomyelinase converts the sphingomyelin in the outer surface into ceramide, by hydrolytic cleavage of the phosphocholine head group. Unlike sphingomyelin, which is perfectly miscible with phosphatidylcholine, the produced ceramide readily segregates (within ~10 s) into brightly fluorescent microdomains (Fig. 4A). The latter is driven by intramolecular hydrogen bonding.30 Subsequently, the domains invaginate and form ‘‘endocytotic-like’’ vesicles into the inner space of the GUV (Fig. 4B). One of the interesting possibilities available when using GUVs is microinjection into vesicle inner space. This requires a thin, long micropipette tip (inner=outer diameter, 0.1=0.2 m), and the use of a vibration isolation table. As expected, a number of vesicles burst in the process of inserting 30
I. Pascher, Biochim. Biophys. Acta 455, 433 (1976).
[2]
giant liposomes in studies on membrane domain formation
21
Fig. 4. Sphingomyelinase (Bacillus cereus) was applied in the vicinity of the outer membrane of a GUV composed of SOPC, N-palmitoyl–sphingomyelin, and BODIPY– sphingomyelin (0.75 : 0.20 : 0.05 molar ratio, respectively). In a few tenths of seconds brightly fluorescent spots appeared in the membrane (A). On further incubation these spots were invaginated as smaller vesicles into the interior of the GUV (B). Note that only a small portion of the GUV is shown. Scale bar in (B) corresponds to 100 m.
the micropipette through the bilayer. However, the success rate is reasonable. A GUV with a micropipette tip inside of it is shown in Fig. 5. Aspects to Be Aware of
It should be emphasized that although some theoretical possibilities have been proposed,31 the exact mechanisms of liposome electroformation are still not understood entirely. Each electroformation case, that is, any particular lipid composition and buffer, needs to be considered individually and takes a few trials before setting an efficient protocol. This is not a complicated process, because the operating person can observe the vesicle formation directly and control the processes. The vesicles obtained by electroformation are spherical and when attached to the electrodes lack thermal undulations. Accordingly, the bilayer is under small yet finite tension. The impact of the latter on the bilayer properties is not known yet. If well-isolated and relaxed GUVs are demanded, the suspension should be taken (gently) out of the preparation chamber and transferred into another working chamber. However, once the conditions have been worked out the electroformation method is highly reproducible and fast. The choice of lipids warrants consideration. As 31
M. I. Angelova and D. S. Dimitrov, Prog. Colloid Polymer Sci. 76, 59 (1988).
22
methods of liposome preparation
[2]
Fig. 5. A still fluorescence image of a GUV composed of SOPC, N-palmitoyl– sphingomyelin, and BODIPY–sphingomyelin (0.75:0.20:0.05 molar ratio, respectively) showing a micropipette embedded in the interior of the vesicle.
expected on the basis of augmented van der Waals interactions,4 more stable GUVs are formed when using lipids with longer saturated acyl chains. For instance, SOPC is preferred over 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC). When fluorescence microscopy or
[3]
preparing small and large unilamellar liposomes
23
spectroscopy studies on GUVs are to be performed, the choice of fluorescent moiety is important. Obviously, the quantum yield of the fluorophore should be high and it should be stable against photobleaching. In addition, a small fluorescent moiety is preferable in order to reduce steric perturbation of the lipid packing in the GUV membrane. The most critical factors in the efficient formation of GUVs are the thickness of the initial dried lipid film, and the applied voltage and frequency. Likewise, the duration of the exposure to the ac field varies and must be found experimentally. Some useful values can be found in Fischer et al.32 and can be used as guidelines when initiating studies with a new system. 32
A. Fischer, P. L. Luisi, T. Oberholzer, and P. Walde, in ‘‘Giant Vesicles’’ (P. L. Luisi and P. Walde, eds.), p. 37. John Wiley & Sons, New York, 1999.
[3]
Preparation and Quantitation of Small Unilamellar Liposomes and Large Unilamellar Reverse-Phase Evaporation Liposomes By Nejat Du¨zgu¨nes,
Introduction
Since the publication of the first article on the preparation and characterization of multilamellar liposomes,1 numerous methods have been developed to generate liposomes of different size and characteristics. Here we describe the preparation of two types of liposomes: (1) small unilamellar liposomes prepared by bath sonication, (2) large unilamellar liposomes prepared by reverse-phase evaporation. Preparation of Small Unilamellar Liposomes
Small unilamellar liposomes were developed by Papahadjopoulos and Miller2 and characterized thoroughly by Huang3 and Suurkuusk et al.4 For a typical preparation, for example, to encapsulate a fluorescent 1
A. D. Bangham, M. M. Standish, and J. C. Watkins, J. Mol. Biol. 13, 238 (1965). D. Papahadjopoulos and N. Miller, Biochim. Biophys. Acta 135, 624 (1967). 3 C. Huang, Biochemistry 8, 344 (1969). 4 J. Suurkuusk, B. R. Lentz, Y. Barenholz, R. L. Biltonen, and T. E. Thompson, Biochemistry 15, 1393 (1976). 2
METHODS IN ENZYMOLOGY, VOL. 367
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