Langmuir 1995,11, 3685-3689
3685
Induced Morphological Changes in Synthetic Giant Vesicles: Growth, Fusion, Undulation, Excretion, Wounding, and Healing F. M. Menger* and S. J. Lee Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received April 28, 1995. In Final Form: June 29, 1995@ Giant unilamellar vesicles, prepared by hydrating a synthetic lipid, are visible under phase-contrast microscopy. Additives injected into or onto the vesicles induce various morphological changes that have been recorded photographically. For example, K V I 2 creates a large, slowly-healing hole in the vesicle surface to form a solvent-filled“nanocup”. Sodium cholate (a bile salt) also injures the vesicle surface, but the defect is smaller and heals rapidly. Sodium acetate induces vesicle fusion, a process explainable by the Svetina-Zeks mechanism. Poly(viny1 alcohol) causes filament-connected vesicles (but not isolated vesicles)to fuse. This observation leads to the speculationthat many fusion experiments with submicroscopic vesicles might also reflect, unknowingly, the presence of intervesicular filaments. Severe osmotic stress, as provided by 0.1 M NaBr, forces the vesicles to undulate vigorously. Finally, injection of a fluorescent dye into the vesicles allows, via fluorescent microscopy, the detection of outward diffusion by the dye. Giant vesicles provide a particularly valuable membrane model because, unlike submicroscopic vesicles on which the bulk of bilayer research has thus far been focused, the results are not affected by an unnatural membrane curvature. Moreover, morphological changes can be monitored as a function of membrane composition and experimental conditions without relying on indirect spectroscopic methods.
Introduction A substantial fraction of lipid-bilayer research has relied vpon either small unilamellar vesicles (SWs,300- 1000 A diameter) or large unilamellar vesicles (LWs, 10002000 A diameter). SWs and L W s have provided valuable information on membranes via methodologies such as NMR,l fluorescence,2and kinetics.3 Yet there is an inherent limitation to S W and L W systems: they do not readily lend themselves to studying undulation, healing, budding, and other manifestations of cell-like behavior. Such morphological changes are best observed and monitored by eye, and for this reason we have begun studying “giant unilamellar vesicles” (GWs). Our G W s , composed mainly of an “unnatural” lipid called DDAB, are 10-500 pm in diameter and visible under the light microscope. Chemically-induced aggregation, budding,
DDAB
birthing, and foraging of DDAB G W s have already been de~cribed.~,*~ A review of these results, and of G W technology in general, will appear elsewhere.6 The present report focuses mainly upon morphological changes occurring when G W s are chemically and physically perturbed by micr~injection.~ @
Abstract published in Advance ACS Abstracts, September 15,
1995. ( 1 )Tsai, T-C.;Jiang, R-T.; Tsai, M-D. Biochemistry 1984,23,5565. (2) Roseman, M. A.; Thompson, T. E. Biochemistry 1980, 19,439. ( 3 )Battacharya, S.; Moss, R. A.; Ringsdorf, H.; Simon, J. J. Am. Chem. SOC.1993,115,3812. ( 4 )Menger, F. M.; Balachander, N. J. Am. Chem. SOC.1992, 114, 5862. ( 5 ) Menger, F. M.; Gabrielson, K. J.Am. Chem. SOC.1994,116,1567. (6) Menger, F. M.; Gabrielson, K. To be published in Angew. Chem.
(7) For additional information on G W systems, see the following references and citations therein: Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Znt. Ed. En& 1988,27, 113. Sackmann, E.; Duwe, H.-P.; Engelhardt,H. Faraday Discuss. Chem. SOC.1986,81,281. Wick, R.; Walde, P.; Luisi, P. L. J. Am. Chem. SOC.1995, 117, 1435.
Figure 1. Injection of water by micropipet that has not penetrated the vesicle. If the pipet were removed, the pocket would disappear and the vesicle would reestablish its original spherical shape.
Experimental Section All work was performed with DDAB containing5%cholesterol (whichwas found to stabilizethe GWs such that the preparations are usable for 2 days at 20 “C and for 1 week at 8 “0.Thus, DDAB plus cholesterol was dissolved in CHCldMeOH and the solvent removed under reduced pressure. The admixture was then sonicated in water and lyopholized to produce a white fluffy powder. Approximately 0.1 mg of powder was smeared with a spatula inside a 14 mm i.d. O-ring cemented to a microscope slide. After addingdeionizedwater (ca.0.45mL)to fill the O-ring, we carefully placed a coverslip onto the O-ring such that no air bubbles were trapped beneath it. The slide was turned upside down for 3 h to allow the lipid to hydrate into G W s at 20 “C (close to the optimum temperature). Invertingthe slide during the hydration period was necessarybecause otherwisethe vesicles floated up to the coverslip and were lost when we removed the coverslip of the uprighted slide to permit micropipet manipulations. High-quality G W s with suitable size and unilamellarity numbered from 0 to 15 per sample.
0743-7463/95/24 11-3685$09.00/0 0 1995 American Chemical Society
Menger and Lee
3686 Langmuir, Vol. 11, No. 10, 1995 I
I
1i
Figure 2. Expansion of a giant vesicle as water was injected within it.
Figure 3. A hole formed on the surface of a giant vesicle by external injection of KI plus 12 to create a “nanocup”. Vesicles were examined at lOOx under phase-contrast or epifluorescence illumination using a Leitz Laborlux S microscope connected, in sequence, to a Dage-MTI CCD-72 solidstate camera, a Panasonic AG-1960 SVHS, a Hamamatsu Argus-10 image processor, and a Hitachi black-and-white monitor. In addition, a MTI-Gen-I1 Sys image intensifier was used with the fluorescence microscopy. The vesicle images, processed with the aid of Image-Pro Plus software on a PC workstation, were printed on a Tetronix Phaser 440 dye sublimation printer. Micropipets, fabricated from model G-1 microcapillaries on a Narishige pipet-puller, were siliconized with dichlorodimethylsilane. Tips of the micropipets averaged 2 pm in diameter. A Nikon PLI-188 picoinjector, set a t 10 psi and 10-900 ms injection time (depending upon the particular pipet, the desired volume, and the viscosity of the solution), was used to inject into and onto the giant vesicles. A typical injection consisted of about 50 pL. Giant vesicles a t least 200 pm in diameter were selected from an array of smaller vesicles, tubes, clusters, and multilamellar vesicles. Lack of opacity under phase-contrast conditions was an important criterion in choosing vesicles that are regarded as single-walled structures. Opaque vesicles, of a type found frequently in both our samples and in the literature: were (8)Sackmann, E.; Sprenga, I.; Noppl, D.; Kas, J.; Dobereiner, H.-G. Biophys. J. 1993,65,1396.
Figure4. Formation and healingof a defect created by injection of sodium cholate. strictly avoided. Our review on G W technology6 discusses further the problem of differentiating unilamellar and multilamellar vesicles. Vesicles smaller than 100 pm possessed bilayer membranes that were too rugged to penetrate easily with micropipets. All morphological changes reported herein were observed repeatedly in multiple experiments. Scale bars in the photomicrographs are 100 pm in length throughout.
Results and Discussion Micropipets loaded with pure water are far more difficult to inject than those loaded with aqueous solutions. Figure 1 shows how a DDABkholesterol G W invaginates in response to a n unsuccessful attempt to inject water. Persistent prodding can, however, lead to insertion of a water-filled micropipet into a G W (Figure 2). When the water was injected into the vesicle, the vesicle expanded greatly in volume. Apparently, unstructured lipid material adhering to the GUV, seen in Figure 2, supplies material needed for the G W to grow in response to the increased internal pressure.
Changes in Synthetic Giant Vesicles
Langmuir, Vol. 11, No. 10, 1995 3687
Figure 5. Vesicle fusion induced by sodium acetate.
Figure 6. Vesicle fusion occurring when an interconnecting filament was exposed to poly(viny1 alcohol).
DDABkholesterol G W s are readilyinjured by reagents externally injected to within 50 pm of the vesicles. Sometimes the lesions heal themselves rapidly, whereas
in other cases the healingprocess is sluggish. An example of the latter case is shown in Figure 3. A large hole developed in the G W upon adding an aqueous solution
Menger and Lee
3688 Langmuir, Vol. 11, No. 10, 1995 -
--.1‘1--r?----“‘--
,w-
.
?
Figure 7. Undulation of a giant vesicle when exposed to 0.1 M NaBr.
of 3 mM KI and 1 mM 12 (3 mM KI or NaI functioned similarly). Insoluble particulate matter (presumably the 13- salt of the lipid) is visible at the lip of the circular defect, accounting in part for the fact that healing takes place slowly. This, incidentally, constitutes the first synthesis of “nanocups”, the practical uses of which must a t present be left to the imagination. Rapid healing of a relatively small defect is given in Figure 4. Thus, injection of 5 mM sodium cholate (a bile salt surfactant) caused the GUV to break open and release a portion of its contents. In less than 10 s (the total time period for the four photos), the vesicle resealed itself into a smaller but fully intact vesicle. Higher concentrations of sodium cholate led to the total and permanent destruction of giant vesicles, whereas lower concentrations produced smaller holes or no holes at all. Figure 5 shows G W fusion induced by 0.1 mM sodium acetate injected in the vicinity of two vesicles. The entire fusion process required less than 5 s after application of the acetate. As discussed p r e v i ~ u s l ythe , ~ acetate effect can be understood in terms of two notions: (a) Strongly hydrated anions, such as acetate, bind relatively loosely to cationic surfaces and (b) according to the Svetina-Zeks model,g the coupled leaflets in a bilayer can act independently. Thus, when excess acetate is added externally to the DDABkholesterol vesicles, acetate exchanges with the bromide counterion to produce an outer leaflet that is more highly dissociated from its counterions. Owing to the resulting headgroup- headgroup repulsion, the outer leaflet expands relative to the inner one, thereby weakening the overall bilayer packing and promoting fusion and other molecular reorganizations. All G W preparations contain a few vesicles t h a t are joined by filaments (Figure 6 ) . As seen in the photomicrographs, the vesicles happened to be “oligolamellar” &e., filled with nonconcentric small vesicles), but unilamellar vesicles were similarly interconnected. Such vesicles fuse together upon exposure to 0.3 mM poly(viny1 alcohol) (MW = 9000) injected to within 50 ,um of a filament. Exposing a connecting filament to polymer caused the vesicles to instantly pull together and fuse into an elongated structure. The new vesicle then transformed into a spherical shape, the entire sequence taking less than 10 s. Fusion failed with nonconnected vesicles which were, instead, destroyed when sufficent polymer was applied. These results are important because they show that fusion properties of SWs and L W s might (unknowingly) depend on fibrous connectivities among the vesicles. Severe osmotic stress, created by external injection of 0.1 M NaBr, caused the giant vesicles to undulate rapidly. (9) Svetina, S.; Zeks, B. Biomed. Biochim. Acta 1983,42, 86.
Figure 8. Epifluorescence photomicographs of a vesicle into which a fluorescent dye had been injected. Fading of the dye due to outward diffusion took placeover three h from upper left to bottom right.
Figure 7 captures one particular instant in the vesicle motion. After about 30 s, the vesicle ceased its distortions and became spherical again. Ionic parity between the outside and inside environments had thus been achieved, or alternatively, the salt had become too diluted in proximity of the vesicle to exert an effect. Finally, we wish to report that injection into giant vesicles offers a new method for studying diffusion processes through synthetic membranes. Figure 8 shows a vesicle into which 0.5 mM of a fluorescent dye,1° drawn below, had been injected. The dye is water-soluble and
HO CH3
cationic in order to inhibit its binding to the cationic lipid. Every 5 min the vesicle (tracked by phase-contrast microscopy) was irradiated for 2 s during which the resulting fluorescence was recorded. This schedule is preferred to constant irradiation that might give rise to photobleaching. Figure 8 shows the vesicle 0, 1,2, and 3 h after injection. Clearly, the dye is escaping the vesicle by passing either through defects or through the bilayer itself. We are currently carrying out similar experiments with fluorescent labeled proteins and nucleotides to learn _________~
(lO)Rosenberry, T. L.; Bernhard, S. A. Biochemistry 1971, 10, 4114.
Langmuir, Vol. 11, No. 10, 1995 3689
Changes in Synthetic Giant Vesicles more about the transport of biomolecules across model membranes. It should be noted that giant vesicles are Darticularlv valuable membrane modzs because, unlike-with SUV; and L w s , results are not affected by an unnatural membrane curvature. Moreover. morDholoeica1 changes " can be monitored as a function of membrane composition
and experimental conditions without relying on indirect spectroscopic methods.
Acknowledgment. This work was supported by the National Institutes of
u
LA950337S
