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Adhesion of Giant Liposomes as Observed by Light Microscopy F. M. Menger,* J. S. Keiper, and S. J. Lee Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received April 1, 1997. In Final Form: June 12, 1997X Giant liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol are imparted with cationic or anionic charge by incorporation of 2-20% cationic or anionic lipids. Although two giant liposomes of like charge do not adhere, two giant liposomes of opposite charge will (depending upon the % of ionic lipid) either adhere slowly on contact, “snap together” instantly, or adhere with severe membrane distortions and even membrane layering. It was also observed that the bursting of a cationic liposome attached to an anionic liposome can be stimulated by adhesion of a second cationic liposome to the anionic liposome at a site distant from the first adhesion site. Addition of a cationic or anionic surfactant to two adhered liposomes of opposite charge causes the two liposomes to drift apart. The effect, however, is only transitory because the surfactant can redistribute itself into the interior of the liposome, thereby diminishing the surfactant’s contribution to the surface charge. Ca2+ induces adhesion between two noncharged liposomes, but here too the effect is only temporary since the Ca2+ can diffuse into the interliposomal liquid.
Introduction Liposomes are being aggressively evaluated as therapeutic agents and drug delivery vehicles for a growing range of medical applications. Cancer therapy,1 gene delivery,2 and treatment of lung disorders3 are among the many areas in which the utilization of liposomes is being pursued to improve medical care. Liposomes are especially attractive for potential medical usage because of their simple and rapid formulation, biocompatibility and low toxicity, and their ability to incorporate or compartmentalize drugs; the latter can enhance a drug’s efficacy by either extending its presence in the body via sustained release or binding to specific targets.4 In the design and formulation of liposomes for in vivo usage, one must be concerned with contact between the liposomes and between the liposomes and bioparticles such as cells. Optimally, the liposomes should repel each other and bind only with the target cells. Two possible contact interactions that can occur are adhesion (with the structures conjoined but retaining separate inner compartments) and fusion (in which the particles merge to share a common inner compartment). These phenomena, and the elucidation of their respective mechanisms, have been the subject for long-standing interest and debate among those involved in liposome research.5-10 Most work has been performed on submicroscopic liposomes (40200 nm), primarily using spectroscopic techniques to assess the dynamic features of adhesion or fusion, along X
Abstract published in Advance ACS Abstracts, August 1, 1997.
(1) Lasic, D. D.; Martin, F., Eds. Stealth Liposomes; CRC Press: Boca Raton, 1995. (2) Lasic, D. D.; Strey, H.; Stuart, M. C. A.; Podgornik, R.; Frederik, P. M. J. Am. Chem. Soc. 1997, 119, 832. Ra¨dler, J. O.; Koltover, I.; Slditt, T.; Safinya, C. R. Science 1997, 275, 810. (3) Perkins, W. R.; Dause, R. B.; Parente, R. A.; Minchey, S. R.; Neuman, K. C.; Gruner, S. M.; Taraschi, T. F.; Janoff, A. S. Science 1996, 273, 330. (4) Lasic, D. D.; Needham, D. Chem. Rev. 1995, 95, 2601. (5) Stamatatos, L.; Leventis, R.; Zuckermann, M. J.; Silvius, J. R. Biochemistry 1988, 27, 3917. (6) Niles, W. D.; Cohen, F. S. Ann. N. Y. Acad. Sci. 1991, 635, 273. (7) Marchi-Artzner, V.; Jullien, L.; Belloni, L.; Raison, D.; Lacombe, L.; Lehn, J.-M. J. Phys. Chem. 1996, 100, 13844. (8) Duzgunes, N.; Wilschut, J.; Fraley, R.; Papahadjopolous, D. Biochim. Biophys. Acta 1981, 642, 182. (9) Rupert, L. A. M.; Hoekstra, D.; Engberts, J. B. F. N. J. Colloid Interface Sci. 1987, 120, 125. (10) Leckband, D. E.; Helm, C. A.; Israelachvili, J. Biochemistry 1993, 32, 1127.
S0743-7463(97)00341-7 CCC: $14.00
with electron microscopy (EM) to provide “before and after” views of the structures. Direct real-time observation of adhesion or fusion between individually isolated liposomes in such experiments, however, is impossible. As part of our continuing interest in the cytomimetic properties of synthetic membranes, we have undertaken the study of giant vesicles and liposomes.11-13 These structures, having diameters greater than 1 µm and curvature similar to cells, can be directly observed with an optical microscope and individually isolated and manipulated using “cytological” techniques. Such methodology provides a complementary approach to the widespread spectroscopic and EM studies of submicroscopic liposomes by capturing liposomal interactions in real time on videotape. In the present work, we apply video-enhanced optical microscopy to confront two significant problems of liposomal adhesion/fusion research: (a) the behavior of oppositely-charged liposomes5-7 and (b) the behavior of liposomes in the presence of multivalent salts.8-10 These subjects address issues that have direct relevance to the burgeoning field of therapeutic liposomes, e.g., the interaction between cationic liposome-DNA complexes and the negatively-charged membranes of cells, and the consequences of high local salt concentration on liposomes during circulation. In addition, the techniques and experiments detailed below should provide the basis for further exploitation of optical microscopy in the observation of dynamic liposomal processes previously studied only indirectly. As will be seen, our results are descriptive in nature. This is not necessarily a failing since all science begins this way. In the particular case here, the hope is that membrane behavior, as observed under the light microscope, will ultimately provide the proper setting for quantitation and prediction. Experimental Section 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG), 1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1(11) Menger, F. M.; Gabrielson, K. D. Angew. Chem. Int. Ed. Engl. 1995, 34, 2091. (12) Menger, F. M.; Lee, S. J. Langmuir 1995, 11, 3685. (13) Menger, F. M.; Lee, S. J.; Keiper, J. S. Langmuir 1996, 12, 4479.
© 1997 American Chemical Society
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Langmuir, Vol. 13, No. 17, 1997 4615 Scheme 1. Lipid Structures
glycerol)] (sodium salt) (DLPG), 1-palmitoyl-2-oleoyl-sn-glycero3-[phospho-L-serine] (sodium salt) (POPS), and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphate (sodium salt) (POPA) were purchased from Avanti Polar Lipids and used as received (either in powder form or in CHCl3 solutions). Cholesterol (99%+, white powder) was purchased from Sigma and used as received, and didodecyldimethylammonium bromide (DDAB, white powder) was purchased from Kodak and recrystallized from ethanol. Structures of the lipids are shown in Scheme 1. Powder mixtures of POPC/cholesterol, POPC/POPG/cholesterol, POPC/DLPG/cholesterol, POPC/POPS/cholesterol, POPC/ POPA/cholesterol, and POPC/DDAB/cholesterol of varying mole percentages were all prepared using the following procedure. Appropriate amounts of lipid, or solutions of lipid in CHCl3, were dissolved in CHCl3/MeOH and hand-mixed, and the solvent was removed under reduced pressure. MilliQ purified water was then added to the dry films, and the resulting solutions were vortexed. Subsequent freezing and lyophilization afforded white fluffy powders. Liposome formation was initiated by smearing small amounts of the desired lipid mixture (0.1 mg or less) with a spatula inside teflon O-rings cemented on standard microscope slides. MilliQ purified water (approximately 0.5 mL/slide) was added to the slides, and the O-rings were covered with glass cover slips. Samples were allowed to hydrate for at least 30 min at 20 °C (the ambient working temperature for all work) and were only used when sufficient liposome formation had occurred. Holding pipettes and injection pipettes were prepared from Narshige G-1 capillary tubes using a Sutter P-97 horizontal pipette puller or Narshige PB-7 vertical pipette puller. Holding pipettes were further polished with a Narshige MF-9 microforge. Liposomes were manipulated using Narshige micromanipulation gear, connected to a Nikon PLT-188 picoinjector. To examine the samples, phase-contrast microscopy was performed using a Nikon Diaphot TMD microscope in tandem with a Dage-MTI CCD-72 solid-state camera, Panasonic AG-1960 SVHS VCR, Hamamatsu Argus-10 image processor, and a Sony black-andwhite monitor. This system was connected to a Micron Millenia 166 PC workstation equipped with Image-Pro Plus image processing software, and images were printed using a Tektronix Phaser 440 color printer. The procedure developed for the study of adhesion between oppositely charged liposomes is represented schematically in Figure 1. Anionic liposomes (POPC/anionic lipid/cholesterol) are shaded gray, and cationic liposomes (POPC/DDAB/cholesterol) are open. Step A shows the liposomes following hydration of the respective lipid mixtures in purified water. Since the cationic liposomes were to be transferred to the anionic liposome slide (the “working” slide), only a small region (∼1/3) of the anionic slide was smeared with lipid mixture. In general, few aggregate structures formed in the “clear” area of the anionic slide, and this portion is considered “liposome-free”. Cationic liposomes in
Figure 1. Schematic depiction of the liposome transfer process and subsequent liposome-liposome contact. aqueous solution were gently drawn from the O-ring well into an unmodified Narshige G-1 capillary pipette attached to a Narshige hand-controlled microsyringe, as portrayed in the top slide in step B. The volume of solution pulled into the pipette, ranging from approximately 25-70 µL, contained liposomes numbering from a few to dozens. After the cationic liposomes were secured in the pipette, the pipette was lifted out of solution and momentarily dipped in purified water to rinse off the sides of the pipette, thereby removing any lamellar material on the glass. Attention was then turned to the anionic liposome sample, as a polished holding pipette (outer tip diameter ∼30-40 µm; inner tip diameter ∼2-3 µm) attached to the Nikon picoinjector was used to capture an anionic liposome via gentle suction, as shown in the lower slide of step B. The held liposome was then carefully pulled to the liposome-free region of the slide prior to adhesion experiments with transferred cationic liposomes. Step C depicts the positioning of the transfer pipette, filled with cationic liposome solution, near the held anionic liposome in the “liposomefree” area of the anionic liposome slide. This was followed by the gentle release of cationic liposomes into the O-ring well (Step D). Care was required here; injecting the solution with excessive force led to cationic liposomes spreading throughout the sample, mixing with anionic liposomes and making them indistinguishable from each other. The cationic liposomes underwent no observable morphological changes or undulation upon introduction into the new solution. After injection, the held anionic liposome was pushed against one of the free cationic liposomes to observe the presence or absence of adhesion (Step E). If desirable, an additional component was introduced in the vicinity (i.e., 10-50 µm) of the paired liposomes by means of an injection pipette attached to the picoinjector.
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Figure 2. Contact between a held POPC/DDAB+ (98/2 mol %) liposome and a free POPC/POPA- (95/5 mol %) liposome. Bar ) 25 µm.
Figure 3. Contact between two POPC/cholesterol (88/12 mol %) liposomes. Bar ) 25 µm.
Results and Discussion Adhesion between Oppositely-Charged Giant Liposomes. Our lipid mixtures usually contained three components: (a) a neutral phospholipid, (b) anionic or cationic lipid, and (c) cholesterol. The ionic lipid will henceforth be designated by placing it in the center of a trio of names along with its formal charge to underscore the anionic or cationic nature of the membrane. The molar percentages of the three components will be given with the percentage of ionic lipid again in the center of a threenumber sequence and in bold type for emphasis. After exhaustive screening of giant liposome-forming properties of neutral phospholipids, POPC was chosen to serve as the base phospholipid for our systems.14 Initial studies of induced contact between oppositely-charged giant liposomes were carried out using liposomes composed of POPC/POPA- (anionic) and POPC/DDAB+ (cationic). Unfortunately, the POPC/POPA- mixtures provided mostly multilamellar or “oligovesicular” structures (i.e., liposomes containing smaller nonconcentric liposomes) which proved too soft and deformable for use in these studies. Figure 2 depicts contact between a held POPC/DDAB+ (98/2 mol %) liposome and a “free” POPC/POPA- (95/5 mol %) liposome. Note that as the held liposome is pulled away, the anionic liposome retains its distorted morphology. To obtain giant liposomes that maintained structural fidelity, 12 mol % cholesterol was added to all ensuing lipid mixtures. Cholesterol is well-known to stabilize liposome bilayers,15 and we have previously demonstrated that POPC/cholesterol mixtures provide spherical liposomes suitable for manipulation.13 Two such liposomes, composed of POPC/cholesterol (88/12 mol %), are shown in Figure 3. The nonionic giant liposomes were purchased against each other, and no lasting adhesion or fusion was seen to occur, only a momentary distortion of the membranes before they are completely pulled apart and the structures regain their original spherical form. The same behavior was observed for two cationic or two anionic (14) Lee, S. J. Ph.D. Thesis, Emory University, Atlanta, GA, 1996. (15) Evans, E.; Needham, D. J. Phys. Chem. 1987, 91, 4219.
liposomes, indicating that contact-induced adhesion does not occur between membranes of like composition. The giant liposomes in Figure 3 and subsequent figures are of a type frequently categorized as “unilamellar” in that onion-like layers do not completely fill the liposomes’ lumen. We cannot, however, specify the number of bilayers in the liposome shells. Fortunately, membrane adhesion, being a property of the outermost membrane leaflet, was independent of the apparent membrane thickness. In the ensuing discussion, we will demonstrate that adhesion does indeed materialize between liposomes of opposite charge. It is thus important to understand our criteria for adhesion. When a held liposome is moved into contact with a free liposome and the two “adhere”, this is evident from (a) a notable increase in the common surface area shared by the two liposomes involving distortion from a spherical shape and (b) an inability to separate the two liposomes by pulling the held liposome away from the free liposome with the aid of the micromanipulator. Previous studies of submicroscopic liposomes of opposite charge determined that contact results in adhesion, not fusion.5-7 Similarly, contact between oppositely-charged giant liposomes leads to membrane adhesion, without fusion of aqueous compartments. Table 1 lists the results of induced contact between a variety of oppositely-charged liposome systems. All observations were reproducible with samples prepared de novo. POPC/DDAB+/cholesterol liposomes were always used as our cationic liposomes, whereas several anionic lipids were used to construct the anionic liposomes. The nature of adhesion between oppositely-charged liposomes varied with the mole percentage of ionic lipid admixed with the POPC and cholesterol. Contact between oppositely-charged liposomes containing only 2 mol % charged lipid resulted in either no adhesion (with POPA-, POPS-, and DLPG-) or adhesion that required a finite period of contact before it occurred (with POPG-). Figure 4 shows the latter case where nearly 3 s of contact was necessary to generate adhesion between a held POPC/
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Figure 4. Adhesion between a held POPC/POPG-/cholesterol (86/2/12 mol %) liposome and a free POPC/DDAB+/cholesterol (86/2/12 mol %) liposome. Time ∼3 s; bar ) 25 µm. Table 1. Summary of Contact Interactions between Oppositely-Charged Liposomes
anionic lipid none POPA
POPS
POPG
DLPG
anionic liposome POPC/anionic lipid/cholesterol (mol %)
cationic liposome POPC/DDAB/ cholesterol (mol %)
result
88/0/12 86/2/12 79/9/12 68/20/12 68/20/12 86/2/12 86/2/12 79/9/12 68/20/12 86/2/12 86/2/12 86/2/12 79/9/12 68/20/12 86/2/12 79/9/12 68/20/12
79/9/12 86/2/12 79/9/12 68/20/12 79/9/12 86/2/12 68/20/12 79/9/12 68/20/12 86/2/12 79/9/12 68/20/12 79/9/12 68/20/12 86/2/12 79/9/12 68/20/12
no adhesion no adhesion adhesion adhesion adhesion no adhesion adhesion adhesion adhesion adhesion adhesion adhesion adhesion adhesion no adhesion adhesion adhesion
POPG-/cholesterol (86/2/12 mol %) liposome and a freefloating POPC/DDAB+/cholesterol (86/2/12 mol %) liposome. The liposomes came to share a common membrane area (note the dark interfacial region between the structures in the final micrograph of Figure 4), without ever forming a single aqueous compartment. Moreover, the held anionic liposome could not be pulled away from the adhered cationic liposome. Following adhesion, the cationic liposome either slowly explored the surface of its neighbor (perhaps under the influence of adventitious currents in the water) or the entire structure rotated on the tip of the pipette. In either case, the cationic liposome reached the glass pipette after a few minutes and nestled against it, at which point the experiment was terminated. As seen in Figure 4 and elsewhere, it is the held liposome that maintains its spherical shape, while the mobile liposome becomes distorted upon adhesion. We surmise that the 0.1-0.2 kP suction pressure imposed upon the held liposome rigidifies the membrane relative to that of the untouched liposome. Increasing the mole percentage of both lipids to 9 mol % yielded instant and unmistakable adhesion upon contact between oppositely-charged liposomes. Figure 5 portrays adhesion between a held POPC/POPA-/cholesterol (79/ 9/12 mol %) liposome and a free POPC/DDAB+/cholesterol (79/9/12 mol %) liposome. Within 1 s of contact, the liposomes “snapped together” in an adhesive complex. This complex was stable for over 5 min, until the adhered liposome rotated around to touch the pipette, causing it to burst. Oppositely-charged liposomes containing 20 mol % ionic lipid also adhered instantaneously, but the subsequent membrane dynamics were more pronounced. Figure 6
shows progressive interaction between a POPC/POPS/ cholesterol (68/20/12 mol %) liposome and a POPC/DDAB+/ cholesterol (68/20/12 mol %) liposome. In the 5 s after adhesion, the area of shared membrane increased until the held anionic liposome deformed to the eventual point of collapse. It appears that the held liposome was attempting to maximize the region of contact between the oppositely-charged surfaces. The held liposome burst following the sequence shown in Figure 6. Similarly, Figure 7 depicts adhesion of a held POPC/ DLPG-/cholesterol (68/20/12 mol %) liposome and a free POPC/DDAB+/cholesterol (68/20/12 mol %) liposome. After the liposomes adhered instantly, the larger cationic liposome began to surround the smaller anionic liposome, again maximizing the shared area between the oppositelycharged membranes. The interaction in this case, however, was strong enough to induce the collapse of the adhered cationic liposome, whose membrane then encapsulated the anionic liposome (note the increase in membrane thickness). This layering of liposomes is a very interesting phenomena whose details are under current investigation and will be reported later. In summary, it appears that by increasing the electrostatic attraction between oppositely-charged giant liposomes (i.e., by increasing the mole percentage of the charged lipid), the interactions gravitate from slow adhesion to instantaneous adhesion to extreme liposome distortions and even membrane layering. It is important to mention that, among the several anionic lipids, POPG- produced liposomes that were consistently the most stable to manipulation and the adhesion process. It is possible that the 1-glycerol group of PGs may participate in hydrogen bonding that enforces the integrity of the liposomes to suction as well as the rigors of liposome-liposome adhesion. Liposomal dimers of POPC/POPG-/cholesterol and POPC/DDAB+/cholesterol occasionally survived intact (without bursting or collapsing) for over 10 min, even at 20 mol % ionic lipid. These systems provided us a sufficient window of opportunity to further probe adhered oppositely-charged liposomes in experiments described below. Attempts to study the adhesion of multiple liposomes were undertaken next. Figure 8 shows two such trials. In Figure 8A, a POPC/DDAB+/cholesterol (79/9/12 mol %) oligovesicular liposome was attached to a held POPC/ POPG-/cholesterol (79/9/12 mol %) unilamellar liposome, after which the complex was brought into the vicinity of another cationic liposome. Adhesion occurred within 1 s of contact, leading to the three liposome configurations pictured in the final micrograph. Within about 10 min, the smaller of the adhered liposomes came to rest against the pipette. There were no noticeable changes in the adhesion between the held anionic liposome and the previously adhered cationic liposome. Another observed scenario, however, is shown in Figure 8B, where adhesion
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Figure 5. Adhesion between a held POPC/POPA-/cholesterol (79/9/12 mol %) liposome and a free POPC/DDAB+/cholesterol (79/9/12 mol %) liposome. Time ∼1 s; bar ) 25 µm.
Figure 6. Adhesion between a held POPC/POPS-/cholesterol (68/20/12 mol %) liposome and a free POPC/DDAB+/cholesterol (68/20/12 mol %) liposome. Time ∼5 s; bar ) 25 µm.
Figure 7. Adhesion between a held POPC/DLPG-/cholesterol (68/20/12 mol %) liposome and a free POPC/DDAB+/cholesterol (68/20/12 mol %) liposome. Time ∼5 s; bar ) 25 µm.
of a second liposome leads to the breakdown of the existing complex. Thus, a POPC/DDAB+/cholesterol (68/20/12 mol %) oligovesicular liposome strongly adhered to a held POPC/POPG-/cholesterol (68/20/12 mol %) oligovesicular liposome. After 2 s of contact between the held anionic liposome with a second free-floating cationic liposome, adhesion occurred a second time. But on this occasion the previously adhered oligovesicular liposome burst, leaving its inner contents (liposomes smaller than 5 µm in a diameter) stuck to the surface of the held liposome. The simultaneous bursting of one bound cationic liposome upon adhesion of another to a common anionic liposome is probably not coincidental as it was observed several times. Perhaps the juncture of the second “guest” liposome somehow weakens the contact area of the first with the anionic liposome even though the binding sites are far apart. This “long-range” communication will be the subject of additional scrutiny. We also became intrigued by the question of what would occur if another reagent (i.e., salt or detergent) was introduced in the vicinity of two adhered oppositelycharged liposomes. Injection of 0.1 M NaCl or 0.1 M sodium acetate at the interface of adhered liposomes had no noticeable effect upon the system. In contrast, injection of surfactant solution near the interface of adhered oppositely-charged liposomes was found to reversibly
disconnect two adhered liposomes. Figure 9A depicts separation induced by 1 mM sodium dodecyl sulfate (SDS) upon adhered POPC/POPG-/cholesterol (68/20/12 mol %, held) and POPC/DDAB+/cholesterol (68/20/12 mol %) liposomes. Multiple injections (of approximately 700 fL each) of the detergent solution over a span of about 10 s led to cationic liposome to completely peel away from the anionic liposome as a deformed sphere. Upon cessation of the injections, the liposomes readhered upon contact. Likewise, 1 mM cetyltrimethylammonium bromide (CTAB) induced reversible separation of adhered liposomes, as shown in Figure 9B. Over approximately 5 s, multiple injections of CTAB solution affected reduction in the shared membrane region between a held POPC/POPG-/ cholesterol (79/9/12 mol %) liposome and an adhered POPC/DDAB+/cholesterol (79/9/12 mol %) liposome. When the two liposomes were pushed together again (not shown), they readhered. A likely explanation can be offered for the departure of the two adhered liposomes upon exposure to the surfactant: The surfactant progressively incorporates into the bilayer (with the SDS binding preferentially to the cationic liposome and the CTAB preferring the anionic liposome). By this means, both liposomes assume the same charge as the added surfactant and, therefore, repel each other. Why, then, should the liposomes subsequently adhere
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Figure 8. (A) Successful “double adhesion” between a held POPC/POPG-/cholesterol (79/9/12 mol %) liposome and free POPC/ DDAB+/cholesterol (79/9/12 mol %) liposomes. Bar ) 25 micrometers. (B) Disruption of an adhered POPC/DDAB+/cholesterol (68/20/12 mol %) liposome upon adhesion of another cationic liposome to the held POPC/POPG-/cholesterol (68/20/12 mol %) liposome. Time ∼3 s; bar ) 25 µm.
Figure 9. (A) Separation between adhered oppositely-charged liposomes induced by 1 mM SDS solution. Time ∼10 s; bar ) 25 µm. (B) Separation between adhered oppositely-charged liposomes induced by 1 mM CTAB solution. Time ∼5 s; bar ) 25 µm.
when pushed together? We surmise that the surfactant can redistribute itself rapidly into the inner layers of the membranes, thereby diminishing its effective surface concentration. In this connection, we should mention our collaborative work (based on electrophoresis, dynamic light scattering, conductometry, and fluorescence spectroscopy) affirming the ability of ionic surfactants to enter the bilayer membranes and alter their electrical charge.16 Effect of Multivalent Salts on POPC/Cholesterol Giant Liposomes. Much research has focused on the binding properties of multivalent salts to phospholipid (16) Yaraslavov, A. A.; Udalykh, O. Y.; Kabanov, V. A.; Menger, F. M. Chem. Eur. J. 1997, 3, 690.
membranes of various compositions.17-19 Studies have revealed that the divalent calcium cation, for example, induces adhesion or fusion between opposed phospholipid bilayers via a much debated, but still not fully understood, mechanism.8-10 What is certain is that Ca2+-induced adhesion or fusion of liposomes is highly dependent on the lipid composition, particularly on the phospholipid headgroups. Submicroscopic liposomes, serving as cell models, have been used for most Ca2+ adhesion and fusion investigations. But giant liposomes, with size and cur(17) Casal, H. L.; Martin, A.; Mantsch, H. H.; Paltauf, F.; Hauser, H. Biochemistry 1987, 26, 7395. (18) Feigenson, G. W. Biochemistry 1989, 28, 1270. (19) Kwon, K. O.; Kim, M. J.; Abe, M.; Ishinomori, T.; Ogino, K. Langmuir 1994, 10, 1415.
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Figure 10. Contact between POPC/cholesterol (90/10 mol %) liposomes. In the absence of Ca2+, no adhesion occured despite contact of 20 s, and the structures were easily pulled apart (micrographs A-C). Injection of 0.1 M CaCl2 via an injection micropipette (lower left-hand corner) induced adhesion within 2 s of renewed contact (micrograph D). Thin filaments which sometimes accompany giant liposome preparations are seen in the micrographs. Bar ) 50 micrometers.
Figure 11. Attempt at inducing adhesion between two POPC/POPG-/cholesterol (86/2/12 mol %) liposomes with multiple injections of 0.1 M CaCl2 solution. Bar ) 25 µm.
vature comparable to biological cells, may constitute better models to examine such cation-induced interactions. Using micromanipulation gear, giant liposomes can be isolated and examined for contact interactions in the absence and presence of multivalent salts such as CaCl2. Microinjection of 0.1 M CaCl2 solution in the vicinity (d < 200 µm) of neutral POPC/cholesterol (90/10 mol %) liposomes brought about immediate adhesion upon contact. Figure 10 shows a typical sequence. In the absence of Ca2+, a held liposome was pushed against a free liposome for 20 s and easily pulled apart, despite a slight cohesion (micrographs A-C). A micropipette was then introduced approximately 75 µm from the held liposome, and after a single injection of 0.1 M CaCl2 solution, contact between the liposomes led to adhesion (micrograph D). Over the course of 30 s, the liposomes lost the strong adhesion and could be pulled apart. This loss of adhesion could be due to diffusion of Ca2+ away from the interfacial region. In support of this conjecture, injection of further solution of 0.1 M CaCl2 solution renewed adhesion between the separated liposomes. This cycle could be repeated many times. (Luisi and co-workers recently reported reversible adhesion between POPC giant liposomes upon microinjection of Hen-egg lysozyme solution.20 The authors attribute the adhesion to the presence of the lysozyme. In light of our observations, however, the adhesion may well have been assisted by the presence of CaCl2 (5 mM) used to activate the lysozyme). Note that reversible Ca2+ binding has, to our knowledge, never been observed with small vesicles (40-200 nm) because these vesicles must be bathed in a uniform and invariant Ca2+ environment. Since CaCl2 microinjection resulted in adhesion between POPC/cholesterol liposomes, we also investigated if other MX2 and MX3 salts could also act as adhesion agents. Using the same experimental protocol, 0.1 M MgCl2 solution did not lead to immediate adhesion between POPC/cholesterol (90/10 mol %) liposomes. To induce
adhesion, repeated injection (>5 injections) of the MgCl2 solution was required, and the adhesion diminished very quickly over 20 s. Similarly, 0.1 M solutions of BaCl2, CdCl2, CuCl2, TmCl3, and EuCl3 only caused weak adhesion relative to the effect of CaCl2. Liposomes carrying net charges were also evaluated for possible Ca2+-induced adhesion. POPC/DDAB+/ cholesterol (86/2/12 mol %) liposomes did not adhere upon nearby injection of 0.1 M CaCl2. Presumably, 2 mol % DDAB+ either provides sufficient repulsion to the Ca2+ ions or “ties” up enough negatively-charged phosphate moieties of the phosphocholine headgroups, to inhibit adhesion. Surprisingly, however, multiple injections of 0.1 M CaCl2 solution at POPC/POPG-/cholesterol liposomes of 68/20/12, 79/9/12, and 86/2/12 mol % also did not afford adhesion upon contact (Figure 11). Macdonald and Seelig have found that addition of POPG- to POPC bilayers significantly increases the Ca2+-binding capacity of the bilayer membranes.21 In the present work, Ca2+ may become fully occupied in “intra-liposomal” binding to anionic headgroups so that “inter-liposomal” cross-linking is impeded. It is also possible that Ca2+ imparts a cationic charge upon the formerly anionic liposomes, so that the liposomes fail to adhere for electrostatic reasons. Systematic investigation of giant liposomes by light microscopy is a new field, and as is usually true for new fields, each observation raises far more questions than it answers. The work described in the present article suffices, however, to demonstrate the potential inherent to membrane research when membranes engaged in dynamic processes can be directly observed as they are modified in composition and physically or chemically perturbed. We anticipate that the field will yield a rich harvest in the future. Acknowledgment. This work was supported by the National Institutes of Health. LA970341Z
(20) Wick, R.; Angelova, M. I.; Walde, P.; Luisi, P. L. Chem. Biol. 1996, 3, 105.
(21) Macdonald, P. M.; Seelig, J. Biochemistry 1987, 26, 1231.
