Langmuir 1990, 6, 1326-1329
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Measurements of Forces Involved in Vesicle Adhesion Using Freeze-Fracture Electron Microscopy Stuart M. Bailey, Shivkumar Chiruvolu, Jacob N. Israelachvili, and Joseph A. N. Zasadzinski’ Department of Chemical and Nuclear Engineering, University of California, Santa Barbara, California 93106 Received March 22, 1990. In Final Form: May 8, 1990
Vesicle adhesion was investigatedby freezefracture electron microscopy. Membrane stresses were impaed by osmotic gradients to assess the relative importance of repulsive thermal undulation and attractive hydrophobic forces on vesicle membrane interactions. The adhesion energy was found to be proportional to the membrane tension, but its magnitude could not be explained from quenching undulation forces since the adhesion energy rose to well above the maximum possible van der Waals contribution. The increased adhesion energy was the result of increased hydrophobic interactions, which result whenever bilayers are stretched so that their hydrocarbon interiors become more exposed to the aqueous phase. Elucidation of the forces involved in the adhesion of bilayers is necessary to improve understanding of cell adhesion and help realize the applications of ve~icles.l-~ One reason for this lack of understanding is that a number of forces, some of which have not been generally recognized, play a role in these interactions. In addition to the wellknown attractive van der Waals and repulsive electrostatic double-layer and “hydration” forces,eS the importance of repulsive thermal “undulation” forces+12 and the recently is only now proposed attractive “hydrophobic” f0rces1~J~ being appreciated. The influence of hydrophobic and undulation forces on adhesion and fusion is not yet understood, especially in free v e s i c l e ~ . ~To ~ J aid ~ this understanding, we present electron micrographs of freezefracture replicas of adhering egg phosphatidylcholine (PC) vesicles under varied states of membrane tension. We show that the adhesion energy increases in bilayers under increased osmotic tension to values exceeding anything that
* To whom correspondence should be addressed.
(1) Hui, S. W.; Stewart,T. P.; Boni, L. T.; Yeagle, P. L. Science 1981, 212,921. Verkleij, A. J.; Mombera, C.; Leunissen-Bijvelt, J.; Ververgaert, P. H. Nature 1979,279,162. Hope, M. S.;Wong, K. F.; Cullis, P. R. J. Electron. Microsc. Tech. 1989,13,277. (2) Szoka, F., Jr.; Papahadjopoulos, D. Annu. Reu. Biophys. Bioeng. 1980. ~... , 9. -,467. (3)Fendler, J. H. Science 1984,223,888.Ringdorf, H. Angew. Chem., Int. Ed. Engl. 1981,20,305. (4)Gamon, B. L.;Virden, J. W.; Berg. J. C. J. Colloid Interface Sci. 1989,132,125. (5)Kaler, E. W.;Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989,245,1371. (6) Chernomordik, L. V.; Melikyan, G. B.; Chizmadzhev, Y. A. Biochim. Biophys. Acto 1987,906,309. (7)Rand, R. P.; Parsegian, V. A. Annu. Rev. Physiol. 1986,48,201. (8)Israelachvili, J. N. Intermolecular and Surface Forces: Academic Press: London, 1985. (9)Helfrich, W. 2.Naturforsch. 1978,33a,305. (10)Evans, E. A.; Parseaian, V. A. Proc. Natl. Acad. Sci. U.S.A. 1986. 83,7132. (11) Safinya, C. R.; Roux, D.; Smith, G. S.; Sinha, S . K.; Dimon, P.; Clark, N. A.; Bellocq, A. M. Phys. Reu. Lett. 1986,57,2718. (12)Servuas, R. M.; Helfrich, W. In Physics of Complex and Supramolecular Fluids; Safran, S. A., Clark, N. A., Eds.; Wiley: New York, 1987;pp 85-100. Servuss, R. M.; Helfrich, W. J. Phys. (Les Ulis, Fr.) 1989,50,809. (13)Israelachvili, J. N.; Pashley, R. M. Nature 1982,300,341. Pashley, R. M.; McGuiggan, P. M.; Ninham, B. w.; Evans, D. F. Science 1985,229,1088. Claeason, P. M.; Blom, C. E.; Herder, P. C.; Ninham, B. W. J. Colloid Interface Sci. 1986,114,234. (14)Helm, C. A.;Israelachvili, J. N.; McGuiggan, P. M. Science 1989, 246. - - - - 919.
(15)Mahanty, J.;Ninham, B. W. Dispersion Forces; Academic Press: New York, 1976.
0743-7463/90/2406-1326$02.50/0
could be explained by van der Waals forces. This enhanced vesicle adhesion under stress is therefore not the result of quenched undulation forces, and we show that it is likely due to an increased hydrophobic attraction. Our results also reconcile recent surface force measurements between bilayers14 with optical microscopy on highly swollen bilayers.12 For membranes of thickness 6 separated by an aqueous layer of thickness d, the interaction energy per unit area is the s u m of the attractive van der Waals and hydrophobic forces and of the repulsive hydration, electrostatic, and undulation forces:*ll
E = EvdW + Ehydrophobie + Ehydration + + The first term, the van der Waals attraction, is8
-[1% d2
AH 1 EvdW = +
1
(d
+ 26)’
-“-I + (d
6)2
(l)
(2)
A H is t h e Hamaker constant, which is relatively independent of the details of the bilayer or solution composition. For egg lecithin bilayers in aqueous solution, AH is about (1-6) X erg.12 Ehydrophobic has only recently been postulated to be an important interaction between bilayers. Between purely hydrophobic surfaces, this interaction is exponential with a decay length of about 1nm.13J6J7 The hydrophobic force can dominate the van der Waals force; however, there is as yet no theoretical description of the hydrophobic interaction. In tension-free bilayers in water, the hydrophobic regions of the bilayers are shielded from the aqueous phase, and there is little hydrophobic contribution to their interactions.16 However, recent work14has shown that the hydrophobic force is important between bilayers of increasing “hydrophobicity”. For lipid bilayers, this happens when the lipid head groups are forced to occupy a larger area than at equlibrium, resulting in greater exposure of the hydrocarbon portions of the molecules. Experimentally, this is accomplished by osmotic swelling of free surfactant vesicles, as was done here, by depositing (16)Marra, J.; Israelachvili,J. N. Biochemistry 1986,24,4608.Marra,
J. J. Colloid Interface Sci. 1986,107,446;1986,109,ll.Marra, J. Bio-
phys. J . 1986,50,815. (17) Israelachvili, J. N. In Physics of Complex and Supramolecular Fluids; Safran, S.A., Clark, N. A., Eds.; Wiley: New York, 1987;pp 101114.
0 1990 American Chemical Society
Langmuir, Vol. 6, No. 7, 1990 1327
Letters
Langmuir-Blodgett films a t surface pressures below equilibrium, or by depleting (thinning) adsorbed bilayen.14 This increased exposure of the hydrocarbon tails leads to a greater attraction and adhesion between bilayers. The remaining three interactions are repulsive and balance the van der Waals and hydrophobic interactions. The fiist is the hydration repulsion, which is the dominant repulsion a t small separation^:^*^ Ehyd = Hoexp(-dlXd (3) T h e next term, Eelec, is the screened electrostatic interaction, which does not arise for the uncharged zwitterionic vesicles examined here. The final term, the undulation interaction, is a steric repulsion that arises from thermal fluctuations and has the form9-12
(4) For egg PC bilayers, k,, the bending modulus, is 2 X 10-l2 erg,l2 and the undulations are small. (If k , is small compared to kT, undulation forces can dominate the interaction).s-l' Undulation forces can be reduced by putting bilayers under tension, e.g., by osmotic swelling of vesicles,which reduces the amplitude of the undulations that lead to repulsion.12 Although there has been much interest in the interactions of vesicles and membranes, experimental results for thermodynamic properties, such as the adhesion energy between vesicles, are rare. Previous experimental work has relied on the estimation of the membrane tension of a limited number of giant vesicles or swollen multimembranes (typically egg PC) observed by optical microscopy,and the adhesion energy was analyzed by using a modified form of the Young equation.12J8 By using smaller, monodisperse, unilamellar vesicles under known membrane tension induced by osmotic gradients, and then imaged by electron microscopy, we have been able to observe and analyze dozens of adhering vesicles. Unilamellar egg PC vesicles2J9with a mean diameter of 76 nm were prepared in either 10 or 20 mmol of glucosewater solution by repeated freeze-thaw cycles followed by repeated high-pressure extrusion (Lipex Biomembranes, Vancouver, BC) through 0.1-pm fiiters (Nucleopore,Pleasanton, CA).20 The initial lipid concentration was 100 mg/ mL in the 10-mmol glucose solution and 200 mg/mL in the 20-mmol glucose solution. Vesicles prepared in this way are spherical and fairly monodisperse, with a standard deviation of about 15 nm from the mean diameter.20 The samples were maintained at a temperature of 38 "C, well above the gel-liquid crystalline temperature of egg PC. An osmotic gradient was induced in the more concentrated lipid solution by adding an equal volume of water. The resultant lipid concentration was 100 mg/mL, and the solution exterior to the vesicles was 10 mmol of glucose; however, the solution trapped in the vesicle interior was still 20 mmol. For an ideal solution, the osmotic pressure across a vesicle membrane is
AP = RTAC (5) where hp is the difference in pressure between the inside ~~
(18) Evans, E. A. Biophys. J. 1980,31,425. Kwok, R.; Evans, E. A. Biophys. J . 1981,35,637. Evans, E. A.; ParSegian, V. A. Ann. N.Y. Acad. Sci. 1983, 416, 13. Evans, E.; Metcalfe, M. Biophys. J. 1984, 46, 423. (19) Egg Lecithin (egg phosphatidylcholine, egg PC) in chloroform/ methanol of >99% purity was purchased from Sigma (St. Louis, MO) and used without further purification. Water was filtered through activated charcoal, deionized, then distilled before we. Dextrose (D-glucose) was purchased from Fisher Scientific (Fairlawn, NJ). (20) Mayer, L. D.; Hope,M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986,858,161.
Figure 1. Model adhesion of two spheroidal vesicles. The adhering pair is characterizedby the radius r and contact angle
e.
and outside of the vesicle, R is the gas constant, T i s the temperature, and AC is the difference between the concentrations of the solute on the outside and the inside of the vesicle. Vesicles were extruded and kept at the same temperature of 38 "C throughout the experiments which were done within 24 h to minimize leakage of glucose from the vesicles. The permeability of glucose through lecithin bilayers is 7 X 10-l2 cm/s,21 resulting in a half-life for the applied osmotic gradient of about 50 h. The lateral tension, u, is related to the osmotic pressure by1* u = APr/2 (6) where r is the radius of the vesicle. This assumes that the vesicles were initially spherical (and not under tension), the state of minimum bending and elastic energy for small vesicles, which is consistent with the micrographs. This assumption is not a significant limitation to calculating membrane tension of the osmotically stressed vesicles but could contribute to the uncertainty in calculating the tension of the vesicles that were not under osmotic stress (see eq 8 below). The area expansion that a vesicle undergoes due to an osmotic pressure gradient isls
(7)
K is the area elastic modulus, about 140 erg/cm2 for egg PC,23A is the final, stretched vesicle area, and A0 is the initial, unstretched vesicle area. Egg PC vesicles are observed to rupture (lyse) if they are swollen more than about 4 % above their unstretched area, which is equivalent to a lateral tension of about 3-4 dyn/cm.= The maximum membrane tension in our experiments corresponds to an area increase of 0.4 % ,much less than that required to lyse the vesicles. To calculate the adhesion energy from measured contact angles, we m u m e that two identical spherical vesicles come into contact and remain spheroidal except for a flat contact as shown in Figure 1. The membrane area remains constant as the time for water permeation is fast (on the order of a few tenths of a second), and the elastic modulus for membrane stretching is large (140 erg/cm2) compared to our measured adhesion energy (I5 000 K/s) from a temperature of 38 o c .
(10)
in which Bi is the Biot modulus of the sample (ht/2k, where h i s the heat-transfer coefficient, t is the sample thickness, and k is the sample thermal conductivity) and T,- T,is the difference between the sample (38 "C) and cryogen (-190 " C ) temperatures. For a typical sample, Bi = 0.01 - 0.02, and the maximum gradient across the sample is of order 5 O C . As a result, across a typical vesicle diameter of about 0.1 cm there will be almost no spatial temperature gradient. Hence, we expect no rearrangements of the contacts due to temperature gradient induced changes in lipid composition. Also, in previous work, we have shown that these cooling rates are sufficient to maintain the room temperature distribution and orientation of similarly sized particles in aqueous solutions,22so we expect no freezinginduced aggregation. The frozen specimens were fractured at Torr and -170 "C in a Reichert-Jung Cryofract instrument, and then the fracture surfaces were replicated by evaporating 1.5 nm of platinum a t a 45O angle followed by 15 nm of carbon deposited normal to the fracture surface. Samples were brought to room temperature and pressure and then cleaned in distilled water and chloroethanol and mounted on gold electron microscope grids. Electron micrographs were taken a t 80 kV with a JEOL lOOCXII transmission electron microscope a n d photographically enlarged. Shadows (absence of platinum) appear light in the prints. Only the contact angles of similarly sized vesicles with symmetric shadows, oriented with their line of contact parallel to the direction of platinum shadowing, were measured. This ensures that the vesicles were fractured near their centers and that the shadowed region did not interfere with the measurements. In all cases, the images showed isolated spherical vesicles randomly distributed across the replica. Vesicles not fractured near their centers appear smaller and often seem elliptical in shape; however, this is due to the relative orientation of the fracture plane with respect to the vesicles (24)
Figure 2. Freeze-fracture electron microscope image of two egg PC vesicles in adhesive contact. The contact angles are 3 5 O and 36". The sample contained 100 mg/mL egg PC in 10 mM glucose
. 1
Figure 3. Two egg PC LUVs in adhesive contact in the presence of a n osmotic pressure g r a d i e n t . T h e i n t e r i o r glucose concentrationwas 20 mmol, and the exterior concentration wm 10 mmol. The contact angles are 49' and 43O. The sample was rapidly frozen (>E OOO K/s) from a temperature of 38 "C.
and the distorting effects of the shadows. There is no indication of aggregation due to ice crystallization, and the bilayer surfaces are smooth and homogeneous. This is consistent with earlier work that shows this technique does not result in artifacts of sample preparation.22 A small fraction of vesicles exhibited symmetric adhesionsas shown in Figure 2 for the case of no applied osmotic gradient and Figure 3 for the applied osmotic gradient. There appeared to be more adhesive contacts in the samples under the applied osmotic gradient, but this has not yet been examined quantitatively. The presence of an osmotic gradient seemed to facilitate adhesion but was not a necessary condition, as the samples under no osmotic stress aggregated into large multilayered liposomes that were easily visible in the sample vials after several days. The contact angles in Figure 2 for the nonosmotically swollen vesicles are = 35" and B2 = 36". The contact
Langmuir, Vol. 6, No. 7, 1990 1329
Letters
lo
h
e
Es
6
L
4
0
2
0 1 5 20 2 5 30 3 5 6 0 4 5 50 5 5 60 6 5 7 0
Contact Angles
Figure 4. Histogram of measured contact angles of osmotically swollen and nonswollen vesicles. Average contact angle for osmotidly swollen vesicles is 4 5 O and for nonosmotidly swollen vesicles is 3 6 O . angles for the osmotically swollen vesicle pair in Figure 3 a r e 6, = 49' a n d 6 2 = 4 3 % . T h e error in this measurement was *4O. Approximately 50 contacts of both types were evaluated and graphed as a histogram of the measured contact angles in Figure 4. The nonswollen vesicles had contact angles ranging between 15O and 53O. In contrast, the contact angles of the swollen vesicles ranged from 33' to 6 7 O , with most between 40" and 50". The average contact angles between pairs of nonswollen and swollen vesicles were 36" and 45", respectively. We observed no 'contact rounding" as described by Servuss and Helfrich,'* most likely due to the relatively high bilayer tensions and large adhesion energies in our system of small (30 pm), low-tension vesicles and multilayer~.Contact rounding has not generally been observed in systems of small unilamellar vesicles undergoing adhesion.' For the nonswollen vesicles (0 = 36O and Ci = 10 mmol), the membrane tension u (due to the slight increase in solute concentration which results from the small volume dyn/cm and the corresponding contraction) is 6.0 X erg/cm2. For the adhesion energy W = 2.3 X osmotically stressed vesicles (0 = 45' and Ci = 20 mmol), u = 0.52 dyn/cm and W = 0.30 erg/cm2. The adhesion energy is plotted against the membrane tension in Figure 5, along with earlier results obtained by Servuss and Helfriehl2 using giant vesicles and multilayers. Over a range of tensions, the adhesion energy increases linearly, which from eq 9 is consistent with the observations that the average contact angle is always in the range 35-45O. Our results are also consistent with those of Evans and Metcalfe,'8 who measured an adhesion energy of 0.015 erg/ cm2 over a range of tensions of 10-2-10-1 dyn/cm. Also shown in Figure 5 is the range of van der Waals attraction expected in this system hy using eq 2 with bilayers of thickness d = 4 nm separated by 6 = 2-2.5 nm of water. The values of d and 6 were chosen from measured equilibrium spacings of multilamellar egg lecithin in excess water and from adhesive contacts in surface force meas~ements.'~ In the absence of other attractive forces,
-6
-5
-3 -2 Log (0) (dyneslcm)
-4
-1
Figure 5. Logarithmic plot of measured adhesion energy M membrane tension. The shaded area is the expected maximum adhesion that could result from van der Waals forces alone. If the increased adhesion energy was a result of only quenching undulation repulsion, then the cuwe should asymptotically approach the shaded region. An additional attractiveinteraction, the hydrophobic interaction, is necessary to explain the large adhesion energies. the only interaction leading to adhesion is the van der Waals interaction. If this were true, we would expect that as membrane tension increased, the undulation repulsion would be removed, and the adhesion energy should asymptotically approach the van der Waals limit. From OUT data, this is clearly not the case; in fact, the measured adhesion energies rise to well above the van der Waals interaction. A possible explanation is that the hydrophobic interaction contributes to the adhesion energy of free (but stressed) bilayer vesicles. As the membrane is put under increased tension, the area per lipid molecule is increased proportionately (see eq 7),resulting in greater contact of the hydrocarbon bilayer interior with the aqueous phase. This leads to an increased hydrophobicity of the bilayer surface and hence an increase in the magnitude (and range, see ref 14) of the hydrophobic interaction. Figure 5 confirms surface force measurements" and shows that measurements of forces between mica-deposited lipid layers have a direct bearing on the interactions of free vesicles in solution. It also shows that the interactions between membranes are highly dependent on their state of tension. Other recent studies have also implicated the hydrophobic interaction in the adhesion and fusion of membranes.2"" The modification of membrane tension, and through this tension intermembrane interaction via the hydrophobic force, might be a general mechanism by which cells control specific adhesion sites via alterations in membrane composition, protein conformation, or lipid fluidity. (25) Maezawa, S.;Yoshimura, T.;Hong, K.;Dfizg~bes,N.;Papahad-
D.:Bioehemistrv 1989.28. 1422. .ioooulm. .
l2fi) ~ l w i k H.; . Nil&, H.;Sve-n. K. E.; Askmdahl, A,; Nihan. U. R.; LundstrBm, 1. J . Collord lnrwface SCL 1988, 125, 139. 127) Stepmann. T.; Nir. S.; Wilsehut. J.; Boxhemutry 1985,28,1698. 128) H'wdburv. D. J.: Hall. J. I? Uioohvs. J . 1988.54. 1053.
(29) We thank-Dr. W. Helfich and DL E : Evans for & b y enlightening discussions on membrane interactions and Dr. C. Helm for ongoing collaborations on hydrophobic forces. We acknowledge f i e i a l support from the National Science Foundation under Grants CBT86-57444 (J.A.N.Z.) and CBT87-21741(J.N.I., J.A.N.Z., S.M.B., and S.C.)
