Biophysical Journal 87:1013-1033 (2004)
© 2004 The Biophysical Society
The Influence of Short-Chain Alcohols on Interfacial Tension, Mechanical Properties, Area/Molecule, and Permeability of Fluid Lipid Bilayers
Hung V. Ly and
Marjorie L. Longo
Department of Chemical Engineering and Material Science, University of California, Davis, California
Correspondence: Address reprint requests to Marjorie L. Longo, Tel.: 530-754-6348; Fax: 530-752-1031; E-mail: mllongo{at}ucdavis.edu.
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ABSTRACT
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We used micropipette aspiration to directly measure the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2-oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols at alcohol concentrations ranging from 0.1 to 9.8 M. The order of effectiveness in decreasing mechanical properties and increasing area per molecule was butanol>propanol>ethanol>methanol, although the lysis strain was invariant to alcohol chain-length. Quantitatively, the trend in area compressibility modulus follows Traube's rule of interfacial tension reduction, i.e., for each additional alcohol CH2 group, the concentration required to reach the same area compressibility modulus was reduced roughly by a factor of 3. We convert our area compressibility data into interfacial tension values to: confirm that Traube's rule is followed for bilayers; show that alcohols decrease the interfacial tension of bilayer-water interfaces less effectively than oil-water interfaces; determine the partition coefficients and standard Gibbs adsorption energy per CH2 group for adsorption of alcohol into the lipid headgroup region; and predict the increase in area per headgroup as well as the critical radius and line tension of a membrane pore for each concentration and chain-length of alcohol. The area expansion predictions were confirmed by direct measurements of the area expansion of vesicles exposed to flowing alcohol solutions. These measurements were fitted to a membrane kinetic model to find membrane permeability coefficients of short-chain alcohols. Taken together, the evidence presented here supports a view that alcohol partitioning into the bilayer headgroup region, with enhanced partitioning as the chain-length of the alcohol increases, results in chain-length-dependent interfacial tension reduction with concomitant chain-length-dependent reduction in mechanical moduli and membrane thickness.
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INTRODUCTION
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The interaction of short-chain alcohols with biological membranes forms an important area of exploration because of the role of alcohols in metabolism, membrane fusion, drug delivery, alcohol toxicity, alcohol tolerance, and general anesthesia. Of particular interest is fundamental understanding of the molecular mechanism of action of short-chain alcohols on the lipid portions of biomembranes. To this end, model systems of known lipid composition such as unilamellar vesicles or supported bilayers have served as ideal platforms to elucidate the details of the effect of short-chain alcohols on the physical and thermodynamic properties of lipid membranes.
The emerging paradigm is that short-chain alcohols (given their amphiphilic nature) primarily confine themselves to the hydrophilic headgroup region instead of the hydrocarbon core as demonstrated by nuclear magnetic resonance (NMR; Barry and Gawrisch, 1994
), nuclear Overhauser effect spectroscopy (Feller et al., 2002; Holte and Gawrisch, 1997), fluorescence spectroscopy (Rottenberg, 1992), and Fourier-transform infrared studies (Chiou et al., 1992). Their location in the headgroup region disturbs the natural microstructure of the lipid membrane and is apparently responsible for observed increases in membrane fluidity or disorder (Chin and Goldstein, 1977), increases in the membrane lipid lateral mobility (Chen et al., 1996), decreases in the main phase transition temperature (Rowe, 1985), and the formation of an interdigitated gel phase (Slater and Huang, 1988). Past works have also shown that alcohols increase membrane permeability (Komatsu and Okada, 1995, 1997), induce shape transformations of vesicles (Angelova et al., 1999), and influence membrane thermodynamic parameters (Rowe et al., 1998; Trandum et al., 2000; Westh and Trandum, 1999; Westh et al., 2001). Other important physical properties that short-chain alcohols may affect, but which need exploring, are mechanical properties and thickness, which are closely tied to integral membrane protein activities and cell shape. A close relationship between mechanical stress and activities in membrane-embedded proteins has been established with mechanical sensitive ion channels (Sukharev et al., 1997), gramicidin (Goulian et al., 1998), and the meta-I to meta-II transition in rhodopsin (Mitchell and Litman, 1999, 2000). Theoretical works exist relating the structure, function, and surface arrangement of membrane-bounded proteins and enzymes to membrane mechanics and thickness (Cantor 1997a,b; Dan et al., 1994; Dan and Safran, 1998). Shape and stability of cells and liposomes are regulated by mechanics as demonstrated by observed shape deformations of erythrocytes (Evans, 1989) and neutrophils (Tsai et al., 1993) and vesicle budding-fission-fusion events involved in vesicle-mediated material transport (Sackmann, 1994). Certainly, a quantification of the perturbation of these mechanical properties and thickness by short-chain alcohols would be relevant in understanding the general mechanisms behind alcohol's adverse action on biological functions.
Until recently, quantitative studies of the effect of alcohol on membrane mechanics and thickness of fluid-phase membranes were unconsidered in literature. We have recently reported how ethanol can modify the mechanical properties and decrease the membrane thickness (from measuring the area expansion of fluid-phase lipid vesicles with the inherent assumption that lipid chain volume is conserved; Ly et al., 2002). Motivated by this earlier study, we now explore a homologous series of n-alcohols from methanol to butanol to understand the role of acyl chain-length on membrane mechanics and thickness. As before, we applied the micropipette aspiration technique, pioneered by Evans and Needham (for general reviews of the technique see Needham and Zhelev, 1996, 2000), to directly measure these mechanical properties (bending modulus, area compressibility modulus, lysis tension, and lysis strain) and area expansion of individual giant unilamellar vesicles (GUVs) in a series of alcohol/water mixtures over a range of concentrations (
0.1 to 9.8 M). We chose 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC) mixed with a minute amount of 1-stearoyl-2-oleoyl-phosphatidylserine (SOPS) as our model membrane because of its similarities to the natural fluidity and thickness of biological membranes. Although each alcohol in our series differs from its neighbor by one CH2 group, we observed quantifiable variations in mechanical properties and area expansion of SOPC membranes exposed to aqueous solutions containing methanol through butanol. We relate this effect to lowering of interfacial tension by showing that the area compressibility data follows Traube's Rule, which predicts that for each CH2 group in an alcohol molecule, three-times-lower alcohol concentration will be required to reach the same interfacial tension between a hydrophobic and water/alcohol solution (Traube, 1891). We then convert area compressibility to interfacial tension to verify and show for the first time that bilayers adhere to Traube's rule, determine partitioning and Gibbs free energy of adsorption of each alcohol into the lipid headgroup region, and predict the increase in the area per headgroup and decrease in line tension of a membrane pore due to interfacial tension reduction by each alcohol (methanol through butanol). Previous to this work, the partitioning and Gibbs energy values had not been determined in this way. We used flow-pipette experiments to directly measure the increase in area per headgroup and membrane permeability for bilayers exposed to short-chain alcohols. We believe that this is the first measurement of short-chain alcohol permeability of a model membrane. Finally, we relate line tension reduction (determined by interfacial tension reduction) to the observed lowered breaking strength (lysis tension) of the membrane.
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EXPERIMENTAL PROCEDURE
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Materials
SOPC and SOPS were purchased in chloroform from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Ethanol (200 Proof) was bought from Gold Shield Chemical (Hayward, CA). Methanol, 1-propanol, and 1-butanol, sucrose, and glucose of high grade were bought from Sigma-Aldrich (St. Louis, MO). Chloroform was purchased from Fisher Scientific (Fairlawn, NJ). Bovine serum albumin (fraction V, low heavy metals) was purchased from Calbiochem (San Diego, CA). Deionized water used was produced by a Barnstead nanopure water system (Dubuque, IA) and had a resistivity of 18.1 M
/cm.
Giant vesicle preparation
Our electroformation protocol to grow GUVs of SOPC and SOPS (99.5:0.5 mol %) is similar in nature to other practitioners of this technique (Angelova et al., 1992; Shoemaker and Vanderlick, 2003). We began with painting 50 µl of an SOPC and SOPS (99.5:0.5 mol %) solution in chloroform/methanol (2:1 volume ratio, 0.5 mg/ml) evenly on two parallel platinum wires (diameter 1 mm) held 3 mm apart in an open-center Teflon block. We used a slow-flowing nitrogen stream to quickly evaporate the solvent from the wires, and afterward placed the block under vacuum for at least 2 h to remove any trace solvent. We sealed the open center on both sides with SurfaSil-coated (Pierce, Rockford, IL) coverslips with vacuum grease. We used a side opening on the block to fill the open volume slowly with 100 mM sucrose and applied an AC field (3 volts peak-to-peak) across the wire at 10 Hz for 30 min, 3 Hz for 15 min, 1 Hz for 7 min, and 0.5 Hz for 7 min. GUVs formed on the electrodes, and we gently harvested and used them within a few days.
Micropipette aspiration setup
Our micropipette aspiration setup included an inverted Nikon Diaphot 300 microscope (Nikon, Melville, NY) equipped with a 40x Hoffman modulation contrast objective (Modulation Optics, Greenvale, NY), a high resolution charge-coupled device camera (Dage MTI, Michigan City, IN), a manometer slider with linear encoder (Velmex, Bloomfield, NY), a video overlay box (Polvision, Western Australia), and an S-VHS recorder (Sony SVO-9500MD). Pressure readings from the slider were overlayed onto vesicle images. We controlled the position of the micropipette with a micromanipulator (model MHW-3, Narishige, Japan). Micropipettes were made from pulling capillary glass tubings (Fredrick and Dimmock, Millville, NJ) into thin tapered shafts with a glass puller (David Kopf Instruments, Tujunga, CA). We fractured the tips flat with inner diameters of 6–8 µm with a microforge (Stoelting, Wood Dale, IL) and then coated the tips with SurfaSil (Pierce) to passivate the surface. We filled the pipettes with 100 mM glucose, 0.02% total wt. albumin solution. Albumin was necessary to reduce static charge buildup on the glass surfaces that could prematurely lyse the vesicles.
The sample chamber, used to house the vesicles in alcohol/water mixtures, was constructed out of two Surfasil-coated microscope slides separated by two small rectangular Plexiglas spacers. We filled the 1-ml cavity with an alcohol/water mixture containing 
105 mM glucose. Alcohol and the osmotic imbalance (between the exterior glucose and interior sucrose concentrations) can change the surface area/volume of the vesicles. We desired slightly deflated vesicles for aspiration so less glucose (105–70 mM) was included in the alcohol/water mixtures as the alcohol concentration increased from 0 to 10 M. We added 10 µl solution of electroformed vesicles enclosing 100 mM sucrose into the chamber. On the timescale that we waited to start aspiration of a vesicle, >5 min, the alcohol concentration outside the vesicle had equilibrated with the concentration inside the vesicle, as will be shown in the results. We easily found vesicles lying on the bottom of the chamber due to the density difference between sucrose and glucose. We inserted an aspiration pipette into one of the open sides of the chamber and a flow pipette into the other when we were conducting flow experiments. Evaporation during the experiments was virtually eliminated by enclosing the sample chamber in a modified petri dish containing a thin layer of an alcohol/water mixture with small openings for pipettes and a viewing port for the objective.
Micropipette aspiration experiments
We performed the micropipette aspiration technique to measure the mechanical properties of vesicles in alcohol/water mixtures at room temperature. We aspirated individual vesicles inside a glass micropipette, and the applied suction pressure, 
P, was related to the induced isotropic membrane tension, 
, as follows (Evans and Needham, 1987),
 | (1) |
where Rp is the radius of the pipette and Rv is the radius of the vesicle. As we increased the suction pressure, the projection length, L, increased (Fig. 1). The change in projection length, L, is related to the observed or apparent area strain, 
, where = (A–Ao)/Ao, with Ao being the membrane area of the vesicle measured at an initial low tension state and A being the membrane area after pressurization to a higher tension state. Through simple geometric arguments, the relationship reduces to (Evans and Needham, 1987)
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FIGURE 1 Video micrographs of an SOPC vesicle aspirated inside a micropipette in an aqueous bathing solution without alcohol. An increase, L, in projection length, L, is observed when the applied membrane tension, , is increased from (A) 0.3 mN/m to (B) 3.4 mN/m. (C) When the same vesicle, held at 0.3 mN/m, is exposed to a flowing stream of an alcohol/water solution of the same osmolarity as the aqueous bathing solution, an increase, L, in projection length, L, is observed. The transmembrane exchange scheme of alcohol from a flow pipette to a vesicle is shown. The value Cout denotes alcohol concentration inside the flow pipette; Cads is the total surface density of adsorbed alcohol molecules in the membrane; Cin denotes the alcohol concentration inside the vesicle; and kon and koff are the rate constants for adsorption and desorption, respectively.
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For aspiration, we chose vesicles with diameters between 20 and 40 µm that were slightly deflated. The excess membrane area aided in forming a small visible projection inside the pipette at an initial low tension. In all experiments, vesicles were prestressed beyond 1 mN/m for 1–2 s to smooth out any folds or tethers in the membrane and relaxed back to a desired low tension. We increased the suction pressures in a stepwise manner and waited 5 s after each step to provide good images for later analysis. For convenience, we performed separate micropipette aspiration experiments in the low and high tension regimes although at times we did aspirate vesicles through the full range. We aspirated vesicles from 0.001 to 0.5 mN/m to determine their bending moduli and aspirated other vesicles from 0.5 mN/m until rupture to determine their area compressibility moduli, lysis tensions, and lysis strains at rupture. We video-recorded the aspiration experiments onto tapes and afterward measured the diameters of the vesicles, the projection lengths, and applied pressures with image analysis software (ATI Multimedia Center, Marlborough, MA). We applied the equations discussed below to determine the mechanical properties.
The mechanics of thin materials show that increases in comes from two modes of deformations of a vesicle under aspiration (Evans and Rawicz, 1990; Rawicz et al., 2000),
 | (3) |
where A is the membrane area, kc is the bending modulus, KA is the direct area compressibility modulus, kB is the Boltzmann's constant, T is absolute temperature, and co is a constant (0.1) that depends on the type of modes (spherical harmonics or plane waves) used to describe surface undulations. Thermal shape undulations of flaccid vesicles are readily seen under a microscope, and the first term represents the smoothing out of these bending undulations. The second term represents direct stretching of the area per lipid molecule. We ignore any contribution in from enhanced partitioning of molecules into the membrane as tension increases. A thermodynamic analysis of alcohol partitioning (presented in the Appendix) shows that the increase in surface density of alcohol molecules in the membrane is small and would have negligible effect on the strain during the mechanical pressurization of vesicles until failure.
Experimentally, we calculated the bending modulus from plotting ln() versus in the tension range of 0.001 to 0.5 mN/m. Presented in Fig. 2 is a typical plot of natural log tension-strain behavior of SOPC vesicles in a 7.4 M methanol/water mixture. In this low tension regime, the logarithmic term dominates and shows that almost all increases in come from smoothing out thermal undulations, and the bending modulus is simply the product of the slope and kbT/8
.
We calculated the area compressibility modulus by plotting versus in the high tension regime ( > 0.5 mN/m). In this regime, the linear term, /KA, dominates as illustrated in Fig. 2 for a vesicle in a 7.4 M methanol/water mixture. By convention, the slope is known as the apparent area compressibility modulus, Kapp (Rawicz et al., 2000). This modulus includes a small contribution from the logarithmic term because even at the highest tension, subvisible thermal undulations persist. We determined the direct area compressibility modulus, KA, by subtracting the logarithmic contribution from to get the direct area strain, dir. The appropriate contribution, (i), for each ith value of that we subtracted out is (Rawicz et al., 2000)
 | (4) |
where kc,avg is the average bending modulus and (1) is the initial low tension state, usually experimentally set at 1 mN/m. KA is the revised slope from plotting versus dir (square points in Fig. 2). In some cases where measured values of kc,avg were unavailable, we used linear interpolated values, but we note that varying the bending modulus at ±20% generally changed the average KA values by ±5%.
In the micropipette aspiration experiments, we subjected vesicles to a range of incremental alcohol concentrations where significant effects on the bending and area compressibility moduli could be observed. Alcohol concentrations were experimentally measured in volume percentages, but they are presented here in molar concentrations to compare correctly the mechanical properties of SOPC membranes as a function of alcohol chain-length. For the bending modulus experiments, the concentrations (M) and number of experiments performed at each concentration (in parentheses) were: 0 (32), 2.47 (9), 4.94 (7), and 7.41 (11) for methanol; 1.70 (6) and 3.41 (12) for ethanol; 0.39 (6), 0.78 (14), and 1.30 (12) for propanol; and 0.22 (11) and 0.55 (10) for butanol. For the area compressibility modulus experiments, the concentrations (M) and number of experiments performed at each concentration (in parentheses) were: 0 (42), 2.47 (16), 4.94 (15), 7.41 (17), and 9.88 (12) for methanol; 1.70 (10), 2.56 (11), and 3.41 (17) for ethanol; 0.19 (10), 0.39 (10), 0.78 (19), and 1.30 (19) for propanol; and 0.11 (12), 0.33 (13), and 0.55 (6) for butanol.
Flow-membrane area expansion experiments
We performed flow experiments to measure membrane area expansion of vesicles caused from alcohol exposure. We aspirated individual vesicles into a micropipette (7–9 µm in diameter) at a low tension of 1 mN/m in a 105 mM glucose mixture. Afterward, we placed a flow pipette (100–150 µm in diameter) directly in front of the aspirated vesicle (see Fig. 1 C) to deliver a constant flow of 105 mM glucose, alcohol/water mixture around the vesicle. We video-recorded the dynamic growth of the projection length until the projection length reached a plateau, the projection broke away from the vesicle, or the vesicle collapsed. We subsequently analyzed the tapes to convert the dynamic growth, L, of the projection length into an apparent area expansion, (A/Ao)exp, where (A/Ao)exp = (A–Ao)/Ao, with Ao being the initial surface area of the vesicle before alcohol exposure and A being the surface area after exposure. Values for (A/Ao)exp are determined through Eq. 2. For vesicles with stable projection growth, we only observed changes in the projection length for (A/Ao)exp values up to 0.15, and the radius of the outer portion of the vesicle remained relatively unchanged within a resolution limit of 0.5 µm.
We made the flow pipettes from drawing capillary glass tubings into constant diameter shafts with the glass puller. We fractured the pipettes for diameters between 100 and 150 µm with the microforge, and the tips were coated with SurfaSil. The flow pipette was connected directly to a syringe with plastic tubing and filled with the desired alcohol/water mixtures. We set the syringe pump (KD Scientific Model 200, New Hope, PA) to drive the flow at a constant velocity of 450 µm/s. The flow velocity was measured by observing polystyrene microspheres (Polysciences; 3 µm in diameter) flowing out of the pipette.
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RESULTS
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Methanol, ethanol, and propanol are miscible in water in all proportions whereas butanol has a limited solubility of 1 M (Brown et al., 2000; Raina et al., 2001b). For all experiments, the maximum alcohol concentration we used for methanol, ethanol, propanol, and butanol were 9.8 M, 3.4 M, 1.3 M, and 0.55 M, respectively. Below this limit, SOPC vesicles in these alcohol/water mixtures were sufficiently stable for micropipette aspiration. Although alcohols can increase the permeability of the membrane to solutes (Ingram, 1990; Mansure et al., 1994), we did not observe any appearances of swollen or ghost vesicles in alcohol/water mixtures during the experimental timescale of a few hours, meaning that no significant amount of glucose or sucrose transferred across the membrane. (Swollen vesicles would be seen if glucose permeated into the vesicle's interior, concomitantly with water. Ghost vesicles are semitransparent due to the loss index of refraction from mixing of exterior glucose and interior sucrose.)
Bending and area compressibility moduli measurements
Presented in Fig. 3 are the bending moduli obtained for the membranes of SOPC vesicles in alcohol/water/glucose mixtures using micropipette aspiration. There is an obvious dependence on acyl chain-length and concentration of alcohol. This is evident by the separation between the curves and the initial steepness in the curves, respectively. For a given concentration, butanol decreased the bending modulus the most followed by the other alcohols in sequence. The average kc value decreased from 0.8 x 10–19 J to 0.4 x 10–19 J (a 50% reduction) as the alcohol concentration reached the high limit in our studied concentration range. Our bending modulus results qualitatively agree with the works of Safinya et al. (1989), who demonstrated that the bending modulus of fluid-phase dimyristoylphosphatidylcholine (DMPC) decreased upon addition of pentanol.
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FIGURE 3 Average bending modulus, kc, values of SOPC vesicles in alcohol/water mixtures: methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles). Bars indicate 1 SD. Differences between control and alcohol-exposed vesicles were statistically significant (P < 0.05) as evaluated by Student's t-test ( = 0.05) except for values at 1.70 M of ethanol and 0.39 M of propanol.
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Fig. 4 contains plots of ln() versus for individual vesicles in four alcohol/water mixtures in the low tension regime of 0.001–0.5 mN/m with individual Ao values set at 0.001 mN/m. We chose data in which the bending modulus of each line is representative of the population average value found in Fig. 3. As we demonstrated previously with ethanol, we generally observe that ln() is linearly proportional to until a crossover tension of 0.5 mN/m for all four alcohols. The linearity indicates that the bending modulus and alcohol interactions with the membrane are independent of the amount of membrane fluctuations that were smoothed out in this low tension regime.
Presented in Fig. 5 are the apparent and true area compressibility moduli values (Kapp and KA) obtained for the membranes of SOPC vesicles in alcohol/water/glucose mixtures using micropipette aspiration. Obtained from the raw data (Fig. 7), Kapp represents both direct stretching and the smoothing of bending fluctuations whereas KA only includes direct stretching of the bilayer. For both of these quantities, there are obvious dependences on acyl chain-length and concentration of the alcohol. For all four alcohols, the average values of Kapp decreased from 200 mN/m to 100 mN/m (a 50% reduction) and average values of KA decreased from 230 mN/m to 150 mN/m or less (a 35% reduction) as alcohol concentration reached the high limit. Independent of alcohol type, we observed the difference between KA and Kapp slightly widens as the alcohol concentration increases. This reflects the significant role of the logarithmic term in Eq. 3 as the magnitude of the bending modulus decreases with increasing alcohol concentration. Since Kapp and KA are dependent mechanical properties, we expect all points in Fig. 5 to collapse on the same curve if we plot KA versus Kapp, regardless of which alcohol interacts with the SOPC membrane. This is evident in Fig. 6, and it demonstrates the accuracy and reproducibility of our mechanical measurements.
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FIGURE 5 Average area compressibility modulus, KA (solid marks) and Kapp (open marks), of SOPC vesicles in alcohol/water mixtures. Symbols are methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles). Bar indicates 1 SD. For clarity, only one representative error bar of all the measurements is shown (all error bars were equal or less than this one). Differences between control and alcohol-exposed vesicles were statistically significant (P < 0.05) as evaluated by Student's t-test ( = 0.05).
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FIGURE 6 Correlation between KA and Kapp values from Fig. 5 at various alcohol/water mixtures: methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles).
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From the area compressibility data in Fig. 5, it can be seen easily that at any particular KA value, the concentration of alcohol required to reach that KA value was reduced by approximately a factor of 3 for each additional CH2 group. For example, at a KA value of 140 mN/m, the concentrations (M) for methanol, ethanol, propanol, and butanol are 9.88, 3.41, 0.78, and 0.3, respectively. Interestingly, this finding agrees with Traube's rule, a general rule for the expected interfacial energy of a hydrophobic-water/alcohol interface due to alcohol adsorption at the interface (Traube, 1891)—a point that we will discuss in detail in the next section.
Fig. 7 contains plots of versus for individual vesicles in four alcohol/water/glucose mixtures in the high tension regime ( > 0.5 mN/m) with individual Ao values set at 0.5 mN/m. We chose data in which the area compressibility modulus of each line is representative of the population average value found in Fig. 5. The versus for individual vesicles is linear in the high tension regime up to the rupture tension for all four alcohols (see Fig. 7) and is still linear when subvisible thermal undulations were removed from (data not shown). The linearity indicates that the area compressibility modulus is constant up to the lysis strain of 0.04, and within this range of areal strain, the alcohol concentration in the membrane remains relatively unchanged as the membrane is being laterally stretched (as expected from the discussion in the Appendix). The linearity we observed for strains up to 0.04 for all the surface-active alcohols qualitatively agrees with the work of Santore et al. (2002), who showed that surfactant (Pluronic L31) binding to unilamellar polymeric (OE7) vesicles exhibited a linear behavior for strains up to 0.05. However, polymeric vesicles are more stretchable than lipid membranes, and they showed that nonlinearity occurred for strains between 0.05 and 0.20, possibly due to significant exposure of the hydrophobic membrane core and subsequent enhanced adsorption capacity for surfactant molecules.
Flow experiments to observe bilayer area expansion
When aspirated vesicles in an aqueous solution without alcohol were exposed to an alcohol/water/glucose mixture (by a flow pipette), we generally observed that the projection quickly increased within <1 min. The change in projection length, L, was converted to an area expansion, (A/Ao)exp, where the increase in membrane area from flow is normalized by the initial membrane area without flow (through Eq. 2).
Fig. 8 shows example time evolutions of the increase and decrease in (A/Ao)exp for SOPC membranes exposed to (and removed from) a flowing stream of methanol, propanol, and butanol. For all alcohol/water mixtures, the major increase and decrease in (A/Ao)exp occurred within <30 s and the process was reversible.
For (A/Ao)exp below 0.05, we generally observed that the projections reached stable plateaus, indicating that the internal alcohol concentration had equilibrated with the exterior flow alcohol concentration. For (A/Ao)exp between 0.05 and 0.15, some projections reached stable plateaus whereas others broke away from the vesicle or the vesicle collapsed or flew away. For (A/Ao)exp above 0.15, we generally did not observe any stable projection growth. These observations show that as the membrane expands rapidly beyond 0.05, there is a higher probability that the projection will pinch and break away from the main body of the vesicle. For vesicles with projections that reach plateaus, we never observed any retraction of the projections. This shows that short-chain alcohols, within the experimental range of concentrations, did not induce membrane pores large enough for external glucose to filter into the vesicle's interior with a co-transport of water. If pores were to form, we would observe vesicle swelling and a retraction of the projection as seen in the works of Longo et al. (1998, 1997) with virus fusion peptide and works of Needham and Zhelev (1995) with lysolipid.
When aspirated vesicles in an aqueous solution without alcohol were exposed to a flow of the same solution, we expected the projection length to remain constant. However, we observed that the projection grew slightly, and the growth appeared proportional with flow velocities between 100 to 500 µm/s. When the flow was removed, the projection returned to its original position. With the flow set at 450 µm/s, a positive applied pressure of 330 ± 18 Pa (or an effective of –0.88 ± 0.07 mN/m) was required to cause the projection to return to its original position. Presently we do not know the cause behind the growth and attribute it to general flow effects. We have ruled out folds or small tethers incorporating into the membrane of the aspirated vesicle because we prestressed vesicles above 2 mN/m for 1–2 s and relaxed them to a holding tension of 1 mN/m to remove such entities. We have also ruled out flow pressure because the pressure drop across the vesicle for flow around a sphere at low Reynolds number is negligible, <30 Pa (or an effective -value below 0.05 mN/m) for the flow velocities we were utilizing. For all the experimental data we present here, we settled on a fast flow velocity of 450 µm/s (much higher than what we used in our previous work on ethanol) to ensure that the local alcohol concentration on the surface of the vesicles was uniform and of similar concentration as in the flow pipette. At this velocity, the flow-induced projection growth was typically small and corresponded to a spike of (A/Ao)exp of 0.022 ± 0.005 within 1 or 2 s of exposure.
Lysis tension and lysis strain
Presented in Fig. 9, A and B, are the direct lysis area strain, dir-lyse, and lysis tension, lyse, of membranes of SOPC vesicles in alcohol/water/glucose mixtures using micropipette aspiration at a fairly fast 0.1 mN/m/s tension ramp. The value lyse is the tensile strength of the membrane at the point of rupture. The apparent lysis strain, lyse, is the corresponding fractional increase in membrane area at rupture. The value dir-lyse was obtained by subtracting the contribution due to smoothing of thermal shape undulations, (i), from lyse using Eq. 4. Hence, dir-lyse accounts for direct lateral stretching of the area per lipid molecules in the membrane before membrane failure. The value dir-lyse in Fig. 9 A has no clear dependence on acyl chain-length or alcohol concentration, and the value averages 0.036 ± 0.006. Fig. 9 A generally shows that the SOPC membrane cannot be stretched beyond 0.05, consistent with previous findings (Evans and Needham, 1987). Consequently, the KA values dictate the lyse values since lyse is given by KAdir-lys. Therefore, lys and KA show the same downward trends as acyl chain-length and concentration of alcohol are increased as evidenced by the striking similarity between Fig. 5 and Fig. 9 B. The lys values are decreased by as much as 50%, once again demonstrating the severe membrane destabilization effect of alcohol.
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FIGURE 9 (A) Average direct lysis area strain, dir-lyse, of SOPC vesicles in alcohol/water mixtures obtained after subtracting out from the apparent lysis area strain, lyse, the area increases due to smoothing out thermal shape undulations. Differences between control and alcohol-exposed vesicles were statistically significant (P < 0.05) as evaluated by Student's t-test ( = 0.05) except for values at 2.47 M of methanol, 1.30 M of propanol, 0.33 M, and 0.55 M of butanol. (B) Average lysis tension, lyse, values of SOPC vesicles in alcohol/water mixtures. Differences between control and alcohol-exposed vesicles were statistically significant (P < 0.05) as evaluated by Student's t-test ( = 0.05), except for values at 2.47 M and 4.94 M of methanol and 0.11 M of butanol. Symbols are methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles). Values are based on 10 vesicles or more. Bars indicate 1 SD.
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DISCUSSION
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Bilayer interfacial tension, area per headgroup, and membrane thickness
The mechanical aspiration results presented in this study show, via decreases in the bending and area compressibility moduli, that less work is required to smooth out thermal undulations or stretch the area per lipid of a bilayer in an alcohol/water mixture compared to water alone. Certainly, this reflects a change in the intermolecular forces between the lipid membrane and water phase, or stated another way, an alcohol-induced decrease in the interfacial tension of each leaflet of the bilayer where this decrease is a net result of the alcohol's interactions with the membrane, the water's interactions with the membrane, and the alcohol's interactions with water. As noted elsewhere (Jaehnig, 1996; Roux, 1996), the interfacial tension of a bilayer is not an experimentally obtainable quantity, but we previously demonstrated with our work on ethanol that simple theoretical models can be applied to estimate interfacial tension, 
, of a bilayer from the elasticity measurements (Ly et al., 2002). It is worth noting here that no complete theory exists that relates the interfacial tension of a bilayer to its mechanical properties. Rawicz et al. (2000) proposed = KA/6 by modeling a lipid monolayer as an idealized polymer brush where the surface pressure in each monolayer depends on the free energy of chain extension. The well-known Israelachvili-Mitchel-Ninham (IMN) model (Israelachvili et al., 1980) predicted = KA/4, from balancing the attractive interfacial tension of a lipid monolayer with a repulsive term dominated by steric interactions, hydration forces, and electrostatics. Both models show that in each monolayer and KA are related by a constant factor, either 4 or 6. The limitations of the idealized polymer brush in approximating the free energy of chain extension as a Gaussian-harmonic and ignoring interactions from the headgroup, interface, and chemical variations in lipid types have been carefully discussed by Rawicz et al. (2000). Despite these limitations, the model has given useful insights into the origins of bilayer elasticity and thickness, and it has correctly predicted the surface pressure-area isotherm measured for an SOPC monolayer, as well as the area/headgroup of an SOPC molecule in a bilayer. From our elasticity measurements of SOPC membranes in water, the Rawicz's model gives an estimated -value of 38 ± 3 mN/m, which predicts an SOPC headgroup area of 58 Å2 ± 2 Å2 (from a surface pressure-area isotherm which is discussed in more detail below in this section). This headgroup size is more consistent with the deuterium NMR measurement (Koenig et al., 1997) of 61.4 ± 0.2 Å2 compared to the IMN estimate of a -value of 57 ± 4 mN/m that predicts a headgroup area of 51 Å2 ± 2 Å2. For this reason, we prefer to apply the Rawicz's relation of = KA/6 to estimate the interfacial tension values from our area compressibility modulus measurements. However, qualitatively, the use of either model will not change the interpretation of our results presented here in terms of alcohol adsorption to the membrane, and the quantitative values based on the Rawicz's model are approximations, given the simplicity of the model.
Fig. 10 A show the -values versus alcohol concentration we obtained from applying the Rawicz's model ( = KA/6) to our average KA data for SOPC membranes. For comparison, -values of alkane-water/alcohol interfaces from the data of Bartell and Davis (1941) and Rivera et al. (2003) are plotted alongside. In comparison to the alkane -values, the bilayer -values decrease much less as alcohol concentration is increased. In addition, the bilayer -values do not parallel the alkane lines, showing that modeling a bilayer as alkane-like may lead to an overestimate of the reduction in the bilayer interfacial tension. However, the alkane system as a model for the bilayer captures the general trend of the dependence of acyl chain-length and concentration of alcohol on bilayer interfacial tension. The differences in interfacial tension values between the bilayer and alkane are attributed to physical differences in the interfaces and the amount of alcohol adsorbed (as will be discussed later in quantitative detail).
Plotting bilayer -values against the log of the alcohol concentration for all the alcohols (as shown in Fig. 10 B) reveals clearly the dependence of on the acyl chain-length of the alcohol. The successive curves are displaced by nearly equal intervals of 0.5 for both the bilayer and alkane interfaces. This finding agrees with Traube's rule that for each additional CH2 group, the concentration required to reach the same interfacial tension was reduced roughly by a factor of 3 (Traube 1891). Langmuir related this difference in concentration to a Gibbs free energy of 2.67 kJ/mol to bring one CH2 group from the bulk aqueous phase to a hydrophobic interface (Langmuir, 1917). Overall, Figs. 10, 5, and 3 show that alcohol decreases the bilayer interfacial tension and concomitantly weakens mechanical cohesion, with the effect more pronounced as acyl chain-length increases.
From the difference in -values between vesicles exposed to water and vesicles exposed to alcohol/water mixtures, we can predict the increase in area per lipid molecule. This entails the use of a surface pressure-area isotherm. We note that the surface pressure, 
, of each monolayer leaflet in a tension-free vesicle is balanced by its interfacial tension, , or = (Evans and Waugh, 1977). Thus, in Fig. 11, we can overlay our -data onto a fit of versus area per molecule data for an SOPC monolayer at the air-water interface from the works of Smaby et al. (1994) and obtain estimates for the area-per-molecule values. For all four types of alcohol molecules, we have plotted the -values onto the SOPC-water monolayer isotherm to obtain area-per-molecule estimates instead of using an SOPC-alcohol-water monolayer isotherm. We assume in the liquid-expanded state of the SOPC-water monolayer where the effective area per SOPC lipid molecule ranges from 60 to 80 Å2 that the headgroup region of the SOPC lipid can accommodate the presence of the alcohol molecules without changing the effective area per lipid molecule. This stems from the fact that the limiting areas of the SOPC lipid and alcohol molecules are 40 Å2 (Smaby et al., 1994) and 18 Å2 (Haydon and Taylor, 1960), respectively; the alcohol/lipid ratio is 2 at the high alcohol limits (from Figs. 11 and 13); and the acyl chains of the alcohols are sufficiently short (less than the maximum length of 0.63 nm for butanol in the all-trans state) that they reside in the headgroup region (0.5 nm) without penetrating inside the hydrocarbon core of the SOPC lipids (Feller et al., 2002; Ueda and Yoshida, 1999). To our knowledge, the SOPC-alcohol-water monolayer isotherms have not been measured. This would be a worthwhile future venture to investigate if the SOPC pressure-area curve, upon addition of alcohols in the subphase, shifts away or crosses the standard curve (no alcohol in the subphase) and to test our assumption that the area per SOPC lipid molecule is approximately the same in either an SOPC-water or SOPC-alcohol-water isotherm for a specific alcohol concentration at a particular surface pressure.
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FIGURE 13 Total alcohol surface density versus concentration for the four alcohol types: methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles). Values at the SOPC bilayer-water interface are solid marks whereas values at the alkane-water interface are open marks. For comparison, the surface density of a saturated butanol monolayer is represented by the dashed line.
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Using a different method of analysis, independent of the assumption of = KA/6, we can predict the area per SOPC lipid molecule of unilamellar vesicles in specific alcohol/water mixtures. In this method, we convert the pressure-area per molecule isotherm of an SOPC monolayer into an elasticity modulus/area per molecule isotherm. By definition, the elasticity modulus, E, of a monolayer is 
where Ah is the area per lipid molecule (Adamson and Gast, 1997), and for comparison to KA, E must be multiplied by a factor of 2 since a bilayer consists of two monolayers. Rawicz et al. (2000) showed that the measured SOPC isotherm of Smaby et al. (1994) could be fitted accurately to an equation of state of the form 
where Ac is the limiting lipid area of 0.4 nm2 and qo is a constant value of 33 nm–2. We applied the elastic modulus definition to this equation of state to generate a plot of 2E vs. Ah. When we compare our measured KA values of SOPC vesicles in alcohol/water mixtures to the derived 2E values, the predictions of area per SOPC lipid molecule are identical to the values estimated by the previous method based on the polymer brush model's general relation of = KA/6. This derives from the fact that, in the area per SOPC molecule range of interest on the -Ah isotherm, the value of 
is always 3 .
We next convert the area/molecule values to direct area expansion, (A/Ao)dir (the increase in area/molecule normalized by the area/molecule with no alcohol present) and plot (A/Ao)dir versus alcohol concentration in Fig. 12 A. We see a clear dependence of (A/Ao)dir on both alcohol concentration and acyl chain-length, with increases in (A/Ao)dir up to 25%. By assuming lipid chain volume is conserved in fluid membranes, we can relate the increase in area/molecule to a corresponding decrease in membrane thickness. For clarity, we show the results separately in Fig. 12 B based on x-ray diffraction measurement of 4 nm (peak to peak) for the thickness of an SOPC membrane in an aqueous solution with a deformable hydrocarbon core of 3 nm (Rawicz et al., 2000).
Our prediction that the headgroup area per lipid molecule increases with alcohol concentration with an accompanying decrease in membrane thickness implies that the lipid chains become more disordered or "looser" packed with a higher degree of gauche conformations. This correlates positively with reported measurements of enhanced membrane fluidity in the presence of alcohol (Chin and Goldstein, 1977). The increase in area per molecule that we predict may also explain the observed faster lateral diffusion motion of lipids in neural crest cells in the presence of alcohol (Chen et al., 1996). According to the free volume theory (Almeida et al., 1992), an increase in the lipid headgroup area would result in a larger free area for lipid diffusion; hence larger lipid diffusion coefficients are probably caused by the "looser" packing of the bilayer in the presence of ethanol compared to the lamellae without alcohol.
Bilayer area expansion from alcohol flows
Our predictions that the lipid membrane will expand and thin as vesicles are exposed to alcohol/water mixtures were confirmed independently through flow experiments. For simplicity, we assumed the membrane area expansion observed upon exposure to alcohol is the sum of three effects although the effects may not be truly independent from one other:
 | (5) |
The first term is the increase in area per molecule from a decrease in interfacial tension of the outer and inner leaflets of the bilayer. This term should be compared to the direct area expansion predictions in Fig. 12 A. For vesicles held at a constant tension, the second term accounts for the increase in mechanical deformation (strain) as mechanical properties (bending and area compressibility moduli) change, but this term is neglectfully small. The last term reflects any flow-induced effects on the membrane area, but we note that this term may not necessarily be independent from (kc, KA) if the unknown cause for (flow) is of a mechanical origin. We ignore any contribution in area expansion from the acyl chains of the alcohols since they are reported to not penetrate into the hydrocarbon core (Chiou et al., 1992). Therefore, to compare our measured flow area expansion numbers, (A/Ao)exp, to our predicted direct area expansion values, (A/Ao)dir, we subtracted (kc, KA) and (flow) of 0.022 from the equilibrium (A/Ao)exp values to get the direct equilibrium area expansion numbers, (A/Ao)exp-dir. We estimated (kc, KA) with the equation
 | (6) |
where is the holding tension, o is the tension at zero strain (estimated to be 0.004 mN/m from where the vs. line typically crosses the y axis in the mechanical properties experiments), kc,alcohol is the average bending modulus of vesicles in a specific alcohol/water mixture, kc,water is the average bending modulus of vesicles in an aqueous mixture without alcohol, KA,alcohol is the average area compressibility modulus of vesicles in a specific alcohol/water mixture, and KA,water is the average area compressibility modulus of vesicles in an aqueous mixture without alcohol. This deformation contribution is very small and averages <0.005. (Our previous article, Ly, 2002, stated that (kc, KA) or (A/A0%) values were 0.02, 0.06, 0.06, and 0.09 for vesicles exposed to 5%, 10%, 15%, and 20% (vol:vol) ethanol/water solutions, respectively. Instead, they should read 0.0012, 0.0013, 0.0022, and 0.0045, respectively.)
The direct equilibrium area expansion (A/Ao)exp-dir values for vesicles with stable projection changes are plotted in Fig. 12 A for comparison with the predicted (A/Ao)dir values obtained from the relation = KA/6. It can be seen that the values are within experimental error of each other, demonstrating that membrane area expansion indeed is derived from decreases in interfacial tension for vesicles exposed to alcohol/water mixtures.
Surface excess
With the establishment that the homologous series of n-alcohols from methanol to butanol decreases the interfacial tension of the bilayer-water interface, we proceed to apply the fundamental theory of Gibbs adsorption (Adamson and Gast, 1997; Chattoraj and Birdi, 1984) to predict quantitatively the amount of alcohol accumulated at the bilayer-water interface in excess of the bulk, from decreases in interfacial tension. For perspective, surface enrichment of methanol, ethanol, propanol, and butanol at the air-water interface (as predicted by the Gibbs equation from surface tension measurements) are experimentally confirmed by x-ray grazing, neutron reflectivity, and mass spectrometry (Li et al., 1993, 1996; Raina et al., 2001a,b). The Gibbs equation of adsorption in concentrated solution for two immiscible phases is
 | (7) |
where 
ex is the surface excess of alcohol molecules, a is the activity coefficient times mole fraction of alcohol in the aqueous phase with the standard state defined by pure alcohol, is the interfacial tension, and RT is the thermal energy per mole. We determined the activity coefficients by fitting the two-suffix Margules equations to experimental vapor-liquid equilibrium data of alcohols and water. (We readily found data for methanol and ethanol, but we had difficulties finding n-propanol and n-butanol data at 25°C and settled on n-propanol at 30°C and 2-butanol at 25°C; Chu, 1956; Gmehling et al., 1977.) We evaluated the derivative d/da for the SOPC bilayer by successfully fitting -values versus activity to exponential functions. The total alcohol surface density values (surface excess density plus surface bulk-phase density) for the bilayer-water interface (and alkane-water interface for comparison; Bartell and Davis, 1941; Rivera et al., 2003) are presented in Fig. 13 as functions of acyl chain-length and concentration of the alcohol. The dashed line represents a saturated butanol monolayer using the estimated surface area of 18 Å2 per alcohol molecule (Haydon and Taylor, 1960). (Ethanol and propanol have similar headgroup areas, but methanol has a slightly smaller area of 16 Å2.) As expected from the difference in interfacial tension behavior between the bilayer and alkane interfaces (Fig. 8), we see in Fig. 13 distinct separations of the curves which clearly shows that alcohol adsorbs much less at the interface of the bilayer than the alkane by almost twofold. Presumably, the hydrophobic effect drives alcohol adsorption at the interface to minimize hydrophobic exposure of the interface to water as well as the hydrophobic surface of the alcohols. Since the alkane has a bare hydrophobic interface whereas the bilayer has an interface of mixed hydrophobic (from the upper segments of the lipid hydrocarbon tails) and hydrophilic (from the polar headgroups) characteristics, the driving force for alcohol adsorption at the interface of the alkane would be greater than the bilayer's.
From the total surface density values presented in Fig. 13, we calculated the partition coefficient, Kx, in terms of mole fraction as defined by the equation
 | (8) |
where xa,b is the mole fraction of alcohol in the bilayer and xa,w is the mole fraction of alcohol in the water phase (Rowe et al., 1998). In calculating xa,b as the ratio of the surface density of alcohol over the sum of surface density of alcohol and surface density of lipid molecules (from the estimated area per lipid molecule in Fig. 11), we note that our estimated Kx values would be slightly less than the true values since we do not account for the small amount of alcohols that may reside in the core of the bilayer. Nonetheless, the Kx values of 9.2, 23.8, 99.1, and 237.7 obtained in the limit of dilute concentrations for methanol, ethanol, propanol, and butanol, respectively, are in general agreement with reported values of 7.8, 16.6, 49.3, and 119.0 as measured by centrifugation of alcohol in DMPC liposomes (and converted from molalities unit; Katz and Diamond, 1974). Additionally, our Kx value of 237.7 for butanol is similar to other published values where titration calorimetry measured 170–183 for dipalmitoyl phosphocholine (DPPC; Zhang and Rowe, 1992) and C-13 nuclear magnetic resonance reported 270–800 for DPPC and 186–600 for DMPC (Rowe et al., 1987) in their fluid-phase states. Note that the difference between each number in the series roughly follows Traube's rule of a factor of 3, indicating that alcohol partitioning is at the root of the Traube's rule effect.
Gibbs free energy of adsorption
The adsorption of alcohol at the bilayer-water interface has long been considered to be primarily driven by the hydrophobic effect (Rowe et al., 1998). The main characteristics of the hydrophobic effect are an overall increase in the entropy of the system, an anomalous heat capacity change, and a standard Gibbs free energy of transfer, Go, that is proportional to the acyl chain-length. Traditionally, Go has been calculated on the basis of the partition coefficient, Kx, of alcohol in the bilayer and water phase, i.e.,
 | (9) |
As we will demonstrate, Go values can be independently (or alternatively) obtained from interfacial tension values. The use of interfacial tension values to calculate Go has been readily applied in air-liquid (Gillap et al., 1968), liquid-liquid (Apostoluk and Szymanowski, 1998), and Langmuir monolayer studies (Heywang et al., 2001), and to our knowledge, we are the first to apply this concept to a bilayer (presumably because interfacial tension values of a bilayer and micelles are not directly measurable). Many forms of Go in terms of interfacial tension values are reported in literature, but for comparison with measured Go values of adsorption of short-chain alcohols to an oil-water interface, we chose the following common form with the standard state defined with respect to an infinitely dilute solution,
 | (10) |
where = o
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ABSTRACT
INTRODUCTION
