The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers

Abstract

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 CH(2) 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 CH(2) 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.

FIGURE 1

Video micrographs of an SOPC vesicle aspirated inside a micropipette in an aqueous bathing solution without alcohol. An increase, ΔL, in projection length, L, is observed when the applied membrane tension, τ, 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, ΔL, in projection length, L, is observed. The transmembrane exchange scheme of alcohol from a flow pipette to a vesicle is shown. The value C_out denotes alcohol concentration inside the flow pipette; C_ads is the total surface density of adsorbed alcohol molecules in the membrane; C_in denotes the alcohol concentration inside the vesicle; and k_on and k_off are the rate constants for adsorption and desorption, respectively.

FIGURE 2

Tension-strain measurements for an SOPC vesicle in a 7.4 M methanol/water solution. The points (circles in left curve) from plotting the natural log of the tension, τ, against area strain, α, is linear in the low-tension regime (0.001–0.5 mN/m). The same points (circles in right curve) plotted with τ against α is nearly linear in the high-tension regime (>0.5 mN/m). Subtracting out contribution from smoothing out subvisible thermal shape undulations from α in the high tension regime gives the direct area strain, α_dir, and the replotted points (squares) shifts the line to the left.

FIGURE 3

Average bending modulus, k_c, 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 (α = 0.05) except for values at 1.70 M of ethanol and 0.39 M of propanol.

FIGURE 4

Natural log tension, τ, versus apparent area strain, α, plotted for individual SOPC vesicles in various alcohol/water mixtures near the high concentration limit: 7.4 M methanol (diamonds), 3.4 M ethanol (squares), 1.3 M propanol (triangles), and 0.55 M butanol (circles). For clarity, the propanol and butanol curves are slightly displaced from their y-intercepts of −5.8 and −5.6, respectively.

FIGURE 5

Average area compressibility modulus, K_A (solid marks) and K_app (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 (α = 0.05).

FIGURE 6

Correlation between K_A and K_app values from Fig. 5 at various alcohol/water mixtures: methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles).

FIGURE 7

Tension, τ, versus apparent area strain, α, of individual SOPC vesicles in four alcohol/water mixtures near the high limit: 7.4 M methanol (diamonds), 3.4 M ethanol (squares), 1.3 M propanol (triangles), and 0.55 butanol (circles). For clarity, the curves are slightly displaced apart.

FIGURE 8

Single vesicles, weakly aspirated inside a micropipette at ∼1 mN/m in an alcohol-free solution, were exposed to a flow pipette (∼100 μm in diameter) that delivered a constant alcohol/water mixture stream of the same osmolarity as the alcohol-free solution at desired alcohol concentration. The time course of vesicle membrane expansion was tracked and analyzed to obtain the equilibrium expansion and apparent permeability coefficient of alcohol transport across the membrane. The removal of the flow pipette results in membrane retraction, showing the reversible dynamics of alcohol transport across the membrane. Symbols are 4.9 M methanol (diamonds), 0.39 M propanol (triangles), and 0.11 M butanol (circles).

FIGURE 9

(A) Average direct lysis area strain, α_dir-lyse, of SOPC vesicles in alcohol/water mixtures obtained after subtracting out from the apparent lysis area strain, α_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 (α = 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, τ_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 (α = 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.

FIGURE 10

(A) Interfacial tension, γ, values versus alcohol concentration for the four alcohol/water mixtures: methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles). Values at the SOPC bilayer-water interface (solid marks) are from the K_A/6 relation, and values at the alkane-water interface (open marks) are reprinted with permission from Bartell and Davis (1941) (Copyright 1941 American Chemical Society) and Rivera et al. (2003) (Copyright 2003 by the American Physical Society). The single error bar is representative of all error bars. (B) The interfacial tension values are replotted against the log of concentration to show roughly equal spacing of 0.5 for all the curves.

FIGURE 11

Surface pressure-area per lipid molecule isotherm of an SOPC monolayer at the air-water interface up to its collapse pressure of ∼45 mN/m (reprinted with permission from Smaby et al., 1994; Copyright 1994 American Chemical Society). Plotted onto the line are γ-values for SOPC vesicles in a butanol/water mixture (given in Fig. 10 based on γ = K_A/6). From the x axis, area per molecule is estimated to predict direct area expansion plotted in Fig. 12. Bars indicate 1 SD.

FIGURE 12

(A) Predicted (ΔA/A_o)_dir and measured (ΔA/A_o)_exp-dir direct membrane area expansion of SOPC vesicles from alcohol/water exposure: methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles). Predicted values (open marks) came from the line in Fig. 11. Measured values (solid marks) came from flow pipette experiments when area expansion of vesicles reached equilibrium conditions. (B) Assuming constant membrane volume, corresponding decreases in membrane thickness from the predicted and measured direct area expansion were calculated. Measured values are based on six vesicles or more. Bars indicate 1 SD.

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.

FIGURE 14

Plot of the standard Gibbs free energy of adsorption of alcohols from an infinitely diluted water phase to an SOPC bilayer-water interface (solid marks) and a petroleum ether-water interface (open marks) (reprinted with permission from Haydon and Taylor, 1960; Copyright 1960 by the Royal Society) against chain length.

FIGURE 15

Plot showing to a first-order approximation a linear relationship between the predicted membrane expansion (from interfacial tension reduction) and alcohol surface density. The predicted membrane expansion values were obtained from Fig. 12 A. The alcohol surface density values came from Fig. 13.

FIGURE 16

Ratio of bulk alcohol concentration over surface density versus bulk alcohol concentration from values found in Fig. 13. The linearity of the points shows that adsorption/desorption of alcohol molecules into the headgroup regions of the bilayers follow the Langmuir adsorption model.

FIGURE 17

Direct membrane area expansion versus time of a vesicle exposed to a 0.39 M propanol/water mixture. (A) Experimental area expansion data (ΔA/A_o)_exp-dir (circles) are fitted to a kinetic model (ΔA/A_o)_mod with a k_off value of 1200 s⁻¹ and a q-value of 3.05 Ų/molecules. The second curve below is the model's prediction of the alcohol concentration inside the vesicle, C_in, used for calculating permeability coefficient, P. (B) Curves for different k_off values from the kinetic model's predictions are drawn for comparison. The top line corresponds to 10⁴ followed by 10³, 10², and 10 s⁻¹ with the best-fitted line at ∼1200 s⁻¹.

FIGURE 18

From the values of C_in in Fig. 17 A, the permeability coefficient of a vesicle in a 0.39 M propanol/water mixture is computed to be 1.9 × 10⁻⁴ cm/s by multiplying the slope with D_v/6 where D_v = 33 μm is the vesicle's diameter.

FIGURE 19

Plot of membrane line tension versus lysis tension. The slope gives a critical radius of 6.5 nm. Symbols are methanol (diamonds), ethanol (squares), propanol (triangles), and butanol (circles).