Permeability coefficients (Pm) across planar egg lecithin/decane bilayers and bulk hydrocarbon/water partition coefficients (Kw-->hc) have been measured for 24 solutes with molecular volumes, V, varying by a factor of 22 and Pm values varying by a factor of 10(7) to explore the chemical nature of the bilayer barrier and the effects of permeant size on permeability. A proper bulk solvent which correctly mimics the microenvironment of the barrier domain was sought. Changes in Pm/Kw-->hc were then ascribed to size-dependent partitioning and/or size-dependent diffusivity. The diffusion coefficient-size dependency was described by Dbarrier = Do/Vn. When n-decane was used as a reference solvent, the correlation between log Pm/Kw-->hc and log V was poor (r = 0.56) with most of the lipophilic (hydrophilic) permeants lying below (above) the regression line. Correlations improved significantly (r = 0.87 and 0.90, respectively) with more polarizable solvents, 1-hexadecene and 1,9-decadiene. Values of the size selectivity parameter n were sensitive to the reference solvent (n = 0.8 +/- 0.3, 1.2 +/- 0.1 and 1.4 +/- 0.2, respectively, for decane, hexadecene, and decadiene). Decadiene was selected as the most suitable reference solvent. The value for n in bilayer transport is higher than that for bulk diffusion in decane (n = 0.74 +/- 0.10), confirming the steep dependence of bilayer permeability on molecular size. Statistical mechanical theory recently developed by the authors suggests that a component of this steep size dependence may reside in size-dependent solute partitioning into the ordered chain region of bilayers. This theory, combined with the above diffusion model, yielded the relationship, Pm/Kw-->hc = D(o)exp(-alpha V)Vn. A fit of the experimental data to this model gave the best fit (r = 0.93) with alpha = 0.0053 +/- 0.0021 and n = 0.8 +/- 0.3, suggesting that both diffusion and partitioning mechanisms may play a role in determining the size dependence of lipid bilayer permeabilities.

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.

The chemical selectivities of the transport barriers in lipid bilayers varying in composition and phase structure (gel-phase DPPC and DHPC bilayers and liquid-crystalline DPPC/CHOL/50:50 mol% bilayers) have been investigated by determining functional group contributions to transport of a series of alpha-substituted p-toluic acid analogs obtained in vesicle efflux experiments. Linear free energy relationships are established between the free energies of transfer for this series of compounds from water to the barrier domain and corresponding values for their transfer from water into six model bulk solvents (hexadecane, hexadecene, decadiene, chlorobutane, butyl ether, and octanol) determined in partitioning experiments to compare the barrier microenvironment to that in these model solvents. The barrier microenvironment in all bilayers studied is substantially more hydrophobic than octanol, thus establishing the location of the barrier beyond the hydrated headgroup interfacial region, as the interface is expected to be more hydrophilic than octanol. The chemical nature of the barrier domain microenvironment varies with bilayer phase structure. The barrier regions in non-interdigitated DPPC and interdigitated DHPC gel-phase bilayers exhibit some degree of hydrogen-bond acceptor capacity as may occur if these domains lie in the vicinity of the ester/ether linkages between the headgroups and the acyl chains. Intercalation of 50 mol% cholesterol into DPPC bilayers, which induces a phase transition to a liquid-crystalline phase, substantially increases the apparent barrier domain hydrophobicity relative to gel-phase bilayers to a nonhydrogen bonding, hydrocarbonlike environment resembling hexadecene. This result, combined with similar observations in liquid-crystalline egg-PC bilayers (J. Pharm. Sci. (1994), 83:1511-1518), supports the notion that the transition from the gel-phase to liquid-crystalline phase shifts the barrier domain further into the bilayer interior (i.e., deeper within the ordered chain region).

Relationships between the permeability coefficient (PHA) and partition coefficient (K m/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated. Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening. Bilayer surface density, sigma, was varied over a range of 0.5-0.9 by changing cholesterol concentration and temperature. The temperature dependence of PHA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (Ea) of 22 +/- 3 kcal/mole over a cholesterol mole fraction range of 0.00-0.40. This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (delta H degree = 4.8 +/- 0.8 kcal/mole) and the calculated Ea (= 8.0 kcal/mole) assuming a "bulk phase" permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane. These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining Ea. Values of 1n PHA decrease linearly with the normalized phospholipid surface density with a slope of kappa = -12.4 +/- 1.1 (r = 0.90). Correction of PHA for those temperature effects considered to be independent of lipid chain order (i.e., enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation (kappa = -11.7 +/- 0.5 (r = 0.96)). The temperature dependence of Km/w is substantially smaller than that for PHA and dependent on cholesterol composition. Values of 1n K m/w decrease linearly with the surface density with a slope of kappa = -4.6 +/- 0.3 (r = 0.95), which is 2.7-fold smaller than the slope of the plot of 1n PHA vs. sigma. Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature.

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