title: Development of a combined NMR paramagnetic ion-induced line-broadening/dynamic light scattering method for permeability measurements across lipid bilayer membranes.
abstract: A combined method using NMR line-broadening for permeant lifetime determination and dynamic light scattering for vesicle size determination has been developed for the measurement of permeability coefficients of ionizable permeants across phospholipid:cholesterol large unilamellar vesicles. The method has been validated by examining its reproducibility and the influence of various factors that might affect the permeability measurements. The vesicle hydrodynamic diameter was varied between 0.1 and 0.2 micron by extruding multilamellar vesicles through polycarbonate membranes with different pore sizes (0.03-0.2 microns). For these large unilamellar vesicles, the normalized size distributions analyzed by the CONTIN method had standard deviations < 0.36, which led to errors in permeability coefficients < 10% as predicted from a theoretical model developed here. The permeability coefficient for acetic acid is independent of its concentration, vesicle hydrodynamic diameter, the concentration of Pr3+, and ionic strength over the ranges 0.01-0.2 M, 0.1-0.2 microns, 0.004-0.04 M, and 0.03-0.3, respectively. Membrane/water and decane/water partition coefficient measurements of acetic acid indicate that the effects of permeant binding onto the bilayer membrane and self-association are negligible within the permeant concentration range 0.01-0.2 M. The addition of Pr3+ ions induces vesicle fusion with rates increasing with temperature and decreasing with cholesterol concentration in the membranes. While the intravesicular resonance intensity for acetic acid decreases continuously with time due to vesicle fusion under certain conditions, the corresponding line width and chemical shift remain constant over the same period, highlighting an important advantage of this NMR method over those based on detecting net flux in response to a concentration gradient as there is no means in the latter experiments of discerning vesicle leakiness from passive diffusion rates. The effective chemical nature of a dimristoylphosphatidylcholine:cholesterol bilayer barrier microenvironment was explored by comparing the transport of two permeants, D-(-)-mandelic acid and phenylacetic acid, to their relative bulk solvent/water partition coefficients using three reference solvents (n-decane, 1,9-decadiene, and isoamyl alcohol). Using the NMR line-broadening method, the permeability coefficients for these two permeants were determined to be (2.9 +/- 0.4) x 10(-4) cm/s and (3.9 +/- 0.7) x 10(-2) cm/s, respectively, at 294 K and Xchol = 0.3. The incremental free energy of transport for the additional OH group in D-(-)-mandelic acid, delta delta G0 = +2.9 kcal/mol, resembles most closely that for the transfer of this group from water to 1,9-decadiene, suggesting that the barrier domain resides in the acyl chain region and is slightly more polar/polarizable than a saturated hydrocarbon, possibly due to the presence of a double bond in cholesterol and/or the proximity of the barrier domain to the hydrophilic interface.
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City 84112, USA.
authors.forename: T X
authors.initials: TX
authors.lastname: Xiang
authors.forename: B D
authors.initials: BD
authors.lastname: Anderson
mesh_terms: D000085:Acetates / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010539:Permeability; D012542:Scattering, Radiation
chemical_list: D000085:Acetates; D008051:Lipid Bilayers; D002784:Cholesterol; D004134:Dimyristoylphosphatidylcholine