Pharmaceutical Research

, Volume 24, Issue 8, pp 1457–1472 | Cite as

Comparing the Lipid Membrane Affinity and Permeation of Drug-like Acids: The Intriguing Effects of Cholesterol and Charged Lipids

  • Anita V. Thomae
  • Tamara Koch
  • Christian Panse
  • Heidi Wunderli-Allenspach
  • Stefanie D. KrämerEmail author
Research Paper



Lipid bilayers regulate the passage of solutes into and between cellular compartments. A general prerequisite for this passage is the partitioning of the solute into the bilayer. We investigated the relationship between bilayer partitioning and permeation of three drug-like acids in liposomal systems consisting of phosphatidylcholine alone or mixed with cholesterol or charged lipids.

Materials and Methods

Bilayer partitioning was determined by equilibrium dialysis. Bilayer permeation was studied with a luminescence assay which is based on the energy transfer of the permeant to intraliposomal terbium(III).


The influence of the lipid composition on the pH-dependent membrane affinity was in accordance with the membrane rigidity and possible electrostatic interactions between the acids and the lipids. However, there was no direct relationship between membrane affinity and permeation. This seeming discrepancy was closer analyzed with numerical simulations of the permeation process based on the single rate constants for partitioning and translocation. The simulations were in line with our experimental findings.


Depending on the single rate constants and on the geometry of the system, lipid bilayer permeation may positively, negatively or not correlate with the bilayer affinity of the permeant.

Key words

cholesterol lipid bilayer liposome membrane affinity membrane permeation 



Anthranilic acid


aromatic carboxylic acid




3,5-Dichlorbenzoic acid






Salicylic acid




2-Hydroxynicotinic acid



We would like to thank Maja Günthert for experimental support as well as Knud Zabrocki and Marian Brandau (Martin-Luther-University Halle, Germany) for their advice concerning the mathematical solution of the differential equation system.

Supplementary material

11095_2007_9263_MOESM1_ESM.doc (3.9 mb)
Supplement 1 Electronic supplementary material (4mb)


  1. 1.
    G. D. Eytan. Mechanism of multidrug resistance in relation to passive membrane permeation. Biomed. Pharmacother. 59:90–97 (2005).PubMedCrossRefGoogle Scholar
  2. 2.
    S. D. Krämer. Lipid bilayers in ADME: Permeation barriers and distribution compartments. In B. Testa, S. D. Krämer, H. Wunderli-Allenspach, and G. Folkers (eds.), Pharmacokinetic Profiling in Drug Research: Biological, Physicochemical and Computational Strategies, Wiley-VCH, Weinheim, 2005.Google Scholar
  3. 3.
    P. Lauger, R. Benz, G. Stark, E. Bamberg, P. C. Jordan, A. Fahr, and W. Brock. Relaxation studies of ion transport systems in lipid bilayer membranes. Q. Rev. Biophys. 14:513–598 (1981).PubMedGoogle Scholar
  4. 4.
    P. T. Mayer, T.-X. Xiang, R. Niemi, and B. D. Anderson. A hydrophobicity scale for the lipid bilayer barrier domain from peptide permeabilities: nonadditivities in residue contributions. Biochemistry 42:1624–1636 (2003).PubMedCrossRefGoogle Scholar
  5. 5.
    F. Frézard and A. Garnier-Suillerot. Permeability of lipid bilayer to anthracycline derivatives. Role of the bilayer composition and of the temperature. Biochim. Biophys. Acta 1389:13–22 (1998).PubMedGoogle Scholar
  6. 6.
    S. D. Krämer, A. Braun, C. Jakits-Deiser, and H. Wunderli-Allenspach. Towards the predictability of drug-lipid membrane interactions: the pH-dependent affinity of propanolol to phosphatidylinositol containing liposomes. Pharm. Res. 15:739–744 (1998).PubMedCrossRefGoogle Scholar
  7. 7.
    M. Marenchino, A. L. Alpstäg-Wöhrle, B. Christen, H. Wunderli-Allenspach, and S. D. Krämer. [alpha]-tocopherol influences the lipid membrane affinity of desipramine in a pH-dependent manner. Eur. J. Pharm. Sci. 21:313–321 (2004).PubMedCrossRefGoogle Scholar
  8. 8.
    S. D. Krämer, C. Jakits-Deiser, and H. Wunderli-Allenspach. Free fatty acids cause pH-dependent changes in drug-lipid membrane interactions around physiological pH. Pharm. Res. 14:827–832 (1997).PubMedCrossRefGoogle Scholar
  9. 9.
    J. Gutknecht and D. C. Tosteson. Diffusion of weak acids through lipid bilayer membranes. Role of chemical reactions in the unstirred layer. Science 182:1258–1261 (1982).CrossRefGoogle Scholar
  10. 10.
    P. A. Shore, B. B. Brodie, and C. A. Hogben. The gastric secretion of drugs: a pH partition hypothesis. J. Pharmacol. Exp. Ther. 119:361–369 (1957).PubMedGoogle Scholar
  11. 11.
    T.-X. Xiang and B. D. Anderson. Substituent contributions to the transport of substituted p-toluic acids across lipid bilayer membranes. J. Pharm. Sci. 83:1511–1518 (1994).PubMedCrossRefGoogle Scholar
  12. 12.
    K. A. Youdim, A. Avdeef, and N. J. Abbott. in vitro trans-monolayer permeability calculations: often forgotten assumptions. Drug Discov. Today 8:997–1003 (2003).PubMedCrossRefGoogle Scholar
  13. 13.
    L. X. Yu, E. Lipka, J. R. Crison, and G. L. Amidon. Transport approaches to the biopharmaceutical design of oral drug delivery systems: prediction of intestinal absorption. Adv. Drug Deliv. Rev. 19:359–376 (1996).PubMedCrossRefGoogle Scholar
  14. 14.
    A. V. Thomae, H. Wunderli-Allenspach, and S. D. Krämer. Permeation of aromatic carboxylic acids across lipid bilayers: the pH-partition hypothesis revisited. Biophys. J. 89:1802–1811 (2005).PubMedCrossRefGoogle Scholar
  15. 15.
    J. H. Ipsen, G. Karlstrom, O. G. Mouritsen, H. Wennerstrom, and M. J. Zuckermann. Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta 905:162–172 (1987).PubMedCrossRefGoogle Scholar
  16. 16.
    D. Kurad, G. Jeschke, and D. Marsh. Lateral ordering of lipid chains in cholesterol-containing membranes: high-field spin-label EPR. Biophys. J. 86:264–271 (2004).PubMedGoogle Scholar
  17. 17.
    O. G. Mouritsen and M. J. Zuckermann. What’s so special about Cholesterol? Lipids 39:1101–1113 (2004).PubMedCrossRefGoogle Scholar
  18. 18.
    M. J. Hope, M. B. Bally, G. Webb, and P. R. Cullis. Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812:55–65 (1985).CrossRefGoogle Scholar
  19. 19.
    S. D. Krämer and H. Wunderli-Allenspach. No entry for TAT(44–57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides. Biochim. Biophys. Acta 1609:161–169 (2003).PubMedCrossRefGoogle Scholar
  20. 20.
    S. D. Krämer, J. A. Hurley, D. J. Begley, and N. J. Abbott. Lipids in blood–brain barrier models in vitro I: TLC and HPLC for the analysis of lipid classes and long polyunsaturated fatty acids. In Vitro Cell. Dev. Biol. Anim. 38:557–565 (2002).CrossRefGoogle Scholar
  21. 21.
    R. Singh, M. Ajagbe, S. Bhamidipati, Z. Ahmad, and I. Ahmad. A rapid isocratic high-performance liquid chromatography method for determination of cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine in liposome-based drug formulations. J. Chromatogr., A 1073:347–353 (2005).CrossRefGoogle Scholar
  22. 22.
    G. M. Pauletti and H. Wunderli-Allenspach. Partition coefficients in vitro: artificial membranes as a standardized distribution model. Eur. J. Pharm. Sci. 1:273–282 (1994).CrossRefGoogle Scholar
  23. 23.
    A. Martin, P. Bustamante, and A. H. C. Chun. Physical Pharmacy, Lea & Febiger, Philadelphia, 1993.Google Scholar
  24. 24.
    B. W. Kernighan and D. M. Ritchie. The C Programming Language, Prentice Hall, New Jersey, 1988.Google Scholar
  25. 25.
    C. Huang and J. T. Mason. Geometric packing constraints in egg phosphatidylcholine vesicles. Proc. Nat’l. Acad. Sci. U. S. A. 75:308–310 (1978).CrossRefGoogle Scholar
  26. 26.
    M. Trumbore, D. W. Chester, J. Moring, D. Rhodes, and L. G. Herbette. Structure and location of amiodarone in a membrane bilayer as determined by molecular mechanics and quantitative x-ray diffraction. Biophys. J. 54:535–543 (1988).PubMedGoogle Scholar
  27. 27.
    Y. N. Antonenko, G. A. Denisov, and P. Pohl. Weak acid transport across bilayer lipid membrane in the presence of buffers. Theoretical and experimental pH profiles in the unstirred layers. Biophys. J. 64:1701–1710 (1993).PubMedGoogle Scholar
  28. 28.
    A. Avdeef, P. Artursson, S. Neuhoff, L. Lazorova, J. Grasjo, and S. Tavelin. Caco-2 permeability of weakly basic drugs predicted with the double-sink PAMPA pKa(flux) method. Eur. J. Pharm. Sci. 24:333–349 (2005).PubMedCrossRefGoogle Scholar
  29. 29.
    S. M. Saparov, Y. N. Antonenko, and P. Pohl. A new model of weak acid permeation through membranes revisited: does Overton still rule? Biophys. J. 90:L86–L88 (2006).PubMedCrossRefGoogle Scholar
  30. 30.
    S. D. Krämer. Liposome/water partitioning: theory, techniques and applications. In B. Testa, G. Folkers, and R. Guy (eds.), Pharmacokinetic Profiling in Drug Research: Biological, Physicochemical and Computational Strategies, Wiley-VCH, Weinheim, 2001.Google Scholar
  31. 31.
    A. Kusumi, W. K. Subczynski, M. Pasenkiewicz-Gierula, J. S. Hyde, and H. Merkle. Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase. Biochim. Biophys. Acta 854:307–317 (1986).PubMedCrossRefGoogle Scholar
  32. 32.
    J. A. Urbina, S. Pekerar, H. B. Le, J. Patterson, B. Montez, and E. Oldfield. Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and lanosterol: a comparative study using 2H-, 13C- and 31P-NMR spectroscopy. Biochim. Biophys. Acta 1238:163–176 (1995).PubMedCrossRefGoogle Scholar
  33. 33.
    J. R. Henriksen, A. C. Rowat, E. Brief, Y.-W. Hsueh, J. L. Thewalt, M. J. Zuckermann, and J. H. Ipsen. Universal behaviour of membranes with sterols. Biophys. J. 90:1639–1649 (2005).PubMedCrossRefGoogle Scholar
  34. 34.
    C. Lagerquist, F. Beigi, A. Karlen, H. Lennernas, and P. Lundahl. Effects of cholesterol and model transmembrane proteins on drug partitioning into lipid bilayers as analysed by immobilized-liposome chromatography. J. Pharm. Pharmacol. 53:1477–1487 (2001).PubMedCrossRefGoogle Scholar
  35. 35.
    J. De Gier, J. G. Mandersloot, and L. L. M. Van Deenen. Lipid composition and permeability of liposomes. Biochim. Biophys. Acta 150:666–675 (1968).PubMedCrossRefGoogle Scholar
  36. 36.
    P. L. Yeagle, W. C. Hutton, C. Huang, and R. B. Martin. Phospholipid head-group conformations; intermolecular interactions and cholesterol effects. Biochem. 16:4344–4349 (1977).CrossRefGoogle Scholar
  37. 37.
    E. Corvera, O. G. Mouritsen, M. A. Singer, and M. J. Zuckermann. The permeability and the effect of acyl-chain length for phospholipid bilayers containing cholesterol: theory and experiment. Biochim. Biophys. Acta 1107:261–270 (1992).PubMedCrossRefGoogle Scholar
  38. 38.
    O. G. Mouritsen and K. Jorgensen. Dynamical order and disorder in lipid bilayers. Chem. Phys. Lipids 73:3–25 (1994).PubMedCrossRefGoogle Scholar
  39. 39.
    T. Parasassi, A. M. Giusti, M. Raimondi, and E. Gratton. Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations. Biophys. J. 68:1895–1902 (1995).PubMedCrossRefGoogle Scholar
  40. 40.
    L. Miao, M. Nielsen, J. Thewalt, J. H. Ipsen, M. Bloom, M. J. Zuckermann, and O. G. Mouritsen. From lanosterol to cholesterol: structural evolution and differential effects on lipid bilayers. Biophys. J. 82:1429–1444 (2002).PubMedGoogle Scholar
  41. 41.
    M. Ptak, M. Egret-Charlier, A. Sanson, and O. Bouloussa. A NMR study of the ionization of fatty acids, fatty amines and N-acylamino acids incorporated in phosphatidylcholine vesicles. Biochim. Biophys. Acta 600:387–397 (1980).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Anita V. Thomae
    • 1
  • Tamara Koch
    • 1
  • Christian Panse
    • 2
  • Heidi Wunderli-Allenspach
    • 1
  • Stefanie D. Krämer
    • 1
    Email author
  1. 1.Institute of Pharmaceutical SciencesDepartment of Chemistry and Applied Biosciences, ETH ZurichZurichSwitzerland
  2. 2.Functional Genomics CenterUNI/ETH ZurichZurichSwitzerland

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