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Miscibility Studies on Amphiphilic Calix[4]arene-Natural Phospholipid Mixed Films Patrick Shahgaldian and Anthony W. Coleman* Institut de Biologie et Chimie des Prote´ ines, CNRS-UMR 5086, 7 passage du Vercors, 69367 Lyon, Cedex 07, France Received September 16, 2002. In Final Form: April 17, 2003 The miscibility of two amphiphilic calix[4]arenes, p-dodecanoylcalix[4]arene and 25,27-bis-dihydroxyphosphoryloxy-tetradodecanoyl-calix[4]arene, in mixed monolayers with four phospholipids, 1,2-dipalmitoylsn-glycero-3-phosphatidic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphoserine, has been studied by the Langmuir film method and Brewster angle microscopy (BAM). For p-dodecanoylcalix[4]arene, the mixed layers are immiscible except for those with high mole fraction of DPPC. For 25,27-bis-dihydroxyphosphoryloxy-tetradodecanoyl-calix[4]arene, the mixed layers are miscible in all proportions. Studies of the additivity of the mean molecular areas show strong deviation from ideal behavior. The BAM images show that film morphology in mixed layers differs from single-component films.
Introduction The use of colloidal transporter systems for the transmembrane passage of bioactive molecules requires interaction between the components of the colloids and the components of the cell membrane. The structural integrity of the cell membrane1 is ensured by a lipid bilayer composed of a wide range of phospholipids, glycolipids, and cholesterol. The structure is a fluid mixture of miscible and clustered zones of the lipids,2 it is evident that the components of the colloidal transport systems should not cause major perturbation or destruction of this complex matrix. Hence knowledge of the interactions between synthetic and natural lipids is a key point in the development of new transport systems. As noted above, the behavior of membrane lipids is complex; the miscibility of membrane components in mono- and bilayers is one of the most widely studied systems in the physical chemistry and biophysics of interfacial systems. The behavior of mixed layers of phospholipids,3 lipid-cholesterol,4 lipidpeptide,5 and lipid-proteins6 have been observed as binary, ternary, and more complex mixtures by a wide range of techniques ranging from static and dynamic isotherm studies, differential scanning calorimetry,7 X-ray diffraction,8 small-angle X-ray scattering,9 and atomic force microscopy10 through to neutron studies.11 The introduction of Brewster angle microscopy (BAM) has permitted the direct optical visualization of the phase * To whom correspondence may be addressed. E-mail:
[email protected]. (1) Gennis, R. B. Biomembranes: Molecular Structure and Fonction; Springer-Verlag: New York, 1989. (2) Rietveld, A.; Simons, K. Biochim. Biophys. Acta 1998, 1376, 467. (3) Dorfler, H. D. Colloid Polym. Sci. 2000, 278, 130. (4) Ramstedt, B.; Slotte, J. P. Biophys. J. 1999, 76, 908. (5) Macho, M. I. S.; Gonzalez, A. G.; Varela, A. S. J. Colloid Interface Sci. 2001, 235, 241. (6) Maget-Dana, R.; Ptak, M. Biophys. J. 1997, 73, 2527. (7) Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M. Biochim. Biophys. Acta 2001, 1512, 27. (8) Nakano, M.; Inoue, R.; Koda, M.; Baba, T.; Matsunaga, H.; Natori, T.; Handa, T. Langmuir 2000, 16, 7156. (9) Ohta, N.; Hatta, I. Chem. Phys. Lipids 2002, 115, 93. (10) Feng, S.-S.; Gong, K.; Chew, J. Langmuir 2002, 18, 4061. (11) Knoll, W.; Schmidt, G.; Roetzer, H.; Henkel, T.; Pfeiffer, W.; Sackmann, E.; Mittler-Neher, S.; Spinke, J. Chem. Phys. Lipids 1991, 57, 363.
behavior of monolayers at the air-water interface and is a potent tool for the study of mixed films.12 Many synthetic amphiphilic molecules13 have been synthesized to mimic the behavior of natural phospholipids. Supramolecular variants have been based on varying skeletons such as cyclodextrins,14 crown ethers,15 and calixarenes.16 Calixarenes, macrocyclic oligomers produced by the base-catalyzed reaction of p-tert-butylphenol and formaldehyde,17 are useful skeletons for the construction of amphiphilic molecules. They, and the resorcinarenes, have been widely studied for their recognition properties at the air-water interface.18-25 Such films have also been transferred onto solid surfaces and used as sensors for volatile molecules in the gas phase, for ions, and for organic molecules in solution.26-31 Regen (12) Mobius, D. Curr. Opin. Colloid Interface Sci. 1998, 3, 137. (13) Chevalier, Y. Curr. Opin. Colloid Interface Sci. 2002, 7, 3. (14) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Press: Dordrecht, 1988. (15) Crown Ethers and Cryptands; Gokel, G. W., Ed.; The Royal Society of Chemistry: Cambridge, 1991. (16) Gutsche, C. D. Calixarenes Revisited; The Royal Society of Chemistry: Cambridge, 1998. (17) Gutsche, C. D.; Muthukrishnan, R. J. Org. Chem. 1978, 43, 4905. (18) Shahgaldian, P.; Coleman, A. W. Langmuir 2001, 17, 6851. (19) Capuzzi, G.; Frantini, E.; Dei, L.; LoNostro, P.; Casnati, A.; Gilles, R.; Baglioni, P. Colloı¨ds Surf., A 2000, 167, 105. (20) Dei, L.; Casnati, A.; Nostro, P. L.; Baglioni, P. Langmuir 1995, 11, 1268. (21) He, W.; Liu, F.; Ye, Z.; Zhang, Y.; Guo, Z.; Zhu, L.; Zhai, X.; Li, J. Langmuir 2001, 17, 1143. (22) Houel, E.; Lazar, A.; Da Silva, E.; Coleman, A. W.; Solovyov, A.; Cherenok, S.; Kalchenko, V. I. Langmuir 2002, 18, 1374. (23) Lo Nostro, P.; Casnati, A.; Bossoletti, L.; Dei, L.; Baglioni, P. Colloı¨ds Surf., A 1996, 116, 203. (24) Prus, P.; Pietraszkiewicz, M.; Bilewicz, R. Mater. Sci. Eng., C 2001, C18, 157. (25) Tyson, J. C.; Moore, J. L.; Hughes, K. D.; Collard, D. M. Langmuir 1997, 13, 2068. (26) Kim, J. H.; Park, J. H.; Xuan, G.; Kang, S. Chem. Sens. 2001, 17, 72. (27) Kim, J. H.; Kim, Y. G.; Lee, K. H.; Kang, S. W.; Koh, K. N. Synth. Met. 2001, 117, 145. (28) Nabok, A. V.; Hassan, A. K.; Ray, A. K. J. Mater. Chem. 2000, 10, 189. (29) Hassan, A. K.; Nabok, A. V.; Ray, A. K.; Davis, F.; Stirling, C. J. M. Thin Solid Films 1998, 327, 686. (30) Dei, L.; LoNostro, P.; Capuzzi, G.; Baglioni, P. Langmuir 1998, 14, 4143. (31) Nabok, A. V.; Hassan, A. K.; Ray, A. K.; Omar, O.; Kalchenko, V. I. Sens. Actuators, B 1997, B45, 115.
10.1021/la026556c CCC: $25.00 © 2003 American Chemical Society Published on Web 05/17/2003
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Shahgaldian and Coleman π-A measurements were carried out in a Teflon trough of 400 mL. Calixarene/phospholipid mixtures were deposited as appropriated volumes with a micropipet (Gilson) at the air-water interface. Thirty minutes was allowed for the solvent evaporation and equilibration. Isotherms were carried out on a Langmuir type balance (Nima Technology). Compressions were performed continuously at a rate of 20 cm2/min from 510 to 50 cm2. Each sample was run at least three times to ensure reproducibility of results (deviations of area and pressure were less than 3%). BAM was carried out using a NFT Mini BAM, on the Nima film balance. Compression rates were the same as for π-A measurements. Image size is 4 × 6 mm and resolution 1 < 20%, while 2 is miscible in all proportions with all the phospholipids. BAM imaging shows that the phase behavior is modified in both miscible and immiscible mixtures. Experimental Section All solvents (HPLC grade) were purchased from Acros Organics (France) and used without further purification. DPPE, DPPC, DPPA, and DPPS were purchased from Fluka with a degree of purity g99% and used without purification. Subphases were prepared with deionized water purified with a Millipore MiliQ water system in order to obtain a resistivity of at least 18 MΩ cm. 1 and 2 have been synthesized as previously described.35 Spreading solutions were prepared by dissolving a known quantity (5-6 mg) of the relevant calix[4]arene in 5 mL of chloroform and stored at -15 °C to prevent solvent evaporation. Solutions of lipids were prepared at the same molar concentration, solutions of DPPE and DPPS were dissolved in a mixture of chloroform/methanol (3:2). (32) Conner, M. D.; Janout, V.; Kudelka, I.; Dedek, P.; Zhu, J.; Regen, S. L. Langmuir 1993, 9, 2389. (33) Conner, M.; Janout, V.; Regen, S. L. J. Am. Chem. Soc. 1993, 115, 5, 1178. (34) Zhang, L.-H.; Hendel, R. A.; Cozzi, P. G.; Regen, S. L. J. Am. Chem. Soc. 1999, 121, 1621. (35) Shahgaldian, P.; Coleman, A. W.; Kalchenko, V. I. Tetrahedron Lett. 2001, 42, 577. (36) Shahgaldian, P.; Cesario, M.; Goreloff, P.; Coleman, A. W. J. Chem. Soc., Chem. Commun 2002, 326.
π-A Isotherms. The π-A isotherm of 1 has previously been reported and shows a collapse pressure of 15.2 mN/m with an apparent molecular area of 120 Å2. The compression isotherm of 2 shows an apparent molecular area of 116 Å2, slightly lower than that of 1, while the collapse pressure increases significantly to 35.5 mN/m. The addition of the two negatively charged headgroups, in 2, to the basic skeleton, considerably increases the monolayer stability. Evidence concerning the mixing properties of 1 and 2 comes from the dependence of the collapse pressures with the molar proportions in the mixed films, shown in Figure 2 for 1 and Figure 3 for 2.With regard to the mixtures of 1 with the phospholipids, for DPPE, DPPA, and DPPS, the collapse pressure is constant at all values of XPL-1 below 90%, and then at XPL-1 ) 100% the collapse pressure jumps to the value corresponding to the collapse pressure of the phospholipids. Hence, total immiscibility is observed in these cases. For the mixture DPPC-1, the collapse pressure is constant at 15.7 ((0.5) mN/m until XDPPC-1 ) 0.4, jumps to 47 mN/m for XDPPC-1 ) 0.78, and for proportions above follows the curve of ideal mixing behavior. Thus the two molecules are miscible in the range of XDPPC-1 ) 0.78-1. For 2, in all mixed systems with the four phospholipids, the plots of collapse pressure against mole fraction of the mixture show a linear dependence. This behavior corresponds to a total miscibility of the two components in all proportions. The nature of the miscibility can be further investigated from the variation of mean molecular area, Am, versus the molar ratio of phospholipids (PL)/calixarene (CA), XPL-CA, at various pressures. For monolayers of two-component mixtures, which are formed by immiscible components or in the case of miscible components which do not interact, or in the case in which 1-2 interactions are identical to the 2-1 and 2-2 interactions, i.e., for ideal miscibility, Am, is additive.37
Am ) X1A1 + X2A2 where Ai is the mean molecular area of the pure component i film at the relevant surface pressure and Xi is the mole fraction of the component i in the mixed film. The variations of two representative systems, 1-DPPE and 2-DPPE are given in Figures 4 and 5; the variations for mixtures of 1 and 2 with DPPA, DPPC, and DPPS are given as Supporting Information. It is evident that there is deviation from the ideal behavior in all cases. The size of the deviation, the form of the curves, and mole fraction for which the maximum deviation occurs depend strongly on both the nature of the amphiphilic calixarene and the nature of phospholipids in the mixed layer. The dotted lines illustrate the additive (37) Goodrich, F. C. Proc. Int. Cong. Surf. Act., 2nd 1957, 1, 33. (38) Hoenig, D.; Moebius, D. Thin Solid Films 1992, 210-211, 64.
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Figure 2. Collapse pressure of the mixed monolayers as a function of molar fraction of PL-1 (a, DPPA-1; b, DPPC-1; c, DPPE-1; d, DPPS-1): theoretical values for a miscible mixed film (‚ ‚ ‚); experimental values (b).
relationship; positive deviations correspond to repulsive molecular interactions between the components of the mixed layer while negative ones are characteristic of contraction of a two-component film due to attractive forces between the molecules. In the following analysis all numerical values of the deviations of area are given at a surface pressure of 5 mN/m. With regard to DPPE, 1 shows only a single small deviation from the ideal curve, with a maximum value at XDPPE-1 ) 0.1 and an area superior to the ideal value of 15 Å2. For the mixture 2-DPPE, there is a large single maximum of 25 Å2 in the deviation from ideal behavior at XDPPE-2 ) 0.75. With both 1 and 2, mixtures with DPPS show double positive maxima deviation from ideal behavior. Maximum deviations for 1 are observed at XDPPS-1 ) 0.30 and 0.70, whereas for 2 the maximum deviation occur for XDPPS-2 at 0.25 and 0.75. The deviations are 25 and 17 Å2 for XDPPS-1 ) 0.30 and 0.70, respectively, whereas for XDPPS-2 ) 0.25 and 0.75, the positive deviations in the areas are 11 and 13 Å2, respectively. In the mixtures with DPPA, both 1 and 2 show both double maxima in the deviation from the ideal, at XDPPA-1 ) 0.4 and 0.8 (with deviation values of 7 and 5 Å2, respectively) and XDPPA-2 ) 0.25 and 0.75 (with deviation values of 9 and 10 Å2, respectively). With regard to DPPC, 1 shows a slight positive deviation from the ideal at around XDPPC-1 ) 0.80, with deviation value of 11 Å2, whereas 2 shows a negative deviation for XDPPC-2 ) 0.5 at high surface pressures. At lower pressures
the deviation changes to positive with a double maxima, at XDPPC-2 ) 0.25 and 0.75, with deviation values of 19 and 12 Å2, respectively, at a pressure of 5 mN/m. All observed deviations are significantly greater than the experimental error of (3 Å2. Thus while 2 is miscible with the four phospholipids, the mixtures show clear deviation from ideal mixing, depending on the nature of the lipid headgroup. 1 is immiscible with all four phospholipids in a mixed layer but again shows deviation from ideal behavior. Except for the case of mixtures of 2-DPPC at high surface pressure, all deviations are positive in nature and hence arise from repulsive interactions. Undoubtedly, the large disparities in the molecular areas contribute to rearrangements in the packing of the molecules at the surface, which may explain this effect. Brewster Angle Microscopy. In Figure 6 are given BAM images of pure films of 1, 2, and DPPA at zero surface pressure, before compression. Images obtained at higher pressures (0.5 mN/m and at the film collapse) are given as Supporting Information. The observed phases for DPPA are in agreement with those observed by Minones et al.39 and are included for clarity of discussion. At zero pressure, the film of 1 shows a mixture of gas and liquid phases, with the presence of a significant number of circular holes in the liquid phase. At higher pressures, the images show a homogeneous film (39) Minones, J., Jr.; Rodriguez Patino, J. M.; Minones, J.; Dynarowicz-Latka, P.; Carrera, C. J. Colloid Interface Sci. 2002, 249, 388.
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Figure 3. Collapse pressure as a function of molar fraction of PL-2 (a, DPPA-2; b, DPPC-2; c, DPPE-2; d, DPPS-2): theoretical values for a miscible mixed film (‚ ‚ ‚); experimental values (b).
Figure 4. Mean molecular area as a function of molar fraction of DPPE for mixed films of DPPE-1 measured at 5 (2), 10 (1), and 15 (9) mN/m. Theoretical variations are given as dashed lines.
with no evidence, at the experimental resolution, of a liquid expanded (LE)/liquid condensed (LC) phase change. This is in agreement with the CS-1 value of 115 mN/m, which implies the presence of only a LC phase.
CS-1 ) -A(∂π/∂A) In the case of 2, at 0 surface pressure, interestingly a highly nonreflective but totally homogeneous phase is
Figure 5. Mean molecular area as a function of molar fraction of DPPE for mixed film of DPPE-2 measured at 5 (b), 10 (9), 15 (2), 20 (1), and 25 (() mN/m. Theoretical variations are given as dashed lines.
observed. Apparently, in this rare case, a pure gas phase is observed. Increase of pressure above 0 mN/m results in an immediate phase change and a homogeneous liquid phase is observed. At the collapse, the film becomes inhomogeneous with dark and light phases corresponding to the formation of multilayers. In Figure 7 are given the BAM images of the mixtures of 1-DPPA and 2-DPPA.
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Figure 7. BAM images of (a) the mixed film of DPPA-1 (1:1) and (b) the mixed film of DPPA-2 (1:1) before compression. Scale bars indicate 800 µm.
Figure 6. BAM images of a monolayer of 1 (a), 2 (b), and DPPA (c) before compression. Scale bars indicate 800 µm.
For the mixture 1-DPPA, a highly granulated film is observed at 0 mN/m. The appearance is totally different from either pure components or the mixture. For pressures of 0.5 mN/m, again the behavior of the mixed layer and the two pure components is totally different. Broad bands of different phases are apparently present. At the collapse, a relatively homogeneous phase with granulation is observed. In the case of the mixture DPPA-2, it is interesting to note that even before the compression an irregular twodimensional foam is formed. When the pressure increases, the film becomes homogeneous with light circular concentric rings, At the film collapse these rings disappear while some holes appear in the film.
Conclusion The study of mixed monolayers between p-dodecanoylcalix[4]arene and 25,27-bis-dihydroxy-phosphoryloxy-tetradodecanoyl-calix[4]arene with four phospholipids, dipalmitoylphosphatidyl-choline (DPPC), di-palmitoylphosphatidic acid (DPPA), di-palmitoylphosphatidyl-serine (DPPS), and dipalmitoylphosphatidyl-ethanolamine (DPPE), show that p-dodecanoylcalix[4]arene is immiscible in all proportions with DPPA, DPPE, and DPPS and miscible with DPPC only at high mole fractions of the phospholipid. The presence of two polar phosphate groups to the amphiphilic skeleton leads to miscible mixed systems with the four phospholipids. The mixing as determined for the additivity of molecular areas is nonideal in all cases, while BAM shows the mixed layers behave differently from layers of either of the pure components. Acknowledgment. P.S. acknowledges financial support from the FRM. We thank NFT for their help with the BAM. Supporting Information Available: Variations of mean molecular area as a function of molar fraction of PL for mixed films of DPPA-1, DPPC-1, DPPS-1, DPPA-2, DPPC2, and DPPS-2 at various pressures and BAM images of 1, 2, and DPPA obtained at 0.5 mN/m and at film collapse. This material is available free of charge via the Internet at http://pubs.acs.org. LA026556C
