Langmuir 1992,8, 2479-2485
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Mixed Monolayers and Cast Films of Acyl Ester and Acylamino Phospholipids David W. Grainger,**+ J. Sunamoto,S K. Akiyoshi,S M. Goto,%and K. Knutsonll Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-1999, Department of Polymer Chemistry, Kyoto University, Kyoto 606, Japan, Kanagawa Academy of Science and Technology, Kawasaki 213, Japan, and Department of Pharmaceutics, University of Utah, Salt Lake City, Utah 84112 Received October 23, 1991. In Final Form: June 22, 1992
Mixed lipid f h s of dimyristoylphosphatidylcholine and its amide-containing analog, l,a-bis(myristoylamido)-1,2-deoxyphosphatidylcholine(DDPC) were investigated, both as mixed monolayers at the air-water interface and solvent-cast films on solid surfaces. Monolayer isotherms of these films at the air-water interface at various temperatures and at various compositions revealed diffuse first-order phase transitions for these systems and phase-mixed behavior, with the addition of more DDPC leading to more diffuse transitions and expanded films. Mixing diagrams show positive deviations from ideal mixing that disappear at higher lateral surface pressures, indicating stearic constraints l i i i t mixing at lower surface pressures. Fluorescence microscopy of these films at the air-water interface shows coexistence behavior in the phase-transition region consistent with phase mixing. Fourier transform IR studies of solvent-cast films show evidence of hydrogen bonding between the two-component lipids strongly contributing to this phase behavior.
Introduction The nonrandom anisotropic arrangement of both lipid and protein componenta of the cell membrane is a strong indicator of how critical the molecular architecture of the membrane assembly is to its function. Molecular-scale interactions involved in receptor function, energy and signal transduction, channel and shuttle dynamics, and membrane barrier properties are dependent upon precise organization of membrane-bound components. Slight disturbances in or perturbations of membrane organization result in suboptimal performance or cessation of function. The mechanisms of structural control that lead to these structure-function relationships in biomembrane aggregates constitute an intriguing research theme. Design criteria found in natural membrane paradigms shall prove useful in constructing stable synthetic membranes and ultrathin films tailored for specific technological requirementa.112 The fundamental requirement to create, predict, and control molecular architecture in thin films and membranes, both to understand cell membrane function and to design functional, synthetic biomimetic interfaces, has prompted us to study membrane models that show microstructuring capabilitiese3v4The present study investigates the behavior of mixed lipid membranes of a typical phospholipid, dimyristoylphosphatidylcholine(DMPC), and a related synthetic phospholipid derivative that contains amide-linked alkyl chains instead of ester acyl chains. This compound, 1,2-bis(myristoylamido)-l,2deoxyphosphatidylcholine (DDPC), has recently been extensively characterized in aqueous dispersions with Institute of Science and Technology. Kyoto University. 8 Kanagawa Academy of Science and Technology. 11 University of Utah. (1) Ringdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Znt. Ed. Engl. 198& 27, 113. (2) Ahlere, M.; MtUler, W.; Reichert, A.; Ringdorf, H.; Venzmer, J. + Oregon Graduate t
Angew. Chem., Int. Ed. Engl. 1990,29, 1269. (3) Grainger, D. W.; Reichert, A,; Ringsdorf, H.; Saleese, C.; Davies, D.; Lloyd, J. B. Biochim. Biophys. Acta 1990,1022, 146. (4) Sunamoto,J.:Goto. M.;Iwamoto.K.; Kond0.H.; Sato, T. Biochim. Biophye. Acta 1990,1024,209.
DMPC using calorimetry$ deuterium NMR,SFTTR,6 and membrane protein reconstitution studies.' The focus of these studies has been characterization of molecular-level hydrogen bonding interactions between DDPC and DMPC in membrane systems that act to stabilize/alter phospholipid bilayer properties. In particular, evidence for specific stabilization and reconstitution efficiencies of the integral membrane protein, human erythrocyte glycophorin, by DDPC-protein interactions in bilayers was presented. The current study attempta to correlate microstructural properties of pure and mixed lipid monolayers of DMPC and DDPC as well as desiccated cast f i s of these compounds with bilayer models proposed from these previous studies.
Experimental Methods Monolayer Isotherms. 1,2-Bis(myristoylamido)-1,2-deoxyphosphatidylcholine (DDPC) was synthesized and purified as described previ~usly.~Dimyristoylphosphatidylcholine (DMPC)was used as supplied (Sigma Chemical) after assessing purity by thin-layer chromatography. DDPC was dissolved in HPLC-grade dichloromethane/ethanol solvent mixtures (91); DMPC was dissolved in pure chloroform. Monolayers of each lipid were spread on subphases of purified water (twicedistilled, Millipore filtered, 18 MQ resistivity) at various temperatures in a thermostated, computer-controlled monolayer film balance described previously.8 After spreading, solvent was allowed to evaporate for 3-5 min depending on temperature and, subsequently, isothermally compressed at various constant rates (525 Az/(molecule/min))to generate surface pressure-area diagrams. Different compression speeds did not alter any feature of the curves other than the "sharpness"of the onset of the liquid expanded (LE)-liquid condensed (LC)phase transitionroll-over. Mixed monolayers containingvarious DPPC:DMPC ratios were spread from premixed binary solutions. These binary solutions were created in triplicate from common, pure DMPC and DPPC stock solutions and measured independently. All isotherms shown represent one of at least three identical curves for each mixture;variances for each mixture are -0.2 mN m-l for surface (5) Sunamoto, J.; Goto, M.; Akiyoshi, K. Chem. Lett. 1990,8, 1249. (6) Sunamoto, J.; Nagai, K.;Goto,M.; Lindman, B. Biochim. Biophys. Acta 1990,1024, 220.
(7) Kawai, T.; Umemura, J.; Takenaka, T.; Goto, M.; Sunamoto, J. Langmuir 1988, 4, 449. (8)Albrecht, 0. Thin Solid Films 1983,99, 227.
0743-7463/92/2408-2419$03.00/00 1992 American Chemical Society
2480 Langmuir, Vol. 8,No. 10, 1992
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Molecular Area (A*/motecute) Figure 1. Isotherms for pure DDPC monolayers on pure water for a series of temperatures: (a) 2 "C, (b) 5 4C, (c) 10 "C, (d) 15 OC, (e) 17 "C, (f) 20 "C. See text for experimental details. pressures and 0.5 A2/moleculeforaverage molecular areas (width of recording pen). Temperature control of the substrate and ambient film balance environment was to -0.2 "C. Fluorescence Microscopy of Lipid Monolayers. A Langmuir trough interfaced with a fluorescence microscopeswas used to observe lipid monolayer morphologiesfor DMPC, DDPC, and various DMPC/DDPC mixtures on pure water subphases at 4 "C. A sulforhodamine-labeled lipid probe baaed on dioctadecylamine'O was incorporated into portions of each lipid solution used for monolayer isotherm measurements at a level of 0.5 mol % . This fluorescent probe partitions preferentially into the LE phase of the monolayer and was shown to mix completely with the fluid phaae lipide at this concentration. Each lipid monolayer was compressed into its LE/LC transition region and observed by fluorescent microscopy as a function of surface pressure. Fourier Transform Infrared Spectroscopy of Solution Cast Lipid Films. Stock solutions of DMPC (3 mg/mL, HPLC grade chloroform) were mixed with stock solutions of DDPC (3 mg/mL, chloroform/ethanol, 2.3:l) in ratios to yield mixed solutions of the same stoichiometry used in the monolayer measurements. Pure and mixed films of DMPC and DDPC were cast onto zinc selenide (ZnSe, Harrick Scientific, Ossing, NY) crystals from these solutions. Solvent was evaporated ambiently until dry and subsequently desiccated under vacuum (IO4 Torr) at room temperature to remove residual solvent. Infrared spectra for each film were obtained with a Digilab FTS 20/80 (Digilab Instruments, BioRad, Cambridge, MA) spectrometer (0.05 or 1.0 cm-l resolution, 1024 scans, nitrogen purge, narrow band MCT detector, triangular apodization). Spectral manipulations were performed with SpectraCalc (Galactic Industries, Salem, NH) and Digilab data manipulation packages. Band position was determined by calculating the center of gravity of each band at 95% of the band height. The algorithms used for these calculations are described in the literature.11 Deconvolution (Digilab package) employed a 20.0-cm-l band peak halfwidth with a K factor varying from 1.8 to 2.2.
Rseults Isotherms for pure DDPC on pure water at various temperatures are shown in Figure 1. All curves exhibit a common lift-off point (liquid expanded state onset) at about 90 A2 per molecule. At the lowest temperature studied (2"C),pure DDPC showsaliquid expanded/liquid condensed (LE/LC) transition at 16.5 mN m-1 and the highest apparent collapse pressure of the series near 67 mN m-l. As temperature for each isotherm is increased, (9) Meller, P. Rev. Sci. Instrum. 1988,59, 2225. (10) Ahlere, M.; Grainger, D. W.; Ringedorf, H.; Saleeee, C. Submitted for publication in Bioconj. Chem. (11) Cameron, D. G.;Kauppinen, J. K.; Moffat, D. J.; Mantach, H. H. Appl. Spectrosc. 1982,36, 245.
0
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Temperalure ("C)
Figure 2. Calculated enthalpy of transition values, Qt,based on the calculation from the two-dimensional Clapeyron equation versus temperature for pure DDPC isotherms. Data taken from Figure 1.
the slope of the LE/LC transition becomes increasingly negative and ita width decreased consistentlyuntil a critical point is reached near 17 "C. Moreover, apparent collapse pressures decrease quite dramaticallywith increasing temperatures; the isotherm at 20 "C collapses near a surface pressure of 43 mN m-l. With the observed LE/LC transition treated as diffuse first-order, the twedimensional Clapeyron equation can be used to calculate experimental heats of transition, Qt12 (drt/dT- dr"/dT), = ASJAA = QJTAA where d r J d T is the change in the transition surface pressure onset with temperature, dro/dTis the change in subphase surface tension with temperature,13ASt and Qt are the respectiveentropy andenthalpy changes associated with the transition, and T is the temperature of the transition. Figure 2 shows the relationship between calculated values Qt and temperature for pure DDPC isotherms at the various temperatures. The plot demonstrates a linear correlation (r2= 0.99) between Qt and isotherm temperature, with an intercept at the X axis (Qt = 0) of about 18 OC. Isotherms of mixed DMPC/DDPC monolayers at 2 OC on water are shown in Figure 3. All curves have a common lift off point, near 90A2 per molecule, indicating identical onset of the liquid expanded region. Pure DMPC exhibits a long, nearly horizontal LE/LC transition at 7 mN m-1, with no apparent LC/solid condensed (SC) transition observed before collapse at a surface pressure of 61.5 mN m-l and limiting molecular area of about 41 A2 per molecule. This isotherm is quantitatively different from that reported for DMPC on isotonic saline/Tris buffer, pH 8.9 at 5 "C,8 although the general isotherm features are similar. The DMPC isotherm on the buffered, high ionic strength subphase shows an LE onset (lift off) at 100 A2 molecule, a horizontal first-order phase transition at 10 mN m-l, and similar collapse pressure of 62 mN m-l (12) Phillips, M. C.; Chapman, D. Bioehim. Biophys. Acta 1968,163, 301. (13)Jasper, J. J. Phys. Chem. Ref. Data 1972, 1, 841.
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Molecular Area (@/molecule) Figure 3. Isothermsfor mixed moriolayers of DDPC and DMPC on pure water as a function of mole percent DDPC in the film. DDPC/DMPC molar ratios were as follows: (a) 0 1 DMPC; (b) 28; (c)46;(d)5:5;(e) 6:4;(082; (g) 1:O. See text for experimental details.
at a limiting molecular area of 38 A2per molecule. The DDPC isotherm shown is identical to that shown in Figure 1 at a temperature of 2 "C. A common onset point of the LE phase for both DDPC and DMPC indicates similar head group areas for these two species under these conditions, although calorimetry data suggest different head group hydration shells in bil a y e r ~ . DDPC ~ shows a significantly higher LE/LC transition pressure at smaller average molecular areas compared to the DMPC isotherm (15mN m-l vs 7 mN m-l, 58 vs 63 A2per molecule, respectively). Also significant is a much higher apparent collapse pressure for DDPC (67 mN m-l) than for DMPC and any of the mixtures. With increasing DDPC content in the mixed monolayers, the isotherms start to shift away from the pure DMPC curve to positions intermediate to both pure compound isotherms. However, two mixed compositions highest in DDPC content-the DMPC/DDPC 4 6 and 2:8 mixed monolayers-deviate from what might be typified as ideal behavior. Both curves show LE/LC transitions at greater surface pressures than either of the pure compounds (16.6 and 18.2 mN m-l, respectively). All of the mixtures show collapse pressures and limiting molecular areas comparable to those for pure DMPC at this temperature, regardless of the ratio of DMPC/DDPC in the mixed monolayers. Moreover, the mixed DMPC/ DDPC monolayers all demonstrate an observable LE/LC transition at surface pressures higher than pure DMPC, indicuting some sort of limited organizational cooperativity between lipid microdomains in transition in the monolayer. However, none of these mixed monolayer transitions exhibit a horizontal slope that approximates a pure firstorder transition. Mean molecular area plots for these mixtures (Figure 4) demonstrate the deviations from ideal mixing or phase separation in DMPC/DDPC mixed monolayers. All plots exhibit positive deviations from ideal behavior, indicating repulsive interactions between film components and expansion of mixed films to larger molecular areas at all mixed compositions. One interesting feature is the prominent progressive shift of the positive deviation maximum toward the molecular area of pure DDPC as a function of increasing DDPC content and increasing surface pressure. This is coupled with an increasing tendency of the mixtures to adapt ideal behavior (additivity) a t both higher DMPC contents and higher surface pressures, despite the existence of large positive deviations a t the same surface pressures but a t lower DMPC contents.
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DDPC mol%
Figure 4. Mean molecular area mixing diagrams for DDPC/ DMPC mixed monolayers as a function of surface pressure. Data were taken from Figure 3. Lateral surface pressure was (a) 5,(b) 7.5, (c) 12.5,(d) 15, (e) 17.5, (0 20,and (g) 25 mN m-1,
FluorescenceMicroscopy. Observation of both pure and mixed monolayers of DMPC and DDPC in their respective LE/LC transition regions at 4 "C provided some morphologkal information on their respective transitions. Figure 5A shows a monolayer of DMPC at approximately 10 mN m-l on pure water. Large dark lipid domains represent LC phase DMPC domains coexisting within a matrix of bright, LE phase DMPC. The domains generally exhibit a long range hexagonal ordering across the surface. Figure 5B shows a DDPC monolayer under the same conditions except at a surface pressure of 20mN m-l. Dark domains of LC phase DDPC are much smaller and much more numerous within the LE DDPC phase. Mixtures of DMPC and DDPC (not shown) displayed morphological behaviors intermediate to these two extremes. That is, DMPC-rich mixed monolayers exhibit larger LC domains which are smaller than domains seen for pure DMPC and decrease in size and increase in number consistently as a function of increasing DDPC content. Fourier Transform Infrared Spectroscopy of Cast Mixed Lipid Films. The CH2 asymmetric and symmetric C-H stretching vibrations of the two pure phospholipids, DMPC and DDPC, absorb at 2918 and 2850 cm-1, respectively (Figure 6). The centers of gravity of the C-H stretching bands are indicative of the most prevalent conformation along the lipid alkylchains. The alkyl chains of the two phospholipids exist predominantly in the trans c~nformation'~J~ in these cast films at these conditions. Mixtures of the two phospholipids did not influence the band positions (conformation) of the alkyl chains significantly in any manner consistent with film composition. Mobility along the alkyl chains is reflected in the C-H stretching bandwidths.1k1s DDPC CH2 asymmetric bands (14) Mantach, H. H.; Madec, C. Biochemistry 1987, 26, 4045. (15) Casal, H. L.; Cameron, D. G.; Smith, I. C. P.; Mantach, H. H. Biochemistry 1980,19, 444. (16)Cfuneron, D. G.; Martin, A.; Moffat, D. J.; Mantach, H. H. Biochemrstry 1986,24,4355. (17) Cameron, D. G.; Mantach, H. H. Biophys. J. 1982,38, 175. (18) Casal, H. L.; Cameron, D. G.; Mantach, H. H. Can. J. Chem. 1983, 61, 1736.
Grainger et al.
2482 Langmuir, Vol. 8, No. 10, 1992
(A)
(B)
Liquid Analog Lipid Phase
Solid Analog Phase Lipid Solid Domains
. Figure 5. Fluorescence micrographs of DMPC and DDPC monolayers at the air-water interface in the phase transition region at 4 "C on pure water. A sulforhodamine-containinglipid at a concentration of 0.5 mol % was used as an imaging probe (see Experimental Section for details): (A) pure DMPC in the phase transition region at 10 mN m-l; (B)pure DDPC in the phase transition region at 20 mN m-l.
the acyl region in equimolar mixed films may be due to intermolecular hydrogen binding is addressed below. The DDPC Amide I C==O stretching band absorbed at 1640 cm-I and N-H stretching band near 3300 cm-1, indicating essentially complete hydrogen bonding? The DMPC ester C=O stretching band absorbed a t 1739cm-l. Figure 7 illustrates the shift in the Amide I band from 1644to 1659for the DDPC/DMPC mixtures as the DDPC content decreased from 80 to 20 70. The shift toward higher wavenumbers reflects decreased amideamide ( C 4 - N H) hydrogen bonding as a result of increased amide-ester secondary interactions across the polar head plane. The weak, symmetric band (N-H stretching) absorbing near 3300 cm-I became weaker and broadened toward higher wavenumbers with decreasing DDPC content in the DDPC/DMPC mixtures from 80 to 20% as a result of decreased N-H bonding (Figure 8). 2950
2900
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WAVENUMBERS (em-1)
Figure 6. FTlR absorbance spectra for (A) pure DDPC, (B) DDPC:DMPC 2:8, (C) DDPC:DMPC 46, (D) DDPC:DMPC 5:5, (E)DDPC:DMPC 6:4, (F)DDPC:DMPC 82,and (G)pure DMPC cast filmson ZnSe IATR crystals (see text for experimental details and interpretation). Spectral range was 3000-2800 cm-l.
(bandwidth 15.1 cm-l) were slightly broader than DMPC bands (bandwidth 12.8 cm-l) suggesting some increased mobility along the alkyl chaim. The 4:6 DDPC/DMPC and 6:4 DDPC/DMPC mixtures exhibited the greatest mobilities (bandwidths 18.6 and 16.9 cm-l, respectively) while the 1:l DDPC:DMPC mixtures were the least mobile (bandwidth 11.8 cm-l). That this restricted mobility of
Discussion Surface pressure area diagrams for DDPC at lower temperatures (shown in Figure 1) demonstrate a main phase transition (a phase coexistence region), defined by a nearhorizontal linear region between two discontinuities in slope of the isotherm. A completely horizontal slope in this region as would be expected for classical first-order behavior is not observed, nor it is achievable at very slow compression rates. The length of this linear region corresponding to the transition converges toward zero with increasingtemperature and is identically zero by definition a t the critical temperature, T,,which is between 17 and
' Langmuir, Vol. 8,No.10, 1992 2483
Mixed Lipid Film 1665 25
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Figure 7. Shifta in infrared Amide I absorbance band between 1640 and 1660 cm-1 as a function of DDPC content in mixed, cast
films of lipids on ZnSe IR crystals (see text for details).
WAVENUMBERS (cm-1)
Figure 8. FTIR absorbance spectra for (A) pure DDPC, (B) DDPC/DMPC 28, (C) DDPC/DMPC 4:6, (D) DDPC/DMPC 55. (E)DDPC/DMPC 64,(F)DDPC/DMPC 8 2 , and (G) pure D&C cast f i i on ZnSe 1A"k crystals (seetext for experimental details and interpretation). Spectral range was 3600-2800 cm-l.
20 OC from the isothermal data. Moreover, the slope of
the transition and its increasingly ambiguous onset with increasing temperature make interpretation of phase behavior complex. We have invoked the procedure introduced by Albrecht and co-workersl9 using compressibility as a function of molecular area to detect onsets of phase transitions and collapse points in the various monolayer systems reported here. Values for d 4 d T and AT for evaluation of Qt in the Clapeyron equation were derived from discontinuities (minima and maxima) in compressibility plots of these monolayers. The intercept of the Qt veraua temperature plots with the x axis near 18 "C in Figure 2, that is, where the heat of transition is zero, (19) Albracht, 0.;Gruler, H.; Sackmann, E. J. Phys. (Paris) 1978,39, 301.
02
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Figure 9. Phase diagram for DDPC and DMPC mixed systems
in monolayers taken from compressibility analysis of isotherm data (see text for details).
indicates a critical point and is identical to T, values derived by DSC studies of bilayer dispersions for DDPC in water.4 This correspondence between monolayer and bilayer critical states has been reported before,12*20i21 but the phenomenological basis for this apparent relationship between the two lipid transitions remains speculative (cf. ref 19). The phase diagram for DMPC and DDPC based on monolayer transitions detected by compressibility analysis is shown in Figure 9. The diagram shows three distinct regions of homogeneous states: homogeneously mixed LE state; homogeneously mixed LC condensed state; a homogeneous bulk phase. Although the LE solid-phase transition was not detectable by compressibility analyses, a heterogeneous two-phase region is apparent, defined by boundaries of the LE-LC phase transition. The region signifies considerable interaction between the two components in the mixed monolayers. The shape of the twophase region is not classically defined (e.g., neither pure cigar-shaped nor positive azeotrope), but comparison of this diagram with mean molecular area curves shown in Figure 4 can yield some interpretations of miscibility. The monolayer state from zero to 6.3 mN m-l is an expanded one-phase region, and miscibility of both components is nearly ideal in this surface pressure range, exhibiting simple additivity in the mean molecular area diagrams. Between 7 and 20 mN m-l, the monolayer mixtures exhibit substantial positive deviations. Mean molecular areas in the expanded one-phase state are much larger than the additivity rule predicts. Mean molecular area plots at lower surface pressures (7-13 mN m-l) show ideal miscibility at most compositions. However, ideal miscibility behavior changes at higher surface pressures such that additivity is again observed only with increasing DMPC content as surface pressure increases. That is, positive deviations from additivity (20) Nagle, J. F. J. Membr. Biol. 1976, 27, 233. (21) Hui,S. W.;Cowden,M.;Papahadjopoulos,D.;Parsons,D.F.Biochim. Eiophys. Acta 1975,582, 263.
2484 Langmuir, Vol. 8,No.10, 1992
yielding expanded films move to higher DDPC contents with increasing surface pressure. Within the heterogeneous two-phase region, mixed monolayers containing up to 50 mol 9% DMPC are ideally miscible but deviate sharply to yield miscible expanded layers with increasing DDPC content. At 60 mol 9% DDPC and higher, the mean molecular areas are much larger than either the expanded one-phaseor condensed one-phase states. This indicates, as seen in the phase diagram, that the heterogeneoustwophase region contains both miscible expanded phases and condensed phases in coexistence. At 15 mN m-l, for example, the mean molecular area plots show complex behavior seen before in the heptadecanoic acid-hexadecy1 acetate system.22 Here, the monolayer shows both positive and negative deviations: a condensed one-phase region, a two-phase region composed of mixed expanded and condensed phases, an expanded one-phase region, a two-phase region of coexisting condensed and expanded phases, and another condensed one-phase region. At 25 mN m-l and greater surface pressures, the mixed monolayer systems exhibit ideal, miscible behavior. Previous experiments on the DDPC/DMPC system in aqueous lamellar dispersions,”7 indicate miscibility of these components across all compositions. Moreover, hydrogen bonding type interactions between DDPC amide groupsand DMPC ester groups were proposed to stabilize DDPC/DMPC m i ~ t u r e s .Also, ~ ~ ~DDPC was evidenced to yield intra- and intermolecular hydrogen bonds in pure DDPD dispersions, both with and without water.7 Although our first impression was that mixed monolayers of these compounds might demonstrate altered phase transitions and condensed films as a result of in-plane intralayer hydrogen bonding, the observation of positive deviations in mixing behavior (monolayer expansion) is not at all inconsistent with such microstructuring. Positive deviations have been proposed to be due to steric constrainta in the mixing of binary phases, though those binary mixtures may be miscible across all prop0rtions.~3In fact, all evidence assembled to date on the DMPC/DDPC system indicates miscibility of the two components. Excess free energies of mixing (AGE) were determined for each mixture using the Goodrich methodz4as applied by Bacon and Barne~.~5 This method involves integrating under each surface pressure-area curve for both mixtures and pure components to evaluate integrals in this expression
s,”
s,”
A G ~ ,= A,, a= A, a= - x 2 JOT A, a= where is the mean molecular area in the mixed film, A1 and Az are the molecular areas for the pure films, and XI and x2 are the molar fractions of monolayer Components DMPC and DDPC, respectively. The analysis is based on the assumption that the contribution from high molecular area (low pressure) data is not significant to the outcome of the analysis.26 The practical lower limit of the integrations was set to the surface pressure onset where monolayer components may be regarded to mix ideally.27 Excess free-energy data calculated from surface pressurearea data appear in Figure 10. At all surface pressures (22) Matuo, H.; Motomura, K.; Matuura, R. Chem. Phys. Lipids 1981, 28,385. (23) Matuo, H.; Motomura, K.; Matuura, R. Chem. Phys. Lipids 1982, 30, 363. (24) Goodrich, F. C. Proceedings of the 2nd International Congress on Surface Activity; Butterworth London, 1957; Vol. 1, p 85. (25) Bacon, K. J.; Barnes, G. T. J. Colloid Interface Sci. 1978,67,70. (26) Costin, 1. S.; Barnes, G . T. J.Colloid Interface Sci. 1975,51,106. (27) Pogano,R. E.; Gershfeld, N. L. J. Colloid Interface Sci. 1972,41,
311.
Grainger et al. 0’02
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Composition (mole% DDPC)
Figure 10. Excess free energies of monolayer mixing as a function
of monolayer composition. DMPC/DDPC mixed monolayers on pure water at 2 ‘c were as follows: H, 5; 0 , 10; 0 , 20;A, 30; 0, 40 mN/m. Maximum error on energy analyses is represented by bar at lower right.
studied, excess free energies are nearly symmetrical with respect to mole fraction. Values for all compositions at low surfaces pressures (5 and 10 mN m-l) are negative, while data from higher surface pressures are positive. While negative ACEmcurves indicate spontaneous mixing at low surface pressures, positive curves at higher surface pressures suggest partial phase separation.26 However, given the relatively small magnitude of the changes in interaction (0.1 kJ/mol), the change from negative to positive AGE, with increasing surface pressure could easily result from packing constraints (entropic contributions AGE,) becoming more important at reduced molecular areas. This is also evidenced in the characteristic changes seen in the mixing diagrams (Figure 4) and in the FTIR data of the acyl region as a function of DDPC content. Despite the slightly positive excess free energy of mixing indicative of small unfavorable interactions at higher surface pressures, mixing at 25 mN m-l and greater surface pressures is ideal. Since the energy differences between positive and negative excess free energy are small, all data taken together are consistent with bidimensional mixing and small unfavorable interactions resulting from DDPC chain packing constraints at small average molecular areas. With both head groups and chain lengths identical, the one apparent explanation for positive deviations lies in the structure of the amide-linked alkyl chain region in DDPC and related ester-amide interaction in the mixed monolayers. Direct intermolecular hydrogen bonds between adjacent DDPC molecules or between DDPC and DMPC molecules is one possible mode of interaction, although such networking might be expected to produce condensed films. Inclusion of a water molecule bridge
Mixed Lipid F i l m
between amide hydrogens and any carbonyl group of the lipid monolayer (cf. refs 4 and 7 and Figure 6) is another possibility that would create hydrogen-bonded networks in monolayers considerably more expanded. These effects in combination with the inherently greater molecular area requirement of pure DDPC monolayers are consistent with a steric type of interaction that expands the mixed system. The shift of positive deviations of mean molecular areas to higher DDPC contentawith increasing surface pressure indicates the correspondence of film expansion to DDPC content and the limiting conformational constraints of the amide-containing lipid at the surface. DDPC’s increased molecular area requirements at the interface indicate a possible nonparallel splaying of the DDPC alkyl chains in the monolayer expanded state as well as a distinctly different, less compact solid condensed state geometry near collapse. FTIR evidence from the mixed cast films indicates a higher mobility (gauche conformer content) in the acyl region of DDPC over DMPC that is consistent with this interpretation. This suggests that restrictive chain-chain interactions with DDPC may be limiting network formation within the monolayer. Such a steric constraint induced by the amide linkage could explain the shift of the phase transitions for the DPMC/ DDPC 4:6 and 2:8 mixtures above that for pure DDPC, even though collapse areas for all mixtures are less than those of pure DDPC. Introduction of excess DDPC (over 50 mol % ) to mixed monolayers with DMPC is observed to exacerbate packing mismatch and disrupt intermolecular association with DMPC, leading to monolayer expansion at higher DDPC contents (Figure4)and higher LE/LC transition pressures (Figure 3). Increasing monolayer surface pressure by lateral compression forces component mixing and lipid monolayer restructuring so that all mixed curves demonstrate common SC and collapse behavior, independent of composition. We maintain that such behavior is consistent with higher disorder inherent in the DDPC amide-linked hydrophobic chain and region, leading to the observed larger molecular area requirements and disrupted by hydrogen bonding at lower surface pressures. That DDPC does not readily form small unilamellar vesicles4 and demonstrates a lower phase transition temperature in multilamellar dispersions4are consistent with such geometry. Deuterium NMR data6 has demonstrated reductions in order parameters in mixed vesicles of DMPC and DDPC over their pure component systems. Additionally, FTIR evidence for DDPC in water7 shows a relativity higher percentage of gauche conformers and disorder in the acyl region below its melting temperature compared to DMPC, demonstrating that the replacement of ester with amide linkages disrupts both thermotropic and packing behavior in lyotropic phases.
Langmuir, Vol. 8, No. 10, 1992 2486
Fluorescent microscopy of the mixed monolayers indicates that all mixtures as well as the pure compounds exhibit a coexistence region where the LE (bright matrix) and LC phases (dark domains) can be visually distinguished. Moreover,condensed phases in all cases are round domains (within the resolution of optical detection), increasing in frequency and decreasing in size with increasing DDPC content in the layer. This indicates that DDPC may be acting as a surface-or edge-active “impurity” in the DMPC layer, decreasing the energy needed to form critical nuclei for LC domain growth, increasing domain frequency in the coexistenceregion, and decreasingaverage domain sizes. Becausethere exists no convincingargument for phase separation, LC domains seen in mixed cases are most likely heterogeneous mixtures of DMPC and DDPC as discussed for the phase diagram.
Summary Mixed lipid monolayers of DMPC and its acylamido analog,DDPC, exhibit positive deviations from additivity, though all evidence suggests that these mixtures are miscible in all proportions. Monolayer expansion is observed in DDPC-rich mixed systems and is proposed to be due to the increased molecular area requirements of DDPC. Hydrogen bonded networks that have been previously reported for mixed bilayer systems of those two lipids are also evidenced here in cast films using FTIR and probably contribute to monolayer miscibility at all surface pressures. As DMPC content in the f i i s increases, FTIR band analysis provides support for increasing C=O*-N-H bonds supplantingamide-amide interactions across the polar head group plane. Positive deviations from ideal mixing are absent at higher lateral surface pressures, indicating that steric constraints limiting DMPC/DDPC interactions at lower surface preesures are overcome by increasing film compression. Fluorescence microscopy studies of these mixed lipid monolayers at the air-water interface were able to visualize the liquid expanded-liquid condensed coexistence regions and indicated that DDPC acts to decrease the size and increase the number of solid analog domains during phase transition. Acknowledgment. We thank Professor Christian Salesse and Professor Helmut Ringsdorf for helpful discussion, support, and suggestions. Financial support from the U.S. Department of Energy, Basic Energy Sciences Program (D.W.G.) and from a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (J.S.,K.A., M.G.) is gratefully acknowledged. Registry No. DMPC, 13699-48-4; DDPC, 18656-38-7.
