Langmuir 1992,8, 619-623
619
Molecular Interactions in Langmuir-Blodgett Films of Phospholipid and Fatty Acid Mixtures D. G. Zhut and M. C. Petty Molecular Electronics Research Group, School of Engineering and Applied Sciences, University of Durham, Durham DH1 3LE, U.K.
H. Ancelin and J. Yarwoodt Department of Chemistry, University of Durham, Durham DHl 3LE, U.K. Received April 24,1991. In Final Form: October 29, 1991
We report an infrared spectroscopic study of the molecular interactions in Langmuir-Blodgett films of mixtures of 22-tricosenoic acid and the phospholipid dipalmitoylphosphatidylethanolamine (DPPE). We demonstrate how head group interactions and alkyl chain conformations and ordering are affected by mixing the two components in different proportions. No particular correlation between structure on the substrate and thermodynamic behavior on the subphase was found. The two techniques are clearly sensitive to different aspects of the detailed microscopic interactions.
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1. Introduction
Naturally occurringbiological membranes are comprised of lipids, proteins, and carbohydrates,together with water. Many of these components (in particular, the lipids) are known to form condensed monolayers at the airlwater interface.' Moreover, a number of these materials may be deposited directly onto solid supports using the technique originally reported by Langmuir and extensively applied by Blodgett.2 Such Langmuir-Blodgett (LB) films may form the basis for artificial biological structures and, in this respect, have been attracting some recent interest. Lipids which have been investigated using the LB technique include pho~phatidylcholine,~ phosphatidylethan~lamine:.~ phosphatidic acid,5I6and ch~lesterol.~JJ However, a number of these materials, in particular the phosphatidylcholines and phosphatidylethanolamines, are difficult to build up in multilayer structures of more than one or two layers. However, we have discovered that highquality films may invariably be produced if the lipid is mixed with a simple long-chain fatty acid. In this work, we report on the deposition of mixtures of dipalmitoylphosphatidylethanolamine (DPPE) and 22-tricosenoic acid (22TA).
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DPPE mixtures: (i) pure 22TA, (ii) 9 1 22TADPPE, (iii) 41, (iv) 3:1, (v) 21, (vi) 1:1, (vii) 1:2, (viii) pure DPPE. (b) Mole fraction versus area per molecule at 30 mN m-l for 22TAIDPPE.
2. Experimental Section The lipidmaterialwaspurchasedfromSigmaandusedwithout further purification. The 22TA was kindly provided by Dr. A. Barraud (CEN Saclay, France). Solutions were obtained by dissolving the fatty acid in chloroform (BDG,Aristar grade) and the DPPE in a 41 volume ratio of chloroformand methanol (BDH, Aristar grade) to a concentration of approximately 1 mg cm-3. ~
+ Current
address: Shanghai Institute of Metallurgy, Academic
Sinica, Shanghai, China. (1) Gainea, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interactions; Wiley-Interscience: New York, 1966. (2) Blodgett, K . B. J. Am. Chem. SOC.1935,57, 1007.
(3) Tav1or.D. - . M.: Mahboubian-J0nes.M.G. B. Thin Solid Films 1982. 87, 167.
(4)Green, J. P.; Philips, M. C.; Shipley, G. G. Biochim. Biophys. Acta 1973,330,243. ( 5 ) Cui, D. F.; Haworth, V. A.; Petty, M. C.; Ancelin, H.; Yarwood, J. Thrn Solid Fclms 1990,192, 391.
(6) Howarth, V. A.; Petty, M. C.; Ancelin, H.; Yarwood, J. Vib. Spectrosc. 1990, 1, 29. (7) Wei, L. Y.; Woo, B. Y. Biophys. J. 1973,19,877. (8) Hasmonay, H.; Caillaud, M.; Dupeyrat, M. Biochem. Biophys.Res. Commun. 1979,89, 338.
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Figure 2. Collapse surface pressure versus mole fraction DPPE; subphase pH 5.6.
The two solutionswere then mixed in the appropriateproportions to obtain the required spreading solutions. The mixture was
then spread onto the surfaceof carefullypurified water (obtained by reverse osmosis/deionization/filtrationand ultraviolet sterilization) contained in a constant perimeter barrier LB trough.9 The subphase was buffered (at pH 5.6) using ammonium hydroxide (BDH,Aristar); no additional material was added to the subphase for the experiments described in this paper.
0743-746319212408-0619$03.00/0 0 1992 American Chemical Society
620 Langmuir, Vol. 8, No. 2,1992
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Figure 3. Infrared spectra of two monolayers of pure 224ricosenoic acid (22TA) on hydrophobic silicon. The monolayers were transferred onto silicon attenuated total reflection (Am)crystals, as described in some of our previous work.6.6 The crystals were prepared with a hydrophobic surface by refluxing in a 2% solution of dimethyldichlorosilane in 1,1,1trichloroethane. In all experiments described two monolayers were deposited (with a deposition ratio of 1.0 0.1). Infrared spectra were obtained on a Mattson Sirius 100Fourier transform spectrometer equipped with a liquid nitrogen cooled cadmium mercury telluride detector. All data were collected at a spectral resolution of 4 cm-l. Spectral subtraction of pure 22TA or DPPE was performed using spectra recorded on the pure materials in LB multilayers, but weighted accordingto the relative proportions of the materials present in the mixtures.
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(9) Petty, M. C. In Polymer Surfaces and Interfaces; Feast, W. J., Munro, H. S., Eds.;Wiley: New York, 1987; p 163.
3. Results and Discussion 3.1. Monolayer Formation. The surface pressure versus area isotherms, measured at a compression rate of approximately nm2molecule-' s-l and a temperature of 20 f 2 "C,for a series of different mixtures of the phospholipid and fatty acid are shown in Figure la; Figure l b shows how the area per molecule, measured at a surface pressure of 30 mN m-l, varies with composition; the error bars reflect the differences in the areas measured upon subsequent expansion and recompression of the floating monolayer. In order to study the miscibility of the two components in the monolayer, we have monitored the collapse pressure as a function of composition. If the materials were
Langmuir, Vol. 8, No. 2, 1992 621
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immiscible in each other, then the surface phase rule1would require that the collapse pressure was invariant as the monolayer composition was changed. (To be strictly correct, the equilibrium spreading pressure should be investigated as a function of composition; however, as this measurement presents difficulties, the collapse pressure may be more conveniently used.') Figure 2 shows the results of our study. Clearly, the collapse pressure does vary with the monolayer composition, exhibiting a distinct maximum corresponding to a mixture containing 20-30 7% of the phospholipid. This represents a situation where the areas occupied by the 22TA and the PED are approximately equal (the area per molecule of the (double chain) lipid is approximately twice that of the simple fatty acid).
3.2. Infrared Spectroscopy. The ATR infrared spectra for two layers of the 22TA and the DPPE are given in Figures 3 and 4, respectively. In the case of the mixed films, four spectral regions have been examined in detail. Each provides information about a particular chemical moiety in ihe LB film. a. ,(PO23 Region near 1200 cm-l. The --02PCH2CH2NH3+group is expected to give riseloto bands at 1220 cm-l (v,(POz-)) and 1560 cm-l (Q(NH~+)).A strong band at 1200 cm-l and a much weaker (broad) band at -1550 cm-l are observed in Figure 4B. We may thus safely assume that, in all the films examined, the chlorine group is zwit-
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(10)Bellamy,L.J.InfraredSpectroscopyof Complex Molecules;Chap man and Hall: London, 1975.
622 Langmuir, Vol. 8, No.2,1992
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Figure 6. Variation in acid v(C=O) band shape and intensity for 22TA as a function of 22TAIDPPE mixed multilayers. The 22TADPPE ratios are (A) 1:0, (B)9:1, (C) 41, (D) 2:1, (E) 1:1,
and (F)1:2.
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terionic. As the mole ratio of 22TAto DPPE in the mixture is increased (i.e., as the proportion of the fatty acid is increased) the va(P02-) band at 1220 cm-' becomes less and less well-defined (Figure 5 ) as expected. The DPPE molecules are separated and subjected to a wider range of P02--.RCOOH interactions. These new "environments" give rise to bands in the 1200-cm-' region and in the 1170-1180-~m-~region. At the molecular ratio 22TA: DPPE = 2:l (curve D)-corresponding approximately to the maximum in the collapse pressure (Figure 2)-there are two prominent bands a t 1220 and 1200 cm-l (with a weaker 1180-cm-' band). These two have approximately equal intensities, probably corresponding to roughly equal environments in which the numbers of hydrocarbon chains from the DPPE and 22TA are equal. In this situation the head group interactions (which will depend on the average head group distances) would appear to be of two types-possibly one "lateral" P02--.RCOOH interaction and one "facing" interaction. At higher proportions of 22TA the DPPE molecules will become progressivelymore isolated from the RCOOH groups, and this may be indicated by the appearance of a prominent band at 1260 cm-l. b. T h e Acid v(C=O) Region of 22-Tricosenoic Acid near 1700 cm-l. Figure 6 again shows the results of changing the 22TA:DPPE ratio. As this ratio decreases (i.e., as the amount of DPPE is increased) the 22TA molecules are separated, the number of 22TA molecules facing each other in cyclic dimers drops, and the intensity of the cyclic dimer band" at 1705 cm-I decreases relative to that due to the monomer acid (C=O) band at 1750 cm-'. There are various other hydrogen-bonded C=O-HO species also present (in the 1680-1700-~m-~ region) with populations which depend, as expected, on the proportions of the fatty acid and phospholipid in the mixture. No particular spectral shape is observed for the 2:l ratio-near the collapse pressure maximum (Figure 2)-dthough the new band a t 1712 cm-' does appear particularly prominently. This band, which may be due to sideways dimers,ll may reflect the dominance of regions where, on average, two 22TA molecules lie side by side. On the other hand, as the proportion of 22TA decreases the monomer acid v(C=O) band a t 1748 cm-' rises rapidly. This indicates that the 22TA molecules are being separated from each
other and a significantproportion are not hydrogen-bonded to either other acid molecules or DPPE. It would appear that with a 1:2 ratio of 22TA/DPPE about 50 7% of the acid molecules are in such a situation (assuming that the extinction coefficients axe not very sensitive to environment). c. Thev(C=O) Region of DPPE near 1740cm-l. The pure DPPE spectrum in Figure 4 shows an intense lipid ester v(C=O) band at 1737 cm-' (somewhat lower than reported previously12). As the amount of 22TA is increased this band decreases relative to a "new" band which arises near 1720 cm-' (Figure 7). (Since 22TA absorption does not show a peak near 1720 cm-', this band is not due to incomplete subtraction of the fatty acid spectrum). This feature is almost certainly due to interaction phenomena within the LB film in which the DPPE ester C-0 groups are situated. One possibility is that the v(C0) groups feel different environments-leading to two different bands. It is not clear from our spectra what type of environmental distribution is involved. One possibility is that the v(C0) groups are involved in hydrogen bonding to the tricosenoic acid-which would reduce the v(C=O) band frequency.
(11)Davies, G. H.; Yarwood, J. Spectrochim. Acta 1987,43A, 1619.
(12)Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986,2,96.
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Figure 7. Variation in ester v(C=O) band shape and intensity for DPPE as a function of 22TAIDPPE mixed multilayers. The 22TADPPE ratios are (A) 01, (B)1:2, (C) 1:1, (D) 2:1, (E) 41, and (F)9:l.
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However, since it is not obvious that the 22TA head groups could interact with the DPPE C=O groups in these films, we prefer an explanation in terms of a change in the environment of the DPPE chains themselves. The most likely situation is one in which the proportion of Sn-1 and Sn-2 carbonyl groups (arising from two different conform a t i o n ~about ~ ~ the C2-C1 bond of the C&2C1(--V)O structural unit of the two ester groups) changes as the amount of 22TA increases. It would appear that the proportion of gauche conformers at the ester group (the band near 1720 cm-l) increases as the amount of DPPE is reduced. This would appear to be consistent with less well-ordered packing of the DPPE molecules at low mole fractions of lipid. But there seems to be only a gradual change in the direction as the mole fraction of lipid is reduced. Indeed, none of the spectra reported so far clearly indicate that the observed collapse pressure maximum in Figure 2 is related to structural features associated with head group interactions. We must now consider the information derivable from the stretching bands associated with the hydrocarbon tails. d. The v(CH2)Region 2850-2940 cm-l.All the spectra collected in this work were obtained by total attenuated reflection (ATR) which couples transition dipoles (TD) both perpendicular and parallel to the substrate. However, ATR spectra of long chains show strong va(CH2) and v,(CH2) bands at -2920 and -2850 cm-'-which, for -(CHZ)~-chains aligned perpendicular to the substrate, have their transition dipolesparallel to the substrate.14J6J7 The V,(CH~)group TD should be largely parallel to the substrate under these circumstances and have a strong ATR spectrum. Examination of our spectra in Figures 3 and 4 (for the pure materials) shows that these expectations are largely fulfilled. The va(CH3)band is, however, absent from the tricosenoic acid spectrum (Figure 3A), as expected. This may indicate greater chain tilting than for the DPPE (which,it should be noted, has two end methyl groups per molecule). The va and v,(CH2) bands are near 2918 and 2850 cm-l (respectively)in almost all our spectra. These values are indicative16of well-ordered hydrocarbon chains. On changing the proportions of these materials there are some real changes in the relative intensity of the va and v8(CH2)bands. The variation is shown in Figure 8 for DPPE chains (data obtained by subtraction of 22TA spectra). It would appear that vJv8 is the greatest for small amounts of DPPE. Variation in this ratio is associated12J4J5with tilting of the backbone plane of the 4 H 2 - groups along the hydrocarbon chain. For ATR (13) Lotta, T.I.; Laakkonen, L. J.; Virtanen, J. A.; Kinnunen, P. K. J. Chem. Phys. Lipids 1988,46,1. (14) Rabolt. J. F.:Burns, F. C.: Schlotter,N. E.: Swalen, J. D. J . Chem.
Phys. 1983, 78, 946. (15) Rabe, J. P.; Robolt, J. F.; Brown, C. A.; Swalen, J. D. J. Chem. Phys. 1986,84,4096. (16) Mitchell, M.L.; Dluhy, R. A. Mikrochim. Acta 1988,l (Vol. II), 349. (17) Rabolt, J. F.; Swalen, J. D. In Fourier Transform Infrared Spectroscopy; Basile, Ferraro, Eds.; Academic Press: New York, 1985; Vol. 4, Chapter 7.
Langmuir, Vol. 8, No. 2, 1992 623
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spectra it is expected that a smaller vJv8 ratio (i.e., a reduced va band intensity) is associated with tilting (since for a tilted plane the va(CH2) TD has a larger component perpendicular to the substrate). Thus, it may be that DPPE chain tilting occurs more readily when there is a higher proportion of it in the mixture (except for the pure material), implying that the molecules of DPPE may be aligned more readily with the 22TA for relatively small proportions of DPPE. Such an alignment at low DPPE concentrations may well be associated with a maximum in the collapse pressure for a 2:l TADPPE ratio. However, one could not claim any special correlation between the data displayed in Figures 2 and 8.
4. Conclusions Examination of the vibrational spectra of mixed monolayers of 22-tricosenoicacid (22TA) and dipalmitoylphosphatidylethanolamine (DPPE) shows that the head group interactions of these two surfactants alter considerably across the concentration range. In particular, the longchain fatty acid molecules became separated from their acid neighbors at high DPPE concentrations, although hydrogen-bonded speciesare present over the whole range. The ester group v(C=O) bands of the DPPE reflect changing chain environments-probably via conformational changes. As the concentration of DPPE decreases, the degree of well-ordered packing decreases and a larger percentage of gauche conformers at the ester group are incorporated. Moreover, there appear to be two principal phospholipid head group environments which are roughly equally populated at a ratio 22TA:DPPE of 2:l (at which the collapse pressure on the subphase shows a maximum and the number of hydrocarbon chains from each component is equal). Despite the observed changes in the tilting of such chains with 22TADPPE ratio, there appears to be no direct correlation between the behavior on the subphase and molecular ordering and alignment on the substrate. Registry No. DPPE, 3026-45-7; ZZTA, 65119-95-1.
