Langmuir 1997, 13, 5521-5523
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New Insights into the Phase Behavior of Lipopolymer Monolayers at the Air/Water Interface. IRRAS Study of a Polyoxazoline Lipopolymer T. R. Baekmark,*,† T. Wiesenthal,† P. Kuhn,‡ T. M. Bayerl,§ O. Nuyken,‡ and R. Merkel† Institut fu¨ r Biophysik, E22, and Lehrstuhl fu¨ r Makromolekulare Stoffe, Technische Universita¨ t Mu¨ nchen, D-85748 Garching bei Mu¨ nchen, Germany, and Institut fu¨ r Experimentelle Physik, V, Universita¨ t Wu¨ rzburg, D-97047 Wu¨ rzburg, Germany Received June 10, 1997. In Final Form: August 14, 1997X Lipopolymers are lipids with a polymer chain covalently attached to the lipid. We studied the infrared reflection absorption behavior as a function of molecular area of Langmuir monolayers of an ether lipopolymer, DC18Gly-M35 (35 monomer units). A plateau region was observed in the monolayer isotherm. Within this region the CH2 asymmetric and symmetric stretching mode absorptions shifted toward lower absorption frequencies (6 and 4 cm-1, respectively). This indicates that the plateau is accompanied by strong local ordering in the lipopolymer, which contradicts previous suggestions that the plateau correlates with a mushroom-brush transition in the polymer.
1. Introduction For steric stabilization of vesicles1
and as model systems for cell membranes2,3 lipopolymers have been the object of much research activity in recent years. Lipopolymers are lipids (typically phospholipids) with their headgroups attached to polymers of varying sizes, functionality, and/ or hydrophilicity. Recently, lipopolymers (and their mixtures with different phospholipids) were subject to monolayer studies using classical film balance techniques.4-9 The lipopolymers were found to show surface activity due to their amphiphilic nature and to exhibit a complex phase behavior of the monolayers,4,6 depending upon the molecular structure of the lipid tail and the hydrophilicity of the polymer chain. Up to two different plateau regimes have been observed in the lipopolymer monolayer isotherms:4-9 one which has its origin in the desorption of the polymer chain from the air-water interface4,6 and a second of a more uncertain origin. Evidence exists to interpret the latter transition either as a transition within the polymer,5,6 the famous mushroom-brush transition of Alexander and de Gennes,10,11 or alternatively as a transition within the lipid hydrocarbon chains, most likely the classical LE to LC transition common to phospholipid monolayers.8,9,12 * Corresponding author. E-mail:
[email protected]. † Institut fu ¨ r Biophysik, E22, Technische Universita¨t Mu¨nchen. ‡ Lehrstuhl fu ¨ r Makromolekulare Stoffe, Technische Universita¨t Mu¨nchen. § Institut fu ¨ r Experimentelle Physik V, Universita¨t Wu¨rzburg. X Abstract published in Advance ACS Abstracts, October 1, 1997. (1) Lasic, D. D.; Papahadjopoulos, D. Curr. Opin. Solid State Mater. Sci. 1996, 1, 392. (2) Noppl-Simson, D. A.; Needham, D. Biophys. J. 1996, 70, 1391. (3) Sackmann, E. Science 1996, 271, 43. (4) Bu¨rner, H.; Winterhalter, M.; Benz, R. J. Colloid Interface Sci. 1994, 168, 183. (5) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Biophys. J. 1994, 66, 1479. (6) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11, 3975. Correction: Langmuir 1996, 12, 4980. (7) Winterhalter, M.; Bu¨rner, H.; Marzinka, S.; Kasianowicz, J. J. Biophys. J. 1995, 69, 1372. (8) Albersdo¨rfer, A. Diploma Thesis. The Technical University of Mu¨nchen, 1996. (9) Naumann, C.; Frank, C. W.; Knoll, W. To be published. (10) Alexander, S. J. Phys. (Paris) 1977, 38, 983. (11) de Gennes, P. G. J. Phys. (Paris) 1976, 37, 1445. Erratum: J. Phys. (Paris) 1977, 38, 426.
S0743-7463(97)00610-0 CCC: $14.00
We investigated the monolayer behavior of a novel lipopolymer, DC18Gly-M35 (cf. Figure 1), synthesized by us. Due to the complete solubility of the polymer part of the lipopolymer in water13 (the lipid serves to anchor the polymer at the surface), no polymer desorption transition is observed for this lipopolymer. However, a plateau region is still observed in the isotherms at elevated lateral pressures. Also, we performed infrared reflection absorption spectroscopy (IRRAS)14-17 on the lipopolymer. We monitored the change in the CH2 symmetric and asymmetric stretching mode absorption as a function of molecular area over the full range of lateral pressures where the lipopolymer forms stable monolayers. A remarkable lowering of the stretching frequencies is observed within the plateau region. 2. Experimental Section The polymer part of DC18Gly-M35 is poly(2-methyl-2-oxazoline) with a polydispersity index of 1.17. The lipopolymer was dissolved in chloroform (Aldrich, HPLC grade) and gently (by use of a microsyringe) deposited on the water surface (Millipore, MilliQ-System, Molsheim, France, R g 18 MΩ‚cm). The film balance was custom-built to fit the measurement compartment of the spectrometer. It is equipped with a Wilhelmy system (paper plate)18 and has a compression ratio of 4. Thus the isotherm shown in Figure 2 is composed of three individual isotherms. We used a compression rate of 9.2 mm2/s at a temperature of 18 ( 0.5 °C. The barrier position was fixed during the IRRAS measurements (30 min). IRRA spectra were obtained on a Nicolet (Madison, WI) 60 SXR FTIR spectrometer, working in the midinfrared region and equipped with an external reflection unit (SPECAC, Orpington, U.K.). Spectra were recorded at an angle of incidence of 20° normal to the surface.15 For each spectrum 4500 interferograms (electronic amplification factor of 8, wavenumber resolution of 8 cm-1) were coadded. The interferograms were Fourier transformed using a triangular apodization function and two levels of zero filling. The resulting normalized spectra were calculated (12) Gonc¸ alves da Silva, A. M.; Filipe, E. J. M.; d’Oliveira, J. M. R.; Martinho, J. M. G. Langmuir 1996, 12, 6547. (13) Chen, C. H.; Wilson, J.; Chen, W.; Davis, R. M.; Riffle, J. S. Polymer 1994, 35, 3587. (14) Dluhy, R. A.; Cornell,, D. G. J. Phys. Chem. 1985, 89, 3195. (15) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373. (16) Mitchell, M. L.; Dluhy, R. A. J. Am. Chem. Soc. 1988, 110, 712. (17) Flach, C. R.; Brauner, J. W.; Mendelsohn, R. Appl. Spectrosc. 1993, 47 (7), 982. (18) Meunier, J. In Liquids at Interfaces; Charvolin, J., Joanny, J. F., Zinn-Justin, J., Eds.; North Holland: Amsterdam, 1990.
© 1997 American Chemical Society
5522 Langmuir, Vol. 13, No. 21, 1997
Letters
Figure 1. Molecular structure of the lipopolymer DC18GlyM35.
Figure 3. IRRA spectra of DC18Gly-M35 in the absorption region of the CH2 symmetric and asymmetric stretching modes, measured at 20 mN/m (full line), in the plateau region at 24 mN/m (dotted line), and at 30 mN/m (dashed line).
Figure 2. Monolayer isotherm of DC18Gly-M35 at 18 °C (full line). Open circles represent average pressure and area during IRRAS measurements. Insert: Plateau region of the isotherm. from the ratio of the monolayer spectrum to the pure water interface spectrum. With a Nicolet software package (Omnic) a first-order baseline correction was applied to the raw data, and peak positions at maximum absorbance of the CH2 stretching mode absorption bands were determined.
3. Results The lipopolymer DC18Gly-M35 is a saturated ether lipid with a poly(methyl oxazoline) group attached as the third leg on the glycerol backbone of the lipid (cf. Figure 1). The steric configuration is s,n, as in common phospholipids. It builds very stable monolayers at the airwater interface, up to a lateral pressure of some 40 mN/m (cf. Figure 2). A plateau is observed in the pressure region between about 22.5 and 25 mN/m. Although the plateau is not as flat as what is common to phospholipids like e.g. DPPC,19 very slow compression rates (lower than 0.05 mm2/s) significantly flatten the plateau (results not shown). Figure 3 shows representative IRRA spectra obtained at 20, 24, and 30 mN/m in the absorption region of the asymmetric (=2920 cm-1) and the symmetric (=2850 cm-1) CH2 stretching modes. We find the intensity of the signal to depend significantly on the surface density of the lipopolymer. Thus we were not able to measure any reliable FTIR signal below about 10 mN/m. In Figure 4 we have plotted the frequency of the CH2 stretching mode absorption peaks at maximum absorption as a function of the molecular area. Three regions of change in the CH2 stretching energy can be observed in both graphs: two regions of slight reduction in absorption energy with reduced area (above (19) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. 1978, 39, 301.
Figure 4. Frequency of the CH2 stretching mode for DC18Gly-M35 at maximum peak absorption versus molecular area: (A) asymmetric stretching mode absorption; (B) symmetric stretching mode absorption; (b) measured position of the absorption peak maximum. In both graphs, the broken vertical lines indicate the approximate limits of the plateau region as deduced from the isotherm. The full lines represent best line fits to the data points. Error bars represent typical errors.
and below the monolayer plateau) and a region of rapid reduction in absorption energy with reduced area, corresponding to the plateau region. The full lines in Figure 4 represent best line fits to the data points in the three different regions. The best line fits in the regions of slight absorption energy reduction have (within experiment error) identical slopes (1.8 ( 0.1 cm-1/nm2). Similarly, in the rapid absorption energy reduction region the slopes have identical values (12 ( 1 cm-1/nm2).
Letters
4. Discussion In common phospholipids the position of the CH2 stretching mode absorption is sensitive to the molecular order in the immediate vicinity of the CH2 groups.14,20 However, a quantitative determination of the conformational order is not possible. Using IRRAS, we have been able to follow the change in the symmetric and asymmetric CH2 stretching mode absorption (cf. Figure 4) while reducing the molecular area. We did our measurements in a manner analogous to the IRRAS experiments on monolayers of DPPC and other phospholipids by Dluhy and co-workers.14-16 We observe a rapid decrease in absorption energy, at molecular areas corresponding to the plateau in the DC18Gly-M35 isotherm. The reduction in the antisymmetric stretching mode is 6 cm-1, and that in the symmetric mode is 4 cm-1. These reductions are comparable to or even slightly larger than the reductions reported for phospholipids undergoing the LE/LC phase transition16,17 or for aqueous dispersions of phospholipid vesicles undergoing their main phase transition.20,21 Normally, this decrease is assigned to conformational ordering in the acyl chains.16,17,20,21 The absolute values for the stretching frequencies (cf. Figure 4) are comparable to the literature values for phospholipids. Our measurements therefore clearly show that in the plateau region there is a significant change in the molecular order of the CH2 groups of the lipopolymer. Nevertheless, a direct comparison of the lipopolymer with phospholipids may seem unreasonable: Though DC18Gly-M35 contains two alkyl chains, the differences in chemical structure are significant. Only 31% of the CH2 groups of DC18Gly-M35 are contained in the alkyl chains (cf. Figure 1) compared to e.g. 87.5% in DPPC. A plateau at elevated lateral pressures, as observed in the isotherm of DC18Gly-M35, is a common feature of a number of lipopolymers with saturated acyl chains.4-7,9 Also common to these lipopolymers is that the plateau is found at very large molecular areas, typically a factor of 3-6 larger than what is observed for the LE/LC transition in phospholipids of the same lipid acyl chain length.6 Two plateaus were observed in monolayer isotherms of the lipopolymers DC18PE-EO45 and DC18PE-EO110:6 One correlated with the desorption of the polymer, poly(ethylene oxide), from the water surface4,6 and a second one resembling the one found here. It was found5,6 that the thickness of a DC18PE-EO45 monolayer scales according to the Alexander-de Gennes model10,11 for polymer mushrooms below the upper transition and for polymer brushes above it. Therefore it was concluded6 that the upper plateau in the two PEO lipopolymers correlates with the mushroom-brush transition of Alexander and de Gennes.10,11 (20) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213. (21) Blume, A. Curr. Opin. Colloid Interface Sci. 1996, 1, 64.
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Our data contradicts this earlier conclusion for the following reason: In the Alexander-de Gennes model10,11 it is assumed that the monomers have the same chemical environment in both conformations.22 In the mushroom conformation, the polymer conformation is that of a classical Flory SAW polymer in a good solvent. In the brush conformation, the polymer exists as a string of blobs, inside of which the polymer behaves as a Flory SAW polymer. The individual monomers therefore have the same chemical environment in both conformations. Due to the significant change in the absorption frequencies within the plateau region, this is not the case here. Instead there must be a change in the molecular order in the vicinity of the CH2 groups. Furthermore, recent experimental23 and theoretical24 work shows that the transition from mushroom to brush is continuous, contrary to the first-order phase transition predicted by Alexander.10 Thus no plateau should be observed in the monolayer isotherm due to a mushroombrush transition. We cannot infer from our data if the frequency shift is caused by CH2 groups in the alkyl chains or in the polymer tail of the lipopolymer or both. There are indications that the hydrocarbon chains of the lipid moiety are important for the existence of the observed plateau. In PEOcontaining lipopolymers where the acyl chains are unsaturated8 or where the acyl chains are short, C14 or less,9 this plateau disappears. Also, no plateau above the desorption transition of PEO is observed in monolayer isotherms of polystyrene/PEO diblock copolymers, where the PEO block is the largest.12 Together, these findings strongly indicate that the plateau correlates with the ordering of the lipid hydrocarbon chains. This interpretation is easily reconciled with the IRRAS data presented here. Presently, we are synthesizing a methyloxazoline lipopolymer with fully deuterated alkyl chains. This should enable us to discern clearly between the lipid and the polymer part of the lipopolymer by use of IRRAS, thereby clarifying which part of the lipopolymer is undergoing the structural ordering observed. Acknowledgment. We thank Drs. C. Naumann, C. W. Frank, and W. Knoll for sharing their monolayer results of DC14PE-EO45 and DC16PE-EO45 with us prior to publication. T.R.B. thanks the Danish Research Academy for financial support. This work was funded by the Deutsche Forschungsgemeinschaft (SFB 266). We are grateful to Prof. E. Sackmann for the possibility to perform this study in his laboratory and for helpful discussions. LA970610L (22) de Gennes, P. G. Adv. Colloid Interface Sci. 1987, 27, 189. (23) Kent, M. S.; Lee, L. T.; Factor, B. F.; Rondelez, F.; Smith, G. S. J. Chem. Phys. 1995, 103, 2320. (24) Carignano, M. A.; Szleifer, I. Macromolecules 1995, 28, 3197.
