Direct Evidence for a Lipid Alkyl Chain Ordering Transition in Poly

A. Wurlitzer, E. Politsch, S. Huebner, P. Krüger, M. Weygand, K. Kjaer, P. Hommes, O. Nuyken, G. Cevc, and M. Lösche. Macromolecules 2001 34 (5), 13...
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Langmuir 1999, 15, 6837-6844

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Direct Evidence for a Lipid Alkyl Chain Ordering Transition in Poly(ethylene oxide) Lipopolymer Monolayers at the Air-Water Interface Obtained from Infrared Reflection Absorption Spectroscopy T. Wiesenthal, T. R. Baekmark, and R. Merkel* Institut fu¨ r Biophysik, E22, Technische Universita¨ t Mu¨ nchen, D-85748 Garching bei Mu¨ nchen, Germany Received February 9, 1999. In Final Form: May 24, 1999 Lipopolymers are lipids with a polymer chain covalently attached to the lipid. At the air-water interface such lipopolymers may undergo up to two monolayer phase transitions. One is correlated with desorption of the polymer group from the surface (the so-called pancake-mushroom transition) and a second is “native” to lipopolymers in the sense that it can only be observed when lipid and polymer are present in the same molecule. In this study we present direct evidence from infrared spectroscopy showing that the “native” transition, although requiring the presence of the polymeric headgroup, is a transition solely within the lipid alkyl chains. This transition correlates to a strong reduction of the number of gauche isomers within the lipid alkyl chains. We used two poly(ethylene oxide) lipopolymers which differed only in the lipid alkyl chains. These were either fully deuterated or fully protonated. The polymer chains were protonated in both cases. Within the protonated lipopolymer, a strong ordering of the CH2 groups was observed during the “native” transition. Within the deuterated lipopolymer, the exclusive deuteration of the alkyl chains allowed us to distinguish directly between lipid and polymer. We observed that the polymer CH2 spectra remained unchanged when the lipopolymer monolayer was compressed. As it was possible to build the subtraction spectra between the two lipopolymers, we could unambiguously identify the lipid component of the protonated lipopolymer in the spectra. It was found that the strong ordering of the methylene groups occurs solely within the lipid alkyl chains.

1. Introduction Lipopolymers, alternatively called polymer lipids, are lipids (typically phospholipids) with headgroup attached polymers of varying sizes, functionality, and/or hydrophilicity. The most well known class of lipopolymers are the so-called PEG lipids and their different derivatives. These PEG lipids are a series of phosphatidylethanolamine lipids with their headgroup coupled to poly(ethylene oxide) (cf., e.g., refs 1 and 2). Also, a number of other lipopolymers have been described in the literature. These include, e.g., ether lipids linked to a number of different polyoxazolines,1,3,4 cholesterol/poly(ethylene oxide) conjugates,5 or even so-called “harmonica” lipids, where both ends of the polymer chain are covalently linked to a lipid.6,7 Such lipopolymersareusedindrugdeliveryandcelltransfection.1,2,7-9 They are also good model molecules for glycolipids and the cellular glycocalix.1,2,10 Monolayer studies at the air-water interface of pure lipopolymers and their mixtures with a number of * Corresponding author. E-mail: [email protected]. (1) Lasic, D. D.; Needham, D. Chem. Rev. 1995, 95, 2601. (2) Lasic, D. D.; Papahadjopoulos, D. Curr. Opin. Solid State Mater. Sci. 1996, 1, 392. (3) Baekmark, T. R.; Wiesenthal, T.; Kuhn, P.; Bayerl, T. M.; Nuyken, O.; Merkel, R. Langmuir 1997, 13, 5521. (4) Baekmark, T. R.; Wiesenthal, T.; Kuhn, P.; Albersdo¨rfer, A.; Nuyken, O.; Merkel, R. Langmuir 1999, 15, 3616. (5) Beugin, S.; Edwards, K.; Karlsson, G.; Ollivon, M.; Lesieur, S. Biophys. J. 1998, 74, 3198. (6) Frey, W.; Schneider, J.; Ringsdorf, H.; Sackmann, E. Macromolecules 1987, 20, 1312. (7) Simon, J.; Ku¨hner, M.; Ringsdorf, H.; Sackmann, E. Chem. Phys. Lipids 1995, 76, 241. (8) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171. (9) Lasic, D. D.; Papahadjopoulos, D. Science 1995, 267, 1275. (10) Sackmann, E. Science 1996, 271, 43.

phospholipids have received much attention as well.3,4,6,11-18 Through these works it is now well established that in pure lipopolymer monolayer isotherms up to two phase transitions can be observed. A so-called pancakemushroom transition if the polymer part of the lipopolymer is only moderately water soluble and a second transition which involves both lipid and polymer parts of the lipopolymers and therefore is native to lipopolymers.4 This “native” transition occurs at higher lateral pressures than the pancake-mushroom transition. Compared to common phospholipids, the “native” transition is observed at surprisingly large molecular areas. Originally13 the “native” lipopolymer transition was proposed to be a polymer transition, the famous mushroom-brush transition of Alexander and de Gennes.19,20 But recent infrared reflection absorption spectroscopy (IRRAS) studies3 showed that this could not be the case. Instead film balance work by Naumann et al.18 and Gonc¸ alves da Silva et al.21 and IRRAS and film balance work by Baekmark et al.4 clearly showed that the lipid (11) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Biophys. J. 1994, 66, 1479. (12) Bu¨rner, H.; Winterhalter, M.; Benz, R. J. Colloid Interface Sci. 1994, 168, 183. (13) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11, 3975; 1996, 12, 4980. (14) Winterhalter, M.; Bu¨rner, H.; Marzinka, S.; Kasianowicz, J. J. Biophys. J. 1995, 69, 1372. (15) Rosilio, V.; Albrecht, G.; Baszkin, A.; Okumura, Y.; Sunamoto, J. Chem. Lett. 1996, 8, 657. (16) Rosilio, V.; Albrecht, G.; Okumura, Y.; Sunamoto, J.; Baszkin, A. Langmuir 1996, 12, 2544. (17) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Israelachvili, J. N.; Smith, G. S. J. Phys. Chem. B 1997, 101, 3122. (18) Naumann, C.; Brooks, C. F.; Fuller, G. G.; Knoll, W.; Frank, C. W. Manuscript in preparation. (19) Alexander, S. J. Phys. (Paris) 1977, 38, 983. (20) de Gennes, P. G. J. Phys. (Paris) 1976, 37, 1445; 1977, 38, 426.

10.1021/la9901332 CCC: $15.00 © 1999 American Chemical Society Published on Web 08/07/1999

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Figure 1. Molecular structure of the lipopolymers used in the present study. The two lipopolymers, h70-EO110 and d70-EO110, differ from each other only within the shaded area, where h70EO110 is fully protonated whereas d70-EO110 is fully deuterated. A common name for the two lipopolymers is DSPE-EO110.

chains of the lipopolymers must be involved in the molecular processes occurring during the native lipopolymer transition. However, none of these experimental methods has unambiguously been able to clarify the nature of the “native” transition. IRRAS measurements showed3,4 that there is a strong local ordering of the methylene groups within lipopolymers undergoing the “native” transition. It was observed that the number of gauche isomers goes down drastically during the “native” transition. But the studies have been incomplete so far, due to the presence of methylene groups in the polymer as well as in the lipid. Thus it has not been possible to establish if both polymer and lipid, or only one of these, would undergo the observed molecular-scale ordering during the “native” transition. However, the data published so far indicate strongly that the molecular ordering is limited to the lipid tails. To account for the origin of the “native” transition, we suggested4 that a process of lipid alkyl chain condensation coupled to a strong reduction in the number of gauche isomers within these supply the driving force for the “native” transition. In the present study we present direct evidence that the strong local ordering of the methylene groups observed previously is occurring exclusively within the lipid part of the lipopolymers. To reach this conclusion we compared infrared reflection absorption spectra of two almost identical lipopolymers. The spectra were taken in situ at the air-water interface. Both lipopolymers were DSPEEO110’s. One (h70-EO110) was fully protonated whereas the second (d70-EO110) contained completely deuterated lipid alkyl chains. In both cases the poly(ethylene oxide) headgroup (PEO) was protonated. The deuteration of d70EO110’s lipid alkyl chains allowed us to distinguish clearly between lipid and polymer in the IRRAS experiments. We found for both lipopolymers that during the “native” transition the local molecular order increased exclusively within the lipid alkyl chains whereas the polymer CH2 groups remained unaffected. This gives direct spectroscopic evidence for a lipid alkyl chain ordering during this transition. 2. Materials and Methods In the present study we compare the monolayer behavior of two almost identical lipopolymers (Figure 1). The lipid alkyl chains of one lipopolymer (h70-EO110) were fully protonated, but those of the second lipopolymer (d70-EO110) were completely deuterated.22 In both cases the poly(ethylene oxide) (PEO) polymeric headgroups of the lipopolymers were protonated. (21) Gonc¸ alves da Silva, A. M.; Filipe, E. J. M.; d’Oliveira, J. M. R.; Martinho, J. M. G. Langmuir 1996, 12, 6547. (22) In many previous publications by various authors (cf., e.g., refs 2 and 11) the lipopolymer h70-EO110 has been termed either PEG5000lipid or DSPE-EO110, a nomenclature we have also used (cf. refs 4 and 13). Majewski et al.,17 who also reported results obtained using a lipid alkyl chain deuterated lipopolymer, used the nomenclature DSPEEO45 without any indication from the nomenclature that the lipid alkyl chains were deuterated. In the present study we use the nomenclature h70-EO110 and d70-EO110, that the reader may easily remember the difference between the two lipopolymers. But the commonly used nomenclature should be kept in mind.

Wiesenthal et al. Chemically,22 both lipid endgroups were DSPEs (L-R-distearoylphosphatidylethanolamine). The polymer chains had a polydispersity of 1.1. Both lipopolymers were purchased from Avanti Polar Lipids (Alabaster, AL) and were used as received. For use in the experiments, the lipopolymers were dissolved in chloroform (Aldrich, HPLC grade) and gently (by use of a microsyringe) deposited on the water surface (Millipore water, Milli-Q-System, Molsheim, France, R g 18 MΩ cm). To calibrate the film balance23 we used arachidic acid from Avanti Polar Lipids (Alabaster, AL). The film balance experiments were performed as described previously.3,4 After each compression step we waited 10 min. This relaxation period was followed by the actual IRRAS measurement which lasted 20 min. Including these periods, the total average compression rate in our experiments was 15 cm2/h. The IRRAS experiments were done according to the concepts of Dluhy, Mendelsohn, and co-workers.24-28 IRRA spectra were obtained on a Nicolet (Madison, WI) 60 SXR FTIR spectrometer, working in the mid-infrared region (Globar infrared source). As detector we used a liquid nitrogen cooled MCT detector (mercury, cadmium, teluride). The infrared beam was guided onto the water surface by an external reflection unit (SPECAC, Orpington, U.K.). Spectra were measured at an angle of incidence of 20° normal to the surface.25 To obtain a good signal to noise ratio, we coadded 4500 interferograms within 20 min using an electronic amplification factor of 16 and a wavenumber resolution of 8 cm-1. Before spreading the monolayers, we measured the background spectra of the pure water surface. The interferograms were Fourier transformed using a triangular apodization function and two levels of zero filling. The resulting normalized spectra were calculated from the ratio of the monolayer spectrum to the pure water interface spectrum. With a Nicolet software package (Omnic) first-order baseline corrections were applied to the raw data in the methylene (CH2 and CD2) as well as the C-O-C stretching mode vibration regions. With the same software, the peak positions at maximum absorbance29 of the methylene stretching mode absorption bands were determined. According to the literature,30,31 the absorption peak containing the C-O-C stretching mode bands is composed of no less than four single absorption bands. We have, where the intensity of the C-O-C peak allowed, decomposed this peak into its constituting bands, using the Multi-Gauss-Fit procedures contained as part of the standard mathematical software IGOR, version 3.11 (WaveMetrics Inc., Oregon, USA).

3. Results The lipopolymers h70-EO110 and d70-EO110 are saturated phospholipids with a PEO polymeric group attached to the lipid headgroup (cf. Figure 1). In Figure 2 we present the monolayer isotherms of both lipopolymers together with the monolayer isotherm of pure DSPE. Both lipopolymers build very stable monolayers at the air-water interface up to about 40 mN/m. Each lipopolymer shows two plateau-like regions in the isotherms. No plateau regions are observed in DSPE at the temperatures of our measurements. In the lipopolymer monolayer isotherms one plateau region is visible at intermediate lateral pressures (about 10 mN/m) but very large molecular areas, which persist over some 8 nm2. This plateau region has (23) Meunier, J. In Liquids at Interfaces; Charvolin, J., Joanny, J. F., Zinn-Justin, J., Eds.; North Holland: Amsterdam, 1990. (24) Dluhy, R. A.; Cornell, D. G. J. Phys. Chem. 1985, 89, 3195. (25) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373. (26) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. Rev. Phys. Chem. 1995, 46, 305. (27) Mitchell, M. L.; Dluhy, R. A. J. Am. Chem. Soc. 1988, 110, 712. (28) Flach, C. R.; Brauner, J. W.; Mendelsohn, R. Appl. Spectrosc. 1993, 47 (7), 982. (29) To obtain the reported peak positions at maximum absorption, we progressed in the following way. The center of the peak was determined at the base and at the top of the peak and weighed equally strong. This weighed value is the reported peak position at maximum absorption. In this way, we are able to compensate for peak asymmetries due to the experimental resolution. (30) Yoshihara, T.; Tadokoro, H.; Murahashi, S. J. Phys. Chem. 1964, 41, 2902. (31) Enriquez, E. P.; Granick, S. Colloids Surf., A 1996, 113, 11.

Lipopolymer Phase Transitions

Figure 2. Monolayer isotherms of h70-EO110 (full line) measured at 20 °C and of d70-EO110 (stippled line) measured at 12 °C. The inset shows the plateau corresponding to the “native” lipopolymer transition for the two lipopolymers, with πnative indicating the onset of this transition. For comparison the monolayer isotherm of DSPE measured at 20 °C is added to the figure.

its origin in a compression-induced desorption from the water surface of initially adsorbed PEO.13,21,32,33 Here the polymer goes from a flat shape (pancake-like) into a spherical shape (mushroom-like). This transition is usually called the pancake-mushroom transition. The second plateau region is observed at high lateral pressures and persists over approximately 0.5 nm2. Compared to the monolayer isotherm of DSPE, this second plateau region is still found at very large molecular areas. The highpressure regions of the two lipopolymer isotherms (above 25 mN/m) fall onto the same curve. This is expected, as we have previously shown,4,13 since the terminal areas of the lipopolymer isotherms are determined by the size of the polymeric headgroup, here 110 monomers in each lipopolymer. We have previously established4 how the lateral pressure at which the “native” transition has its onset is influenced by the choice of lipid, polymer length, and temperature. This is also the case for h70-EO110 and d70EO110. While the polymer length is the same in both cases, the lipids differ, and we observe the onset of the “native” transition for d70-EO110 at 20 °C at a higher lateral pressure than for h70-EO110. To bring the two isotherms to coincide in the same lateral pressure region of the phase diagram, it was necessary to lower the temperature at which we measured the isotherm of d70-EO110 by 8 °C, from 20 to 12 °C. The result is quite interesting. As mentioned, there is a strong reduction of the number of gauche isomers within the methylene groups during the “native” lipopolymer transition. It is well known for common lipids34-36 that the complete deuteration of the lipid alkyl chains results in an increase of the number of gauche isomers. For the LE to LC phase transition of (32) Kim, M. W.; Cao, B. H. Europhys. Lett. 1993, 24, 229. (33) Cao, B. H.; Kim, M. W. Faraday Discuss. 1994 (1995), 98, 245. (34) Mo¨hwald, H. Phospholipid Handbook, 1st ed.; Cevc, G., Ed.; Marcel Dekker, Inc.: New York, 1993. (35) Mendelsohn, R.; Davies, M. A.; Brauner, J. W.; Schuster, H. F.; Dluhy, R. A. Biochemistry 1989, 28, 8934. (36) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213.

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common phospholipids34-36 this leads to an offset of the LE/LC transition corresponding to a temperature difference35,36 of 6-8 °C. While the “native” lipopolymer transition is not identical to the LE/LC transition,4 the film balance data presented in Figure 2 give a strong indication for the coupling of a lipid alkyl chain ordering process to the “native” lipopolymer transition observed in the monolayer isotherms. However, the difference in temperature between the two lipopolymer isotherms also influences the lateral pressure at which the PEO chains desorb from the surface.13,21 This is the reason why the pancake-mushroom transition for d70-EO110 appears at about 1 mN/m lower than that for h70-EO110 in the isotherms of Figure 2. Figure 3 shows representative IRRA spectra in the CH2 stretching mode absorption region for the lipopolymers (A) h70-EO110 and (B) d70-EO110. The spectra in (C) are the subtraction spectra C ) A - B. To obtain C, spectra A and B were taken at identical lateral pressure and molecular area. For performing the subtraction, one has to assume that the state of phase within the polymer is the same in both cases, even so the two lipopolymers were investigated at different temperatures. The question, if the temperature difference may be neglected, shall be discussed below. However, since the intensities of the absorbing bands of the polymer are only very weakly influenced by temperature,37 when changing the temperature from 12 to 20 °C, we may confidently expect the polymers to absorb equally strong at the two temperatures. Since the lipid alkyl chains of d70-EO110 do not absorb in the ν(CH2) region, it is not necessary to make any assumptions concerning the phase state of the lipid alkyl chains, as the lipid alkyl chains of d70-EO110 and h70EO110 are not compared with each other. In the spectral region of the CH2 stretching absorption, three different stretching modes can be discerned. In Figure 3 these are denominated a, b, and c. (a) is the alkyl asymmetric stretching vibration νas(CH2)alk whereas (b) is an asymmetric ether dependent stretching vibration νas(CH2)eth of PEO. (c) is the lipid alkyl symmetric stretching vibration νs(CH2)alk.26,30,31 In the IRRA spectra of h70-EO110 (Figure 3A), all three absorptions can be observed, especially above the “native” lipopolymer transition. The IRRA spectra for d70-EO110 (Figure 3B) in the ν(CH2) region are in good agreement with the literature spectra30,31,38 of PEO within this spectral region. In contrast to alkanes, for PEO the symmetric alkyl stretching vibration is absent in the infrared absorption spectra whereas two asymmetric stretching vibrations can be observed. Due to the deuteration of the alkyl chains within this lipopolymer we naturally do not observe any contribution to the CH2 stretching absorptions from the lipid. Instead we observe the ν(CD2)alk absorption in the spectral region26 between 2080 and 2200 cm-1 (cf. Figure 4). Here we are able to discern the CD2 asymmetric stretching vibration (peak a), whereas the symmetric CD2 peak (peak b) is close to the detection limit. The spectral results of building the subtraction spectra (Figure 3C) are most satisfying. For example, the νas(CH2)alk absorption in Figure 3A (peak a) was broad at all lateral pressures, and below the “native” transition difficult to discern. Now this peak is also strong and clearly visible below the “native” transition. But the strongest effect is on the νs(CH2)alk absorption (peak c). Previously (37) Griffiths, P. R.; de Haseth, J. A. Fourier Transform Infrared Spectrometry, 1st ed.; John Wiley & Sons: New York, 1986. (38) Lin, K.-J.; Parsons, J. L. Macromolecules 1969, 2, 529.

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Figure 4. Representative IRRA spectra in the CD2 stretching mode absorption region of the lipopolymer d70-EO110 measured during (dotted line) and above (full line) the “native” lipopolymer transition. The lettering a and b refer to the two identifiable peaks26 within this spectral region: (a) νas(CD2) at approximately 2193 cm-1, and (b) νs(CD2) at approximately 2090 cm-1.

Figure 3. Representative IRRA spectra in the CH2 stretching mode absorption region of the lipopolymers (A) h70-EO110, (B) d70-EO110 measured below (dotted line), during (full line), and above (dashed line) the “native” lipopolymer transition. (C) Subtraction spectra C ) A - B. The lettering a, b, and c refer to the three identifiable peaks26,30,31 within this spectral region: (a) νas(CH2)alk at approximately 2920 cm-1, (b) νas(CH2)eth at approximately 2885 cm-1, and (c) νs(CH2)alk at approximately 2855 cm-1.

this peak was almost completely covered by the PEO spectrum, only discernible as a shoulder above and during the “native” transition (and with some goodwill, also below the “native” transition). In the subtraction spectra, the νs(CH2)alk absorption is visible as a peak even below the “native” transition. Above and during the “native” transition, this absorption is observed as strong and sharp. Figure 5 shows a representative IRRA spectrum for the two lipopolymers above the “native” transition in the frequency region from 1025 to 1200 cm-1. The intense and broad absorption peak observed in this spectral region

Figure 5. Representative IRRA spectrum of the polymeric headgroup (PEO) of the lipopolymers in the C-O-C stretching mode absorption region measured above the “native” lipopolymer transition: O, raw data points. The resulting four bands obtained by deconvoluting the ν(COC) band by fitting a fourpeak Gaussian model to the raw data are as follows: fully drawn curve, [νas(COC) + rs(CH2)]; dashed curve, νas(COC); dotted curve, νs(COC); dash/dotted curve, [ν(CC) - νas(COC)]. In assigning the bands, we follow the literature.30,31 The fat, fully drawn curve shows the resulting sum curve of the fit; see also Table 1. The general shape of the ν(COC) band is characteristic of PEO in an amorphous, noncrystalline state.38,39

is correlated to various vibration modes within the PEO headgroup.30,31,38,39 It should also be possible26 to observe a reflection-absorption from the PO2- symmetric stretch at 1090 cm-1. Due to the estimated low signal intensity of this absorption band compared to the relatively strong and broad PEO absorption, we cannot identify this PO2band in our spectra. (39) Dissanayake, M. A. K. L.; Frech, R. Macromolecules 1995, 28, 5312.

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Table 1. Band Assignment and Average Peak Position of the Resulting Four Peaks Obtained by Fitting a Four-Peak Gaussian Model to Our IRRAS Data in the Spectral Region of the ν(COC) Absorptiona band assignment30,31

average position (cm-1)

positions from ref 31 (cm-1)

νas(COC) + rs(CH2) νas(COC) νs(COC) ν(CC) - νas(COC)

1064 ( 5 1087 ( 5 1110 ( 3 1138 ( 3

1061 1078 1104 1134

a ν denotes stretching, r rocking motion. For comparison, we included the results obtained by Enriquez and Granick.31 These results agree with ours if one takes into account the uncertainty of the fits.

According to the literature30,31 the ν(COC) band is composed of no less than four subpeaks, of which two at about 1087 and 1110 cm-1 correspond to the ν(COC) asymmetric and symmetric stretching mode absorptions, respectively.31 We deconvoluted the polymer ν(COC) band into its constituting four subpeaks using a Gaussian band shape model. In Table 1 we list the average peak positions at maximum absorption as fitted by us and by Enriquez and Granick.31 Our results correlate well with those presented in the literature. For h70-EO110 they are identical to the results which we have already presented4 for h70-EO45 (DSPE-EO45) although this latter lipopolymer has a shorter polymer chain and therefore absorbs less strongly. The possibility of deconvoluting the ν(COC) peak is of special interest when doing s,p-polarized IRRAS experiments. In this case31 one may obtain information on the conformational state of PEO, especially on the presence of helical structures. As the question of helicality in PEO is still unresolved,40-42 IRRAS offers a new access to clarify this question. But even without the extra information obtainable from the IRRAS experiment using a polarized beam, one can extract valuable information38,39 through the observation of the general shape of the ν(COC) band. Its general shape is broad and without structure at all lateral pressures above the pancake-mushroom transition. It does not even change during the “native” transition. This observation is valid for both of the lipopolymers on which we report here, irrespective of temperature. This spectral shape is characteristic of a noncrystalline, amorphous PEO structure.38,39 In the case of a crystalline structure the mentioned four subbands of the ν(COC) band would show up as sharp, spikelike, and resolvable peaks instead of the present broad band. In Figures 6 and 7 we plot the frequency at maximum absorption versus molecular area for the asymmetric (Figure 6) and symmetric (Figure 7) methylene stretching mode absorptions of d70-EO110 and h70-EO110. In Figure 8 we show the position of the two asymmetric methylene stretching mode absorptions of poly(ethylene oxide). The measured peak positions in Figures 6 and 7 belong to (A) ν(CD2) of d70-EO110 and (B) ν(CH2)alk of h70-EO110 obtained from the IRRAS spectra. (C) shows the peak positions determined by building the subtraction spectra. Due to the decrease in the signal to noise ratio when building the subtraction spectra (cf. Figure 3C), a number of peaks observable in the region of high molecular area disappear in the subtraction spectra (compare Figures 6B and 7B with 6C and 7C). (40) Kjellander, R.; Florin, E. J. Chem. Soc., Faraday. Trans. 1 1981, 77, 2053. (41) Israelachvili, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8378. (42) Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8399.

Figure 6. Frequency at maximum absorption versus molecular area for (A) the CD2 and (B) the CH2 asymmetric stretching mode absorptions of d70-EO110 and h70-EO110, respectively. (C) shows the results from the subtraction spectra, i.e., for the lipid alkyl νas(CH2) absorption of h70-EO110: b, measured position of the absorption peak maximum. In all plots, the broken vertical lines indicate the limits of the “native” transition regions as deduced from the isotherms.

The ν(CD2) measurements for d70-EO110 are hindered by low signal intensity. Therefore we only evaluated ν(CD2) peaks which were stronger than 2.5 times the average noise level, the latter being about 0.1 mRAU. For νas(CD2) we observe a decrease in the number of gauche isomers with decreasing area. On the basis of our results it is difficult to establish clearly if this decrease only takes place during the “native” transition or if it is a continuous process. However the decrease is stronger than what is expected if the decrease in the absorption frequency would only be coupled to the decrease in molecular area. For h70-EO45 (DSPE-EO45) we have previously4 established this “background” ordering with decreasing molecular area to be about 1.3 cm-1/nm2. Here we observe a reduction of 2.4 cm-1/nm2. Not a large difference, but it indicates that there is a contribution to the frequency shift which is not

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Figure 8. Frequency at maximum absorption versus molecular area for the two asymmetric CH2 stretching mode absorptions observable in poly(ethylene oxide): O, results obtained for d70EO110; b, results obtained for h70-EO110. Due to the overlap of the polymer and lipid alkyl νas(CH2)alk absorptions in the 2920 cm-1 region, no separate polymer CH2 peak can be observed here for h70-EO110. In both plots, the broken vertical lines indicate the limits of the “native” transition regions as deduced from the isotherms.

Figure 7. Frequency at maximum absorption versus molecular area for (A) the CD2 and (B) the alkyl CH2 symmetric stretching mode absorptions of d70-EO110 and h70-EO110, respectively. (C) shows the results from the subtraction spectra, i.e., for the lipid alkyl νs(CH2) absorption of h70-EO110: b, measured position of the absorption peak maximum. In all plots, the broken vertical lines indicate the limits of the “native” transition regions as deduced from the isotherms.

just due to the forced closer packing of the lipid chains at the interface when the molecular area is reduced. For the νas(CH2)alk peak of h70-EO110 (Figure 6B) we observe three regions of change in frequency with de(43) The constant region above the “native” transition appears less constant in Figure 6 than it actually is. This is due to visual contortion. In IRRAS experiments one traditionally plots the change in frequency versus molecular area. This is a natural thing to do, as it reflects the experimental reality. Also it emphasizes regions of strong change in frequency with changing area. However, at small molecular areas where the lateral pressure may change strongly, the data points for the frequency values visually lie closer to each other than at large molecular areas, even though the data points may, as is the case here, be far apart in lateral pressure. In a plot of lateral pressure versus wavenumber, the opposite would be the case. Here the large area region would become compressed whereas the high-pressure region would be elongated. Actually, the experimental scatter is low at high lateral pressures (good signal intensity), and therefore the frequency results are more accurately determined at high lateral pressures than at low.

creasing molecular area. Two regions of a rather constant position of the absorption peak are located at molecular areas above and below the “native” transition,43 and one region of strong reduction in absorption energy with decreasing molecular area closely coincides with the “native” transition as observed in the isotherms. The frequency change during this intermediate region is linear with a slope of 5.8 cm-1/nm2. This is comparable to the result we have obtained previously4 for h70-EO45 (DSPEEO45), where we found a value of 6 cm-1/nm2. For the symmetric stretching absorption of h70-EO110 instead, we observe (Figure 7B) two different but practically constant absorption frequency regions with decreasing area. Here a jump occurs in the absorption frequency of about 5 cm-1 from 2857.7 ( 1.4 cm-1 down to a value of 2852.0 ( 0.5 cm-1 during the “native” transition. The results for both symmetric and asymmetric vibration modes clearly show that there is a strong reduction in the number of the gauche isomers of the alkyl CH2 groups occurring almost exclusively during the “native” transition. The results obtained from building the subtraction spectra (Figures 6C and 7C) are comparable with one important difference. Any effect observed here may unambiguously be assigned to occurrences exclusively within the lipid alkyl chains, since the absorption contribution by any other CH2 group is annihilated by the subtraction of the background spectra. However, due to the broad CH2 spectra of PEO (cf. Figure 3) and to the decreased signal to noise ratio in the subtraction spectra,

Lipopolymer Phase Transitions

the experimental scatter is increased compared to the results for h70-EO110. For the νas(CH2)alk absorption peaks (Figure 6C), again we observe two different regions of constant frequency, one above and one below the “native” transition indicated by vertical lines.43 Between these two regions the frequency of maximum absorption changes linearly with molecular area with a rate of 5.6 cm-1/nm2. This is in agreement with our previous results.4 The νs(CH2)alk exhibits a very similar dependence on the molecular area (Figure 7C). However, we do not observe any absorption peaks below the “native” transition due to the low signal intensity. During the transition we observe a rate of 6.5 cm-1/nm2, while the peak position is again constant above the transition. The results presented in Figure 8 show the position of the two asymmetric methylene stretching vibration bands of poly(ethylene oxide). The band located around 2887 cm-1 (Figure 8A) is an ether-dependent methylene vibration,30,31 whereas the band located around 2920 cm-1 (Figure 8B) is the common alkyl-dependent methylene asymmetric stretching vibration.26,30,31 Due to the overlap of the polymer and the lipid alkyl-dependent ν(CH2) bands, we are only able to observe both polymer methylene peaks separately in the case of d70-EO110. For the νas(CH2)eth band around 2887 cm-1 we find its position for h70-EO110 to be very constant (2887.1 ( 0.8 cm-1) whereas the position for the deuterated lipopolymer scatter more (2885.3 ( 2.7 cm-1). For the alkyl-dependent νas(CH2)alk band free of an overlaying lipid peak (d70-EO110) the peak position still scatter although less strongly (2920.4 ( 1.4 cm-1). This scatter has its origin in fluctuations in the baseline of the spectra. For sharp peaks like the lipid methylene ν(CH2) peaks, such fluctuations in the baseline influence the peak positions only little, but for broad bands like the PEO methylene spectra, it can strongly influence the peak positions leading to the large scatter observed. We do not have reliable spectroscopic data in the isotherm region of the pancake-mushroom transition. We are therefore unable to conclude if hydration of the polymer leads to any shift in the absorption frequency of the methylene groups of PEO. But the spectroscopic results presented in Figure 8 unambiguously show that the local molecular order of neither of the two polymer CH2 groups is influenced by the lateral pressure and that the polymer CH2 groups do not undergo any ordering during the “native” transition. This is also supported by the noncrystalline bandshape of PEO presented in Figure 5. 4. Discussion In the introduction we postulated that our experimental results allow us to conclude beyond doubt that the strong ordering of the CH2 groups within the lipopolymers occurring during the “native” transition occurs solely within the lipid chains. A comparison of Figures 6 and 7 with Figure 8 shows that this is indeed the case. The data presented in Figure 8 show how the methylene stretching absorptions for PEO scatter around a mean value, whereas the data obtained from the original spectra (especially the symmetric methylene band, which is unique to the lipid) and the subtraction spectra show how the lipid alkyl chains increase their local order during the “native” transition. This directly proves our postulate. However, we left a loose end above when we compared the polymer spectra of the two lipopolymers directly to build the subtraction spectra, irrespective of the 8 °C difference in temperature. The assumption is nontrivial, but whether it holds or not does not influence our results. The same information which is obtained through the

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subtraction spectra is contained in the symmetric methylene band due to its unique assignment to the lipid alkyl chains of h70-EO110. But, does our assumption hold? As a reminder: To build the subtraction spectra, we assumed that the same lateral pressure and molecular area correspond to the same state of phase within the polymer, irrespective of temperature. This requires basically that the PEO must not undergo any phase transitions upon heating from 12 to 20 °C, and it must remain equally hydrated at the two temperatures. The latter is the case, as soon as the lipopolymers have undergone the pancakemushroom transition. The phase diagram of PEO in water40 does not show any miscibility gap in the temperature interval from 12 to 20 °C, nor do our spectroscopic results show any indication of demixing (see the discussion of Figures 5 and 8). And to the best of our knowledge,40,41 no phase transitions upon changing the temperature from 12 to 20 °C in fully hydrated PEO have ever been reported in the literature. Therefore we feel confident that building the subtraction spectra is possible and that the obtained results are valid. The lipid alkyl chains order, not the polymers. At this point let us consider the implications of our findings. Our present experiments resolve a major problem in the interpretation of our earlier IRRAS measurements of lipopolymers at the air-water interface.3,4 Only now we are able to distinguish directly between polymer and lipid in the lipopolymer. Previously we only observed the sum spectra of polymer and lipid, i.e. the spectra corresponding to Figure 3A. Thus, the experimental results presented here allow us to show that the ordering observed is due solely to the lipid alkyl chains reducing their number of gauche isomers. Since the PEO does not order during the “native” transition, logically the ordering observed in the sum spectra of h70-EO110 is also the effect of this lipid chain ordering (cf. also Figure 7B). Further, the absolute values for the measured peak positions of ν(CH2) for h70EO110 from sum spectra and subtraction spectra are very similar. Another important implication which follows from the IRRAS results for PEO (Figure 8) concerns the shorter lipopolymer h70-EO45 (DSPE-EO45) and two chemically different poly(2-oxazoline) lipopolymers whose IRRAS spectra we have reported earlier.4 We have observed how the molecular order within the CH2 groups of these lipopolymers increased with decreasing molecular area, i.e., the number of gauche isomers decreased during the “native” lipopolymer transition. Since we show in the present work that there is no ordering of the methylene groups in PEO during the “native” transition, we may therefore conclude that the observed ordering process in h70-EO45 as in h70-EO110 occurs exclusively within the lipid chains. Furthermore, and this is important, we demonstrated in our previous work4 that for the investigated lipopolymers showing the “native” lipopolymer transition, this “native” transition was identical for the different lipopolymers. The consequence is that also for the poly(2-oxazoline) lipopolymers undergoing the “native” transition, this involves ordering of the lipopolymer lipid chains without accompanying polymer ordering. In a previous publication we proposed the hypothesis that the “native” lipopolymer transition is a lateral condensation process of the lipid moieties.4 Our present data do not allow us to expand this model. However, it is now unambiguously proven that the basic assumption underlying this model is correct. The lipid alkyl chains of the lipopolymers really undergo an ordering transition. This constitutes an important step forward toward understanding the phase behavior of lipopolymer mono-

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layers at the air-water interface, an understanding one may hope to expand through application of structuresensitive techniques like X-ray and/or neutron reflectometry and scattering. 5. Some Important Abbreviations DSPE ) L-R-distearoylphosphatidylethanolamine h70 ) DSPE containing fully protonated alkyl chains d70 ) DSPE containing fully deuterated alkyl chains PE ) phosphatidylethanolamine PEO ) poly(ethylene oxide) EON ) poly(ethylene oxide) of monomer number N IRRAS ) infrared reflection absorption spectroscopy mRAU ) 10-3 reflectance-absorbance units ν ) stretching vibration motion as/s ) asymmetric/symmetric alk/eth ) alkyl/ether

Wiesenthal et al. νas/s(CH2)alk/eth ) methylene stretching mode vibration νas/s(COC) ) C-O-C stretching mode vibration of PEO

Acknowledgment. We thank Drs. C. Naumann, C. W. Frank, and W. Knoll for sharing their monolayer results of DMPE-EO45 (h62-EO45) and DPPE-EO45 (h66-EO45) with us prior to publication. Professor T. Bayerl and Dr. A. Boulbitch are thanked for many enlightening discussions. Professor G. Cevc is thanked for the gift of h70EO110 (DSPE-EO110). 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 Professor E. Sackmann for the possibility to perform this study in his laboratory and for helpful discussions. LA9901332