Temperature-Dependent Aggregation Behavior of Symmetric Long

and Institut für Pharmazeutische Chemie, MLU Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4,. 06120 Halle/Saale, Germany. ReceiVed October 18, 2005...
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Langmuir 2006, 22, 2668-2675

Temperature-Dependent Aggregation Behavior of Symmetric Long-Chain Bolaamphiphiles at the Air-Water Interface Karen Ko¨hler,†,‡ Annette Meister,† Bodo Dobner,§ Simon Drescher,§ Friederike Ziethe,§ and Alfred Blume*,† Institut fu¨r Physikalische Chemie, MLU Halle-Wittenberg, Mu¨hlpforte 1, D-06108 Halle/Saale, Germany, and Institut fu¨r Pharmazeutische Chemie, MLU Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle/Saale, Germany ReceiVed October 18, 2005. In Final Form: January 24, 2006 The behavior of the symmetric long-chain bolaamphiphiles dotriacontane-1,32-diyl bis[2-(trimethylammonio)ethyl phosphate] (PC-C32-PC), and dotriacontane-1,32-diyl bis[2-(dimethylammonio)ethyl phosphate] (Me2PE-C32Me2PE) at the air-water interface was investigated by means of temperature-dependent film-balance measurements and Brewster angle microscopy. Upon compression of the monolayer the isotherms show a strong surface pressure increase. We assume that at high pressure the monolayer consists of molecules in a reversed U-shaped conformation. At an area of 0.9-1.1 nm2 per molecule a plateau is reached for both bolaamphiphiles, which marks the beginning of an aggregate formation on the water surface. The plateau pressure increases with increasing temperature. For PC-C32-PC at 6.7 °C curved shorter fibrous domains with a diameter of 20-30 µm are seen on the water surface, whereas at 29.2 °C stripelike domains with a thickness of 200-500 µm are observed. Isotherms recorded within this temperature range show a characteristic break within the steep slope marking a region where a mixture or a hybrid form of both structures exists. Me2PE-C32-Me2PE in its zwitterionic state at low pH forms microcrystals on the water surface, whose formation is kinetically retarded. Depending on the temperature, the aqueous subphase is more or less homogeneously covered with a crystalline-like film. In contrast, no aggregates are observed at pH 10 when the bolaamphiphile is negatively charged.

Introduction Organized molecular films at the air-water interface as well as on solid substrates find application in a variety of different fields, e.g., nanotechnology,1 material science,2 and biomimetic chemistry.3 Regarding the behavior at the air-water interface, monofunctional amphiphilic molecules are the most commonly used model compounds which have been extensively studied. In a monolayer of amphiphiles, the hydrophilic headgroups are immersed in the water and the hydrophobic tails jut out into the air. Compared to the many investigations on amphiphilic molecules with one polar group, only a few studies are devoted to the behavior of bolaamphiphiles at the air-water interface. Bolaamphiphiles consist of two polar headgroups covalently linked by one or more hydrophobic chains (for a recent review see ref 4). The original interest in these molecules stems from the bipolar ether lipids of archaebacterial membranes.5,6 These natural bolaamphiphiles and similar synthetic analogues can form monolayer lipid membranes (MLMs), which are stable at extreme conditions, such as high temperature,7 low pH,8 and high ionic strength. By altering the length, cross-sectional area, and nature * To whom correspondence should be addressed. E-mail: [email protected]. † Institut fu ¨ r Physikalische Chemie. ‡ Current address: Max-Planck Institute of Colloids and Interfaces, 14424 Golm/Potsdam, Germany. § Institut fu ¨ r Pharmazeutische Chemie. (1) Weissbuch, I.; Baxter, P. N. W.; Cohen, S.; Cohen, H.; Kjaer, K.; Howes, P. B.; Als-Nielsen, J.; Hanan, G. S.; Schubert, U. S.; Lehn, J.-M.; Leiserowitz, L.; Lahav, M. J. Am. Chem. Soc. 1998, 120, 4850-4860. (2) Swift, J. A.; Pivovar, A. M.; Reynolds, A. M.; Ward, M. D. J. Am. Chem. Soc. 1998, 120, 5887-5894. (3) Sengupta, K.; Schilling, J.; Marx, S.; Fischer, M.; Bacher, A.; Sackmann, E. Langmuir 2003, 19, 1775-1781. (4) Fuhrhop, J.-H.; Wang, T. Chem. ReV. 2004, 104, 2901-2937. (5) Kates, M.; Kushner, D. J.; Matheson, A. T. The Biochemistry of Archaea (Archaebacteria); Elsevier: Amsterdam, London, New York, Tokyo, 1993. (6) Nishihara, M.; Morii, H.; Koga, Y. J. Biochem. 1987, 101, 1007-1015. (7) Kim, J.-M.; Thompson, D. H. Langmuir 1992, 8, 7-644.

of the hydrophobic or hydrophilic parts, a great variety of unique aggregation morphologies is found in addition to lamellar membrane structures.9-14 Recently, monolayer investigations of bolaamphiphiles have attracted considerable interest since these films provide a fundamental model to understand the molecular orientation and packing mode of bipolar molecules at the airwater interface. Basically, two possibilities arise for the orientation of these molecules in monolayers; either one or both polar headgroups are immersed in the water. In the case that only one headgroup is in contact with water, only the hydration energy of one headgroup is released and the stretched molecules have a parallel or tilted orientation with respect to the surface normal. If both hydrophilic chain ends are immersed in water, the bolaamphiphiles can either lie flat on the water surface or form a reversed U-shaped conformation. Some evidence about the actual conformation of the molecules can be obtained from the molecular area at the film collapse. For most investigated bolaamphiphiles the reversed U-shaped conformation is observed,7,15-20 whereas a monolayer of perpendicularly oriented molecules was (8) Fuhrhop, J.-H.; Liman, U.; Koesling, V. J. Am. Chem. Soc. 1988, 110, 6840-6845. (9) Sirieix, J.; Lauth-de Viguerie, N.; Riviere, M.; Lattes, A. Langmuir 2000, 16, 9221-9224. (10) Thompson, D. H.; Wong, K. F.; Humphry-Baker, R.; Wheeler, J. J.; Kim, J.-M.; Rananavare, S. B. J. Am. Chem. Soc. 1992, 114, 9035-9042. (11) Iwaura, I.; Yoshida, K.; Masuda, M.; Yase, K.; Shimizu, T. Chem. Mater. 2002, 14, 3047-3053. (12) Estroff, L. A.; Hamilton, A. D. Angew. Chem., Int. Ed. 2000, 39, 34473450. (13) Estroff, L. A.; Leiserowitz, L.; Addadi, L.; Weiner, S.; Hamilton, A. D. AdV. Mater. 2003, 15, 38-42. (14) Shimizu, T.; Iwaura, R.; Masuda, M.; Hanada, T.; Yase, K. J. Am. Chem. Soc. 2001, 123, 5947-5955. (15) Patwardhan, A. P.; Thompson, D. H. Langmuir 2000, 16, 10340-10350. (16) Lee, J.; Joo, H.; Youm, S. G.; Song, S.-H.; Jung, S.; Sohn, D. Langmuir 2003, 19, 4652-4657. (17) Di Meglio, C.; Rananavare, S. B.; Svenson, S.; Thompson, D. H. Langmuir 2000, 16, 128-133. (18) Liu, M.; Cai, J. Langmuir 2000, 16, 2899-2901.

10.1021/la052798b CCC: $33.50 © 2006 American Chemical Society Published on Web 02/17/2006

Aggregation BehaVior of Long-Chain Bolaamphiphiles

Figure 1. Chemical structure of the bolaamphiphiles PC-C32-PC with R ) CH3 and Me2PE-C32-Me2PE with R ) H (at pH 5) consisting of two bulky headgroups connected by a C32 alkyl chain.

only reported for bolaamphiphiles with a very short or an extremely rigid hydrophobic region.15,18,19,21 However, an orientation perpendicular to the water surface can also be induced by decreasing the molecular area of a film where the molecules initially have a reversed U-shaped or horseshoe conformation.22 Recently we reported on the remarkable self-assembly behavior of the symmetric long-chain bolaamphiphile dotriacontane1,32-diyl bis[2-(trimethylammonio)ethyl phosphate] (PC-C32PC).23,24 In the meantime we have also studied an analogue compound with a slightly changed headgroup structure, namely, the dimethylammonio analogue dotriacontane-1,32-diyl bis[2(dimethylammonio)ethyl phosphate] (Me2PE-C32-Me2PE) (Figure 1).25 Both bolaamphiphiles gel water very efficiently in their zwitterionic state at concentrations below 1 mg mL-1 by forming a dense network of helically structured fibrils. Within these fibrils it is assumed that the extended molecules are arranged next to each other, but because of the larger space requirements of the headgroups compared to the small cross-sectional area of the alkyl chain, they are slightly twisted relative to each other. Such an arrangement leads to the formation of the observed helical fibers whose diameter corresponds roughly to the length of extended bolaamphiphile molecules. In the case of PC-C32-PC this self-assembly process seems to be solely driven by hydrophobic interactions between the long alkyl chains. The interesting molecular architecture with its packing restrictions and the exceptional aggregation behavior in aqueous suspension raised the question about the arrangement of these bolaamphiphiles at the air-water interface. In the present study we provide a detailed analysis of the surface pressure (π)-molecular area (A) isotherms at different temperatures combined with Brewster angle microscopy that enables the detection of morphological changes within the monolayer, e.g., homogeneity, orientation, defects, or domain formation. Materials and Methods Materials. PC-C32-PC and Me2PE-C32-Me2PE were synthesized as described previously.23,26 Chloroform and methanol were purchased from Roth (Karlsruhe, Germany). Sodium acetate, acetic acid (100%), sodium carbonate, and sodium hydrogencarbonate (p.a.) were purchased from Merck (Darmstadt, Germany), Riedel-de Hae¨n (19) Fuhrhop, J.-H.; Krull, M.; Schulz, A.; Mo¨bius, D. Langmuir 1990, 6, 497-505. (20) Jonkheijm, P.; Fransen, M.; Schenning, A. P. H. J.; Meijer, E. W. J. Chem. Soc., Perkin Trans. 2 2001, 1280-1286. (21) Bo¨hme, P.; Hicke, H.-G.; Boettcher, C.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1995, 117, 5824-5828. (22) Vogel, V.; Mo¨bius, D. Thin Solid Films 1985, 132, 205-219. (23) Ko¨hler, K.; Fo¨rster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A. Angew. Chem., Int. Ed. 2004, 43, 245-247. (24) Ko¨hler, K.; Fo¨rster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A. J. Am. Chem. Soc. 2004, 126, 16804-16813.

Langmuir, Vol. 22, No. 6, 2006 2669 (Seelze, Germany), Acros (New Jersey), and Solvay Alkali GmbH (Rheinsberg, Germany), respectively. Film Balance Measurements. The π-A isotherms were recorded using a Wilhelmy film balance with a thermostated Teflon trough (Riegler and Kirstein GmbH, Berlin, Germany). Typically, 40 µL of a 0.3 mM bolaamphiphile solution in a mixture of chloroform and methanol (3:1) was spread on the subphase. After 10 min of solvent evaporation and temperature equilibration the π-A isotherms were recorded at a constant compression speed of 286 mm2 min-1. For the measurements of PC-C32-PC the subphase was water, whereas for the pH-sensitive Me2PE-C32-Me2PE it consisted of a 10 mM buffer solution of pH 5 or 10. At pH 5 Me2PE-C32-Me2PE is in its zwitterionic state, and at pH 10 the phosphate groups are negatively charged.25 Brewster Angle Microscopy (BAM). The morphology changes of the monolayer film were observed using a MiniBAM instrument (Nanofilm Technology GmbH, Go¨ttingen, Germany). To avoid scattering of light from the trough bottom, a black glass plate was put in the trough below the microscope. A high-power diode laser (30 mW, 688 nm) was used as the source of light, and the images were taken using the integrated CCD camera. The lateral resolution of the microscope is approximately 20 µm.

Results and Discussion PC-C32-PC. Surface Pressure-Area Isotherms. The π-A isotherms of PC-C32-PC on pure water at different temperatures are plotted on the left of Figure 2. All isotherms exhibit a long surface pressure increase with an onset at a large molecular area of about 2.5 nm2. By further compression a plateau is reached between 0.9 and 1.0 nm2 per molecule depending on the temperature of the subphase. Below a molecular area of 0.4 nm2 the pressure increases a little bit within the plateau region. Because of the restricted compression ratio of the used Langmuir trough, the surface films could only be measured up to a molecular area of 0.25 nm2 per molecule. Stopping the compression in the plateau region results in a sudden drop of the surface pressure. By contrast, at larger molecular areas within the range of the steep pressure increase, the pressure stays constant for a much longer time. The right graph of Figure 2 shows the dependence of the surface pressure at the onset of the plateau region between 0.9 and 1.0 nm2 per molecule as a function of temperature. The plateau pressure increases with increasing temperature and approaches a limiting pressure of about 24.5 mN m-1. The opposite was observed for bixin bolaamphiphiles19 and R,ω-13,16-dimethyloctacosanedioate dimethyl ester.16 In the case of bixin the behavior was explained by the enthalpy for the conformational transition of the linear (parallel to the air-water interface) to reversed U-shape, whereas in the latter case a highly temperature-dependent hydration of the headgroups, and thus a change from the horizontal to the erected orientation initiated by increasing temperatures, was assumed. The isotherms between 11 and 20 °C exhibit a characteristic break at an area of approximately 1.1 nm2 per molecule. In this temperature range the pressure increases just before the plateau, and this increase becomes steeper at higher temperature. At 25 °C the second step cannot be observed anymore. By extrapolating the linear part of the surface pressure increase to zero, a limiting area of approximately 1.4 nm2 is obtained. This value corresponds to more than twice the cross-sectional area requirements for a phosphocholine headgroup, and it indicates a possible horseshoelike conformation of the bolaamphiphile with both headgroups in the water. The comparison with the limiting molecular area (25) Ko¨hler, K.; Meister, A.; Fo¨rster, G.; Dobner, B.; Drescher, S.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Hause, G.; Blume, A. Soft Matter 2006, 2, 77-86. (26) Ziethe, F. Ph.D. Thesis, University of Halle-Wittenberg, Germany, 2003.

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Figure 2. π-A isotherms of PC-C32-PC at different temperatures (top), and the temperature dependence of π at the plateau (bottom; the line is only a guide to the eyes).

of 0.57 nm2 per molecule at 25 °C for the double-chained phospholipid l,2-dipalmitoyl-glycero-3-phosphocholine (DPPC), which bears additionally the glycerol backbone, supports this interpretation.27 Our own preliminary experiments using X-ray reflectivity and infrared reflection absorption spectroscopy (IRRAS) of the bolaamphiphile films give further support that a horseshoe-like conformation is adopted at this pressure (unpublished results). Similar molecular area values for long-chain phosphocholine bolaamphiphiles were reported by Di Meglio et al.17 Therefore, the isotherms of PC-C32-PC can be interpreted as follows: At large molecular areas and a surface pressure of almost zero, the bolaamphiphile molecules lie isolated and in an extended conformation on the water surface with the alkyl chain almost parallel to the water surface. Upon compression, the molecules come in contact with each other and the long flexible C32 chains start to bend upward. At a certain molecular area, i.e., the beginning of the pressure plateau, a horseshoe-like conformation exists. Further approach of the polar headgroups becomes energetically unfavorable due to repulsion and the strong bending of the chains. As a consequence the formation of multilayers or other 3D aggregates and/or a possible squeeze-out of these structures into the subphase or above the air-water surface can take place when the area is reduced. The beginning of the plateau marks the transition to nonmonolayer structures, i.e., 3D aggregates of various shapes. The onset pressure at the beginning of the plateau region increases

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with temperature. For the transition from liquid-expanded (LE) to liquid-condensed (LC) monolayers the onset pressure also increases with temperature. However, for the bolaamphiphiles the transition to 3D structures always occurs at constant molecular area, whereas for the LE-LC transition the molecular area at the onset of the plateau decreases with increasing temperature. This is due to the fact that the LE-LC transition vanishes above the critical temperature; i.e., it shows critical behavior. In contrast the transition to 3D structures shows no critical behavior. Here, the thermal fluctuations at higher temperature just lead to an increase in pressure for the conversion to the 3D aggregate structures. That in the plateau region these structures are formed is indicated by the strong drop in surface pressure when the compression is stopped and by the results of our BAM measurements (see below). The shape of the π-A isotherms of PC-C32-PC plotted in Figure 2 seems to be characteristic for bolaamphiphiles, since for similar phosphocholine derivatives17 as well as for some tetraether bolaamphiphiles7,15,28 curves with a long-range pressure increase and a subsequent plateau starting from molecular areas of 1.2-0.8 nm2 are observed. In all these cases a reversed U-shaped conformation of the bipolar compounds at the airwater interface is assumed with a transition to a collapsed structure in the plateau region. Brewster Angle Microscopy. To get a more detailed view of the behavior of PC-C32-PC at the air-water interface, the amphiphile films were investigated at different temperatures by means of BAM. Within the region of the pressure increase at large molecular areas no domains could be observed by BAM at all temperatures; only a black water surface was seen. At 6.7 °C shortly after the plateau pressure is reached the formation of small rodlike domains starts (Figure 3A), which have a width of 20-30 µm and grow during further compression in one direction (Figure 3B) until they form curved fibrous domains with a length of several millimeters (Figure 3C,D). These flexible fibrous domains aggregate, and are irregularly distributed over the water surface, so that there are aggregate-free regions as well. The structures formed at the air-water interface at 29 °C differ significantly from the aggregates formed at 6.7 °C. When the film is compressed to an area of 0.7 nm2 per molecule, isolated aggregates with low contrast in the form of stripes with a thickness of 200-500 µm and a length of several tens of millimeters appear on the water surface. With decreasing molecular area, these stripes appear clearer due to a better contrast (Figure 4C). In comparison to the thinner curved fibrous domains observed at 6.7 °C these stripes seem to be much more rigid. To detect the transition temperature at which both structures interconvert, the domain formation in the plateau region of the surface pressure-area isotherm was followed at different temperatures between 7 and 29 °C, and the structures seen at 0.45 nm2 per molecule were compared. Therefore, the isotherms at every temperature were measured separately, since a halt at constant area per molecule within the plateau region leads to a dramatic drop of surface pressure as already mentioned above. At 9.8 °C no oblong structures are observed, but bright grainy domains surrounded by some haze appear (Figure 4A). After a further temperature increase to 11.1 °C, the grainy aggregates are no longer seen and blurred stripes form out of the haze (Figure 4B). At higher temperatures the stripes become clearer until they look like the ones shown in Figure 4C. (27) Rolland, J.-P.; Santaella, C.; Vierling, P. Chem. Phys. Lipids 1996, 79, 71-77. (28) Fuhrhop, J.-H.; David, H.-H.; Mathieu, J.; Liman, U.; Winter, H.-J.; Boekema, E. J. Am. Chem. Soc. 1986, 108, 1785-1791.

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Figure 3. BAM images of PC-C32-PC at 6.7 °C and a molecular area of (A) 0.90 nm2, (B) 0.60 nm2, (C) 0.45 nm2, and (D) 0.45 nm2.

In accordance with the assumptions made from the π-A isotherms, PC-C32-PC does not form a monomolecular film within the plateau region of the isotherm, but large aggregate structures of diverse form and nature. The arrangement of the bolaamphiphile molecules within the domains is still not clear due to their large dimensions compared to the size of the individual molecules and the size of the 6-7 nm thick fibers observed in bulk water.23,24 AFM investigations on transferred films to mica gave no clue about the structure of the aggregates. The temperature-dependent aggregate structures observed with BAM can be correlated with the breakpoints in the π-A isotherms. At 6.7 and 29.2 °C either short fibrous domains or broader stripelike domains, respectively, exist at low molecular areas, and the isotherms at these temperatures do not show an additional breakpoint before the plateau region. The breakpoints of the π-A curves of the temperatures in between (11.1, 15.6, and 20.2 °C) point accordingly to the existence of either a mixture of both aggregate types or incompletely formed structures. If this interpretation is correct, then the first breakpoint at a molecular area of about 1.1 nm2 (the beginning of the plateau of the isotherm at 6.7 °C) indicates the formation of the short fibrous domains shown in Figure 3. The second breakpoint, i.e., the beginning of the plateau region of these isotherms, characterizes obviously the formation of the broader stripelike domains (Figure 4C). With increasing temperature this transition is shifted to higher molecular areas until at 24.9 °C the first breakpoint vanishes and only stripelike aggregates are observed. A comparison with the behavior of PC-C32-PC in bulk solution shows that the formation of different types of large aggregates at the air-water surface is not connected with the transitions observed in the bulk system.

In this temperature range only stiff nanofibers were observed in the solution, and no transition was seen up to 40 °C.24 Me2PE-C32-Me2PE. Surface Pressure-Area Isotherms. The pKa value for the phosphate group of Me2PE-C32-Me2PE was determined to be 3.3 and for the dimethylammonio group to be 6.5.25 Accordingly, at pH 5 both functional groups are charged and Me2PE-C32-Me2PE is in a zwitterionic state. The investigations were first carried out using a 10 mM acetate buffer solution at pH 5 as the subphase. The left graph of Figure 5 shows the π-A isotherms at different temperatures. As in the case of PC-C32-PC, Me2PE-C32-Me2PE exhibits a long-range surface pressure increase at large molecular areas followed by a region where the pressure is essentially constant, the plateau region. However, some differences can be detected in the isotherms of the two zwitterionic bolalipids. The onset of the surface pressure increase of Me2PE-C32-Me2PE is found at around 2 nm2 per molecule, and is thus shifted to lower molecular areas. Furthermore, at a constant temperature much lower plateau pressures are found for Me2PE-C32-Me2PE in comparison to its trimethylamino analogue PC-C32-PC. For example, at 20 °C the surface pressure at the beginning of the plateau is 14.3 mN m-1 for Me2PE-C32-Me2PE, whereas it is 20.3 mN m-1 for PCC32-PC under the same conditions. On the right side of Figure 5 the temperature dependence of the plateau pressure at the beginning of the plateau is plotted. As can be seen the plateau pressure increases linearly with increasing temperature. None of the isotherms of Me2PE-C32-Me2PE show a second breakpoint within the investigated temperature range, but characteristic overshoots at the beginning of the plateau instead. A similar behavior was observed for the bolaamphiphiles 1,1′-di-O-

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Figure 5. π-A isotherms of Me2PE-C32-Me2PE on 10 mM acetate buffer, pH 5, as the subphase at different temperatures (top), and the temperature dependence of the surface pressure at the plateau (bottom; the line is only a guide to the eyes). The π-A isotherm on 10 mM carbonate buffer, pH 10, as the subphase at 20 °C is presented for comparison.

Figure 4. BAM images of PC-C32-PC at a molecular area of 0.45 nm2 and a temperature of (A) 9.8 °C, (B) 11.1 °C, and (C) 29.2 °C.

eicosamethylene-2,2′-di-O-decylbis(glycero-3-phosphocholine),17 methylbixin,19 and 1,16-bis(acrylacyloxy)hexadecane diester.29 For the diester the surface pressure decrease marks the collapse of the monomolecular film and the beginning of the formation of crystalline islands. The same is expected for methylbixin, where the monolayer composed of stiff, horizontally oriented molecules is not compressible by vertical folding or uplifting. As already assumed for PC-C32-PC, at large molecular areas (>2 nm2) the molecules of Me2PE-C32-Me2PE probably lie in a stretched or slightly bent conformation horizontally on the water surface. With further compression the interactions between the molecules increase, the alkyl chain bends, and the molecules form a reversed U-shaped conformation. The beginning of the

plateau between 1 and 1.1 nm2 per molecule is found at a molecular area similar to that for PC-C32-PC, and the limiting molecular area of approximately 1.3 nm2 is only slightly lower. The overshoot of pressure seen at the beginning of the plateau could indicate the beginning of a metastable region followed by crystallization or formation of aggregates. At the maximum pressure new structures form spontaneously, resulting in a decrease of surface pressure to an equilibrium value. Within the investigated temperature range all π-A isotherms exhibit an identical shape, which points to the existence of the same type of aggregates on the air-water interface between 6.7 and 33.6 °C. Figure 5 also shows the π-A isotherm of Me2PE-C32-Me2PE on 10 mM carbonate buffer solution at pH 10 as the subphase for comparison. At 20°C this isotherm exhibits an extremely shallow surface pressure increase starting at a molecular area larger than 3.2 nm2. This behavior is likely due to the repulsion of the negatively charged headgroups in addition to the unfavorable bending of the alkyl chains upon compression. The surface pressure at the beginning of the plateau is 19.2 mN m-1, and the corresponding molecular area is found to be similar (1.0-1.1 nm2) to the values observed for the other bolalipids. (29) Dubault, A.; Casagrande, C.; Veyssie, M.; Caille, A.; Zuckermann, M. J. J. Colloid Interface Sci. 1978, 64 290-299.

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Figure 6. BAM images of Me2PE-C32-Me2PE on 10 mM acetate buffer, pH 5, as the subphase at 6.7 °C and a molecular area of (A) 0.82 nm2, (B) 0.70 nm2, (C) 0.50 nm2, (D) 0.38 nm2, and (E) 0.35 nm2.

Brewster Angle Microscopy. Figure 6 shows the BAM images of the π-A isotherm at 6.7 °C. Within the plateau region at a molecular area of about 0.85 nm2 per molecule the formation of a homogeneous granular film can be observed (Figure 6A). With further compression and thus decreasing molecular area this film can be seen more clearly because of increased contrast (Figure 6B). Obviously, the film consists of small microcrystals, which are formed along the plateau. The kinetically inhibited formation of these crystals could explain the observed characteristic overshoots of the surface pressure before the beginning of the plateau. Below a molecular area of about 0.5 nm2 darker areas appear on the initially uniformly covered water surface (Figure 6C). These darker domains spread out with further compression of the film, and black domains become visible (Figure 6D). The black color indicates that probably holes are

formed in the film. Further compression to 0.35 nm2 per molecule leads to a collapse of the film structure. Clearly, cracks can be seen in Figure 6E, indicating the solid crystalline-like structure of the film. Although we expected for Me2PE-C32-Me2PE the same type of aggregates at the air-water interface within the investigated temperature range due to the similar shapes of the π-A isotherms, clear differences in the domain formation are observed at temperatures higher than 6.7 °C. At 29 °C no homogeneous film is formed at the beginning of the plateau, but a granular banded film consisting of darker and lighter stripelike domains (Figure 7A). This structure does not change much with decreasing molecular area; only the contrast between the different regions of the film is enhanced under BAM, and the small microcrystals seem to coalesce to bigger structures (Figure 7B,C).

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C32-Me2PE. Whereas at pH 5 the above-mentioned domain structures are observed, at pH 10 no formation of surface aggregates could be found despite the fact that the isotherm also shows a plateau region where the formation of aggregated structures can be expected. Because of the negatively charged headgroups of Me2PE-C32-Me2PE at pH 10, it is possible that these aggregates are squeezed out into the subphase because of their better solubility.

Conclusions

Figure 7. BAM images of Me2PE-C32-Me2PE on 10 mM acetate buffer, pH 5, as the subphase at 29.2 °C and a molecular area of (A) 0.82 nm2, (B) 0.55 nm2, and (C) 0.28 nm2.

Between 7 and 29 °C a continuous conversion between the domains shown in Figures 6 and 7 is observed. Compared to PC-C32-PC, the dimethylammonio analogue Me2PE-C32Me2PE forms completely different domain structures at the airwater interface; only before the plateau region a similar reversed U-shaped conformation of the bolaamphiphiles seems to be evident. These differences can be explained by the possibility of Me2PE-C32-Me2PE to form intermolecular hydrogen bonds between the headgroups, whereas for PC-C32-PC this is not possible due to a missing hydrogen bond donor. It is interesting to note that the pH of the subphase strongly influences the formation of aggregate structures for Me2PE-

Due to their molecular architecture with different space requirements between the large headgroups and the long hydrocarbon chain the symmetric bolaamphiphiles PC-C32-PC and Me2PE-C32-Me2PE proved to have not only a remarkable self-assembly behavior in bulk water, but also interesting temperature-dependent aggregation properties at the air-water interface. The shapes of the surface pressure-area isotherms of the investigated bolalipids are at first glance very similar. At large molecular areas the bipolar molecules probably form a homogeneous monolayer and adopt a reversed U-shaped conformation when the surface pressure increases up to the plateau region at a limiting molecular area of about 1.3-1.4 nm2. At lower areas in the plateau region the surface pressure stays constant for both of the bipolar molecules. Within this region PC-C32-PC and Me2PE-C32-Me2PE show, depending on the temperature, a characteristic domain formation on the water surface. The plateau pressure for Me2PE-C32-Me2PE is lower at all temperatures compared to that for PC-C32-PC. This is caused by the higher tendency of Me2PE-C32-Me2PE to form 3D aggregates due to the additional attractive interaction via hydrogen bonds between headgroups. Using Brewster angle microscopy, short fibrous domains with a thickness of 20-30 µm can be observed within the plateau region of the isotherm of PC-C32-PC at 6.7 °C. In contrast, at 29.2 °C at the same molecular area 200-500 µm broad stripelike domains are found on the water surface. An intermediate form between these two domain types exists at temperatures within this range. The isotherms show a breakpoint shortly before the plateau marking the formation of the fibers, whereas the actual beginning of the plateau of these isotherms marks the formation of the stripe structures. With increasing temperature the plateau pressure increases until it reaches a maximum above around 40 °C. Also for Me2PE-C32-Me2PE the plateau pressure increases with increasing temperature, but linearly within the investigated temperature range. At the beginning of the isotherms measured on a subphase of pH 5 a characteristic overshoot of the surface pressure due to a kinetically retarded formation of microcrystals is observed. BAM images taken within the plateau region of Me2PE-C32-Me2PE at pH 5 show a more or less complete film of microcrystals on the water surface. The coverage is less complete with increasing temperature. The H-bonding possibility of Me2PE-C32-Me2PE is lost when the pH is increased to 10. In addition, the now negatively charged headgroups repel each other. These two effects prevent large-scale aggregate formation so that no domains can be observed using BAM. Compared to most other bolaamphiphiles described in the literature, the chemical structures of PC-C32-PC and Me2PEC32-Me2PE have as a unique property the long C32 alkane chain between the two headgroups. Similar bolaamphiphiles described by Di Meglio17 have C16 or C20 chains and an additional glycerol linkage between the phosphocholine headgroup and the chain. These compounds, however, show completely different behavior in the bulk as they form lamellar phases and vesicles. Also, their monolayer behavior is different.

Aggregation BehaVior of Long-Chain Bolaamphiphiles

The measurements show the delicate balance between attractive and repulsive forces in monomolecular films of our bolaamphiphiles as apparent from the strong influence of temperature on the domain formation. The structures found at the air-water interface are different from those observed in bulk solution where mostly a network of fibers is seen at low temperature and the increase in temperature leads to a breakdown of the network and also the fibers at higher temperature. Whether the fibrous aggregates observed at the surface are composed of fibrils with 3-5 nm diameter similar to those observed in the bulk is unclear at present. It may well be that the air-water interface serves as

Langmuir, Vol. 22, No. 6, 2006 2675

a template to orient the fibrils so that they can form larger sheetlike structures as observed before for PC-C32-PC at higher concentration using freeze-fracture EM.24 Further experiments are needed to understand this remarkable self-assembly behavior at the airwater surface and to clarify the arrangement of the molecules within these aggregate structures. Acknowledgment. This work was supported by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft. LA052798B