Bolaamphiphilic Phosphocholines: Structure and Phase Behavior in

Kim, J.-M.; Thompson, D. H. Langmuir 1992, 8, 637. ...... Mingotaud, A.-F.; Mingotaud, C.; Patterson, L. K. Handbook of Monolayers; Academic Press: Sa...
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Bolaamphiphilic Phosphocholines: Structure and Phase Behavior in Aqueous Media† Ciro Di Meglio,‡,§ Shankar B. Rananavare,§ So¨nke Svenson,‡ and David H. Thompson*,‡ Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393, and Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science & Technology, 20000 NW Walker Road, Beaverton, Oregon 97006-8921 Received May 24, 1999. In Final Form: November 3, 1999 The phase behavior of 1,1′-di-O-eicosamethylene-2,2′-di-O-decylbis(glycero-3-phosphocholine) (1) and 1,1′-di-O-hexadecamethylene-2,2′-di-O-octylbis(glycero-3-phosphocholine) (2) was studied at the air-water interface and in aqueous dispersions using monolayer film balance, small-angle X-ray scattering, Raman spectroscopy, and cryo-TEM techniques. The limiting molecular areas (20 °C, pH 6.8) were observed to be 172 Å2 for 2 and 105 Å2 for 1, indicating a U-shaped configuration of the long polymethylene chains at the air-water interface. The larger molecular area of 2 relative to 1 at the air-water interface suggests that the alkyl chains of 2 are less ordered than those of 1 at similar pressures. The extrapolated d spacings for 1 from SAXS experiments were 32 Å in the liquid crystalline lamellar phase and 40 Å in the gel phase where the hydrocarbon chains are in their all trans configuration. The extrapolated d spacings for 2 were 27 and 33 Å in the liquid crystalline and gel phases, respectively. DSC analysis revealed phase transition temperatures of 1 and 2 in excess water at 15.3 and -23.5 °C, respectively, with observed transition enthalpies of 9.1 and 2.9 kJ/mol, respectively. These values are in good agreement with Raman spectroscopy experiments that detect alkyl melting transitions near these temperatures. Addition of 30 mol % cholesterol to both bolaamphiphile samples lowered the observed transition temperatures (1, 9.5 °C; 2, -25.1 °C) and enthalpies (1, 5.7 kJ/mol; 2, 2.5 kJ/mol), suggesting that cholesterol reduces the transition cooperativity. Extruded samples of pure 1 in buffer (30 mg/mL in 20 mM Tris, pH 8) produced vesicle structures that were variable in size (0.15-1.5 µm), with multilamellar and nonspherical morphologies apparent. Incorporation of cholesterol in the extruded samples (7:3 1/Chol), however, gave stable spherical unilamellar vesicles with a mean diameter of 857 ( 237 Å. Similar results were observed for dispersions of 2 and 7:3 2/Chol. These results suggest that stable, unilamellar monolayer membrane vesicles can be formed from bolaamphiphiles with relatively short (C16 and C20) membrane-spanning alkyl chains.

Introduction Surfactants bearing hydrophilic substituents at the Rand ω-positions of a hydrophobe, so-called “bolaform amphiphiles”1,2 (also called bolaamphiphiles, bolas, or “gemini” surfactants3), have recently been the subject of numerous reports. While many of these studies describe the synthesis of phosphatidyl,4-14 amino acid,15-17 carbo* Corresponding author. Fax: 765-494-0386. E-mail: davethom@ chem.purdue.edu. † Part of the Special Issue “Clifford A. Bunton: From Reaction Mechanisms to Association Colloids; Crucial Contributions to Physical Organic Chemistry”. ‡ Purdue University. § Oregon Graduate Institute of Science & Technology. (1) Fuhrhop, J.-H.; Fritsch, D. Acc. Chem. Res. 1986, 19, 130. (2) Escamilla, G. H.; Newkome, G. R. Angew. Chem., Int. Ed. Engl. 1994, 33, 1937. (3) Zana, R. Curr. Opin. Colloid Interface Sci. 1996, 1, 566. (4) Yamauchi, K.; Moriya, A.; Kinoshita, M. Biochim. Biophys. Acta 1989, 1003, 151. (5) Yamauchi, K.; Sakamoto, Y.; Moriya, A.; Yamada, K.; Hosokawa, T.; Higuchi, T.; Kinoshita, M. J. Am. Chem. Soc. 1990, 112, 3188. (6) Kim, J.-M.; Thompson, D. H. Langmuir 1992, 8, 637. (7) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.; Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600. (8) Berkowitz, W. F.; Pan, D.; Bittman, R. Tetrahedron Lett. 1993, 34, 4297. (9) Thompson, D. H.; Svendsen, C. B.; Di Meglio, C.; Anderson, V. C. J. Org. Chem. 1994, 59, 2945. (10) Yamauchi, K.; Togawa, K.; Kinoshita, M. J. Biochem. 1996, 119, 115. (11) Eguchi, T.; Ibaragi, K.; Kakinuma, K. J. Org. Chem. 1998, 63, 2689. (12) Arakawa, K.; Eguchi, T.; Kakinuma, K. J. Org. Chem. 1998, 63, 4741.

hydrate,18-24 reactive,25 or novel hydrophobic linker26-29 bolas, comparatively few of these efforts have focused on the unique structural and physical properties displayed by this class of bioinspired materials. Interest in bolaform amphiphiles within our group has centered on their (13) Svenson, S.; Thompson, D. H. J. Org. Chem. 1998, 63, 7180. (14) Patwardhan, A.; Thompson, D. H. Org. Lett. 1999, 1, 241. (15) Fuhrhop, J.-H.; Spiroski, D.; Boettcher, C. J. Am. Chem. Soc. 1993, 115, 1600. (16) Schade, B.; Hubert, V.; Andre, C.; Luger, P.; Fuhrhop, J.-H. J. Peptide Res. 1997, 49, 363. (17) Kogiso, M.; Masuda, M.; Shimizu, T. Supramol. Chem. 1998, 9, 183. (18) Andre, D.; Luger, P.; Nehmzow, D.; Fuhrhop, J.-H. Carbohydr. Res. 1994, 261, 1. (19) Brisset, F.; Garelli-Calvet, R.; Azema, J.; Chebli, C.; Rico-Lattes, I.; Lattes, A.; Moisand, A. New J. Chem. 1996, 20, 595. (20) Pestman, J. M.; Terpstra, K. R.; Stuart, M. C. A.; van Doren, H. A.; Brisson, A.; Kellogg, R. M.; Engberts, J. B. F. N. Langmuir 1997, 13, 6857. (21) Bertho, J.-N.; Coue´, A.; Ewing, D. F.; Goodby, J. W.; Letellier, P.; Mackenzie, G.; Plusquellec, D. Carbohydr. Res. 1997, 300, 341. (22) Shimizu, T.; Masuda, M. J. Am. Chem. Soc. 1997, 119, 2812. (23) Rivaux, Y., Noiret, N., Patin, H. New J. Chem. 1998, 22, 857. (24) Auze´ly-Velty, R.; Benvegnu, T.; Plusquellec, D.; Mackenzie, G.; Haley, J. A.; Goodby, J. W. Angew. Chem., Int. Ed. Engl. 1998, 37, 2511. (25) Boehme, P.; Hicke, H.-G.; Boettcher, C.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1995, 117, 5824. (26) Moss, R. A.; Li, J. M. J. Am. Chem. Soc. 1992, 114, 9227. (27) Bhattacharya, S.; De, S.; Subramanian, M. J. Org. Chem. 1998, 63, 7640. (28) Unusual diols with an H-shaped hydrocarbon domain have recently been isolated from a thermophilic methanogen: Morii, H.; Eguchi, T.; Nishihara, M.; Kakinuma, K.; Ko¨nig, H.; Koga, Y. Biochim. Biophys. Acta 1998, 1390, 339. (29) Bolaamphiphiles with regioisomeric glycerol backbones have been prepared: Eguchi, T.; Kano, H.; Kakinuma, K. Chem. Commun. 1996, 365.

10.1021/la990627z CCC: $19.00 © 2000 American Chemical Society Published on Web 12/11/1999

Bolaamphiphilic Phosphocholines

Figure 1. Structures of 1,1′-di-O-eicosamethylene-2,2′-di-Odecylbis(glycero-3-phosphocholine) (1) and 1,1′-di-O-hexadecamethylene-2,2′-di-O-octylbis(glycero-3-phosphocholine) (2).

possible use as a host monolayer membrane matrix for coupled ion and electron-transfer30-34 and supported membrane devices. Previously reported efforts from this laboratory30,31 utilized bisglycerolphosphate-based bolaamphiphiles with C16 and C20 membrane-spanning chains. Dispersions of these materials, however, proved difficult to handle, since their sensitivity to acidic pH’s and multivalent cations provided an extremely narrow range of solution conditions for studying their photochemical properties. By recasting the headgroup of these phospholipids as the phosphocholine derivatives, it was believed that more stable vesicle suspensions would be obtained. This paper describes the physical properties of synthetic bolaform amphiphiles13,14 with eicosane (1) and hexadecane (2) chains that couple two glycerophosphocholine headgroups. Experimental Methods Materials. The bolaamphile lipids (Figure 1) used in this work were prepared as described previously and gave satisfactory analytical data.9,13 Purified water, obtained from a Millipore 18 MΩ system, was used as the subphase for monolayer experiments and for all other solution preparations. All other materials were obtained from Aldrich (g98% pure) and used as received. Monolayer Measurements. The monolayer film balance used was a KSV 3000 trough (KSV Instruments, Helsinki, Finland) milled from a solid block of Teflon to provide a 712.44 cm2 surface area over a 1.1 L total subphase volume. The density of the monolayer film was controlled by a mobile hydrophilic barrier which swept the interface by means of a microstepped DC motor. The surface pressure was measured with a sandblasted platinum Wilhelmy plate which was dipped in 100% ethanol, heated with a bunsen burner, and suspended from an electronic microbalance after cooling. The Wilhelmy plate was maintained in the null position by an electronic negative feedback loop. Surface pressure was determined digitally using the restoring force on the plate and a computer-stored calibration constant. Each experiment utilized chloroform solutions (40.0 µL) of 1 (1.02 mg/mL) or 2 (0.68 mg/mL) spread onto water at 20 °C using a microsyringe with solvent evaporation for ∼ 1 h prior to compression. A compression rate of -6.0 cm2/min was used. The π-A isotherms were measured three times for each sample and were exactly reproducible. Differential Scanning Calorimetry (DSC). Phase-transition temperatures and enthalpies were determined using a Perkin-Elmer DSC-7 with a TAC7/PC instrument controller (30) Thompson, D. H.; Kim, J.-M. Mater. Res. Soc. Symp. Proc. 1992, 277, 93. (31) Thompson, D. H.; Kim, J.-M.; Di Meglio, C. SPIE Proc. 1993, 1853, 142. (32) Komatsu, T.; Yamada, K.; Tsuchida, E.; Siggel, U.; Bo¨ttcher, C.; Fuhrhop, J.-H. Langmuir 1996, 12, 6242. (33) Tsuchida, E.; Komatsu, T.; Fuhrhop, J.-H. Polym. Adv. Technol. 1998, 9, 569. (34) Komatsu, T.; Yanagimoto, T.; Tsuchida, E.; Siggel, U.; Fuhrhop, J.-H. J. Phys. Chem. B 1998, 102, 6759.

Langmuir, Vol. 16, No. 1, 2000 129 interfaced to an IBM PS/2 Model 50 Z computer. Decane (Tm ) -29.7 °C) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Tm ) 41 °C) were used as calibration standards. Powder samples of bolaamphiphiles were weighed into aluminum pans (6-7 mg), 50 µL of Millipore water was added, and the pan was sealed. Thermograms were collected in both heating and cooling modes using a scan rate of 1.0 °C/min. The temperature ranges scanned were -40 to 60 °C for 1 and -40 to -10 °C for 2. Typically, data from four thermograms were collected to determine the average values for the transition temperature (Tc) and the transition enthalpy (∆H). Small-Angle X-ray Scattering Studies (SAXS). These experiments were performed using a Phillips XRG-2500 generator operating at 35 kV and 20 ma with a sealed fine focus copper tube. The X-ray beam was monochromatized (l ) 1.54 Å) using a β-nickel filter and a pinhole collimator. The instrument was calibrated using lead stearate as a standard. The lipid/water mixtures were bath sonicated for 1 day and equilibrated for 5 days prior to insertion into Mark capillaries (1 mm o.d.) within a temperature-regulated ((0.25 °C) chamber. The diffraction patterns were collected using a linear position sensitive detector (spatial resolution of 92 µm) interfaced to a personal computer through a Nuclear Data multichannel analyzer. The sampledetector distance (30 cm) was adjusted to permit detection of small angle diffraction peaks. Vesicle Preparation. Lipid samples of 1 (35 mg) or 7:3 1/Chol (30 mg of 1, 0.0279 mmol; 4.6 mg of cholesterol, 0.012 mmol) were dissolved in ∼2 mL of 2:1 chloroform/methanol in a cryovial and evaporated with a stream of nitrogen gas. Buffer (1.0 mL, 20 mM Tris, pH 8) was then added and the suspension hydrated using five cycles of freezing (LN2)/heating above the phase transition/vortexing for 30 s. This emulsion was extruded 10 times through two stacked polycarbonate membranes (0.08 µm) with a thermobarrel Extruder35 and used immediately unless otherwise noted. Cryogenic-TEM Experiments. A drop of the lipid dispersion was transferred to a 400 mesh gold grid and lightly blotted with filter paper from the opposite side of the grid from which the drop was applied. The sample grid was then rapidly plunged into a liquid propane bath cooled to -195 °C and the sample transported, using a Reichert-Jung Model KF80 apparatus, to a Gatan 126 LN2-cooled cryostage within a Zeiss EM 10C electron microscope operating at 60 kV. Micrographs were recorded at 12500× magnification on areas that best represented the overall sample appearance. The photographs were then magnified (2.5×), digitized using a Lacie Silverscan II interfaced with a Power Mac 6100/60w, and analyzed using Adobe Photoshop. The observed scaling factor was determined by correlating the microscope calibration bar with optically distinct features. Particle size estimates were based on populations of 100-125 particles. Dynamic Light Scattering (DLS). Extruded bolaamphiphile samples were diluted, typically ∼1:20, prior to DLS measurements. Vesicle size distributions were determined using a Nicomp Model 200 Laser Particle Sizer (Nicomp Instruments, Goleta, CA) which employed a 5 mW He Ne laser operating at 632.8 nm.

Results and Discussion Lyotropic Phase Behavior. Monolayer Experiments. The limiting molecular area was determined to be 172 Å2 for 2, whereas the limiting molecular area for 1 was 105 Å2 (Figure 2). Compound 1 exhibits a limiting molecular area that is approximately twice that found for 1,2dieicosanoyl-sn-glycerophosphocholine, while 2 displays a limiting molecular area that is roughly twice that found for 1,2-dihexadecanoyl-sn-glycerophosphocholine and 1,2dioctanoyl-sn-glycerophosphocholine.39 Since 1 and 2 both (35) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55. (36) Moss, R. A.; Fujita, T.; Okumura, Y. Langmuir 1991, 7, 2415. (37) De Rosa, M. Thin Solid Films 1996, 284-285, 13. (38) Fyles, T. M.; Zeng, B. J. Org. Chem. 1998, 63, 8337. (39) Mingotaud, A.-F.; Mingotaud, C.; Patterson, L. K. Handbook of Monolayers; Academic Press: San Diego, CA, 1993; Vol. 1, pp 778-804.

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Figure 2. Pressure-area curves for monolayers of 1 and 2 (20 °C, pH 6.8). Each experiment utilized chloroform solutions (40.0 µL) of 1 (1.02 mg/mL) or 2 (0.68 mg/mL) spread onto water using a microsyringe with solvent evaporation for ∼1 h prior to compression. A compression rate of -6.0 cm2/min was used. The π-A isotherms were measured three times for each sample and were exactly reproducible.

have two glycerophosphocholine headgroups, the limiting molecular areas for 1 and 2 are consistent with a U-shaped configuration of the alkyl chain tethering the two glycerol backbones, as has been observed for more closely related flexible bolaamphiphiles.6,36-38,40 The observation of a greater area for 2 than 1 suggests that 2 has less ordered chains (i.e. more fluid-like character) under these experimental conditions, a result of fewer hydrophobic interactions that limit molecular packing in the liquid-expanded state. These observations are similar to those reported by this laboratory for bisglycerophosphate-based bolas possessing C16 and C20 chains.6 Small-Angle X-ray Scattering. Preparations of 1 in quartz capillaries gave rise to X-ray reflections in the lowangle region with measured d spacings that varied as a function of water concentration (Figure 3a and Supporting Information Table 1). From pure lipid up to 25% water, the d spacing did not vary significantly. The scattering patterns in this composition regime did not exhibit second or higher order peaks, indicative of gel-phase lipid at room temperature. Higher order peaks began to appear in samples containing >25% water. The long spacings in the low-angle region occurred in a 1:2:3 ratio, indicative of a lamellar structure. The water-dependent, one-dimensional repeat distance d100 characterizes the small-angle X-ray reflection spacings in a series of stacked lamellae. The observed repeat distances increased with increasing water content between 50% and 65% water until a limiting value was reached at ∼75% water. After this point, water no longer caused interlamellar swelling; further addition of water simply contributed to the excess free (intervesicular) water phase. The effective lamellar thickness dl, was calculated41 from this composition-dependent d spacing data.42 These values were then used to estimate the molecular area A0, within the lamellar phase at various (40) Elongated molecular conformations at the air-water interface (i.e. perpendicular to the aqueous subphase) have only been reported for less polar and relatively rigid bisaroyl azide bolas.25 (41) Luzzati, V.; Gulik-Krzwicki, T.; Tardieu, A. Nature 1968. 218, 1031. (42) (a) The effective bilayer thickness dl was calculated using the formula dl ) d100/[1 + (vw/vl)(1 - c)/c], where d100 is the unidimensional lamellar repeat distance and c is the weight fraction of the lipid at limiting hydration. The values vl and vw are the partial specific volumes for the lipid and the interlamellar water, respectively. (b) Marsh, D. Handbook of Lipid Bilayers; CRC Press, Inc.: Boca Raton, FL, 1990; pp 163-173.

Figure 3. Phase diagrams of 1 (a) and 2 (b) determined by small-angle X-ray scattering as described in the Experimental Section. The vertical lines in the diagrams are intended to indicate approximate phase boundaries between the gel, lamellar, and vesicular states of the bolaamphiphile dispersions in water at various water/lipid ratios.

lipid/water ratios.43 The extrapolated d spacing for 1 in the liquid crystalline lamellar phase is 32 Å. This differs from the 40 Å d spacing of the gel-phase lipid, where the hydrocarbon chains are in their crystalline, all trans, conformation, since (i) the molten chains contribute less to the lamellar thickness due to increased gauche conformations and (ii) linear extrapolation of d spacing values assumes that all the water is involved in unidirectional swelling, which neglects the contributions from headgroup hydration. The latter effect could lead to an underestimation of the membrane thickness, particularly at low water contents. The measured d spacings as a function of water concentration for 2 appear in Figure 3b (the calculated dl and A0 parameters are summarized in Supporting Information Table 2). Unlike 1, the gel phase of 2 disappears below 10% water concentration; however, the crosssectional area at an equivalent water concentration is comparable (44 Å versus 48 Å). The lamellar phase exists (43) (a) The molecular area was obtained from the lipid thickness, dl by the equation A0 ) zMvl/(NAdl), where M is the lipid molecular weight, NA is Avogadro’s number, and z is 1 for monolayer lamella and 2 for bilayer structures. (b) Marsh, D. General Features Of Phospholipid Phase-Transitions. Chem. Phys. Lipids 1991, 57, 109.

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Table 1. Phase Transition Temperatures of Phosphocholine Bolaamphiphile Samples Determined by DSC lipid onset Tc (°C) composition heating cycle

∆H (kJ/mol)

onset Tc (°C) cooling cycle

∆H (kJ/mol)

15.3 ( 0.5 9.5 ( 0.4 -23.5 ( 2.6 -25.1 ( 2.2

9.1 ( 0.5 5.7 ( 0.4 2.9 ( 1.5 2.5 ( 1.2

14.0 ( 1.0 13.6 ( 1.0 -23.4 ( 1.5 -24.8 ( 0.2

-9.2 ( 0.1 -6.0 ( 0.6 -1.3 ( 0.6 -0.6 ( 0.5

1 7:3 1/Chol 2 7:3 2/Chol

within a narrow regime between 10 and 20% water. The cross-sectional area of 2 in the lamellar phase is significantly lower (52-57 Å2) than that of 1, presumably due to a lower extent of headgroup hydration. An increase in chain ordering relative to 1 is suggested by the larger incremental length per methylene group for 2. Assuming that these bolaamphiphiles adopt a membrane-spanning orientation like their structurally similar bisglycerophosphate derivatives44 and other structurally related analogues,26,45,46 this increase in segment length and low extent of hydration suggest that the alkyl chains of 2 are fully extended (i.e. trans conformation), producing a rigid hydrophobic domain. Since the hydrophobic interactions should be weaker for 2 relative to 1, we infer from these observations that the interfacial tension acting on both headgroups of bolaamphiphiles in their membrane-spanning conformation will stretch the molecules and reduce the number of gauche conformations in the chains. This effect should be more significant for 2, since interactions between the hexadecamethylene chains will provide a weaker opposing force than the corresponding eicosamethylene analogue. Phase Transition Temperatures. Differential Scanning Calorimetry. A phase transition of hydrated 1 at 15.3 °C, with a transition enthalpy of 9.1 kJ/mol, was observed during the heating cycle of the DSC analysis (Table1). The thermal parameters acquired during the cooling cycles of these samples were in good agreement with these values. Addition of 30 mol % cholesterol to the sample of 1 lowered both the observed transition temperature (9.5 °C) and the enthalpy (5.7 kJ/mol), suggesting a reduced cooperativity in the transition. The shorter alkyl chains of 2 also reduced the cooperativity, leading to lower observed phase transition temperatures and enthalpies: -23.5 °C and 2.9 kJ/ mol for pure 2 and -25.1 °C and 2.5 kJ/mol for 7:3 2/Chol. These values are considerably lower than those found for the related monopolar phospholipids 1,2-dihexadecyl-snglycero-3-phosphocholine (Tc, 43 °C, ∆H, 38 kJ/mol) and 1,2-dieicosyl-sn-glycero-3-phosphocholine (Tc, 68 °C, ∆H, 59 kJ/mol),47 reflecting a weaker hydrophobic cohesion of the bolaamphiphiles due to water interpenetration at both ends of the lyophobic unit. Raman Spectroscopy. Analysis of Raman spectra recorded at various temperatures, using the ratio method48 of peak intensities at 2936 and 2883 cm-1 (Figure 4a and b), indicates that the phase transitions observed in the DSC experiments of 1 and 2 are alkyl chain-melting transitions. The phase transition temperatures obtained (44) 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. (45) Bhattacharya, S.; De, S.; George, S. K. Chem. Commun. 1997, 2287. (46) Gliozzi, A.; Robello, M.; Relini, A.; Accardo, G. Biochim. Biophys. Acta 1994, 1189, 96. (47) Quantities reported for the hexadecyl derivative were averaged over four different published values, whereas values for the eicosyl derivative were estimated; see: Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990; pp 138 and 149. (48) Levin, I. W. In Advances in Infrared and Raman Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Wiley: Heyden, 1984; Vol. 11, pp 1-48.

Figure 4. Alkyl chain melting transitions of 1 (a) and 2 (b) determined using the ratio method of Raman scattering intensities.48 The transitions were taken as the point of major inflection in the ratio plots, with the apparent Tc of 1 occurring at ∼19 °C and the Tc of 2 occurring at approximately -20 °C.

by this method (19 °C for 1 and -20 °C for 2) are in reasonable agreement with those determined by DSC. The ratio values are also lower than those observed for conventional phospholipid dispersions, strongly suggesting that significant populations of U-shaped conformers do not exist in the bola samples, in good agreement with the conclusions drawn from the SAXS experiments (see above). Bisphosphatidic acid bola dispersions were also found to contain few U-shaped conformers on the basis of SAXS and Raman experiments.44 Phase morphology. Cryo-TEM and Dynamic Light Scattering. Samples of 1 in buffer (30 mg/mL in 20 mM Tris, pH 8) could not be extruded at room temperature; however, they could be successfully processed at 60 °C. The resulting samples were kept in a 60 °C bath until imaged by cryo-TEM (∼2 h elapsed between the extrusion and imaging steps). The vesicle structures observed (Figure 5a) were variable in size, with multilamellar and nonspherical morphologies apparent. Despite the fact that

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Figure 5. (a) Cryo-transmission electron micrograph of extruded 1 in 20 mM Tris buffer, pH 8.0. Scale bar: 100 nm. (b) Cryotransmission electron micrograph of extruded 7:3 1/Chol in 20 mM Tris buffer, pH 8.0. Scale bar: 100 nm. (c) Vesicle diameter histogram of extruded 7:3 1/Chol determined from analysis of cryo-TEM images.

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the samples of 1 were extruded through 0.08 µm filters, the observed range of particle sizes was 0.15-1.5 µm. It is presumed that small vesicles were formed during the extrusion process, which rapidly fused to relieve excessive membrane curvature; however, it is also possible that the larger structures observed were produced during lipid hydration and simply deformed upon passage through the extrusion membrane pores. The images of 7:3 1/Chol vesicles (Figure 5b) clearly show spherical unilamellar vesicles. Unlike the pure 1 case, these systems did not show significant populations of vesicles with dimensions >100 nm. Figure 5c shows the diameter distribution for 7:3 1/Chol dispersions. The consistency of mean particle sizes, as determined by dynamic light scattering, in freshly extruded and aged (3 days) samples (mean diameter ) 857 ( 237 and 817 ( 195 Å, respectively) indicates that no significant vesicle diameter changes occur during this period. Extruded samples of 7:3 2/Chol also produced vesicles with similar diameters by DLS analysis (data not shown). The results from the microscopy experiments indicate that polydisperse nonspherical vesicle structures exist for pure 1, with sizes in some cases exceeding 1 µm. Planar lamellae and very large vesicles, similar to those observed for bisglycerophosphatidic acid bolas,44 would be expected for a symmetrical bipolar lipid like 1. This is especially true if the population of U-shaped species at the outer vesicle surface is small.49 Conversely, cholesterol-containing bolaamphiphile dispersions are capable of forming stable spherical vesicles, presumably by adopting an asymmetric distribution of this dopant at opposing membrane interfaces, thus allowing the formation of highly curved surfaces. Conclusions The results of our monolayer experiments indicate that a U-shaped conformation of bolaamphiphiles is present at the air-water interface. The highly ordered alkyl chains indicated by the SAXS and Raman experiments, however, (49) An asymmetric distribution of U-shaped conformations, with a greater proportion of them necessary on the outer surface to accommodate the surface area mismatch between the inner and outer surfaces, is required to form highly curved vesicles with diameters e 0.1 µm.

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suggest a type of packing which would favor lamellar structures with low curvature. The latter data strongly suggest the presence of an elongated membrane-spanning conformation in aqueous dispersions of liquid crystalline and gel-phase glycerophosphocholine bolaamphiphiles, rather than the U-shaped configuration predicted on the basis of the monolayer data. The melting transition temperatures and enthalpies of 1 and 2 are substantially lower than their monopolar counterparts of similar alkyl chain lengths, suggesting that water interpenetration at both membrane-water interfaces diminishes the hydrophobic cohesion of bolaamphiphile membranes. Cryo-TEM and DLS experiments show that stable unilamellar vesicles of ∼830 Å diameter form from 7:3 mixtures of bola/Chol. SAXS experiments reveal hydrated lamellar thicknesses of pure liquid crystalline bolaamphiphile membranes of 32 and 27 Å for 1 and 2, respectively. Taken together, these results suggest that vesicles formed from these materials should be capable of serving as a host matrix for insertion of the ion channel gramicidin A. The sodium ion permeability of these monolayer membrane vesicles in the presence and absence of gramicidin A will be described elsewhere.50 Acknowledgment. The authors would like to thank Professor Pieter R. Cullis for providing access to the University of British Columbia Microscope Facility (cryoTEM experiments) and Professor Dor Ben-Amotz for his efforts in helping us acquire the Raman data. The technical expertise of Kevin Maloney (monolayer measurements) and Jeff Wheeler (cryo-TEM experiments) is also acknowledged. Support by the National Science Foundation (Grant MCB-9319099) is greatly appreciated. Supporting Information Available: Tables of X-ray parameters for 1 and 2 and temperature-dependent Raman spectra. This material is available free of charge via the Internet at http://pubs.acs.org. LA990627Z (50) Patwardhan, A.; Di Meglio, C.; Rananavare, S. B.; Svenson, S.; Haynes, R.; Thompson, D. H. Unpublished results.