Langmuir 2006, 22, 1973-1975
1973
Articles Nanoscale Ripples in Self-Assembled Lipid Tubules Nidhi Mahajan,† Yue Zhao, Tianbao Du,‡ and Jiyu Fang* AdVanced Materials Processing and Analysis Center and Department of Mechanical, Materials, and Aerospace Engineering, UniVersity of Central Florida, Orlando, Florida 32816 ReceiVed June 29, 2005. In Final Form: December 14, 2005 Self-assembled cylindrical tubules of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) have been studied by atomic force microscopy in both the height and amplitude modes. Nanoscale ripple structures in the cylindrical lipid tubules are clearly resolved in amplitude mode images. The periodicity of the ripples is found to be 200 ( 30 nm for tubules with diameters in the range from 200 to 650 nm. The angle of the ripples with respect to the equator of the tubules shows a bimodal distribution with centers at ∼28° and ∼5°.
Introduction It is well-known that lipid bilayer membranes are flexible and can form a variety of different phases. One of the most intriguing phases is the ripple phase, which is characterized by corrugations with defined periodicities.1 It is generally believed that ripples result from a periodic local curvature in lipid bilayers. Different reasons for the origin of this periodic local curvature have been proposed, including electrostatic coupling between water molecules and the polar lipid headgroups,2 coupling between membrane curvature and molecular tilt,3 and generation of linear arrays of the fluid state.4 The ripple phase has been observed in a number of lipid bilayer systems, including multilamellar lipid bilayers,5-7 multilamellar liposomes,8-11 and supported lipid bilayers.12-15 It has been fascinating to a broad range of researchers in soft materials and biophysics as an example of periodically modulated phases. Lipid tubules are hollow cylindrical supramolecular structures formed by rolled-up lipid bilayers.16-17 The hollow cylindrical lipid tubules have been extensively characterized with electron microscopy (EM), but little is known about ripple structures * To whom correspondence should be addressed. E-mail: jfang@ mail.ucf.edu. † Present address: Amgen Inc., Thousand Oaks, CA. ‡ Present address: Applied Materials Inc., Sunnyvale, CA. (1) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta 2000, 1469, 159. (2) Doniach, S. J. Chem. Phys. 1979, 70, 4587. (3) Lubensky, T. C.; MacKintosh, F. C. Phys. ReV. Lett. 1993, 71, 1565. (4) Heimburg, T. Biophys. J. 2000, 78, 1154. (5) Woodward, J. T.; Zasadzinski, J. A. N. Biophys. J. 1997, 72, 964. (6) Cunningham, B. A.; Brown, A. D.; Wolfe, D. H.; Williams, W. P.; Brain, A. Phys. ReV. E 1998, 58, 3662. (7) Katsaras, J. S.; Tristram-Nagle, S.; Liu, Y.; Headrick, R. L.; Fontes, E.; Mason, P. C.; Nagle. J. F. Phys. ReV. E 2000, 61, 5668. (8) Copeland, B. R.; McConnell, H. M. Biochim. Biophys. Acta 1980, 599, 95. (9) Zasadzinski, J. A. N. Biochim. Biophys. Acta 1988, 946, 235. (10) Brown, R. E.; Anderson, W. H.; Kulkarni, V. S. Biophys. J. 1995, 68, 1396. (11) Raudino, A.; Castelli, F.; Briganti, G.; Cametti, C. J. Chem. Phys. 2001, 115, 8238. (12) Mou, J. X.; Yang, J.; Shao, Z. F. Biochemistry 1994, 33, 4439. (13) Fang, Y.; Yang, J. J. Phys. Chem. 1996, 100, 15614. (14) Leidy, C.; Kaasgaard, T.; Crowe, J. H.; Mouritsen, O. G.; Jørgensen, K. Biophys. J. 2002, 83, 2625. (15) Kaasgaard, T.; Leidy, C.; Crowe, J. H.; Mouritsen, O. G.; Jørgensen, K. Biophys. J. 2003, 85, 350. (16) Schnur, J. M. Science 1993, 262, 1669. (17) Shimizu, T.; Masuda, M.; Minamikawa, H. Chem. ReV. 2005, 105, 1401.
with a few exceptions. Yager et al.18 reported a ripple structure with a periodicity of 100 nm in an earlier freeze-fracture EM study of the tubules of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero3-phosphocholine (DC8,9PC). The angle of the ripple with respect to the equator of the tubules was found to be ∼60°, but later EM studies of DC8,9PC tubules show no ripples at the same resolution. More recently, Lauf et al.19 observed a ripple structure in the freeze-fracture EM images of the nanotubes of 1,2-dimyristoylsn-glycero-3-phosphatidylcholine (DMPC). They found that the ripples have a period of about 23 nm and run along the equator of the tubes. Atomic force microscopy (AFM) has been proven to be a powerful technique for studying the structure and kinetics of the ripple phase in a supported planar lipid bilayer.12-15 Recent AFM studies of cylindrical lipid tubules have shown some detailed information on molecular packing20,21 and helical markings,22-23 but ripple structures in the lipid tubules are not observed by AFM. In this paper, we present AFM studies of DC8,9PC tubules in both the height and amplitude modes. Nanoscale ripple structures in the cylindrical tubules, which are not visible in the height mode, are clearly resolved in the amplitude mode. The periodicity of the ripples is found to be 200 ( 30 nm for tubules with diameters in the range from 200 to 650 nm. The angle of the ripples with respect to the equator of the tubules shows a bimodal distribution with centers at ∼28° and ∼5°. Materials and Methods Lipid tubules were prepared by dissolving DC8,9PC (Avanti Polar Lipids, Alabaster, AL) in ethanol/water (70:30, v/v) at a concentration of 5 mg/mL and temperature of 60 °C and then slowly cooling the solution to room temperature at a rate of ∼0.5 °C/min. The polymerization of the tubule suspension was performed with UV (18) Yager, P.; Schoen, P. E.; Davies, C.; Price, R.; Singh, A. Biophys. J. 1985, 48, 899. (19) Lauf, U.; Fahr, A.; Westesent, K.; Ulrich, A. S. ChemPhysChem 2004, 5, 1246. (20) Shimizu, T.; Ohnishi, S.; Kogiso, M. Angew. Chem., Int. Ed. 1998, 37, 3260. (21) Song, J.; Cheng, Q.; Kopta, S.; Stevens, R. C. J. Am. Chem. Soc. 2001, 123, 3205. (22) Thomas, B. N.; Lindemann, C. M.; Corcoran, R. C.; Cotant, C. L.; Kirsch, J. E.; Persichini, P. J. J. Am. Chem. Soc. 2002, 124, 1227. (23) Mahajan, N.; Fang, J. Y. Langmuir 2005, 21, 3153.
10.1021/la051751n CCC: $33.50 © 2006 American Chemical Society Published on Web 02/03/2006
1974 Langmuir, Vol. 22, No. 5, 2006
Mahajan et al.
Figure 1. Height mode AFM images of a twisted stripe (a), a helical ribbon (b), and a partially closed tubule (c) on glass substrates. These images were taken after the samples were dried in air at room temperature for a day. The pitch angle of the helical ribbon is marked in (b). Figure 3. Diameter of the cylindrical tubules versus the periodicity of the ripples. The diameter was estimated from the measured height of cylindrical tubules with helical ripples.
Figure 2. Height (a) and amplitude (b) mode AFM images of DC8,9PC tubules on glass substrates. These images were taken simultaneously after the tubules were dried in air at room temperature for a day. irradiation (254 nm) for 20 min at room temperature. A drop of the tubule solution was placed on a cleaned glass substrate and dried in air at room temperature. An atomic force microscope (Dimension 3100, Digital Instruments) was used to characterize structures of the tubules adsorbed on the glass substrate. Silicon nitride cantilevers (Nanosensors) with a normal spring constant of about 30 N/m and a resonant frequency of about 260 kHz were used. The size of the cantilever tips (radius of curvature) is about 20 nm according to the manufacturer. The cantilever was excited just below its resonant frequency. All AFM measurements were performed in the tapping mode at a scan rate of 0.5 Hz in air under ambient conditions.
Results and Discussion When a drop of tubule solution was dried on a glass substrate, we observed a few twisted bilayer stripes (Figure 1a), helical ribbons (Figure 1b), and partially closed cylindrical tubules (Figure 1c). They represent intermediates during the formation of hollow cylindrical tubules. The measured thickness of the twisted bilayer stripes is about 54 nm. This corresponds to a stack of eight lipid bilayers because X-ray diffraction shows that the thickness of the lipid bilayer in the lipid tubules is about 6.6 nm.24 The helix of the ribbon shown in Figure 1b is turning from right to left and is noted as right-handed. The pitch angle of the helical ribbon is measured to be about 28°. It can be inferred from the partially closed tubule shown in Figure 1c that the formation of cylindrical tubules is a result of the closing of the ribbon gaps. Figure 2a shows a height mode AFM image of DC8,9PC tubules on a glass substrate. The external surface of the tubules appears to be considerably smooth and has a cylindrical shape, suggesting that the tubules are not compressed at the tip force employed for scanning and deformed by the adsorption. The apparent width of the tubules in the AFM image is broadened by the finite size of the AFM tip. Figure 2b is an amplitude mode image of the cylindrical tubules that was taken simultaneously with the height (24) Thomas, B. N.; Safinya, C. R.; Plano, R. J.; Clark, N. A. Science 1995, 267, 1635.
mode image during the scanning. We find that the image resolution in the amplitude mode is better than that in the height mode. The nanoscale ripple structures in the cylindrical tubules, which are not detected in the height mode, are clearly resolved in the amplitude mode. The sensitivity of the amplitude mode in imaging the nanoscale ripple structures of planar lipid bilayers was also reported in the literature.14,15 The ripple structure in the cylindrical lipid tubules is stable during the repeated scans when the loading force applied on the tip is low. The analysis of a large number of AFM images of DC8,9PC tubules shows that ∼7% of the total tubules have helical ripples. Because the nanoscale ripples in the cylindrical tubules are not visible in the height model image, the amplitudes of the ripples are unknown. The periodicity of the ripples was measured from the amplitude mode images as a function of the diameter of the cylindrical tubules with helical ripples. The diameter of the cylindrical tubules was estimated from their heights in the corresponding height mode images. The results are plotted in Figure 3. We find that there is no strong correlation between the periodicity and the diameter. The periodicity of the ripples is 200 ( 30 nm for cylindrical tubules with diameters in the range from 200 to 650 nm. It should be pointed out that the measured periodicity of the ripples is twice that reported in the earlier freeze-fracture EM study.17 The doubling of the periodicity is unlikely due to the effect of the tip size because the radius of the tip used in our measurements is about 20 nm, which is about 10 times smaller than the periodicity of the ripples. The difference in the periodicity might be related to the methods of sample preparation. For the freeze-fracture EM study,17 the sample was frozen in nitrogen and then fractured and replicated with 2 nm Pt-C films. Only the ripples, which align perpendicular to the shadowing direction, were visualized in the freeze-fracture EM study. In the AFM studies, the tubules were dried on glass substrates and imaged in air at room temperature. The analysis of AFM images of the cylindrical tubules with ripple structures shows that the angles of the helical ripples away from the equator of the tubules have a bimodal distribution with two centers at ∼28° and ∼5°, respectively (Figure 4). After being dried in air at room temperature for a week, some of the tubules deform on glass substrates through flattening (Figure 5a). The deformed tubules are about 195 nm high with a flattened top surface. Despite the significant deformation, there are no cracks and breaks observed on the surface of the flattened tubules. The helical markings, which are the edges of helical bilayer ribbons wrapped around the inner tubule core, are visible in the flattened tubules. The pitch angle of the helical ribbons away from the equator of the tubule is about 28°. Figure 5b is an amplitude mode image of the flattened tubules that was taken
Nanoscale Ripples in Self-Assembled Lipid Tubules
Figure 4. Histogram of the angles of the helical ripples away from the equator of the cylindrical tubules (n ) 24) with the fitted Gaussian distribution.
Figure 5. Height (a) and amplitude (b) mode AFM images of flattened DC8,9PC tubules on glass substrates. These images were taken simultaneously after the tubules were dried in air at room temperature for a week. The helical marking is inset in (a).
simultaneously with the height mode image during the scanning. The nanoscale ripples in the flattened tubules, which are not detected in the height mode image, are visible in the amplitude mode image. The periodicity of the ripples in the flattened tubules is similar to that in the cylindrical tubules. It is clear from the corresponding height mode image (Figure 5a) that the nanoscale ripples shown in Figure 5b run at the same angle as the helical bilayer ribbons, so the bimodal distribution of the ripple angles shown in Figure 4 suggests that the helical ribbons might have two distinct pitch angles. Recently, Lvov et al.25 used the selective adsorption of charged nanoparticles to decorate the edges of the helical ribbons forming the tubules of 1,2-bis(pentacosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,11PC) mixed with 2% 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphohydroxyethano(DC8,9PEOH). They found that the pitch angles away from the equator of the tubules were grouped around three values: 17°, 28°, and 42°. The most common pitch angle is about 28°, which agrees with the high angle of the helical ripples observed here. So far, low-pitch (5°) helical ribbons have not been reported in studies of lipid tubules. X-ray diffraction24,26 showed that the acyl chains are highly ordered and tilted in the DC8,9PC tubules. Theoretical models based on chiral interactions, coupled with molecular tilt, have been used to explain the formation of the cylindrical tubules.27-29 In these models, the chiral molecules do not pack parallel to their
Langmuir, Vol. 22, No. 5, 2006 1975
neighbors, but rather at a nonzero angle with respect to their neighbors. This chiral packing causes the twisting of a bilayer stripe and leads to the formation of a cylindrical tubule, which agrees with what we observe here (Figure 1). The model by Selinger et al.29 suggests that the tubules can have both uniform and modulated tilt states. In the uniform tilt state, the tubule has a constant direction of the molecule tilt with respect to the equator of the tubule. In the modulated tilt state, a striped modulation of the molecular tilt direction winds around the tubule in a helical fashion. The stripes are separated by sharp defect lines. This modulated tilt state is stable as long as the free-energy gain from the chiral interaction favoring the bend of the molecular tilt director and the coupling between the curvature and the splay of the molecular tilt director exceeds the free-energy cost of the defect lines. They further predict that the modulated molecular tilt can induce a ripple structure in the cylindrical tubules. Several experiments have suggested that the lipid tubules have some kind of modulated tilt structure. The deposition of charged polymers and nanoparticles in the lipid tubules reveals that tubules have a helical charge distribution,25 which is consistent with the modulated tilt structure because a periodic bend in the tilt direction can lead to a periodic splay in the electrostatic polarization vector, and hence to a periodic modulation of the charge density. In a recent publication,30 we imaged the DC8,9PC tubules with liquidcrystal optical amplification and found that the DC8,9PC tubules can induce both uniform and modulated tilt orders of 4-pentyl4′-cyanobiphenyl (5CB) liquid crystal. To further test whether the modulated tilt order of 5CB reflects the modulated tilt state of the lipid tubules, we studied the orientation of 5CB on the well-characterized tilt stripes in a Langmuir monolayer of pentadecanoic acid,31-32 which have been suggested to be analogous to the tilt stripes predicted in the lipid tubules, but in a planar rather than a cylindrical geometry. The liquid-crystal image of the tilt stripes in the monolayer is found to be similar to that in the tubule. The details will be discussed in a separate paper. The consistency gives us confidence to conclude that the lipid tubules have the modulated tilt state. The analysis of a large number of liquid-crystal images shows that ∼10% of the total tubules have the modulated tilt state, which is close to the percentage of ripple tubules observed here, so we infer that the observed nanoscale ripples might be induced by the modulated molecular tilt in the lipid tubules, which is predicted by current theories. In summary, we present AFM studies of cylindrical DC8,9PC tubules. Nanoscale ripple structures are observed in ∼7% of the total lipid tubules. We find that the periodicity of the ripples is 200 ( 30 nm for tubules with diameters in the range from 200 to 650 nm. The angle of the ripples away from the equator of the tubule shows a bimodal distribution with two distinct centers at ∼28° and ∼5°. We expect to gain a better understanding of the nature of the nanoscale ripple phase by studying the cylindrical tubules produced with different lipid molecules and their mixtures. The understanding of the ripple structures in lipid tubules is critical for studying molecular self-assembly during tubule formation and controlling the surface properties of the lipid tubules. LA051751N
(25) Lvov, Y. M.; Price, R. R.; Selinger, J. V.; Singh, A.; Spector, M. S.; Schnur, J. M. Langmuir 2000, 16, 5932. (26) Caffrey, M.; Hogan, J.; Rudolph, A. S. Biochemstry 1991, 30. 2134. (27) Helfrich, W.; Prost, J. Phys. ReV. A 1988, 38, 3065. (28) Ou-Yang, Z. C.; Liu, J. X. Phys. ReV. A 1991, 43, 6826. (29) Selinger, J. V.; Spector, M. S.; Schnur, J. M. J. Phys. Chem. B 2001, 105, 7157.
(30) Zhao, Y.; Mahajan, N.; Lu, R.; Fang, J. Y. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7438. (31) Ruiz-Garcia, J.; Qiu, X.; Tsao, M. W.; Marshall, G.; Knobler, C. M.; Overbeck, G. A.; Mobius, D. J. Phys. Chem. 1993, 97, 6955. (32) Schwartz, D. K.; Ruiz-Garcia, J.; Qiu, X.; Selinger, J. V.; Knobler, C. M. Physica A 1994, 204, 606.
