Growth of Giant Membrane Lobes Mechanically Driven by Wetting

Dec 15, 2004 - We report on the growth of giant membrane lobes that is mechanically driven by wetting fronts of phospholipid membranes at water-solid ...
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Langmuir 2005, 21, 537-544

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Growth of Giant Membrane Lobes Mechanically Driven by Wetting Fronts of Phospholipid Membranes at Water-Solid Interfaces Kenji Suzuki* and Hiroshi Masuhara* Department of Applied Physics, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received February 18, 2004. In Final Form: July 4, 2004 We report on the growth of giant membrane lobes that is mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces and a strategy to control the two-dimensional structure of the membrane lobes on a solid surface. The growth of giant membrane lobes was observed on a singlelipid bilayer which spread from a lump of phospholipid deposited on a silica-glass substrate or an oxidized silicon wafer in aqueous solutions of NaCl, KCl, MgCl2, or CaCl2 at relatively high salt concentrations. Most of the membrane lobes were very flat unilamellar tubes elongating from the lump of phospholipid, and their length reached 1 mm in 5 h. Experimental findings clearly indicate that the membrane lobes are adherent to the surface of the single-lipid bilayer and are mechanically elongated from the lump of phospholipid by the sliding motion of the single-lipid bilayer. We could control the two-dimensional structure of the membrane lobes on the substrate by controlling the spreading direction of the single-lipid bilayer using Pt micropatterns that were deposited on the smooth surface of the oxidized silicon wafer.

Introduction Phospholipid membranes have been studied as model biomembranes1 and have been used as a convenient material for such applications as reconstituted membranes and as delivery vehicles for drugs and genetic materials.2,3 Most of the basic properties and functions of phospholipid membranes have been examined using phospholipid membranes with simple geometries: planar bilayer lipid membranes supported on solid surfaces,4-8 and spherical vesicles (liposomes) dispersed in aqueous solutions.1-3,9 Recently, to further explore the functionalities of phospholipid membranes, the creation of more-complex microstructures of phospholipid membranes immobilized at water-solid interfaces have received much attention and have been demonstrated by utilizing microscopic techniques: vesicle fusion onto micropatterned surfaces,10-14 microcontact printing or blotting of lipid bilayers,15,16 and micromanipulation of liposomes.17-20 As suggested re(1) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238. (2) Gregoriadis, G., Ed. Liposome Technology Volume II; CRC Press: Boca Raton, FL, 1984. (3) Gregoriadis, G., Ed. Liposome Technology Volume III; CRC Press: Boca Raton, FL, 1984. (4) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (5) Merkel, R.; Sackmann, E.; Evans E. J. Phys. (Paris) 1989, 50, 1535. (6) Bayerl, T. M.; Bloom, M. Biophys. J. 1990, 58, 357. (7) Czaja, C.; Jekutsch G.; Rothernha¨usler, B.; Gaub, H. E. Biosensers; VCH: Weinhem, New York, 1987. (8) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (9) Gregoriadis, G., Ed. Liposome Technology Volume I; CRC Press: Boca Raton, FL, 1984. (10) Stelzle, M.; Miehlich R.; Sackmann E. Biophy. J. 1992, 63, 1346. (11) Groves, J. T.; Wu¨lfing C.; Boxer, S. G. Biophy. J. 1996, 71, 2716. (12) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651. (13) Oudenaarden, A.; Boxer, S. G. Science 1999, 285, 1046. (14) Groves, J. T.; Ulman, N.; Cremer, P. S.; Boxer, S. G. Langmuir 1998, 14, 3347. (15) Kung, L. A.; Kam, L.; Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 6773. (16) Hovis, J. S.; Boxer, S. G. Langmuir 2001, 17, 3400. (17) Evans, E.; Browman, H.: Leung, A.; Needham, D.; Tirrell, D. Science 1996, 273, 933.

peatedly in these studies, water-solid interfaces work not only as a support medium for the resulting microstructures but also as an environment that induces dynamical shape transformations of the phospholipid membranes due to membrane-solid interactions. Therefore, to realize a variety of functional microstructures at water-solid interfaces, it is important to search for new shape transformation phenomena and to take advantage of them. Recently, Ra¨dler and co-workers have studied the hydration processes of lumps of phospholipid at watersolid interfaces, and they have found two fundamentally different spreading mechanisms of phospholipid membranes on the water-solid interfaces: (1) the sliding of a single-lipid bilayer on hydrophilic solid surfaces and (2) the rolling (i.e., a tank-tread-like rolling process) of membrane lobes on a dehydrated surface.21 These shape transformations of phospholipid membranes have been regarded as wetting phenomena driven by gains in free energy due to the formation of membrane-solid coupling. The sliding of a single-lipid bilayer has been intensely studied in relation to the formation of supported planar bilayer lipid membranes that have potential applications in biocompatible coatings and sensoric interfaces.21-23 However, there have been few studies that focus on the spreading of nonplanar structures of phospholipid membranes, including the rolling of the membrane lobes, although such structures would be very advantageous for creating novel nonplanar microstructures at water-solid interfaces. In this study, we have investigated the wetting phenomena of phospholipid membranes on silica-glass sub(18) Karlsson, A.; Karlsson, R.; Karlsson, M.; Cans, A.-S.; Stro¨mberg, A.; Ryttsen, F.; Orwar, O. Nature 2001, 409, 150. (19) Karlsson, M.; Sott, K.; Cans, A.-S.; Karlsson, A.; Karlsson, R.; Orwar, O. Langmuir 2001, 17, 6754. (20) Karlsson, M.; Sott, K.; Davidson, M.; Cans, A.-S.; Linderholm, P.; Chiu, D.; Orwar, O. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11573. (21) Ra¨dler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539. (22) Nissen, J.; Gritsch, S.; Wiegand, G.; Ra¨dler, J. O. Eur. Phys. J. B 1999, 10, 335. (23) Nissen, J.; Jacobs, K.; Ra¨dler, J. O. Phys. Rev. Lett. 2001, 86, 1904.

10.1021/la040027m CCC: $30.25 © 2005 American Chemical Society Published on Web 12/15/2004

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strates and oxidized silicon wafers systematically varying salt concentrations and we have found a new growth mechanism of membrane lobes that occurs on a singlelipid bilayer spreading on a water-solid interface at relatively high salt concentrations. We used fluorescence microscopy and microspectroscopy to reveal that the most feasible structure of the resulting membrane lobe was a flat unilamellar tube that adheres to the underlying single-lipid bilayer. According to the growth dynamics of the membrane lobes, it was clarified that their growth mechanism clearly differs from the rolling of the membrane lobes or the double-bilayer lobes that had been reported previously.21,22,24 On the basis of these results, we have controlled the two-dimensional structures of the membrane lobes by utilizing micropatterns fabricated on smooth solid surfaces. Since the growth of the membrane lobes enables us to construct novel, nonplanar microstructures of phospholipid membranes on solid supports, it is of great importance to explore further the functionalities of the phospholipid membranes. Experimental Section Materials. The phospholipids used in this study were 99% L-R phosphatidylcholine from egg yolk (99%Egg-PC) (Sigma, P-2772) and 60% L-R phosphatidylcholine from egg yolk (60%Egg-PC) (Sigma, P-5394). The latter contains approximately 71% phosphatidylcholine (PC), 21% phosphatidylethanolamine (PE), and 8% phosphatidylserine (PS) and is more negatively charged than the former in aqueous solutions at pH 8.25 Perylene (Nacalai GR-26614), a commonly used fluorescence probe for membranes,26,27 was used after purification by sublimation. Optically flat silica-glass substrates (Sigma Koki, OPSQ) and n-type (111) silicon wafers (Nilaco, SI-500443) were used after various surface treatments that will be described later. All of the aqueous solutions of the salts, such as NaCl, KCl, MgCl2, and CaCl2, were prepared using a buffer solution of 10mM Tris-HCl (pH 8) and were filtered through membrane filters (Whatman, PURADISC 25PP). The aqueous solutions were bubbled with N2 gas for 1h to reduce the dissolved oxygen that enhances the photobleaching of perylene fluorescence under a fluorescence microscope and were stored at 25 °C. Surface Treatments of Solid Substrates. The silicaglass substrates were washed with a surfactant (Nacalai Tesque, SCATT) and rinsed with distilled water. The washed silica-glass substrates were immersed in an SC1 solution (NH4OH:H2O2:H2O ) 1:1:10)28 at 80 °C for 15 min and immersed in an aqueous solution of 5% HF for 5 min to reduce the sharpness of any scratches on the substrates. The silica-glass substrates were then rinsed with distilled water in an ultrasonic cleaner and finally dried at 60 °C prior to use. The n-type silicon wafers were washed with the surfactant and rinsed with distilled water, and they were also immersed in the aqueous solution of HF to remove the native oxide on the surfaces of the silicon wafers along with contaminants. The silicon wafers were immersed in the SC1 solution at 80 °C for 15 min to form an oxide layer of less than 1 nm in thickness.28 The oxidized silicon wafers were rinsed with pure water and finally dried at 60 °C prior to use. To prepare clean surfaces on oxidized silicon wafers with Pt micropatterns, n-type silicon wafers were oxidized in an electric furnace at 1100 °C. The thickness of the resulting oxide layer on the silicon wafers was about 40 nm. Pt micropatterns were deposited on the oxidized silicon wafers using a focused ion beam system (FIE, Dual Beam 253 system). A gallium ion beam was scanned on the oxidized silicon wafers in a vacuum chamber into (24) Sackmann, E. Science 1996, 271, 43. (25) Dimitrov, D. S.; Angelova, M. I. Prog. Colloid Polym. Sci. 1987, 73, 48. (26) Johnson, D. A.; Nguyen, B.; Bohorquez, A. F.; Valenzuela, C. F. Biophys. Chem. 1999, 79, 1. (27) Khan, T. K.; Chong, P. L.-G. Biophys. J. 2000, 78, 1390. (28) Kern, W.; Puotinen, D. A. RCA Rev. 1970, 31, 207.

Suzuki and Masuhara which a gaseous organic Pt compound was introduced. Assisted with the gallium ion beam, the gas was decomposed into a film of Pt at the scanned areas. After the Pt deposition, the patterned, oxidized silicon wafers were immersed in HF (0.5%) to completely remove the oxide layers on the Pt-free areas, and then the substrates were immersed in an SC1 solution to produce a chemical oxide of less than 1 nm in thickness on the bare silicon surfaces in the Pt-free areas. The resulting micropatterns consisted of Pt lines with a height of ca. 0.16 µm and a width of ca. 1.6 µm. Finally, the resulting oxidized silicon wafers with the Pt micropatterns were rinsed with distilled water and dried at 60 °C prior to use. Hydration of Lumps of Phospholipid on Substrates. A chloroform solution of phospholipid (7.2 mg/mL) and perylene was prepared in a ratio of ca. [perylene]: [lipid] ) 1:200. Next, 2 µL of the chloroform solution was painted onto a glass rod with a diameter of about 0.1 mm in 1 min in air using a microsyringe. Some of the solvent evaporated during the process, and thin films of lipid were left on the surface of the glass rod. Immediately after that, the glass rod was stored in a desiccator connected to a rotary vacuum pump for 1 h to remove the solvent completely. The dried lipid films were stamped onto the clean surfaces of the silica-glass substrates or the oxidized silicon wafers to deposit a line of phospholipid on the substrates as a lipid source. The substrates were placed into a well on a slide glass and the well was filled with an aqueous solution of a salt by dropping a sufficiently large droplet of the aqueous solution onto the substrate. The well was then covered with a cover slip and was placed on the stage of an incident-light fluorescence microscope (Olympus, BX-50). Fluorescence Microscopy and Microscopic Spectroscopy. The wetting phenomena of phospholipid membranes at the three-phase boundary of the water, the lipid, and the solid were observed using the fluorescence microscope with 10, 20, and 40 × objectives lenses (Olympus, Uplan Apo). Using a dichroic mirror unit (Olympus, U-MNV), perylene molecules that were doped in each of the lipid bilayers were excited by an emission line at 404.7 nm from a high-pressure mercury lamp (Ushio, USH-102D) and the fluorescence above 455 nm was observed though the fluorescence microscope. The fluorescence images were captured using a CCD camera (Olympus, DP-50) that was included on the fluorescence microscope. Fluorescence microspectroscopic measurements were performed using a multichannel spectrophotometer (Hamamatsu Photonics, PMA-10) that was equipped on the microscope. The measured areas were selected using the microscope aperture. All of the experiments were performed in a clean room at 25 ( 1 °C. Turbidity Measurements. Multilamellar vesicles (MLV) were used to investigate the salt aggregation of liposomes. The chloroform solution of the phospholipid was evaporated in a round-bottom flask, which was then evacuated overnight. The dried lipid films that were left on the walls of the flask were suspended in a buffer solution of 10 mM Tris-HCl (pH 8) by vortexing for several minutes to prepare a liposome suspension. The liposome suspension was then treated with a probe sonicator for just 2 min to suppress the rapid sedimentation of the liposome. At this point, the lipid concentration was typically around 10 mg/mL and the liposome suspension still remained quite turbid, suggesting the existence of MLV. The average diameter of MLV from the 99% Egg-PC was probably larger than that of the 60% Egg-PC since the liposome suspension of the 99% Egg-PC was more turbid than that of the 60% Egg-PC. 50 µL of the liposome suspension was mixed completely with 500 µL of a buffer solution with NaCl, KCl, MgCl2, CaCl2, or with no added salts, and each of the mixtures was stored at 25 ( 1 °C for 1 h. Each mixture was then shaken gently and the turbidity of the mixtures was measured at 620 nm using a UV-vis spectrometer (Hitachi, U-2000). Atomic Force Microscope (AFM) Observations. The surface topography of the substrates was imaged using an AFM (Digital Instrument, Nanoscope IIIa) in tapping mode using silicon cantilevers with a normal spring constant of 40 N/m (Nanosensors, NCH-10V). The images were captured at a scan angle of 0° and at a scan rate of 0.5 Hz.

Growth of Giant Membrane Lobes

Figure 1. Fluorescence images of lipid membranes spreading from lines of 99% Egg-PC on silica-glass substrates in aqueous solutions of NaCl. (a) 0.01 mM NaCl after 5-10 min, (b) 10 mM NaCl after 5-10 min, (c) 100 mM NaCl after 5-10 min, (d) 1000 mM NaCl after 5-10 min, and (e) 100 mM NaCl after 5 h. The phospholipid membranes spread to both sides from the line of phospholipid, though we only observed one side in the photographs.

Results and Discussion Growth of Membrane Lobes on a Single-Lipid Bilayer. Figure 1 depicts the spreading of 99% Egg-PC observed on silica-glass substrates for various NaCl concentrations. The brightest regions at the left-hand corners of the fluorescence images correspond to lines of phospholipid deposited on the silica-glass substrates, and the phospholipid is spreading from the left to the right sides of each photograph. As shown in Figure 1a and b, only the single-lipid bilayers spread from the lipid sources covering the bare water-solid interfaces at relatively low NaCl concentrations (0.01-10 mM), as already reported in the pervious papers.21-23 In contrast, as shown in Figure 1c and d, fingerlike membrane lobes are growing on the spreading single-lipid bilayers at relatively high NaCl concentrations (100 and 1000 mM). It is clear that the membrane lobes are adherent to the surface of the singlelipid bilayer since no membrane lobes were observed to leave the surface of the single-lipid bilayers, even if the substrates were gently agitated in the aqueous solutions using a pair of tweezers. When there was a sufficient amount of lipid on the substrates, the membrane lobes grew into giant membrane lobes of over 1 mm in length over the course of 5 h, as shown in Figure 1e. As time elapsed, the width of the membrane lobes also increased due to fusion between mutually adjacent membrane lobes, although fusion did not necessarily occur between two membrane lobes that were touching each other. All of the images of the membrane lobes could be in focus, even for the 40 × objective lens (N. A. ≈ 0.85). This means that the height of the membrane lobes is less than the focal depth ( T2. This slowing down of the singlelipid bilayer may indicate the exhaustion of the lipid source since the line of phospholipids had already became a line of discrete islands of phospholipids at t ) T2. With regard to the membrane lobes, the velocity of the fronts of the double-bilayer lobes is roughly in accord with that of the single-lipid bilayers, as shown in Figure 4a. Indeed, as typically shown at t ) T2, the velocities of the fronts of the double-bilayer lobes and of the single-lipid bilayer slowed down simultaneously. The fronts of the double lobes were sometimes stopped and then start to move again after a short rest, as clearly shown at t ) T1. This short rest period gives rise to the dispersion in Llobe among the individual membrane lobes and is probably due to the surface roughness of the substrate.

Growth of Giant Membrane Lobes

Figure 5. Motion of the membrane lobes upon collision of two fronts of single-lipid bilayers of 99% Egg-PC spreading on a silica-glass substrate in an aqueous solution of 100 mM NaCl. (a)-(d) Successive fluorescence images of the lipid membranes (the initial image was captured 11 min after the immersion of the substrate in 100 mM NaCl solution). (e) Position-time relationship of the front position of the single-lipid bilayer on the left-hand side (9), of the double-bilayer lobe on the left hand side (O), and of the single-lipid bilayer on the right-hand side ((); all of them were measured along the white line indicated in (a).

To investigate the relationship between the motion of the membrane lobes and that of the single-lipid bilayers in detail, we have observed the motion of the fronts of the membrane lobes when a collision between two fronts of single-lipid bilayers occurred. The successive photographs in Figure 5a-d show the collision behavior of the fronts of the membrane lobes and Figure 5e shows a positiontime relationship representing the positions of the fronts of the two single-lipid bilayers and the double-bilayer lobe measured along the white line indicated in Figure 5a. The motion of the double-bilayer lobe stopped when the collision occurred. This indicates that the most-dominant component of the motion of the double-bilayer lobes on the substrate is simply determined by the motion of the surface of the single-lipid bilayers and that the membrane lobes do not proceed by themselves on the single-lipid bilayer. Indeed, as already shown in Figure 1c-e, each of the membrane lobes proceeded on a straight path in the direction in which the single-lipid bilayers are spreading (i.e., the direction from the left to the right corners in each fluorescence image). On the basis of these experimental results, we propose a mechanism for the growth of the membrane lobes, as follows. The spreading of the single-lipid bilayer can be understood in terms of a wetting phenomenon driven by the gain in free energy by the formation of membranesolid coupling, as previously reported.21-23 In contrast, the growth of the membrane lobes is not due to the wetting phenomena driven by the gain in free energy by the formation of membrane-membrane coupling between a membrane lobe and a single-lipid bilayer since the membrane lobes do not proceed by themselves on the

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single-lipid bilayer. Considering that the lobes were adherent to the surface of the single-lipid bilayer at relatively high salt concentrations, it is reasonable to say that the motion of the single-lipid bilayer mechanically drives the growth of the membrane lobes to cause the elongation of the membrane lobes from the lump of phospholipid fixed on the substrate. The growth mechanism of the membrane lobes clearly differs from the rolling of the membrane lobes (i.e., the tank-tread-like rolling process, in which the membrane lobes proceed by themselves) reported previously21,22,24 and also from the growth mechanism of the myelin figures.30-34 Finally, we discuss how the added NaCl induces the growth of the membrane lobes. As shown in Figure 1b, the membrane lobes can be generated even for a 10 mM NaCl solution, but they will never grow. This means that the membrane lobes slip on the single-lipid bilayer, inhibiting further growth. Therefore, the adhesion of the membrane lobes onto the single-lipid bilayer might play an indispensable role in the growth of the membrane lobes. Since there have already been numerous studies on the salt-induced aggregation of liposomes, where the addition of NaCl, as well as KCl, MgCl2, and CaCl2, has been thought to promote adhesion between lipid membranes,35-37 it is reasonable to suppose that the added NaCl promotes the adhesion of the membrane lobes onto the single-lipid bilayer to induce the growth of the membrane lobes. Further supporting information is shown in the next section. Note that in this report we focus our attention on the growth of the membrane lobes; the generation of the membrane lobes is another problem. Growth of Membrane Lobes under Various Salt Concentrations. In this section, we demonstrate that the growth of membrane lobes can generally be observed in aqueous solutions of KCl, MgCl2, and CaCl2, as well as NaCl, and show that the salt-induced growth of the membrane lobes is closely related to the salt-induced aggregation of liposomes. We first investigated the wetting phenomena of phospholipid membranes in aqueous solutions of NaCl, KCl, MgCl2, and CaCl2, systematically varying the salt concentrations. As a result, it was clarified that all of these salts can cause the growth of membrane lobes on spreading single-lipid bilayers. Figure 6a-f depict typical examples of growing membrane lobes composed of 99% Egg-PC or 60% Egg-PC. To sum up our observations over wide ranges of salt concentrations, we used the ratio of SL (the area occupied with membrane lobes on the single-lipid bilayer) to SB (the total area of the single-lipid bilayer) to represent the amount of growing membrane lobes on a single-lipid bilayer and plotted it against the salt concentrations, as shown in Figure 7. There is a threshold salt concentration above which the growth of the membrane lobes occurs for each system, though there is a drop in SL/SB around 10 mM for 99% Egg-PC in aqueous solutions of MgCl2 or CaCl2. The threshold salt concentration is generally higher for 60% Egg-PC than for 99% Egg-PC, reflecting the difference in lipid composition which affects various forces between the lipid membranes, such as repulsive doublelayer and hydration forces.38 Even in the systems that exhibit a drop in SL/SB, the generation of the membrane (35) Ohki, S.; Du¨zgu¨nes¸ , N.; Leonards, K. Biochemstry 1982, 21, 2127. (36) Ohki, S.; Roy S.; Oshima, H.; Leonards, K. Biochemistry 1984, 23, 6126. (37) Nagata, M.; Yotsuyanagi, T.; Ikeda, K. Chem. Pharm. Bull. 1986, 34, 1391. (38) Israelachivili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992; p 395.

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Figure 6. Fluorescence images of lipid membranes of 99% Egg-PC or 60% Egg-PC spreading on silica-glass substrates in aqueous solutions of various salts captured at 5-10 min after the immersion of the substrates into the salt solutions; (a) 99% Egg-PC in 100 mM KCl, (b) 99% Egg-PC in 1 mM MgCl2, (c) 99% Egg-PC in 10 mM MgCl2, (d) 99% Egg-PC in 100mM MgCl2, (e) 99% Egg-PC in 1 mM CaCl2, and (f) 60% Egg-PC in 100 mM NaCl.

Figure 7. The dependence of SL/SB on the salt concentration for 99% Egg-PC (left) and 60% Egg-PC (right). The ratio SL/SB was estimated by analyzing the fluorescence images of lipid membranes spreading on silica-glass substrates captured at 5-10 min after the immersion of the substrates into the salt solutions (See text).

lobes was found to occur immediately after the immersion of the substrate into the aqueous solutions, although the growing membrane lobes suddenly shrink in width after a few minutes and become string-like lipid aggregates, as typically shown in Figure 6c. The drops in the value of SL/SB are due to the shrinkage of the membrane lobes. In addition to the wetting phenomena, we have investigated the salt-induced aggregation of liposomes in aqueous solutions of NaCl, KCl, MgCl2, and CaCl2 while systematically varying the salt concentrations. The turbidity (i.e., the absorbance at 620 nm) of liposome suspensions mixed with dense salt solutions was measured to detect the salt-induced aggregation of liposomes and is plotted against the final salt concentrations in Figure 8.

Suzuki and Masuhara

Figure 8. The dependence of turbidity (i.e., absorbance at 620 nm) on the salt concentration for 99% Egg-PC (left) and 60% Egg-PC (right). The broken horizontal lines indicate the turbidity of the liposome dispersion mixed with the buffer solution without added salts.

Here, the horizontal broken lines indicate the turbidity of a liposome suspension mixed with a buffer solution without added salts, and the rise in turbidity above the broken line reflects the liposome aggregation induced by the added salts.35-37 Comparing Figure 7 with Figure 8, it is clear that the growth of the membrane lobes occurs at salt concentrations where there is an evident rise in turbidity, although the rise in turbidity did not necessarily involve the growth of the membrane lobes. This means that the salt-induced growth of membrane lobes is closely related to the saltinduced aggregation of the liposomes and strongly supports the notion that the added salts promote the adhesion of the membrane lobes onto the single-lipid bilayer to induce the growth of the membrane lobes. With regard to the drops in the values of SL/SB, the shrinkage of the membrane lobes can be explained by considering the influence of divalent cations on the repeat distance of the lamellar phase of the neutral phospholipid, as reported previously.39 For the DPPC/water system, Inoko and co-workers found that, while the lamellae do not swell in monovalent salt solutions, they do swell by a few tens of nanometers in MgCl2 and CaCl2 solutions ranging from 1 to 100 mM, due to a long-range repulsive double-layer force brought about by the binding of the divalent cations onto the neutral membrane surface. In our systems, similar repulsive double-layer force might have become significant a few minutes after the immersion of the substrates into MgCl2 or CaCl2 solutions in the case of 99% Egg-PC. Such a repulsive double-layer force possibly destabilizes the adhesion of the membrane lobes onto the single-lipid bilayer to cause the shrinkage of the membrane lobes. As a matter of fact, there seems to be no salt-induced aggregation of liposomes for systems of 99% Egg-PC in 10 mM MgCl2 or CaCl2 solutions, probably reflecting the long-range repulsive double-layer force. In this section, we show the generality of the saltinduced growth of the membrane lobes in aqueous solutions of salt. Since the salt-induced growth of the membrane lobes reflects the transition from kinetic to static friction between the lipid membranes, it might prove (39) Inoko, Y.; Yamaguchi, T.; Furuya, K.; Mitsui, T. Biochim. Biophys. Acta 1975, 413, 24.

Growth of Giant Membrane Lobes

Figure 9. Fluorescence images of the membrane lobes of 99% Egg-PC on an oxidized silicon wafer, with an oxide layer of < 1 nm, immersed in 100mM NaCl solutions. (a) The fronts of the membrane lobes, where the membrane lobes spread from the left to the right-hand side of the image; the corresponding single-lipid bilayer cannot be seen in the image. Even in this system, fusions between mutually adjacent membrane lobes were often observed after long durations of contact with each other. (b) Some of the fronts of the membrane lobes. (c) The same fronts of the membrane lobes excited by more-intense light, where the single-lipid bilayer can be seen in front of the membrane lobes.

to be one of the most interesting subjects in the field of colloid science. In addition to its scientific importance, the salt-induced growth of the membrane lobes is of great importance for the creation of novel supported membrane systems since the membrane lobes can grow in various physiological salt solutions, which would permit us to use the membrane lobes for a variety of biological applications. Growth of Giant Membrane Lobes on Micropatterned Surfaces. In this section, we describe a strategy for controlling the two-dimensional structures of the membrane lobes by utilizing micropatterns on a smooth substrate since the two-dimensional structures of membrane lobes shown in the above sections were very disordered. An oxidized silicon wafer was chosen as the smooth substrate since its surface is chemically almost the same as the silica-glass substrates. We first investigated salt-induced growth of membrane lobes on the oxidized silicon wafer and spreading behavior of singlelipid bilayer on simple Pt micropatterns deposited on the substrates. We then demonstrated control of the twodimensional structure of the membrane lobes by utilizing more complex Pt micropatterns. Figure 9a shows the salt-induced growth of membrane lobes on smooth, oxidized silicon wafers, where the rootmean-square (RMS) roughness of the oxidized silicon wafers measured by AFM was about 0.2 nm, while that of silica-glass substrates was around 3.3 nm. In this fluorescence image, no preceding single-lipid bilayer could be seen in front of the membrane lobes, and the same holds true for the enlarged image of the front of the membrane lobes shown in Figure 9b. However, this is merely due to the low contrast of the fluorescence image of the single-lipid bilayer and does not confirm its absence. In fact, the preceding single-lipid bilayer did become visible (as shown in Figure 9c) when it was excited by stronger excitation light. The low contrast of the single-lipid bilayer on the oxidized silicon wafer can be explained by optical interference in front of the SiO2/Si interface.40-44 Since

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Figure 10. The spreading of a single-lipid bilayer of 99% EggPC at a Pt gate on an oxidized silicon wafer with an oxide layer of < 1 nm in 100 mM NaCl. (a) AFM image of a Pt gate on an oxidized silicon wafer. (b) Topography of Pt line measured along the broken line in (a). (c) Fluorescence image of a single-lipid bilayer spreading through a Pt gate in 100mM NaCl solution. The sample is illuminated not only by fluorescence excitation light λ ) 404.7 nm but also by an illumination lamp to visualize the Pt gate by its scattering effect.

the surface of the oxidized silicon wafer is very close to a SiO2/Si interface that corresponds to a node point of the standing waves of the excitation light and of the emission light, it is harder to excite perylene molecules in the singlelipid bilayer than those in the double-bilayer lobes that are further away from the SiO2/Si interface. Comparing Figure 9a with Figure 1c-d, the number of membrane lobes generated per unit length along the line of the phospholipids on the oxidized silicon wafer was clearly larger than that on the silica-glass substrates. This may be due to the differences in surface roughness between the substrates since the chemical composition of the oxide layers on the silicon wafers is almost the same as that on the silica-glass substrates. Figure 10a shows an AFM image of a simple Pt micropattern (i.e., a Pt gate, which is composed of a pair of Pt lines) deposited on the smooth surface of an oxidized silicon wafer. As shown in Figure 10b, the height and the width of each of the Pt lines were about 0.16 and 1.58 µm, respectively. Figure 10c shows a fluorescence image of a single-lipid bilayer spreading through the Pt gate, where the bilayer was excited with the same power of excitation light to that of Figure 9c. The single-lipid bilayer clearly formed a semicircular front at the Pt gate, indicating that the Pt lines work as barriers for the single-lipid bilayer. A similar barrier effect against the single-lipid bilayer has already been reported for hydrophobic Al2O3 deposited on glass substrates.23 This experimental result indicates (40) Born M.; Wolf E. Principles of Optics; Pergamon Press: London, 1959; p 278. (41) Lambacher, A.; Fromherz, P. Appl. Phys. A 1996, 63, 207. (42) Braun, D.; Fromherz, P. Appl. Phys. A 1997, 65, 341. (43) Iwanaga, Y.; Braun, D.; Fromherz, P. Eur. Biophys. J. 2001, 30, 17. (44) Lambacher, A.; Fromherz, P. J. Phys. Chem. B 2001, 105, 343.

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of the adhering vesicles clearly differ from those of the membrane lobes; the adhering vesicles take an almost spherical or “spindle adopted morphology” on the striped patterns, while the membrane lobes form very flat structures. Compared with the system of giant unilamellar vesicles, from a practical point of view, the system of the membrane lobes have some advantages in terms of the creation of large-scale microstructures since the membrane lobes are continuously supplied with lipid molecules from the lipid source, in contrast to the giant unilamellar vesicles whose diameters are normally several tens of micrometers. A ‘long-stretch’ of membrane lobes might be suitable for the creation of microchannels for transportation and separation of biomolecules in lipid bilayers.10-14

Figure 11. Spreading of the membrane lobes on Pt micropatterns prepared on oxidized silicon wafers with oxide layers of < 1 nm. (a) Optical microscope image of a Pt micropattern on an oxidized silicon wafer, where the dark lines correspond to the Pt lines. (b) Fluorescence image of membrane lobes of 99% Egg-PC on Pt micropatterns 5 h after the immersion of the substrate into 100 mM NaCl; a line of phospholipid has been deposited to the left-hand side of the Pt micropattern, though it can not be seen in the image.

that the spreading direction of the single-lipid bilayer can be controlled using micropatterns comprising of Pt lines. Figure 11a shows an incident-light optical microscopic image of a more-complex Pt micropattern deposited on an oxidized silicon wafer. Figure 11b shows a fluorescence image of the resulting membrane lobes observed on the Pt micropattern 5 h after the immersion of the substrate into an aqueous solution of NaCl; all of the membrane lobes had already stopped advancing when the image was captured. As typically shown in this image, all of the membrane lobes proceeded completely along the Pt-free lanes surrounded by the Pt lines, and some of them even branched at the T-junctions. Each of the fronts of the membrane lobes had stopped before arriving at the end of their respective lanes. This suggests that the preceding single-lipid bilayers had also stopped due to collisions with Pt lines at the end of each lane. In this section, it is demonstrated that the two-dimensional structures of the membrane lobes can be effectively controlled by simply utilizing micropatterns on smooth substrates. Recently, Bernard et al. have reported a dynamic process for the spreading of giant unilamellar vesicles along striped patterns on chemically decorated surfaces.45 However, the resulting structures

Conclusions We observed a new growth behavior for membrane lobes on a single-lipid bilayer that spreads from a lump of phospholipid deposited on a hydrophilic solid surface in an aqueous salt solution. The sliding motion of the singlelipid bilayer mechanically drives the growth of the membrane lobes due to adhesion between the lipid bilayers. The experimental results strongly support the notion that the added salts promote the adhesion between the lipid membranes. From a practical point of view, the membrane lobes are easy to prepare, are available in various physiological salt solutions, and are free from direct interactions with the solid surface. Therefore, the membrane lobes are of great advantage to technological, as well as biological, applications as a novel supported membrane system, especially as a binding matrix for large transmembrane proteins. Since the two-dimensional structures of the membrane lobes can be easily controlled by utilizing micropatterned substrates, a system that is composed of a lipid source and a designed surface for the growth of the membrane lobes can provide us with a variety of two-dimensional structures of membrane lobes to further explore the functionality of phospholipid membranes. Acknowledgment. This work is partly supported by “Bio-Medical Cluster Project in Saito (North Part of Osaka Prefecture)” from the Ministry of Education, Culture, Sports, Science and Technology, and by a Grant-in-Aid for Scientific Research “KAKENHI” (S) from the Japan Society for the Promotion of Science. LA040027M (45) Bernard, A. L.; Guedeau-Boudeville, M. A.; Sandre, O.; Palacin, S.; di Meglio, J. M.; Jullien, L. Langmuir 2000, 16, 6801.