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Temperature-Dependent Vesicle Response to Surface Topography Susan D. Gillmor,*,‡,† Julia J. Heetderks,§,† and Paul S. Weiss*,† Department of Chemistry, George Washington UniVersity, 725 21st Street, N.W., Washington, DC 20052, Departments of Chemistry and Physics, The PennsylVania State UniVersity, 104 DaVey Laboratory, UniVersity Park, PennsylVania 16802-6300 ReceiVed: February 16, 2009; ReVised Manuscript ReceiVed: June 18, 2009
We have examined the deformation of vesicles on surface topography. From known vesicle shape dynamics, we expect temperature to play a major role; however, when cooling vesicles in the presence of a low-permeability solute, the mechanism of deflation is not clear. We investigate giant unilamellar vesicles on topography to quantify their cooling over a range of temperatures. The volume constraints point to a transient defect pore model as being responsible for vesicle deflation over the temperature range studied. Introduction With a lipid vesicle model, we probe topographic effects on the lipid permeability and shape rearrangement that occur in response to surface topography. By illuminating the compliant response of vesicles to the substrate, we have focused selectively on the lipid response without chemical or protein adhesion. We have found that vesicles at physiological temperatures faithfully conform to surface topography. Several research groups have demonstrated supported lipid bilayer compliance to surfaces on the submicrometer scale.1-5 Nanoparticle and supported bilayer investigations suggest that bilayers encapsulate particles of 20 nm and greater radius.6 Below this 20-nm threshold, supported bilayers form small pores or drape over nanoparticles. Our study probes how closed vesicles in the presence of a low-permeability solute accommodate topography on the micrometer and submicrometer scale. We use a single-phase, single-lipid component vesicle and investigate its response to temperature and the topography that it encounters. The lipid we use, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), does not exhibit a phase transition in the temperature range studied (20-55 °C). We place giant unilamellar vesicles on planar and patterned substrates at three temperatures (20, 37, and 55 °C) and analyze their deformation as a function of temperature. In the cooling process, we expect the surface area to shrink, as detailed by Seifert and Petrache.7,8 However, we observe excess surface area and reduced volume, which allows vesicles to undulate on surface topography, following the temperature variation. Tethering and electronic perturbation of vesicles are the traditional methods to measure line tension and bending rigidity.9-15 However, in several papers and preparations, the vesicles are intentionally dehydrated13,14 or tested in a glucose/ sucrose sugar gradient.9-12,15 In strong DC fields, above the lysis tension (0.6 N/m2),13,16 vesicles form transient pores.10,13,16 Riske and Dimova have characterized macropores (0.5-5 µm) that are optically resolvable and their lifetimes (∼10 ms).10 Electroporation allows solution from inside a vesicle to leak and * Corresponding author. E-mail: (S. D. G.)
[email protected] and (P. S. W.)
[email protected]. ‡ George Washington University. † The Pennsylvania State University. § Current address: Department of Materials Science and Engineering, University of Maryland, College Park, Maryland 20742.
creates excess surface area. Similarly, dehydration removes interior water, creating excess surface area, enabling mechanical measurements without vesicle rupture. Our system keeps vesicles in equimolar sucrose solution. We vary the substrate topography to test vesicle shape transformation and bilayer compliance. Materials and Methods Materials. The following materials were used as purchased: phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine from Avanti Polar Lipids (Alabaster, AL); the fluorescent probe N-(6tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylammonium salt (TRITC) from Molecular Probes (Eugene, OR); chloroform (VWR; West Chester, PA) and methanol (VWR). All water is filtered before use through a Barnstead Nanopure filter and has a resistivity greater than 18.0 MΩ-cm (Boston, MA). Vesicle Formation. We form our giant unilamellar vesicles through an electroformation technique developed by Angelova et al.17-19 and modified by D’Onofrio et al.20 Briefly, two platinum wires are anchored in a polycarbonate plug spaced 3 mm apart. Our lipid solution consists of 450 µL of chloroform, 20 µL of 10 mg/mL DOPC, and 2.0 µL of 100 µg/mL TRITC. Several 2 µL drops of the lipid solution are applied along each wire at discrete locations with the pendant drops hanging toward the second wire. Once dried in air, the wires are placed in vacuum for at least 2 h to form a lipid cake. An aqueous solution of 50 mM sucrose is heated to at least 60 °C, above the lipid transition temperature.21,22 The electrodes are then connected in parallel to a function generator (HP33120A, Hewlett-Packard, Palo Alto, CA) via a wire harness. An initial AC field of 50 mV and 10 Hz is applied across the electrodes. The sucrose solution is added to a 1-mm path-length cuvette (EW-83301-00, Cole-Palmer, Vernon Hills, IL), and the electrode plug is inserted into the cuvette chamber. The assembly is sealed with microcrystalline wax (BW-431, Blended Waxes, Oshkosh, WI). The assembly is incubated at 55 °C in an oven during the electroformation process. The voltage is increased by 50 mV every 5 min until the potential reaches 600 mV, which is maintained for 3 h. Next, the frequency is lowered to 4 Hz for 30 min to dislodge the vesicles from the wires.17-19 Finally, the formation chamber is disconnected from the function generator, removed from the incubator, and allowed to cool to
10.1021/jp901428c CCC: $40.75 2009 American Chemical Society Published on Web 07/27/2009
Vesicle Response to Surface Topography room temperature in an insulated box with a block of aluminum at 55 °C that acts to increase the thermal mass to slow the equilibration process. Surface Preparation. Vesicles are deposited onto planar and patterned surfaces as they cool to the appropriate temperature. For the planar experiments, the vesicles are deposited and sealed onto microscope coverslips without further modification (micro cover square glasses No. 1, VWR). Our patterned substrates are 10 µm pillars and wells of SU-8 formed through routine photolithography as described in the manufacturer’s protocol. Round coverslips (micro cover round glasses No. 1, VWR) are cleaned using a detergent wash of ICN (7X Laboratory Detergent, MP Biomedicals, Irvine, CA), rinsed in a stream of water for 5 min, and dried in a 55 °C oven. Photoresist (SU-8 resist, MicroChem, Newton, MA) is spun onto the glass coverslips with a progressive spin of 500 rpm for 5 s followed by 3000 rpm for 40 s. The softbake of the resist is performed in a stepwise fashion on a 65 °C hot plate for 60 s, followed by a 100 °C bake on a hot plate for 60 s and allowing it to cool down on a 65 °C hot plate for 60 s before lithography. We expose the substrates to 12 mW/cm2 on a Karl Suss MA6 contact aligner for 10 s followed by a postbake identical to the softbake before development in the SU-8 developer (SU-8 developer, MicroChem, Newton, MA). Similar SU-8 preparations are used in microfluidics without modification.23,24 The vesicles are then deposited and sealed onto the topographically patterned substrate identically to the planar samples. Matrix Preparation. The peptide matrix substrate is formed as instructed by the manufacturer. After 30 min of sonication and 10 min of centrifugation, the PuraMatrix peptide hydrogel (BD Biosciences, Bedford, MA) is mixed in equal parts with 50 mM sucrose solution and placed on a clean coverslip. After washing three times with 50 mM sucrose and ascertaining the pH to be approximately 7, the substrate is placed in the oven, and the vesicles are mounted onto the matrix surface at 55 °C and sealed. The vesicle and matrix sample is incubated at 55 °C for 20 min before being placed in an insulated box with a block of aluminum (also at 55 °C), which acts as a thermal mass to slow the cooling to room temperature for ∼18 h. Microscopes. We used an inverted Nikon Ellipse TE300 microscope, an Olympus FluoView 300 confocal microscope, and a LMS Pascal Zeiss confocal microscope for imaging. For confocal work, we used water immersion lenses. Results When we placed vesicles on planar surfaces at different temperatures, we observed different rates of deformation. Figures 1-3 show typical examples of vesicles in the three different temperature ranges. In Figure 1, the vesicle was fully cooled from its preparation temperature of 55 °C to room temperature (20-23 °C) before placement on the surface. All vesicles were prepared in sucrose solution, which has low bilayer permeability. From the equatorial XY slice and the adjacent XZ and YZ planes, the vesicle appeared spherical, exhibiting no bending to accommodate the surface. We have quantified its spherical shape in Table 1. The vesicle’s contact with the glass substrate was limited to a small tip of the sphere. The small contact area reflected the minimal deformation of the vesicle at 20 °C. For our physiological temperature experiments, we cooled the vesicles from their preparation temperature of 55 to 37 °C, placed the vesicles on the 37 °C surface, and allowed the sample to cool to 20 °C, the imaging temperature. A typical vesicle response is shown in Figure 2. These vesicles were prepared in
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Figure 1. Example of vesicle deformation on a planar surface at 20 °C. The images represent several viewpoints of the same vesicle seated on a planar substrate. The stacked images adjacent to the equatorial slice are oriented such that the abutting substrate edge corresponds to the equatorial image edge. The equatorial slice shows the vesicle in the XY plane at its maximum radius. From the XZ and YZ views reconstructed from the stack of XY slices, the vesicle was found to be resting on the surface with minimal deformation. When the contact XY slice was examined, the tip of the vesicle was found to be resting on the surface. All scale bars correspond to 10 µm.
Figure 2. Example of vesicle deformation on a planar surface at 37 °C. The vesicle was found to be resting deformed in the threedimensional image reconstruction. In the XY equatorial slice here, we observed a regular, circular outline of the vesicle, but the YZ and XZ views illustrated the flattening of the vesicle next to the substrate. The contact XY plane showed the flat bottom of the vesicle as it rested on the planar substrate. All scale bars correspond to 10 µm.
sucrose solution. The equatorial XY slice showed a round vesicle, but the XZ and YZ planes showed deformation of the vesicle at the interface with the substrate. The vesicle deformed partially, and instead of only a small spot touching the surface, which was the case at 20 °C, a much larger area of the vesicle was in contact with the surface. The vesicle no longer maintained maximum volume, as quantified in Table 1. It suggests that the vesicle deflated to rest on the planar substrate. The 55 °C vesicles in sucrose solution were not cooled from their preparation temperature and were placed at 55 °C on a 55 °C surface and then cooled to the imaging temperature of 20 °C. As illustrated in Figure 3, images showed significant departures from the round shape of the 20 °C vesicles (cf. Figure 1). The equatorial XY slices were not circles, but were irregularly shaped. Their departures from spherical are detailed in Table 1. Similarly, the XZ and YZ planes did not show spherical vesicles, but squat ellipsoids. The contact area with the planar surface was comparable to the equatorial cross-sectional area instead of decreasing to form a closed sphere. The deformed vesicles’ low profiles were departures from the regular spheres exemplified in Figure 1.
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Gillmor et al.
SAratio )
Figure 3. Example of vesicle deformation on a planar surface at 55 °C. Similar to 37 °C vesicles, when these vesicles were placed on the planar substrate at 55 °C, the liposomes flattened. The squat shape was evident when viewing along the XZ and YZ planes. In addition, the contact area was similar in size to the equatorial slice of the liposome. All scale bars correspond to 5 µm.
TABLE 1: Vesicle Deformation at Three Different Temperatures Characterized Using Vratio and SAratioa temp, °C
Vratio range
Vratio mean
SAratio range
SAratio mean
20 37 55
1.02-0.92 0.95-0.83 0.89-0.79
1.00 ( 0.03 0.90 ( 0.04 0.85 ( 0.05
0.98-1.07 1.04-1.11 1.08-1.17
1.01 ( 0.03 1.03 ( 0.03 1.11 ( 0.04
a From the surface area of each vesicle, a maximum volume is calculated and compared to the actual volume of that vesicle.
To quantify this general description, we analyzed the surface area and volume of each vesicle at the three temperatures. We have defined our system as vesicles in contact with the surface, not free-floating. We also establish baseline vesicles introduced to the surface after fully cooling in the preparation assembly. Free-floating vesicles formed via electropreparation have been generally characterized as rigid and spherical.16 In this cooling study, we have chosen to concentrate on surface-deformed vesicles, since we are able to control and to characterize the glass surfaces. Free-floating vesicles, while generally spherical, may be deformed through a variety of means, such as temperature variation,8,25,26 osmolarity,13,14 and electrical fields.10,16 Cooling vesicles only contracts the bilayer and does not lead to excess surface area and deformed shapes.7,8 From Berndl et al.,25 we find a ratio of the observed volume divided by the calculated volume from the surface area, as detailed in the Supporting Information. Simply put, from our estimate of the surface area, we are able to calculate a radius of the corresponding sphere (SAvesicle ) 4πR02). We then find the volume (V0) from R0 (V0 ) (4π/3)R03) and compare it with the observed vesicle volume (Vvesicle) to find:
Vratio )
Vvesicle V0
(1)
The Vratio allows us to compare the different deformations observed at 20, 37, and 55 °C. For a sphere, Vratio ) 1. We have also quantified the data with SAratio, similar to Vratio. It compares the expected surface area from the volume to the calculated surface area of the vesicle to quantify the excess surface area. We define RV as the calculated radius of the observed volume (RV )[(3/4π)Vvesicle]1/3).
SAvesicle 3Vvesicle RV
(
)
(2)
Our experimental data analysis is based on vesicle deformation on planar substrates, where we can accurately assess the surface area and volume of a given vesicle. In Table 1, we see the range and the average of the deformation in terms of Vratio and SAratio. These measurements quantify the increase in vesicle deformation with temperature. The range of deformation overlaps between the temperature regimes. Spontaneous vesicle rupturing on the surface has also been observed at all three temperatures, and any correlation between the deformation range and rupturing has yet to be determined. Seifert, Lipowsky, and Brendl have shown greater deviations of Vratio in their investigations, analyzing small temperature increases and vesicle shape dynamics.8,25-27 This study focuses on cooling and a larger temperature range. When we analyze the energetic forces acting on the vesicle as it deflates, we find that we are close to the lysing tension. When we apply this increase in deformation as a function of temperature, we see the compliance difference at 20 and 37 °C. Vesicles in sucrose solution at 20 °C were placed on 10 µm pillars with a 20 µm pitch and 1-2 µm depth; these sat on the pillars with minimal deformation to accommodate the surface topography. In Figure 4a, all three views (XY, XZ, YZ) of a typical vesicle showed a spherical object that rested on the corners of four pillars, with little, if any, deformation to conform to the topographically patterned surface. In contrast, the vesicle in Figure 4b, placed on the substrate at 37 °C, pinched between two pillars to fill the valley fully. In the XZ cutaway, which is located at the dotted line in the XY slice, the vesicle edge drapes conformally between pillars. In the YZ cutaway, the vesicle follows the topography of the pillar, matching the XY slice. When we examined the individual XY slices in the z-stack of the confocal image (Figure 4c), we also found that the bilayer rested on the tops of the pillars. Figure 4c and d shows individual XY slices in different z-positions of the vesicle. In the lower position of Figure 4c, we observed the filled valley between the pillars, whereas the higher z-position of Figure 4d revealed the pillars. The bilayer follows the contours of the surface, faithfully undulating along the pillar walls, valleys, and peaks. To test the compliance of this simple system, we incubate vesicles in sucrose solution on a PuraMatrix peptide hydrogel at 55 °C for 20 min. The vesicle deformed to accommodate the complex structure, and the high-resolution image of the vesicle surface shows undulations of the bilayer to curve around matrix tendrils below 1 µm in diameter. In the YZ plane of Figure 5a, the vesicle has a hemispherical profile with a large protrusion to form an incomplete circle. The XZ plane appears blurred due to undulations on the vesicle surface, in contrast to the sharp edges of Figures 1-4. When we examine the cutaway views in Figure 5b, the XY view reveals a large divot, which is also shown in the YZ cut-away. In the YZ cutaway, the profile is clearer than the reconstructed image of Figure 5a. The XZ cutaway shows several indentations into the vesicle, consistent with the XY and YZ cutaways. The irregular hemisphere is apparent in the YZ cutaway. Finally, Figure 5c shows a ruptured vesicle that illuminated the PuraMatrix structure. Discussion Given the data sets of vesicles at different temperatures, we examine their shape transformations. The vesicles at 37 and 55 °C exhibit excess area and squat ellipsoid shapes.
Vesicle Response to Surface Topography
Figure 4. Comparison between vesicles placed on a topographically patterned surface at 20 and at 37 °C. (a) A vesicle was placed on the surface at 20 °C and showed little evidence of conforming to the surface. It maintained its spherical shape on top of the 10 µm pillars. (b) When a vesicle was placed on the patterned surface at 37 °C, the vesicle exhibited much different behavior. The liposome deflated sufficiently to allow the bilayer to conform faithfully to the pillars. The lines correspond to the XZ and YZ planes shown in the adjacent images. In the YZ plane, we observed a dip corresponding to the valley between the pillars. The XZ plane showed the vesicle accommodating a pillar as the bilayer bows upward. (c, d) From the deformed vesicle in b, the individual XY slices were examined in greater detail. From these slices, the valleys were in focus initially in image (c), corresponding to the bilayer seated in the space in between the pillars. In the XY slice corresponding to the tops of the pillars (d), the bilayer sat on the pillars, a perfect complement to the XY slice in (c). The schematic illustrates the varying z focus for images (c) and (d) and also shows the entanglement of the vesicle on the surface. Compliance on patterned surfaces has been previously documented using supported lipid bilayers.1,3-5,28,29 All scale bars correspond to 20 µm.
From Seifert and Petrache,7,8 we expect the lipid headgroup to reduce its area as the vesicle cools from 55 and 37 to 20 °C. According to Petrache, PC lipid headgroups expand ∼10% over a 20 °C range for various PC lipids within the LR phase. Conversely, for cooling, the surface area will decrease. As the surface area decreases, the volume therefore also decreases.
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Figure 5. Vesicle supported in PuraMatrix. A vesicle was enmeshed in a three-dimensional flexible peptide matrix, similar to a collagen extracellular matrix. (a) Three-dimensional reconstruction of the vesicles revealed a highly deformed vesicle with significant undulations to accommodate the surrounding matrix, indicating the compliance of the vesicle to the available space in the peptide scaffold. When the surface of the vesicle was examined, many small-scale undulations were evident where it accommodated the individual tendrils of the matrix. When viewed in profile, there were several deviations from spherical. In the YZ reconstruction, the vesicle had a curved, inverted bowl profile, instead of a full spherical shape, with a large protrusion. In the XZ reconstruction, the surface undulations rendered the view blurry, in contrast to the sharp edges of Figures 1-4. (b) In the cutaway views, the edges were blurred due to the undulations on the surface of the vesicle, shown in (a). The XZ cutaway revealed indentations along the profile not evident in the three-dimensional reconstruction. (c) The PuraMatrix is visible after a vesicle ruptured and labeled the tendrils with the lipid dye. As evident in the image, the scaffold formed by PuraMatrix was complex with small ropelike tendrils, forcing the vesicles to conform to the rich topography with feature sizes larger and smaller than 1 µm.
From Seifert, we find that predicting the volume decrease is not trivial. Bilayer thermal expansivity is an order of magnitude larger than that of the aqueous fluid.8,30 In the case of a temperature increase, as Seifert analyzed, this difference leads to an excess surface area and a plethora of shapes from the (originally) spherical vesicle. For a temperature decrease, this thermal contraction of the bilayer will shrink the surface area
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by 8-10% over the temperature range, suggesting that the enclosed volume must decrease by ∼15%. In contrast, the temperature change will lead to an only 1% volume change of the aqueous solution (FH2O ) 0.998 21 g/mL at 20 °C, and FH2O ) 0.988 21 g/mL at 50 °C).31 From these differences in volume and surface area thermal properties, we identify a driving impetus for volume change in or rupture of the vesicle. We expect the encapsulated solution volume decrease to follow one of two mechanisms: (1) simple diffusion across the bilayer or (2) compromise in integrity of the bilayer (transient pores, tears in the bilayer). Although the most likely actor in the decrease in vesicle volume would seem to be transmembrane diffusion, our analysis of the system does not support this mechanism. At 25 °C, water permeability across the membrane is ∼3 × 10-3 cm/s.32 However, sucrose at 25 °C has a significantly lower permeability of ∼8 × 10-14 cm/s.33 Since the DOPC membrane does not undergo a phase transition, the sucrose permeability will rise slightly due to temperature, but will remain at the same order of magnitude (∼1 × 10-13 cm/s). The inability of sucrose to diffuse across the lipid bilayer effectively maintains the volume of the vesicle. Without proteins to control the solute movement actively, the interior and exterior solutions must have identical concentrations (50 mM sucrose). Water molecules exchange across the bilayer,32,34 but any net loss of water would increase the concentration of sucrose inside the vesicle and create an osmotic gradient, which would then drive water into the interior of the vesicle. Therefore, transmembrane diffusion does not appear to be the determining factor for the vesicle deformation process. Given the temperature reduction and the surface area contraction, we expect a rise in membrane instabilities or transmembrane pores to form and to alleviate the volume pressure. From pipet aspiration experiments, the energy required to form a pore is 20-2400kBT.35-39 Water compressibility is 4.45 × 1011 Pa for this temperature range.31 Stretching elasticity and bending rigidity are 2.4 × 10-3 N and 20kBT (or 10-7 pN-m, or 10-20 N-m), respectively,16 whereas the lysis tension is 0.6 N/m2.13 When we compare these forces on a vesicle of typical size, we find that transient pores are energetically the least costly pathway. For example, from Raphael et al.,38 we choose a vesicle of radius 9 µm and calculate its perimeter (P ) 2πr) to be 56 µm, and its surface area (SA ) 4πr2) to be 1000 µm2. On this size scale, the water compressibility requires a force of 44.5 N to decrease in volume, whereas the lysis tension 6.0 × 10-10 N (tension times the surface area) and the bending rigidity exerts 1.8 × 10-15 N (bending rigidity divided by perimeter or arc length). From these balancing forces (stretching, 2.4 × 10-3 N; bending 1.8 × 10-15 N, lysis tension 6.0 × 10-10 N, and water compressibility 44.5 N), the aqueous solution is not compressible. Forming a pore to allow the exit of aqueous solution from the vesicle costs 20-2400kBT or 10-7-10-5 pN-m or 5.6 × 10-11-5.6 × 10-9 N, which is the same energetic range as the lysis tension. The low bending rigidity and lysis tension create an energy pathway for volume decrease via transient pores. For free-floating vesicles, as the volume decreases, the need for further volume reduction beyond that of a sphere is not necessary. However, a vesicle on a surface would experience additional pressure from the hard support, and our temperature studies document this deformation in the form of a surface area excess of ∼8-15% for 37 and 55 °C. Under those conditions, we observe overdeflation of vesicles, which we do not see for fully cooled vesicles resting on surfaces. We have not yet investigated surface chemistry effects, although we anticipate
Gillmor et al. that these will be of great interest.40-44 Certainly, protein-coated surfaces could promote spreading and increase deformation. When we examine vesicles in contact with topographically rich surfaces, we observe faithful compliance of the bilayer to the underlying support surface, which is consistent with findings in supported bilayer investigations.1-5 Surface interactions add additional complexity to the volume dynamics. Whereas DOPC vesicles must deflate to conform to the surface, the minimum energy shape of the vesicle is then based on solute behavior, surface tension between the lipid head groups, water, and the glass surface. Any energetic gain due to surface adhesion must counterbalance the energy cost of curvature and the possible fission of the vesicle to maximize lipid-to-glass contact.45 We have modeled the behavior of the lipid compliance to morphology for fluid-disordered vesicles. Our system concentrates on vesicles in sucrose solution, which does not permeate the bilayer as quickly as water or other chemical species. We note that behavior of liposomes in the gel phase (Lβ) might prove distinct from that observed here. The line tensions and fluidity of coexistence of LR and Lo is of particular interest due to its biological relevance. Although phase domains appear distinct via optical microscopy, recent theoretical findings suggest a diffuse boundary between the two states and low line tension forces between the phases (3.5 pN).46 However, Baumgart and Keller have measured line tensions between the phase domains (LR and Lo) below 0.1 pN close to the phase transition.47-49 These low separations between the phase boundaries suggest that the boundaries are not the weak seam for transient pore formation. Instead, the overall difference in bending rigidity would favor pore formation in the LR over the Lo domains, since LR domains have lower bending rigidity than Lo domains. Molecular packing mismatches evident in LR and Lβ domain boundaries have been the site for pore formation in the case of phase transitions.28,50-53 Conclusions Our study has highlighted the conformability of fluiddisordered lipid vesicles under specific temperature conditions. We have investigated LR-phase vesicle response to controlled topographies as a function of temperature. We have quantified their behavior and have explored a solute-dependent model for volume deflation. Once the solute crosses the lipid bilayer, the vesicles deflate and readily conform to complex surface topographies. To accommodate the low permeability of sucrose across the bilayer, transient-defect pores allow sucrose to bypass the lipid barrier and to deflate the vesicle. The defect model has been developed to study the release of inner leaflet compression via lipid flipping, and in our case, it may serve a dual purpose to allow solutes to pass through the aqueous pore instead of the hydrophobic bilayer core. Acknowledgment. The authors thank Professors E. D. Sheets and Q. Du for many useful discussions, Professor A. A. Heikel for use of his Olympus confocal microscope, J. Brenner of Zeiss for use of a demonstration Zeiss confocal microscope, and the Center for Quantitative Cell Analysis in the Huck Institutes of Life Sciences. In addition, Professor R. M. Raphael provided many helpful suggestions in the data interpretation. We also thank the Center for Nanoscale Science, a National Science Foundation Materials Research Science and Engineering Center, for financial support. Supporting Information Available: Vesicle volume and surface area calculations. This material is available free of charge via the Internet at http://pubs.acs.org.
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