J. Phys. Chem. B 2008, 112, 5175-5181
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Influence of Nanotopography on Phospholipid Bilayer Formation on Silicon Dioxide Indriati Pfeiffer,*,† Bastien Seantier,† Sarunas Petronis,† Duncan Sutherland,‡ Bengt Kasemo,† and Michael Za1 ch† Department of Applied Physics, Chalmers UniVersity of Technology, SE-41296 Gothenburg, Sweden, and iNANO Center, UniVersity of Aarhus, Ny Munkegade, Building 521, 8000 Aarhus C, Denmark ReceiVed: NoVember 5, 2007; In Final Form: January 25, 2008
We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicleto-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipidmembrane-based applications using rough or topographically structured surfaces.
Introduction Biological membranes play an important role as the physical barrier between the interior of cells and their extracellular environment as well as between different compartments within a cell. The membranes allow selective transport of ions and molecules, as well as actively participate in inter- and intracellular communications involved in living processes via embedded membrane proteins. These membranes are highly dynamic and complex. They are composed of amphiphilic lipid molecules, which self-assemble into a double, continuous fluid lipid membrane, a so-called bilayer, and proteins and peptides associated with the membrane that serve different functions.1 For systematic investigation and understanding of such a complex system, simple and reproducible model membranes are needed, so that properties and functions of individual membrane components or an ensemble of selected constituents can be studied. The formation of supported phospholipid bilayers (SPBs) on various surfaces has been thoroughly investigated for a variety of lipid chemistries, not only because SPBs resemble the cell membrane, but also owing to their potential in biotechnological applications such as biosensors,2-4 bioMEMS,5-7 and immunoassay development.8,9 The two most common ways to form SPBs on hydrophilic surfaces are either to utilize the LangmuirBlodgett transfer technique10,11 or via the adsorption and spontaneous rupture of small unilamellar lipid vesicles (SUVs).12-14 The latter technique is favorable due to its simplicity and the potential to incorporate different biomolecules during the vesicle * Author to whom correspondence should be addressed. Present address: Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany. Tel: +49-(0)6131-379566. Fax: +49-(0)6131379100. E-mail:
[email protected]. † Chalmers University of Technology. ‡ University of Aarhus.
preparation step, thereby enabling the immobilization of these biomolecules into subsequently formed SPBs.15 The adsorption of SUVs and their (eventual) transformation to SPBs are governed by a number of key parameters, such as vesicles size, lipid chemistry and charge, surface chemistry and charge, temperature, as well as the nature and concentration of ions in the buffer solution, as characterized using different surfacesensitive analytical techniques.14,16-21 One of these powerful characterization tools is the quartz crystal microbalance with dissipation monitoring (QCM-D), which enables real-time monitoring of vesicle adsorption and bilayer formation. In particular, the method allows one to distinguish the spontaneous rupture of adsorbing vesicles from adsorption of intact vesicles.22 As previously reported in our work and that of others, both quantitative and qualitative data of the effect of the various parameters involved in SPB formation can be obtained by combining the QCM-D with complementary techniques, such as atomic force microscopy (AFM),23-26 surface plasmon resonance (SPR),25,27,28 ellipsometry,26 and fluorescence microscopy.29 These combined approaches reveal significant information concerning the mechanism (pathway) of vesicle adsorption and SPB formation23,30,31 as well as the long-term stability and the lateral mobility of SPBs.32,33 Four pathways have hitherto been identified:14,19,23,25,26,34,35 (i) repulsive interaction between vesicles and the surface, which prevents vesicles from adsorbing and completely disables SPB formation; (ii) weakly attractive interaction, allowing vesicles to adsorb, but not causing their rupture; (iii) strongly attractive interaction, leading to adsorption and spontaneous rupture of individual vesicles; and (iv) an intermediate case between pathways (ii) and (iii), where the attractive interaction is such that individual vesicles do not rupture, but the combined vesicle-surface and vesicle-vesicle interaction in sufficiently large ensembles of vesicles (referred to as “critical vesicle coverage”) leads to
10.1021/jp710614m CCC: $40.75 © 2008 American Chemical Society Published on Web 03/28/2008
5176 J. Phys. Chem. B, Vol. 112, No. 16, 2008 vesicle rupture. The latter pathway (iv) has been found to be operative in the system considered here, i.e., POPC vesicles on SiO2 surfaces.25,34 Although significant understanding of the processes underlying SPB formation from vesicle solution has been established, these fundamental studies have almost exclusively been done on relatively flat, hydrophilic surfaces (e.g., mica, glass, and SiO2) or on surfaces with ill-defined and/or noncharacterized roughness. In spite of the fact that many biotechnological applications may require more or less rough surfaces, the role of surface topography in SPB formation has not been thoroughly investigated and is still poorly understood.36,37 Previous studies from several research groups have shown that surface roughness induces changes during protein adsorption and cell attachment on different surfaces.38-41 These results raise the question whether surface topography can induce differences in vesiclesurface interaction and, consequently, in the mechanism and the kinetics of vesicle adsorption and lipid membrane formation. This study addresses the effect of well-controlled and wellcharacterized nanoscale topography on vesicle adsorption and SPB formation as a function of vesicle size and compares the results to unstructured, flat surfaces. The nanostructured SiO2 surfaces (pitted surfaces), with two different topographical length scales, namely 110 and 190 nm pit diameter, were fabricated on top of SiO2-coated QCM-D sensor crystals using the colloidal lithography technique.42,43 These nanostructured as well as unstructured SiO2-coated QCM-D sensor surfaces were then exposed to POPC lipid vesicles with two different nominal diameters, 30 and 100 nm. The vesicle adsorption and bilayer formation processes were characterized in real-time with the QCM-D technique, and AFM was employed to capture snapshots of the lateral arrangement of vesicles and bilayer for selected choices of vesicle size, pit diameter, and exposure time. Materials and Methods Materials. All buffers used in the experiments contained 10 mM tris(hydroxymethylamino)methane (Tris) and 100 mM NaCl, both obtained from Sigma Aldrich. The buffer pH was adjusted to 8.0 by addition of HCl. The phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the form of lyophilized powder and the polycarbonate membranes with pore sizes of 30 and 100 nm were purchased from Avanti Polar Lipid, Alabaster, AL. SiO2-coated QCM-D sensor crystals with 5 MHz resonance frequency were obtained from Q-sense AB, Gothenburg, Sweden. Polystyrene particle suspensions with particle mean diameters of 107 and 190 nm were acquired from Interfacial Dynamic Corp., Portland, Oregon. Poly(diallyldimethylammonium) chloride (PDDA) 20% w/w, MW ) 200 000350 000, and poly (sodium 4-styrenesulfonate) (PSS), MW ) 70 000, were purchased from Aldrich. Chloroform was obtained from Merck, and sodium dodecyl sulfate (SDS) was obtained from Sigma Aldrich. Oxide-sharpened silicon nitride AFM tips of the MSCT-AUNM type were purchased from Veeco Europe. Vesicle Preparation. POPC lipids (5 mg) were dissolved in chloroform in a round-bottomed flask. The chloroform was evaporated using a flow of N2 gas for 1 h while a thin dried lipid film formed on the wall of the flask. Buffer (1 mL) was added to the dry lipid film to achieve a lipid concentration of 5 mg/mL. In order to form unilamellar vesicles with a nominal size of 100 nm, the extrusion method was applied by passing the solution back and forth 11 times through a polycarbonate membrane with a matching pore size, yielding 160 ( 47 nm vesicles as determined with a Brookhaven BI-90 automatic particle sizer. In the case of 30 nm nominal size, the suspension
Pfeiffer et al. TABLE 1: Characteristic Features of Nanostructured SiO2 Crystals pit diameter (nm)
pit depth (nm)
pit coverage (%)
(calcd) total pit perimeter per unit area (µm/µm2)
110 190
25 25
11.8 11.0
4.29 2.32
was first passed through a 100 nm and then a 30 nm pore size membrane for 11 times each. The size distribution of the resulting vesicles in solution was 50 ( 22 nm. Nanofabrication of Pitted Surfaces. Colloidal lithography,43 based on electrostatic self-assembly of charged nanoparticles, thin film deposition, and lift-off, was used to create nanoscale pitted surfaces on SiO2-coated QCM-D sensor crystals. First, two types of polyelectrolytes, PDDA and PSS, both with a concentration of 0.2% w/w in water, were used to create a charged triple layer of PDDA/PSS/PDDA on the sensor surface. Thereafter, the monosized polystyrene particle suspension (107 or 190 nm size, 2.5% v/v) was deposited on top of the polyelectrolyte layer and left to adsorb for 60 s. The excess of colloidal particles was rinsed with MilliQ water and dried under N2, leaving a monolayer of uniformly dispersed colloidal particles on the surface. Using e-beam evaporation in an AVAC HVC-600 deposition system, a thin Ti adhesion layer (1 nm) was deposited on top of the particle-covered SiO2 sensor crystals, followed by deposition of a 24-nm-thick layer of SiO2. The polystyrene particles served as a mask, preventing thin film deposition under them during this process. The particles were then removed from the surface by tape-stripping, leaving 25nm-deep pits with a diameter corresponding to the particle size (see Table 1). Preparation of Surfaces. Prior to each measurement, the QCM-D sensor crystals were cleaned by dipping them overnight in 0.4% w/w SDS solution, followed by rinsing two times with MilliQ water and a 10-min treatment in a UV-ozone chamber. This treatment removed organic contaminants from the surfaces, as has been checked for selected samples by X-ray photoelectron spectroscopy (XPS). QCM-D Experiments. QCM-D experiments were performed using Q-sense D-300 instruments (Q-sense AB, Gothenburg, Sweden). A nanotopography-modified QCM-D sensor crystal was mounted to the cell, which provides a fast nonperturbing exchange of stagnant liquid. The temperature was set to 22 °C and controlled using Peltier elements. Since the resonant frequency (f) of the QCM-D sensor crystal depends on the total oscillating mass, a change in the oscillating mass, for example, due to the adsorption of a lipid film on the sensor crystal, can be detected as a change in f. If the adsorbed film is thin, rigid, firmly attached, and evenly distributed, there is a linear relationship between the adsorbed mass and change in frequency
∆m ) -
CQCM ∆f n
where CQCM is 17.7 ng cm-2 Hz-1 for a 5 MHz sensor crystal and n ) 1, 3, 5, ... is the overtone number. The QCM-D technique also provides information about changes in dissipation (D), which are related to viscous losses in the adsorbed film. Changes in f and D are measured simultaneously by recording the frequency and decay time of the crystal oscillation while the driving voltage is (intermittently) switched off. Data were collected using Q-Soft 301 software (Q-sense AB), and all data for the frequency shift presented here are normalized to the response of a 5 MHz sensor crystal. Each set of parameters in
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Figure 1. QCM-D curves, showing the temporal changes in frequency (∆f, black) and dissipation (∆D, blue), both recorded at the third overtone, upon adsorption of (A) 30 nm vesicles and (B) 100 nm vesicles onto a flat, SiO2-coated sensor crystal (solid line), a nanostructured SiO2 crystal with 190 nm pits (dotted line), and a nanostructured SiO2 crystal with 110 nm pits (dashed line). The same data but plotted as dissipation change vs frequency change is shown in (C) for adsorption of 30 nm vesicles and in (D) for 100 nm vesicles onto a flat (solid line), a 190 nm pitted (dotted line), and a 110 nm pitted (dashed line) SiO2 crystal. Each set of parameters measured by QCM-D has been repeated five times, with standard deviations for 30 and 100 nm vesicles within (2% and (6%, respectively.
this study has been measured five times, with highly reproducible results (the standard deviations for 30 and 100 nm vesicles are within (2% and (6%, respectively). AFM Experiments. A PicoSPM microscope with a largearea scanner (80 × 80 µm2), both from Molecular Imaging/ Agilent, and a homemade, O-ring-sealed liquid cell able to accommodate QCM-D sensor crystals were used for all our AFM experiments. QCM-D sensor crystals were prepared and cleaned according to the procedures described above and then exposed to a lipid vesicle solution directly in the AFM liquid cell. Once the desired exposure time had been reached, the vesicle solution was exchanged with buffer by rinsing more than five times using two pipettes. After an equilibration period of typically 1 h (required for drifts to drop below an acceptable level), images were acquired in soft constant-force contact mode, with the force setpoint continuously being adjusted in order to minimize the imaging force. Only cantilevers with force constants k e 0.03 N/m (according to specification) were used. The scan speed was adjusted to 1-2 lines/s, and feedback gains were tuned toward optimal tracking while strictly avoiding any feedback oscillations. Topography images were plane-fitted (manual tilting, followed by applying a “max. flatness tilt”) and median-filtered (3 × 3 pixels) using the Scanning Probe Image Processor, SPIP (Image Metrology Inc.), which was also used to extract cross-sectional profiles and histograms from the images. Gaussian fits to the histogram peaks were obtained using IgorPro software (WaveMetrics Inc.) in order to determine peak positions.
Results and Discussion It has previously been demonstrated that the adsorption kinetics of POPC vesicles onto a flat SiO2 surface is qualitatively independent of vesicle size in the SUV regime (L ∼ 25-200 nm).14,19 However, as shown in Figure 1, QCM-D signals recorded on nanostructured surfaces provide clear evidence that vesicle size, in relation to the topographical length scale, has a profound influence on the kinetics and mechanism of vesicle adsorption and bilayer formation on nanostructured surfaces. Upon exposure of 30 nm vesicles to flat surfaces (solid line in Figure 1A), frequency (∆f) and dissipation (∆D) signals display a time-dependent evolution, which is characteristic of the adsorption of intact vesicles (t < tmin), followed by rupture of these vesicles to form SPBs once a critical vesicle coverage has been reached (t g tmin).44 Upon adding nanotopography to the sensor surface, the surface coverage of vesicles needed to trigger rupture of vesicles (∆fmin) and the time required to achieve this critical vesicular coverage (tmin) are shifted to lower frequencies and longer times, respectively; i.e., bilayer formation is hampered. The shifts are more pronounced for the surface with smaller pits, which has a larger total pit perimeter per unit area than the surface with larger pits (cf. Table 1). Since the surface coverage of pits is nearly identical for the two samples, we conclude that pit edges play a major role. The vesicle rupturing and bilayer formation processes beyond the critical vesicular coverage were also observed to depend on pit diameter. Upon decreasing the pit size, longer time is needed from tmin to reach equilibrium (teq), and slightly different equilibrium
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Figure 2. Plots summarizing the average response of three important parameters derived from QCM-D: (A) frequency minimum, -∆fmin; (B) time required to reach frequency minimum, tmin; and (C) time required to reach equilibrium, tequilitrium, as a function of the total pit perimeter per unit area (pit size) and vesicle size.
frequency shifts (∆feq) are derived from QCM-D measurements for different pit diameters. To assess the effect of surface topography for different vesicle sizes, the experiment was repeated with a 100 nm lipid vesicle solution with the same concentration as above. As shown by the QCM-D reference measurement on a flat surface (Figure 1B, solid line), ∆fmin is much lower and ∆Dmin is much higher for the adsorption of 100 nm vesicles than for 30 nm vesicles (Figure 1A). The values obtained here are in good agreement with a previous report, which showed that the decrease of ∆fmin and the increase of ∆Dmin are linear with increasing vesicle diameter.19 For topographically structured surfaces, the values of ∆fmin and tmin were shifted to higher frequency and shorter time with increasing total perimeter of the pits, i.e., bilayer formation was facilitated by the nanostructures. This behavior is in remarkable contrast to what we observed for 30 nm vesicles, where the shifts occurred in opposite directions. Additionally, we observed that teq and ∆feq were essentially independent of topography in the case of 100 nm vesicles, again in contrast to the 30 nm vesicles. It is worthwhile to mention that independent of vesicles size and surface topography, all QCM-D data collected here display ∆feq and ∆Deq (frequency and dissipation shifts in equilibrium) in the region between 25.5 and 28.5 Hz and 0 to 0.2 × 10-6, respectively. These variations are within the range of (5% standard deviation from the average value (26 Hz)22 observed for vesicles on a flat SiO2 surface (see the gray, shaded areas in Figure 1), where complete bilayer formation is accomplished. Thus, we assume that complete SPB formation took place asymptotically in each measurement. Moreover, this is evidence indicating that the QCM-D measurements made using flat and nanostructured sensor surfaces are comparable, i.e., the nanoscale pits induce a surface roughness that is below the magnitude that would affect the QCM-D signal.45 In order to better understand the details of vesicle adsorption and the vesicle-to-bilayer transformation, we have replotted the QCM-D data as ∆D versus ∆f for the two vesicle sizes (Figure 1, parts C and D, respectively). This representation allows one to qualitatively distinguish intact vesicles from bilayer and to extract qualitative information regarding vesicle deformation.19 While the frequency signal includes contributions from vesicles, bilayer, and coupled water, the dissipation signal is mainly associated with vesicles, with more deformed (flattened) vesicles displaying a smaller dissipation shift than less deformed vesicles of the same nominal size. In the ∆D versus ∆f representation, adsorption of intact vesicles in the low-coverage regime would be seen as a straight line, the slope of which depends on vesicle size and the degree of vesicle deformation.19 We indeed find
straight lines for all combinations of vesicle size and surface topography in this part of the curves. Remarkably, however, the slopes for 100 nm vesicle adsorption onto flat and nanostructured surfaces are significantly different, with a smaller slope being observed on the nanostructured surfaces (Figure 1D). We conclude that vesicles on the nanostructured surfaces are (on average) more deformed than vesicles on the flat surface. An alternative explanation is that a significant number of (isolated) vesicles rupture before the critical vesicle coverage has been reached, since ruptured vesicles do not contribute to the D value. However, AFM does not show any evidence of this being the case (see Figure 4 and discussion below). For the 30 nm vesicles (Figure 1C), the slopes for flat and nanostructured surfaces are identical within measurement error. Considering the comparably low dissipation signal associated with these smaller vesicles, we can, however, neither exclude nor confirm the presence of a similar effect (more deformed vesicles on structured surfaces) as found for the 100 nm vesicles. Using the parameters ∆fmin, tmin, and teq, the behavior of 30 and 100 nm vesicles on three topographically different surfaces (flat and 110 and 190 nm pit diameter) is summarized in Figure 2. It is evident that a decrease in the total pit perimeter per unit area gives opposite responses in all three parameters for 30 nm vesicles compared to 100 nm vesicles. However, the effect is more pronounced for ∆fmin and tmin (Figure 2A,B) and weaker for teq (especially for 100 nm vesicles; Figure 2C). In our measurements using 30 nm vesicles, the decrease in ∆fmin and increase in tmin for larger total pit perimeter suggests that more lipid vesicles need to be deposited onto the nanostructured surfaces in order to induce complete bilayer formation. In the opposite case for 100 nm vesicles, fewer vesicles are required and surface topography seems to catalyze bilayer formation. To support our hypotheses derived from the QCM-D measurements and to gain additional insight, we used contact-mode AFM to obtain images of the lateral arrangement of lipid vesicles and bilayers on the surface for selected conditions, namely a 190 nm pitted surface in combination with 30 and 100 nm vesicles. We chose to specifically address the issues of vesicle adsorption, deformation, and rupture in the region prior to ∆fmin and tmin, since the most intriguing effects of surface topography seem to occur in this time/exposure/coverage regime. For this purpose, the pitted surface was exposed to a solution of 30 or 100 nm lipid vesicles for 50 or 90 s, respectively, before it was thoroughly rinsed with buffer to prevent further vesicle adsorption. For flat SiO2 surfaces, using combined QCM-D and AFM measurements and 30 nm vesicles, we have previously shown that the region prior to ∆fmin and tmin is dominated by the adsorption and accumulation of intact vesicles on the surface,
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Figure 3. (A) AFM image of a 190 nm pitted SiO2 surface after 50 s exposure to 30 nm vesicles (z-range 85 nm), showing various features characteristic of vesicle adsorption onto nanostructured surfaces: (a) formation of bilayer patches, (b) adsorption of vesicles to the bottom and/or the wall of a pit, (c) vesicle adsorbed across a pit edge with a deformation comparable to vesicles adsorbed onto flat surface, (d) strongly deformed vesicle adsorbed across a pit edge, (e) strongly deformed vesicle at the periphery of a pit, and (f) vesicle adsorbed far away from any pits. The cross-sectional profile along the dashed line x′, which is plotted in B, indicates the presence of a bilayer as confirmed by a characteristic height of ≈4-5 nm. The dashed line y′ indicates a cross-section across two pits as plotted in C. It discloses the formation of a bilayer at the bottom of one of the pits, as deduced from a ≈4 nm difference in pit depth. This information is visible in the image histogram of A, which is plotted in E. This histogram displays two small peaks (inset) with a height difference of 24.7 and 20.8 nm to the main peak. The former peak corresponds well to the depth of empty pits as derived from the histogram (D) of an image taken on the same surface before vesicle adsorption, while the latter peak derives from pits with a bilayer-covered bottom consistent with a ≈4 nm reduced depth.
with essentially no vesicle rupture and bilayer formation being detectable up to the critical coverage.23,25 However, the AFM image in Figure 3A, which was acquired on the topographically structured surface after exposure to 30 nm vesicles, shows clear evidence for the formation of bilayer patches (feature a) already in the subcritical coverage regime. The presence of a bilayer is confirmed by the cross sectional profile x′, which displays a characteristic feature height of ≈4-5 nm (Figure 3B). Interestingly, these bilayer patches are typically located in the vicinity of pits; i.e., the occurrence of bilayer patches in the low-coverage regime seems to be intimately coupled to the (edges of the) pits. Furthermore, the cross sectional profile y′ across two neighboring pits (Figure 3C) shows clear evidence of bilayer formation at the bottom of some pits, as deduced from the depth of these two pits differing by around 4 nm, i.e., a characteristic bilayer thickness. This can also be seen by comparing the histograms of images taken before and after vesicles adsorption (Figure 3, parts D and E, respectively; see figure caption for details). Such “trapped” bilayer patches are observed in about 10% of all pits for the chosen exposure conditions. These findings strengthen the notion that the pathway of vesicle adsorption and rupture, which leads to the formation of a lipid bilayer, is significantly altered on the topographically structured surface as compared to a flat surface. In particular, surface topography induces the rupture of individual, isolated 30 nm
vesicles, without the need of a cooperative effect between several neighboring, interacting vesicles to induce rupture (which is required on a flat surface).25,34,35 Judging from the overall delayed kinetics for 30 nm vesicles, the rupture of such isolated vesicles does, however, not seem to catalyze the rupture of further vesicles (such an “autocatalytic” reaction mechanism has been invoked for the case of a flat surface23,25). This is likely related to the lack of vesicles in close enough proximity to the rupturing vesicle in this low-coverage regime (or for a vesicle located inside a pit), in combination with the rather confined “spreading zone” after rupture of these small vesicles. Bilayer patches originating from the rupture of isolated vesicles at the edges and on the bottom of the pits likely do not catalyze the rupture of further vesicles, because the bilayer edge stabilizes and thus becomes inert after some equilibration time (note that the autocatalytic mechanism described for flat surfaces requires an adVancing bilayer edge). Such preformed, isolated bilayer patches may thus be considered as “abandoned” lipid material not driving autocatalytic bilayer formation.46 This might to some extent explain the need for additional lipid vesicles to complete bilayer formation, as expressed by the deeper frequency minimum found for nanostructured surfaces. Another factor leading to a higher critical coverage for 30 nm vesicles on nanostructured surfaces might be the “disrupting” effect of the pits, which has previously been observed by Rossetti et al.47 As opposed to a flat surface, the local (on a micrometer length
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Figure 4. (A) AFM image of a 190 nm pitted SiO2 surface after 90 s exposure to 100 nm vesicles (z-range 85 nm), showing various characteristic features: (i) small vesicles adsorbed into a pit, (ii) strongly deformed vesicles at the periphery of a pit or (iii) adsorbed across a pit edge, (iv) larger vesicles that seem to be masking an underlying pit, and (v) vesicles that are adsorbed a distance away from the pits and show a deformation comparable to a flat surface. The cross-sectional profiles in B illustrate that vesicle (iii) is much more strongly deformed (i.e., has a smaller height-to-width ratio) than vesicle (iv). No bilayer patches can be identified.
scale) vesicle coverage is limited on the pitted surface due to the bilayer patches in and around the pits being nonadherent for further vesicles. Thus, for a cooperative effect to occur, a much higher global critical coverage is required to result in rupture and bilayer formation at some (statistical) location on the sample. The AFM image in Figure 3A also provides interesting clues to where vesicle adsorption occurs. We find that vesicles may adsorb onto the areas in between the pits, into the pits (feature b), and across the edges of the pits (c and d), as well as at the periphery of the pits (e). It is interesting to note that the degree of deformation of adsorbed vesicles varies depending on their adsorption site. An extraordinarily strong deformation (compared to a flat surface, cf. for instance feature f) was frequently observed for vesicles lying inside a pit (b), stretching across the edge of a pit (d, yet not c), or being located at the periphery of a pit (e). According to the theoretical framework developed by Seifert and Lipowsky,48,49 we argue that this strong deformation is caused by the additional decrease in adhesion energy caused by spreading of a fractional area of the vesicle in direct surface contact compensating the additional increase in energy required to bend the vesicle across the edge of a pit. Since vesicles in such a configuration are in a highly stressed state, they are likely to be much more susceptible to rupture than vesicles on a flat surface, which would explain the occurrence of bilayer patches in the vicinity of the pits. For 100 nm vesicles (Figure 4), we find some similarities but also some important differences compared to 30 nm vesicles. A striking difference between the 30 and 100 nm vesicles is the much broader size distribution of the latter, as mentioned in the Materials and Methods section and directly evident from Figure 4. While some vesicles are small enough to fit into the pits (feature i in Figure 4A), most pits are empty. As for the 30 nm vesicles, we find strongly deformed vesicles at the perimeter of a pit (feature ii) or stretching across the edge of a pit (feature iii). The cross-section in Figure 4B illustrates that feature iii, which stretches across a pit edge, has a much smaller heightto-width ratio (i.e., is more strongly deformed) than the neighboring vesicle (feature iv), which is located at some distance from the pit. In some cases we find (large) vesicles, which seem to completely mask a pit (feature v). Note that feature v is not a bilayer patch, as indicated by its height clearly
exceeding the typical thickness of a bilayer (14-15 vs 4-5 nm, cf. Figure 4C). In fact, we did not observe any bilayer patches for 100 nm vesicles, neither in the pits nor in the vicinity of the pits, for short vesicle exposures (t < tmin); i.e., individual vesicles did not rupture spontaneously due to the presence of the pits. This observation is in line with the QCM results, and it explains why bilayer formation is enhanced by the pits for larger vesicles. Vesicles adsorbing in the vicinity of a pit display a strong degree of deformation, which facilitates the occurrence of vesicle rupture via a cooperative effect involving many vesicles (but which by itself does not lead to rupture). Note this subtle difference related to vesicle size: while the adsorption of an isolated 30 nm vesicle across a pit edge may lead to strong enough deformation to cause its rupture, the 100 nm vesicles do not rupture spontaneously, even when adsorbed across a pit edge. They are, however, in a highly prestressed state, which facilities rupture upon interaction with neighboring vesicles, thus explaining the lower critical coverage observed for nanostructured surfaces. This difference might be related to the larger vesicles being somewhat more flexible than the 30 nm vesicles due to their larger (average) radius of curvature. Conclusions In summary, we have shown that nanotopography significantly alters the kinetics and the mechanism of the vesicle-tobilayer transformation of POPC vesicles on SiO2, although the end result is a complete bilayer in all considered cases. We have identified very subtle effects of vesicle size, namely that nanotopography can promote or hamper bilayer formation in the case of 100 and 30 nm vesicles, respectively. The effect of nanotopography is related to vesicles in the vicinity of the pits being more deformed than vesicles on a flat surface. While this extra deformation is strong enough to cause rupture of isolated 30 nm vesicles, it merely makes 100 nm vesicles more susceptible to rupture (but does not actually lead to rupture of isolated vesicles). It would thus be interesting to investigate the behavior of vesicle solutions with an intentionally broad or bimodal size distribution. Other ongoing work in our group scrutinizes the effect of chemical nanostructuring, in addition to topographical nanostructuring, on SPB formation.
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