Mechanical Stability and Lubrication by Phosphatidylcholine

Verberne , G.; Schroeder , A.; Halperin , G.; Barenholz , Y.; Etsion , I. Liposomes as potential biolubricant additives for wear reduction in human sy...
7 downloads 0 Views 552KB Size
Download PDF
Article pubs.acs.org/Langmuir

Mechanical Stability and Lubrication by Phosphatidylcholine Boundary Layers in the Vesicular and in the Extended Lamellar Phases Raya Sorkin, Yael Dror, Nir Kampf, and Jacob Klein* Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel ABSTRACT: The lubrication properties of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) extended supported bilayers were studied and compared to those of surface-attached DSPC small unilamellar vesicles (liposomes) in order to elucidate the effect of phospholipid geometrical packaging on the lubrication and mechanical properties of these boundary layers. The topography and response to the nanoindentation of bilayer- and liposomecovered surfaces were studied by an atomic force microscope (AFM). In parallel, normal and shear (frictional) forces between two opposing surfaces bearing DSPC vesicles/bilayers across water were studied with the surface force balance (SFB). A correlation between nanomechanical performance in the AFM and stability and lubrication in the SFB was observed. Bilayers were readily punctured by the AFM tip and exhibited substantial hysteresis between approach and retraction curves, whereas liposomes were not punctured and exhibited purely elastic behavior. At the same time, SFB measurements showed that bilayers are less stable and less efficient lubricants compared to liposomes. Bilayers provided efficient lubrication with very low friction coefficients, 0.002−0.008 up to pressures of more then 50 atm. However, bilayers were less robust and tended to detach from the surface as a result of shear, leading to high friction for subsequent approaches at the same contact position. In contrast, liposomes showed reversible and reproducible behavior under shear and compression, exhibiting ultralow friction coefficients of μ ≈ 10−4 for pressures as high as 180 atm. This is attributed to the increased mechanical stability of the self-closed, closely packed liposomes, which we believe results from the more defect-free nature of the finitely sized vesicles.

1. INTRODUCTION

lubrication by PCs were conducted with different lipid configurations and utilized different experimental methods. Trunfio-Sfarghiu et al. studied lipid bilayers and multilayers of solid ordered (SO) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and liquid disordered (LD) 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) by using a tribometer.13 Other groups have also studied friction between lipid bilayers in both SO and LD phase, both in water and in salt solutions by means of lateral force microscopy.14,15 Liposomes composed of phospholipids were also studied, and their abilities to lubricate excised cartilage surfaces were assessed using a tribometer.5,16

Phosphatidylcholine (PC) liposomes and bilayers, along with their many biological functions,1,2 have recently been revealed as efficient lubricating agents that enable substantial friction reduction in aqueous systems.3−6 This friction reduction is achieved by the hydration lubrication mechanism enabled by the highly hydrated PC headgroups, having up to 15 water molecules in the primary hydration shell for the case of liposomes in the solid ordered (SO) phase and even more in the liquid disordered (LD) phase.7−10 In this mechanism, the hydration layers about the close-packed PC headgroups exposed at the outer bilayer surfaces are both tenaciously attached and at the same time undergo rapid relaxation that ensures a fluidlike response to shear, thereby leading to very low friction coefficients.11,12 Studies that demonstrated © 2014 American Chemical Society

Received: January 31, 2014 Revised: April 6, 2014 Published: April 7, 2014 5005

dx.doi.org/10.1021/la500420u | Langmuir 2014, 30, 5005−5014

Langmuir

Article

are the same, so there is no difference in acyl chain length or saturation. In parallel to the SFB study of the friction behavior under load, we also perform nanomechanical measurements in the AFM in order to study the mechanical stability and resistance of the two different morphologies and examine their correlation to the lubrication performance. A striking difference between the mechanical properties and lubrication behavior of the two morphologies is revealed: supported bilayers in the extended lamellar phase are readily punctured by the AFM tip and exhibit substantial hysteresis between approach and retraction curves whereas liposomes that are not punctured exhibit purely elastic behavior. At the same time, SFB measurements show that bilayers are less stable and significantly less efficient lubricants than are liposomes.

The most striking lubrication performance under physiologically high pressures with ultralow friction coefficients (μ ≈ 10−4) was recently demonstrated using the surface force balance (SFB).3,4,6 Lubrication by phospholipids (PL) may be physiologically relevant, as they are ubiquitous in living systems and are believed to play a role in the friction reduction of articulating joints.17−21 Both multilayer structures22 and spherical liposome-resembling structures23 were reported to exist in vivo. As lubrication performance is studied under load, it is also important to examine the mechanical properties of the lubricant, which will influence its ability to withstand pressures. The lubrication ability of lipid layers was shown to improve with mechanical stability and resistance to indentation; therefore, frictional studies are often conducted in parallel with AFM nanomechanical measurements.13,24 Separately, elaborate nanomechanical studies of supported lipid bilayers and liposomes have also been conducted using AFM to measure the Young’s modulus (in the case of elastic deformation) or the breakthrough force required to penetrate the layers.25−32 In most studies, a similar behavior is observed, with typical force plots where at a certain threshold force the tip breaks through the bilayer and punctures it, reaching the underlying hard surface as a result. Such force plots are obtained with lipids of varying headgroups and lipid chains.30 Egg PC liposomes and bilayers were shown to be punctured by the AFM tips,27,28 as were the SO-phase 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) and LD-phase DOPCsupported bilayers.26 An increase in the acyl chain length was shown to increase the breakthrough force required to penetrate the bilayers.25 When the nanomechanical response of multilayers of lipids was studied, some plots without tip penetration were observed, with a very elastic, hysteresis-less response.29 No study, however, compared the nanomechanical properties of SO-phase bilayers to SO-phase liposomes, which is of particular interest in view of the extremely efficient boundary lubrication by the latter. In this study, we perform a detailed nanomechanical study of DSPC bilayers and DSPC liposomes in order to examine the correlation between mechanical properties and the lubrication performance of the two lipid structures. We try to answer the question, are bilayers incorporated into liposomes better from a lubrication point of view than extended, supported bilayers in the lamellar phase, and if so, why? We have previously revealed striking differences in the boundary lubrication performance of PC lipids as the acyl chain lengths were varied;6 when liposomes are adsorbed to mica surfaces, which are then rinsed and studied in the SFB, the lubrication performance is improved with the increase in the acyl chain length. This difference in the lubrication performance is correlated to a geometrical difference in the lipid configuration on the supporting substrate (mica): liposomes composed of the longer-chained DSPC (18:0) lipids maintain their self-closed vesicular structure, whereas 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC, 14:0) liposomes rupture, forming an extended lamellar phase. It appears, therefore, that the mechanical stability of the self-closed shape of the surfaceattached, close-packed liposomes is an important factor in their lubrication performance. We here extend our previous study and perform a detailed comparison between bilayers and liposomes composed of the same DSPC lipid, which are in the SO phase at room temperature (Tm = 55 °C). All parameters are kept constant apart from the extended structure; the lipids

2. MATERIALS AND METHODS 2.1. Liposome Preparation. DSPC was purchased from Lipoid (Ludwigshafen, Germany). Single unilamelar vesicles (SUVs) were prepared using standard approaches.4,33 Briefly, lipids were dispersed in water, bath-sonicated for 5 min, and homogenized for 5 min at the appropriate temperature above the main phase transition of each lipid in order to obtain dispersed multilamellar vesicles (MLV). Next, the MLVs were progressively downsized using an extruder (Northern Lipids Inc., Burnaby, BC, Canada) with polycarbonate filters having defined pore sizes starting with 400 nm (3 cycles) and 100 nm (4 cycles) and ending with 50 nm (10 cycles). The liposomal size distribution (by volume) was determined in pure water using a Viscotek 802 DLS. Subsequent to their adsorption on the surface, samples were also characterized by AFM. The water used throughout both for the liposome preparation and subsequent measurements was highly purified (so-called conductivity) water from a Barnstead NanoPure system with a total organic content (TOC) of ca. 1 ppb and a resistivity of 18.2 MΩ. 2.2. Preparation of Liposome- and Bilayer-Coated Surfaces. Liposome-covered mica surfaces were prepared as follows. Freshly cleaved mica (mounted on cylindrical lenses for use in the SFB, see below) was placed in a 0.1−0.3 mM DSPC-SUV dispersion prepared with Barnstead purified conductivity water. Incubation was at room temperature. After overnight incubation, the surfaces were rinsed to remove excess material by placing them in a beaker containing 300 mL of water for 30 min, along with a delicate shaking motion. For SFB bilayer experiments, surfaces were heated in the oven for 1 h at 55 °C. This temperature is below the transition temperature for supported DSPC layers (as supported bilayers have been shown to have substantially higher transition temperatures than dispersed liposomes34); however, it was sufficient to induce the fusion of the liposomes and the formation of a continuous lamellar phase, as was confirmed by AFM scans. Surfaces prepared for AFM nanomechanical measurments were prepared similarly; however, the incubation was in a 0.1 mM SUV liposome dispersion. This resulted in the formation of a layer with some holes but prevented the accretion of excess material on top of the bilayers and allowed the acquisition of well-defined force plots. We note that surfaces prepared for SFB experiments passed through an air−water interface, whereas for AFM measurements during the entire preparation care was taken that the samples did not cross the air−water interface. 2.3. Atomic Force Microscopy (AFM). Imaging of surfaces and nanomechanical measurements were carried out with an MFP-3D SA (AFM) instrument (Asylum Research, Santa Barbara, CA). Scanning in tapping mode in conductivity water was conducted using a silicon nitride V-shaped 115-μm-long cantilever having a nominal spring constant of 0.35 N/m with a pyramidal silicon nitride tip with a nominal radius of 20 nm (NP, Bruker). Force plots were obtained using the same type of tip but with a new tip utilized for the force mapping only, in order to avoid tip contamination as a result of imaging. Force−distance curves are obtained by monitoring the cantilever deflection and the vertical expansion of the z piezo to which the 5006

dx.doi.org/10.1021/la500420u | Langmuir 2014, 30, 5005−5014

Langmuir

Article

Figure 1. AFM images of bilayer- and liposome-covered surfaces. (A) A DSPC bilayer. The thickness of the bilayer may be measured from the cross section, as shown in the inset. (B) A 3D representation of a different bilayer-covered surface. Samples A and B were adsorbed from a 0.1 mM DSPC SUV dispersion. In C and D, DSPC SUV-covered mica surfaces are shown. It is likely that the AFM tip has dislodged or removed liposomes during the scan. It is also likely that the liposome thickness as revealed by the cross section is significantly smaller than that for unperturbed liposomes due to compression by the AFM tip (see text). (E) DSPC bilayer obtained by adsorption from a 0.3 mM liposome dispersion. The high white areas are excess material on top of the bilayer. Scale bars are 200 nm for A−D and 500 nm for E.

F = kdInvOLS

cantilever is connected, called Zsnsr, measured by a linear variable differential transformer. Prior to performing nanomechanical measurements, the photodiode optical lever sensitivity is measured (characterized by inverse optical lever sensitivity, InvOLS, (nm/V)) and the cantilever spring constant k is determined using the equipartition theorem (thermal noise)35 so that the deflection signal from the photodiode (V) can be converted to deflection (nm) and force, F, as

(1)

where d is the deflection signal (V). Force maps were captured in relative trigger mode using a force trigger of 5 nN. This means that the tip would press on the surface until a force of 5 nN, relative to the baseline before force application, was reached. This trigger value was chosen after higher forces were examined (up to 50 nN) to ensure that penetrations of the tip through the layer are not missed due to 5007

dx.doi.org/10.1021/la500420u | Langmuir 2014, 30, 5005−5014

Langmuir

Article

Figure 2. Characteristic plots of force vs piezo elongation distance, (A, B) and force versus indentation (C and inset to B). The steep green curve (1) was obtained on bare mica, the black curve (2) was obtained on a DSPC-liposome covered surface, and the red curve (3) was obtained on a DSPC bilayer-coated surface. There is a clear penetration of the tip through the bilayer, which was not observed for the liposomes. In B, the same plots as in A are shown, with the addition of another plot (4) obtained on liposomes where a higher trigger force was used. No penetration was observed for triggers of up to 50 nN. The inset in B shows a force vs indentation graph for plot (4). Both approach (■) and retraction (+) plots are shown in A and are also marked by arrows for the red curves (3). For curves 1 and 2, the approach and retraction plots are completely overlapping and hence barely distinguishable. Only approach curves are shown in C. (D) A histogram of the breakthrough forces obtained for DSPC bilayers. The histogram is based on 410 plots. A clear bimodal distribution is observed, with breakthrough forces of 1.6 ± 0.5 and 3.8 ± 0.6 nN. The inset shows typical plots for the two breakthrough events. As indicated in the inset, the bimodal distribution in the breakthrough force is correlated with the overall indentation associated with the penetration. temperature-stabilized rooms (±0.2 °C about the chosen temperature, ca. 25 °C).

insufficient force.36 Force plots are presented as either force−distance or force−indentation plots. The indentation of the tip into a soft sample is obtained by subtracting the deflection value from the Zsnsr value.29 To obtain a large number of force plots at different locations on the sample, the function of force maps was used: over an area of 2 × 2 or 5 × 5 μm2, 16 × 16 force plots were taken, so 1 force map produced 256 force curves. For each sample, several force maps at different locations were acquired. Usually for each system studied at least two samples were analyzed. The force rate used was 1 Hz, and the approach rate was 200 or 400 nm/s. 2.4. Surface Force Balance (SFB). The SFB technique and detailed procedures for the measurement of normal (Fn) and shear (Fs) forces between molecularly smooth mica surfaces, half-silvered on their backsides, in a crossed cylinder configuration (mean radius of curvature R), and the closest separation D have been described in detail previously.37 D is optimally measured to ±2−3 Å by monitoring the wavelength of optical interference fringes of equal chromatic order (FECO). Shear profiles were taken by directly measuring the response of the lower surface to lateral motion applied to the upper surface. Lateral amplitudes Δx0 in the range of 200−1200 nm and shear velocities vs in the range of 10−600 nm/s were applied. Shear force profiles were taken simultaneously with normal force profiles by applying lateral motion at several separations at progressively increasing normal loads. All experiments in the SFB, as well as AFM scans and force plots, were carried out with the surfaces immersed in conductivity water (no added salt or liposomes). The results presented here are based on three separate bilayer experiments and two liposome experiments (which are described in detail in ref 6), each with several different contact points between the interacting surfaces, carried out in

3. RESULTS AND DISCUSSION 3.1. AFM Imaging. Mica surfaces covered with DSPC liposomes or bilayers were prepared and imaged in the AFM. The coverage of the surface by liposomes/bilayers was optimized by changing the concentration in the adsorption dispersion so that minimal accretion of excess material was observed (as these perturbed the nanomechanical measurments). In Figure 1, typical AFM height scans are shown. In Figure 1A,B, DSPC bilayers are shown. The scans are of two different samples, and plot B is a 3D representation of a height scan. It can be seen that not the entire surface is covered, while a cross section reveals a characteristic bilayer thickness of ∼4 nm, in agreement with the literature38 (Figure 1A, inset). Figure 1C,D shows two characteristic liposome-covered samples used for nanomechanical studies. The lower inset in Figure 1C shows details of a cryo-SEM scan which depicts that a flattened, close-packed liposome layer is found below the top layer, in agreement with earlier studies.4,6 This image differs from the one obtained by AFM likely because the top liposome layer is removed by the tip during the scan, as are also some liposomes from the close-packed layer adjacent to the mica; this results in a sparser liposome layer compared to the closepacking revealed by cryo-SEM imaging.4,6 A cross section through one liposome indicates a thickness of ∼8 nm; this 5008

dx.doi.org/10.1021/la500420u | Langmuir 2014, 30, 5005−5014

Langmuir

Article

corresponding force- indentation curve, where indentation is the extent of sample deformation by the tip, as illustrated in the inset of Figure 2C (obtained by subtracting the deflection value from the Zsenr value). The steepest green curve (1 in the figure) was obtained on bare mica and does not exhibit indentation, as indeed expected from a hard surface. There is also no hysteresis, and the approach and retraction curves are indistinguishable. The parabolic black curve (2 in the figure) was obtained on a liposome sample, and the red curve (3 in the figure) was obtained on a bilayer sample. The liposomes exhibit very elastic behavior, without any hysteresis, as both approach and retraction curves are the same, within the scatter, indicating that there is little energy dissipation or irreversible deformation up to the maximal pressure applied to the liposome. The bilayers, however, show very different behavior: it can be seen in Figure 2A that at the very beginning (from right to left) the red curve follows the black one precisely until a critical force is reached that allows for penetration of the tip through the bilayer. The retraction curve is very different from the approach curve, with substantial hysteresis between the two. It should be emphasized that until the critical force is reached the force plots are similar because the basic interaction between the tip and the headgroup is the same at that stage for both bilayer and liposome. A remark about the extent of indentation should be made; it can be seen that total indentation until the hard surface is reached in the bilayer case (red plot Figure 2C) is 6.5 nm. This is higher than the total bilayer thickness, as measured from the cross section in the scans (Figure 1A). There are a number of possible explanations for this: first, the zero point is arbitrarily chosen as the point of the onset of forces; however, there are not necessarily only steric forces, as electrostatic double-layer repulsion or hydrodynamic effects might also contribute at the onset of repulsion. We may try to estimate these. Although the Derjaguin approximation for the electrostatic double layer repulsion (Fes) between a sphere (radius Rt) and a flat area of similar surface potential ψ0 (say 100 mV42,43) would not apply well since the Rt (20 nm) of the AFM tip is comparable to the tip−surface separation D (on the order of nanometers), we may use it as a rough estimate. According to this44

value is likely to be much lower than the unperturbed liposome height due to compression by the AFM tip during a scan.4,39,40 One strong indication of such liposome compresssion by the tip is obtained from hydrodynamic measurments of a very similar liposome layer adsorbed on mica in the SFB, revealing a thickness of 21 ± 2 nm per uncompressed layer.4 Such discrepancies in the height arising from strong tip compression can in principle be minimized in a number of ways, by suitable settings of the AFM, for example, by changing the set point ratio (where a small change can lead to a large effect), or by driving the cantilever close to its resonance frequency or using small drive amplitudes.41 We remark on the “bumps” observed at the liposome centers in Figure 1C,D. Such bumps were observed on many, though not all, liposomes imaged with the AFM (though they were not observed in cryo-SEM images of the same liposomes); we do not have a good explanation for them and believe that they may be an artifact of the scanning tip compression of the soft liposomes. We should also bear in mind that the width/shape of objects is overestimated/altered due to the shape of the tip and that AFM images should in principle be deconvoluted to obtain the real shape (e.g., Borkent et al.41), though we estimate that this is not an important effect in our system. It should be noted that for SFB measurements, samples were initially incubated with a higher concentration of liposomes, as described in the Materials and Methods section. This resulted in surfaces with substantially fewer holes and more accreted layers of excess material (Figure 1E). This was done in order to obtain more complete layers with fewer defects. 3.2. AFM Nanomechanical Study. Samples that were mostly uniform and well-defined when imaged in the AFM, and without accreted material, were then measured by AFM force spectroscopy. New probes were used for each sample to avoid contamination. Force plots (1024) were acquired for the DSPC liposome-covered sample, and 640 force plots were acquired for two different DSPC bilayer-covered samples in two locations on each sample. Data reported in Figure 2 are all based on measurements where the noise level in the AFM signal was sufficiently low to enable a clear determination of the force versus distance profiles (e.g., Figure 2A). The mechanical response to the nanoindentation of DSPC liposomes and bilayers shows a qualitative difference between the two morphologies. In the case of bilayers, discontinuities in the form of kinks in the force plots are observed, as in Figure 2A,C, due to the puncturing of the layer by the AFM tip; these are absent in the DSPC liposomes. DSPC bilayers were shown to be punctured by the AFM tip in 56% of the cases, and another 24% of the plots exhibited substantial hysteresis without a clear penetration event indicated by a kink. The remaining 20% of the plots looked like bare mica plots, as expected due to incomplete coverage of the surface (see Figure 1A,B). In these plots, no indentation was observed as all of the motion of the z piezo was translated directly to the tip deflection, as would be the case for any hard surface. In contrast to this, the DSPC liposomes exhibited a very elastic response with neither a kink nor hysteresis in 97% of the cases, even up to trigger levels of 50 nN (as opposed to 5 nN for the bilayers). Figure 2 shows several types of typical force plots for the two cases. Zero separation is defined manually at the point of onset of forces. For each type of plot, both approach (square symbols) and retraction (plus symbols) curves are shown. The direction of the tip movement is depicted by arrows for clarity. Figure 2A is a force versus Zsenr curve, while Figure 2C is the

⎛ eΨ ⎞ Fes = 128πR tCkBTκ −1 tanh2⎜ 0 ⎟ exp( −κD) ⎝ kBT ⎠

(2) −5

where C is the electrolyte concentration (taken as 10 M in conductivity water42,43), e the charge on the electron, kB is Boltzmann’s constant, T is the temperature (298 K), κ−1 is the Debye length (ca. 90 nm at this C value), and D is the separation between the surfaces (taken as the tip−mica separation, say 5 nm; the Fes value is insensitive to this). Putting in values, we find Fes ≈ 10 pN, some 2 orders of magnitude smaller than the nN range of the measured indentation forces. Likewise, we find that the hydrodynamic force Fhyd also should not have a substantial effect. We estimate it using45

Fhyd = 6πR t 2η

Ḋ D

(3)

where Rt is the radius of the tip curvature, D is the distance between the two surfaces (say 2 nm), and Ḋ is the approach velocity (400 nm/s). With these values, we find Fhyd ≈ 4 pN, which is again very small compared to the values of breakthrough forces measured. 5009

dx.doi.org/10.1021/la500420u | Langmuir 2014, 30, 5005−5014

Langmuir

Article

bilayers are defect-free over the area of a liposome. Thus, they may be better able to withstand the tip pressure relative to the extended lamellar phase. This is because defects in the lamellarphase supported bilayers, seen for example in Figure 1, may have an extended effect, resulting in an overall weaker structure which is more prone to rupture, even when the tip is not pressing on the defect itself. These nanoindentation indications concerning the robustness of the layers correlate well with the SFB results, as shown below. A remark is in order concerning the extent of indentation of the surface-attached liposomes. Even for the highest loads applied, up to ca. 50 nN (which is more than an order of magnitude higher than the breakthrough loads for the case of the extended bilayers), the indentation is only some 8 nm (Figure 2B inset). This is consistent with the unperturbed thickness of the surface-attached DSPC-SUVs, which is some 20 nm as revealed by hydrodynamic measurements on similar liposomes4 and shows that even at such an indentation the liposome thickness on the mica exceeds that of a double bilayer. This implies that the liposome bilayers remain undamaged. It also supports our inference that the 8 nm or so liposome thickness indicated by the AFM micrographs (Figure 1C,D) is smaller than the unperturbed thickness and may be viewed as an artifact arising from compression by the AFM tip. 3.3. SFB Results. 3.3.1. Normal Interactions between DSPC-Bilayer-Coated Mica Surfaces. Figure 3 shows normal forces F(D)/R as a function of separation D between two

The second possibility, which we believe is closer to the real situation (see also below), is that some material is adsorbed on the AFM tip and that this adsorbed layer is indented as well, in addition to the measured sample, and would therefore increase the indentation length before the tip touches the mica. In Figure 2D, the distribution of breakthrough forces obtained for bilayers is shown. A clear bimodal distribution is observed, with breakthrough forces of 1.6 ± 0.5 and 3.8 ± 0.6 nN. One possible explanation of the bimodal behavior is lipid adsorption to the tip (see above). It is possible that some material is intermittently adsorbed and desorbed from the tip in the time course of the measurements. For example, it may be removed upon penetration and adsorbed again when the bare tip next interpenetrates the bilayer. Such an extra layer on the tip will require the application of a roughly 2-fold-larger breakthrough force, as may be seen by a simple consideration: We assume that a given indentation δy (with y standing for yield) of a bilayer (of unperturbed thickness L) is required before its elastic response fails and breakthrough/interpenetration by the (clean) tip takes place, corresponding to a bilayer yield strain of εy; clearly εy = (δy/L). If we assume that the effective bilayer elastic modulus is K and that the tip radius pressing on the bilayer is Rt, then, in the elastic regime, contact mechanics tells us46 that the force Fy required for an indentation δy is given by Fy ≈ R1/2Kδy3/2 (this approximation assumes a semi-infinite bilayer thickness). If now the tip becomes coated with a bilayer (as a result of the previous penetration of the mica-supported bilayer, conjectured earlier), then as it presses down it will effectively be compressing two stacked bilayers rather than one bilayer for the case of a “clean” tip. Since the modulus of two supported stacked bilayers is similar to that of a single bilayer, the extent of indentation by the tip required for the critical yield strain of the two stacked bilayers is then just double that required for a single layer so that the normal force required for the yield and interpenetration needs to be ∼23/2 ≈ 2.8 times higher. This accounts for the binodal nature of the force required for yield shown in Figure 2D, where the ratio of the maxima of the two yield forces is ca. 2.4 ± 0.5, and also supports the explanation of the interpenetration distance (Figure 2C) sometimes being larger than a single bilayer thickness. There is a striking difference between interpenetration by the AFM tip of the bilayers in their extended lamellar phase (Figure 2A) and the purely elastic response, with no penetration of the bilayers, when liposomes are pressed by the tip, even up to much higher normal forces (Figure 2B). This may be attributed to a number of effects. We recall that a careful determination of the thickness of similar liposomes adsorbed close-packed on mica revealed their thickness to be ca. 20 nm4 rather than that of the much thinner liposomes shown in Figure 1, which are likely substantially compressed by the AFM tip during the scan. Since the double-bilayer thickness of the DSPC-SUVs is some 10 nm, this implies that they are filled with water. Thus they are much “softer” than the supported bilayers in the extended lamellar phase and would be expected to respond elastically to initial deformation. Even once they are indented so that the AFM tip is within some 10 nm of the mica substrate (the thickness of a double bilayer), the contact area of the tip with the double bilayer would be much larger than for a single supported bilayer. Thus, the normal stress acting to rupture the bilayers would be much lower, for a given normal force, in the case of the liposomes. Finally, we believe that the closed nature and finite size of the DSPC-SUVs make it more likely that the

Figure 3. Normalized Fn(D)/R vs distance D profiles between DSPC bilayer-coated mica surfaces (formed as described in the text) across conductivity water. Filled symbols are first approaches, empty symbols are second approaches, and crossed symbols correspond to the separation of the surfaces, increasing D. The final separation between the surfaces for all profiles is 11 ± 2 nm. In A, shear was applied for all approaches, whereas in B no shear was applied. The shaded band in B indicates the range of second approach and separation profiles from A. The inset in A shows force plots for a symmetrical liposome system for comparison, with a shaded band marking the range of first approaches for bilayers, from plot A. 5010

dx.doi.org/10.1021/la500420u | Langmuir 2014, 30, 5005−5014

Langmuir

Article

DSPC bilayer-covered mica surfaces. For the contact points presented in A, shear profiles were taken at different D values while measuring the normal force profiles. In the normal force profiles, it can be seen that most out profiles (crossed symbols) as well as subsequent approaches (open symbols) exhibited much higher forces with an onset at longer separation distances compared to first-approach profiles (corresponding full symbols). This is a strong indication that the surfaces have been damaged, most likely due to the detachment of material from the surface as a result of shearing and “debris” formation during shear, leading to greater repulsion, as previously described for DPPC multilayers.6 Two of the contact points (squares and diamonds) showed better resistance to loads, possibly due to better coverage of the surface. Subsequent approaches are more inward for these contact positions compared to the first approaches, which is attributed to the squeezing out of excess material. In the contact points of Figure 3B, no shear was applied. It can be clearly seen that subsequent approaches are more inward then first approaches for all contact points examined. We attribute this to the removal of excess material upon first approach. The subsequent approaches in Figure 3A are shown here as a shaded band for comparison. Compression was applied to a maximal normalized force of 1.7 N/m, corresponding to pressure P ≈ 54 atm over the contact area, at which the final hard wall separation was Df = 11 ± 2 nm. The inset in Figure 3A shows normal force profiles for DSPC liposomes (taken from ref 6). The shaded band is the range of first approach profiles shown in Figure 3A, added for comparison. In contrast to DSPC bilayers, the DSPC liposomes exhibit reversible and reproducible behavior, with subsequent approaches and decompression profiles that are very similar to those of first approaches to contact. This behavior, we believe, results from the greater stability and robustness to compression and shear of DSPC liposomes on the mica surface. In contrast, DSPC bilayers, unlike liposomes, are clearly detached from the surface upon shear application, as can be seen from comparing Figure 3A,B. This difference likely results from the larger number of defects in the bilayers compared to the number in liposomes, which renders them less robust to shear under compression. 3.3.2. Shear Forces between DSPC-Bilayer-Coated Mica Surfaces. A typical shear profile is shown in Figure 4. In the upper trace, the applied back-and-forth motion of the upper surface is shown, while all of the other traces represent the shear force Fs transmitted to the lower surface at increasing pressures. Little response to the shear motion above the noise level was observed down to compressions of 10 ± 1 nm and mean pressures corresponding to 29 atm. With further increases in the pressure (at the same D values), a detectable sliding friction was observed, which increased with increasing pressure but remained low until abrupt rigid coupling of the surfaces was observed at 51 atm. This means that the applied shear amplitude was not sufficient to achieve sliding of the lower surface under these conditions; hence both surfaces moved together as the static friction could not be overcome at these conditions. Under similar experimental conditions (sliding velocities), DSPC liposomes could withstand pressures as high as 180 atm6 while still sliding freely. We emphasize that even at pressures sufficiently high (>30 atm) that rigid coupling of the bilayers was reached (e.g., Figure 4, trace g), we saw no evidence of yielding of the bilayers or the squeezing out of material (e.g., Becker et al.47), but nonetheless damage must have occurred. This is because, following separation, subse-

Figure 4. Typical shear force vs time traces for the symmetrical DSPC bilayer system, taken directly from the SFB. The top trace (a) is the applied back-and-forth lateral motion (Δx) of the top surface, while traces b−f are the frictional forces, Fs, between the sliding surfaces.

quent approaches to contact showed high friction forces due to irreversible damage to the layers. If, however, surfaces were not compressed beyond 30 atm and were not sheared, then subsequent approaches showed similar friction behavior as first approaches to contact. A summary of frictional versus normal forces is shown in Figure 5 for the DSPC-bilayers system, with the corresponding

Figure 5. Friction forces Fs as a function of applied loads Fn between two DSPC bilayer-coated mica surfaces. Full symbols, first approaches; empty symbols, second approaches at corresponding contact positions. Friction coefficients and maximal applied pressures prior to rigid coupling appear in the caption.

friction coefficients (μ = dFs/dFn) for the different profiles. It can be seen that the initial friction forces are very low but then increase with further increases in load. For instance, for the triangle data set, the friction force for the first three data points is within the noise level (