Effect of Cholesterol on the Stability and Lubrication Efficiency of

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Effect of cholesterol on the stability and lubrication efficiency of Phosphatidylcholine surface layers Raya Sorkin, Nir Kampf, and Jacob Klein Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01521 • Publication Date (Web): 30 Jun 2017 Downloaded from http://pubs.acs.org on July 2, 2017

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Effect of cholesterol on the stability and lubrication efficiency of Phosphatidylcholine surface layers #

Raya Sorkin* , Nir Kampf and Jacob Klein*

Weizmann Institute of Science, Materials and Interfaces Department, Rehovot 76100, Israel #

Current address: VU University of Amsterdam, Department of Physics and Astronomy, 1081HV Amsterdam, The Netherlands *Corresponding authors, E-mail: [email protected], [email protected]

Abstract Lubrication properties of saturated PC lipid vesicles containing high cholesterol content under high loads were examined by detailed Surface Force Balance measurements of normal and shear forces between two surface-attached lipid layers. Forces between two opposing mica surfaces bearing distearoyl phosphatidylcholine (PC) (DSPC) small unilamellar vesicles (SUVs) or liposomes, or bilayers, with varying cholesterol content were measured across water, while dimyristoyl PC (DMPC), dipalmitoyl PC (DPPC) and DSPC SUVs containing 40% cholesterol were measured across liposome dispersions of SUVs of the same lipid composition as the adsorbed layers. The results clearly demonstrate decreased stability and resistance to normal load with the increase in cholesterol content of DSPC SUVs. Friction coefficients between two 10% cholesterol PC-bilayers were in the same range as for 40% cholesterol bilayers (µ~10-3), indicating that cholesterol has a more substantial effect on mechanical properties of a bilayer than on its lubrication performance. We further find that the lubrication efficiency of DMPC and DPPC with 40% cholesterol is superior to DSPC 40% cholesterol, most likely due to enhanced hydration-lubrication in these systems. We previously found that when experiments are performed in the presence of a

lipid reservoir, layers can self-heal and therefore their robustness is less important at such conditions. We conclude that the effect of cholesterol in decreasing stability is more pronounced than its effect on hydration, but the stability is, in turn, less important when a lipid reservoir is present. This study complements our previous work and sheds light on the effect of cholesterol, a prominent and important physiological lipid, on the mechanical and lubrication properties of gel-phase lipid layers.

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Introduction Lipids are essential building blocks of all living matter, and they

have important

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physiological roles quite apart from constituting cell membranes . In the context of biological lubrication, phospholipids are known to be present in synovial fluid2, 3, 4, 5 and cartilage tissue3, 4, 6, 7 and have been proposed3, 4, 5, 8, 9, 10 to play a role in physiological lubrication as in lungs, cartilage and elsewhere. A major phospholipid family identified on the cartilage surface is that of phosphotidylcholines (PCs), with large variability in fatty acid chain lengths and degrees of saturation7. Out of the saturated fatty acids, saturated C16 and C18 chains (indicating the presence of DPPC and DSPC) are a major fraction (30%). Many other non-lipid components are also present in synovial joints11, 12, and a synergetic activity of these components with the PC lipids might be responsible for the unique tribological properties of articulating cartilage 13, 14, 15. We have previously studied the boundary lubrication properties enabled by phospholipids at physiologically high pressures16,

17

and demonstrated exceptionally low friction

coefficients. We further studied such lubrication across liposome dispersions18 in order to better emulate physiological conditions where lipid reservoirs in the form of liposomes are present. It is of interest to extend these studies to examine the effect of cholesetrol, a major lipid component prevalent in the human body. It is found in eukaryotic membranes at concentrations around 20-30%, and even as high as 50% and 70% in the membranes of certain cell types. It is known to affect the phase state and mechanical properties of lipids 19, 20, 21

. Moreover it is known to be present in synovial fluid22 , so that following the insight

obtained in our previous studies concerning the effects of different lipid structures16, 17, here we examine the effect of cholesterol incorporation into the PC SUVs in their role in lubrication.

Outstanding lubrication by PC liposomes under high pressure is attributed mainly to a mechanism called hydration lubrication23,

24, 25

. According to this mechanism, water

molecules are tenaciously attached to the highly hydrated PC groups but at the same time rapidly interchange with other water molecules present in the bulk behaving in a fluid-like manner under shear, as long as the shear rate is lower than the relaxation rate of the hydration layer (~109 sec-1 23, 24, 26) This mechanism is very efficient in PC headgroups due to their high hydration level (up to 15 water molecules in the primary hydration shell 2, 27, 28, 29

). Several indications suggest that cholesterol influences lipid hydration

friction reduction by phospholipid boundary layers

30, 31, 32, 33, 34

. As

is facilitated by the hydration


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lubrication mechanism, cholesterol might therefore have a substantial effect on lubrication capabilities. It should be noted that the reports in the literature are not consistent with respect to whether cholesterol should increase

30, 31, 32

or decrease

33, 34

hydration. In either

case, however, it is expected to affect hydration lubrication.

In this study, therefore, we sought to examine the effect of cholesterol addition on the lubrication abilities of PC SUVs. We first conducted experiments with DSPC SUVs consisting of different cholesterol content in pure water conditions, to examine the effect of different extents of cholesterol incorporation on lipid SUVs that have previously demonstrated very good lubrication16, 17. Next we worked with single lipid component PC SUVs of different acyl chain lengths (14:0, 16:0, 18:0), all with a high cholesterol content of 40%. These latter measurements were conducted across SUV dispersions, in order to compare the results to our previous experiments with the same series of lipids without cholesterol that were also measured in dispersion, as well as to better emulate physiological conditions18 where cholesterol is present in synovial fluid22. The surface of articular cartilage has been found to contain 32% Sphingomyelin, 41% PC and 27% Phosphatidylethanolamine (PE)7, therefore we intend to study lipid systems of increased complexity in the future. We aim to gradually increase the complexity of the systems studied, and also propose to examine mixtures of lipids.

Materials and methods

Materials The PC lipids, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC,14:0), 1,2dipalmitoyl-sn-glycero-3 phosphatidylcholine (DPPC 16:0) and 1,2-distearoyl-sn-glycero3- phosphatidylcholine (DSPC, 18:0), were purchased from Lipoid (Ludwigshafen, Germany). The main SO-to-LD transition temperatures of unsupported bilayers of these PCs are 240C, 410C and 540C respectively 35. The CMC values are: 6nM for DMPC, 0.46 nM for DPPC36. Cholesterol was purchased from Sigma Aldrich. Ultrapure (conductivity) water was obtained by treating tap water with an activated charcoal filter followed by a Barnstead NanoPure system. The water specific resistivity was higher than 18.2 MΩ cm, and the concentration of total organic compounds (TOC) was below 1ppb (henceforth


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conductivity water). The mica was ruby muscovite, grade 1, supplied by S&J Trading Inc., NY. Shell “EPON 1004” resin was used to glue the mica sheets to the quarz lenses.

Liposome preparation Single unilamelar vesicles (SUVs) were prepared using standard approaches 28, 37. Briefly, phospholipids were mixed with the appropriate amount of cholesterol (either 10% or 40% molar) and dissolved in a glass tube with a 3:1 chloroform methanol solution. Then they were dried under a N2 flow overnight, following drying in a desiccator. Next, 10 ml of water was added and multilamellar vesicles were obtained by vortex mixing and bathsonication for 20-30 min while heating 10°C above the main phase transition temperature, Tm. For all preparations,

the MLVs were progressively downsized using an extruder

(Northern lipid Inc, Burnaby, BC, Canada) through polycarbonate filters having defined pore sizes starting with 400nm (3 cycles), 100nm (4 cycles) and ending with 50nm (10 cycles). Liposomal size distribution (by volume) was determined in pure water using a Viscotek 802 DLS. Subsequent to their adsorption on the mica surface, samples were also characterized by AFM. The DLS-derived dimensions of the solution-dispersed liposomes were in line with those estimated from AFM and cryo-SEM micrographs of the surfaceattached vesicles (see later), though the latter had a spread of dimensions on the surfaces due to the adsorption process 28.

Sample preparation For both AFM scans and SFB measurements, freshly cleaved mica (mounted on cylindrical lenses for use in the SFB, see below) was placed in a 0.26 mM SUV dispersion prepared with conductivity water. For the first set of experiments, mica surfaces were incubated with DSPC SUVs containing varying cholesterol content (either 10%, 15% or 40% cholesterol, molar percentage). Following overnight incubation, surfaces were gently rinsed by placing the sample in 300ml of conductivity water, and next mounted in the SFB instrument. For the second set of experiments, SUV dispersions of the different lipids, DMPC, DPPC and DSPC, each containing 40% cholesterol, were added to water-immersed freshly cleaved mica surfaces mounted in the SFB to a final concentration of 0.3 mM. Measurements were conducted starting from 30min following addition of the liposome dispersion.


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Atomic force microscopy (AFM) Imaging of surfaces was 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 115µm long cantilever having a nominal spring constant of 0.35 N/m with a pyramidal silicon nitride tip with a nominal radius of 2nm (SNL, Bruker).

Surface Force Balance (SFB) The procedures of the mica- SFB technique have been described in detail previously

38

.

Briefly, the measurement of normal (Fn) and shear (Fs) forces was conducted between two back-silvered atomically-smooth mica surfaces, in a cross cylinder configuration (mean radius of curvature, R), by monitoring the bending of two orthogonal leaf springs, a vertical spring and a horizontal spring. The bending of the horizontal spring is determined using multiple beam interferometry; the separation of closest approach, D, is optimally measured to ±2-3Å by monitoring the wavelength of optical interference fringes of equal chromatic order (FECO). The bending of the vertical springs which provides a direct measure of the shear forces is monitored by an air-gap capacitor.

Normal force profiles Fn(D)/R and shear traces Fs(t) were recorded in the same approach and separation cycles, as the surfaces were progressively compressed. Fn(D)/R is, in the Derjaguin approximation, proportional to the interaction energy per unit area between two flat parallel surfaces obeying the same force law, and is a means of normalizing results obtained using different curvature surfaces

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. At each surface separation, both during

compression and upon separation of the surfaces, shear motion was applied for one minute. Shear profiles were taken by directly measuring the response of the lower surface to lateral motion applied to the upper surface. Lateral amplitudes, ∆x0, in the range of 200-1200 nm and shear velocities vs in the range 10-600 nm/sec were applied to the upper mica surface. Shear force traces – revealing the frictional forces between the sliding surfaces - were measured simultaneously with normal force profiles by applying lateral motion at several separations at progressively increasing (or decreasing) normal loads (and corresponding surface separations). Sliding friction forces Fs are measured from the plateau regime of the shear-force vs. time traces (shown later), where Fs is independent of the shear amplitude. The magnitude of the weakest shear forces which may be comparable with the noise in the signals is determined either by filtering the signal about the drive frequency, or by fast


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Fourier transform of the data to yield Fs at the drive frequency, see e.g. refs.

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23, 24

; the two

approaches yield similar values of Fs. The results described here are based on 8 independent experiments overall (and a further experiment for (DSPC/15% cholesterol content) not shown here), each with at least three different contact points between the interacting surfaces, with several approach and retraction profiles at each contact point, carried out in temperature-stabilized rooms of T=25±0.2ºC.

The mean pressure, P, between the compressed surfaces can be directly evaluated from the dimensions of the flattened area A (obtained from directly measuring the radius a of the flattened contact area at the tips of the interference fringes) as P=Fn/A= Fn/πa2. The flattening of contacting, non-adhering surfaces can be also evaluated from Hertzian contact mechanics40, and can then be used for pressure evaluation. This method is preferred for cases of small flattening (small a). Detailed explanations of the mean pressure evaluation for similar SFB measurements are given elsewhere17.

Results and Discussion

DSPC with varying cholesterol content Surface force experiments were conducted with DSPC containing 10%, 15% and 40% cholesterol. We focus our discussion on the extreme cases of 10% and 40% cholesterol, while the results for the 15% system, not shown here, are broadly closer to those of the 10% cholesterol case (differences are noted in the text). Normal force profiles for these two systems are shown in figure 1, (A) and (C). For the 10% system, the profiles are generally similar to those of cholesterol-free DSPC and HSPC

17, 28

Fn(D) increases roughly exponentially with

decreasing D at large separations and more sharply on closer approach, attributed to onset of steric interactions between opposing liposome layers (Figure 1 (A)). Significant repulsion forces (>1 mN/m) commence already at ca. 100nm, likely corresponding to a flattened SUV layer with an overlayer of loosely attached SUVs, as previously observed 17, 28

. Then, upon further compression, the repulsion increases as the excess SUV layers are

squeezed out. This squeeze-out of excess SUVs is responsible for the shorter range repulsion of the second approach profiles relative to first approaches, as seen in figure 1(A). Upon compression, mean contact pressures P of up to 100 atm could be reached before rigid coupling of the surfaces. “Rigid coupling” is an indication that the maximal applied shear force between the surfaces, Ks∆x0, was lower than the sliding friction force, Fs, and so


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could not induce sliding of the upper surface past the lower one, so that Fs could not be measured. The final distance obtained, at which the surfaces cannot be further compressed up to the maximal loads applied, is 22±2.5 nm, roughly corresponding to 2 confined fullycompressed liposome layers (i.e. 4 bilayers in all). We note that similar profiles were obtained for the 15% cholesterol case in water, with the important difference that at high pressures the surfaces approached to a ‘hard wall’ separation of 11±2 nm, corresponding to two bilayers, in contrast to the final separation of the 10% system which is exactly double that. It seems, therefore, that the addition of merely another 5% of cholesterol renders the liposomes less robust and stable, causing them to rupture under pressure and enable expulsion of lipids leading to a final layer thickness of one bilayer (on each surface). This effect is well in line with the reduced mechanical stability as a result of an increase in cholesterol concentration that was observed in AFM nanomechanical study (Salome Mielke et al, unpublished data). An AFM scan of the surfaces after experiment confirmed the presence of liposomes on the surface, as shown in figure 1(B).

Addition of 40% cholesterol induces a substantial change compared to the 10% cholesterol system both in the interactions and in the surface structure. As can be seen in figure 1(C), the onset of forces is at much lower separations than for the 40% cholesterol case, below 30nm.

This lack of long-range steric repulsion implies that the initial surface layer

configuration, before the application of pressure, is different than for the 10% system. The hard wall separation is at 12.5±2.5 nm, which corresponds to somewhat over 2 bilayers. An AFM scan of the surface after the SFB experiment supports this, showing a ‘holey’ bilayer sparsely covered by an additional ~2nm monolayer is observed (figure 1(D)). In interpreting the relevance of the AFM micrograph to the structure of the lipid/cholesterol surface layer, two points should be borne in mind. Firstly, the lipid-coated surfaces are withdrawn through an air-water interface en route from the SFB to the AFM cell, which may remove an upper leaflet leaving hydrophobic tails exposed (and indeed the mica surfaces emerge only partly wetted). Secondly, even in tapping mode imaging, the surface structure of the 40%-cholesterol-containing DSPC may be perturbed by the tip. Our best attribution is that the DSPC-SUVs layers on the mica rupture under compression and shear to form structures that are between 1 – 2 bilayers on each mica, exposing their hydrated phosphocholine groups while under water. At the highest compressions, hemi-fusion may occur over the whole or, more probably, part of the contact region, expelling a bilayer, and resulting in adhesion and thus high friction between the surfaces at sufficiently high


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pressures (see next section on shear forces). In line with this, there was a jump out upon separation of the surfaces due to the adhesion forces between the hemi-fused patches following the highest compression and shear. Interfacial energy of γ = -10.5 + 4.3 mJ/m2 was estimated from the JKR relation40. Fpulloff = 3πRγ, where R is the mean radius of curvature of the uncompressed surfaces and Fpulloff is the SFB-determined force required to separate the surfaces. This value compares with a value γ ≈ 50 mJ/m2 for the alkane/water interface41. This suggests either that, if pulloff occurs at the hemi-fusion sites, such sites are present only in patches covering some 20% of the contact area (taking the surface energy at the lipid-acyl-chains/water interface to be comparable to that of the alkane/water interface); or, quite likely, that the pull-off occurs at the weaker lipid-headgroup/mica interface (for which γ ≈ 14±4 mJ/m2 ( R. Goldberg, J. Klein (unpublished data)). (A)

(B)

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Figure 1: Normalized Fn(D)/R versus distance D profiles between DSPC SUV coated mica surfaces across conductivity water, prepared by adsorption of DSPC SUVs containing (A) 10% and (C) 40% cholesterol. Filled symbols are first approaches, empty symbols are second approaches and crossed symbols correspond to separation of the surfaces- increasing D. The results are of two different experiments (black and blue


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symbols) for (A) and (C).The final separation between the surfaces is 22±2.5 nm (A), 12.5±2.5 nm (C), corresponding to 2 liposome layers and 2-3 bilayers respectively. (The insets show the final separation distances in more detail, on a linear scale.) (B) and (D) are tapping-mode AFM scans of surfaces after the SFB experiments, revealing a liposome coated and bilayer+monolayer coated surfaces, respectively, in agreement with the SFB data (see text). A cross-section of the surface in (D).

We further examined the shear forces between the DSPC-cholesterol layers. Typical shear trace results are shown inset to fig. 2B. Friction forces Fs as a function of applied loads Fn between two DSPC coated mica surfaces with varying cholesterol percentage are shown in figure 2 as indicated. Friction coefficients µ = (Fs/Fn) at the extrema data points indicated, and maximal pressures at these points prior to rigid coupling of the surfaces are given. In the 10% cholesterol system larger variation is observed compared to the 40% system. The reason for this is may lie in the larger heterogeneity of the surface layers, as can be seen in the AFM micrographs of figure 1: loose SUV over-layers may be abundant in some contact areas more than in others and such overlayers would increase the friction coefficient due to increased viscoelastic dissipation15 (A)

(B)

∆x= 874 nm

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Figure 2: Friction forces Fs as a function of applied loads Fn between two DSPC SUV/bilayer coated mica surfaces with varying cholesterol percentage: 10% in (A) and 40% in (B). Full symbols- first approaches, empty symbols- second approaches at corresponding contact positions. Friction coefficients and maximal achieved pressures appear in the plots. Inset in (B) are typical traces from which the data is extracted: the top trace is the back and forth applied lateral motion, while the lower traces are the sliding friction transmitted between the surfaces at the compressions shown.


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A summary of all the data ranges, in comparison to previously studied cholesterol-free DSPC liposomes17 and bilayers16 is given in figure 3. The range of friction coefficients for all cholesterol containing systems, as well as for the DSPC bilayer system, is similar, while low cholesterol-content systems are able to withstand higher pressures: maximal pressures of ~100 atm were observed for the DSPC 10% cholesterol system, while for the 40% cholesterol system the maximal pressure at which there was good lubrication is ~60 atm. The pristine DSPC liposomes, however, clearly have lower friction coefficients, at least one order of magnitude lower than all the other systems. Nonetheless, all the described systems provide efficient lubrication with low friction coefficients of 10-3-10-4. For detailed information on friction of DSPC the reader is referred to our previous work17.

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DSPC 10% Chol, µ=5x10-4-10-2 DSPC 15% Chol, µ=1.2-5.4x10-3 DSPC 40% Chol, µ=1-4x10-3 DSPC SUVs, µ=7x10-5-1.5x10-4 DSPC BL, µ=2-8x10-3

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Figure 3: Range of friction coefficients for different boundary lubrication systems.

It is clearly seen that an increase in the cholesterol content from 10% to 40% reduces the robustness of the boundary lubrication of the system, as the surface layers at 40%-chol stop providing efficient lubrication at lower loads (and thus pressures) compared to the 10% or 15% cholesterol-content systems. Indeed the lubrication behavior is very similar to the


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previously studied DSPC BL (bilayer) system16. We attribute this to reduced mechanical strength and robustness arising from the increase in cholesterol content (seen directly in the spontaneous rupture of vesicles to bilayers, fig. 1D); in a parallel nanomechanical AFM study we also found that cholesterol addition reduces the mechanical strength of DSPC bilayers (ref Salome to be published). We have previously observed that robustness of boundary lubricant layers in pure water conditions are one key factor in their high pressure lubrication performance17, and that strong correlation exists between nanomechanical performance in the AFM and stability and lubrication in the SFB for PC systems studied in pure water

16

. Therefore, we believe that cholesterol addition reduces strength and

robustness of DSPC bilayers and hence impairs their lubrication performance. Another important effect of cholesterol addition is that it introduces defects to the bilayer. We have previously observed that increase in density of defects also decreases the efficiency of lubrication16, and we believe that a similar principle applies in the cholesterol case. Moreover, cholesterol associated ‘defects’ (domain boundaries) are known to be associated with membrane fusion42, which is in line with our results for the DMPC and DPPC 40% cholesterol systems, as described below.

2. Liposomes of varying acyl chain length with 40% Cholesterol measured in liposome dispersions

Normal and shear forces across SUV dispersions of DMPC, DPPC and DSPC, each with 40% cholesterol content were measured, to further examine the effect of cholesterol on PC SUV lubrication in conditions that are more physiologically relevant, as there is ample evidence that different physiological fluids

43, 44, 45

, including the synovial fluid (SF)

46

,

contain lipids or lipid vesicles. It is, therefore, possible that the presence of lipids including cholesterol in the surrounding SF is contributing to the stability of cartilage-attached lipid layers, and thus may play a role in their function as boundary lubricants14, 15. This part is a natural continuation of our previous study on lubrication by DMPC, DPPC and DSPC SUVs18 in dispersions of the respective liposomes, where new insights into the effect of lipids in the reservoir on lubrication as well as membrane fusion were obtained,

as

elaborated later in the next.

Figure 4 shows normal forces Fn(D)/R as a function of separation D between two mica surfaces coated with SUVs of (A) DMPC (B) DPPC (C) DSPC, each with 40% cholesterol, across the corresponding SUV dispersions at 0.3 mM. For the DMPC system, Fn(D)


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increases roughly exponentially with decreasing D at large separations, and more sharply on closer approach where steric interactions between opposing bilayers presumably set on. Upon further compression, a sudden jump in occurs from 7.5±1 nm to 3.5±1nm, roughly corresponding to a change from two bilayers to one, as can be seen in the inset of figure 4 (A) (the thickness of one DMPC bilayer was reported47 to be 3.5+0.1nm). This is attributed to hemi-fusion of the bilayers (with the expulsion of a bilayer). Subsequent approaches to contact are similar to the first approaches: hemifusion occurs again on approach, as apparent from the change in distance. From preliminary measurements of jumps-out upon separation of the compressed surfaces, we could estimate from the JKR relation40 an interfacial energy of ≈4 mJ/m2, slightly lower than for the pure DMPC system17. For the DPPC system, similar behavior is observed, also with a jump into contact, from 8±1nm to 4±1nm, as seen in the inset to figure 4(B). For the DSPC system the final separation is 11±2.5, and no hemifusion events with transition from two bilayers into one are observed. We do however observe a transition from ~20nm to ~10 nm in the first approach profiles, corresponding, most likely, to rupture of the closed vesicle structures and squeezing out of two excess lipid bilayers which are not immediately attached to the mica surface. The second approach profiles for DSPC are characterized by onset of steric repulsion at bigger separations compared to first approaches (possibly due to rearrangement of the surface layers), however the final distances are similar. The DSPC system is reversible and reproducible in terms of the friction behavior, as will be shown next.

From the normal profiles for the 40% cholesterol containing liposomes, we learn that hemifusion of two bilayes into one occurs in the DMPC-40% cholesterol and DPPC-40% cholesterol systems, as described above. This is a very interesting observation, not observed for the same systems in the absence of cholesterol18. When measurements are performed across pristine SUV dispersions of the same lipids (DMPC and DPPC), the final separations in the force profiles (at similar or higher pressures) correspond to two bilayers, and no hemifusion is observed18. This is a direct demonstration of the contribution of the added cholesterol to bilayer hemifusion. The importance of cholesterol in membrane fusion events, e.g. during virus entry into cells, has been previously described 48, 49, 50, 51, 52, 53, 54, 55. Possibly, cholesterol introduces packing defects, and thus promotes hemifusion, as fusion occurs as a result of mutual membrane proximity and exposure of hydrophobic regions 56.


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10000

Fn/R (mN/m)

(A)

Fn/R (mN/m)

1000 100

4000 3000 2000 1000

10

0 0

2

1

4 6 8 D (nm)

10 12

0.1 0.01 0

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(B)

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Fn /R (mN/m)

1000 Fn/R (mN/m)

100 D (nm)

10000 8000 6000 4000 2000 0

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(C) 10000 Fn/R (mN/m)

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1000 100 10

12000 10000 8000 6000 4000 2000 0 -2000 0

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5 10 15 20 25 30 35 40 D (nm)

1 0.1 0.01 0

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200 300 D (nm)

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Figure 4 : Force Fn(D) versus distance D profiles between (A) DMPC (B) DPPC (C) DSPC, each with 40% Cholesterol, SUV coated mica surfaces across the corresponding SUV dispersions (0.3 mM) normalized as


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Fn(D)/R. The zero separation is defined with respect to air calibration. Filled symbols indicate first approach to contact and empty symbols are the corresponding second approach. Insets show the final separation distances and jumps-in for DMPC and DPPC on an expanded scale. For DMPC: Jump in from 7.5±1 to 3.5±1. For DPPC: Jump in from 8±1 to 4±1 DSPC: final distance 11±2.5. Red arrows mark jump from ~20nm to ~10 nm, which may result from rupture of the closed vesicles structures and squeezing out of two excess lipid bilayers which are not immediately attached to the mica surface.

Shear forces and friction coefficients

Typical shear profiles are shown in figure 5 for (A) DMPC 40% cholesterol, (B) DPPC 40% cholesterol and (C) DSPC 40% cholesterol. A schematic drawing of the hemifusion process observed as jumps in DMPC and DPPC systems is depicted in figure 5 (D). All measurements above were performed in SUV dispersions. For the DMPC 40% cholesterol system it can be seen that until pressures as high as 70 atm, very low friction forces are recorded. There is a trend of a slight increase in the friction force (see figure 5(A)), however the forces are very low. Then, there is a sudden jump-in from 7.5±1 nm to 3.5±1nm, corresponding to a change from two bilayers to one (See schematic depiction of this in figure 5(D)). This jump-in is accompanied by an abrupt increase in friction force leading to rigid coupling of the surfaces (figure 5(A)). Similarly, for the DPPC case very low friction force, close to the noise level, is recorded until pressures as high as 80 atm (figure 5(B)). Then upon further slight increase in pressure, a sudden and dramatic increase in the friction force is observed (one order of magnitude increase in the friction force.) This is accompanied by a jump in from 9 nm to 4.5 nm, corresponding to a change from two bilayers to one. (The average between the profiles gives values of jump in from 8±1nm to 4±1nm. See schematic depiction of this in figure 5(D)). At this point the surfaces still slide past each other, however upon further compression there is no more sliding and the surfaces are rigidly coupled (under the given experimental conditions.) The behavior of the DSPC-40% Cholesterol system is rather different; the friction force increases gradually and no jumps- in and rearrangement of the layers occurs (figure 5 (C)). Another important difference between the three systems is in the friction behavior upon subsequent approaches to contact; for the DMPC system, subsequent approaches to contact are very similar to first approaches, and are also characterized by very low friction coefficients. For the DPPC system, good lubrication was observed for subsequent approached in ~30% of the cases. This is in line with our previous observation that healing


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Langmuir

of DPPC surface layers is less efficient compared with DMPC18. As seen in the normal profiles in figure 4(B), subsequent approaches to contact of the profiles marked by open triangles and open pentagons are characterized by bigger final separations, ~30nm instead of ~4nm, implying debris formation of irregular material as a result of shear. Accordingly with this, the friction forces for subsequent approaches were high. For the DPSC system, the behavior was reversible and reproducible, with low friction coefficients in all cases for both first and subsequent approaches to contact.

(A)

(B) ∆x= 870nm

1 sec

1 sec

∆x= 620nm

Fs < 1.5 µN, D= 8 + 1 nm, P= 26 atm

Fs=3.5 µN, D= 10 + 1 nm, P= 22 atm

Fs= 2µN, D=8 + 1 nm, P= 54atm Fs= 3.9 µN, D= 9+ 1 nm, P=80 atm

40µN

Fs= 3.3 µN, D=8 + 1 nm, P= 61atm

40µN Fs= 31 µN, D=4.5 + 1 nm, P=83 atm

D= 4.5+ 1nm, P= 87 atm

D=4 + 1 nm, P= 68 atm

(C)

(D)

∆x= 775nm

1 sec

a

(D)

Fs= 8 µN, D= 30 + 1 nm, P= 33 atm Fs= 11 µN, D= 21+ 1 nm, P=40 atm 40µN Fs= 14 µN, D=12.5 + 1 nm, P=52 atm

b

Fs= 16 µN, D= 10 + 1nm, P= 62 atm

c Fs= 23.5 µN, D= 10+1 nm, P=76 atm

Figure 5: Typical shear traces for (A) DMPC-40% Cholesterol SUVs (B) DPPC-40% Cholesterol SUVs and (C) DSPC-40% Cholesterol SUVs, with SUVs in dispersion at 0.26 mM concentration (which is more than an order of magnitude higher than the CMC concentration of all the lipids studied36). (D) Schematic representation of the transition from two bilayers to one as observed in (A) and (B): initially, the two lenses


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are covered by one bilayer each (a) . Next, the upper surface is pressed towards the lower surface and a separation corresponding to two bilayers is observed (b). At this stage the surfaces still slide past each other with very low µ. Following further compression, a jump is observed to a distance corresponding to one bilayer (as two monolayers are squeezed out), at which point the surfaces are coupled at the experimental conditions and no sliding is observed.

Friction forces Fs as a function of applied loads Fn for the three different systems are given in figure 6. For DMPC 40% cholesterol measured in the SUV dispersion, friction coefficients of 6x10-5- 1.3x10-4 were obtained. The friction force increases gradually with the load, but remains very low, below 5 µN. This is the case for both first and subsequent approaches to contact, indicating that healing of the layers occurred after separation. For DPPC 40% cholesterol measured in the SUV dispersion, friction force is within the noise level up until pressures as high as 80 atm, with friction coefficients µ

Effect of Cholesterol on the Stability and Lubrication Efficiency of

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