Surface-Active Lipid Linings under Shear LoadâA ... - ACS Publications

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Surface Active Lipid Linings under Shear Load - A Combined In-situ Neutron Reflectivity and ATR-FTIR Study Felicitas Schwörer, Marcus Trapp, Matthias M Ballauff, Reiner Dahint, and Roland Steitz Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01678 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on October 7, 2015

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Surface Active Lipid Linings under Shear Load - A Combined In-situ Neutron Reflectivity and ATR-FTIR Study

Felicitas Schwörer#, Marcus Trapp$, Matthias Ballauff$, Reiner Dahint# and Roland Steitz$,*

#

Ruprecht-Karls-Universität Heidelberg, Applied Physical Chemistry, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany

$

Helmholtz-Zentrum Berlin, Institute for Soft Matter and Functional Materials, Hahn-MeitnerPlatz 1, 14109 Berlin, Germany

*Corresponding author

Abstract: We study shear effects in solid-supported lipid membrane stacks by simultaneous combined in-situ neutron reflectivity (NR) and attenuated total reflection Fourier transform


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infrared spectroscopy (ATR-FTIR). The stacks mimic the terminal surface active lipid coatings (SAPLs) on cartilage in mammalian joints. Piles of 11 bilayer membranes of 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) are immobilized at the interface of the solid silicon support and the liquid D2O backing phase. We replace the natural hyaluronic acid (HA) component of synovial fluid by a synthetic substitute, namely poly(allylamine hydrochloride) (PAH) at identical concentration. We find the oligolamellar DMPC bilayer films strongly interacting with PAH resulting in a drastic increase of the membranes d-spacing (by a factor of ~5). Onset of shear causes a buckling-like deformation of the DMPC bilayers perpendicular to the applied shear field. With increasing shear rate we observe substantially enhanced water fractions in the membrane slabs which we attribute to increasing fragmentation caused by Kelvin-Helmholtz-like instabilities parallel to the applied shear field. Both effects are in line with recent theoretical predictions on shear-induced instabilities of lipid bilayer membranes in water [I. Hanasaki, J. H. Walther, S. Kawano, and P. Koumoutsakos, Physical Review E 82, 051602 (2010)]. With the applied shear the interfacial lipid linings transform from their gel state Pβ’ to their fluid state Lα. Although in chain-molten state with reduced bending rigidity the lipid layers do not detach from their solid support. We hold steric bridging of the fragmented lipid bilayer membranes by PAH molecules responsible for the unexpected mechanical stability of the DMPC linings.

KEYWORDS neutron reflectivity, ATR-FTIR, interface, lipids, shear


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1. INTRODUCTION Shear forces naturally occurring in the joints of humans and other mammalians can have severe impact on their health. In combination with increased friction in the joint(s) they may cause osteoarthritis (OA) with clinical symptoms ranging from joint pain to the occurrence of local inflammation and bruising. OA is one of the most common joint diseases and a leading cause of chronic disability.1 In a mammalian joint the opposing bone ends are covered by a thin layer of cartilage, which in turn is decorated with an even thinner, oligolamellar film of surface active phospholipid (SAPL) bilayers.2 The articulating surfaces operate in a closed compartment, the socalled synovial membrane, which is filled with the synovial fluid. Under progressive wear conditions the terminal lipid layers may be damaged, the articular cartilage degenerated and the bone ends altered, resulting in OA.3 Various clinical studies have shown that the synovial fluid of patients suffering from OA contains HA at reduced concentration and molecular weight.4, 5 HA is a major component of the joint fluid and held responsible for friction-diminishing and protective properties of the lubricating fluid. It is believed that HA, adsorbed to the lipid-coated cartilage, forms a protective layer against wear under operational conditions of the joint.6 Manual therapy and the administration of analgesic drugs are first-line treatments of OA.7 Besides these primary options local joint injections of HA or mixtures of HA and SAPLs are applied as a second or thirdline cure in a procedure called viscosupplementation (VS).8, 9 VS leads to temporary pain relief and increased joint mobility which typically lasts over a period of three months.8 It is claimed that VS improves the lubrication properties of the synovial fluid.8 The underlying mechanisms are not known in detail and the effectiveness of VS is subject to current medical research.10 The SAPLs that are dissolved in the synovial fluid and further cover the articular cartilage are assumed to play a key role in reducing friction. Simultaneous intra-articular injection of HA and phospholipids showed better protection against joint degeneration than intra-articular injection of


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HA alone.11 The finding supports earlier results by Hills and Gale et al., who attributed a significant role in joint lubrication to the terminal, cartilage decorating lipid layers.2, 12 Mechanisms of joint lubrication have been under heavy debate over the past 50 years. Respective models deal with hydrodynamic and elastohydrodynamic, weeping, boosted, boundary and biphasic boundary lubrication.13-17 All models have in common that the individual components of the synovial fluid in a joint are supposed to work in a synergistic manner. Zooming in on the cartilage level the respective boundary lubricant is claimed to consist of the surface-active phospholipids and/or of macromolecular components of the synovial fluid.2, 11, 18-28 For a very simplified model system comprising solid-supported oligolamellar stacks of 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer membranes against water and solutions of HA in water we observed a tremendous swelling (by a factor of ~4) and a stabilization of the lipid linings upon incubation with HA at 39 °C, i.e. well above the main phase transition temperature, 𝑇𝑚 , of the lipids. By in-situ neutron reflectivity (NR) measurements we followed the kinetics of swelling and registered equilibrium after 43 days.29 The on-board ATR-FTIR (attenuated total reflection Fourier transform infrared spectroscopy) set-up allowed us to investigate in parallel the phase state of the swollen DMPC linings.30 The individual lipid membranes of the swollen hydrogel coatings that were formed by interaction with HA changed from their gel to their fluid state upon increasing temperature, but surprisingly did not detach from their solid support as was observed for the pure reference system, i.e. solid-supported oligolamellar DMPC bilayers in contact with pure water.31 The apparent question arising in the context of joint lubrication and protection is on the mechanical stability and respective alterations of the DMPC hydrogel coating. For the very reason we developed a dedicated sample cell for combined in-situ NR and ATR-FTIR measurements


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under shear load. With this sample cell we investigated oligolamellar stacks of DMPC bilayer membranes on silicon support against an aqueous solution of a synthetic polyelectrolyte, namely poly(allylamine hydrochloride) (PAH), in D2O as a function of applied shear rate from 0 to 25714 s-1, i.e. in the range relevant to physiological conditions32, 33. The reason for substituting HA (𝑀𝑤 = 769 kDa) by PAH (𝑀𝑤 = 15 kDa) was to accelerate the kinetics of hydrogel layer formation at the solid-liquid interface. 2. EXPERIMENTAL SECTION 2.1. Overall features of the shear cell The home-made shear apparatus is based on a motor-driven rotating cone mounted on a shaft and brought in close contact with the substrate under study. Both a sketch of the set-up and design drawings of the unit are shown in Fig. 1. To provide a constant shear rate across the sample, the distance between the cone and the substrate increases linearly with increasing distance from the rotation center. In this way, a potential enhancement of shear due to the radially increasing rotational speed is compensated by the larger distance between surface and rotor. The rotor is housed in a cylindrical liquid cell with its shaft sealed against the environment by lock-rings. Rotor and housing are made from aluminum. The diameter of the rotating cone is 43 mm and its opening angle 177.2°. A Si block, 60 mm in diameter and 10 mm thick, is deposited on the gasket (O-ring, Viton, 50 mm inner diameter) of the housing and tightly screwed against the sealing by the use of a hollow cylindrical aluminum block to form the cover lid of the liquid cell. The distance between the tip of the rotor and the substrate is 0.5 mm. Note that Si is transparent for both neutrons in the selected wavelength regime (1.8 – 12.2 Å) and IR radiation from about 1500 – 6000 cm-1, thus allowing for simultaneous measurements as detailed below. A closed water circuit running through the outer


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shell of the liquid cell and the interior of the cylindrical aluminum block, mounted on top of the Si substrate, is connected to an external water bath to adjust temperature between 5 and 60 °C with an accuracy of 0.1 °C. Temperature is monitored by a Pt100 thermistor located in the immediate vicinity of the sample. An AC servo motor (DSD 036 S44 U 60-31 356/0750, Baumüller, Nürnberg, Germany) is used to drive the rotor. Its speed may be varied between 0 and 6000 rpm with a maximum torque of 1.2 Nm at the lowest and 0.9 Nm at the highest velocity. According to the specifications of the supplier, speed droop is 4% at 6 rpm and 1% for velocities higher than 24 rpm. A torque sensor (DRFL-I1, ETH

a)

b)

c)


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Fig. 1: a) Schematic view of the shear cell. The rotating cone exerts shear stress on the immobilized lipid layer. IR analysis is performed in an ATR configuration with total reflection at the siliconliquid interface. The neutron beam (green rectangle) is perpendicular to the IR beam and reflected at the same interface at shallow angle. b) Photo of the shear cell in operation at BioRef. c) Design drawing of the shear cell.

Messtechnik, Gschwend, Germany) with a measurement range up to 1 Nm and a maximum error of 0.001 Nm is mounted in between the shafts of the servo motor and the rotating cone. By this means, the exerted shear stress  = 3𝑀⁄(2𝜋𝑅 3 ) may be monitored. M denotes the measured torque and R the radius of the cone. According to 𝛾̇ = 2𝜋𝑛/𝜃 with 𝑛 = 𝑟𝑝𝑚/60 in 1/s and 𝜃 = (180°-177.2°)/2pi/180, the cone angle in radians, the applicable shear rates may be varied between 0 and 2.57104 s-1. In order to minimize mechanical vibration at the sample position and to compensate for potential minimal misalignment, motor, torque sensor and rotor are connected via elastomeric clutches. A special feature of the shear cell is that simultaneous NR and ATR-FTIR measurements may be performed to provide detailed information on both the structure and molecular conformation of substrate-bound films under applied mechanical stress. For this purpose, Si substrates with polished side planes cut at an angle of 45° were used. The beveled side planes served as entrance and exit windows for the IR beam. For the chosen dimensions of the crystal, the IR beam was 3 times totally internally reflected at the substrate-liquid interface before leaving the substrate. The beam diameter was set to 4 mm. Attenuation of the IR beam may be further reduced by diminishing the thickness of the Si blocks. This will also increase the number of internal reflections and thus the sensitivity of the measurement. Neutrons entered the Si substrate at 90° with respect to the direction of the IR beam and were reflected at the substrateliquid interface as well.


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2.2. Sample preparation Disc-shaped silicon substrates with polished front and back sides (60 mm radius, 10 mm thickness, rms roughness < 0.6 nm) were supplied by Siliciumbearbeitung Holm (Tann/Ndb., Germany) and Sil’tronix (Archamps, France). For combined, simultaneous measurements of NR and ATR-FTIR, Si substrates with 45° cut polished side planes were used. DMPC was purchased from Avanti Polar Lipids, Inc., Alabaster, Alabama, US. D2O (purity = 99.9 atom % D) and PAH (𝑀𝑤 =15 kDa in case of samples S3, P4 and 58 kDa in case of samples S1, P3) were from Sigma-Aldrich. All chemicals were used as received. The substrates were rinsed with chloroform (Merck, Uvasol® grade), cleaned for 1 hour in an ethanol bath (absolute puriss. p.a., Sigma-Aldrich) and subsequently washed with ethanol and chloroform. Oligolamellar lipid films were prepared as first described by Mennicke and Salditt on the pre-cleaned silicon discs by spin-coating from 10 mg/mL lipid solutions in chloroform at a speed of 500 rpm for 1 sec followed by a speed of 4000 rpm for 60 sec, using a spin-coater (Model 6708D, SCS, US).34 Prior to the neutron experiments the dry samples were characterized by X-ray reflectometry (XRR) utilizing a home-built set-up (see also Fig. 2).35

100 10-1

X-ray reflectivity

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10-2 10-3 10-4 10-5 10-6 0.00

0.04

0.08

0.12

0.16

0.20

Q (Å-1)

Fig. 2: X-ray reflectivity of sample P4 against air. From the position of the first order Bragg peak, the lamellar 𝒅-spacing and from the positions of the Kiessig oscillations the overall thickness 𝑫


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of the coating were deduced: 𝒅 = 𝟐𝝅/𝑸𝑩 = 𝟐𝝅/(𝟎. 𝟏𝟏𝟔𝟎 ± 𝟎. 𝟎𝟎𝟎𝟑) Å−𝟏 = (54.2 ± 0.1) Å and 𝑫 = 𝟐𝝅/𝑸𝑲 = 𝟐𝝅/((𝟎. 𝟏𝟎𝟑 − 𝟎. 𝟎𝟗𝟑) ± 𝟎. 𝟎𝟎𝟎𝟑) Å−𝟏 = (628 ± 19) Å. The ratio 𝑫/𝒅 gives the total number 𝑵 of bilayers and is 11.6 ± 0.4 for sample P4.

The viscosities of the PAH/D2O solutions utilized in this work were measured with a commercial Anton-Paar MCR301 rheometer (Graz, Austria) in concentric cylinder geometry. The values are listed in Table 1. Table 1: Viscosity 𝜂 ± Δ𝜂 of PAH/D2O solutions (3 mg/mL) at 20°C PAH+D2O

15 kDa

58 kDa

shear rate [1/s]

𝜂 [Pa.s]

1.76E-03

3.56E-03

10-316

Δ𝜂 [Pa.s]

0.01E-03

0.18E-03

10-316

2.3. Neutron reflectivity and ATR-FTIR NR measurements were performed at the time-of-flight reflectometer BioRef at the HelmholtzZentrum Berlin (HZB).30 A chopper speed of 25 Hz allowed to cover a scattering vector 𝑄 range from 0.009 to 0.227 Å-1 with two angular settings ( = 0.5° and 1.9°, respectively). 𝑄 is dependent on wavelength 𝜆 and scattering angle 𝜃 via 𝑄 = 4𝜋 𝑠𝑖𝑛(𝜃)/𝜆. A constant wavelength resolution

/ was achieved by operating the frame defining choppers in “optical blind” mode.36 In the experiments described here / was set to 7% for samples P2, P3, S1 and S3 and 1% for sample P4 with the angular resolution / adjusted accordingly. The 𝑄 resolution, 𝑄/𝑄, set on the instrument was thus 9.9% for samples P2, P3, S1, and 1.4% for P4 and S3. The reflected neutrons were recorded in /2 geometry by a position sensitive area detector (PSD) with active area of 300 x 300 mm² filled with 3He. Typical measuring times were 6 h per shear rate and run. The


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neutron footprint on the sample was adjusted by diaphragms in the incident beam for each angle such as to avoid over-illumination of the sample and was 64 x 36 mm² ( = 0.5°) and 48 x 36 mm² ( = 1.9°), respectively. A Bruker Vertex 70 FT-IR spectrometer with DTGS detector was mounted at the sample position of the instrument in order to allow for simultaneous recording of ATR-FTIR spectra.30 128 scans were summed up for each shear rate. The spectral resolution of the spectrometer was set to 4 cm-1. 2.4. Data reduction and analysis Neutron data reduction and normalization was performed using an in-house developed software suite available at BioRef. The raw data were normalized to the direct beam and binned with a constant step width of 9.9% for P2, P3, S1, and 1.4% for sample P4 and S3. For data fitting we used the program suite Motofit.37 The employed model was based on the one used by Kreuzer et al. and consisted of one DMPC bilayer adsorbed (a) to the silicon substrate, a stack of 𝑁 − 2 lipid bilayers forming the core (c) of the oligolamellar lining and one outer bilayer (o) exposed to the liquid backing phase.29 Each bilayer was at maximum described by one hydrophobic tail region and two individual hydrophilic head regions on either side of the tail region. The bilayers are separated by interstitial aqueous layers. The total number 𝑁 of bilayers was extracted from XRR characterization of the dry samples. 3. RESULTS Fig. 3 displays the NR curves and IR spectra of the solid-supported oligolamellar DMPC linings against D2O (P2) or against a solution of 3 mg/mL PAH in D2O (P3) at 20°C as a function of applied shear load. The NR curves of the reference system P2 against pure D2O (Fig. 3a), although recorded in the limited 𝑄-range 0.06-0.15 Å-1 and at low resolution (∆𝜆⁄𝜆 = 7%), cover already


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the main response. Starting from a system with a lamellar spacing of 𝑑 = 2𝜋⁄𝑄𝐵 = 2𝜋⁄0.1113 Å−1 = 56 ± 2 Å at rest (0 s-1) the lipid lining equilibrated after turning on shear to a lamellar spacing of 65 ± 2 Å at 129 s-1. Increasing shear load induced a reduction in Bragg peak intensity at fixed position in 𝑄. Raising shear rate from 129 s-1 to 257 s-1 lowered the peak intensity by a factor of 2. The DMPC lining P3 incubated with PAH in D2O behaved differently. At first, in the recorded 𝑄-window of the NR curve (0.03-0.10 Å-1; ∆𝜆⁄𝜆 = 7%) there are 3 pronounced Bragg peaks rather than 1 (Fig. 3b). According to 𝑄𝑛 = (2𝜋⁄𝑑 ) ∙ 𝑛 , where 𝑄𝑛 is the position of the 𝑛-th Bragg peak, we extracted a lamellar spacing 𝑑 of 314 ± 5 Å at rest. Hence, upon interaction with PAH the spacing between the individual DMPC bilayers increased by a factor of ~5. Applying shear to this system reduced the Bragg peak intensities but did not shift their positions any further. In contrast to the reference system P2, the PAH-swollen DMPC lining P3 did not show any alteration at low shear (129 s-1). A 4-fold decrease in intensity of the first Bragg peak at 0.043 Å-1 was observed upon increasing shear from 257 to 2571 s-1. From linear interpolation a reduction of Bragg peak intensity by a factor of 2 was reached at a shear rate of 1029 s-1 (not measured). Hence, interaction with PAH did not only swell the lipid coating but also increased its mechanical stability by a factor of 1029/257 = 4 with respect to applied shear. Similar observations were made in the simultaneously recorded IR-spectra in the wavenumber range 2800-3100 cm-1 (Fig. 3c, d). For both, the symmetric and asymmetric CH2-stretching vibration at 𝜈𝑠 ~2850 cm-1 and 𝜈𝑎𝑠 ~2921 cm-1, respectively, the loss in IR absorbance sets in earlier and was faster for the reference system (compare Fig. 3c with Fig. 3d). In addition, the absorbance peak positions shifted by ∆𝜈𝑠 ~2 cm-1 and ∆𝜈𝑎𝑠 ~3-4 cm-1, respectively, for an increase in shear rate from 0 to 12857 s-1 for both P2 and P3.


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Figure 4 depicts a more extended investigation on system P4 consisting of a stack of 11 DMPC bilayers on silicon support incubated with 3 mg/mL PAH in D2O at 20 °C. The neutron reflectivity of P4 as a function of shear load is gathered in Fig. 4a. The sample equilibrated 3-7 hours after changing shear. Subsequent runs at constant shear rate did not show further alterations in reflectivity. At rest (0 s-1) the reflectivity pattern displays four well-pronounced Bragg peaks from which the lamellar spacing 𝑑 of the repeat unit (bilayer membrane + interstitial water layer) was extracted to (288 ± 4) Å. a)

b) 0 s-1 129 s-1 257 s-1 2571 s-1 12857 s-1

Neutron Reflectivity


10-2

10-3

10

10-3

10-4

-4

0.06

0.08

0.10

0.12

0 s-1 129 s-1 257 s-1 2571 s-1 6429 s-1 12857 s-1

10-2

D2O

PAH/D2O

0.14

0.04

Q [Å-1]

0.06

0.08

0.10

Q [Å-1]

d) 0 s-1 129 s-1 257 s-1 2571 s-1 12857 s-1

D2O -0.06

-0.08

-0.10 2800

2900

3000

0 s-1 129 s-1 257 s-1 2571 s-1 6429 s-1 12857 s-1

PAH/D2O

IR Absorbance [a.u.]

c)

IR Absorbance [a.u.]

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-0.16

-0.18

-0.20 2800

Wavenumber [cm-1]


2900

Wavenumber [cm-1]

3000

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Fig. 3: Neutron reflectivity curves (a, b), and ATR-FTIR spectra (c, d) of oligolamellar DMPC stacks immobilized at the solid-liquid interface against D2O (a, c) or against a solution of PAH (𝑴𝑾 = 58 kDa, 3 mg/mL) in D2O (b, d) at 20°C.

With increasing shear load the Bragg peak positions of the DMPC lining incubated with the solution of PAH in D2O moved outwards (i.e. to larger Q) and decreased in intensity. At the highest shear rate (25714 s-1) only the first and second order Bragg peaks remained detectable with significantly reduced intensity (Fig. 4a). The repeat spacing, 𝑑, of the DMPC-lining under maximum load (25714 s-1) shrank from (288 ± 4) to (249 ± 9) Å, i.e. to 86% of its original value. The solid lines in Fig. 4a are the best global fits to the experimental data based on a simplified model of the DMPC lining. This model consisted of the silicon fronting (through which the neutron beam assessed the solid-liquid interface), the native silicon oxide layer (box 1), the first adsorbed DMPC bilayer (box 2, headgroups and tail regions not resolved), the film core [2 boxes: interstitial aqueous layer (box 3) + DMPC bilayer (box 4)], repeated 𝑁 − 2 times, the outermost interstitial aqueous layer (box 5) and the outermost DMPC bilayer (box 6) facing the aqueous liquid backing in that order. Globally fixed parameters were the number of bilayers, 𝑁 = 11, from the analysis of the XRR pattern of the lipid lining against air, measured prior to the neutron experiment (see Fig. 2), the scattering length densities of silicon fronting, 𝑆𝐿𝐷𝑆𝑖 , and native SiO2 layer, 𝑆𝐿𝐷𝑆𝑖𝑂2 , the scattering length density of any given DMPC bilayer membrane (𝑏𝑙), 𝑆𝐿𝐷𝑏𝑙 , the scattering length density of interstitial water (𝑖𝑤), 𝑆𝐿𝐷𝑖𝑤 (including dissolved PAH), and the scattering length density of the aqueous backing phase (𝑤) 𝑆𝐿𝐷𝑤 (including dissolved PAH). Individual thicknesses, 𝑑𝑖 , and roughness, 𝜎𝑖 , were pre-determined from individual fits, averaged and globally fixed. Table 2 compiles the applied fitting strategy with all its settings.


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The applied fitting strategy with only three variable parameters at a time worked well (note, that 𝑑3 and 𝑑5 are presumed to be equal). There was excellent match of fits and experimental data throughout the shear range exerted on the lipid lining (see Fig. 4a). With the onset of shear (0  257 s-1) the thickness of the bilayer membranes changed from 60 to 78 Å (see Fig. 4b).

Table 2: Parameters and settings of global fitting to NR data Box-No.

Material

𝑑𝑖 [Å]

106 ∙ 𝑆𝐿𝐷𝑖 [Å-2]

𝜑𝑤

𝜎 [Å]

Repeated

Fronting

Si

-

2.07

0

2

1

1

SiO2

13

3.48

0

5

1

2

DMPC

49

0.3a)

0

13

1

v

5.72b)

1

13

DMPC

60c)

0.3

v

13

bilayer, bl

78d)

0.3

v

Interstitial

v

5.72

1

13

1

DMPC

60c)

0.3

v

13

1

bilayer, bl

78d)

0.3

v

Aqueous

-

5.72b)

1

13

1

bilayer, bl 3

Interstitial water, iw

4 5

9

water, iw 6 Backing

phase, w a)

𝑆𝐿𝐷𝐷𝑀𝑃𝐶 = ∑𝑖 𝑏𝑖 /𝑉𝑀 = 3.09610-4 Å/1043 Å3 38;

b)

the reduced 𝑆𝐿𝐷 of interstitial and bulk

water phase of 5.7210-6 Å-2 comes about by 𝜑𝑃𝐴𝐻 = 0.0017 and 𝜑𝐻2𝑂 = 0.094. H2O was brought in through cleaning of the shear cell prior to conducting the shear experiment. c) Applied at rest (0 s-1); d) applied under load (257 - 25714 s-1); 𝑑3 = 𝑑5 ; v = variable; 𝜑𝑤 = volume fraction of water (including PAH) in the respective layer. The 𝑆𝐿𝐷 of layer 𝑖 is given by 𝑆𝐿𝐷𝑖 = 𝜑𝑤 ∙ 𝑆𝐿𝐷𝑤 + (1 − 𝜑𝑤 ) ∙ 𝑆𝐿𝐷𝑏𝑙


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The bilayer thickness did not vary any further up to the highest experimental load. What did vary as a function of applied shear was the thickness of the interstitial water layers and the volume fraction of water within the lipid bilayers. While 𝑑𝑖𝑤 decreased moderately 𝜑𝑤 increased substantially (see Figs. 4b, d). The consequence was a drastic loss of contrast between the individual bilayer membranes and the interstitial water layers, concomitantly with a left shift of bilayers positions.

a)

b)

c)

d)

Fig. 4: Neutron reflectivity curves (a), together with best global fits (solid lines), extracted thickness of building blocks (b), underlying scattering length density profiles (c) and water content within the DMPC bilayers (d) of the solid-supported oligolamellar lipid hydrogel lining P4 (11 DMPC lamellae in total) interacting with PAH in water as a function of applied shear load. In (a) and (c) every second curve is omitted for clarity of presentation. In (d) yellow symbols denote the


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water content (𝝋𝒘,𝒄 ∙ 𝟏𝟎𝟎) in the core (c) bilayers (2 ≤ 𝑵 ≤ 10), blue symbols represent the water content (𝝋𝒘,𝒐 ∙ 𝟏𝟎𝟎) of the outermost (o) lipid bilayer (𝑵 = 11). Fitting was pursued on the basis of ∆𝑸⁄𝑸 = 𝟓% rather than ∆𝑸⁄𝑸 = 𝟏. 𝟒% as set by the instrument indicating undulations (waviness) of the lipid membranes on the micron-millimeter scale.

The extracted 𝑆𝐿𝐷 profiles across the solid-liquid interface as a function of depth 𝑧 are shown in Fig. 4c. It is the loss of contrast that caused the fading of Bragg peak intensities with applied shear. By increasing the water fraction within the bilayer membranes by a factor of ~2 the contrast decreased by a factor of ~2. Close inspection of Figs. 4c, d shows that at low to moderate shear load (0 – 17143 s-1) there was a difference in the water content of core and outer DMPC bilayer membranes, which vanished only at high shear rate (> 17143 s-1) reaching 𝜑𝑤 values of more than 90%. All scattering experiments but one were conducted with narrow diaphragms on the exit side to reduce the high scattering background and thus exclusively focusing on the specular reflectivity. A single experiment was performed with open slits. This experiment revealed additional offspecular scattering (see Fig. 5). The scattering map recorded at rest (Fig. 5a) shows diffuse scattering at high Q and Bragg-sheets intercepting the Bragg peaks in the specular line. The Bragg sheets indicate conformal fluctuations and/or correlated layer distortions in the lamellar stack of lipid membranes. With the onset of shear the Bragg sheets sharpen and the diffuse scattering increases, c.f. Fig. 5b, up to 514 s-1 (maximum) and then decreases again with higher load, see Fig. 5d. We have hints for the lipid layers not recovering from shear-induced alterations. Fig. 6 shows the neutron reflectivity of a DMPC coating (10 bilayers, incubated in 15 kDa PAH in D 2O at a concentration of 3 mg/mL) prior to shear and two weeks after cessation of shear (2571 s -1). From


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the drastic change of the recorded neutron reflectivity curves it is evident that the lipid lining did not survive the structural alterations introduced by the applied shear load. The lamellar structure was lost (no Bragg peaks after cessation of shear) and most of the lipid molecules were sheared off the interface (reduced reflectivity after shear) presumably as lipid vesicles and dispersed in the bulk aqueous phase.

a)

b)

c)

d) 6.8x104

sum intensity [cts]

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6.7x104

6.6x104

6.5x104

6.4x104 0

5000

10000

15000

20000

25000

30000

shear rate [1/s]

Fig. 5: Neutron scattering maps (raw data) from sample S1 (12.5 ± 0.5 DMPC bilayers on silicon support in dry state against air by XRR) incubated with a solution of 58 kDa PAH in D 2O at a concentration of 3 mg/mL as a function of applied shear, a) – c). The wavelength resolution in the NR experiment was 7%. The integrated offspecular scattering at high 𝑄 (i.e. inside the black frame surrounding low wavelength and high offspecular scattering angle 𝛼𝑓 , (a) – (c), increases with


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shear from 0 (a) to 514 s-1 (b) and then decreases again with higher load, (d). The specular line is centered at offspecular scattering angle 𝛼𝑓 = 0 and a function of wavelength only (indicated as blue dotted line in (b)). Note that the Bragg-sheets (streaks of negative slope intercepting the specular line at the Bragg peak positions) sharpen with shear, (a) and (b), and disappear completely at highest shear load, 25714 s-1 (c).

100


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before shear after shear

-1

10

10-2 10-3 10-4 10-5 0.00

0.05

0.10

0.15

0.20

-1

Q [Å ]

Fig. 6: Neutron reflectivity of a solid-supported stack of 10 DMPC bilayer membranes (sample S3) incubated with 15kDa PAH in D2O at a concentration of 3 mg/mL prior to shear load and 19 days after cessation of applied shear. In parallel with the neutron reflectivity investigation we examined the response of the DMPC lining to the applied shear by ATR-FTIR. These measurements gave respective information on the conformation of the lipid molecules in the bilayer membranes. Figure 7 shows the region of the symmetric and asymmetric stretching modes 𝜈𝑠 and 𝜈𝑎𝑠 of CH2-groups of the lipid tails as a function of shear load. The peak positions were extracted from Gaussian fits to the spectra and plotted versus the applied shear rate in Fig. 8. A clear shift to higher wavenumbers is observable for both symmetric as well as asymmetric CH2 stretching.


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-0.10

-0.12

2926 2924 -1

wave number [cm ]

0 1/s 257 1/s 1286 1/s 2571 1/s 3857 1/s 5143 1/s 10286 1/s 15429 1/s

-0.08

IR absorbance [a.u.]

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2922 2920 2854 2853 CH2 symmetric stretching

2852

-0.14 3000

CH2 asymmetric stretching

2851

2950

2900

2850

2800

0

5000

10000

15000

shear rate [1/s]

-1

wavenumber [cm ]

Fig. 7: ATR-FTIR spectra of the DMPC lining Fig. 8: Shift of the peak position of the CH2 P4 in the region of the symmetric and stretching modes as a function of shear rate. asymmetric stretching modes of CH2-groups of the aliphatic lipid tails as function of shear load.

With increasing shear also the IR absorbance changed (see Fig. 9). We recorded a signal loss of ~50% on crossing a shear rate of 12857 s-1, which did not change any further up to 15429 s-1 (the highest shear load for which the absorbance could safely be analyzed by Gaussian fitting).

Fig. 9: Integrated IR absorbance of asymmetric (asym) and symmetric (sym) CH2 stretching vibration and their sum as a function of applied shear load. Data are normalized on measured values at rest (0 s-1).


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4. DISCUSSION In this work we substituted hyaluronic acid (HA) of high molecular weight (𝑀𝑤 =769 kDa), negatively charged, by the synthetic polyelectrolyte PAH of low molecular weight (𝑀𝑤 =15 kDa or 58 kDa), positively charged. The effects of HA(-) and PAH(+) on the solid-supported, zwitterionic DMPC bilayer membranes are rather identical. As in the case of HA, we likewise observe a drastic swelling of the lipid lining by a factor of 4-5. This is to say that also upon interaction with PAH the lipid coating transforms into a heavily swollen hydrogel layer. As in the HA case, we hold electrostatic repulsion responsible for the increase in spacing of the lamellae.29 This repulsion is generated by charging up opposing bilayer membrane surfaces in the stack by PAH adsorbing to the headgroups. Our findings are consistent with a modified DLVO theory by Ohshima, Mitsui and Izumitani taking into account van der Waals-interaction of the lipid membranes, hydration interaction of the membranes and electrostatic interactions caused by ions (Ca2+ in their case) adsorbed to the membrane surfaces.39, 40 Their theory was able to reproduce experimental results and predicted static repeat distances up to 260 Å, which is well in the range we observe here. The somewhat larger spacing of 288 Å of the DMPC + PAH system at rest might be due to a higher number of excess surface charges brought about by the PAH ad-layers. Thickness/composition/resolution without shear. The lamellar lattice constant 𝑑 of multilamellar DMPC bilayer stackings in excess water at 20 °C is 65 Å for aligned and 66 Å for non-aligned systems.41, 42 These values match with the lamellar lattice constant of 65 Å of solidsupported oligolamellar DMPC linings against excess water (see also P2, Fig. 3).31 In all systems the underlying repeat unit consists of one bilayer membrane and one adjacent interlamellar (interstitial) water layer. The thickness of the hydrophobic tails slab of such an aligned DMPC membrane 𝑖 measures 32.4 Å at 21°C (i.e. in the gel state 𝑃ß′ ), the thickness of the water interlayer including the adjacent headgroups of membrane 𝑖 and 𝑖 + 1 reads as 32.1 Å.31 The water interlayer


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can be further subdivided into heads / water / heads slices with dimensions of approximately 10 / 10 / 10 Å with small adjustment on the thickness of lipid tails slab from 32.4 to 34 Å.29 Upon interaction with HA the water interlayer increases drastically from 10  142 Å with additional HA ad-layers, 22 Å in thickness, each with a volume fraction of 𝜑𝐻𝐴 = 0.23, decorating the lipid headgroups from the aqueous side. The lipid tails slab stays virtually unchanged (34  33 Å).29 A very similar situation is observed in the case of PAH interacting with DMPC membranes investigated here. Fits with high resolution discriminating interstitial water layer, PAH ad-layer, lipid heads and lipid tails reveal that the interstitial water layer (D2O + dissolved PAH at a volume fraction of 0.0013) increases from 10  204 Å. Additional PAH ad-layers, 24 Å in thickness, each with a volume fraction of PAH of 0.17, decorate the lipid headgroups from the aqueous side (see Fig. 10). Fits with this high resolution (and zero roughness!) were found identical to the fits of the simplified model (low resolution) described in chapter 3 and Table 2. The latter model discriminates DMPC bilayer membrane(s) (including tails, heads and PAH ad-layers) as one integrated slab from the water interlayer(s). PAH ad- and lipid headgroup layers are taken into account by smearing their 𝑆𝐿𝐷s in the corresponding 𝑧-regions, i.e. by introducing membrane “roughness” of 𝜎 = 13 Å at either side of the lipid membrane (see Fig. 10 and Table 2). The maximum momentum transfer 𝑄𝑚𝑎𝑥 realized in the experiment was 0.12 Å-1. The minimum directly resolved thickness in real space was thus 𝑑𝑚𝑖𝑛 = 2𝜋/𝑄𝑚𝑎𝑥 = 52 Å. It is therefore not surprising that fits with low resolution matched the experimental data as good as fits with high resolution and that for analyzing the experiment the model with low resolution (Table 2) was sufficient throughout.


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6

SLD (10-6 A-2)

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interlayer

interlayer

5

PAH heads

4

high res. low res.

3

tails 1450

1500

1550

1600

distance from interface (A)

Fig. 10: Detailed view on DMPC bilayer membrane number 6 (see also Fig. 4) analyzed by the high (black) and low (blue) resolution model, respectively. The low resolution model was utilized for retrieving the data gathered in Table 2. For further details see text. Shear/structure/conformation. On turning on shear, the integrated thickness of the bilayer membranes of sample P4 increased from 60 to 78 Å where it stayed constant up to the highest experimentally applied shear rate (2.57104 s-1) (see Fig. 4). We interpret this observation as a buckling-like deformation of the bilayer membranes in the direction perpendicular to the applied shear in concordance with recent coarse-grained molecular dynamics simulations of shear-induced instabilities of lipid bilayer membranes in water.43 With the buckling of the membranes, their xyintegrated thickness (z) increases from 𝑑𝑏𝑙,0 to 𝑑𝑏𝑙,𝑠 as does the water fraction in the respective slices (see Fig. 11). Along with the increasing water fraction with shear (see Fig. 4) the average surface charge density of the lipid membranes decreases due to lateral dilution. In consequence, the electrostatic repulsion reduces and the measured thickness of the interstitial water layer, 𝑑𝑖𝑤 , decreases (see Fig. 4). The increase in water fraction in the lipid bilayer membranes can be explained by localized Kelvin-Helmholtz-like instabilities (parallel to the applied shear) which lead to membrane rupture (see Fig. 11), as predicted by the MD-simulations of Hanasaki and coworkers.43 Approaches to fit the corresponding NR curves by the incoherent superposition of the


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reflectivities of fragmented lipid bilayer stacks (𝑅𝑆𝑖/𝑙𝑖𝑝𝑖𝑑 𝑠𝑡𝑎𝑐𝑘/𝑤𝑎𝑡𝑒𝑟 ) and plain, lipid free areas (𝑅𝑆𝑖/𝑤𝑎𝑡𝑒𝑟 ) as 𝑅𝑒𝑥𝑝 = (1 − 𝛼) ∙ 𝑅𝑆𝑖/𝑙𝑖𝑝𝑖𝑑 𝑠𝑡𝑎𝑐𝑘/𝑤𝑎𝑡𝑒𝑟 + 𝛼 ∙ 𝑅𝑆𝑖/𝑤𝑎𝑡𝑒𝑟 failed. Therefore, the lateral separation of bilayer patches (fragments) was smaller than the coherence length of the probing neutron beam, i.e. ≤ 67 µm.44 This was true up to highest experimental shear load. Also from fitting approaches with a reduced number of 𝑁 there was no statistically relevant indication of a loss of bilayer membranes with shear. The experimental reflectivity curve taken at 20571 s-1 could be fitted with 𝑁=8 instead of 𝑁=11 and the reflectivity curve at 25714 s-1 could be matched by applying 𝑁=7 instead of 𝑁=11 in the fitting model, but the improvement in terms of resulting χ2 deviation was marginal, 2 and 4% respectively. The wavelength of the shear induced undulations can be large with respect to the coherence length of the neutron beam. In this case the applied shear field induced increased waviness of the lipid membranes. It can, on the other hand, be smaller than the neutron coherence length. In that case the applied shear force increased the roughness of the lipid bilayers. Waviness in its strict sense would contribute a relaxed resolution ∆𝑄 to the experimentally recorded reflectivity pattern concomitant with an increased average lipid bilayer thickness and an increased water fraction within the bilayers. Roughness -on the other hand- would damp the experimentally recorded reflectivity pattern with 𝑄 while maintaining the bilayers thickness.45 We modified our global model (Table 2) to assess the effects of the applied shear field with respect to waviness and/or roughness: Applying the modified model, now with variable roughness, but bilayer thickness fixed at the value for zero shear, and with preset instrumental resolution, ∆𝑄⁄𝑄 = 1.4%, produced fits which were by a factor of 3.3 worse than those of our global model applied and described in Table 2. In addition we found no systematic variation of bilayer roughness


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with shear. On average the extracted rms-roughness of the lipid membranes was (14.2 ± 2.2) Å and independent of shear. Relaxing the resolution from the instrumentally adjusted value of ∆𝑄⁄𝑄 = 1.4% to ∆𝑄⁄𝑄 = 5%, i.e. introducing waviness into the model of variable roughness but fixed bilayer thickness resulted in fits that were by a factor of 1.2 worse than those of the global model gathered in Table 2. Although there was an increase in roughness from 12 to 15 Å with the onset of shear, there was no further systematic variation with shear. The average extracted rms-roughness from this model was (14.7 ± 1.8) Å. The best fits to the experimental data were achieved with the relaxed resolution, fixed rmsroughness, fixed global values for the lipid membranes thickness, 𝑑𝑏𝑙,0 , at zero shear and, 𝑑𝑏𝑙,𝑠 , at applied shear (cf. Table 2, Fig. 4b and Fig. 10) and the water fraction varied as a function of shear. We therefore conclude that the buckling of the membranes induces an increased waviness of the interface rather than an increased rms-roughness. As outlined in the Results section all experiments but one were conducted with narrow slits on the exit side of the neutron reflectometer to reduce the high scattering background. The single experiment performed with open slits gave hints for the rupture of the oligolamellar lipid membranes stack into bits smaller than the neutron coherence length. The off-specular signal from that experiment, displayed in Fig. 5, shows that the diffuse scattering increases with the imposed shear up to 514 s-1 (maximum) and then decreases again with higher load. We interpret this finding as a shear-induced improvement of the conformal fluctuations and/or correlated layer distortions in the lamellar stack of lipid membranes as visible in the sharpening of the Bragg sheets up to a shear load of 514 s-1, c.f. Fig. 5a and Fig. 5b. Higher shear load then results in the loss of crosslayer correlations (no Bragg sheets visible anymore at highest shear, c.f. Fig. 5c) and also in a loss


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of integrated diffuse scattering intensity 𝐼𝑑𝑖𝑓𝑓 at high 𝑄 (see Fig. 5d). The latter effects correlate with the irreversible loss of bilayer patches from the interface according to 𝐼𝑑𝑖𝑓𝑓 ∝ 𝑁 2 , where 𝑁 is the number of patches.46

In the end, it is the amount of lipid molecules (i.e. their volume fraction) within the individual DMPC bilayer membranes and thus the morphology of the membranes that changes with shear. By NR we measured a 50% increase in water fraction from 45% at 0 s-1 to 95% at 1.54104 s-1 (see Fig. 4). This is consistent with our ATR-FTIR measurements, where we detected a decrease in IR absorbance of the lipid bilayers stack by 50% with shear (see Fig. 9).

Fig. 11: Cartoon of the structural evolution of the lipid bilayer membranes with increasing shear rate from 0 to 25714 s-1. For further details see text.

The interfacial DMPC linings responded to shear by a shift of the CH2 stretching vibrations to higher wave numbers (see Fig. 8). The spectral position of the IR absorbance peaks is dependent on the phase state of the lipids and known to shift by about 2.5 cm-1 when changing from the gel state Pβ’ to the chain-molten, fluid state Lα above the main phase transition temperature 𝑇𝑚 of 24 °C.47 Upon application of shear we also observed a shift in peak position which is of the order of


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3-4 cm-1 although the interfacial DMPC lining is at constant temperature 𝑇 < 𝑇𝑚 . Hence, we conclude that our system shows a shear-induced transition, in line with theoretical predictions which claim shear-induced changes in molecular orientation and increasing membrane area.43, 48 Oligolamellar DMPC linings in their 𝐿𝛼 state were found to detach from their support, when in contact with pure water.29, 31 This behavior is also true for our reference system P2. The interfacial DMPC linings P3 and P4 incubated with PAH from the aqueous phase, although in 𝐿𝛼 state, turned out more stable against shear-induced abrasion. The only way to explain this observation is by postulating restoring forces acting on the fragmented DMPC bilayer membranes. Those forces could be installed by PAH macromolecules bridging and thus interconnecting individual DMPC bilayer membrane fragments across the lipid lining. In addition, the structural quality of the lipid linings seems important. In that respect P3 is a poor quality lining while P4 is a high quality lining, indicated by the fact that the width (FWHM) of the Bragg peaks of P3 is by a factor of 2 larger than that of P4. But, the mechanical stability of P4 (in terms of Bragg peak intensity loss) is by a factor of 2 less (compare Fig. 3b with Fig. 4a). The findings on sample P3 when compared with the observations on sample P4 point towards a non-negligible effect of the molecular weight of the PAH polymer, 58 vs. 15 kDa.

5. CONCLUSIONS With a newly developed shear cell for combined in-situ NR and ATR-FTIR measurements we investigated the response of solid-supported oligolamellar stacks of DMPC bilayer membranes against D2O and very dilute solutions of PAH in D2O to applied shear load. The system under study was meant to mimic the situation of terminal surface active lipid coatings (SAPLs) on cartilage in mammalian joints in a very simplified manner. The oligolamellar, zwitterionic DMPC


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bilayer films interacted strongly with the positively charged PAH resulting in a drastic increase of the d-spacing (by a factor of ~5) of the lipid bilayers. Onset of shear caused a buckling-like deformation of the DMPC membranes perpendicular to the applied shear field. With increasing shear rate we observed substantially enhanced water fractions in the membrane slabs which we attribute to increasing fragmentation caused by Kelvin-Helmholtz-like instabilities parallel to the applied shear field. With shear the interfacial lipid linings transformed from their gel state Pβ’ to their fluid state Lα. Although in chain-molten state with reduced bending rigidity the lipid layers did not detach from their solid support. We hold steric bridging of the fragmented lipid bilayer membranes by PAH molecules responsible for the unexpected mechanical stability of the DMPC lining. The latter is four times higher than the stability of the reference system against plain D2O.

6. ACKNOWLEDGEMENTS We thank the Helmholtz-Zentrum Berlin for providing beamtime and financial support. This research project has further been supported by the German Ministry for Education and Science (BMBF) through contract no. 05KN7VH2.

REFERENCES 1. 2. 3. 4.

Choong, P. F.; Dowsey, M. M., The Grand Challenge – Managing End-Staged Joint Osteoarthritis. Frontiers in Surgery 2014, 1, 9. Hills, B. A., Boundary lubrication in vivo. Proceedings of the Institution of Mechanical Engineers Part H-Journal of Engineering in Medicine 2000, 214, (H1), 83-94. Le Graverand, M.-P. H.; Vignon, E.; Otterness, I.; Hart, D., Early changes in lapine menisci during osteoarthritis development: Part I: cellular and matrix alterations. Osteoarthritis and Cartilage 2001, 9, (1), 56-64. Dahl, L. B.; Dahl, I. M. S.; Engstromlaurent, A.; Granath, K., Concentration and MolecularWeight of Sodium Hyaluronate in Synovial-Fluid from Patients with Rheumatoid-Arthritis and Other Arthropathies. Annals of the Rheumatic Diseases 1985, 44, (12), 817-822.


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5.

6. 7. 8. 9. 10. 11.

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22.

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