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Drastic Swelling of Lipid Oligobilayers by Polyelectrolytes - A Potential Molecular Model for the Internal Structure of Lubricating Films in Mammalian Joints Felicitas Schwörer, Marcus Trapp, Xiao Xu, Olaf Soltwedel, Joachim Dzubiella, Roland Steitz, and Reiner Dahint Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03229 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017
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Drastic Swelling of Lipid Oligobilayers by Polyelectrolytes - A Potential Molecular Model for the Internal Structure of Lubricating Films in Mammalian Joints Felicitas Schwörer1), Marcus Trapp2), Xiao Xu2),3), Olaf Soltwedel4),5), Joachim Dzubiella2),3)*), Roland Steitz2), and Reiner Dahint1)*) 1)
Ruprecht-Karls-Universität Heidelberg, Applied Physical Chemistry, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany
2)
Helmholtz-Zentrum Berlin, Institute for Soft Matter and Functional Materials, HahnMeitner-Platz 1, 14109 Berlin, Germany
3)
Institut für Physik, Humboldt-Universität zu Berlin, Newtonstr. 15, 12159 Berlin
4)
Max Planck Society Outstation at the Heinz-Maier-Leibnitz-Zentrum (MLZ), 85747 Garching, Germany
5)
Physics Department, Technical University Munich, James-Franck-Str. 1, 85747, Munich
*) corresponding authors:
J.D. (theory):
[email protected] R.D. (experiments):
[email protected] -1ACS Paragon Plus Environment
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Abstract Osteoarthritis is the most common arthropathy in Western civilisation. It is primarily caused by the degeneration of lipid-coated cartilage, leading to increased friction in joints. Hyaluronic acid (HA), a negatively charged polysaccharide and the main component of the synovial fluid, is held responsible for joint lubrication. It is believed that HA, adsorbed to the lipid-coated cartilage, forms a protective layer against wear. Studies have shown that the concentration and molecular weight (MW) of HA are reduced in joints suffering from osteoarthritis. Based on these observations, local joint injections of HA or mixtures of HA and surface active phospholipids (SAPLs) have been applied as a medical cure to restore the functionality of the joints in a procedure called viscosupplementation. However, this cure is still disputed and no consensus has been reached with respect to optimum HA concentration and MW. To provide detailed insight in the structural rearrangement of lipid films upon contact with HA or polymeric analogues, we studied the interaction of the polyelectrolyte polyallylamine hydrochloride (PAH) with surface-bound oligobilayers of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) by neutron reflectivity (NR) and ellipsometry. Using this model system we found a drastic swelling of the lipid films as a function of PAH concentration which strength compares to previous studies on HA incubation. In contrast, no significant dependence of film thickness on PAH MW was observed. A detailed picture of the film architecture was developed which inter alia shows that charged PAH is adsorbed to the lipid head groups leading to electrostatic repulsion. The swelling behavior is well explained by the equilibrium of Coulomb and van der Waals interactions in a DLVO-based model. Our detailed structural analysis of the PAH/lipid interfacial layer may help to elucidate the mechanisms of viscosupplementation and derive a structure-function relationship for the lubricating interface in mammalian joints.
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Introduction The World Health Organization (WHO) estimates that about 10% of men and 18% of women aged over 60 suffer from symptomatic osteoarthritis with 80% of them limited in movement, and about 25% with severe limitations in daily life activities.1 The disease is primarily caused by the degeneration of the articular cartilage covering the bone ends of the joints, which operate in a closed compartment, the articular capsule, filled with the synovial fluid. The articular cartilage is in turn decorated with surface active phospholipid (SAPL) bilayers. The synovial fluid contains hyaluronic acid (HA), lubricin, proteinases, and collagenases, out of which the negatively charged polysaccharide HA is the main component. HA, lubricin and phospholipids (PLs) are supposed to reduce friction and protect the cartilage so that osteoarthritis is impeded. Based on the observation that HA concentration and molecular weight (MW) is reduced in diseased joints2-4 a new non-surgery cure called viscosupplementation has been developed, where HA or mixtures of HA and PLs are intraarticularly injected.5-7 While the short-term benefit of viscosupplementation in the treatment of mild to moderate osteoarthritis is recognized to provide comfort and relief pain, its value in the long run has not been approved yet.8 The contribution of HA to the boundary lubrication at an articular cartilage interface is wellestablished, the role of the SAPLs, however, is not.9 HA facilitates the boundary lubrication in a dose-dependent manner, and further in vitro pilot studies indicated that HA adsorbed to the articular surface of bovine osteochondral samples was able to contribute to boundary lubrication even without HA in the test bath.9 The latter studies suggest that HA may reduce friction by being retained at or between the articular cartilage surfaces under relative motion. On the opposite, the role of SAPLs in the boundary lubrication at articular cartilage–cartilage interfaces is still under debate. The SAPL 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a concentration of around 0.35 g/l in its fluid 𝐿𝐿𝛼𝛼 -phase was found to slightly lower friction at a
cartilage–steel interface,10 while Schmidt et al. showed in boundary lubrication tests with fresh
osteochondral samples from the patellofemoral groove of skeletally mature bovine stifle joints that SAPL in the form of DPPC at a physiologic concentration of 200 µg/ml did not significantly lower the kinetic friction coefficient.11 The conducted boundary lubrication experiments so far collected macroscopic information on the reduction of friction by surface-bound HA and SAPLs. However, they did not address the internal architecture of the lubricating interface to potentially establish the underlying structure-3ACS Paragon Plus Environment
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function relationship. Only recently, efforts have been made to investigate the interaction of HA and lipid films on a molecular level and reveal its impact on layer composition and architecture. In dynamic light scattering experiments and QCM-D (quartz crystal microbalance with dissipation monitoring) studies Wang et al. show strong association of HA with vesicles of DPPC and pronounced HA adsorption on solid-supported DPPC bilayers. The structures formed were stable enough to allow for sequential adsorption of DPPC and HA layers.12 From X-ray reflectivity (XRR) experiments on lipid monolayers at the air-water interface Wieland et al. concluded that HA and DPPC interact via electrostatic and hydrophobic interactions. HA of lower MW exhibited stronger interactions with the lipid film and tended to accumulate between single domains of condensed DPPC.13 Ionov et al. reported that the domain shape of such films did not significantly change upon addition of HA to the aqueous subphase. However, an approximately 10% reduction in domain size was observed.14 Measurements on solid-supported DPPC bilayers revealed that both high and low MW HA adsorbs to the headgroup region that is oriented towards the liquid phase. Some distortion of the alkyl chain region suggests that HA penetrates into the lipid tail regime only to minor extent. Moreover, a higher stability of the lipid films with respect to high hydrostatic pressure was observed in the presence of HA.15 In neutron reflectometry (NR) studies focusing on the structure and stability of solid-supported lipid films we have previously shown that aqueous solutions of HA or charged polymeric substitutes induce significant swelling of SAPL oligobilayers and stabilize the interfacial films with respect to increased temperature and mechanical stress.16-19 In these investigations we used as a model system for natural joints a silicon wafer (representing the bone end) which was covered with oligolamellar SAPL bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and incubated that system in an aqueous solution containing HA or alternatively the polyelectrolyte (PE) poly(allylamine hydrochloride) (PAH). A molecular concentration of 3 g/L was selected in close match to the concentration of HA in the synovial fluid of healthy joints (2.85 g/L).4, 20 We found a dramatic swelling of the pre-formed oligolamellar SAPL film at the solid-liquid interface upon interaction with HA over an extended time period of ~ 40 days,17 which could be drastically accelerated by substituting the natural HA (negatively charged) by the synthetic PAH (positively charged) by a factor of ~ 10, i.e. reaching equilibrium already after ~ 4 days of incubation.18 Most importantly, HA and PAH introduce the same principal effects in the SAPL boundary layer. We now investigate the impact of the synthetic PAH on pre-formed DMPC oligobilayers at various PAH concentrations and MWs by combined NR, XRR, ellipsometry and attenuated -4ACS Paragon Plus Environment
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total reflection Fourier transform infrared spectroscopy (ATR-FTIR) studies in order to deduce the relevance of those system parameters on the structure and re-arrangement of the SAPL boundary layers on the molecular scale. The experimental findings are complemented with a theoretical analysis in which we show that the experimentally observed effects can be fully understood and quantitatively described by a PE-driven swelling with equilibrium distance between charged bilayers dependent on PE adsorption as well as PE monomer concentration. Our studies may, therefore, also contribute to a better understanding of the mechanisms of viscosupplementation by providing detailed insight in changes in the internal architecture of friction reducing and cartilage protecting lipid films in human joints upon contact with HA or polymeric substitutes.
Experimental Methods Materials and Samples Samples for ellipsometric studies were prepared on disc-shaped silicon (Si) substrates (76.2 mm diameter, 525 µm thickness) with polished front side supplied by Siegert Wafer GmbH (Aachen, Germany). Prior to analysis they were sliced into pieces of 2 cm x 2 cm. Disc-shaped substrates for NR and IR measurements (60 mm diameter, 10 mm thickness, rms-roughness < 0.6 nm) had polished front and back sides, and were obtained from Siliciumbearbeitung Holm (Tann/Ndb., Germany) or Sil’tronix (Archamps, France). For combined simultaneous NR and ATR-FTIR investigations, Si substrates with 45° cut polished side planes were used. DMPC was purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA), D2O (purity 99.9 atom % D), NaCl (purity 99.5%), and PAH with an average molecular weight MW of 15, 58 and 900 kDa from Sigma-Aldrich (Steinheim, Germany). All chemicals were used as received. Prior to sample preparation, the substrates were rinsed with chloroform (Uvasol® grade, Merck, Darmstadt, Germany), cleaned for 1 h in ethanol (absolute puriss. p.a., Sigma-Aldrich), and subsequently washed with ethanol and chloroform. Oligolamellar lipid films were prepared on the cleaned silicon discs by spin-coating (Model 6708D, SCS, USA) as first described by Mennicke and Salditt21 using lipid solutions in chloroform at a concentration of 10 g/L for NR experiments and 5 g/L for ellipsometric measurements. A rotational speed of 500 rpm was selected for 1 sec followed by 4000 rpm for 60 sec. Prior to the NR experiments the dry samples were characterized by XRR using a home-built set-up.22
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For each MW, NR measurements at 3 g/L PAH, corresponding to the concentration of HA in healthy mammalian joints, have been performed on at least three different lipid samples. Concentration dependent NR studies were conducted on single lipid samples due to the large number of data points, long data acquisition times, and restricted neutron beamtime. For PAH 15 kDa, an additional measurement has been performed at 1 g/L, which was combined with the respective concentration series. In the ellipsometry studies, individual lipid samples were used for each concentration of PAH 15 kDa, while for the other MWs the concentration series was conducted with single samples. Reproducibility was successfully tested for a selection of data points. Experimental Setup and Film Characterization The total thickness of the lipid layers was determined by ellipsometry (Type M44®, J.A. Woollam Co. Inc., Lincoln, USA) at a fixed angle of incidence of 75°. Data were taken at 44 wavelengths in a spectral range from 400 and 800 nm, and evaluated with commercial software (WVASE32™) provided by the manufacturer. In the fitting procedure, the optical constants of the silicon substrate were chosen as suggested by Jellison et al.,23 and the lipid film was modelled as a Cauchy layer. Depending on the experimental situation, either air or water was selected as ambient medium contacting the lipid film. Measurements in water took place in a home-built liquid cell with an internal volume of about 10 mL. The entrance and exit windows for the light beam are made of synthetic fused silica. The liquid cell was connected to a flow system, which facilitates solution exchange by the use of a peristaltic pump (MCP Standard, Ismatec, Wertheim, Germany). Temperature was controlled by an independent water circuit guided through the ground plate of the liquid cell. The PAH solutions had a pH of about 4. Combined NR and ATR-IR measurements were performed at the time-of-flight reflectometer BioRef at the Helmholtz-Zentrum Berlin (HZB), Germany.24-25 A chopper speed of 25 Hz allowed to cover a wave vector transfer, qz, range from 0.009 to 0.227 Å-1 with three angular settings (θ = 0.5°, 1° and 2.8°). In specular reflectivity qz is dependent on wavelength, λ, and scattering angle, θ, via 𝑞𝑞𝑧𝑧 =
4𝜋𝜋sin(𝜃𝜃) 𝜆𝜆
1
A constant wavelength resolution, Δλ/λ, was achieved by operating the frame defining choppers in “optical blind” mode.26 In the experiments described here, Δλ/λ was set to 5% with the -6ACS Paragon Plus Environment
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angular resolution Δθ/θ adjusted accordingly. The qz resolution,
𝛥𝛥𝑞𝑞𝑧𝑧 𝑞𝑞𝑧𝑧
𝛥𝛥𝛥𝛥
𝛥𝛥𝛥𝛥
= �( 𝜆𝜆 )2 + ( 𝜃𝜃 )2 set on
the instrument was thus 7%. The reflected neutrons were recorded in θ/2θ geometry by a position sensitive area detector (PSD) with an active area of 300 mm x 300 mm, filled with 3He. Typical measuring times were 6 h per concentration step. The neutron footprint on the sample was adjusted by diaphragms in the incident beam for each angle to avoid over-illumination of the sample. Neutron data reduction and normalization were performed using an in-house developed software package available at BioRef. The raw data were normalized to the direct beam and binned with a constant step width of 1%. A Vertex 70 FTIR spectrometer (Bruker, Leipzig, Germany) was mounted at the sample position in order to allow for simultaneous recording of NR curves and ATR-FTIR spectra.27 During one NR measurement 128 IR individual scans were summed up for a single measurement. The spectral resolution of the IR spectrometer was set to 2 cm-1. Simultaneous IR and NR measurements took place in a home-built liquid cell with an internal volume of about 3 mL connected to a flow system driven by a peristaltic pump (MCP Standard, Ismatec, Wertheim, Germany). Temperature was measured by a Pt 100 and maintained constant at 20 °C by the use of an external water bath. Additional NR measurements without ATR-FTIR option were performed at NREX at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany.28 NREX is an angle-dispersive monochromatic instrument with a default wavelength of 4.28 Å and a wavelength resolution between 1 and 2%. The sample is aligned horizontally. By tilting the sample the incident angle is varied and reflected neutrons are detected with a 200 mm x 200 mm position sensitive detector. For XRR and NR data fitting we used the program suite Motofit29 which is based on the Parratt algorithm and is implemented in the IGOR Pro software package (WaveMetrics Inc., Portland, USA). The model describing our system is depicted in Figure 1. It consists of the silicon backing, and its native oxide layer, followed by a DMPC lamella, subdivided in lipid heads, tails and heads strata, a potentially adsorbed PAH layer, an interlayer of liquid phase and a second PAH layer at a distance d from top to bottom. This lamellar unit makes up for the distance dlam and is repeated N times. The terminal lamella against the excess liquid phase consists of DMPC heads, tails, heads and potentially a final PAH ad-layer. All relevant lengths and other variables are also introduced. The SAPL boundary layer is characterized experimentally by neutrons and infrared radiation accessing the solid-liquid interface through -7ACS Paragon Plus Environment
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the silicon fronting, by visible light (ellipsometry) accessing the solid-liquid interface through the excess liquid backing, and by X-rays accessing the boundary layer in dry state at the solidair interface through air.
Figure 1: System sketch: From top to bottom the model used to describe the SAPL (+PAH) boundary layer consists of a multilayer set of strata starting with the silicon backing (light grey) and its native oxide layer (dark grey), followed by a DMPC lamella, subdivided in lipid heads (light blue), tails (magenta) and heads strata, a potentially adsorbed PAH layer (green), an interlayer of liquid phase (light green) and a second PAH layer at a distance d from top to bottom. This lamellar unit makes up for the distance 𝑑𝑑𝑙𝑙𝑙𝑙𝑙𝑙 and is repeated N times. The terminal lamella against the excess liquid phase consists of DMPC heads, tails, heads and potentially a final PAH ad-layer. The pink circle represents the size of a PAH blob. For definition of lengths and other variables see text.
Theoretical Methods We complement our experimental studies on the swelling of lipid oligobilayers and PE ad-layer formation with theoretical analysis. For this, we follow the classical ideas of Ohshima and Mitsui30-31 who showed that a Derjaguin-Verwey-Landau-Overbeek (DLVO)-like mechanism for colloidal stabilization32 by electrostatic (ionic) charge adsorption can be employed for a quantitative description of lamellar swelling. In this DLVO picture the electrostatic repulsion between the intrinsically neutral bilayers that become charged due to the ionic adsorption is balanced by attractive Hamaker (van der Waals) interactions, leading to an (in general -8ACS Paragon Plus Environment
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metastable) equilibrium distance between the membranes, much larger than in the dry or pure water reference states. Based on this, Ohshima and Mitsui discussed and quantified the lamellar swelling the first time for calcium ions at DPPC membranes. Later similar ideas were applied for the systematic investigation of ion-specific effects on the swelling for other membranes and surfaces.33-35 We show in this contribution that these ideas can be extended to fully understand and quantitatively describe also the swelling induced by PE adsorption as well as the influence of the PE monomer concentration on the swelling equilibrium. Based on the joint experimental and theoretical analysis we find for all investigated MWs and concentrations that the systems are in a strong adsorption regime with substantial effective surface charge regulation effects, accompanied by a PE adsorption layer thickness on the order of the typical PE blob correlation size. Model and Techniques For our theoretical modeling let us consider the two parallel lipid bilayers in some distance (cf. Figure 1) in the presence of a reservoir of the cationic polyelectrolytes (PAH) of M charged monomers per chain and their counterions both at a monomer concentration c. It is assumed that every monomer of the PE is charged with +1 e. Here we consider two lipid bilayers in our model and thereby assume that the influence of their parallel repeat is comparable small. We also neglect short-range interaction effects, such as hydration repulsion or bilayer undulation3032
as the experimental swelling equilibrium is established at much larger distances in the whole
investigated concentration range than the typical range of these interactions (~ 1 nm), and is thus completely dominated by the DLVO balance mechanism. The bilayers are intrinsically neutral, i.e., carry no surface charge. However, we assume that in a thin adsorption layer of width, δ ≈ dPAH, expected to be of a size of a typical PE correlation length like the blob size (cf. Figure 1),36-37 there is a specific adsorption of PE chains and counterions. The difference in the adsorption between PEs and ions will lead to a non-vanishing surface charge density and consequently a surface potential ϕ0. The result is an electrostatic repulsion between the bilayers, and an equilibrium distance d will result from a balance between attractive Hamaker (van der Waals) and the electrostatic forces. We can write the total interaction energy per bilayer area as30-31 𝑉𝑉𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 (𝑧𝑧) = 𝑉𝑉𝐻𝐻 (𝑧𝑧) + 𝑉𝑉𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 (𝑧𝑧), -9ACS Paragon Plus Environment
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where z is the distance coordinate between the bilayers to be optimized. Typically this DLVO type of energy has the global minimum at the contact (the collapsed, aggregated state) and a secondary minimum at d.32. Hence, the latter is metastable but in most relevant regimes separated by a large energy barrier to the contact state, and the swollen state can be kinetically stable on very long observation time scales. The Hamaker interaction has the attractive form 𝑉𝑉𝐻𝐻 (𝑧𝑧) = −
𝐻𝐻 𝐻𝐻 ≅− 2 12𝜋𝜋(𝑧𝑧 + 2𝛿𝛿) 12𝜋𝜋𝜋𝜋 2
3
where we assumed d >> δ to obtain the result on the right hand side. H is the Hamaker constant to be fitted from the experimental data. The latter is usually on the order of one kBT, the thermal energy, for dense organic materials like lipid bilayers.32-33, 38-39 For the electrostatic part, we use the standard Debye-Hückel (DH) theory for charged planes32 which virtually always applies in the far field,40 𝑉𝑉𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 (𝑧𝑧) = −
𝜅𝜅𝐷𝐷 𝜙𝜙 2 𝑒𝑒𝑒𝑒𝑒𝑒(−𝜅𝜅𝐷𝐷 𝑧𝑧) 2𝜋𝜋𝜆𝜆𝐵𝐵 0
4
where 𝜅𝜅𝐷𝐷 = �8𝜋𝜋𝜆𝜆𝐵𝐵 𝑐𝑐 = 𝜆𝜆−1 𝐷𝐷 defines the inverse Debye screening length at a concentration c of monovalent salt given a Bjerrum length λB = e2/(4πεε0kBT) with 𝜀𝜀𝜀𝜀0 denoting the usual
permittivities. The Debye screening length is the decisive factor that determines the range of the electrostatic repulsion, i.e., the higher the monomer and counterion concentration the shorter the repulsion and less swelling is expected. We assume in the following that PE monomers and counterions both contribute equally to screening, i.e. the salt concentration c in the calculation of 𝜆𝜆𝐷𝐷 is taken as that of the PE monomers. In the pure DH limit of low surface potentials the
linear Grahame relation32 holds between surface potential and the surface charge (built up from adsorption), ϕ0 = 𝜆𝜆𝐷𝐷 σ/ 𝜀𝜀𝜀𝜀0 . The experimental results on the surface coverage of PAH (cf. Fig.
6e) of more than 2 monomers per lipid (with 53.5 Å2 area41) indicate already that the bare surface potential will be beyond the simple DH treatments and we need to employ the results from non-linear theory. In the limit of large charge adsorption and thus large surface charge, in fact, it is well known that charge renormalization effects lead to an effective surface potential 𝜙𝜙𝑒𝑒𝑒𝑒𝑒𝑒 , i.e., the one seen
far away from the surface. Rigorous solutions from the non-linear Poisson-Boltzmann equation show that in this case an effective surface potential of 𝑒𝑒𝑒𝑒𝜙𝜙𝑒𝑒𝑒𝑒𝑒𝑒 = 4𝛾𝛾 is established.44 Here, 1
𝑒𝑒
𝛽𝛽 = 𝑘𝑘 𝑇𝑇, 𝛾𝛾 = √𝑥𝑥 2 + 1 − 𝑥𝑥, with 𝑥𝑥 = 𝜅𝜅𝐷𝐷 𝜆𝜆𝐺𝐺𝐺𝐺 and the Gouy-Chapman length 𝜆𝜆𝐺𝐺𝐺𝐺 = 2𝜋𝜋𝜆𝜆
𝐵𝐵 𝜎𝜎
𝐵𝐵
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. In
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particular, a very high bare surface charge density leads to 𝑥𝑥 → 0. Consequently, in the large
PE adsorption limit the surface potential simply saturates at 𝑒𝑒𝑒𝑒𝜙𝜙𝑚𝑚𝑚𝑚𝑚𝑚 = 4, i.e., a maximum value is established for the far field, independent of the bare built-up surface charge σ (the effective surface charge σeff is again given by the linear Grahame relation above.) In the weak PE adsorption limit, in contrast, the effective surface potential is the 'real' bare surface potential, and the DH eq. 4 holds for all distances and is directly related to charge adsorption. If the PE adsorption happens in the layer with thickness δ
