Probing the Molecular Interactions and Lubrication Mechanisms of

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Probing the Molecular Interactions and Lubrication Mechanisms of Purified Full-length Recombinant Human Proteoglycan 4 (rhPRG4) and Hyaluronic Acid (HA) Jun Huang, Xiaoyong Qiu, Lei Xie, Gregory D. Jay, Tannin A Schmidt, and Hongbo Zeng Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01678 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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Biomacromolecules

Probing the Molecular Interactions and Lubrication Mechanisms of Purified Full-length Recombinant Human Proteoglycan 4 (rhPRG4) and Hyaluronic Acid (HA) Jun Huang,§a,b Xiaoyong Qiu,§b Lei Xie,b Gregory D. Jay,c,d Tannin A. Schmidt,e Hongbo Zeng*b aCenter

for Advanced Jet Engineering Technologies (CaJET), Key Laboratory of High-efficiency and

Clean Mechanical Manufacture (Ministry of Education), Department of Mechanical Engineering, Shandong University, Jingshi Road 17923, Jinan 250061, China bDepartment

of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta,

T6G 1H9, Canada cDepartment

of Emergency Medicine, Warren Alpert Medical School of Brown University,

Providence, RI 02903, USA dDepartment

eBiomedical

of Engineering, Brown University, Providence, RI 02903, USA

Engineering Department, School of Dental Medicine, University of Connecticut Health

Center, 263 Farmington Avenue, Farmington, CT 06030, USA.

*Email: [email protected]; Phone: +1-780-492-1044

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Abstract Probing the adsorption and lubrication behavior of lubricin, also known as Proteoglycan 4 (PRG4), is important for understanding the ultra-low friction of cartilage lubrication. Most previous research has focused on native lubricin either purified from synovial fluid or articular cartilage explant culture media. In this work, the adsorption behavior and lubrication mechanism of full-length recombinant human PRG4 (rhPRG4) on mica as well as the effect of adding hyaluronic acid (HA, a polysaccharide) were systematically investigated using a Surface Forces Apparatus (SFA) technique. A low friction coefficient (µ ~ 0.04) was measured when multi-layer rhPRG4 (~ 31 nm) was confined in between mica surfaces, even when the load increased to ~ 1.2 MPa. Intriguingly, a previously unreported ultra-low friction coefficient (μ < 0.005) was observed at a low sliding velocity (v = 0.14 μm/s) with the applied load P reaching ~ 3.6 MPa when a diluted rhPRG4 solution (~ 90 μg/ml) was used. The distinct friction behavior is likely due to the smooth and more close-packed lubricin coating, as evidenced by the Atomic Force Microscope imaging. Adding HA onto multi-layer rhPRG4 coated mica increased the friction coefficient μ to ~ 0.1; however, the load bearing property increased, indicating potential synergistic effect between rhPRG4 and HA, which was further demonstrated by the weak adhesion observed when separating rhPRG4 coated mica and HA coated aminopropyltriethoxysilane (APTES)-mica. Alternatively, adding pre-mixed rhPRG4-HA on mica had a friction coefficient (μ ~ 0.1) close to that of injecting concentrated rhPRG4 (~ 450 μg/ml) with lower load sustainability. Our results provide fundamental insights into the adsorption and lubrication behavior of lubricin and its interaction with HA, with useful implications for the underlying mechanism of ultra-low friction provided by synovial fluid.

Keywords: Lubricin, hyaluronic acid, surface forces, lubrication

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Biomacromolecules

Introduction Lubricin, a mucin-like, amphiphilic glycoprotein originally discovered in synovial joints and

recently at the ocular surface, functions as a boundary lubricant and protective layer.1-3 Lubricin is capable of reducing the coefficient of friction by binding to either hydrophobic or hydrophilic surfaces.4-8 Appropriate and high levels of recombinant expression of lubricin has proved to be challenging in the past, as such most of the previous research has focused on native lubricin either purified from synovial fluid or media-conditioned articular cartilage explant culture which has repeatedly demonstrated the ability to reduce friction at a variety of biointerfaces (e.g. cartilage-cartilage,9,

10

cornea-eyelid,1,

11

and cornea/eyelid-biomaterial12,

13).

However, it can be

challenging to generate sufficient amounts of highly-purified lubricin (e.g., human PRG4 or bovine PRG4) from synovial fluid, due to the complexity of synovial fluid, which has in part impeded the advances toward the research on the intrinsic role of lubricin and designing of bio-inspired lubricants. Recent technological advances have enabled abundant expression of a full-length recombinant human version of PRG4 (also known as rhPRG4),14 which has exhibited O-linked glycosylation consistent with that of native lubricin, higher order structure (e.g. disulfide-bonded multimers) as well as immunoreactivity at the appropriate apparent molecular weight.14 More importantly, rhPRG4 has functioned as an effective boundary lubricant at both human cornea-polydimethylsiloxane and cartilage-cartilage biointerfaces.14,

15

Despite the tremendous progress that has been made on the

scale production of this type of lubricin (rhPRG4) and macro-tribological study1,

14, 15

as well as

effective use clinically for the treatment of dry eye,16 molecular-level understanding of the lubrication mechanism of rhPRG4 still remains elusive. Hyaluronic Acid (HA) is a linear polysaccharide with alternating units of glucuronic acid and N-acetylglucosamine in synovial fluid and at the surface of articular cartilage.12, 17-19 HA produced by bacteria is already being used in a broad range of biomedical engineering applications, e.g. intra-articular injections as a visco-supplement. Indeed, most research indicates that the major mechanical role of HA is to maintain the high viscosity of synovial fluid; however, research also indicates that HA coating could work as an effective boundary lubricant under static conditions and enhance the anti-wear performance in joint lubrication.20 Grafted HA has been found to function 3



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synergistically with native lubricin (e.g., PRG4) on achieving low friction coefficient.21 Overall, the boundary lubricating properties of HA are often debated, and based on conflicting results at different bio-interfaces and length scales.2,

22-26

Nevertheless, the adsorption process and interaction

mechanisms of HA and lubricin on substrates is still not fully understood. Surface Forces Apparatus (SFA) is a widely used technique for characterizing intermolecular and surface forces (e.g, surface adhesion and lubrication behaviors) for numerous biological and non-biological systems in vapors and liquids with high force sensitivity and distance resolution down to 0.1 nm.27-30 This technique could provide molecular insights into the configuration and lubrication mechanism of lubricin on different surfaces, and the interaction and lubrication behaviors of native lubricin on various types of surfaces have been measured and reported previously.5, 10, 21, 31, 32 In this work, the adsorption behavior and lubrication mechanism of purified full-length recombinant human PRG4 (rhPRG4) on a model substrate (i.e., mica) as well as the interactions between rhPRG4 and HA have been systematically studied by using the SFA technique. It was observed that rhPRG4 could quickly adsorb (< 10 min) onto a mica surface and suppress further adsorption of HA on mica. Surprisingly, super-lubricity (µ < 0.005) was achieved with the normal load reached ~ 3.6 MPa when well anchored rhPRG4 layer were obtained, demonstrating the excellent lubricating properties of rhPRG4. Alternatively, a higher friction coefficient (µ ~ 0.04) was measured between the confined multi-layer rhPRG4 under similar normal load. As such, the formation of close-packed thin rhPRG4 layer is a prerequisite for achieving super-lubricity. Importantly, adding HA enabled a higher sustainability of the multi-layer rhPRG4 surface under higher loads (~ 3.9 MPa) during sliding with the friction coefficient increased slightly to about 0.1. Thus, the rhPRG4 showed a potential synergic effect with HA for achieving higher load bearing property. Normal force measurements also indicated that there was weak adhesion between pre-adsorbed HA layer and rhPRG4-coated mica surface during separation, most probably due to the non-covalent interactions such as polymer chain diffusion, electrostatic interaction, hydrophobic interaction as well as hydrogen bonding. Collectively these results provide fundamental insights into the adsorption and lubrication behavior of lubricin and the polysaccharide HA on model mica

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surfaces, which contribute to the understanding of the underlying mechanism of ultra-low friction provided by synovial fluid in mammalian diarthrodial joints.

2. Experiment and Methods 2.1 Protein preparation Dulbecco's phosphate-buffered saline (1 x DPBS, Fisher Scientific, composing of 2.7 mM KCl, 1.5 mM KH2PO4, 138 mM NaCl and 8.1 mM Na2HPO4·7H2O) was used for solution preparation and force measurement. The full-length rhPRG4 protein was prepared as described previously with the concentration being 450 µg/ml.33,

34

Briefly, the gene encoding the full-length 1404 amino acid

PRG4 protein in humans was inserted into plasmid vectors at Selexis SA (Geneva, Switzerland) for enhanced gene expression in mammalian Chinese hamster ovary (CHO-M) cell lines (Lubris, LLC; Framingham, MA). The modified CHO cells were used to post-translationally glycosylate expressed proteins on a large scale.35 The rhPRG4 clonal cell line was fed-batch cultured in a shake flask containing SFM4CHO medium (Hyclone; Logan, UT) supplemented with 8 mM L-Glutamine, hypoxanthine and thymidine (1 x HT, Life Technologies; Carlsbad, CA). The resultant rhPRG4 rich media was subjected to ultrafiltration/diafiltration and a three-step chromatographic purification process.34 The obtained rhPRG4 has an apparent molecular weight Mw of ~ 460 kDa as assessed by SDS-PAGE and also contains the disulfide-bonded dimeric form with a Mw being ~ 1 MDa.14 More importantly, the obtained rhPRG4 is heavily and variably O-glycosylated, with the glycosidic residue side chains contributing at least 30% or more of its molecular weight. Indeed, rhPRG4 demonstrated reactivity

via

SDS-PAGE

western

blot

that

is

consistent

with

O-linked

n-acetyl

galactosamine-galactose glycosylations, and enzymatic removal of these resulted in the anticipated decrease of rhPRG4's apparent molecular weight.35 HA (~ 1.5 MDa, LifeCore Biomedical, Chaska, MN) was prepared in 1 x DPBS at a physiological concentration of 3.33 mg/ml. For preparing rhPRG4-HA mixture, 0.5 ml of rhPRG4 (450 µg/ml in DPBS buffer) was mixed with 0.5 ml of HA (3.3 mg/ml in DPBS buffer) in a clear plastic centrifuge tube (1.5 ml) using a vortex mixer (Fisher Scientific, USA) for 10 s and then waited for 1 h before injecting into mica surfaces. 5



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2.2 Deposition of APTES on mica via the vapor deposition method Freshly cleaved mica sheets were placed in a vacuum desiccator (diameter of 230 mm, Bel-Art Scienceware) and 0.1 ml of aminopropyltriethoxysilane (APTES) was added into a PTFE container placed in the desiccator. The desiccator was vacuumed for 60 min (~ 80 mm Hg), and then sealed and kept for 4 hour for vapor deposition. Finally, after the desired deposition time was reached, the resulting mica sheets were taken out and washed with deionized water, dried with nitrogen flow and kept in vacuum desiccator before further experiment. For achieving stable HA coatings, those APTES-treated mica surfaces were drop-coated with 3.3 mg/ml HA solution for at least 3 h, and then rinsed with filtered DPBS buffer, noting that the surfaces were kept wet during the whole process. 2.3 Surface force and lubrication measurement A Surface Forces Apparatus equipped with piezoelectric bimorph slider was used to measure the normal force and friction force in the test solutions. The detailed experimental setup using the SFA system and its working principles can be found elsewhere.27, 36-39 To conduct the force measurement, uniform-thin mica sheets

with the thickness of ~ 5 µm were firstly cut into small pieces (2 cm × 2 cm) using hot platinum wire (0.2 mm, 99.99%, Alfa Aesar, Canada) and then attached to another piece of freshly-cleaved, clean mica sheets (15 cm × 20 cm, thickness > 100 µm, also know as backing sheet). Then the whole backing sheet was coated with 50 nm of silver using an electron-beam evaporation deposition tool (Kurt J. Lesker, USA) with a deposition rate less than 1 Å/s. After that, the thin backed-silvered mica sheets of the same thickness were glued onto cylindrical silica disks (R = 2 cm) using either epoxy glue or NOA 81 UV glue (Norland Products, Inc.). Then the two silica discs were mounted into the SFA chamber in a cross-cylinder configuration, of which the surface interaction is equivalent to a sphere with the same radius of R approaching a flat surface when the separation distance (D) is much smaller than R.40, 41 For measurements in aqueous solutions, the desired solution was injected between the two mica surfaces with a 1 ml syringe (Norm-Ject, Fisher Scientific) with sharp needles (BD PrecisionGlide, Fisher Scientific). About

0.1 ml of solution was injected in between the two mica surfaces, mounted in the SFA chamber saturated with water vapor. For friction measurements, the bottom surface was displaced horizontally by the piezoelectric bimorph slider while the upper surface was connected to a vertical double-cantilever spring whose lateral deflection was measured with four strain gauges.

The film thickness and the separation distance between the two surfaces can be determined in situ and in real time by using an optical technique named Multiple Beam Interferometry (MBI) by 6


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employing fringes of equal chromatic order (FECO).38, 42 More importantly, the FECO fringe can also provide direct information about the shape deformation of the interaction region during sliding process. 2.4 Morphology characterization The morphology of the rhPRG4 modified mica surface was characterized with an Atomic Force Microscope (Bruker Dimension Icon, CA, USA) under the peak-force tapping mode. Silicon cantilevers (Bruker Nano) with a nominal resonance frequency of 300 − 400 kHz and spring constant of ~ 40 N/m were used for imaging.

3. Results and Discussion 3.1 Surface interaction and lubrication of rhPRG4 confined between two mica surfaces

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Figure 1. (a) Force-distance profile of mica surfaces after injecting 450 μg/ml rhPRG4-DPBS solution and equilibrating for 30 min with rhPRG4 film (~ 31 nm) being confined between mica surfaces. The black curve indicates approaching while the red curve shows separation, and the inset shows the configuration of SFA experiment during normal/lateral force measurements; (b) friction force f, as a function of normal load F ⊥ for two mica surfaces across rhPRG4-DPBS solution with the shear velocity of 0.14 μm/s; typical shear force versus time traces within the (c) low load regime (d) high load regime; (e) FECO images of mica-mica contact in air before injecting rhPRG4; (f, g) in situ FECO images of mica surfaces under the normal load of (f) 2.4 mN and (g) 10 mN after injecting rhPRG4.

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Biomacromolecules

The normal and friction forces of rhPRG4 confined between two mica surfaces were measured by an SFA equipped with a piezoelectric bimorph slider. Figure 1a shows the force-distance profile on approaching and separation, respectively, for two mica surfaces, obtained after injecting rhPRG4 solution (450 μg/ml) in DPBS buffer for 30 min. Pure repulsion is observed during approaching and separation with the distance, where repulsion starts to increase obviously at ~ 180 nm during approaching and at ~ 170 nm during separation, suggesting a slight hysteresis during loading/unloading process. The measured long-range repulsion could be due to the entropic-steric repulsion between the extended mucin-like polymer chains, since electrostatic force is significantly suppressed when the overall salt concentration reaches 150 mM. The observed hysteresis is different from previously reported results based on extracted-PRG4 from synovial fluid, where pure repulsion with no hysteresis was measured even when the surface were pressed together for 2 h.5 Considering the higher order-structure of rhPRG4,14 the hysteresis could be mainly due to the protein conformation change and chain penetration/interdigitation during loading/unloading process, especially when multi-layers of rhPRG4 are confined between mica surfaces. In addition, the confined rhPRG4 film is measured to be ~ 45 nm when the compressing load reaches 30 mN/m; while the film thickness decreases to ~ 31 nm at higher compression-load (e.g., 120 mN/m, see Figure 1f). Here the zero distance (D = 0) is defined when two mica surface are brought into contact in air (see Figure 1e). The contour length l of lubricin was reported to be 150-220 nm.4,

14

The

repulsion range measured here (~ 180 nm) is comparable to previously reported results for native PRG4 (~ 200 nm);5 however, it is much smaller than twice the contour length of PRG4 layers, suggesting that these proteins are either not to be fully extended or stay in the form of “loop” on mica surface. The friction force f as a function of the applied normal load F⊥ are also presented in Figure 1b. The measured friction coefficient µ is ~ 0.04 within the applied load regime when the sliding velocity is kept at 0.14 μm/s. The coefficient of friction here is close to previously reported results based on native lubricin for low loads (µ ~0.025).5 However, the purified recombinant rhPRG4 here shows low friction coefficient throughout the load regime even when F⊥ reached 10 mN (see Figure 1g, load pressure P ~ 2.6 MPa), which is different from native PRG4 where the measured friction 9



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coefficient quickly increased to more than 0.2 with the load greater than 0.4 MPa.5 Figure 1c shows the typical shear force versus time traces with the normal load of 2.4 mN and 10 mN, respectively. Scattered spikes are observed during sliding, the increased static friction force was measured when the normal load increases, which may be explained by the higher degree of protein-chain penetration at higher load. It is worth noting that the surfaces got stuck and no kinetic friction forces could be measured when the normal load was higher than 14 mN, though no signs of surface damage were observed. The FECO patterns shown in Figure 1f and 1g suggest that the flat mica-mica contact became uneven during sliding. The deformed FECO images indicate a shear-induced clustering or aggregation of rhPRG4 molecules (see the bumps in Figure 1f and 1g). The clustering of rhPRG4 is consistent with the notion that rhPRG4 confined between mica surfaces forms a multi-layer structure and is not stable enough to sustain high load during shearing. Interestingly, the measured friction coefficient did not change much when this aggregation behavior was observed. The normal and lateral force results indicate that rhPRG4 is forming a protective multi-layer structure during confining and sliding, resulting in long-range repulsion during compression and those surface aggregates generated during sliding still maintain good lubrication properties.

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Biomacromolecules

Figure 2. (a) Force-distance profile of mica surfaces after injecting 90 μg/ml rhPRG4 - DPBS solution and equilibrating for 30 min (a thin rhPRG4 layer of ~ 20 nm was confined between mica surfaces). The black curve indicates approaching while the red curve shows separation; (b) friction force f, as a function of normal load F ⊥ for two mica surfaces across rhPRG4-DPBS solution with the shear velocity of 0.14 μm/s; typical shear forces versus time traces when the load being (c) ~ 3mN and (d) 15 mN, respectively; (e, f, g, h) in situ FECO images of rhPRG4 confined between mica surfaces under the normal load of 0.8 mN, 3 mN, 10 mN and 18 mN, respectively; Noting that

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large static friction force is observed at high load (e.g., F ⊥ > 10 mN) when the shear direction reversed. In order to further understand the adsorption and friction behaviors of rhPRG4 on model mica surface, diluted rhPRG4 sample (90 μg/ml rhPRG4 in 0.1 M DPBS buffer) was also injected in between mica surfaces as independent experiments. Interestingly, an unexpected low friction coefficient region was measured and shown in detail in Figure 2. Figure 2a shows the typical force-distance profile obtained after injecting diluted rhPRG4 solution for 30 min. Similar to the case of un-diluted rhPRG4 (450 μg/ml), pure repulsion force was measured during both approaching and separation. However, both the confined film thickness (~ 22 nm) and repulsion range (~ 130 nm) decreased as compared to the force results for the case of higher concentration of rhPRG4 (Figure 1a), suggesting the decreased adsorption amount and changed protein configuration. Surprisingly, the friction results (Figure 2b) shows two regimes during loading process: the friction coefficient μ was less than 0.005 at low load regime (F⊥ < 10 mN, P < 3.6 MPa) and increased to ~ 0.03 for high load regime (F⊥ > 10 mN, P > 3.6 MPa). Figure 2c and 2d show the typical shear force versus time traces with the normal load being 3 mN and 15 mN, respectively. Clearly, for the low load regime (Figure 2e, 2f, F⊥ < 3mN), dynamic sliding with small static friction force was observed. However, a larger static friction force was measured when the normal loads were higher than 10 mN (see the marked grey region shown in Figure 2d), though the FECO image remained flattened. The large static friction force is highly likely due to the penetration of protein chains of the adsorbed lubricin layers that was behaving as polymer brushes during sliding 5, 43 The in situ FECO patterns shown in Figure 2e, 2f, 2g and 2h suggest that the surfaces remain flattened even when the normal load increases to 18 mN (~ 4.5 MPa), which demonstrates the excellent anti-wear property of rhPRG4 confined between mica surfaces for this case. To further understand the origin of the low friction force, the results of friction force as a function of sliding velocity at fixed normal load is presented in Figure S1. Interestingly the friction force was observed to increase with shear velocity: the friction force measured at higher shear velocity (e.g., 6 μm/s) was five times higher than that of lower shear velocity (e.g., 0.02 μm/s). The shear-velocity-dependent friction behavior provides additional evidence that the friction force measured is due to the protein chain interactions during loading 12


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Biomacromolecules

process. The friction results for both high and low concentration of rhPRG4 demonstrate that the purified rhPRG4 exhibits better lubrication performance as compared to native lubricin (e.g., PRG4) purified from synovail fluid. 4, 6 Potential mechanisms for this finding are further discussed below.

Figure 3. Topographic AFM images of (a, b) 450 μg/ml of rhPRG4 coated mica; (d, e) 90 μg/ml of rhPRG4 coated mica in air; (c, f) typical cross section line-scan profiles of rhPRG4 coated mica surfaces derived from the corresponding topographic AFM images shown in (b and e), the black, red and green lines represent three different locations. In order to understand the origin of the different lubrication behavior for the two concentrations of rhPRG4 tested, Atomic Force Microscope imaging on rhPRG4 adsorbed mica surface was conducted. Figure 3 shows the surface morphology of rhPRG4 coated mica surface in air with different concentrations of rhPRG4 after conducting friction measurement. A dense layer of aggregates (see Figure 3a, 3b) was observed on mica surface after friction measurement in 450 μg/ml rhPRG4 solution, and the surface roughness was measured to be 1.9 nm. Apparently there were at least two layers of rhPRG4 adsorbed on mica, with the bottom layer covered with densely packed smaller particles and scattered large particles distributed evenly on top, i.e., multi-layer rhPRG4 film was observed. The line section profiles shown in Figure 3c indicate the peak-peak value is about 6 13



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nm. Conversely, for the case of low concentration (90 μg/ml) of rhPRG4 (Figure 3d-3f), the amount of large particles decreases significantly, and the surface roughness also decreases to less than 1 nm. The section profiles shown in Figure 3f suggest that the peak-peak value also decreases to ~ 3 nm. Thus low concentration of rhPRG4 coated mica surface showed a relatively smooth surface as compared to the case of high concentration of rhPRG4. The obtained AFM results are consistent with the SFA results shown in Figure 1 and Figure 2, where distinct lubrication behavior (i.e., µ was < 0.005 for low concentration of rhPRG4 and ~0.04 for high concentration of rhPRG4 under the same normal load) was observed. Previous reports have indicated potential synergistic interaction between HA and native PRG4 for

enhanced

wear

protection

between

mica-mica21

or

reduced

friction

for

human

cornea-polydimethysiloxane biointerface.13 The rhPRG4 has also demonstrated similar synergism with HA at a cartilage-cartilage interface.15 Thus in this study, HA dissolved in DPBS buffer (3.3 mg/ml) was injected in between the mica surfaces at the same location where the friction measurement of mica-mica across rhPRG4 solution was conducted. The normal force obtained after injecting HA was measured and shown in detail in Figure 4a. It was interesting to observe that after adding HA and equilibrating for 20 min, the confined film thickness increases by only ~ 2 nm, which is much smaller than the hydrodynamic radius of HA with a molecular weight of ~1.5 MDa. The small thickness change suggests that a very small amount of HA molecules were confined in between the mica surfaces after the pre-adsorption of rhPRG4 layer. These results are consistent with previously reported results that lubricin can form an anti-adhesive layer on substrates, protecting the substrates from further adsorption of other chemicals.5, 44 It is hypothesized that the low adsorption amount of HA is due to the anti-adhesive property and electrostatic repulsion between the negatively charged mucin-like domain (the positive charge domains could attach to the negative mica surface by forming loop structure4) and the negatively charged polysaccharide, ultimately leading to the most exclusion of injected HA molecules from the rhPRG4 coated surface during the loading process. Further friction tests shown in Figure 4b suggest that the coefficient of friction, µ, increases slightly to ~ 0.11, while the maximum load that the surfaces can sustain is doubled and increases to 35 mN when the shear velocity is kept at 0.14 µm/s., which is consistent with previous study based on 14


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grafted HA and native PRG4 that enhanced wear protection and lubrication is observed after adding HA.21 For the case of injecting low concentration (90 μg/ml) of rhPRG4, an independent-HA injection experiment also showed that there was no obvious accumulation on pre-adsorbed rhPRG4 surface after adding HA solution. However, the HA could enhance the sustainability of higher normal load on rhPRG4 coated mica without significantly changing the friction force (see supporting data of Figure S2).

Figure 4. Normal force and friction results of rhPRG4 coated mica surfaces after conducting friction measurement followed by injecting HA solution; (a) normal force as a function of separation distance. The inset figure shows the schematic illustration of the possible conformation for rhPRG4 and HA; (b) friction as a function of normal load. 3.2 Normal force and lubrication behaviors of rhPRG4-HA mixture confined between mica surfaces To further understand the potential synergistic effect between rhPRG4 and HA, a pre-mixed rhPRG4-HA solution was directly injected onto fresh mica surfaces for friction measurement (results are shown in Figure 5). It was observed that after injecting the lubricin-HA mixture, a thin layer of film with the thickness of ~ 20 nm was confined between mica surfaces during the approaching-separation process (see Figure 5a). The repulsion range (~ 140 nm) is smaller than the case when rhPRG4 alone was injected into two mica surfaces (Figure 1a). However, unlike the case 15



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of injecting rhPRG4 alone, the approaching and separation curves showed much smaller hysteresis, exhibiting polymer-brush like behavior attributed to the conformation change of rhPRG4 after mixing with HA. Pre-mixing of HA and rhPRG4 in solution could possibly alter the charge distribution of the rhPRG4 since HA is negatively charged while rhPRG4 is positively charge at the two ends, thus the conformation and the amount of adsorbed rhPRG4 from this mixture on mica would also be different from using rhPRG4 alone. Figure 5b shows the measured friction force as a function of applied normal load. Linear fitting shows that the coefficient of friction of rhPRG4+HA µ is ~ 0.10, which is close to that of the case with injecting a solution of rhPRG4 alone at high (450 μg/ml) concentration. Figure 5c shows the normal force results measured after friction testing, and it was found that the film thickened by more than 50% after sliding, which was also demonstrated by the sharp FECO change shown in Figure 5f. The in situ FECO pattern shown in Figure 5e suggested that the flat mica-mica contact also becomes uneven during sliding, which also indicates a shear-induced clustering or aggregation of rhPRG4/HA molecules. The normal force results shown in Figure 4a and 4c together with the FECO change shown in Figure 5f do demonstrate that the rhPRG4+HA mixture coated mica could not sustain high load during sliding.

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Figure 5. Pre-mixed rhPRG4-HA solution after being injected in between two atomically smooth mica surface (a) normal force results (b) friction force results (c) normal force results after shearing at the same position (d-f) in situ FECO image change during loading process, surface is apparently not flat during sliding.

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3.3 Friction tests with HA solution followed by addition of rhPRG4

Figure 6. (a-b) Force-distance profiles obtained after injecting HA solution (3.3 mg/mL in 0.1M DPBS buffer) in between mica surfaces for (a) 5 min and (b) 80 min; (c) Force-distance curves obtained at the same location after injecting HA and followed by injecting 450 μg/ml rhPRG4, (d) friction force f, as a function of normal load F ⊥ for two mica surfaces across HA & rhPRG4 DPBS solution, the shear velocity is 0.14 μm/s, the inset figure shows the in situ FECO image when the 18


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friction force increase significantly, (e) typical friction force (2f) versus time traces with a normal load of 4.2 mN, where the friction force increase significant when surface deformation occurred.

To further explore the effect of HA on the lubrication performance of rhRPG4, another control SFA experiment was conducted by first injecting HA followed by adding rhPRG4 in between the mica surfaces. Figure 6a-6b shows the force-distance profile after injecting HA solution with the soaking time being 5 min and 80 min. It was found that the force-distance profile changes with soaking time, while the confined film thickness was less than 2 nm even after immersing for 80 min, suggesting that HA does not naturally adsorb onto negatively charged mica surfaces. During approaching, the HA molecules was mostly extruded from two mica surfaces, while slightly adhesion force (< 0.5 mN/m) was measured, probably due to the bridging effect of confined HA molecules between mica surfaces. The results are consistent with previous reports on studying the adsorption of HA on mica, where it was found that HA does not naturally adsorb onto hydrophilic mica surface.22 After injecting rhPRG4, the thickness of confined film quickly increased to ~20 nm in less than 10 min. As shown in Figure 6c, small adhesion hysteresis was also measured during the loading-unloading process and the measured range of repulsion also increased to ~ 100 nm. The measured repulsion range is slightly decreased compared to injecting rhPRG4 (450 μg/ml) between fresh mica surfaces, which can be explained by the decreased amount of adsorbed rhPRG4 on mica surface. Interestingly, the lubrication result (Figure 6d) shows that there are also two lubrication regions during sliding. The friction coefficient μ was quite small (~ 0.008) when the exerted load was small (F⊥ < 4.2 mN), and a transition happened when higher load ((F⊥ > 4.2 mN)) was applied, with the friction coefficient quickly increasing to ~ 0.07. The in situ FECO image proved that the surface become uneven when the load was higher than 4.2 mN (see inset of Figure 6d). The friction force vs. time trace (Figure 6e) shows the significant increase of friction due to the aggregation of rhRPG4 molecule on mica surface, and large static-friction force with stick-slip like spikes are measured when the contacted surface deformed. The control experiment based on pre-adsorbed HA surface demonstrates that the formation of rhPRG4 layer on mica could be affected by the free HA, 19



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even though there is no obvious adsorption of HA on hydrophilic mica, which provide indirect evidence for the possible interaction between HA and rhPRG4. 3.4 Normal forces measured for rhPRG4 vs. HA, rhPRG4 vs. rhPRG4, HA vs. HA mica surfaces

Figure 7. Force-distance curves measured between (a) rhPRG4 modified mica vs. HA-modified mica (with APTES buffer layer) surface, (b) rhPRG4 modified mica vs. rhPRG4 modified mica, the inset figures shows the AdG fitting of the approaching force curve, (c) HA-modified mica vs. HA-modified mica (with APTES buffer layer) in 0.1 mM PBS buffer, (d) the Alexander-deGennes model fitting of the approaching force curve shown in (c). To better understand the molecular interactions between rhPRG4 and HA, the normal forces for rhPRG4 vs. HA, rhPRG4 vs. rhPRG4, HA vs. HA mica surfaces were also measured and shown in Figure 7. Noting that in order to prepare a stable HA surface, the mica surfaces were first exposed to 20


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Aminopropyltriethoxysilane (APTES) vapor for 5h and then immersed in HA solution for 3 h to form a stable HA-coated mica surface through electrostatic interaction and amide linkage.21 As shown in Figure 7a, pure repulsion was observed between rhPRG4 and HA during approaching, which is most likely due to the steric repulsion and electrostatic repulsion between the extended polysaccharide chain of HA and O-linked glycosylated mucin domain of rhPRG4. Slight adhesion was observed during separation, suggesting a weak interaction between rhPRG4 and HA. Previously reported results indicate that the native lubricin (PRG4) interacts with HA through cationic regions within the Somatomedin B (SMB) and Hemopexin (PEX) like domains, creating a network of entanglements within the HA matrix.5,

24, 45, 46

Also, other non-covalent interactions such as

hydrophobic interaction between the non-polar N- and C- terminals of lubricin and the non-polar regions of HA could also contribute to the entanglement between lubricin and HA. This adhesion could be mainly caused by non-covalent interactions including polymer chain diffusion, electrostatic interaction between oppositely-charged domains, hydrophobic interaction as well as hydrogen bonding.24 Conversely, pure repulsion was measured during both approaching and separation for the case of rhPRG4 vs. rhPRG4, and the two curves almost overlapped, suggesting the polymer-brush like behavior for rhPRG4-coated mica surfaces (see Figure 7b ). It is well known that the electric double layer will be greatly restrained at the salt concentration of ~ 150 mM, thus the double layer force can be mostly neglected at long separation distances. The force-distance curve for rhPRG4 vs. rhPRG4 was compared to the Alexander-deGennes model,47 as shown in Eq. 1 .

𝐹(𝐷) 𝑅

=

[

16𝜋𝑘𝑇𝐿 3

35𝑠

54

(2𝐿𝐷)

7

74

(2𝐿𝐷 )

+5

]

― 12

(Eq. 1)

In this equation, L stands for the thickness of one extended brush layer and s stands for the distance between grafting sites. Figure 7d indicates that the approaching force curve can be well fitted by brush thickness of L = 65.5 nm and the grafting distance of s = 12.7 nm. This value is in good agreement with a previously reported result based on extracted native human lubricin (PRG4) where L and s were reported to be 65 nm and 14 nm, respectively. The well fitted results indicate that the force between rhPRG4 coated surfaces is mainly due to steric-entropic force between the flexible lubricin chains which plays a dominant role for the relatively low friction during the whole loading 21



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process for friction test. For the case of HA vs. HA, as shown in Figure 7c, only pure repulsion is observed during both approaching and separation, and the repulsion range (~ 90 nm) is comparable with rhPRG4 vs. rhPRG4 and rhPRG4 vs. HA. Considering the high salt concentration of buffer (0.15 M salt) used for measurement, the long-range repulsion mainly come from the steric entropic repulsion between the extended polysaccharide chains. 3.5. Proposed lubrication interaction mechanism

Figure 8 Scheme of (a) few layers lubricin packing densely and (b) multi-layer lubricin confined between mica surfaces, and (c) adding HA resulting in the conformation change and adsorption behavior change of lubricin on mica surface. In previous studies, native lubricin was demonstrated to be an effective lubricant for model surfaces (e.g., mica) by forming a low friction boundary layer composing of brush-like layers of loops and tails elongated away from the surface, and the steric-entropic repulsion between the boundary layers is believed to be the microscopic origin of the lubrication function. However, the lubrication failure occurs at a relatively low load (contact pressure P > 0.4 MPa) and a larger friction coefficient (μ > 0.2) is measured under high salt condition when P > 0.4 MPa (buffer salt concentration >100 mM).5, 6, 48

22


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Unlike the results obtained previously,5, 6 a much lower friction coefficient was measured in this study, noting that DPBS buffer with a total salt concentration of 0.15M was used (chosen in this study for consistency with previous macro-scale cartilage-cartilage tribology tests

14, 15)

thus the

double layer force can be mostly restrained, especially at long separation distances. The SFA results here show that a low friction coefficient (μ < 0.04) is observed across the whole load region (0 – 2.8 MPa) with full length recombinant human lubricin (rhPRG4) being injected. For the case of injecting a lower concentration of rhPRG4 (90 μg/ml), a surprisingly low friction (μ < 0.005) with a small static friction force is achieved at medium loads (P < 3.6 MPa), and the friction coefficient is still below 0.1 at high load (P > 4.5 MPa). The reason for the better performance of rhPRG4 and the lubrication mechanism could be explained as follows. Firstly, it is reported that native lubricin may interact with other components in the synovial fluid, such as lipid and other proteins, making it difficult to obtain highly purified native lubricin (PRG4).9, 31, 44

However, the highly purified rhPRG4 in this study minimized the effect of interaction between

lubricin and other biomolecules that are difficult to remove when purifying from synovial fluid; reducing the competitive-adsorption between rhPRG4 and other components onto mica. The simplified lubrication system also helps better understand the lubrication mechanism. Secondly, the surface property of mica and the charge property of rhPRG4 can also play important roles in the mechanical study. Mica is one type of layered mineral comprising sheets of octahedral

hydroxyl-aluminum sandwiched between two silicon tetrahedral layers,41 and the atomic-smooth mica is negatively charged in 1 × DPBS buffer. Previously reported results indicate that the particular

charge distribution of lubricin (PRG4) can promote the adsorption of the two positively charged globular ends of the molecule, forming “loop” or “tail” structure rather than a random adsorption of all parts of the molecule. Full-length rhPRG4 shares similar protein structure as that of native PRG4 which, when anchored, promotes a polymer-brush-like structure, especially when the isoelectric point of rhPRG4 is ~ pH 4. The “loop/tail” structure with heavily charged and hydrated mucin domains (see Figure 8a, the rhPRG4 molecular contains a large amount of O-link glycosylations) which greatly reduces the friction force during sliding.4-6 Thus a uniform, well anchored rhPRG4 layer, with polymer-brush-like structures can be obtained, which show very good shear resistance 23



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(see Figure 8a, 8b), especially at low concentration of rhPRG4, as demonstrated by the AFM images (see Figure 3) and flat FECO images under high load (see Figure 2). Previous research using neutron reflectometry has proved that the degree of hydration and level of surface coverage play import role for the lubrication performance of lubricin. The very low friction requires the formation of a very specific and highly ordered "telechelic" polymer brush, or molecular loops with sharp brush-brush interface under compression.4, 49 The two friction regions with different values of friction coefficient (Figure 2b), the large static friction force at high load (Figure 2e-h) as well as the shear velocity dependent friction force (Figure S1) support the formation of a well anchored polymer-brush layer on mica after injection of rhPRG4. In the low load regime, good brush-brush lubrication is provided by the hydration layers surrounding the charged mucin domains.5, 50-52 At the critical pressure, the interpenetration of polymer chains suddenly increases and the friction increases. It is worth noting that the well anchored lubricin with very low friction coefficient (μ < 0.005) is seldom observed when studying the adsorption and lubrication of native lubricin (e.g., human lubricin or bovine lubricin). It should be pointed out that the mean pressure on hip joints during the peak of a normal walking cycle is ~ 5 MPa although local pressures of ~ 20 MPa can be achieved.53 For this study, the applied normal load (0 ~ 4 MPa) is close and comparable to that of walking pressure for joint lubrication. The SFA results obtained at low shear rate (0.14 µm/s) can represent the low-shear-rate regime when slow join lubrication occured in our daily life (e.g., slow motions), since the actual sliding speed of cartilage may span a much broader range (from several nm per second to hundreds of mm per second or even higher). For detailed information on the grafting density and conformation of rhPRG4, other techniques such as Neutron reflectometry might be necessary for further study.49 Thirdly, the pre-adsorption of lubricin on mica can inhibit further adsorption of HA, which can lead to the increase of friction coefficient, but these surfaces appear able to sustain a higher normal load (Figure 4 and Figure S2). On the other hand, although HA did not bind to hydrophilic mica surface naturally, the free HA molecule could still affect the adsorption and the conformation of rhPRG4 on mica surface (see Figure 8c), as evidenced by the reduced repulsion range (< 100 nm, see Figure 6c) and lower load bearing property (Figure 6d). These results suggest that there is potential synergistic interaction between rhPRG4 and HA during sliding, which is consistent with recent study based on native lubricin and HA related derivatives.26, 54 24


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4.

Biomacromolecules

Conclusions In this study, systematic evaluation was carried out to characterize the adsorption and lubrication

behavior of the recently available, and clinically effective, full-length recombinant human rhPRG4 on mica as well as the interaction mechanism between rhPRG4 and HA. It was found that rhPRG4 (~ 450 μg/ml) exhibits a low friction coefficient (µ ~ 0.04) when confined between mica surfaces. Intriguingly, ultra-low friction coefficient (μ < 0.005) was observed at low sliding velocity (v = 0.14 μm/s) with the applied load P reaching ~ 3.6 MPa when diluted rhPRG4 solution (~ 90 μg/ml) was injected. Adding HA afterwards enables the higher sustainability of the surface to higher loads, while the friction coefficient increases slightly to ~0.1. Our SFA results demonstrate that the formation of a spontaneously well anchored lubricin layer on mica is the prerequisite for achieving low friction force. Normal force measurements revealed a very weak attraction between pre-adsorbed HA layer and rhPRG4-coated mica surface, which was most probably due to non-covalent interactions between the polymer structures. Our results provide fundamental insight into the adsorption and lubrication behavior of the full-length human lubricin (rhPRG4) and the polysaccharide HA on model mica surfaces, which contribute to the understanding of the underlying mechanism of ultra-low friction for synovial fluid with implications for development of biomimetic lubricants.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The Supporting Information shows the friction force f, as a function of sliding velocity v, at fixed normal load after injecting 90 μg/ml rhPRG4 in 0.1 M DPBS buffer between two mica surfaces. And the measured friction force as a function of normal load for injecting low concentration of rhPRG4 (~ 90 μg/ml) between two mica surfaces, followed with injecting HA solution (~ 3.3 mg/ml).

25



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5. Acknowledgement This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation, the Canada Research Chairs Program (H. Zeng) and the Qilu Young Talented Scholar Program of Shandong University (J. Huang).

Authors contributions: J.H., T.S. and H.Z designed research; J.H., X.Q. and L. X. performed research; J.H., X.Q. and L. X. analyzed data; J.H., X.Q., L.X., G. J. ,T.S and H.Z wrote and revised the paper.

§

J. Huang and X. Qiu contributed equally to this work.

Conflict of Interest: TS and GJ hold patents on rhPRG4 and equity in Lμbris BioPharma, LLC, and TS is a paid consultant for Lμbris BioPharma, LLC. All other authors declare no competing interests.

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Biomacromolecules

TOC GRAPHICS

Probing the Molecular Interactions and Lubrication Mechanisms of Purified Full-length Recombinant Human Proteoglycan 4 (rhPRG4) and Hyaluronic Acid (HA) Jun Huang, Xiaoyong Qiu, Lei Xie, Gregory D. Jay, Tannin A. Schmidt, Hongbo Zeng

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Probing the Molecular Interactions and Lubrication Mechanisms of

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