Poly(Dimethylsiloxane

Jul 28, 2005 - ... specific adsorption behavior at the water/PDMS interface. The lubrication mechanism of the self-mated sliding contact of PDMS by me...
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8344

Langmuir 2005, 21, 8344-8353

Porcine Gastric Mucin (PGM) at the Water/ Poly(Dimethylsiloxane) (PDMS) Interface: Influence of pH and Ionic Strength on Its Conformation, Adsorption, and Aqueous Lubrication Properties Seunghwan Lee,† Markus Mu¨ller,† Kurosch Rezwan,‡ and Nicholas D. Spencer*,† Laboratory for Surface Science and Technology, Department of Materials, Swiss Federal Institute of Technology (ETH-Ho¨ nggerberg), Wolfgang-Pauli-Strasse 10, CH-8093 Zu¨ rich, Switzerland Received March 24, 2005. In Final Form: May 23, 2005 We have investigated the influence of pH and ionic strength on the conformation of porcine gastric mucin (PGM) in bulk aqueous solution, its adsorption behavior onto poly(dimethylsiloxane) (PDMS) surfaces, and its lubricating behavior upon the self-mated sliding contact of a PDMS tribopair by means of circular dichroism (CD) spectroscopy, optical waveguide lightmode spectroscopy (OWLS), and pin-on-disk tribometry, respectively. In a low-concentration regime (1 mg/mL), where the formation of a mucus-gel is generally excluded, PGM is still observed to exhibit effective aqueous lubricating properties under specific conditions of acidic pH and low ionic strength. This behavior was closely correlated with specific conformations in the bulk solution as well as specific adsorption behavior at the water/PDMS interface. The lubrication mechanism of the self-mated sliding contact of PDMS by means of surface modification with PGM is discussed in terms of isoviscous-elastic/soft-elastohydrodynamic lubrication (soft-EHL).

1.Introduction 6

Mucins are high-molecular-weight (>10 Da) glycoproteins that are components of the slimy mucus secretion that covers epithelial cell surfaces.1-3 They are found in various internal organs, including the digestive, respiratory, and reproductive systems of vertebrates and are known to be primarily responsible for the protective and lubricious properties of the viscoelastic mucous barrier.1-3 Although the detailed compositions and structures of mucins are diverse depending on their origin, they display some common features; mucin is composed of a linear peptide backbone and radially arranged oligosaccharide chains (70-80 wt %1-6). The glycosylated regions are enriched with serine or threonine, which are linked to oligosaccharides (typically less than 20 sugar units1,4) via bonding with GalNAc (O-glycosylation),1-3 while the interjacent amino acids usually consist of small residues such as proline, glycine, and alanine.2 The unglycosylated regions, which are normally found at both C and N termini, contain a large number of cysteines and charged amino acids.1-3,5 The characteristic species that determine the electrostatic properties of mucins are present both within the polypeptide backbones, e.g., glutamic acid and aspartic * Author to whom correspondence should be addressed. E-mail: [email protected]. Telephone: +41 1 632 58 50. Fax: +41 1 633 10 27. † Laboratory for Surface Science and Technology. ‡ Institute of Nonmetallic Materials, Department of Materials, Swiss Federal Institute of Technology (ETH-Ho¨nggerberg), Wolfgang-Pauli-Strasse 10, CH-8093 Zu¨rich. (1) Bansil, R.; Stanely, E.; LaMont, J. T. Annu. Rev. Physiol. 1995, 57, 635-657. (2) Strous, G. J.; Dekker, J. Crit. Rev. Biochem. Mol. Biol. 1992, 27, 57-92. (3) Van den Steen, P.; Rudd, P. M.; Dwek, R. A.; Opdenakker, G. Crit. Rev. Biochem. Mol. Biol. 1998 33, 151-208. (4) Turner, B.; Bhaskar, K. R.; Hadzopoulou-Cladaras, M.; Specian, R. D.; La Mont, J. T. Biochem. J. 1995, 308, 89-96. (5) Silberberg A. Biorheology 1987, 24, 605-614. (6) Cao, X.; Bansil, R.; Bhaskar, K. R.; Turner, B. S.; LaMont, T.; Niu, N.; Afdhal, N. H. Biophys. J. 1999, 76, 1250-1258.

acid residues (pKa ≈ 4),6 as well as in the oligosaccharide side chains, e.g., sialic acid residues (pKa ≈ 2.6) and sulfate groups (pKa ≈ 1).7 Many unique properties of mucins are closely associated with their gel-forming properties. Previous studies have shown that the aggregation of mucins depends on several parameters, including type, purity, and concentration of mucin as well as physiological environments such as pH and ionic strength.1,6-12 At physiological concentration (g 20 mg/mL1), for instance, mucins generally form a gellike structure at low pH, which is particularly important for gastric mucins in order to protect the gastric epithelial surfaces during HCl secretion. Intra- and/or intermolecular disulfide bonding has been suggested as the main driving force for such behavior because the treatment of mucins with cysteine-specific enzymes or reducing agents such as dithiolthreitol (DTT) prevents the aggregation of mucins.1,8 Meanwhile, electrostatic interaction, often involving carbohydrate side chains,8,10 has also been argued as being a contributing factor to the aggregation of mucins because the addition of high-concentration salt has also been observed to prevent the formation of gellike structures.8 At low concentrations, however, mucins generally do not exhibit such extensive aggregation, regardless of pH and ionic strength; for instance, porcine gastric mucin (PGM) solution is characterized as “dilute” at concentrations lower than 1.5 mg/mL and as “semidilute” between 1.5 and 4.4 mg/mL.7 However, mucins undergo some interesting conformational changes upon modification of the aqueous environment. For instance, (7) Waigh T. A.; Papagiannopoulos, A.; Voice, A.; Bansil, R.; Unwin, A. P.; Dewhurst, C. D.; Turner, B.; Afdhal N. Langmuir 2002, 18, 71887195. (8) Bhaskar, K. R.; Gong D.; Bansil R.; Pajevic, S.; Hamilton J. A.; Turner, B. S.; La Mont, T. Am. J. Physiol. 1991, 261, G827-G832. (9) Davies, J. M.; Viney, C. Thermochim. Acta 1998, 315, 39-49. (10) Sellers, L. A.; Allen, A. Carbohydr. Res. 1988, 178, 92-110. (11) Kocevar-Nared, J.; Kritl J.; Smid-Korbar, J. Biomaterials 1997, 18, 677-681. (12) Durrer C.; Irache J. M.; Duchene, D.; Ponchel, G. J. Colloid Interface Sci. 1995, 170, 555-561.

10.1021/la050779w CCC: $30.25 © 2005 American Chemical Society Published on Web 07/28/2005

PGM at the Water/PDMS interface

according to the dynamic light scattering study by X. Cao et al.,6 the hydrodynamic radius, RH, of PGM was observed to increase with decreasing pH at concentrations lower than 5 mg/mL, which was attributed to the conformational change from a random coil at pH g 4 to an anisotropic, extended conformation at pH < 4. In this work, we are particularly interested in mucin’s lubricating properties at an elastomeric sliding contact in an aqueous environment. As with its other protective properties, the high viscosity of mucus-gel is also believed to play a significant role in the lubricating behavior of mucins. However, some previous studies have shown that mucin exhibited effective boundary-lubricating properties even at low concentrations, where gel formation is generally excluded.13 This behavior is closely associated with the mucin’s structure, adsorption behavior, and consequent conformation at the liquid/solid interface; while the unglycosylated, hydrophobic regions of mucins bind onto hydrophobic surfaces, the glycosylated, hydrophilic regions stretch out into aqueous media because of favorable interactions with water.12,14-19 This conformation of mucins at the water/hydrophobic interface in fact resembles that of amphiphilic copolymers, e.g., poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO).20 In previous studies,21,22 we have shown that PEO-PPO-PEO can behave as an effective additive for the aqueous lubrication of a hydrophobic elastomer, e.g., poly(dimethylsiloxane) (PDMS). Similar aqueous lubricating properties can be expected from mucin as a result of its similar conformation at the water/hydrophobic interface. However, it is also clear that the detailed adsorption and lubricating behavior of mucin would be more complicated due to its polyanionic characteristics, which may exhibit a strong dependence on both the pH and the ionic strength of the mucin solution. In this study, we have employed PGM as a test mucin and self-mated PDMS as a tribopair to investigate mucin’s aqueous lubricating properties in an elastomeric tribosystem. The selection of PDMS was mainly motivated by its elasticity (elasticity modulus, typically e 2 MPa) and consequent resemblance to biomechanical contacts. The pH- and ionic-strength-dependent lubricating properties of PGM have been correlated both with its conformation in bulk aqueous solution and its adsorption behavior at the water/PDMS interface, studied by means of circular dichroism (CD) and optical waveguide lightmode spectroscopies (OWLS), respectively. 2. Materials and Method 2.1. Biopolymers. The biopolymers employed in this work include porcine gastric mucin (PGM), dextran, and hyaluronic acid sodium salt (HA). PGM (Type II: crude, from porcine stomach, bound sialic acids 1%) and dextran (average MW 850011 000) were purchased from Sigma (St. Louis, MO), and HA (13) Cassin, G.; Heinrich, E.; Spikes, H. A. Tribol. Lett. 2001, 11, 95-102. (14) Shi, L.; Caldwell, K. D. J. Colloid Interface Sci. 2000, 224, 372381. (15) Perez, E.; Proust, J. E. J. Colloid Interface Sci. 1987, 118, 182191. (16) Malmsten, M.; Blomberg, E.; Claesson P.; Carlstedt, I.; Ljusegren, I. J. Colloid Interface Sci. 1992, 151, 579-590. (17) Baszkin, A.; Proust, J. E.; Boissonnade, M. M. Biomaterials 1984, 5, 175-179. (18) Castillo, E. J.; Koenig, J. L.; Anderson, J. M.; Jentoft, N. Biomaterials 1986, 7, 9-16. (19) Lindh, L.; Glantz, P.-O.; Carlstedt, I.; Wickstro¨m, C.; Arnebrant, T. Colloids Surf., B 2002, 25, 139-146. (20) Alexandridis, P.; Hatton, T. A. Colloids Surf., A 1995, 96, 1-46. (21) Lee, S.; Iten, R.; Mu¨ller, M.; Spencer, N. D. Macromolecules 2004, 37, 8349-8356. (22) Lee, S.; Spencer, N. D. Tribol. Int. 2005. In press.

Langmuir, Vol. 21, No. 18, 2005 8345 (from Streptococcus equi sp.) was purchased from Fluka (Switzerland). All biopolymers were used as received. 2.2. Buffer Solutions. Buffer solutions were prepared by dissolving 1 mM KH2PO4 (Sigma, St. Louis, MO) in distilled water and the pH was adjusted by 36-38% HCl (J. T. Baker, Switzerland) or 6 M KOH (Merck, Switzerland). For a group of buffer solutions with varying pH (2, 4, 7, 10, 12), 0.1 M KCl (Fluka, Switzerland) was added to maintain the ionic strength nearly constant. For pH 2, 7, and 12, another group of buffer solution was prepared by varying ionic strength (KCl, 0, 0.01, 0.1, and 1.0 M for each pH). Biopolymers were freshly dissolved in aqueous buffer solutions at a concentration of 1 mg/mL prior to all measurements. 2.3. Zeta (ζ) Potential Measurements. The isoelectric point (IEP) of PGM has been characterized by means of ζ potential measurements as a function of pH. In this approach, we have dispersed particles with a well-defined specific surface area and IEP into a series of PGM solutions with various concentrations. TiO2 and Al2O3 have been employed as particles. Given that the concentration of particles is fixed, the shift of the IEP of the suspension was monitored as a function of PGM concentration. TiO2 powder (Kronos 1171, anatase >99%, density 3.8 g/cm3) was purchased and annealed for 4 h at 400 °C to remove organic residues. Al2O3 powder was purchased from Taimei (TM-DAR, alumina >99.99%, density 3.98 g/cm3) and annealed under the same conditions. The specific surface area was measured by the BET method using a NOVA 1000 device (Quantachrome, Boyton Beach, FL). The specific surface area of TiO2 was found to be 6.37 m2/g, whereas the specific surface area of Al2O3 was 13.37 m2/g. The spherical particle morphology was verified by scanning electron microscopy (SEM). For the reference measurements, two 40 mL suspensions with TiO2 and Al2O3 (1% v/v) were prepared and sonicated with an ultrasound horn for 5 min. After sonication, the suspension was left under stirring conditions for 2 h, after which the ζ potential was measured as a function of pH. The acid and base solutions used to change the pH were 0.1 N HNO3 and 0.1 N KOH, respectively. The ζ potential was measured by means of an electroacoustic technique23 using a DT 1200 device (Dispersion Technology Inc., Bedford Hills, NY). The device is equipped with an automatic titration unit. For each measuring point, an equilibration time of 30 s was allowed. The PGM suspensions were prepared in a similar way except that, to predissolve the PGM, 20 mL of water were subtracted from the total suspension volume. The suspension was prepared with approximately half the amount of water, ultrasonicated, and then the 20-mL PGM solution was added. After stirring for 2 h, the ζ potential measurements were started. The amount of PGM needed was normalized to the powder surface area to make the measurements comparable. Three PGM suspensions with 250, 500, and 1000 ng of PGM with respect to unit area (cm2) of the oxide particles were prepared. 2.4. Viscosity Measurements. The viscosities of PGM solutions with varying pH and ionic strength were measured using a UDS 2000 Rheometer in Couette geometry (Physica Messtechnik, Ostfildern, Germany). The viscosity was obtained from the slope of the linear part (from 102 to 103 s-1) of the shear strain versus shear rate plot. 2.5. Circular Dichroism (CD). Circular dichroism (CD) spectra were recorded using a spectropolarimeter (JASCO J-715 Omnilab, Tokyo, Japan). A cuvette of size 10 mm × 1 mm (path length d ) 1 mm) was used for far-UV-region measurements (wavelength, 195-250 nm) and a size of 10 mm × 10 mm (path length d ) 10 mm) was used for near-UV-region measurements (wavelength, 250-350 nm). The scanning speed was 20 nm/m, response 2 s, bandwidth 2.0 nm, accumulation 5, and the data pitch 0.2 nm. For each pH and ionic strength, polymer-free buffer solution was measured first to set the baseline. 2.6. Optical Waveguide Lightmode Spectroscopy (OWLS). Optical waveguide lightmode spectroscopy (OWLS) is based on grating-assisted in-coupling of a He-Ne laser into a planar waveguide coating (200-nm thick Si0.75Ti0.25O2 waveguiding layer on 1-mm thick AF 45 glass, MicroVacuum Ltd, Budapest, (23) Rezwan, K.; Meier, L. P.; Rezwan, M.; Vo¨ro¨s, J.; Textor, M.; Gauckler, L. J. Langmuir 2004, 20, 10055-10061.

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Figure 1. ζ potential measurements of (a) PGM-TiO2 and (b) PGM-Al2O3 suspensions in water as a function of pH. Hungary). In this approach, adsorbed mass is obtained by probing the change in refractive index in the vicinity of the surface as a result of adsorption of molecules from solution. This method is highly sensitive out to a distance of ∼200 nm from the surface of a waveguide. Furthermore, a measurement-time resolution of 3 s allows the in situ, real-time study of adsorption kinetics. All OWLS experiments were carried out in a BIOS-I instrument (ASI AG, Zu¨rich, Switzerland) using a Kalrez O-ring and flowthrough cell (8 mm × 2 mm × 1 mm, Dupont, Wilmington, DE). More detailed information on the operational principles of OWLS is to be found in the literature.21,24,25 In this work, the waveguides were coated with an ultrathin PDMS film using a spin-coater in order to characterize the adsorption behavior of PGM and other biopolymers onto PDMS surfaces in aqueous buffer solutions. The base and curing agent of a commercial silicon elastomer (SYLGARD 184 elstomer kit, Dow Corning, Midland, MI) were dissolved in hexane at a ratio of 10:3 (total concentration, 0.5% w/w) and spin-coated onto a waveguide. After spin coating at 2000 rpm for 40 s, the waveguides were cured in an oven at ∼70 °C overnight. The thickness of the thin PDMS film generated by this method was characterized by ellipsometry and found to be ∼30 nm. The static water contact angle was 100°((2°). The PDMS-coated waveguide was first exposed to buffer solution until a stable baseline was obtained. Then, biopolymer solution was injected into the flow cell. After rinsing the flow cell with buffer solution, adsorbed mass density data were calculated according to de Feijter’s equations.26 A typical refractive index increment (dn/dc) value for protein, 0.182 cm3/g,27-29 was used for the calculation of all biopolymer adsorption masses. 2.7. Pin-on-Disk Tribometry and Tribopair. The lubricating properties of PGM solution have been characterized by means of pin-on-disk tribometry (CSM, Neuchaˆtel, Switzerland).21,22 In this approach, a loaded pin was allowed to form a contact with a disk, and the sliding friction forces between them were measured while rotating the disk at controlled speeds. The load was controlled by dead weight and the sliding speed by a motor underneath the disk. The friction force generated during sliding contact was monitored by a strain gauge. The raw data thus consists of friction forces measured as a function of time or the number of rotations over a fixed track. By varying load or speed, load- and/or speed-dependent frictional behavior of a given tribosystem can be investigated. In this work, sliding friction forces for the self-mated sliding of PDMS lubricated by PGM or other biopolymer solutions at various values of pH and ionic strength have been measured as a function of speed (from 1 to 100 mm/s). The applied load was 1 N throughout this work. Some measured and calculated (24) Kurrat, R.; Textor, M.; Ramsden, J. J.; Bo¨ni, P.; Spencer, N. D. Rev. Sci. Instrum. 1997, 68, 2172-2176. (25) Ho¨o¨k, F.; Vo¨ro¨s, J.; Rodahl, M.; Kurrat, R.; Bo¨ni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf., B 2002, 24, 155-170. (26) Ramsden, J. J. J. Stat. Phys. 1993, 73, 835-877. (27) Kenausis, G. L.; Vo¨ro¨s, J.; Elbert, D. L.; Huang, N.-P.; Hofer, R.; Ruiz-Taylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 104, 3298-3309. (28) Huang, N.-P.; Vo¨ro¨s, J.; De Paul, S. M.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 220-230. (29) Pasche, S.; De Paul, S. M.; Vo¨ro¨s, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 19, 9216-9225.

mechanical parameters for the tribopair are presented in Table 2. The average friction over a defined number of rotations (20) was obtained at each speed, and thus the coefficient of friction, µ (µ ) friction/load), versus speed plot was obtained. The influence of pH and ionic strength on the lubrication properties of PGM have been characterized by comparing µ versus speed plots obtained at each condition. In some cases, the friction forces in the initial few rotations showed a characteristic change (“runningin” behavior), depending on the pH and ionic strength of the PGM buffer solution employed, yet exhibited a steady kinetic friction force, Fk, after no more than 5 rotations. For µ versus speed plots, the latter half of the total number of rotations (11th to 20th) was taken into account to eliminate the “running-in” effect. Nevertheless, the “running-in” period often contains significant information on the tribological behavior of a given system, and thus was also carefully examined. Friction measurements were started 30 min after the polymer solutions were transferred into the cell in which the pin/disk contact was to occur, to allow the adsorption of the biopolymers onto the tribopair surfaces. PDMS has been employed for both pin and disk materials using the same commercial silicon elastomer kit used for the PDMS ultrathin film coating on the OWLS waveguide described above. The base and curing agent were thoroughly mixed at a 10:1 ratio (w/w), and the foam generated during mixing removed using a gentle vacuum. Then the mixture was transferred into the molds for pin and disk and cured in an oven at ∼70 °C overnight. A commercial polystyrene cell culture plate with roundshaped wells (96 MicroWell Plates, NUNCLON Delta Surface, Roskilde, Denmark) served as the mold for the hemispherical PDMS pin, and a home-machined aluminum plate with flat wells was used as the mold for the flat PDMS disk. The dimensions of the pin and disk fabricated in this way were 3 mm (radius) for the pin and 30 mm (diameter) × 5 mm (thickness) for the disk. The static water contact angle measured on the PDMS disk was 110°(( 2°). The roughness of the PDMS disk (air side during curing process) was characterized as Ra ∼ 0.5 nm/100 µm2 by atomic force microscopy (Dimension 3000, Digital Instruments, Santa Barbara, CA). The roughness of the pin was estimated as Ra ∼ 2 nm/100 µm2 by measuring the morphology of the polystyrene template with AFM. Finally, it is noted that one pair of PDMS surfaces was used for only one measurement and was replaced by another pair for the next measurements to avoid cross-contamination. In a control experiment, the PDMS tribopair was oxygenplasma-treated for one min in a plasma cleaner/sterilizer PDC32G instrument (Harrick, Ossining, NY). Following oxygenplasma treatment, the water contact angle of the PDMS surface (ox-PDMS) was measured to be lower than 3°.

3. Results 3.1. ζ Potential Measurements. The results for the pH dependence of ζ potentials of the PGM-particle suspensions are presented in Figure 1. With increasing PGM concentration, the shift of IEP of the suspensions was monitored. Assuming that the surface coverage of the oxide particles with PGM is gradually increasing with increasing PGM concentration, the IEP of the suspension, which is initially determined by surface charge properties

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Figure 3. Near-UV CD spectra of PGM solution (1 mg/mL) as a function of ionic strength at pH 2, 7, and 12. Figure 2. Near-UV CD spectra of PGM solution (1 mg/mL) as a function of pH. Table 1. Viscosity of PGM Solution at Various Values of pH and Ionic Strength PGM concentrated (mg/mL)

pH

KCl concentrated (M)

viscosity (×10-4 Pa s-1)

1 1 1 1 1 1 1 1 1 1 1 1

2 2 2 2 7 7 7 7 12 12 12 12

0 0.01 0.1 1.0 0 0.01 0.1 1.0 0 0.01 0.1 1.0

9.1 9.2 9.1 9.3 9.2 9.1 9.2 9.1 9.2 9.2 9.0 9.1

of the native particles, would be progressively dominated by that of PGM. While the IEP of the PGM-TiO2 suspension remained virtually unchanged as a function of PGM concentration (∼2), the IEP of PGM-Al2O3 suspension showed a drastic change as a function of PGM concentration (from ∼9 for native Al2O3 particles to 2-3 for the highest concentration of PGM). This observation suggests that the IEP of PGM lies roughly between 2 and 3. This is consistent with a previous ζ potential measurement study of PGM in which carboxylate- and amino-groupfunctionalized polystyrene latex particles were employed for the suspension.12 3.2. Viscosity. The viscosity of PGM solution was measured as a function of pH and ionic strength. The results are summarized in Table 1. Under all conditions investigated, the viscosity of 1 mg/mL PGM solution was close to that of pure water (∼9 × 10-4 Pa‚s) and the values indistinguishable from each other. 3.3. Circular Dichroism (CD) Spectroscopy. FarUV (195-250 nm) CD spectra were initially obtained. At pH 7 (no KCl, total I ) 0.001 M), a broad spectrum with a negative minimum at ∼205 nm and a slightly positive shoulder at ∼225 nm was recorded (data not shown). However, the two characteristic negative minima (208 and 222 nm) expected for an R-helical protein structure or single negative minimum (215 nm) for β-sheet structure,30 respectively, were not observed. The far-UV CD spectra obtained at pH 2 and 12 (no KCl) were indistinguishable from that obtained at pH 7. Near-UV (250-350 nm) CD spectra of PGM solution as a function of pH are shown in Figure 2. For these buffer solutions, KCl was added to maintain the total ionic strength nearly constant (I ) ∼0.1 M). With respect to the spectrum at pH 7, a drastic change in the spectra was (30) Tiffany, M. L.; Krimm, S. Biopolymers 1969, 8, 347-359.

Figure 4. Representative plot of mass uptake vs time for the adsorption of PGM onto a hydrophobic PDMS surface by means of the OWLS technique (pH 2, 7, 12, and 0.1 M KCl for all cases).

observed by varying pH; by changing pH in the acidic direction, the positive maximum peak wavelength of ∼270 nm at pH 7 shifted to ∼274 nm (pH 4) and ∼288 nm (pH 2) with apparently decreasing intensity. By changing pH in the alkaline direction, however, the maximum peak position remained at ∼270 nm (pH 10) or shifted to ∼276 nm (pH 12) without a significant reduction in intensity. Near-UV CD spectra were also obtained at pH 2, 7, and 12 at various values of ionic strength (0, 0.01, 0.1, and 1.0 M KCl). The results are presented in Figure 3. Given that the pH of PGM solution is fixed, the variation of ionic strength did not influence the near-UV CD spectra. 3.4. Optical Waveguide Lightmode Spectroscopy (OWLS). In Figure 4, representative plots of the mass uptake versus time for PGM solution at three different pHs, 2, 7, 12 (KCl ) 0.1 M), are presented. Upon exposure of a PDMS-coated waveguide to a PGM solution, at t ) 0 min, the mass uptake showed a rapid increase in the initial 10 min, followed by a slower, yet continuous increase, even after 30 min. To keep the adsorption time identical with that for the tribological measurements, the flow-cell was rinsed with buffer solution at t ) 30 min. The mass of adsorbed PGM was obtained by monitoring the mass at t ) 40 min. At this specific ionic strength (total I ) ∼0.1 M for all three pHs), the adsorbed amount of PGM from pH 7 buffer solution (262.7 ( 38.7 ng/cm2) was distinctly higher than that from either pH 2 (122.0 ( 37.0 ng/cm2) or pH 12 (109.7 ( 11.7 ng/cm2) buffer solutions (100 ng/cm2 corresponds to 1 mg/m2). The average and standard deviation were obtained from three separate measurements. The OWLS measurements were further carried out as a function of ionic strength (KCl, 0, 0.01, 0.1, and 1.0 M) at three different pH values, 2, 7, and 12. At each pH, the mass of adsorbed PGM is plotted against the total ionic strength in Figure 5. At pH 7, the mass of adsorbed PGM showed an increasing trend at low and medium ionic strength (maximum 262.7 ( 38.7 ng/cm2 at

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Figure 5. Adsorbed mass of PGM vs ionic strength at pH 2, 7, and 12 as characterized by the OWLS technique.

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Figure 7. µ vs speed plots of the self-mated sliding of PDMS lubricated by a PGM solution at pH 7 with varying ionic strength.

Figure 6. Adsorbed mass of hyaluronic acid (HA) and dextran onto PDMS surface from pH 7 and pH 2 aqueous solutions (no additional KCl). The corresponding masses of PGM for each condition are also presented for comparison.

Figure 8. µ vs speed plots of the self-mated sliding of PDMS lubricated by a PGM solution at pH 2 with varying ionic strength.

KCl 0.1 M, as mentioned above), yet exhibited a drastic drop (49.1 ( 18.6 ng/cm2) at the highest salt concentration (1.0 M KCl). Meanwhile, at pH 2, the mass of adsorbed PGM was ∼120 ng/cm2 and showed no significant change except at the highest ionic strength (I ) 1.01 M), where the adsorbed mass dropped to roughly 30% (38.7 ( 12.7 ng/cm2) of the values at lower ionic strengths (I e 0.1 M). Finally, at pH 12, the mass of adsorbed PGM showed a continuously increasing trend with increasing ionic strength (maximum 132.0 ( 35.5 ng/cm2 at KCl 1.0 M) and did not show a decrease even at the highest salt concentration. In Figure 6, the adsorbed mass of HA and dextran at pH 7 and 2 obtained by the same experimental protocol as with PGM are presented. Buffer solutions with no additional KCl were used at both pH values. For comparison, the adsorbed masses of PGM under the corresponding conditions are also presented. The adsorbed masses of HA for pH 7 and 2 were 4.1 and 0.7 ng/cm2, respectively, and those for dextran were 10.5 and 1.2 ng/ cm2, respectively, which are distinctively lower than those of PGM under the corresponding conditions (40.9 and 115.2 ng/cm2 on average for pH 7 and 2, respectively). 3.5. Pin-on-Disk Tribometry. The lubricating properties of the buffer solutions were initially characterized, including those with three pH values, 2, 7, and 12, and varying salt concentrations, 0, 0.01, 0.1, and 1.0 M KCl at each pH. The µ versus speed plots for KCl-free buffer solutions showed a monotonic increase of µ with increasing speed (µ ) from ∼1 to ∼2 within the speed range of 1-100 mm/s, see Figure 7 to 9) apart from a slight decrease of µ observed at the highest speed under pH 12 conditions (see Figure 9). Furthermore, no noticeable influence of added KCl was observed for each pH when no polymer was added. It is noted that the frictional behavior of PDMS/ PDMS sliding under lubrication by polymer-free aqueous

buffer solution was indistinguishable from that measured under ambient conditions. The lubricating properties of PGM solution at pH 7 are presented as a function of ionic strength in Figure 7. By adding PGM (1 mg/mL) into the KCl-free buffer solution, the µ showed a noticeable reduction at the high-speed regime (for instance, µ changed from 1.83 to 0.81 at 100 mm/s), yet remained virtually indistinguishable from that of buffer solution in the low-speed regime (10 mm/s), which is similar to PGM solution under the same conditions. More importantly, however, changing pH from 7 to 2 did not induce any significant difference for either dextran or HA solutions.

Finally, the lubrication of PDMS/PDMS sliding contact by means of PGM-adsorption was compared with oxygenplasma treatment of a PDMS surface. As shown in Figure 12, the surface modification of PDMS via oxygen-plasma treatment resulted in a similarly enhanced lubrication effect, both at pH 2 and 7, to that observed upon the adsorption of PGM at pH 2. 4. Discussion 4.1. A Comment on the Purity of PGM. In terms of the sample source, previous studies on PGM can be divided into two groups. First, PGM is obtained by direct scraping from the pig stomach epithelium, then purified in the laboratory; isopycnic density gradient centrifugation in CsCl/guanidium chloride is the most common purification method.4,6,8,10,16 A drawback of this approach is the highly denaturing character of the solvents used,2,9 in addition to potential diversity in details of the method. An alternative approach is to use commercially available PGM (Sigma).7,9,12 This approach can offer a standard for different laboratories, but the uncertainty in purity and purification procedure remains problematic; potential impurities include other components in mucus gel such as salts, immunoglobulins, and secreted proteins etc.2 A more detailed discussion on the influence of purification of mucins is available in the literature.2,9 In the present work, we have employed as-received commercial PGM, following the argument by Davies et al.9 that additional purification may further degrade molecular weight and alter functionality relative to the native condition. For this reason, the experimental results and interpretation presented in this study may, strictly speaking, be compatible only with the previous studies that have employed commercial PGM.7,9,12 To date, the most well-known effect of the purification of mucin is the degraded viscoelastic behavior of commercial PGM compared with natural pig gastric gel that has not received any chemical treatment.11 This issue, however, is of minor importance in this work

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because we deal with the low-concentration regime (1 mg/ mL), where gel-forming behavior is intrinsically negligible, regardless of the purity. 4.2. Conformation of PGM in Bulk Aqueous Solution. As mentioned in the Introduction, PGM has been known to exhibit drastic changes in its conformation, depending on changes in pH and/or ionic strength;1-3 the most distinctive feature is its pronounced aggregation or gelation, especially at low pH, when the concentration of PGM is sufficiently high. In a dilute solution, as in this work (1 mg/mL), however, PGM has been reported to exist predominantly as nonassociated macromolecular species, yet exhibit interesting changes in conformation as a function of solution parameters.6 Viscosity measurements (Table 1) are supportive for this argument, in that the viscosity of PGM solution was similar to that of distilled water and remained virtually unaltered over a wide range of pH (2-12) and ionic strength (0.001-1.0 M for each pH). Meanwhile, the ζ potential measurements revealed that the IEP of PGM lies between 2 and 3, suggesting that the charge characteristics of PGM are significantly different as pH is varied. More relevant information on the conformation of PGM in bulk solution was obtained from CD spectroscopy. As shown in the Results, while the far-UV CD spectra implied only the absence of a distinctive secondary polypeptide backbone structure, the near-UV CD spectra revealed a strong dependence of both the position and intensity of maximum positive peaks on pH (Figure 2). Although a quantitative analytical methodology is much less developed for near-UV CD spectroscopy than for far-UV CD, it is generally agreed that near-UV CD spectra reflect the tertiary structure associated with the surroundings of amino acids containing aromatic residues, e.g., tyrosine, tryptophane, and phenylalanine.34-37 Because these amino acid residues reside in unglycosylated polypeptide regions, the strong positive peak at ∼270 nm at pH 7 (Figure 2) can be ascribed to the tertiary structure of those regions, more specifically of tyrosines;38 polypeptide chains generally tend to fold, such that the hydrophobic residues are concentrated inside the folded structure, while the hydrophilic or polar residues are at the surface of a protein, to optimize its interaction with water. With decreasing pH, the tendency to maintain such a folded structure will be diminished because of the protonation of the polar residues. The distinctive loss of the peak at ∼270 nm with decreasing pH is well correlated with the expected loss of the tertiary structure of the unglycosylated polypeptide region of PGM in an acidic environment; the rapid rotation of aromatic groups following exposure to water may blur the chirality of the R-carbons needed for near-UV CD signals.39 The absence of an apparent change in the nearUV CD spectra of PGM as a function of the ionic strength change (Figure 3) suggests that the change of the tertiary (31) Dedinaite, A.; Bastardo, L. Langmuir 2002, 18, 9383-9392. (32) Lindh, L.; Glantz, P.-O.; Carlstedt, I.; Wickstro¨m, C.; Arnebrant, T. Colloids Surf., B 2002, 25, 139-146 (33) Shi, L.; Ardehali, R.; Caldwell, K. D.; Valint, P. Colloids Surf., B 2000, 17, 229-239. (34) Aromatic and Cystine Side-Chain Circular Dichroism in Proteins. In Circular Dichroism and the Conformational Analysis of Biomolecules; Fasman, G. D., Ed.; Plenum Press: New York, 1996; Chapter 4. (35) Kelly, S. M.; Price, N. C. Curr. Protein Pept. Sci. 2000, 1, 349384. (36) Woody, R. W. Biopolymers 1978, 17, 1451-1467. (37) Sreerama, N.; Manning, M. C.; Powers, M. E.; Zhang, J. X.; Goldenberg, D. P.; Woody, R. W. Biochemistry 1999, 38, 10814-10822. (38) Engel, M. F. M.; van Mierlo, C. P. M.; Visser, A. J. W. G. J. Biol. Chem. 2002, 277, 10922-10930. (39) Dolgikh, D. A.; Gilmanshin, R. I., Brazhnikov, E. V.; Bychkova, V. E.; Semisotnov, G. V.; Venyanimov, S. Yu.; Ptitsyn. O. B. FEBS Lett. 1981, 136, 311-315.

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structure by pH is mainly of nonpolar origin, such as hydrophobic interaction. Although a direct correlation cannot be ascertained at this stage, the results of the nearUV CD spectra as pH is decreased are in good agreement with the previous dynamic scattering study by X. Cao et al.,6 in that a more anisotropic or unfolded conformation of PGM prevails at low pH. On the other hand, increasing pH above 7 resulted in a much less pronounced change in both the position and intensity of the peak at ∼270 nm. This observation is also well correlated with the expected maintenance of the polar residues under alkaline conditions and suggests a less distinctive change in the native conformation of PGM at pH values higher than 7. 4.3. Adsorption of PGM onto PDMS Surface. As already mentioned in the Introduction, it has been suggested that the adsorption of mucins onto hydrophobic surfaces occurs in a way that the unglycosylated polypeptide backbone segments of mucins act as binding sites through their hydrophobic interactions with the surface, while hydrophilic carbohydrate chains may dangle out to interact with water. Such an adsorption mechanism is consistent with the observation that the adsorbed amounts of polysaccharides are distinctly lower than that of PGM (Figure 6) at both pH 2 and 7 in this work; both polysaccharides, HA (negatively charged) and dextran (neutral), lack effective anchoring groups (polypeptides) for adsorption onto a hydrophobic surface. While the near-UV CD spectra of PGM showed a critical dependence on pH (see Figures 2 and 3), its adsorption behavior onto a PDMS surface showed a more complex dependence on both pH and ionic strength (Figure 5). The pronounced influence of ionic strength in adsorption is mainly due to the confinement of PGM molecules at the liquid/solid interface during adsorption, rendering the intermolecular interaction between PGMs stronger. In the low ionic strength regime (I e ∼0.1 M), the screening effect by the added salts appears to play a major role; for the adsorption of charged species onto nonpolar substrates, the addition of salts can suppress the repulsion between preadsorbed and/or approaching adsorbates at the liquid/ solid interface, and thus facilitates the adsorption. This behavior is not only observed for proteins,40-43 but also from various synthetic polyelectrolytes.44-46 The facts that the adsorption of PGM is not influenced by salt concentration when PGM has almost no net charge (pH 2, see Figure 5) and that the screening by added salt is more effective when PGM is less charged (i.e., more effect at pH 7 than pH 12, also see Figure 5) are consistent with this view. For pH 12, where PGM is most highly charged in this work, the screening effect by added salts appears to persist to the highest salt concentration (1.0 M) and results in a continuous increase in adsorbed mass (Figure 5). However, when PGM is only moderately charged (pH 7) or close to neutral (pH 2), the addition of the corresponding salts results in a reduction of the adsorbed mass (Figure 5); the adsorbed masses at the highest salt concentration (1.0 M) are, in fact, the smallest values at both pH 2 and 7, which (40) Ho¨o¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. J. Colloid Interface Sci. 1998, 208, 63-67. (41) Fragneto, G.; Su, T. J.; Lu, J. R.; Thomas, R. K.; Rennie, A. R. Phys. Chem. Chem. Phys. 2000, 2, 5214-5221. (42) Luey, J.-K.; McGuire, J.; Sproull, R. D. J. Colloid Interface Sci. 1991, 143, 489-500. (43) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-340. (44) Go¨bel, J. G.; Besseling, N. A. M.; Cohen Stuart, M. A.; Poncet, C. J. Colloid Interface Sci. 1999, 209, 129-135. (45) Abraham, T.; Giasson, S.; Gohy, J. F.; Je´rome, R.; Mu¨ller, B.; Stamm, M. Macromolecules 2000, 33, 6051-6059. (46) Abraham, T. Polymer 2002, 43, 849-855.

PGM at the Water/PDMS interface

may reflect the improved stability of PGM in bulk solution under those conditions. In the case of proteins, the saltinduced stability is generally expected when the interaction of water with salts is stronger than with proteins, i.e., in the “salting-out” regime,47-50 and it constitutes the physicochemical basis for the salt-induced precipitation (purification) of proteins. The mechanism of the saltingout effect is generally ascribed to the increase of surface tension of water by added salts;51 with increasing surface tension of water, it becomes more difficult to create a cavity in water to fit nonpolar molecules, and thus their solubility is decreased. For proteins, the propensity to expose the hydrophobic part is thus further decreased in the saltingout regime, and eventually the driving force to adsorb onto hydrophobic surfaces is diminished. It should be noted that proteins that are stabilized by means of the saltingout effect are precipitated in their native, rather than denatured, conformations. Considering the significance of the “naked” polypeptide region in the adsorption process of PGM onto hydrophobic surfaces as discussed above, it is plausible that the salting-out mechanism contributes to the reduced adsorption of PGM at high salt concentrations. In addition, given that the aggregation of PGM at high concentration and low pH is associated with hydrophobic interactions, the salting-out mechanism may also be responsible for the interruption of such aggregation in the presence of high salt concentrations.8 Further studies are needed to understand the details of this behavior because of the higher complexity in the structure and composition of mucins compared with proteins. Because of the complex influence of salts on the adsorption behavior of PGM, the relative adsorbed mass at different pHs also varies as a function of ionic strength. For instance, at low ionic strength (I e ∼0.01 M), the adsorbed PGM mass reaches its maximum at pH 2, which is closest to its IEP (Figure 5). This behavior is similar to that of most proteins, which exhibit their maximum adsorption onto nonpolar surfaces at around their IEP, mainly because of weaker electrostatic repulsion in this condition.43 This further supports the polypeptide-led adsorption mechanism discussed above. However, the trend in adsorbed mass at pH 2 and 7 is reversed when the screening effect is optimized, e.g., when I ) ∼0.1 M (Figure 5). This is important because PGM displays the most contrasting conformation under these two pH conditions in bulk solution (Figure 2), and thus it may directly influence the adsorption behavior of PGM onto hydrophobic surfaces. At pH 2, the tertiary structure of “naked” polypeptide region of PGM is interrupted (Figure 2), and thus hydrophobic residues are more exposed into the bulk aqueous solution even before they encounter the hydrophobic surface. At pH 7, on the other hand, the strong tertiary structure and charge properties of PGM (Figures 1 and 2) suggest that the hydrophobic residues are exposed only when they encounter the hydrophobic PDMS surface. This can cause several differences in the adsorption behavior of PGM onto hydrophobic surfaces. First, there is a higher probability at pH 2 that the adsorption of PGM occurs through multiple binding, resulting from the interaction between anchoring groups and consequent cohesion either in bulk solution and/or at the water/PDMS interface. Second, in the same context, the area occupied by per PGM molecule (or per adsorbed mass) would be (47) Baldwin, R. Biophys. J. 1996, 71, 2056-2063. (48) Arakawa, T.; Timasheff, S. N. Biochemistry 1984, 23, 59125923. (49) Lin, T.-Y.; Timasheff, S. N. Protein Sci. 1996, 5, 372-381. (50) Ghose, S.; Mattiasson, B. Biotechnol. Appl. Biochem. 1993, 18, 311-320. (51) Leberman, R.; Soper, A. K. Nature 1995, 378, 364-366.

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greater at pH 2, and the binding force would be stronger for the multiple binding characteristics. On the other hand, while PGM can be more highly packed at the liquid/solid interface at pH 7 due to the smaller area required for the adsorption of single PGM molecule, the binding force is expected to be weaker due to the lack of internal cohesion. In fact, the influence of “pre-unfolding” of a protein on its adsorption behavior onto hydrophobic surfaces has shown a similar tendency; a previous OWLS study by Heuberger et al. has shown that human serum albumin (HSA) adsorbs from Ringer’s solution onto hydrophobic poly(ethylene) surfaces more readily (i.e., stronger binding, larger area per molecule, and lower adsorbed mass) when they are already unfolded by heat treatment than when they retain native conformation.52 When the PGM-coated PDMS surfaces are subjected to mechanical perturbation, such as interfacial shear motion, the difference in the conformation of PGM at pH 2 and 7 and the strength or stability of the binding onto surfaces are more clearly manifested, as will be discussed in the following section. 4.4. Lubrication Properties of PGM; Influence of pH and Ionic Strength. As with the adsorption behavior of PGM onto a PDMS surface, the lubricating properties of PGM at the self-mated sliding interface of PDMS in an aqueous environment were observed to be highly dependent upon both pH and ionic strength. At pH 2, the relationship between the adsorbed amount of PGM and the lubricating capabilities is most straightforward. Consistent with the roughly constant adsorbed mass (∼120 ng/cm2) at ionic strengths equal to or lower than ∼0.1 M, the measured µ values were consistently low, while a drop of adsorbed mass at the highest ionic strength (I ) ∼1.0 M) exactly coincides with the degraded lubricating properties (see Figures 5 and 8). In other words, within a certain ionic strength range, PGM can act as an excellent aqueous lubricant additive at pH 2 (see below). At pH 7, however, PGM, regardless of its varying adsorbed amounts at different ionic strengths, did not lead to any significant lubricating effect. This observation suggests that the significant amount of adsorption onto the PDMS surface is a necessary, but not a sufficient condition for effective aqueous lubrication within the speed range selected in this work. Obviously, the conformation of PGM at the liquid/solid interface also plays a very significant role. As discussed in the previous section, the difference between the conformation at the liquid/solid interface of PGM at two different pHs, 2 and 7, can be summarized by the presence/absence of internal cohesion among the anchoring groups (hydrophobic species) and the consequent strength of binding onto the PDMS substrate surface. This difference is most clearly manifested during the runningin process in the friction measurements (Figure 10); because there was no apparent mechanical damage on the PDMS substrate surface, the stepwise increase of friction forces with repeated sliding in the first few rotations can be ascribed to the shear-induced desorption of the adsorbed PGM under these conditions. The lubrication properties at pH 12 are most complicated; because the near-UV CD spectrum of PGM at pH 12 is closer to that at pH 7 than that at pH 2, the conformation of PGM in bulk solution as well as at the liquid/solid interface at pH 12 is also expected to more closely resemble those at 7 than at 2. Furthermore, the adsorbed amount of PGM onto a PDMS surface is even smaller at pH 12 than at pH 7 for I e ∼0.1 M (see Figure 5). Thus, it is somewhat puzzling that PGM exhibits a (52) Heuberger, M. P.; Widmer, M. R.; Zobeley, E.; Glockshuber, R.; Spencer, N. D. Biomaterials 2005, 26, 1165-1173.

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Table 2. Measured and Calculated Mechanical Properties of the Tribosystem (Point contact Between PDMS Pin on PDMS Disk) and the Calculated Values of the Dimensionless Parameters According to Hamrock Et Al.57,58 for the Lubricated Contacts in This Studya definition elasticity modulus, E

Measured

reduced contact modulus, E′

E′ ) 2

Poisson ratio, ν load, w radius of pin, Rx

Measured

(

values

)

1 - ν12 1 - ν22 + E1 E2

Measured

x 3

3wRx 4E′

2 × 106 Pa 2.67 × 106 Pa 0.5 1N 3 × 10-3 m 9.45 × 10-4 m

contact radius, a

a)

maximum contact pressure, P0

P0 )

mean contact pressure P h

2 P h ≈ P0 3

viscosity of lubricant, η0 pressure-coefficient of viscosity of lubricant, ξ entrainment speed, u () half of the sliding speed for pure sliding)

ref 13 ref 13

dimensionless speed parameter, U

U)

η0u E′Rx

5.63 × 10-11

dimensionless load parameter, W

W)

w E′R2x

4.17 × 10-2

dimensionless material parameter, G

G ) ξE′

dimensionless elasticity parameter, gE

gE )

W8/3 U2

6.59 × 1016

dimensionless viscosity parameter, gV

gV )

GW3 U2

2.19 × 1013

3

1 π

x

3wE′2 2R2x

3.37 × 105 Pa

2.25 × 105 Pa 9 × 10-4 Pa‚s 3.6 × 10-10 Pa-1 0.5 × 10-3 m‚s-1

9.6 × 10-4

a For the calculation of mechanical parameters, a Hertzian model was employed. The viscosity value of water was used for the calculation of all PGM solutions because the difference was negligible, as shown in Table 1. The dimensionless parameters shown are the cases for the lowest sliding speed, 1 × 10-3 ms-1, and both the elasticity parameter, gE, and viscosity parameter, gV, show a decreasing trend with increasing speed; for the entire speed range, the combination gE and gV is expected to yield isoviscous-elastic lubrication in the map of elastohydrodynamic lubrication regimes.57,58

lubricating effect (see Figure 9). Two possibilities are suggested as the origin for this effect; first, the strong alkaline environment at pH 12 may induce an unfolding of the polypeptide region of PGM to some extent, and thus facilitate the stable anchoring of PGM onto the PDMS surface. As previously mentioned, the tertiary structure of PGM probed by near-UV CD spectroscopy at pH 12 is closer to that at pH 7 than at pH 2, yet unlike at pH 10, is still somewhat shifted from its native conformation (Figure 2). Second, when two surfaces carrying the same charges are slid against each other in an aqueous environment, the electrostatic repulsion between them contributes to the reduction of interfacial friction forces. This phenomenon is frequently observed for rigid oxide materials53,54 and for soft materials such as gels.55,56 While the adsorbed mass of PGM or negative charges accumulated at the surface at pH 12 is proportional to the ionic strength (Figure 5), the repulsion between the charged surface would be inversely proportional to the ionic strength. This, or the combination of the two factors mentioned above, may account for the unique lubricating (53) Kelshall, G. H.; Zhu, Y.; Spikes, H. A. J. Chem. Soc., Faraday Trans. 1993, 89, 267-272. (54) Marti, A.; Ha¨hner, G.; Spencer, N. D. Langmuir 1995, 11, 46324635. (55) Gong, J. P.; Kagata, G.; Osada, Y. J. Phys. Chem. B 1999, 103, 6007-6014. (56) Gong, J. P.; Osada, Y. Prog. Polym. Sci. 2002, 27, 3-38.

properties of PGM at pH 12, with an optimum (the lowest µ values) at I ≈ 0.1 M. 4.5. Lubrication Properties of PGM; Soft-EHL Mechanism. As mentioned in the Introduction, the selection of a silicone rubber (PDMS) to fabricate the tribopair in this work was mainly motivated by its low elasticity and consequent relevance to the mechanical contacts between many organs in human or animal bodies. As is well-known, water is typically a poor lubricant for most engineering systems because of its extremely low pressure-coefficient of viscosity (increase of the viscosity of lubricants according to the increase of contact pressure).21,22 However, when an elastomer is involved in the tribopair, this feature is not a barrier toward generating a lubricating film because the isoviscous-elastic lubrication (or soft-EHL) mechanism is expected to become dominant.57,58 In the soft-EHL regime, the deformation of contacting bodies even under low load is significant, yet the increase of the pressure within the contacting area is negligible. Thus, liquids with a low pressure-coefficient of viscosity, such as water, can readily form and maintain an effective lubricating film. As shown in Table 2, the combination of the bulk mechanical properties of the tribopair, PDMS for both pin and disk, and the bulk (57) Hamrock B. J.; Dowson, D. Proc. Leeds-Lyon Symp. Tribol. 1979, 5, 22-27. (58) Esfahanian, M.; Hamrock, B. J. Tribol. Trans. 1991, 34, 628632.

PGM at the Water/PDMS interface

rheological properties of the lubricant, water, is expected to provide soft-EHL lubrication under the measurement conditions of speed and load used in this study. Activation of the soft-EHL mechanism is, however, often not realized despite adequate bulk mechanical properties of the tribosystem; as in the case of the self-mated sliding of PDMS tribopair in polymer-free aqueous buffer solutions, the µ was observed to be generally higher than 1. This discrepancy is attributed to the highly adhesive properties of PDMS surfaces,21,22 and the hydrophilization of PDMS surface via oxygen-plasma treatment or surface-coating with amphiphilic copolymers effectively removes such adhesion, thus facilitating the soft-EHL mechanism. As shown in Figure 12, the enhanced aqueous lubricating effect on the sliding of PDMS/PDMS via oxygen-plasma treatment is indistinguishable from that of PGM adsorption at pH 2 under the experimental conditions of load and speed used in this study. Previous studies have shown that copolymers incorporating poly(ethylene oxide) (PEO) (or poly(ethylene glycol) (PEG)), e.g., poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO),21 or poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG)22 are also effective additives for the aqueous lubrication of PDMS under similar measurement conditions. As mentioned in the Introduction, mucin’s structure can be simplified as an amphiphilic block copolymer, consisting of hydrophilic (heavily glycosylated region) and hydrophobic (“naked” polypeptide region) blocks, and thus its lubricating mechanism can be considered in the same context, especially when compared with PEO-PPO-PEO; while the hydrophobic block, either polypeptides or PPO, functions as an anchoring group, the hydrophilic block, either PEO chains or clusters of oligosaccharides, reduce or eliminate the hydrophobic interaction and thus facilitate the entrainment of the lubricant, water, into the sliding interface. In addition, the fact that the amount of adsorption alone does not guarantee effective lubrication for either PGM and PEO-PPO-PEO21 should be specifically noted; for both cases, stable anchoring of the polymers onto the tribopair surface was observed to be a more important factor for effective aqueous lubrication than the actual amount of adsorbed material. Meanwhile, the presence of polar species on both blocks of PGM renders its dependence on pH and ionic strength more complicated than that of PEO-PPO-PEO. This feature may, however, further improve the lubricating properties due to the electrostatic repulsion operating between the opposing surfaces.53-56,59 5. Summary In this work, we have investigated the influence of pH and ionic strength on the conformation of PGM in bulk (59) Raviv, U.; Giasson, S.; Kampf, N.; Gohy, J. F.; Jerome, R.; Klein, J. Nature 2003, 163-165.

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aqueous solution, the adsorption behavior of PGM onto hydrophobic PDMS surfaces, and the lubricating behavior of PGM in the self-mated sliding contact of PDMS. NearUV CD spectroscopy has revealed the most distinctive change of the tertiary structure of the unglycosylated region of PGM to occur when pH was changed to the acidic direction; the strong indications of tertiary structure observed in neutral conditions appeared to be mostly disrupted upon acidification. This change was interpreted to reflect the exposure of hydrophobic aromatic residues of the unglycosylated polypeptide regions. Although unglycosylated polypeptide regions constitute only a minor portion of PGM, adsorption onto PDMS surface appears to be mainly driven by those regions. Under the conditions where the ionic strength was lower than ∼0.1 M, the observed adsorption behavior of PGM onto PDMS was understood in terms of the general “screening effect” by added salts on the adsorption of polyelectrolytes onto nonpolar surfaces; while the adsorption was virtually uninfluenced by the change in ionic strength when PGM was nearly uncharged at pH 2, the adsorbed mass increased with increasing ionic strength when the PGM was negatively charged at pH 7 or 12. This trend persisted up to an ionic strength of ∼1.0 M when the PGM is highly charged at pH 12, whereas distinctively lower adsorbed masses were observed at pH 2 and 7 at the same ionic strength, which was attributed to a “salting-out” effect on the unglycosylated polypeptide regions. The aqueous lubricating properties of PGM for the self-mated sliding of PDMS were most effective at pH 2, where stable anchoring was enabled because of multiple adsorption sites caused by internal cohesion between anchoring groups. At this pH, the lubricating performance was observed to be proportional to the adsorbed amount of PGM. However, at pH 7, despite the large mass adsorbed at the optimal ionic strength, the lubricating effect was generally poor because of the weak anchoring of PGM onto the surface and consequent shear-induced desorption over a few initial sliding cycles. Aqueous lubrication by PGM showed several common features with that of nonionic amphiphilic block copolymers, such as PEOPPO-PEO, while being more complex and showing a strong dependence on pH and ionic strength due to its polyanionic character. Acknowledgment. This work was financially supported by the U.S. Air Force Office of Scientific Research under Contract No. F49620-02-0346. The authors are very grateful to Dr. Eva Zobeley, Molecular Biology and Biophysics, Department of Biology, Swiss Federal Institute of Technology, Zu¨rich (ETH Zurich), for her help in the circular dichroism spectroscopy measurements, and Dr. Kirill Feldman, Institute for Polymers, Department of Materials, Swiss Federal Institute of Technology, Zu¨rich (ETH Zurich), for the elasticity measurements on PDMS. LA050779W