Article pubs.acs.org/Langmuir
Boundary Lubrication by Associative Mucin Xiang Wang, Miao Du,* Hongpeng Han, Yihu Song, and Qiang Zheng MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China S Supporting Information *
ABSTRACT: Mucus lubricants are widely distributed in living organisms. Such lubricants consist of a gel structure constructed by associative mucin. However, limited tribological studies exist on associative mucin fluids. The present research is the first to investigate the frictional behavior of a typical intact vertebrate mucin (loach skin mucin), which can recover the gel structure of mucus via hydrophobic association under physiological conditions (5−10 mg/mL loach skin mucin dissolved in water). Both rough hydrophobic and hydrophilic polydimethylsiloxane (PDMS) rubber plates were used as friction substrates. Up to 10 mg/mL loach skin mucin dissolved in water led to a 10-fold reduction in boundary friction of the two substrates. The boundary-lubricating ability for hydrophilic PDMS decreased with rubbing time, whereas that for hydrophobic PDMS remained constant. The boundary-lubricating abilities of the mucin on hydrophobic PDMS and hydrophilic PDMS showed almost similar responses toward changing concentration or sodium dodecyl sulfate (SDS). The mucin fluids reduced boundary friction coefficients (μ) only at concentrations (c) in which intermucin associations were formed, with a relationship shown as μ ∼ c−0.7. Destroying intermucin associations by SDS largely impaired the boundary-lubricating ability. Results reveal for the first time that intermolecular association of intact mucin in bulk solution largely enhances boundary lubrication, whereas tightly adsorbed layer plays a minor role in the lubrication. This study indicates that associated mucin should contribute considerably to the lubricating ability of biological mucus in vivo.
1. INTRODUCTION Aqueous lubrication with macromolecules has long been studied in man-made and natural systems. For example, two opposing mica surfaces bearing polyelectrolyte or polyzwitterionic brushes in aqueous environments can achieve an extremely low friction coefficient, which is less than 0.001 under pressure of several megapascals because of large hydration forces.1,2 Polymer hydrogels have exhibited very rich and excellent lubricating properties mediated by surface interactions (adsorption or repulsion).3,4 In natural systems, synovial fluid in synergy with cartilage remarkably provides long-term excellent lubrication for animal joints.5−7 Among various lubricants, biological mucus may be the most common in living organisms.8−10 Mucus is a weak biological hydrogel covering various mucosae, such as respiratory,11 digestive,12 and reproductive13 tracts within vertebrates. Mucus contains various substances, including water (>90 wt %), mucin, and other minor components depending on its source.8,14 Mucin, the main nonwater component of mucus, is a large polydisperse glycoprotein with saccharide often accounting for more than 70 wt %.15,16 Mucin consists of a polypeptide backbone linked by a large number of oligosaccharide side chains (typically less than 20 sugar units) via O-glycosylation, hence showing a comblike structure.17 It can form intermolecular associations in water and recover gel structure of mucus under physiological conditions.18,19 Other © 2015 American Chemical Society
nonwater components in mucus can modulate the properties of the gel formed by mucin.20 Considering the ubiquity and importance of mucus lubrication, lubrication with mucous systems has aroused scientific interest, and the lubricating properties of mucin solutions have been investigated.21−26 Lee et al. have found that the strong anchorage of pig gastric mucin on water/ poly(dimethylsiloxane) (PDMS) interface at pH 2.0 leads to a 20-fold reduction in boundary friction coefficient, whereas the weak anchorage at pH 7.0 cannot reduce boundary friction because of shear-induced desorption of the mucin.22 Yakubov et al. have found that a complex adsorption layer of “Orthana” mucin on hydrophobic PDMS surface leads to an inverse proportion of boundary friction coefficient to the thickness of the adsorption layer, which is consistent to a viscous boundary lubrication mechanism. However, the “Orthana” mucin does not lubricate hydrophilic PDMS because of relatively low adsorption mass.23 Chang et al. have reported that mucinlike lubricin adsorbed on either hydrophobic or hydrophilic substrates yields a friction coefficient of ca. 0.2, which results from steric repulsion and adhesion between the opposing adsorption layers.24,27 Long-range steric repulsion has also been Received: October 23, 2014 Revised: April 2, 2015 Published: April 6, 2015 4733
DOI: 10.1021/acs.langmuir.5b00604 Langmuir 2015, 31, 4733−4740
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sweep tests were conducted within linear viscoelastic region as determined by oscillatory strain sweep tests. Steady-State Pyrene Fluorescence Study. Loach skin mucin dissolved in ultrapure water, with concentrations ranging from 0.01 to 1 mg/mL, was subjected to steady-state pyrene fluorescence study by using LS-55 fluorescence spectrophotometer (PerkinElmer, U.S.) and a 10 mm path length quartz cell under room temperature. Approximately 1 mM pyrene in absolute methanol was prepared as stock solution, from which 1 μL was added to 1 mL of mucin sample. After equilibration overnight with shaking, the sample was scanned for emission spectra (λex = 335 nm), which were obtained by averaging three scans and corrected for scatter by using an equivalent blank mucin sample. Adsorption Measurement. The adsorption of loach skin mucin on gold surface coated with a monolayer of hydroxyl- or methylterminated thiol was monitored by both D300 quartz crystal microbalance (QCM-D, Q-Sense, Sweden) and SR7000DC surface plasmon resonance (SPR, Reichert, New York) at room temperature (20 to 25 °C). To prepare the surface with a monolayer of thiol, goldcoated QCM and SPR chips were subjected to the following treatment. First, the chips were cleansed in piranha solutions (98% H2SO4/30% H2O2 = 7/3, v/v) at room temperature to remove any contaminants and washed thoroughly with ultrapure water. The chips were then incubated in 1 mM ethanol solution of 1-dodecanethiol or 11-mercapto-1-undecanol to be CH3- or OH-terminated, washed thoroughly with copious amounts of ethanol, stored in ethanol, and dried in a stream of nitrogen before use. Static contact angles of the methyl- and hydroxyl-terminated gold surfaces were measured in ambient conditions at room temperature, with ultrapure water on at least six different locations on the same sample. The contact angles were reported as average values. For QCM measurement, two cells mounted separately with methyland hydroxyl-terminated gold-coated crystal quartz (fundamental frequency of 4.95 MHz) were linked in series and injected with required liquids at a flow rate of 0.2 mg/mL during experiments. After initial equilibration of the two surfaces at ultrapure water flow, mucin samples dissolved in water at concentrations of 0.04, 0.4, and 5 mg/ mL were injected sequentially with increasing concentration over the two surfaces. Each injection step lasted for 20 min, followed by rinsing with ultrapure water to remove the loosely adsorbed mucin. The shifts of frequency and dissipation from the baseline were monitored. For SPR measurement, a chip was first equilibrated with ultrapure water injected at a flow rate of 10 μL/min. Mucin samples dissolved in water at concentrations of 0.1, 0.4, 2, and 4 mg/mL were then injected sequentially with increasing concentration at the same flow rate for 60 min. Each injection was followed by rinsing with ultrapure water to remove the loosely adsorbed mucin. The response of SPR was monitored.
found between mica and silica surfaces coated by bovine submaxillary gland mucin, thus resulting in an effective friction coefficient as low as 0.03 ± 0.02.25 The above studies have provided substantial information on mucin lubrication. However, a key issue still exists. Previous investigations always focus on nanoscale-thick mucin layers on solid−liquid interfaces adsorbed from nonassociative mucin solutions, whereas mucin in vivo always highly associates into a weak gel of over 100 μm thickness.28 Tribological research on highly associative mucin fluids may elucidate in vivo lubrication with mucin. However, such studies remain limited. Therefore, the current work is the first to investigate the lubricating properties of associative mucin (loach skin mucin). Novel result indicated that an increased extent of intermucin association can decrease boundary friction.
2. MATERIALS AND METHODS Preparation of Mucin Fluids. Mucin was isolated from loach skin mucus in the laboratory using a previously reported isolation method.29 Ultrapure water (resistivity of 18.25 MΩ cm), 0.05 M NaCl or 0.05 M SDS was selected to dissolve purified loach skin mucin. Up to 0.02 wt % NaN3 was added to depress bacteria. After gentle stirring for about 40 h in an ice-water bath, uniform fluids without bubbles were obtained and subjected to tests. For tests requiring a series of concentrations, mucin fluids were prepared by diluting the one with the highest concentration in the solvent. Tribological Test. The lubricating properties of the mucin fluids were investigated using an AR-G2 rotational rheometer (TA Instruments) equipped with a Peltier plate and a plate geometry (radius R = 10 mm) at 20 °C. The Peltier and geometry plates were glued with a PDMS rubber plate on their surfaces. The PDMS plate exhibited a compression modulus of 4 ± 0.5 MPa, thickness of 1.2 mm, and water contact angle of 106° ± 8°. Hydrophilic PDMS surfaces derived from hydrophobic one by air plasma treatment under a power of 250 W for 20 min were also used. Given that plasmatreated PDMS surfaces gradually revert to hydrophobic form when exposed in air for several hours,30,31 the tribological tests on hydrophilic PDMS were performed within 30 min after plasma treatment was stopped. The static water contact angle of the hydrophilic PDMS surface with about 30 min exposure in air was 25° ± 6°. For tribological measurements, about 0.5 mL of the sample was placed on the lower PDMS glued on the Peltier plate. Then, the parallel upper PDMS glued on the geometry plate was driven to move toward the stationary lower PDMS. The distance between the two PDMS was monitored automatically by the rheometer. When the two PDMS were separated, the distance showed a positive value. When the two PDMS just began to contact, the distance was zero. After critical contact, the upper PDMS could still move toward the lower PDMS, which meant that the two PDMS pressed each other, the distance showed a negative value. The normal force between the two mutually pressed PDMS was also automatically monitored by the rheometer. After a certain time of auto adjustment of the upper PDMS, a normal force Fn of 3 N (apparent pressure of about 9.5 KPa) was obtained, with a stable, negative distance between the two PDMS. The rheometer then drove the PDMS glued on the geometry plate to rotate in one direction at a rotating velocity ω and a torque T, while keeping the normal force around 3 N by automatically adjusting the height of the upper PDMS. Friction coefficient μ was obtained as μ = T/RFn.32 To investigate the velocity dependence of friction, rotating velocity was changed stepwise with every single velocity point lasting for 180 s, and the average torque of the last 150 s was adopted to calculate the friction coefficient. Rheological Method. Rheological tests were conducted using the AR-G2 stress-controlled rheometer (TA Instruments) equipped with a 40 mm cone−plate geometry (cone angle of 2°) at 20 °C. After loading samples on the rheometer, 5 min rest without preshear was allowed before the specific rheological tests. All oscillatory frequency
3. RESULTS 3.1. Association and Adsorption of Loach Skin Mucin. We previously reported that loach skin mucin exhibits high purity, low amino acid residue content, large and polydisperse molecular size, and gel-forming property in water.29 In S.I.2 of the Supporting Information, the amino acid and sugar composition (Table S1 and S2 of the Supporting Information), high net negative charge (Figure S1 of the Supporting Information), and linear molecular shape (Figure S2 of the Supporting Information) of the mucin are shown. The properties of loach skin mucin described above characterize those of vertebrate mucin.15 Amino acids (Pro, Ala, Val, Met, Ile, Leu, and Phe) with hydrophobic groups comprise about 40% of total amino acid weight of loach skin mucin (Table S1 of the Supporting Information), hence indicating that hydrophobic interaction is very likely to occur in aqueous solution of the mucin. The intensity ratio of the first (373 nm) to the third (384 nm) 4734
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Langmuir vibronic peak (I1/I3) of pyrene fluorescence as a function of the mucin concentration in water was used to detect hydrophobic associations (Figure 1). The I1/I3 value was around 1.7 as
mucus usually ranges from 5 to 10 mg/mL. Thus, the loach skin mucin completely retained its associative ability after purification. As mentioned above, the loach skin mucin formed intermolecular hydrophobic association in water above the concentration of 0.2 mg/mL. Hence, the formation of the weak gel at 6 mg/mL should be hydrophobically driven, which can be confirmed by the frequency spectrum of 6 mg/mL mucin dissolved in 0.05 M SDS that can break hydrophobic associations of polymers. Up to 6 mg/mL mucin dissolved in 0.05 M SDS characterized a viscous solution, which exhibited a G′ lower than G″ over the entire test frequency range. Its G′ was nearly 2 orders of magnitude lower than the G′ of 6 mg/ mL mucin dissolved in water. Hence, SDS converted 6 mg/mL loach skin mucin into a viscous solution by breaking intermolecular hydrophobic associations. Figures 1 and 2 reveal that the intermolecular hydrophobic associations of loach skin mucin formed above the concentration of 0.2 mg/mL in water and played a key role in the formation of a weak gel at 6 mg/ mL. QCM and SPR were used to investigate adsorption of loach skin mucin from water on both hydrophobic and hydrophilic substrates, which were methyl- (hydrophobic) and hydroxylterminated (hydrophilic) gold surfaces with static water contact angles of 104° ± 7° and 27° ± 3°, respectively. Table 1 lists the
Figure 1. I1/I3 of pyrene in solutions of loach skin mucin dissolved in water at a series of concentrations.
mucin concentration was below 0.05 mg/mL and decreased to around 1.1 as concentration reached 0.2 mg/mL. The dependence of I1/I3 on mucin concentration was typical of hydrophobically modified water-soluble polymers18,33,34 and demonstrated that hydrophobic associations formed in the mucin solution above the concentration of 0.2 mg/mL. On the other hand, 0.2 mg/mL loach skin mucin solution showed shear-thinning behavior with a zero-shear viscosity twice as much as the viscosity of pure water (Figure S3 of the Supporting Information), indicative of a critical overlapping state.35 Therefore, the mucin molecules in water began contact at the concentration of 0.2 mg/mL, leading to hydrophobic intermolecular association above this concentration. Figure 2
Table 1. Frequency Shift of Third overtone (Δf 3/3) of CH3(Hydrophobic) and OH-Terminated (Hydrophilic) Gold Surfaces Adsorbing Mucin from Aqueous Solutions with Concentrations of 0.04, 0.4, and 5 mg/mL
Figure 2. Storage modulus (G′) and loss modulus (G″) of 6 mg/mL loach skin mucin dissolved in water, 6 mg/mL mucin dissolved in 0.05 M SDS, and crude loach skin mucus as functions of angular frequency (ω) during oscillatory frequency sweep tests within linear regions.
frequency shift of third overtone (Δf 3/3) after rinsing of the two surfaces that adsorbed mucin from aqueous solutions of 0.04, 0.4, and 5 mg/mL. The Δf 3/3 of hydrophobic substrate insignificantly varied at the three mucin concentrations with values around −40 Hz. This indicates that the sensed masses by QCM insignificantly differed at the three concentrations. Note that QCM sensed the total mass of dry mucin and water associated and trapped with the mucin. Macakova et al. have verified that mucin adsorbing on hydrophobic surfaces forms a viscoelastic layer containing large amounts of water.36 Following their work36 in which QCM-D data was fitted with a Voigt model to calculate the thickness of the mucin layer, we obtained about a 7 nm adsorption layer thickness of loach skin mucin on the hydrophobic surface at 0.04 mg/mL (S.I.4 of the Supporting Information). The layer thickness was approximately equal to the diameter of the mucin chain, indicating that a monolayer of individual mucin molecules with flat extended conformation adsorbed on the hydrophobic substrate. Considering the high net negative charge of loach skin mucin (Figure S1 of the Supporting Information), its flat extended conformation on the hydrophobic surface should originate from the electrostatic repulsion between its charged units. Above the critical association concentration, that is, at 0.4 and 5 mg/mL, bulk solutions consisted of individual mucin molecules and intermucin associations. However, adsorption of intermucin associations and formation of multilayers were excluded at the two concentrations because these two processes should have caused much more significant decrease in Δf 3/3 if they had
illustrates storage moduli (G′) and loss moduli (G″) of 6 mg/ mL mucin dissolved in water, 6 mg/mL mucin dissolved in 0.05 M SDS, and crude loach skin mucus during oscillatory frequency sweep tests within the linear region. Up to 6 mg/ mL mucin dissolved in water characterized a weak gel, which exhibited a G′ larger than G″over the entire test frequency range (from 0.05 to 10 rad/s). Moreover, its spectrum almost coincided with the spectrum of loach skin mucus, indicating that 6 mg/mL loach skin mucin in water associated into a weak gel like the mucus. Notably, the solid content of loach skin 4735
c (mg/mL)
hydrophobic substrate
hydrophilic substrate
0.04 0.4 5
−40 Hz −37 Hz −39 HZ
none detected none detected none detected
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the mucin chain diameter (∼7 nm). As bulk concentration increased, more individual mucin molecules adsorbed onto unoccupied surface sites and thus R detected by SPR increased. The additional adsorption followed a monolayer mechanism and thus did not increase the thickness of the water film. Considering that a monolayer of intact vertebrate mucin on hydrophobic surface can associate and trap water several ten times of its own weight,36 the unchanged QCM signals (Table 1) should mainly reflect the mass of the unchanged water film. QCM and SPR demonstrated that only a monolayer of individual mucin molecules with flat extended conformation adsorbed on hydrophobic surface after rinsing. This result is consistent with Macakova’s work on salivary mucin adsorbing on hydrophobic surface in water.36 On the other hand, given that a mucin molecule is a multisticker chain that can be as long as 1 μm and brings hydrophobic stickers at both ends,15 intermucin associations of the bulk solutions near the solid/ liquid interface are very likely to be tethered on the hydrophobic surface by their dangling tails before rinsing. QCM measurement showed that the adsorption of loach skin mucin onto OH-terminated (hydrophilic) gold surface did not occur, but more sensitive SPR measurement detected a response of the hydrophilic surface, which was one-sixth of the response of hydrophobic surface (result not shown). Much lower adsorption mass on hydrophilic surface than on hydrophobic surface has also been reported on “Orthana” mucin.23 Generally, loach skin mucin adsorbed on hydrophobic surface as a monolayer of individual flat extended molecules trapping and associating large amounts of water. Adsorption was much weaker onto hydrophilic surface than onto hydrophobic surfaces. The adsorption behavior of loach skin mucin is in good accordance with previous studies on mucin adsorption.23,36 3.2. Boundary Lubrication by Associative Mucin. Vertebrate mucus provides excellent lubricating functions for various biosurfaces such as respiratory, digestive, and reproductive tracts. Notably, mucus in vivo always functions as a highly associative fluid with network structure constructed by mucin.14 In this section, frictional behaviors of associative loach skin mucin fluids lubricating both hydrophobic and hydrophilic PDMS substrates are investigated. Figure 4 shows the friction coefficients of water and 10 mg/ mL loach skin mucin dissolved in water lubricating PDMS surfaces, as velocity increased from 0.01 rad/s to 10 rad/s then decreased back to 0.01 rad/s without separating the rubbing
occurred. Hydrophobic patches of intermucin associations were shielded by surrounding glycosylated chains. Hence, the drive force for intermucin associations to adsorb onto hydrophobic substrates should be much weaker than the drive force for individual mucin molecules in which hydrophobic patches were exposed to water. The monolayer of adsorbed individual mucin rendered the surface with much negative charge. Thus, multilayer adsorption cannot occur because of electrostatic repulsion. Given that QCM signals represented the overall contribution of mucin and water, SPR measurement only detecting the mucin mass was performed. Figure 3 shows the
Figure 3. SPR response (R) of methyl-terminated (hydrophobic) gold surface adsorbing loach skin mucin from aqueous solutions of different concentrations (c).
bulk concentration dependence of the SPR response (R) that was proportional to the mass of dry adsorbed mucin on methylterminated (hydrophobic) gold surface after rinsing. R increased with increasing concentration and can be fitted perfectly by a Langmuir model (R-square 0.999), which indicated that the adsorption of loach skin mucin onto hydrophobic surface followed a monolayer mechanism, consistent with the QCM result. R detected by SPR indicated that the mass of adsorbed mucin increased with bulk concentration, whereas Δf 3/3 detected by QCM showed that the total mass of mucin and water on the surface was almost unchanged with bulk concentration. This should result from the quite good hydrophilicity of loach skin mucin.15,29 At low bulk concentration, although mucin did not cover the surface to the maximum, mucin’s good hydrophilicity enabled the surface to be completely covered by a water film with a thickness equal to
Figure 4. Friction coefficients of (A) hydrophobic and (B) hydrophilic PDMS lubricated by water and 10 mg/mL loach skin mucin dissolved in water, respectively, with rotating velocity increasing from 0.01 to 10 rad/s and then decreasing back to 0.01 rad/s without separating the rubbing surfaces. Black and red symbols represent increased and decreased velocities, respectively. 4736
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skin mucin and the mucin used in previous works are discussed in detail in Discussion. The concentration effect of loach skin mucin on boundary friction was investigated. Figure 6 shows the boundary friction
surfaces. Between hydrophobic surfaces (Figure 4A), friction coefficients showed typical Stribeck behavior, that is, plateau (boundary region) at low velocity followed by decrease (mixed region) with increased velocity. Friction curves during increased and decreased velocities insignificantly differed with each other. Compared with water, 10 mg/mL mucin showed a 10-fold reduction in the boundary friction coefficient and a narrower boundary region. Between hydrophilic surfaces (Figure 4B), the friction coefficient of 10 mg/mL mucin between 0.01 and 0.1 rad/s showed an upward curve from 0.015 to 0.2 during increased velocity, and a plateau around 0.2 during decreased velocity. Above 0.1 rad/s, the friction coefficient showed downward curves without difference between the increased and decreased velocities. Figure 4 indicated that 10 mg/mL loach skin mucin effectively reduced boundary friction coefficients of both hydrophobic and hydrophilic PDMS substrates. Specially, a large hysteresis, which is usually indicative of time effect, is observed in the boundary region of hydrophilic substrates. Figure 5 illustrates the friction coefficients of water and 10 mg/mL mucin dissolved in water lubricating PDMS during five
Figure 6. Concentration dependence of boundary friction coefficients of loach skin mucin dissolved in water lubricating both the hydrophobic and hydrophilic PDMS substrates at a rotating velocity of 0.05 rad/s (boundary region). The friction coefficients were derived by averaging the data during the second to the fourth rotating circle.
coefficients of the mucin lubricating both hydrophobic and hydrophilic PDMS at different concentrations in water. The boundary friction coefficient plotted in Figure 6 is derived from averaging data during the second to fourth rotating circle (125 to 500 s) at a rotating velocity of 0.05 rad/s (boundary region) because the mucin fluid between hydrophilic PDMS exhibited a friction coefficient increasing with rubbing time (Figure 5). A rotating circle meant that, relative to the stationary lower PDMS, the upper PDMS has rotated for an angle of 2π. At a rotating velocity of 0.05 rad/s, a rotating circle lasted 2π/0.05 ≈ 125 s. At low concentrations of less than 0.2 and 0.9 mg/mL for hydrophilic and hydrophobic PDMS, respectively, the boundary friction coefficients slightly changed with increasing concentration. At relatively high concentrations, the boundary friction coefficients sharply decreased with increasing concentration (c), which showed a power law relationship of μ ∼ c−0.7. Loach skin mucin lubricating hydrophobic and hydrophilic substrates showed similar trends of boundary lubrication with increased concentration, with the adsorption on the two substrates being significantly different. This phenomenon is rarely reported in other aqueous polymer lubricants, because functions of boundary lubricants are commonly determined by adsorption layers, which are quite different on substrates of different surface chemistry. On the other hand, fluorescence and rheological results showed that loach skin mucin formed intermolecular associations above 0.2 mg/mL. Therefore, the rapid decay μ ∼ c−0.7 (Figure 6) occurred when mucin formed intermolecular associations, and was accompanied by increase of associated mucin molecules which finally led to gelation. To investigate the role of mucin associations in boundary lubrication, boundary friction of 6 mg/mL loach skin mucin dissolved in different solvents (0.05 M SDS and 0.05 M NaCl) was investigated. SDS can dissociate 6 mg/mL mucin into a viscous solution (Figure 2), whereas NaCl cannot adversely affect the hydrophobic association of polymers.37 A much lower associative extent for 6 mg/mL mucin dissolved in 0.05 M SDS than of that dissolved in 0.05 M NaCl was demonstrated by the zero-shear viscosity of the former being near 2 orders of magnitude lower than the viscosity of the latter (Figure S4 of
Figure 5. Time-dependent boundary friction coefficients of water and 10 mg/mL loach skin mucin dissolved in water lubricating hydrophobic (circle symbols) and hydrophilic (square symbols) PDMS during five consecutive rotating circles at a rotating velocity of 0.05 rad/s (boundary region). A rotating circle needs 2π/0.05 ≈ 125 s, and every value plotted on the figure is derived by averaging the data during the corresponding rotating circle.
consecutive rotation circles (∼10 min) at a rotating velocity of 0.05 rad/s, which was located in the boundary region according to Figure 4. Between hydrophobic PDMS, the boundary friction coefficient of 10 mg/mL mucin remained constant with rotating circle, whereas between hydrophilic PDMS, 10 mg/mL mucin showed a friction coefficient that increased with rotating circle. In both cases, friction coefficients of 10 mg/mL mucin were about 1 order of magnitude lower than the constant friction coefficients of water. The increase of friction coefficient of 10 mg/mL mucin between hydrophilic PDMS corresponded well with the hysteresis in Figure 4B, thereby suggesting a time effect. Figures 4 and 5 demonstrate that 10 mg/mL mucin possesses stable boundary lubricating ability between hydrophobic PDMS and can provide boundary lubricating function for hydrophilic PDMS for tens of minutes. Previous investigations on mucin lubrication have reported that mucin cannot reduce the boundary friction of hydrophilic substrates.23,24 By contrast, the present study on loach skin mucin showed a different result. The differences between loach 4737
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Figure 7. Boundary friction coefficients of (A) hydrophobic and (B) hydrophilic PDMS lubricated by 6 mg/mL loach skin mucin dissolved in 0.05 M NaCl, 6 mg/mL loach skin mucin dissolved in 0.05 M SDS, and pure solvents.
much closer to the in vivo mucin lubricant than the nonassociative commercial mucin solutions. Loach skin mucin properties, including low amino acid content, large and polydisperse size, linear shape, and net negative charge, are typical of intact vertebrate mucin.15 Thus, we believe that the associative mucin fluid investigated in this study can represent in vivo mucin lubricant in the body of vertebrates, which has never been achieved before. Most mucus-covered biosurfaces are soft rough tissues with asperities that can be visualized by naked eyes. Rough PDMS rubber (root-mean-square roughness: 221 ± 44 nm) was used as friction substrate in this study because of its closeness to the biosurfaces in both mechanical property and surface roughness. We believe that the lubricating system of highly associative mucin fluid covering soft rough PDMS rubber is similar to the in vivo lubrication with mucus to a large extent. 4.2. Role of Adsorption and Association in Boundary Lubrication. The frictional behavior of associative mucin fluid is quite different from that of nonassociative mucin solution. Previous studies on nonassociative mucin attributed the origin of mucin lubrication to tightly adsorbed layer.23,24,39 Yakubov et al. found that the boundary friction of hydrophobic PDMS lubricated by “Orthana” mucin is inversely proportional to the thickness of the entanglement-governed adsorbed mucin layer and proved the friction to be the shear stress of the layer (viscous boundary lubrication mechanism).23 Chang et al. found that mucin-like lubricin adopts brush conformation on hydrophobic surface and reduces the friction because of repulsion between the two opposed surfaces bearing lubricin brushes.24,27 Nonassociative mucin was found not to reduce the boundary friction of the hydrophilic surface.23,24 However, in the case of associative mucin fluids, mucin reduced the boundary friction of both hydrophobic and hydrophilic substrates for the first time, with concentration dependence shown as μ ∼ c−0.7. For hydrophobic substrate, the sharp increase in adsorption mass at bulk concentration below 0.9 mg/mL was accompanied only with a very slight decrease in boundary friction, whereas the mild increase in adsorption mass above 0.9 mg/mL was accompanied by steep friction decrease, which is quite different from the result of the nonassociative mucin showing that the decrease in boundary friction is approximately proportional to the increase in adsorption mass.23,24 Loach skin mucin adsorbing onto hydrophobic surface formed a monolayer with the thickness close to the diameter of a mucin chain. Thus, the viscous boundary lubrication mechanism for the “Orthana” mucin23 that ascribes
the Supporting Information). The boundary friction coefficients of the two mucin fluids and their corresponding solvents were measured in angular velocity ranging from 0.01 rad/s to 0.1 rad/s (boundary region), as shown in Figure 7. The 6 mg/mL mucin in 0.05 M NaCl lubricating hydrophobic PDMS (Figure 7A) showed a boundary friction coefficient (ca. 0.3) that was less than 15% of the coefficient (ca. 2.5) of pure 0.05 M NaCl, whereas 6 mg/mL mucin in 0.05 M SDS yielded a coefficient (ca. 0.6) of about 60% of the coefficient of pure 0.05 M SDS (ca. 1.0). When lubricating hydrophilic PDMS (Figure 7B), 6 mg/mL mucin in 0.05 M NaCl showed increased fiction with increased velocity, which was consistent with the data shown in Figure 4B. The average boundary friction coefficient (ca. 0.1) was less than 20% of the coefficient of pure 0.05 M NaCl (ca. 0.55), whereas 6 mg/mL mucin in 0.05 M SDS did not show better boundary lubrication than 0.05 M SDS. Figure 7 reveals that the boundary lubrication of 6 mg/mL loach skin mucin was largely impaired between both hydrophilic and hydrophobic PDMS after intermucin associations were destroyed by SDS.
4. DISCUSSION 4.1. Tribological System. Commercial mucins are usually utilized to investigate the lubricating properties of mucin solutions. For example, Lee et al. used porcine gastric mucin purchased from Sigma to investigate the influence of pH and ionic strength on mucin lubrication,22 and Yakubov et al. used porcine gastric mucin purchased from A/S Orthana Kemisk Fabrik to investigate the viscous boundary lubrication.23 In the present study, loach skin mucin was isolated in the laboratory. A very remarkable difference between loach skin mucin and the commercial mucins is their associative ability. The commercial mucins are degraded during purification;38 hence, their associative ability is much weaker than that of loach skin mucin. For instance, 60 mg/mL commercial “Orthana” mucin in water, which was of the highest concentration in Yakubov’s study,23 exhibited a viscosity that was only six times as much as the viscosity of pure water. Definitely, intermucin association at that condition was very weak (or not existing), and physically associated network similar to crude mucus cannot form. By contrast, 6 mg/mL (physiological concentration) loach skin mucin highly associated into a viscoelastic gel like crude mucus (Figure 2), with a viscosity more than ten thousand times as much as that of pure water. It is well-known that biological mucus in vivo functions as a viscoelastic gel constructed by mucin.15 Thus, associative loach skin mucin fluid should be 4738
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5. CONCLUDING REMARKS Mimicking in vivo lubrication by biological mucus, the frictional behavior of associative mucin fluids lubricating rough PDMS rubber substrates was investigated for the first time. About 10 mg/mL mucin in water associating into a weak gel similar to crude mucus reduced the boundary friction by 90% between both hydrophobic and hydrophilic PDMS, with the friction increasing with rubbing time between hydrophilic PDMS and remaining constant over time between hydrophobic PDMS. The boundary friction reduction can be achieved only at concentration sufficiently high to form intermucin associations, showing a relationship as μ ∼ c−0.7. The boundary-lubricating property was largely impaired when the mucin was dissociated by SDS. Remarkably, the concentration dependence and SDS effect were observed on both hydrophilic and hydrophobic PDMS. In contrast to previous results on nonassociative mucin, the tightly adsorbed layer played a very minor role in boundary lubrication by associative mucin. Furthermore, intermolecular association of the mucin in bulk solution largely enhanced the boundary lubrication. This finding indicates that the lubricating ability of biological mucus in vivo is very likely to originate from associated mucin. To the best of the authors’ knowledge, boundary lubrication enhancement originating from the association in bulk solution rather than the tightly adsorbed layer on the interface has never been reported in the field of aqueous polymer lubricants. Under the inspiration of this study, intermolecular association has been found to enhance the boundary-lubricating ability of several synthetic associative polymers with frictional behavior similar to that of associative mucin. An ongoing study is focused on the synthetic associative polymers, which may provide further insightful interpretation for mucus lubrication.
boundary friction reduction to the increase in adsorption layer thickness is not suitable for loach skin mucin. The loach skin mucin adopted the flat extended conformation on the hydrophobic surface, and a monolayer of flat extended vertebrate mucin adsorbed on hydrophobic substrate has been proved to only slightly enhance the boundary lubrication over a normal force of 2 N.40 After a tribological test of 10 mg/ mL loach skin mucin on a hydrophobic PDMS, replacing the mucin with water gives the hydrophobic PDMS a same boundary friction coefficient as that of another hydrophobic PDMS only lubricated by water (data not shown). The result also indicates that the adsorbed flat extended mucin remained after rinsing does not enhance boundary lubrication. On the basis of the above analysis, the adsorbed monolayer is not the reason for the excellent boundary-lubricating ability of associative loach skin mucin fluid. Bulk solution concentration or SDS changed the lubricating ability of loach skin mucin between hydrophilic substrates in a manner similar to between hydrophobic substrates, although adsorption amount was much lower on hydrophilic surface than on hydrophobic surface. This also proves that the tightly adsorbed layer, that is, the adsorbed mucin after rinsing, plays a very limited role in the boundarylubricating ability of associative loach skin mucin. This finding is sharply in contrast with the result of nonassociative mucin in which tightly adsorbed layers play a decisive role in the lubrication. To the best of the authors’ knowledge, boundary friction reduction with minor influence of adsorption is also rarely found in aqueous polymer lubricants. For example, ordinary nonadsorbing polymer solutions can lower friction in mixed region at higher concentration due to higher viscosity, but no boundary lubrication can be obtained.41 Loach skin mucin dissolved in water provided boundary lubrication only at concentration sufficiently high to form intermolecular associations. As concentration continuously increased with more intermucin associations, boundary friction decreased with the relationship shown as μ ∼ c−0.7. Meanwhile, boundary lubrication was largely impaired after SDS destroyed intermucin associations. Thus, the boundary lubrication originates from intermolecular association of the mucin. This result should be a key to elucidating in vivo lubrication by biological mucus because all in vivo mucus lubricants are highly associative fluids basically constructed by associated mucin. However, the mechanism by which intermucin associations enhance boundary lubrication remains obscure. A plausible mechanism is that intermucin associations trapped in cavities of the rough surface bear some of normal force because of their cross-linked structure when asperities of the opposed surface slide by them. This load-bearing characteristic contributes to the boundary friction reduction. Intact mucin exhibits a rather complex molecular structure and physicochemical parameters.15 Thus, ensuring the lubricating mechanism of the associative mucin is difficult. Fortunately, inspired by that intermolecular association of mucin enhances boundary lubrication, we recently found that several synthetic associative aqueous polymers with much simpler molecular structures and physicochemical parameters demonstrate frictional behavior similar to that of associative mucin. An ongoing investigation is focused on the synthetic associative polymers, which may provide further physical interpretation for boundary lubrication by associative mucin.
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ASSOCIATED CONTENT
S Supporting Information *
Methods and results of the composition, charge, molecular shape, viscosity, and QCM-D data of loach skin mucin. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge Prof. Binyang Du for the valuable discussions on mucin adsorption, Ms. Dandan Li and Mr. He Zhang for the QCM measurement, and Mr. Yang Liu for the SPR measurement. This research is supported by the National Natural Science Foundation of China (Grant 51173164).
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