Interactions between Mucin and Surfactants at Solid− Liquid Interfaces

Langmuir 2002, 18, 9383-9392

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Interactions between Mucin and Surfactants at Solid-Liquid Interfaces Andra De˘ dinaite˘ *,† and Luis Bastardo‡ Unilever Research & Development Port Sunlight, Quarry Road East, Bebington, Wirral CH63 3JW, United Kingdom, and the Department of Chemistry, Surface Chemistry, Royal Institute of Technology, Drottning Kristinas va¨ g 51, SE-100 44 Stockholm, Sweden and Institute for Surface Chemistry, Box 5607, SE-114 86 Stockholm, Sweden Received May 23, 2002. In Final Form: August 2, 2002 The association between mucin and surfactants at the solid-liquid interface has been investigated employing reflectometry. The study is particularly aimed at understanding the removal of preadsorbed mucin layers by surfactant addition. To this end we investigated the effect of three different surfactants, one anionic surfactant, sodium dodecylsulfate (SDS), and two nonionic ones, penta(oxy ethylene) dodecyl ether (C12E5) and n-dodecyl β-D-maltopyranoside (C12-mal). All three surfactants were found to be potent in removing mucin from hydrophobic surfaces. On the other hand, C12-mal was found to have a very limited effect on mucin adsorbed to hydrophilic negatively charged surfaces, whereas the mucin layer was removed by SDS and C12E5. The association between mucin and the three different surfactants was also investigated by means of dynamic light scattering and surface tension measurements. It was concluded that SDS associates readily with mucin above a critical surfactant concentration, about 0.2 cmc, whereas the nonionic surfactants associate with mucin to a very limited degree. The results obtained with the different techniques allow us to propose that C12E5 removes mucin from silica surfaces by competitive adsorption, whereas the removal of mucin by SDS is due to formation of mucin/SDS complexes that have reduced surface affinity and increased water solubility compared to mucin alone.

Introduction The term “mucins” denotes a large group of glycoproteins consisting of a linear polypeptide backbone with alternating naked and densely glycosilated protein fractions. The carbohydrate part in mucins constitutes typically 70-90 wt % and occurs as neutral and acidic, linear and branched oligosaccharide side chains covalently bound to the polypeptide backbone.1 Mucin molecules carry a net negative charge due to the presence of sialic acid residues, but cationic sites are also present. In general terms, mucins are long linear macromolecules consisting of subunits with molecular weight of 0.25-5 million. In the native state the molecular weight of mucin is in the range 1-50 million. A high degree of glycosilation makes the carbohydrate fraction larger than that of the polypeptide.2 Mucins fulfill numerous functions, most of them related to defense and protection of bodily surfaces. They have an important role in controlling the permeability of oral mucosal surfaces.3 The molecular structure of mucins allows them to bind water effectively and thus the presence of mucins on oral surfaces helps to keep them hydrated4 and lubricated.5 There is a considerable amount of data indicating that the carbohydrate moieties of salivary mucins play a major role in the nonimmune clearance of the microbial flora.6 * To whom correspondence should be addressed. † Unilever Research & Development Port Sunlight. ‡ Royal Institute of Technology and Institute for Surface Chemistry. (1) Carlstedt, I.; Sheehan, J. K.; Corfield, A. P.; Gallagher, J. T. Essays Biochem. 1985, 20, 40. (2) Strous, G. J.; Dekker, J. Critical Reviews in Biochemistry and Molecular Biology 1992, 27, 59. (3) Adams, D. J. Dent. Res. 1975, 54, B19. (4) Tabak, L. A.; Levine, M. J.; Mandel, I. D.; Ellison, S. A. J. Oral Pathol. 1982, 11, 1. (5) Mandel, I. D. J. Dent. Res. 1987, 66, 623. (6) Gottschalk, A.; Bhargava, A. S.; Murty, V. L. N. In Glycoproteins: Their Compositions, Structure and Function; Gottschalk, A., Ed.; Elsevier: Amsterdam, 1972; p 810.

A recent work by Shi et al.7,8 demonstrated that by using mucin coatings it is possible to create surfaces with increased resistance to bacterial adhesion. The adsorption of mucins onto various types of surfaces is of great importance in biomaterial applications. Therefore, extensive studies have been carried out in an effort to understand the behavior of mucins in contact with surfaces. Proust and co-workers demonstrated, using a range of surfaces (polyethylene, oxidized polyethylene, silicone, mica, PVP grafted silicone), that it is not possible to attain a real plateau value for BSM adsorption even after adsorption times of 20 h or more.9,10,11 Durrer and co-workers demonstrated with the adsorption of pig gastric mucin (PGM) and BSM on carboxylate- and aminofunctionalized polystyrene latexes (PCM and PAM) over a broad range of pH values (3-7.5) that mucin adsorption is driven not only by electrostatic attraction.12 Malmsten et al.13 investigated the adsorption of rat and pig gastric mucins, RGM and PGM respectively on hydrophobic substrates using ellipsometry and the interferometric surface force apparatus. The authors showed that both types of mucin adsorbed on hydrophobic surfaces, but the adsorbed amount approached equilibrium slowly. Negligible desorption of the mucin was observed upon dilution. Recently, adsorption of size-exclusion chromatography purified BSM on hydrophobic polymeric surfaces (poly(7) Shi, L.; Ardehali, R.; Valint, P.; Caldwell, K. D. Biotechnol. Lett. 2001, 23, 437. (8) Shi, L.; Ardehali, R.; Caldwell, K. D.; Valint, P. Colloids Surf. B-Biointerfaces 2000, 17, 229. (9) Proust, J. E.; Baszkin, A.; Boissonade, M. M. J. Colloid Interface Sci. 1983, 94, 421. (10) Proust, J. E.; Baszkin, A.; Perez, E.; Boissonade, M. M. Colloids Surf. 1984, 10, 43. (11) Proust, J. E.; Proust, J. E.; Baszkin, A.; Boissonade, M. M. Colloids Surf. 1984, 9, 297. (12) Durrer, C.; Irache, J. M.; Duchene, D.; Ponchel, G. J. Colloid Interface Sci. 1995, 170, 555. (13) Malmsten, M.; Blomberg, E.; Claesson, P.; Carlstedt, I.; Ljusegren, I. Journal Colloid Inter. Sci. 1992, 151, 579.

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styrene particles) was studied by the amino acid analysis method and yielded adsorbed amount of BSM at an adsorption plateau equal to 2.3 mg/m2.14 Also in this case the adsorption was reported to be practically irreversible with respect to dilution. In another recent study Lindh et al.15 investigated adsorption of the salivary mucin MUC5B and BSM on hydrophobic and hydrophilic surfaces. Both mucins showed a higher affinity for hydrophobic surfaces than for hydrophilic ones, and an increased ionic strength resulted in an increased adsorption. This was rationalized in terms of a decreased repulsive interaction between charged mucin residues and between such residues and the surface. Vassilakos et al. studied adsorption from saliva onto hydrophilic and hydrophobic surfaces and the effect of anionic and cationic surfactant, SDS and CTAB, respectively, on the adsorbed salivary films.16 Though saliva is a complex liquid, consisting of a mixture of inorganic ions, small organic compounds, proteins, enzymes, and mucins, the mucins make up a major weight fraction of saliva. 17,18 Vassilakos’ studies showed that SDS completely removed salivary films adsorbed on a hydrophilic surface. On the hydrophobic surface SDS caused a reduction of the adsorbed pellicle material but complete removal did not take place. The cationic surfactant, CTAB, induced either reduction of the adsorbed amount or complete removal of the film.19 The effect of surfactants on salivary films formed from six different salivary fractions proved to be complex and dependent on the composition of the films, the charge and the hydrophobicity of the surfactants, and the charge and hydrophobicity of the interfaces. However, it was concluded that anionic surfactant generally is most efficient in removing the salivary films from surfaces.20 Recently, Hahn Berg et al. compared adsorption of salivary protein films on silica and hydroxyapatite surfaces and their removal by SDS.21 The authors found similar adsorbed amounts on both these surfaces. However, the elutability of the films by SDS was different. Full removal from silica took place, whereas a residual layer was found on hydroxyapatite. This was explained as being partly due to the net positive charge of hydroxyapatite. Arnebrant and Whalgren reviewed protein-surfactant interactions in bulk and at solid-liquid interfaces.22 It has been found that the elutability of preadsorbed proteins decreases with factors favoring conformational changes of the protein on the surface. Experimental findings suggest that the surfactant induced elutability of proteins from hydrophilic surfaces correlates with the strength of binding of the surfactant to the protein in solution. With hydrophobic surfaces, removal of protein is similar for the different surfactants, indicating that the removal is due to competitive adsorption by the surfactant. This work puts an emphasis on mucin-surfactant interactions at the solid-liquid interface. We have explored how the removal of preadsorbed mucin by surfac(14) Shi, L.; Caldwell, K. D. J. Colloid Interface Sci. 2000, 224, 372. (15) Lindh, L.; Glantz, P.-O.; Carlstedt, I.; Wickstro¨m, C.; Arnebrant, T. Colloids and Surfaces B-Biointerfaces 2002, 25, 139. (16) Vassilakos, N. PhD Thesis, Lund University, 1992. (17) Rehak, N. N.; Cecco, S. A.; Csako, G. Clin. Chem. Lab. Med. 2000, 38, 335. (18) Metabolism; Altman, P. L., Dittmer, D. S., Eds.; Bethesda, Maryland, 1968. (19) Vassilakos, N.; Arnebrant, T.; Glantz, P.-O. Biofouling 1992, 5, 227. (20) Vassilakos, N.; Arnebrant, T.; Rundergren, J. Acta Ontodontol. Scand. 1992, 50, 179. (21) Hahn Berg, I. C.; Elofsson, U. M.; Joiner, A.; Malmsten, M.; Arnebrant, T. Biofouling 2001, 17, 173. (22) Arnebrant, T.; Wahlgren, M. C. Proteins at Interfaces II 1995, 602, 239.

tant is affected by the nature of the solid surface and by the nature of the polar surfactant headgroup. It will be shown that the removal of preadsorbed mucin from the surface can be understood by considering the mucinsurfactant association in bulk solution and the affinity of the surfactant to the solid surface. We have also to some extent investigated the adsorption from solutions containing both mucin and surfactant. Materials and Methods The bovine submaxillary gland mucin, BSM, catalog number M3895, and the SDS were purchased from Sigma. The BSM, specified by the manufacturer to contain 12% sialic acid, was used as received. The SDS was purified by recrystallization from Milli Q water, repeating the procedure twice. Penta(oxy ethylene) dodecyl ether, C12E5, from Nikko and n-dodecyl β-D-maltopyranoside, C12-mal, from Sigma were used as received. Proanalysis grade NaCl was obtained from Merck. All solutions were made by using water purified with a MilliRO 10 Plus pretreatment unit, followed by purification with a Q-PAK unit. The outgoing water, resistivity >18 Mohm, was filtered through a 0.2 µm filter. All mucin and surfactant solutions contained 30 mM NaCl if not otherwise stated. The mucin solutions prior to use in reflectometry experiments were aged for at least 16 h. No older than 120 h old solutions were used. No significant difference was seen on results obtained over this period. The mucin stock solution was kept at 4 °C in order to obstruct bacterial growth. Surfaces. Silica. One-side-polished silicon wafers were purchased from Aurel GmbH, Germany. The wafers were thermally oxidized in laboratory air at 1000 °C for 1.5 h. This procedure results in an ellipsometrically (El X-04, DRE-Dr. Riss Ellipsometerbau GmbH) measured SiO2 layer thickness of 80-110 nm. The oxidized silicon wafers were subsequently cut into ∼1cm-wide strips and stored in a closed container with laboratory air. The strips were cleaned by red glowing in a natural gas flame (Butane, Super clean, Swan, UK) for about 5 s just before being used in the reflectometer. Hydrophobically Modified Silica. Nonpolar surfaces were prepared by silanation with (3,3-dimethylbutyl)dimethylchlorosilane (97%, ABCR, Karlsruhe) via the gas phase. The surfaces were kept in a silane vapor atmosphere for 20 h and then heated for 40 min in 120 °C and subsequently rinsed in ethanol (ethanol, absolute, Fisher Chemicals) and water. The advancing contact angle of water on the silanized surfaces was 92-95°. Dynamic Light Scattering. The dynamic light scattering instrument used in these sets of experiments is described in detail in, e.g., ref 23. The data collection is based on an ALV5000 digital multiple τ correlator with 288 exponentially spaced channels. The measured intensity autocorrelation function is given by

g(2)(t) ) B(1 + β|g(1)(t)|2)

(1)

where β is a nonideality factor and B is a baseline term. g(1)(t) can be written as the inverse Laplace transform (ILT) of the distribution of relaxation times, τ:

g(1)(t) )

∫ A(τ ) exp(-t/τ) dτ ∞

0

(2)

A constrained regularization of the REPES routine was used to perform the ILT analysis. This routine is essentially similar to CONTIN, except that REPES directly minimizes the sum of the squared differences between experimental and calculated intensity-intensity autocorrelation function g(2)(t) using a nonlinear programming and allows the selection of the smoothing parameter. The relaxation time distributions are represented in the form of τA(τ) versus log τ plots, with τA(τ) in arbitrary units. This provides an equal area representation. Surface Tension Measurements. The surface tension of surfactant solutions in the absence and presence of mucin was (23) Shille´n, K.; Brown, W.; Jonhsen, R. M. Macromolecules 1994, 27, 4825.

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measured with a Kru¨ss K12 tensiometer, employing the Wilhelmy plate method. The measurements were carried out by using a sand-blasted platinum plate to ensure zero contact angle. The plate was cleaned using bichromosulfuric acid prior to each isotherm measurement, rinsed with ethanol, and finally dried with nitrogen gas. The surface tension measurements using surfactant solutions without mucin were carried out by titrating the solution by successive addition of surfactant. However, due to the long equilibration times in the presence of mucin, a different approach was taken for these samples: each surfactant-mucin sample was prepared separately and allowed to equilibrate for 48 h prior to determining the surface tension. Reflectometry. The adsorbed amount of mucin was determined by fixed angle reflectometry in combination with a stagnation point flow cell. The reflectometer setup used in this study, along with the theoretical considerations concerned with the adsorbed amount calculation, have been thoroughly described by Dijt et al.24,25 In short, a linearly polarized monochromatic light (Uniphase He/Ne laser 1108 P, λ ) 632.8 nm) is passed through the 45° glass (BK7 Glass) prism and impinges at the surface at an angle close to the Brewster angle θ (θ ) arctan ns/nsubstrate). Here ns and nsubstrate are the refractive index of solvent and substrate, respectively. The reflected light that passes the prism is split into its parallel (p) and perpendicular (s) components. The ratio between the intensities of these components Ip/Is is the output signal S.

S)

IP IS

(3)

Due to adsorption of material, the refractive index at the surface will change as well as the reflectivity and, consequently, the ratio Ip/Is will change. During the start of an experiment the cell is filled with pure solvent (30 mM NaCl solution in our experiments), and the signal due to the surface without adsorbate, S0, is registered. The increment of the signal due to adsorption, ∆S, is

∆S ) S - S0

(4)

From this change the adsorbed amount is calculated.

Γ)

∆S 1 ∆S ) Q [mg/m2] S0 As S0

(5)

As is a sensitivity factor defined as

As )

d(Rp/Rs) 2 1 [m /mg] dΓ (Rp/Rs)0

(6)

Rp and Rs in eq 6 are the parallel and perpendicular reflectivity, respectively, the symbol 0 indicates the starting situation. As is calculated by using a model description of the system as described by Dijt et al.24 Alternatively, a sensitivity factor can be experimentally determined for each new Si/SiO2 strip by using a surfactant with very well-defined adsorption behavior. This method was adopted in our study. For this purpose we employed C12E5 that has an adsorption plateau of 5.7 µmol/m2 or 2.3 mg/m2 on hydrophilic silica surface at pH ∼6.26 The differences in dn/dc values for the reference adsorbate, C12E5, and the sample under study can be taken into account by using

( )(

Γ ) Γref

)

ref ∆S (dn/dc) ∆Sref (dn/dc)

(7)

In our evaluation, dn/dc ) 0.131, 0.119, 0.142, and 0.159 mL/g for C12E5,26 SDS,27 C12-mal, and mucin, respectively, were used. (24) Dijt, J. C.; Cohen Stuart, M. A.; Fleer, G. J. Advances in Colloid and Interface Sci. 1994, 50, 79. (25) Dijt, J. C. Ph.D. Thesis, Landbouwuniversiteit te Wageningen, 1993. (26) Brinck, J.; Jonsson, B.; Tiberg, F. Langmuir 1998, 14, 1058.

Figure 1. Relaxation time distributions obtained through inverse Laplace transformation of DLS data at an angle of 90°. All solutions contained 25 ppm of BSM and 30 mM NaCl. Measurements were made 48 h after preparing the solutions. The curves correspond to the following situations: no added surfactant ([), surfactant added to a concentration of 1 cmc SDS (0), C12E5 (2), and C12-mal (O). The refractive index of mucin solutions was measured by using a Wyatt Thechnology, Optilab DSP interferometric refractometer. Atomic Force Microscope Imaging. The principles of atomic force microscopy (AFM) have been described in ref 28. In this study we employed a TopoMetrix scanning probe microscope, TMX 2010 controller. AFM images of mucin adsorbed on silica surfaces were obtained in contact mode in the liquid. We used Park Scientific Instruments’ gold-coated sharpened silicone nitride cantilevers, model MSCT-AUHW, with a spring constant of about 0.01 N/m. It should be noted that the images reported here were acquired by using only a small load. If higher loads were used, the surface features were found to be deformed and eventually moved along the surface by the cantilever. The acquired images were analyzed by using SPMLab image analysis software. During image processing, leveling in order to compensate for sample tilt and shading for enhancement of image features were used. A single line editing to remove some noise lines was also employed. Mucin Solutions. Mucin solutions were prepared by dissolving the BSM to a concentration of 25 ppm in 30 mM NaCl. This yielded a solution with pH in the range 6.5-7, which is close to the average pH in unstimulated whole saliva.17

Results Dynamic Light Scattering. We have previously shown that this mucin sample in 30 mM NaCl associate into large aggregates.29 The aggregates are dissolved by addition of sufficient amount of SDS, above ≈0.2 cmc. The deaggregation is a rather slow process at low surfactant concentration. For this reason we here chose to report results only for samples that have been allowed to equilibrate for 48 h. The relaxation time distribution curves for mucin and mucin-surfactant solutions are shown in Figure 1. The mucin concentration was kept constant at 25 ppm and the surfactant concentration was in all cases equal to cmc in the 30 mM NaCl solution, i.e., 3.3 mM for SDS, 0.064 mM for C12E5,30and 0.167 mM for C12-mal.31 The relaxation time distribution for the solutions are essentially monomodal, ignoring the small and not reproducible peaks seen at short relaxation times. Very large aggregates (Rh ≈ 430 nm) are present in the absence of surfactant. These aggregates remain when either of the nonionic surfactants are added or when SDS is added to (27) Nishikido, N.; Shinozaki, M.; Sugihara, G.; Tanaka, M. J. Colloid Interface Sci. 1980, 74, 474. (28) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930. (29) Bastardo, L.; Claesson, P.; Brown, W. Langmuir 2002, 18, 3848. (30) Rosen, M. J. Surfactants and Interfacial Phenomena; John Wiley & Sons: New York, 1989. (31) Hines, J. D.; Thomas, R. K.; Garrett, P. R.; Rennie, G. K.; Penfold, J. J. Phys. Chem. B 1998, 102, 9708.

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Table 1. Hydrodynamic Radius of Mucin in a Range of Different Surfactant Solutionsa surfactant

CSurfactant, mM

Rh, nm

mucin C12E5 C12E5 C12E5 C12-maltoside C12-maltoside C12-maltoside SDS SDS SDS SDS SDS SDS

0.00 0.01 0.07 0.20 0.05 0.17 0.51 0.20 0.50 0.75 1.00 2.00 4.00

430 650 415 430 600 445 385 650 183 83 79 44 44

a All samples contained 25 ppm of mucin and 30 mM of NaCl. The samples were allowed to equilibrate at room temperature for about 2 days. Some minor peaks present are not reported.

concentrations below 0.75 mM (0.22 cmc); see Figure 1 and Table 1. However, at SDS concentrations at and above 0.75 mM a deaggregation process occurs and smaller units are present in solution. This demonstrates that SDS associates with mucin and as a result the aggregates are dissolved. In a previous study we showed that the deaggregation process is affected by the alkyl sulfate chain length and the salt concentration29 and that it could be understood by considering electrostatic and hydrophobic interactions. The most important result in the context of this work is that SDS causes deaggregation, whereas the nonionic surfactants do not. Surface Tension. The dynamic light scattering results demonstrate that SDS associates with mucin molecules in solution. However, they do not prove that the nonionic surfactants do not associate with the mucin, since it is conceivable that such an association would not result in deaggregation. To obtain further information on this subject, we measured the surface tension of mucinsurfactant solutions and compared the results with the surface tension of mucin-free surfactant solutions. The results are shown in Figure 2. To obtain reproducible results we allowed the solutions containing mucin to equilibrate for 48 h prior to measurement. Even so it was difficult to obtain a precise value of the surface tension of the 25 ppm mucin solution without surfactant. The best estimate we can give is 65 ( 2 mN/m. One may speculate that the poor reproducibility is due to inhomogeneity of the mucin layer at the air-water interface. Addition of a small amount of C12E5 (Figure 2a) or C12mal (Figure 2b) results in a lowering of the surface tension to just below 60 mN/m. At the low surfactant region the surface tension of the mucin/surfactant solution is lower than that of the pure surfactant solution, indicating formation of a mixed layer. However, once the nonionic surfactant concentration has reached 0.1 cmc (6.4 × 10-6 M for C12E5 and 1.7 × 10-5 M for C12-mal), the surface tension of the mucin-surfactant solution is indistinguishable from that of the pure surfactant. Hence, the surfactant has displaced the mucin from the air-water interface, demonstrating that no surface-active complexes are formed and that the amount of surfactant bound to the mucin, if any, is low. The surface tension of mucin-SDS solutions is strikingly different compared to that of pure SDS. Addition of a small amount of SDS to the mucin solution results in a surface tension value of about 45 mN/m, demonstrating the presence of surface-active aggregates. The surface

Figure 2. (a) Surface tension isotherms for C12E5 in 30 mM NaCl with (0) and without ([) 25 ppm BSM. (b) Surface tension isotherms for C12-mal in 30 mM NaCl with (0) and without ([) 25 ppm BSM. (c) Surface tension isotherms for SDS in 30 mM NaCl with (0) and without ([) 25 ppm BSM.

Figure 3. Adsorption isotherm of C12E5 on silica surface in 30 mM NaCl. The surfactant concentration is expressed in units of cmc (1 cmc ) 6.4 × 10-5 M).

tension of the mucin-SDS solution remains significantly lower than that of the mucin-free SDS solution until cmc of the pure surfactant is approached. Hence, mucin remains on the surface at least up to the cmc. The surface tension reached above the cmc is, however, the same with and without 25 ppm mucin present in solution. Reflectometry, Adsorption of Pure Components. The adsorbed amounts of C12E5, C12-mal, and SDS onto silica and hydrophobically modified silica at pH 5.5-6 from 30 mM NaCl were determined by using reflectometry. The adsorption isotherm for C12E5 on hydrophilic surface is displayed in Figure 3. No adsorption is detected until the concentration is about half the cmc. At higher concentrations, the adsorption increases rapidly and levels off at 2.3-2.5 mg/m2 once

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Figure 4. Adsorption isotherms of C12-mal (9), C12E5 (O), and SDS (b) on hydrophobically modified silica in 30 mM NaCl. The surfactant concentrations are normalized against the cmc of the respective surfactant.

the cmc has been reached. The shape of the adsorption isotherm indicates a cooperative adsorption and can be described as a self-assembly of the surfactant at the solidliquid interface. The results presented in Figure 3 are very similar to those of Tiberg32 for the same surfactant but without any added salt. The AFM study of Grant et al.33 demonstrated that the adsorbed surfactant layer consists of bilayer aggregates. We noted that hardly any adsorption of C12-mal could be detected up to a concentration of 1.5 cmc, the highest concentration probed. Considering the scatter of the experimental data, this means that the adsorption of this surfactant is below 0.2 mg/m2. It is remarkable that the nonionic C12-mal does not adsorb, whereas the also nonionic C12E5 does. However, this result is in line with previous findings34,35 that demonstrate that sugar-based surfactants adsorb to alumina, titania, and hematite but less so to silica, whereas ethylene oxide based surfactants adsorb strongly to silica.32 It has been proposed that differences in hydrogen bonding between the surfactants and the surface are the reason for this different adsorption behavior. Adsorption of the anionic SDS to the negatively charged silica surface was also beyond the limits of detection up to 1.5 cmc as also reported by, e.g., Joabsson at al.36 The adsorption isotherms of C12E5, C12-mal, and SDS on hydrophobically modified silica are displayed in Figure 4. All the three surfactants readily adsorb on the hydrophobic surface. Adsorption of surfactants starts already at very low concentrations and levels off when a surfactant monolayer has adsorbed. The area per molecule at the adsorption plateau is 48 Å2/molecule for C12E5 and SDS and 49 Å2/molecule for C12-mal. The large molecular weight of BSM makes the adsorption kinetics considerably slower than for the surfactants. The data in Figure 5a show that adsorption equilibrium of mucin from a solution containing 25 ppm BSM and 30 mM NaCl on silica surfaces has not been reached even after 4 h. At this stage the adsorbed amount is about 2 mg/m2. The very long equilibration times are consistent with previous studies using various types of mucin and surfaces.9-11,13,37 This indicates that slow conformational (32) Tiberg, F. J. Chem. Soc., Faraday Trans. 1996, 92, 531. (33) Grant, L. M.; Tiberg, F.; Ducker, W. A. J. Phys. Chem. B 1998, 102, 4288. (34) Zhang, H.; He, H. X.; Mu, T.; Liu, Z. F. Thin Solid Films 1998, 237-239, 778. (35) Zhang, L.; Somasundaran, P.; Mielczarski, J.; Mielczarski, E. J. Colloid Interface Sci., in press. (36) Joabsson, F.; Thuresson, K.; Lindman, B. Langmuir 2001, 17, 1499.

Figure 5. (a) Adsorption of BSM from a 30 mM NaCl solution containing 25 ppm of mucin. The adsorption occurred on a silica surface. The experiment was ended by flushing with 30 mM NaCl without any mucin. The arrow indicates the start of the rinsing step. (b) Amplification of the data at short times. Adsorption of BSM from a 30 mM NaCl solution containing 25 ppm of mucin on a silica surface (9) and on a hydrophobically modified silica surface (O). The solid line represents the kinetics for diffusion-limited adsorption.

changes and/or additional adsorption in an outer layer occur with time. The adsorbed amounts reported in different studies vary substantially, however, depending on the nature of the surface, solution salt concentration, and pH. For instance, Lindh et al.15 found that the adsorbed amount of BSM on silica from a 25 ppm mucin solution containing 50 mM NaCl and 10 mM phosphate buffer was of the order of 0.8 mg/m2, i.e., about 2.5 times lower than in our case (30 mM NaCl, no buffer). Clearly, the adsorption of mucin onto negatively charged hydrophilic silica surfaces depends strongly on salt concentration and pH. This is not surprising considering the large amount of acidic sialic acid groups on mucin and silanol groups on silica. This issue deserves further attention but is beyond the scope of the present investigation. We note, however, that under our conditions the adsorbed amount was consistent between experiments to within 10%. When the cell is rinsed with mucin-free 30 mM NaCl, a very limited desorption is observed. This is a consequence of the high-affinity binding isotherm that leads to a very slow mass transport away from the surface.38 It is of some interest to take a closer look at the adsorption kinetics at short time scales. The relevant data are displayed in Figure 5b. The reflectometry measurements are carried out in a stagnant flow geometry that allows accurate determination of the flux (J) of material to the surface:39,40

J ) 0.776v1/3R-1D2/3(RRe)1/3c

(8)

(37) Perez, E.; Proust, J. E.; Baszkin, A.; Boissonade, M. M. Colloids Surf. 1984, 9, 297. (38) Cohen Stuart, M. A.; Fleer, G. J. Annu. Rev. Mater. Sci. 1996, 463.

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Figure 6. Adsorption-desorption of mucin from a 25 ppm of 30 mM NaCl solution onto silica. Arrows indicate the points where the flushing with 30 mM NaCl and with 1.3 cmc SDS solution starts (1 cmc SDS ) 3.3 × 10-3 M in 30 mM NaCl).

where ν is the kinematic viscosity, R the radius of the inlet tube, D the diffusion coefficient, R a parameter defined by the geometry of the system and the Reynolds number, Re, and c the concentration. The diffusion coefficients in the 25 ppm mucin solution were determined by dynamic light scattering, as described above. We recall that the mucin solution consisted of aggregates with mean hydrodynamic radius of 430 nm. The straight line in Figure 4b is the calculated flux of mucin to the surface using eq 8 with no fitting parameters. The adsorption rate for the hydrophobically modified silica is at short times (up to 60 s) in good agreement with the theoretical line. The fact that the adsorption at short times increases linearly with time demonstrates that the adsorption process is diffusion controlled. However, the quantitative agreement between theory and experiment is to some degree coincidental. The aggregates present in the mucin solution do not all have the same size and the smaller aggregates that diffuse quickest will arrive at the surface first. Hence, one would expect a somewhat more rapid increase in adsorption than indicated by the theoretical line in Figure 5 b, and it is more correct to draw the conclusion that a small adsorption barrier exists. This could be an electrostatic barrier due to the presence of a small negative charge on the hydrophobic surface. The existence of a negative charge on such a surface was demonstrated by Kjellin et al. and explained by the presence of unreacted silanol groups.41 The adsorption rate on the hydrophilic silica is somewhat slower than on the hydrophobic surface. This is partly due to a larger electrostatic repulsion between the negatively charged mucin and the former surface. At longer times (higher adsorbed amounts) the adsorption rate is significantly reduced both on silica and hydrophobized silica. This is caused by an increase in the barrier against further adsorption due to repulsive interactions, both steric and electrostatic, between mucin molecules colliding with the surface and such molecules that are already adsorbed. Reflectometry, Removal of Preadsorbed Mucin by Surfactant. Due to the very long time needed in order to reach equilibrium, Figure 5a, see also ref 37, we established the following experimental protocol. The mucin was allowed to adsorb for 60-70 min from a 25 ppm mucin solution in 30 mM NaCl, then the surface was flushed with mucin-free 30 mM NaCl solution for 30 min, next the surfactant solution was introduced, and finally the surface was rinsed again with 30 mM NaCl. A typical experimental curve is shown in Figure 6. (39) Dabros, T.; van de Ven, T. G. M. Colloid Polym. Sci. 1983, 261, 694. (40) Dijt, C. J.; Cohen Stuart, M. A.; Hofman, J. E.; Fleer, G. J. Colloids Surf. 1990, 51, 141. (41) Kjellin, U. R. M.; Claesson, P. M.; Vulfson, E. N. Langmuir 2001, 17, 1941.

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Figure 7. Fraction of removed mucin from silica surfaces as a function of surfactant concentration. The surfactant concentrations are normalized by the cmc of the respective surfactant. Removal of mucin with addition of SDS (O), C12E5 (9), and C12-mal (2).

Figure 8. Fraction of removed mucin from hydrophobized silica surfaces as a function of surfactant concentration. The surfactant concentrations are normalized by the cmc of the respective surfactant. Removal of mucin with addition of SDS (b), C12E5 (9), and C12-mal (2).

In this particular case the surfactant was SDS and the concentration was equal to 1.3 cmc (1 cmc ) 3.3 mM). We note that addition of SDS results in a rapid removal of a large part of the adsorbed mucin. The fraction of mucin removed from silica surfaces as a function of surfactant concentration and surfactant type is provided in Figure 7. Note that the fraction remaining on the surface was determined after finally rinsing the surface with 30 mM NaCl, which removes any surfactant that eventually has adsorbed to the surface in the previous step (disregarding the possibility of formation of insoluble mucin-surfactant aggregates that are stable in surfactant-free solution). To be able to compare the effect of the surfactants we have normalized the surfactant concentration with the cmc of each surfactant. The surfactants studied all contain a straight C12 hydrocarbon chain, whereas the nature of the polar group differs. SDS has an anionic sulfate headgroup, C12E5, a five-unit-long ethylene oxide chain, and C12-mal, a maltose unit. Clearly, the preadsorbed mucin layer is affected very differently by the different surfactants. It is quite resistant to the presence of C12mal, and less than 10% of the adsorbed mucin is removed at the highest surfactant concentration investigated, 1.5 cmc. On the other hand, SDS is rather efficient in removing the mucin once the concentration is above 0.3 cmc. Nearly all mucin is removed above the cmc of SDS. The mucin layer is not much affected by the presence of C12E5 until the concentration has reached about 0.7 cmc. However, at higher concentration most of the preadsorbed mucin layer is removed by the surfactant. Similar results for removal of mucin preadsorbed on hydrophobized silica surfaces are displayed in Figure 8. In this case all three surfactants are efficient in removing the mucin from the surface and the effectiveness, when considering the surfactant concentration normalized by cmc, is C12E5 > C12-mal > SDS. We also note that the nonionic surfactants are more efficient in removing

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Figure 9. Mucin adsorption to silica surfaces from solutions containing 25 ppm mucin, 30 mM NaCl, and surfactants: SDS (b); C12E5 (9); C12-mal (2).

Figure 10. Mucin adsorption to silica from solutions containing 25 ppm mucin, 30 mM NaCl, and SDS. The surfactant concentration is normalized by the cmc. Adsorption from solutions that were mixed just prior to experiment (9) and from mixtures that were aged for 7 days prior to determining the adsorption (b).

the mucin from hydrophobized silica as compared with silica. Reflectometry, Adsorption from Mucin-Surfactant Mixtures. In another set of experiments, we studied the adsorption onto silica from solutions containing 25 ppm mucin, 30 mM NaCl, and various amounts of surfactant. The results, which were evaluated after flushing with 30 mM NaCl to remove any adsorbed surfactant, are summarized in Figure 9. We note that addition of SDS reduces the mucin adsorption from the mixture and that the reduction in adsorbed amount is rather gradual. Very limited adsorption occurs above the cmc of the surfactant. The adsorption from the mixture containing C12E5 and mucin displays a different behavior. Here, the addition of C12E5 up to a concentration of 0.5 cmc has essentially no effect on the amount of adsorbed mucin. However, at a concentration of 1 cmc hardly any mucin is adsorbed to the surface. Finally, the presence of C12-mal has little effect on the mucin adsorption. We note that the results obtained for adsorption from the mixtures, Figure 9, is completely consistent with the results obtained for removal of preadsorbed mucin by the different surfactants, Figure 7. These results will be discussed further below. The light scattering data for mucin-surfactant systems reported previously29 show that the deaggregation of mucin in SDS solutions well below the cmc is a rather slow process with smaller aggregates being formed after longer equilibration times. No similar effect was observed for the other surfactants. For this reason we found it to be of interest to study the adsorption of mucin from SDSmucin mixtures after different equilibration times. The results, displayed in Figure 10, show that a longer equilibration of the mixture results in a lower amount of mucin adsorbed. These results provide a strong coupling between the deaggregation of mucin by SDS and the reduction of mucin adsorption in the presence of SDS.

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AFM Imaging. The AFM image of the silica surface (not shown) used in the reflectometry study is rather featureless but confirms the smoothness of the substrate, with typical peak-to-trough heights of 1 nm. The AFM images of the surface after adsorption times in the range 30 min to 4 h show a homogeneously coated surface (Figure 11a) with the roughness that is larger than that of bare silica. On the other hand, AFM images recorded after allowing the surface to be in contact with the solution for 19 h display some pronounced surface features, Figure 11b. One immediately notices the presence of large aggregates on the surface. The typical diameter of these aggregates is 300-400 nm with a height of about 80 nm. This size is smaller than the size of the large aggregates observed in the dynamic light scattering study. Nevertheless, we propose that this surface feature originates from adsorption of rather intact large aggregates that are present in bulk solution. The smaller dimensions can be explained by a partial deaggregation and spreading upon adsorption. In fact, this hypothesis is supported by the absence of aggregates on the surface after a short time. We note that the surface topology between these aggregates is similar to that of the surface after 40 min of adsorption. Hence, slow adsorption of mucin aggregates is suggested to be the reason for the slow increase in adsorbed amount with time observed by us and others.37 AFM images recorded after flushing the mucin coated with SDS solution above the cmc (Figure 11c) show that the material between the adsorbed aggregates has been removed and the flat feature of the bare silica has been recovered. However, the large aggregates remain attached to the surface also in the presence of SDS above the cmc. Discussion Mucin Association in Bulk. In a previous report29 we showed that the BSM preparation when dissolved in 30 mM NaCl contained large aggregates of mucin held together by physical bonds. This conclusion was reached based on the fact that mercaptoethanol, which breaks disulfide bonds, had a negligible effect on the aggregate size. The size of the aggregates was also found to decrease only slightly with time during 48 h of equilibration. Initially, approximately 50% of the mucin material was present in aggregates with a hydrodynamic radius of about 720 nm, whereas the remaining mucin was present in significantly smaller aggregates or single coils. After 48 h of equilibration the size distribution of the aggregates was more monomodal (Figure 1), with an average hydrodynamic radius of 430 nm. It was suggested that the aggregation occurred due to association between the less heavily glucosidated regions. Association between Mucin and Surfactants in Bulk. The dynamic light scattering study also showed that addition of SDS could reduce the size of the aggregates and the amount of mucin present in the aggregates to a significant degree. In fact, once the SDS concentration was increased to 0.2 cmc or above, and sufficient time was allowed (longer times were needed for lower SDS concentrations), the aggregates disappeared and the solution contained single mucin molecules decorated with SDS that had a typical hydrodynamic radius of 44-90 nm, see Table 1. Clearly, mucin and SDS associate in bulk solution once the SDS concentration is high enough, and the association breaks the physical bonds, keeping the mucin aggregates together. The increased charge of the mucin-SDS complex as well as direct solvation of mucin-mucin attachment points by SDS will contribute to the deaggregation of the

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Figure 11. (a) AFM image of mucin on SiO2 surface after 40 min of adsorption from a 30 mM NaCl solution containing 25 ppm mucin and (b) after 19 h adsorption. (c) AFM image of the mucin remaining on the surface after flushing with SDS solution above its cmc in 30 mM NaCl.

mucin aggregates. We note that both mucin and SDS are anionic and that the mucin-SDS association is driven by hydrophobic interactions. This latter conclusion is supported by the observation that the alkyl sulfate surfactant has to have a sufficiently large nonpolar chain to affect the mucin aggregates.29 On the other hand, addition of C12E5 or C12-mal to the mucin solution has no dramatic effect on the size of the mucin aggregates even after 48 h (Figure 1). Hence, from the dynamic light scattering data we do not find any evidence for an association between mucin and the nonionic surfactant. The surface tension measurements presented in Figure 2 support the notion that the nonionic

surfactants studied here do not associate with mucin molecules to any significant degree. Adsorption/Desorption of Mucin. The adsorption kinetics of anionic mucin on hydrophobically modified silica surfaces is at short times close to diffusion limited. Hence, in a 30 mM NaCl solution the electrostatic barrier against the initial adsorption is small, but comparison between the adsorption kinetics to hydrophobized silica and silica indicates that such a barrier does exist at least in the case of silica. At higher surface coverage the rate of adsorption is, of course, significantly slower. In fact, we do not see a well-expressed plateau even after 4 h, and others have reported that no plateau is reached for mucin

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adsorption on mica even after 24 h.37 The reason for this very slow approach toward equilibrium is the polydispersity of the sample and the presence of large aggregates. That large aggregates do attach to the surface is confirmed by the AFM images of the mucin-coated surface, Figure 11b. As expected, for a high molecular weight sample with a reasonably high affinity for the surface, the desorption by dilution is very limited (as also observed with BSM and the other type of mucins13,14) during the experimental time scale. This does not mean that the adsorption is irreversible but only that the kinetics of desorption is very slow, which is well understood theoretically.38 Mucin consists of heavily glucosidated and negatively charged regions separated by less glucosidated regions where the protein backbone is exposed. It is not too bold to suggest that it is the “naked” regions of the mucin molecule that adsorb to the silica surface. This statement is based on the observations that sugar units do not have a strong affinity to silica, e.g., C12-mal does not adsorb to silica as shown in this work, and the adsorption of sugarbased polymers on silica has been reported to be low.42 Further, there is an electrostatic repulsion between the glucosidated regions and the silica surface that counteracts the adsorption of these regions. Adsorption of Mucin-SDS Complexes. The adsorption of mucin from solutions containing both mucin and SDS to silica surfaces is significantly reduced compared to the adsorption of mucin from SDS-free solutions, Figures 9 and 10. This demonstrates that the mucin-SDS complexes that are formed in bulk solution have a lower affinity for the surface than the mucin itself. Part of the reason for this is the increased negative charge of the SDS-mucin complex compared to mucin alone, which increases the electrostatic repulsion at the negatively charged surface. Another important reason is that SDS associates with the naked regions of the mucin molecule, i.e., the regions that preferentially adsorb to the silica surface. Hence, we suggest that SDS blocks the adsorption by solvating the adsorbing regions of the mucin. The results reported in Figure 9 further indicate that these regions are not blocked by the nonionic surfactants, which supports the conclusions from DLS and surface tension measurements that the association between mucin and these surfactants is limited or absent. Removal of Preadsorbed Mucin by Surfactants. Surfactants may facilitate removal of polymers by two different mechanisms. First, the surfactant may associate with the polymer and the affinity to the surface for the polymer-surfactant complex may be different than that of the polymer alone. This change in surface affinity can be due to a change in solvency of the polymer-surfactant complex compared to that of the pure polymer or caused by a direct change in the interaction between the polymer and the surface. This is, for instance, the mechanism behind the removal of lysozyme43 and cationic polyelectrolytes44,45 from mica surfaces by addition of SDS. Polymers may also be removed from the surface by surfactants by competitive adsorption between the polymer and the surfactant. This occurs if both the polymer and the surfactant have an affinity for the surface but do not associate with each other. Of course, both mechanisms may be operative at the same time. (42) Weissenborn, P. K.; Warren, L. J.; Dunn, J. G. Colloids Surf. 1995, 99, 29. (43) Fro¨berg, J. C.; Blomberg, E.; Claesson, P. M. Langmuir 1999, 15, 1410. (44) Rojas, O. J.; Neuman, R. D.; Claesson, P. M. J. Colloid Interface Sci. 2001, 237, 104. (45) Rojas, O. J.; Ernstsson, M.; Neuman, R. D.; Claesson, M. P. Langmuir 2002, 18, 1604.

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From Figure 7 we see that SDS is efficient in removing mucin from the silica surface. Since SDS does not adsorb to silica we can rule out competitive adsorption as the mechanism of removal. Instead, SDS associates with mucin and the removal is facilitated by the increased negative charge of the complex formed. Further, we argue that SDS adsorbs to the regions of mucin that have the highest affinity for the silica surface, and thus mucinsurface attachment points are broken by SDS. It should also be pointed out that removal of mucin from the surface by SDS occurs above a critical surfactant concentration (≈0.2-0.3 cmc) and that this surfactant concentration is very similar to the one above which SDS causes deaggregation of mucin aggregates formed in bulk solution. Hence, the critical association concentration between SDS and mucin is very similar in bulk solution and at the silica surface. The explanation for the removal of mucin by C12E5 is different. In this case the surfactant adsorbs to the silica surface, Figure 3, whereas we have no evidence for any surfactant-mucin association. Hence, the likely mechanism of mucin removal is in this case competitive adsorption. This conclusion is further supported by the observation that mucin removal occurs only at such high surfactant concentrations that a significant adsorption of the surfactant on silica takes place, compare Figures 3 and 7. We also note that the surfactant adsorption to silica is cooperative and so is the removal of mucin from the surface by C12E5. C12-mal does not adsorb to silica surfaces to any significant extent, and we have no evidence for any association with mucin. Hence, in this case addition of the surfactant does not affect the adsorbed layer either by competitive adsorption or by modifying the polymersurface affinity. This is the reason C12-mal does not remove mucin from silica surfaces. The situation is different on hydrophobized silica. On this surface C12-mal does adsorb and it has the ability to remove mucin by competitive adsorption, Figure 8. Competitive adsorption also explains the removal of mucin from hydrophobized silica by C12E5. One may speculate that the reason C12E5 is more efficient in removing mucin from hydrophobic surface than C12mal is a larger repulsion between the headgroups and mucin residues in the former case. The observation that the SDS is rather inefficient in removing mucin from hydrophobic surfaces is consistent with the formation of surface-active mucin-SDS complexes as evidenced from surface tension measurements, Figure 2c. Conclusions We have investigated the association between mucin and three different surfactants in bulk solution, at the air-water interface, and at two different solid-liquid interfaces. It is found that mucin and SDS associate once the SDS concentration has reached above about 0.2 cmc. A very similar critical association concentration (cac) is found in bulk solution and at the silica/solution interface. SDS facilitates removal of preadsorbed mucin above the cac, and adsorption from premixed mucin-SDS solutions is reduced above cac. We propose that the mucin-SDS association leads to a reduced surface affinity and to an increased solubility of the complex as compared to mucin alone. However, the mucin-SDS complexes do have a high affinity to the air-water interface and also to some extent to nonpolar surfaces. This suggests that the composition of the complex in bulk and at the nonpolar interfaces is different. We find no evidence for association between C12E5 and mucin in bulk solution or at the air-water

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interface. Nevertheless, C12E5 readily removes mucin from silica surfaces at concentrations above about 0.7 cmc, i.e., at slightly larger concentrations than that where the cooperative adsorption of C12E5 to silica takes place. The mechanism of removal of mucin is different for SDS and for C12E5. In case of C12E5, the desorption is due to competitive adsorption, and the adsorbing surfactant displaces the preadsorbed mucin layer from the surface. Consistent with this explanation it is found that the adsorption from C12E5-mucin mixtures is not affected by the presence of the surfactant until its concentration is high enough to induce adsorption of the surfactant alone. The third surfactant studied, C12-mal, has still a different effect. In this case, we have no evidence for association with mucin in bulk solution or at the air-water interface, and the surfactant does not adsorb to any significant

degree on silica. As a consequence, C12-mal does not displace preadsorbed mucin layers. On the other hand, when mucin is preadsorbed to hydrophobized silica, all three surfactants are able to displace the mucin due to their affinity for the surface. Formation of surface-active mucin-SDS complex, as evidenced from surface tension measurements, is suggested to explain why this surfactant is a relatively inefficient displacer for mucin preadsorbed to nonpolar surfaces. Acknowledgment. This work was carried out within the VINNOVA Competence Centre for Surfactants Based on Natural Products, SNAP. A.D. acknowledges the financial support of a Marie Curie Postdoctoral Fellowship. LA0259813