Fibronectin Displacement at Polymer Surfaces | Langmuir

Nicoletta Giamblanco, Mohammed Yaseen, Genady Zhavnerko, Jian R. Lu, and Giovanni Marletta . Fibronectin Conformation Switch Induced by Coadsorption ...
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Langmuir 2005, 21, 4571-4577

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Fibronectin Displacement at Polymer Surfaces Lars Renner,† Tilo Pompe,*,† Katrin Salchert,† and Carsten Werner†,‡ Leibniz Institute of Polymer Research Dresden & The Max Bergmann Center of Biomaterials Dresden, Hohe Str. 6, 01069 Dresden, Germany, and Department of Mechanical and Industrial Engineering, University of Toronto, 5 King’s College Road, M5S 3G8 Toronto, Ontario, Canada Received December 23, 2004. In Final Form: February 7, 2005 The interactions of fibronectin with thin polymer films are studied in displacement experiments using human serum albumin. Fibronectin adsorption and exchange on two different maleic anhydride copolymer surfaces differing in hydrophobicity and surface charge density have been analyzed by quartz crystal microbalance and laser scanning microscopy with respect to adsorbed amounts, viscoelastic properties, and conformation. Fibronectin is concluded to become attached onto hydrophilic surfaces as a “softer”, less rigid protein layer, in contrast to the more rigid, densely packed layer on hydrophobic surfaces. As a result, the fibronectin conformation is more distorted on the hydrophobic substrates together with remarkably different displacement characteristics in dependence on the adsorbed fibronectin surface concentration and the displacing albumin solution concentration. While the displacement kinetic remains constant for the strongly interacting surface, an acceleration in fibronectin exchange is observed for the weakly interacting surface with increasing fibronectin coverage. For displaced amounts, no change is determined for the hydrophobic substrate, in contrast to the hydrophilic substrate with a decrease of fibronectin exchange with decreasing coverage leading finally to a constant nondisplaceable amount of adsorbed proteins. Furthermore, the variation of the albumin exchange concentration reveals a stronger dependence of the kinetic for the weakly interacting substrate with higher rates at higher albumin concentrations.

Introduction Protein adsorption at solid/liquid interfaces is still not fully understood and influences many important processes in various widespread and emerging technologies. Although numerous published studies have provided dedicated model approaches,1-6 the mechanism of protein adsorption and displacement at interfaces largely remains enigmatic. The interplay of different forces, the variety of protein structures, and their dynamical conformational changes so far have interfered with the applicability of any more general description. At realistic settings, mixtures of different proteins occur in biofluids and, thus, determine the composition of adsorbed protein layers at interfaces. When considering the involved heterodisplacement phenomena, one often refers to the “Vroman effect”, after Vroman et al.,7,8 who pioneered the investigation of the exchange of plasma proteins at interfaces, putting emphasis on the transient maximum in the adsorbed amount of fibrinogen adsorbed from plasma. Although the findings of Vroman were confirmed to represent a frequently observed pattern of size-dependent sequential protein displacement,9 there are numerous reports on protein displacement processes that are not in agreement with the size-dependent displacement. Specifically, the * Corresponding author. E-mail: [email protected]. † Leibniz Institute of Polymer Research Dresden & The Max Bergmann Center of Biomaterials Dresden. ‡ University of Toronto. (1) Haynes, C. A.; Norde, W. Colloids Surf. 1994, B2, 517. (2) Ramsden, J. J. Chem. Soc. Rev. 1995, 73-78. (3) Ramsden, J. J. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; p 321. (4) Ball, V.; Bentaleb, A.; Hemmerle, J.; Voegel, J.-C.; Schaaf, P. Langmuir 1996, 12, 1614. (5) Evans, J. W. Rev. Mod. Phys. 1993, 65, 1281. (6) Calonder, C.; Van Tassel, P. R. Langmuir 2001, 17, 4392. (7) Vroman, L.; Adams, A. L. Surf. Sci. 1969, 16, 438. (8) Vroman, L.; Adams, A. L.; Klings, M. Fed. Proc. 1971, 30, 1494. (9) Norde, W.; Lyklema, J. The Vroman Effect; VSP: Utrecht, The Netherlands, 1992; pp 1-20.

interaction strength between protein and solid surface as well as the protein solution concentration were found to determine the patterns of competitive protein adsorption.10-12 Fibronectin (FN) and human serum albumin (HSA) are important, well-known proteins. FN, a key component of the extracellular matrix (ECM), is a large dimeric glycoprotein triggering cell adhesion and, in turn, undergoes cell-driven assembly in supramolecular fibrils and furthermore provides specific binding sites for various ECM biopolymers.13-16 HSA is the most abundant component of many biofluids serving the transport of various metabolites and the regulation of the osmotic pressure.17-19 Numerous techniques have been elaborated to study the process of protein adsorption, including optical, electrical, gravimetric, and labeling methods.20 The present study combines gravimetric (quartz crystal microbalance with dissipation (QCM-D) measurements) and fluorescent labeling techniques (confocal laser scanning microscopy (cLSM)) to gain a deeper insight into protein-protein interactions at solid/liquid interfaces. The applicability of the QCM-D technique has been described as a powerful (10) Renner, L.; Pompe, T.; Salchert, K.; Werner, C. Langmuir 2004, 20, 2928. (11) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (12) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 17. (13) Bayneux, G.; Baugh, L.; Vogel, V. Cell Biol. 2002, 5193. (14) Oberhauser, A. F.; Badilla-Fernandez, C.; Carrion-Vazquez, M.; Fernandez, J. M. J. Mol. Biol. 2002, 319, 433. (15) Schwarzbauer, J. E.; Sechler, J. L. Curr. Opin. Struct. Biol. 1999, 11, 622. (16) MacDonald, D. E.; Markovic, B.; Allen, M.; Somasundaran, P.; Boskey, A. L. J. Biomed. Mater. Res. 1998, 41, 120. (17) Watanabe, H.; Kragh-Hansen, U.; Tanase, S.; Nakajou, K.; Mitarai, M.; Iwao, Y.; Maruyama, T.; Otagiri, M. Biochem. J. 2001, 357, 269. (18) Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Nat. Struct. Biol. 1998, 5, 827. (19) Sukhishvili, S. A.; Granick, S. J. Chem. Phys. 1999, 110, 10153. (20) Ramsden, J. J. Q. Rev. Biophys. 1993, 27, 41.

10.1021/la046801n CCC: $30.25 © 2005 American Chemical Society Published on Web 03/23/2005

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tool for studies on protein adsorption,21-23 lipid vesicle adsorption,24 as well as swelling behavior,25 providing in addition to the mass increase viscoelastic properties of the layered substrates. Recently, we analyzed the desorption and displacement kinetics of FN and HSA from maleic acid copolymer thin films10 and the impact of the observed differences in the FN anchorage on the behavior of adherent endothelial cells grown in contact with the FN coated polymer substrates.26 It was shown that the physicochemical surface characteristics of the polymer films influence the displacement and the subsequent reorganization of FN by the endothelial cells. FN was displaced more rapidly on the hydrophilic substrates.10 In accordance with these findings, the cells were capable of rearranging FN molecules extensively on hydrophilic surfaces, whereas, on hydrophobic substrates, the rearrangement was restricted due to a higher binding force between protein and surface.26 In the present study, we focused on the displacement of FN by HSA as it depends on the preadsorbed FN surface concentration as well as on the concentration of the HSA solution. The results were expected to unravel general patterns of protein-protein interactions concerning FN in adlayers at solid surfaces with different physicochemical characteristics. To extend the options for the mechanistic interpretation of the obtained data, QCM experiments were further evaluated with respect to the viscoelastic properties of the protein layers on the compared substrates. Materials and Methods Substrates. Thin films of poly(octadecene-alt-maleic anhydride) (POMA) (Polysciences Inc., Warrington, PA) (MW ) 50 000) and poly(propene-alt-maleic anhydride) (PPMA) (Leuna-Werke AG, Germany) (MW ) 39 000) were produced by spin-coating (RC5, Suess Microtec, Garching, Germany) 0.08 and 0.1%, respectively, copolymer solutions in tetrahydrofuran (Fluka, Deisenhofen, Germany) and methylethylketone (Fluka), respectively, on top of glass cover slips. The cover slips have been freshly oxidized before in a mixture of aqueous solutions of ammonia (Acros Organics, Geel, Belgium) and hydrogen peroxide (Merck, Darmstadt, Germany) and were subsequently surface-modified with 3-aminopropyl-dimethylethoxy-silane (ABCR, Karlsruhe, Germany) prior to spin-coating of the copolymer solutions to allow a covalent fixation of the thin copolymer films. Stable covalent binding of the polymer films to the glass carriers was achieved by annealing at 120 °C for 2 h. The polymer films were thoroughly characterized with respect to water contact angle, film thickness, surface roughness, and chemical composition, as published recently.27 Exposure of protein solution was performed with polymer surfaces after autoclaving (steam sterilization). Autoclaving induced hydrolysis of the anhydride moieties to provide a surface bearing exclusively carboxylic acid but no anhydride groups. In this state, the polymer surfaces exhibit hydrophobic (POMA) or hydrophilic (PPMA) properties characterized by water contact angles of 100 and 38°, respectively. This difference primarily originates from the different density of polar groups, namely, carboxylic acid groups, with an estimated density derived from anhydride surface concentrations of 1 × 1014 and 4 × 1014 cm-2, respectively (for details, see ref 27). (21) Otzen, D. E.; Oliveberg, M.; Ho¨o¨k, F. Colloids Surf., B 2003, 29, 67. (22) Ho¨o¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. J. Colloid Interface Sci. 1998, 208, 63. (23) Welle, A. J. Biomater. Sci., Polym. Ed. 2004, 15, 357. (24) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397. (25) Fa¨lt, S.; Wågberg, L.; Vesterlind, E.-L. Langmuir 2003, 19, 7895. (26) Pompe, T.; Markowski, M.; Werner, C. Tissue Eng. 2004, 10, 841. (27) Pompe, T.; Zschoche, S.; Herold, N.; Salchert, K.; Gouzy, M.-F.; Sperling, C.; Werner, C. Biomacromolecules 2003, 4, 1072.

Renner et al. The same cleaning and spin-coating procedure was applied for the coating of quartz crystal sensors (Q-Sense AB, Gothenburg, Sweden), which had been previously covered with a SiO2 layer of 50 nm thickness (GeSiM GmbH, Dresden, Germany). Prior to copolymer film preparation, SiO2 coated quartz crystal sensors were additionally cleaned by mechanical cleaning by CO2 Snowjet followed by an oxygen plasma treatment (Harrick Scientific Products, Ossining, NY) for 100 s at medium energy level. Proteins and Labeling. For fluorescence detection, FN was labeled by the fluorescent dye carboxytetramethylrhodamine (rhodamine). FN, purified from adult human plasma following the protocol of Brew et al.,28 was conjugated with a commercially available labeling kit (Molecular Probes, Eugene, OR). The average degree of FN labeling was 3.8 ((1.1) mol of dye/mol of protein. For protein adsorption experiments, polymer surfaces were immersed in solutions of different concentrations (2.5-50 µg mL-1) of rhodamine-labeled protein in phosphate buffered saline (PBS) (Sigma, Steinheim, Germany) at pH 7.4 for 1 h. Unlabeled HSA was purchased from Sigma (Mu¨nchen, Germany) and stored as stock solution of 10 mg mL-1 at -20 °C. For exchange studies, HSA was diluted in PBS shortly before use and adjusted to concentrations of 50, 100, 500, and 2500 µg mL-1. Unlabeled FN was used for QCM-D measurements freshly diluted to concentrations of 2.5, 10, and 50 µg mL-1 in PBS. Adsorption. In situ adsorption studies were performed for time periods of 1 h with a quartz crystal microbalance including dissipation factor measurement (QCM-D, Q-Sense, Gothenburg, Sweden). The hydrolyzed polymer surfaces (maleic acid form of the copolymers) were exposed to 2.5, 5, 10, and 50 µg mL-1 FN solutions. Data acquisition of frequency and dissipation changes utilized the QSOFT software, and the raw data were further analyzed by the QTOOLS software, based on a model approach of Voinova et al.29 The amount of adsorbed FN was also analyzed by HPLC, as described in detail elsewhere.30 Briefly, the adsorbed FN layers were decomposed into amino acids by acidic vapor phase hydrolysis, fluorescently labeled, and subsequently quantified in a HPLC system (Series 1100, Agilent Technologies, Bo¨blingen, Germany). Displacement Experiments. After 1 h of incubation (at 37 °C) with rhodamine-FN solutions, the surfaces were rinsed three times by PBS followed by immersion in HSA solution to study displacement processes. A fluorescence confocal laser scanning microscope (TCS SP, Leica, Bensheim, Germany) equipped with a 40× oil immersion objective was used to reveal the displacement of FN as a decay of the fluorescence of the substrate surface. Each data point of the kinetics (at time 0, 1, 2, 6, 24, and 48 h) was determined by the average of 10 measured spots. All experiments have been repeated at least three times. Laser intensities were calibrated prior to each measurement by using the InSpeck Orange calibration kit (Molecular Probes, Eugene, OR), as described in detail elsewhere.10 With calibration beads of different intensity, a simple correlation between the photomultiplier gain of the cLSM and the measured intensities could be established to convert the measured relative intensities to an absolute scale. The conversion allows a reliable comparison of the adsorbed protein amounts on both polymer surfaces. Immunofluorescence. After 1 h of incubation with FN solutions of different concentrations (2-20 µg mL-1), the surfaces were rinsed with PBS and stained with primary mouse monoclonal antibodies for the heparin binding domain and the primary cell binding domain (clone: FNH3-8, FN12-8; Takara Biochemicals, Japan). Secondary antibody staining was performed with sheep anti-mouse IgG rhodamine Red-X conjugated antibodies (Jackson Immunoresearch, West Grove, PA). Fluorescence intensities were analyzed by fluorescence laser scanning microscopy as described above. Mean values from 10 measurements of two independent experiments were evaluated. (28) Brew, S. A.; Ingham, K. C. J. Tissue Cult. Methods 1994, 16, 197. (29) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391. (30) Salchert, K.; Pompe, T.; Sperling, C.; Werner, C. J. Chromatogr., A 2003, 1005, 113.

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Langmuir, Vol. 21, No. 10, 2005 4573 Table 1. Calculated Surface Coverage of FN Layers on POMA and PPMA Polymer Filmsa

POMA

PPMA

Figure 1. Dissipation and frequency change versus FN surface coverage. The mean of two independent measurements is shown with mean error.

Results QCM-D Studies. FN layers were adsorbed from solutions of 2.5, 5, 10, and 50 µg mL-1 FN in PBS onto hydrolyzed POMA and PPMA thin films. The adsorption process was monitored by QCM-D. QCM-D allows by measuring changes in resonance frequency and damping characteristics (energy dissipation) of the quartz crystal the determination of changes in mass and viscoelastic properties due to adlayers, respectively. Figure 1 illustrates the overall dissipation and frequency shifts after 1 h of adsorption versus the corresponding FN adsorbed amount determined by HPLC. The dissipation changes on POMA are always lower than those on PPMA substrates, whereas the frequency shifts are higher on POMA. The frequency changes correlate with the adsorbed amount of FN, which is higher on POMA. Analysis of the dissipation changes revealed that FN adsorbed on the hydrophobic POMA surfaces forms a stiffer layer than on the hydrophilic PPMA. Because of the low dissipation change, one could expect a compact protein conformation and strong binding to the surface. Strong hydrophobic interactions apparently cause the tight binding of FN most pronounced at low coverage. At very high coverage, the FN molecules may bind more loosely even on POMA due to protein-protein in-plane interactions and competition of proteins for surface sites, leading to a less tightly bound layer structure with higher dissipation. In contrast, on the hydrophilic PPMA surface, the protein is bound through electrostatic interactions between the carboxylic groups of the copolymer substrate and positively charged domains of FN, which leads to a less tightly bound and more open structure of the protein layer already at low surface coverage. Subsequently, the loose and open substrate contact of the FN molecules may lead to an adsorption regime, where FN molecules are assembled partly on top of each other. As the electrostatic interactions are weaker (partly repulsive) than hydrophobic interactions, the more loosely packed layer on the hydrophilic PPMA results in a higher energy loss, as seen in the dissipation data. These data are supported by AFM measurements31 showing FN adsorbed in a compact form on hydrophobic substrates, while it is assembled in an elongated, less compact manner on hydrophilic surfaces. This interpretation is based on the assumption of no conformational changes of the underlying substrate surface. As for the hydrophilic PPMA surface, a polymer brush swelling behavior was found recently;32 one could (31) Bergkvist, M.; Carlsson, J.; Oscarsson, S. J. Biomed. Mater. Res. 2003, 64, 349. (32) Pompe, T.; Renner, L.; Grimmer, M.; Herold, N.; Werner, C. To be published.

cFN (µg mL-1)

ΓHPLC (ng cm-2)

ΓSauerbrey (ng cm-2)

ΓVoight (ng cm-2)

2.5 5 10 50 2.5 5 10 50

173 205 269 471 80 129 202 409

220 410 688 1250 45 127 265 530

184 448 780 1369 97 186 367 702

a The surface coverage for the Voigt/Kelvin model is calculated from the modeled layer thickness by taking the constant layer density of the model (M ) Fprottprot).

expect changes in the polymer layer properties as well as the protein adsorption pattern during the adsorption process. While from our data set we cannot totally rule out those changes, the linearity of the frequency and dissipation changes (see Figure 1) and comparable measurements under different swelling conditions of the PPMA filmsmeaning solutions of different ionic strengths (data not shown)sindicate negligible effects from those interactions under the given solution conditions at pH 7.4 in PBS. The comparison of the calculated surface coverage from the QCM-D measurements with the HPLC data is shown in Table 1. The values of the Sauerbrey and Voigt/Kelvin approaches are restricted by the boundary conditions of the applied models, assuming nonviscous mass contributions in a vacuum for the Sauerbrey approach and an additional dissipative contribution due to viscosity for the Voigt/Kelvin model (for more details, see ref 29). This may explain the lower values for the adsorbed amounts determined by the Sauerbrey approach. The comparison of the FN surface coverage determined by QCM-D and HPLC further reveals a high water mass content incorporated or coupled to the protein layer, bound as hydration shells to FN molecules (see Table 1), because the surface coverage calculated by the more appropriate Voigt/Kelvin model from the QCM-D datasrepresenting the state of the protein layer in an aqueous environments indicate a higher mass than the HPLC datassolely characterizing the pure protein amount on the surface. In regard to the listed results, in comparison to PPMA, a higher water mass uptake must occur on POMA-bound FN molecules. Interestingly, the dissipation data indicated a higher energy loss in PPMA. As mentioned above, the higher dissipation could indicate a less dense FN layer on PPMA. By calculating the effective density, Feff, of the adsorbed layer from the QCM-D data, this hypothesis is supported. As introduced by Ho¨o¨k et al.,33 it can be calculated for the used system by

Feff )

mlayer mQCM ) , msolv ) mQCM - mHPLC Vlayer mprot msolv + Fprot Fsolv (1)

where the values of mlayer and mprot ()mHPLC) are taken from measurement data: The solvent and protein densities are set to Fsolv ) 1000 kg m-3 and Fprot ) 1370 kg m-3 according to ref 34. By using this equation, the density can be calculated in dependence on the FN solution (33) Ho¨o¨k, F.; Kasemo, B.; Nylander, T.; Fant, C.; Scott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796. (34) Quillin, M. L.; Matthews, B. W. Acta Crystallogr. 2000, D56, 791.

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Figure 2. Effective density, Feff, of the FN layer versus the surface coverage determined by HPLC calculated from the data in Table 1 according to eq 1.

concentration during adsorption, as presented in Figure 2. A strong change in the layer composition is observed for POMA surfaces, in contrast to a smaller change in the density for PPMA surfaces. The decrease in surface density corresponds to the change of coupling of water into the FN layer. Hence, one can conclude again that FN adsorbs on POMA at low coverage in a very compact form with strong conformational changes due to the strong hydrophobic interaction. At higher coverage, it still adsorbs in a more globular form with a strong affinity to the surface. The globular shape, similar to the shape in solution, probably leads to the high water content in the FN layer and the higher surface coverage in comparison to the PPMA surface. On the hydrophilic PPMA, the adsorption of FN was assumed to occur in a more elongated state due to the electrostatic interaction. The lower affinity to the surface does not strongly change the conformation. However, the electrostatic interaction opens the globular structure of the dissolved FN. Thus, less water can be coupled to FN molecules. Furthermore, the electrostatic protein-substrate interaction probably decreases the amount of free charges on the molecule to build up a thicker hydration shell, leading to smaller changes in the effective density. The hypothesis of varying conformational changes with varying surface coverage is supported by estimating the coverage of a dense packed monolayer of FN dimers with a monomer of 60 nm in length and 2.5 nm in diameter. A surface coverage of ∼250 ng cm-2 is determined. As seen in Figure 2, the decrease of the effective layer density stops at about this surface coverage, because above this coverage the adsorbing proteins cannot spread as much and the overlapping of FN molecules during adsorption should increase. By that, the adsorbing FN molecules

Renner et al.

exhibit only a less intense interaction with the polymer substrate, inducing only minor changes of the conformational state of the whole FN molecule. Immunofluorescence of FN Layers. To verify the above assumptions about conformational changes of adsorbed FN molecules on hydrophobic POMA and hydrophilic PPMA, binding of monoclonal antibodies to the heparin binding domain near the C-terminus (clone FNH3-8) and a domain near the primary cell binding domain (clone FN12-8) was measured by confocal laser scanning microscopy. Figure 3 shows the fluorescence intensities in dependence on the FN surface coverage. An influence of the different substrates POMA and PPMA is found again. While for the heparin binding domain (FNH3-8) a slightly lower antibody binding is observed for POMA, the difference is more drastic near the cell binding domain (FN12-8) with a lower increase in antibody binding on POMA. This observation can be interpreted in the following way. Near the cell binding domain, strong conformational changes occur on the hydrophobic POMA substrate, leading to a diminished antibody binding at low coverage. At high coverage, the antibody binding is increased, because of the less drastic conformational changes due to the inhibited spreading and the increased overlapping of the FN molecules as stated above. In contrast, on the hydrophilic PPMA, the antibody can bind unrestricted already at low coverage because of less conformational changes due to the only loose FN-substrate interaction. A similar behavior is true for the heparin binding domain with less pronounced conformational changes. At very high FN coverage (about a theoretical monolayer, 250 ng cm-2), a saturation behavior for the antibody binding is observed probably due to lateral spatial restrictions for the antibody, leading to a decreased binding probability. These experiments are a nice agreement with the conformational analysis of FN and FN fragments on different selfassembled monolayers by immunofluorescence and theoretical calculations showing similar trends.35,36 Displacement of FN. Using the results of QCM-D and immunofluorescence experiments as a platform for further studies, we investigated in detail the behavior of the exchange of preadsorbed FN by HSA molecules to characterize the state of the FN layer on both polymer surfaces. First, rhodamine-labeled FN at different bulk concentrations, as explained in the QCM-D section, has been adsorbed to both substrates. In previous work, it was shown by comparison with experiments with unlabeled FN and HPLC-based amino acid analysis that the fluorescence label did not alter the FN adsorption and displacement behavior.10

Figure 3. Intensities of antibody binding (A, clone FNH3-8; B, clone FN12-8) to FN layers on POMA and PPMA. Mean values and mean errors of two independent experiments are shown.

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Figure 4. FN surface coverage versus time during protein exchange by HSA (50 µg mL-1) on PPMA (A) and POMA (B). The data sets are average values of three independent measurements (mean error shown).

Figure 5. FN surface coverage versus time during protein exchange by HSA rescaled from Figure 4 with respect to HPLC data at initial coverage on PPMA (A) and POMA (B).

As shown in Figure 4A, at low initial surface coverage, FN was hardly displaced by HSA on the hydrophilic PPMA surface. Increasing the initial surface coverage by increasing the coating solution concentration leads to an increase in the relative amount of displaced FN up to 50% for the highest absolute surface coverage of 409 ng cm-2 (see Table 1). In contrast (Figure 4B), FN adsorbed on POMA was displaced up to 30% with no significant difference caused by the initial surface coverage. Only for the lowest surface coverage, a slightly higher relative displacement of the preadsorbed FN species was determined. Plotting the absolute FN surface coverage versus time (Figure 5) for different initial coverages on each substrate indicates that different absolute amounts were displaced on PPMA, while approximately similar amounts were exchanged on POMA surfaces. The comparison of the absolute surface coverage at late stages of displacement on PPMA reveals a limiting nondisplaceable fraction of surface-bound FN which was concluded to be independent from the initial surface coverage. The displacement rate was accomplished by fitting the exponential decay of the observed kinetics. In a common approach, Huetz and co-workers37 suggested a simple kinetics for the description of protein desorption, depending only on the amount of immobilized protein, which is characterized by the differential equation

∂Γ ) -kΓ ∂t

(2)

where Γ and k are the surface coverage and the desorption time constant, respectively. Assuming two species of

immobilized protein, a fast (index A) and a slow (index B) desorbing one, leads to

Γ(t) ) ΓA exp(-kAt) + ΓB exp(-kBt)

(3)

This two-species model has been fitted within the investigated systems in earlier experiments.10,26 A constant nondisplaceable amount was not used in the model in the earlier study because the displacement kinetics indicated no relevance for such a component. As postulated earlier, in this study, a nondisplaceable amount might occur for a low FN coverage on PPMA. By using the model equation, this should manifest itself as a drastic decrease in the second time constant. Figure 6 shows the parameters of the kinetic fits depending on the surface coverage. The regression coefficient of the fits was always R2 > 0.97. For the POMA surface, the time constants of the fast and slow species show no clear trend. However, on the PPMA surface, the slow time constant is 1 order of magnitude lower for low coverage than for high ones, indicating almost no displacement. In further studies, we investigated the behavior of the exchange of preadsorbed FN by HSA molecules depending on the concentration of the HSA solution. First, FN at a bulk concentration of 50 µg mL-1 was adsorbed to the polymer surfaces and subsequently exchanged by HSA bulk solutions of different concentrations (50, 100, 500, (35) Keselowsky, B. G.; Collard, D. M.; Garcia, A. J. J. Biomed. Mater. Res 2003, 66A, 247. (36) Wilson, K.; Stuart, S. J.; Garcia, A.; Latour, R. A., Jr. J. Biomed. Mater. Res. 2004, 69A, 686. (37) Huetz, Ph.; Ball, V.; Voegel, J.-C.; Schaaf, P. Langmuir 1995, 11, 3145.

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Discussion

Figure 6. Time constants of the exponential fit of the FN displacement in dependence on FN surface coverage. Values were determined by fits to the averaged values in Figure 4 with R2 > 0.97.

and 2500 µg mL-1) monitored by confocal laser scanning microscopy (cLSM). The chosen concentrations of 500 and 2500 µg mL-1 correspond to the HSA concentration in cell culture media, where 2 or 10% serum concentrations are applied. In the range of average scatter of the surface coverage, small differences (Figure 7A) were observed in dependence on the HSA concentration on PPMA substrates. Slightly higher amounts (up to 60%) are exchanged at high HSA concentrations. On the hydrophobic POMA surfaces, less FN is displaced in general (up to 40% for 2500 µg mL-1), as shown in Figure 7B. However, the increased FN exchange for higher HSA concentrations is more drastic here in comparison to the PPMA substrate. The kinetics were fitted according to eq 3 with the time constants plotted in Figure 8 (R2 > 0.97). A significant trend is obtained for the slow time constant, kB, showing an increase with increasing HSA concentration. Although the displacement kinetics changes with the HSA concentration, the displaced amount of FN remains constant on the PPMA surface. In contrast, for POMA, the increase in the slow time event correlates with an increased FN exchange for the higher HSA concentrations. This observation can be attributed to the different adsorption characteristic of the FN molecules on PPMA in comparison to POMA. As the kinetics is accelerated for both substrates with an increased amount of competing HSA molecules, on PPMA, the nondisplaceable amount of FN molecules at a low coverage cannot be exchanged in contrast to the weaker adsorbed FN molecules which do overlap surfacebound FN molecules.

In the present study, the influence of different FN surface coverages on the subsequent exchange by HSA molecules was investigated. The FN exchange characteristic was compared on two copolymer substrates with varying physicochemical properties, namely, the density of charged carboxylic acid groups. The different density of maleic acid groups results in a more hydrophilic PPMA surface and a more hydrophobic POMA surface. Within this work, the status of the protein layer assembly was characterized and shown to influence the subsequent displacement by the exchange protein. For the characteristic of the adsorbed FN layer, the following conclusion can be drawn from the QCM-D, HPLC, and immunofluorescence measurements: (i) FN builds a more rigid layer on POMA in a more compact form with stronger conformational changes as the result of the strong hydrophobic interactions with the long alkyl chains of the co-monomer. The more globular structure of the protein molecules allows a high amount of water to be coupled into the FN layer. (ii) On PPMA, the characteristics of the FN layer are more determined by the electrostatic interactions of the carboxylic acid groups, leading to looser binding of molecules in a more open form, however, with less conformational changes. By that, we suppose that FN adsorbs partly overlapping each other and with an in total smaller amount on the substrate surface. Less water is coupled into this FN layer, where the open form of the molecules and the electrostatic origin of their binding are thought to be the main reasons for the smaller coupled hydration shell in comparison to POMA. The different states of FN on both substrates are in agreement with established models and reported data for protein adsorption. Norde et al.11,38 discussed the influence of electrostatic charge as well as hydrophobic properties on the adsorption of various model proteins. In regard to the surface properties, FN attaches with a higher affinity to POMA by exposing the hydrophobic core of the protein toward the surface hydrophobic surface, which results in an increasing binding strength and stronger conformational changes in the secondary protein structure. On PPMA, the electrostatic interactions lead to a change in the tertiary structure by opening the globular form, however, less changes in the secondary structure due to the less intense interaction with the negatively charged carboxylic acid groups. The hypothesized change in the tertiary structure is supported by the observations of Bergkvist et al.,31 revealing elongated FN molecules on hydrophilic surfaces and globular compact molecules on

Figure 7. Displacement of FN by differently concentrated HSA solutions on PPMA (A) and POMA (B). The data sets are average values of three independent measurements (mean error shown).

Fibronectin Displacement at Polymer Surfaces

Figure 8. Time constants of the exponential fit of FN displacement in dependence on HSA solution concentration. Values were determined by fits to the averaged values in Figure 7 with R2 > 0.97.

hydrophobic substrates. Studies on FN and FN-fragment adsorption onto CH3 and COOH terminating self-assembled monolayers35,36 found similar conformational changes in dependence on the substrate surface chemistry. Stronger conformational changes on the more hydrophobic CH3 terminating surfaces were concluded from a lower intensity in binding of different monoclonal antibodies in agreement with molecular dynamics simulations of the structure of the adsorbed protein. As mentioned earlier, the observed changes in the FN adsorption behavior on the hydrophilic PPMA might also be influenced by changes in the properties of the substrate due to the swollen polymer brush characteristic.32 While those effects could not be revealed in the QCM-D experimentssbecause of their possibly compensating charactersswelling experiments and comparable FN adsorption experiments at different ionic strengths (unpublished data) imply a negligible influence, if it is present at all. The different characteristics of the adsorbed FN on the two substrates reflect themselves in modified exchange properties, as found in the displacement studies by HSA molecules: (i) on the hydrophobic POMA substrate, similar amounts are displaced independent of the surface coverage, because every molecule adsorbs in a compact form (38) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87.

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and there is no potential for further spreading due to the strong hydrophobic interaction to the substrate; (ii) the electrostatic interaction on PPMA substrates leads to a less strong interaction and a higher spreading potential. By varying the surface coverage, FN molecules adsorb with a different degree of spreading due to the different space available. This behavior results in a dependence of the exchange characteristic on the surface coverage with less exchange at low surface coverage. This interpretation is based on the well-known RSA model39-41 with relaxation and spreading of adsorbed proteins in dependence on the available space. For POMA, the strong interaction prevents further spreading and the HSA displacement occurs similarly at each surface coverage. However, on PPMA, the FN molecules spread very well at low surface coverage and remain in a nondisplaceable state. Above a surface coverage of ∼100 ng cm-2, the FN molecules cannot fully spread and start to partly overlap each other, resulting in an increase of the exchange probability by the HSA molecules. The displacement studies in dependence on the HSA concentration confirm the above statements concerning the state of the adsorbed FN molecules. While the exchange process was accelerated by a higher HSA concentration on both substrates, the nondisplaceable FN molecules on PPMA led to a limited amount of displaced FN molecules. In summary, the strong correlation of the state of the adsorbed FN molecules with their exchange characteristic in competition with HSA molecules was demonstrated. The combination of QCM-D, immunofluorescence, and fluorescence microscopy allows a detailed characterization of the physicochemical and biological state of the adsorbed proteins together with a quantification of their surface concentration under competing conditions such as culture cell. Acknowledgment. The authors of this study gratefully acknowledge for granting in parts the Bundesministerium fu¨r Bildung, Forschung und Technologie, Berlin, Germany, as BMBF-Kompetenzzentrum fu¨r Materialien im Blut- und Gewebekontakt (Grant No. 03N4022). LA046801N (39) Ewans, J. W. Rev. Mod. Phys. 1993, 65. (40) Schaaf, P.; Talbot, J. Phys. Rev. Lett. 1989, 62, 175. (41) Van Tassel, P. R.; Talbot, J.; Tarjus, G.; Viot, P. Phys. Rev. E 1996, 53, 785.