Fibrinogen Adsorption and Conformational Change on Model Polymers

Apr 14, 2009 - Novel Aspects of Mutual Molecular Rearrangement ... water content, and corresponding protein film structure of fibrinogen adsorbed to a...
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Fibrinogen Adsorption and Conformational Change on Model Polymers: Novel Aspects of Mutual Molecular Rearrangement Mattias Berglin,*,† Emiliano Pinori,† Anders Sellborn,‡ Marcus Andersson,† Mats Hulander,† and Hans Elwing† †

:: :: Department of Cell and Molecular Biology, Interface Biophysics, Goteborg University, Goteborg SE-40530, :: :: Sweden, and ‡Department of Surgery, Sahlgrenska University Hospital, Goteborg University, Goteborg SE-41345, Sweden Received November 6, 2008. Revised Manuscript Received March 9, 2009

By combining quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR), the organic mass, water content, and corresponding protein film structure of fibrinogen adsorbed to acrylic polymeric substrates with varying polymer chain flexibility was investigated. Albumin and immunoglobulin G were included as reference proteins. For fibrinogen, the QCM-D model resulted in decreased adsorbed mass with increased polymer chain flexibility. This stands in contrast to the SPR model, in which the adsorbed mass increased with increased polymer chain flexibility. As the QCM-D model includes the hydrodynamically coupled water, we propose that on the nonflexible polymer significant protein conformational change with water incorporation in the protein film takes place. Fibrinogen maintained a more native conformation on the flexible polymer, probably due to polymer chain rearrangement rather than protein conformational change. In comparison with immunoglobulin G and albumin, polymer chain flexibility had only minor impact on adsorbed mass and protein structure. Understanding the adsorption and corresponding conformational change of a protein together with the mutual rearrangement of the polymer chain upon adsorption not only has implications in biomaterial science but could also increase the efficacy of molecular imprinted polymers (MIPs).

Introduction Protein adsorption onto solid surfaces is important, as it has implications in biotechnology, immunoassays, biosensors, biofouling, and biomaterials, among others.1-6 The driving force for adsorption is well established. For comprehensive reviews, see, for example, the work of Norde and colleagues, Andrade and Hlady, or Mrkisch and Whitesides.7-9 As discussed in their papers, the conformational change upon adsorption is one important driving factor for adsorption. In biomaterial science, this conformational change not only promotes the adsorption but also dictates biological responses on another time scales such as immune complement activation and inflammatory response,10,11 cell recruitment, and cell attachment.12-14 Even though there is importance in many fields, a fundamental understanding of both adsorption kinetics and the corresponding conformational *To whom correspondence should be addressed. E-mail: Mattias.berglin@ cmb.gu.se. Telephone: +46 31 786 25 93. Fax: +46 31 786 25 99. (1) Baty, A. M.; Suci, P. A.; Tyler, B. J.; Geesey, G. G. J. Colloid Interface Sci. 1996, 177, 307–315. (2) Chittur, K. K. Biomaterials 1998, 19, 357–369. (3) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336–6343. (4) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164–1167. (5) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28–60. (6) Sellborn, A.; Andersson, M.; Hedlund, J.; Andersson, J.; Berglin, M.; Elwing, H. Mol. Immunol. 2005, 42, 569–574. (7) Norde, W.; Lyklema, J. J. Biomater. Sci., Polym. Ed. 1990, 2, 183–202. (8) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79, 1–63. (9) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55–78. (10) Lin, Y. S.; Hlady, V.; Janatova, J. Biomaterials 1992, 13, 497–504. (11) Tang, L. P.; Liu, L.; Elwing, H. B. J. Biomed. Mater. Res. 1998, 41, 333–340. (12) Werthen, M.; Sellborn, A.; Kalltorp, M.; Elwing, H.; Thomsen, P. Biomaterials 2001, 22, 827–832. (13) Li, J.; Thielemann, C.; Reuning, U.; Johannsmann, D. Biosens. Bioelectron. 2005, 20, 1333–1340. (14) Wittmer, C. R.; Phelps, J. A.; Saltzman, W. M.; Van Tassel, P. R. Biomaterials 2007, 28, 851–860.

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change is still lacking, which testifies to the complexity of this problem. The adsorbed amount, orientation, and conformational change of fibrinogen has received considerable attention.12,15-19 It has been shown that fibrinogen plays a major role in mediating the adhesion of platelets to implanted surfaces, which could lead to thrombotic events.20-22 One of the milestones in biomaterial research was the discovery by Tang and co-workers that surface-adsorbed fibrinogen initiated the acute inflammatory response.23-25 It was shown that fibrinogen upon adsorption underwent a conformation change exposing two cell binding epitopes, P1 and P2, responsible for attracting inflammatory cells to the site. Tang and co-workers also showed that different materials caused different magnitudes of inflammatory response, probably due to different degrees of conformation change. The biological response to biomaterials is thus strongly correlated to the structure and conformation of fibrinogen. In a recent study, Weber and co-workers used quartz crystal microbalance with dissipation monitoring (QCM-D) for the real time study of fibrinogen adsorption to model biomaterial (15) Sit, P. S.; Marchant, R. E. Surf. Sci. 2001, 491, 421–432. (16) Walivaara, B.; Aronsson, B. O.; Rodahl, M.; Lausmaa, J.; Tengvall, P. Biomaterials 1994, 15, 827–834. (17) Martins, M. C. L.; Ratner, B. D.; Barbosa, M. A. J. Biomed. Mater. Res., Part A 2003, 67A, 158–171. (18) Ishizaki, T.; Saito, N.; Sato, Y.; Takai, O. Surf. Sci. 2007, 601, 3861–3865. (19) Luck, M.; Paulke, B. R.; Schroder, W.; Blunk, T.; Muller, R. H. J. Biomed. Mater. Res. 1998, 39, 478-485. (20) Savage, B.; Shattil, S. J.; Ruggeri, Z. M. J. Biol. Chem. 1992, 267, 11300–11306. (21) Savage, B.; Ruggeri, Z. M. J. Biol. Chem. 1991, 266, 11227–11233. (22) Shiba, E.; Lindon, J. N.; Kushner, L.; Matsueda, G. R.; Hawiger, J.; Kloczewiak, M.; Kudryk, B.; Salzman, E. W. Am. J. Physiol. 1991, 260, C965–C974. (23) Tang, L. P.; Eaton, J. W. Am. J. Clin. Pathol. 1995, 103, 466–471. (24) Hu, W. J.; Eaton, J. W.; Tang, L. P. Blood 2001, 98, 1231–1238. (25) Tang, L. P.; Eaton, J. W. J. Exp. Med. 1993, 178, 2147–2156.

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surfaces.26 In their study, they concluded that by small changes in polymer chemical structure they could affect the adsorption kinetics, the amount of surface bound fibrinogen, and the viscosity of the protein layer. The authors considered differences in biological response as a possible outcome. They also affirmed in their report that the QCM-D technique reveals the surface bound “wet mass”, which is the adsorbed “dry protein” mass plus the hydrodynamically coupled water.27 They also measured overall higher adsorbed mass as compared to other studies using, for example, optical methods such as ellipsometry.28 The discriminating results between acoustic and optical models should not be regarded as problematic but could bring further understanding into the dynamics of protein adsorption and corresponding conformational change. Multitechnique studies are currently state of the art to understand and better predict complex phenomena such as protein adsorption.29-33 To better describe the kinetics and conformational change upon adsorption, a systematical investigation of all possible variables using model surfaces is needed. One such variable might well be the molecular mobility or flexibility. In the field of polymer science, polymer chain flexibility is commonly denoted polymer compliance. It is reasonable to assume that polymer chain mobility would influence the interfacial reactions of solute molecules. The mobility of polymer molecules is expressed via a commonly measured bulk property of polymers, the glass transition temperature (Tg). It should be stressed that in most systems it is difficult to vary Tg without varying the surface functional groups, hydrophobicity, or surface morphology. However, the family of alkyl methacrylate polymers with different length alkyl ester side chains provides a means of being able to vary Tg without greatly altering surface chemistry or wetting characteristics. We have in previous studies used a series of poly(alkyl methacrylates) displaying a wide range of different Tg temperatures.34-36 We have observed both in vitro, that is, immune complement activation, and in vivo correlations such as foreign body capsule formation as a function of polymer flexibility. Our current hypothesis is that this difference in biological response could be mediated through differences in the structure of the initial adsorbed protein layer on the biomaterial surface and especially differences in fibrinogen conformational change. In this study, we have combined the acoustical QCM-D technique with the optical surface plasmon resonance (SPR) technique with the aim to characterize the adsorption of fibrinogen to poly(alkyl methacrylate) model polymers. As reference proteins, albumin and immunoglobulin G were included. (26) Weber, N.; Pesnell, A.; Bolikal, D.; Zeltinger, J.; Kohn, J. Langmuir 2007, 23, 3298–3304. (27) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796–5804. (28) Elwing, H. Biomaterials 1998, 19, 397. (29) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf., B 2002, 24, 155–170. (30) Choi, K. H.; Friedt, J. M.; Frederix, F.; Campitelli, A.; Borghs, G. Appl. Phys. Lett. 2002, 81, 1335–1337. (31) Yang, Q.; Zhang, Y. Y.; Liu, M. L.; Zhang, Y. Q.; Yao, S. Z. Anal. Chim. Acta 2007, 597, 58–66. (32) Zhou, C.; Friedt, J. M.; Angelova, A.; Choi, K. H.; Laureyn, W.; Frederix, F.; Francis, L. A.; Campitelli, A.; Engelborghs, Y.; Borghs, G. Langmuir 2004, 20, 5870–5878. (33) Voros, J. Biophys. J. 2004, 87, 553–561. (34) Berglin, M.; Andersson, M.; Sellborn, A.; Elwing, H. Biomaterials 2004, 25, 4581–4590. (35) Andersson, M.; Hedlund, J.; Berglin, M.; Elwing, H.; Tang, L. J. Mater. Sci.: Mater. Med. 2007, 18, 283–286. (36) Andersson, M.; Suska, F.; Johansson, A.; Berglin, M.; Emanuelsson, L.; Elwing, H.; Thomsen, P. J. Biomed. Mater. Res., Part A 2008, 84A, 652–660.

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Experimental Section Polymeric Substrates. Poly(isobutyl methacrylate) (PIBMA) and poly(butyl methacrylate) (PBMA) were provided by SigmaAldrich (CAS# 9011-15-8 and CAS# 25719-52-2, respectively). PIBMA was dissolved in toluene (Merck, Sweden) to a final concentration of 0.5% (w/v). Poly (lauryl methacrylate) (PLMA) was provided already dissolved in toluene and was diluted with toluene to a final concentration of 0.5% (w/v). Polymeric coatings were spin-coated on quartz crystals and SPR sensor surfaces by applying 50 μL of the polymer solutions at 2000 rpm for 1 min. Characterization of Pristine Coatings. The procedure for applying polymer coatings on the sensor surfaces has been evaluated and published.34 In this study, we only verified the water contact angle before performing protein adsorption studies. We foresee no changes in pristine polymer characteristics. Thus, the surface chemical and morphological results presented in Table 1 are data previously generated but presented in this study as they are important for the discussions. X-ray photoelectron spectroscopy (XPS) was done using a Physical Electronics Quantum 2000 scanning ESCA microprobe. The X-ray source was an Al KR anode with a beam size of 100 μm operating at 20 W/15 kW. All measurements were made at a 45° takeoff angle, and the area analyzed was 0.5  0.5 mm2. Binding energy shifts were resolved using the peak for hydrocarbon (C1s) at 285.0 eV as a reference. A 3 eV electron flood gun was used for charge neutralization during analysis. Sensitivity factors determined for this instrument were used to calculate elemental compositions. Advancing and receding contact angles were determined by the Wilhelmy plate method at 20 °C. The instrument used was a DCA-322 from Cahn (WI). The instrument was operating at 100 μm s-1. Data were evaluated with WinDCA version 1.03 (Cahn, WI). Wettability measurements were made using deionized water (γ = 72.8 mJ/m2). The surface energies (γs) of the poly(alkyl methacrylate) coatings were calculated using an equation-of-state approach formulated by Wulf et al.37 (eq 1): rffiffiffiffiffiffi γsv -βðγlv -γsv Þ2 cos θ ¼ -1 þ 2 e γlv

ð1Þ

where β is a constant determined to 0.0001247 (m2/mJ)2, γlv is the surface tension of the probe liquid, and cos θ is the advancing or receding water contact angle. Cover glass slides covered with polymer were used in these experiments. The surface morphology was examined with a Digital Instruments NanoScope III atomic force microscope fitted with a NanoScope IIIA controller and a Dimension 3000 large sample type G scanner. The coatings were analyzed in tapping mode with standard silicon tips. Analyses were made at a set point ratio (Asp/A0) of 0.7-0.8, where Asp is the set point amplitude and A0 is the free oscillation amplitude of the cantilever. At least an area of 5  5 μm2 was used in the calculation of the rootmean-square roughness (rms).

Protein Adsorption Studies with QCM-D and SPR. Human fibrinogen (Sigma-Aldrich), human albumin (SigmaAldrich), and human immunoglobulin G (Sigma-Aldrich) were dissolved in veronal buffered saline (VBS) buffer to a final concentration of 1 mg/mL. The solutions where kept in a -70 °C freezer until needed. A typical adsorption experiment was carried out as follows: (1) establish a stable baseline with pure VBS buffer for at least 3 min; (2) add protein solution to the sample chamber and observe protein adsorption during 30 min; and (3) wash with VBS buffer for 5 min. The adsorbed mass was calculated from the shift in resonance frequency for the third overtone for the QCM-D experiment and shift in refractive index for the SPR (37) Wulf, M.; Grundke, K.; Kwok, D. Y.; Neumann, A. W. J. Appl. Polym. Sci. 2000, 77, 2493–2504.

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Berglin et al. Table 1. Pristine Polymer Characteristicsa

polymer (no. of carbons)

Tg, °C

C/O ratio (calculated)

C/O ratio (measured)

roughness (rms), nm

surface energy γs (advancing/ receding), mJ/m2

contact angle hysteresis (Θ)

PIBMA (4) 66 4.0 4.2 0.88 28.3/36.5 13.0 PBMA (4) 17 4.0 4.1 0.70 28.3/40.3 22.2 PLMA (12) -70 8.0 8.6 0.70 19.1/40.5 34.6 a Shown is the polymer abbreviation with the number of carbons in alkyl side chain and glass transition temperature (Tg). The surface chemistry was analyzed with XPS, and the carbon to oxygen elemental ratio was calculated based on the repeating unit of the polymer and compared with measured. Surface roughness (root-mean-square roughness, rms) was determined with TM-AFM. The surface energy was calculated from both advancing and receding water contact angles using eq 1. Contact angle hysteresis is the advancing contact angle minus the receding contact angle.

experiment. Calculations are based on well established acoustic38 (eq 2) and optical models (eq 3).27,39 -

Cf Δf ¼ mQCMD nr

ð2Þ

Cf is the mass sensitive constant (e.g., 17.7 ng cm-2 Hz-1 at f = 5 MHz), nr is the shear wavenumber, and mQCM-D is the adsorbed mass per unit area as measured with QCM-D (ng/cm2). In this study, the third resonance was used for all mass calculations. The first resonance is generally disturbed by edge effects due to the way the crystal is mounted. Using higher harmonics could give insight in viscoelastic properties of the protein film but is not further discussed in this paper. It should be mentioned that the propagation depth of the acoustic shear wave could ;be somewhat decreased when the polymer films are applied on the sensor. The thickness of polymer and corresponding protein films are well below the analytical depth (around 200 nm in water), and we foresee minor errors due to the polymer layer. CSPR ΔRU ¼ mSPR

ð3Þ

Equation 3 describes the model used to calculate the SPR adsorbed mass. The constant, CSPR, has been calibrated to be in the range of 6.5  10-2-1.0  10-1 ng/cm2 for a wide range of different proteins.27,39 In this paper, the adsorbed mass was calculated using the same value for all proteins, that is, 6.5  10-2. ΔRU is the measured change in response units (a dimensionless quantity that is proportional to the change in refractive index, Δn, at the interfacial region). It has been shown that the molecular mass of the protein affects the refractive index; that is, lower values were observed for higher molecular mass proteins.33 Using the same model for all proteins is thus not completely correct, but the estimated error is most likely low and is not altering the conclusions presented in this paper. As discussed above, by applying a polymer film onto the sensor, the analytical depth is decreased and that could cause some minor errors in the mass calculations. The error should be minimal but, more importantly, similar for all proteins. In addition to the frequency shift, the QCM-D method determines the shift in damping or dissipation of the crystal. Dissipation measurements are made by periodically switching on and off the AC voltage over the crystal. The decay signal is recorded and fitted to an exponentially damped sinusoidal curve. High damping indicates higher viscoelastic properties of the protein layer. The quartz crystal microbalance with dissipation monitoring (QCM-D) instrument used in this study was a Q-Sense D300 :: (Q-Sense AB, Goteborg, Sweden) with a temperature controlled fluid cell. The instrument was operated at 4 V drive amplitude and automatic resonance frequency tracking. The surface plasmon resonance (SPR) measurements were done using a BIAcore 2000 system (GE Healthcare, Uppsala, Sweden) in a flow cell providing laminar flow, using a flow rate of 10 μL/min. (38) Saurbrey, G. Z. Phys. 1959, 155, 206–22. (39) Wilson, W. D. Science 2002, 295, 2103–2105.

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Both QCM-D and SPR experiments were carried out at a temperature of 22 °C.

Results and Discussion Polymer Characterizations. The general polymer characteristics together with the surface properties of the pristine polymers are summarized in Table 1. As expected, the Tg decreases with increased number of carbons in the alkyl ester side chain, with the highest Tg for PIBMA (66 °C) and the lowest for PLMA (-70 °C). Thus, at 22 °C, the polymer chain mobility of PIBMA will be constrained with no or small chain movements. In contrast, the polymer chains of PLMA will be in constant motion. The Tg of PBMA is almost at the measurement temperature, so the degree of chain movements are difficult to predict for this polymer, but it certainly demonstrates some segmental movements. The XPS results show no deviation from expected stoichiometric composition (Table 1). Hence, no inadvertent surface contaminants due to the preparation of the coated sensor surfaces could be detected. The average roughness (rootmean-square roughness, rms), as determined by tapping mode atomic force microscopy (TM-AFM) ranged from 0.7-0.9 nm for all coatings as can be seen in Table 1. Consequently, differences in response are not likely a result of different surface morphologies. Furthermore, no cracks and other surface imperfections that could affect the response were found with TM-AFM. The solid surface energies of the coatings were calculated from the advancing and receding contact angles (Table 1). The surface energy calculated from advancing contact angles ranged from 28.3 mJ/m2 (PIBMA and PBMA) down to 19.1 mJ/m2 for PLMA. On the other hand, it can be argued that the receding contact angle should be used due to the fact that all the adsorption experiments were carried out in aqueous environment. Using the receding contact angle when calculating the solid surface energy significantly increases the surface energy of the coatings as can be seen in Table 1. For example, the surface energy of PIBMA was calculated to 36.5 mJ/m2 and the surface energy of PLMA was calculated to 40.5 mJ/m2. The difference between the coatings was still minor and should not significantly affect the interpretation of the adsorption results. Presented in the table is also the contact angle hysteresis (advancing minus receding contact angle). Increased level of hysteresis can be explained by higher molecular mobility allowing ester groups in the polymer side chain to orient toward the interface during the measurement. Protein Adsorption Studies. In Figure 1, typical QCM-D adsorption profiles for fibrinogen at 1 mg/mL are shown. The peak after 30 min of adsorption is due to the washing step, and the protein solution in the sample chamber was replaced with VBS buffer. In general, the proteins where strongly bound as low desorption was observed upon replacement of the buffer. The corresponding fibrinogen SPR experiments are shown in Figure 2. In Figure 3, the adsorbed mass for fibrinogen and the reference proteins, albumin and immunoglobulin G, is Langmuir 2009, 25(10), 5602–5608

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Figure 1. Representative fibrinogen adsorption isotherms at 1 mg/mL measured with QCM-D on three different model polymeric substrates, PLMA, PBMA, and PIBMA.

Figure 2. Representative fibrinogen adsorption isotherms at 1 mg/mL measured with SPR on three different model polymeric substrates, PLMA, PBMA, and PIBMA.

summarized. As can be seen, only fibrinogen showed a clear trend in adsorbed mass as a function of polymer chain flexibility. Note that the acoustic QCM-D model and the optical SPR model provided inverse results. The shift in dissipation as a function of polymer flexibility is shown in Figure 4. Flexibility had a profound effect on both fibrinogen and Ig G dissipation but not on albumin dissipation. The adsorbed mass and dissipation results of fibrinogen together with the results of albumin and immunoglobulin G are further discussed below. With albumin, no difference in adsorbed mass as a function of polymer chain flexibility was observed. The mass recorded with QCM-D ranged from 475 ( 93 ng/cm2 for PBMA (medium flexibility) over to 639 ( 65 ng/cm2 for PLMA (high flexibility), which could be compared with 532 ( 46 ng/cm2 on PIBMA (low flexibility). Error is calculated at 95% confidence interval from at least seven measurements. With SPR, the corresponding Langmuir 2009, 25(10), 5602–5608

Figure 3. The average adsorbed mass (95% confidence interval of (top) albumin, (middle) immunoglobulin G, and (bottom) fibrinogen as measured with QCM-D and SPR on three different model polymeric substrates, PIBMA, PBMA, and PLMA. The polymer chain molecular flexibility increases from left (PIBMA) to right (PLMA).

adsorbed mass was 110 ( 10 ng/cm2, 96 ( 15 ng/cm2, and 87 ( 15 ng/cm2 for PBMA, PLMA, and PIBMA, respectively. The acoustic QCM-D model resulted in about 5-6 times higher DOI: 10.1021/la803686m 5605

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Figure 4. Shift in dissipation (95% confidence interval after adsorption of fibrinogen, immunoglobulin G (IgG), and albumin on three different model polymeric substrates, PIBMA, PBMA, and PLMA. The polymer molecular flexibility increases from left (PIBMA) to right (PLMA). Note that the line is added to guide the eye and does not reflect any model as the flexibility is not linear going from PIBMA to PLMA. The flexibility is at least 3 orders of magnitude different.

adsorbed mass compared to the optical SPR model. This is due to the hydrodynamically coupled water, which is included in the QCM-D model. The degree of conformational change is not possible to elucidate based only on the SPR and the QCM-D results, but one can safely assume that it is rather comparable on the three model surfaces. The statement that the same degree of conformational change occurs on the model surfaces is also supported by the observed minor differences in dissipation values, ranging from 2.1  10-6 on PBMA over to 2.4  10-6 and 2.6  10-6 on PLMA and PIBMA, respectively. It can be speculated that albumin undergoes large conformational change on the surfaces based on the SPR control experiment with heat denatured protein (60 °C for 30 min). The adsorbed mass did not change significantly (92 ( 19 ng/cm2 on PLMA) compared to that of native protein. With immunoglobulin G, no difference in adsorbed mass (with neither QCM-D nor SPR) as a function of polymer chain flexibility was seen. Again, the hydrodynamically coupled water resulted in higher adsorbed mass with QCM-D compared to SPR. The adsorbed mass was about twice that observed with albumin (QCM-D: 1018 ( 89, 1016 ( 275, and 936 ( 136 ng/cm2, and SPR: 256 ( 4, 178 ( 52, and 254 ( 21 ng/cm2 for PLMA, PBMA, and PIBMA, respectively). The large differences in dry and wet mass are indicating some degree of conformational change upon adsorption. Interestingly, the dissipation significantly differed between PLMA, PBMA, and PIBMA with values of 4.4  10-6, 3.1  10-6, and 1.9  10-6, respectively (significance calculated based on the average of at least three measurements in a t test assuming equal variance, significance level p < 0.05). One explanation for this result could be that there is more water incorporated into the protein film on PLMA, counteracting eventual decrease in organic mass. The water rich protein film then gives raise to higher dissipation. This hypothesis is enforced by results presented by Voros,33 in which it was showed that a denatured protein film both contained higher amounts of water and resulted in higher dissipation values. It should be stressed that we observed no differences in 5606

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adsorbed mass with SPR, so this hypothesis is not fully supported. Immunoglobulin G is considered to be a rather stable protein, and it could be that the difference in dissipation is not caused by various degrees of conformational change on the model surfaces. Instead, it could be a result of differences in immunoglobulin G alignment or configuration on the surfaces. Hypothetically, on the flexible PLMA surface, one part of the molecule could be responsible for the interaction with the surface, but on the stiff nonflexible PIBMA it could be another part of the molecule that interacts with the surface. This difference in alignment could give raise to differences in water incorporation in the film and differences in dissipation. Finally, another explanation could be that immunoglobulin G is wobbling back and forth on the compliant PLMA surface as the crystal is oscillating. This wobbling, which partly could be caused by the high flexibility in the polymer, results in higher damping of the crystal. This might be tested by increasing the amplitude of the oscillating crystal and following eventual changes in dissipation. It should be stressed that the polymer chain movements need to be in the same time scale as the oscillating crystal if this wobbling should be detected, so this hypothesis is very suggestive and not supported by any experimental evidence. With fibrinogen, the polymer chain flexibility had a profound effect on the adsorbed mass. With QCM-D, the highest adsorbed mass was observed on PIBMA with 2423 ( 103 ng/cm2, which was followed by PBMA with 2021 ( 44 ng/cm2 and PLMA with 1445 ( 261 ng/cm2. Significant effects on surface hydrophobicity on fibrinogen adsorption have been documented40-42 among others, but to our knowledge this is the first time it has been shown on polymer surfaces with differences in molecular flexibility. As mentioned above, the adsorbed mass measured with QCM-D includes the hydrodynamically coupled water. This is worth remembering when comparing the QCM-D with the SPR adsorption results. With SPR, the flexible PLMA polymer achieved the highest amount of fibrinogen with 580 ( 98 ng/cm2. Significantly lower mass was found on the PBMA with 356 ( 10 ng/cm2 and PIBMA with 339 ( 18 ng/cm2 (p < 0.05, t test assuming equal variances). Thus, the acoustical QCM-D model results in 3-8 times higher mass loadings as compared with the optical SPR model. But more interestingly, an inverse trend was observed when comparing the two models. Taking into account the two methods, a hypothetical adsorption model can be formulated. On PIBMA, the fibrinogen film contains less organic mass per unit area together with high amounts of water. It could be that on this surface the protein layer is not fully covering the surface. The molecule is most likely firmly bound with extensive conformational change. Thus, no remodelling and close packing of the protein molecules could take place. This leads to increased roughness, and water could be incorporated in the “holes” found between the fibrinogen molecules. Such water is not hydration water in the strict sense but will still be detected with QCM-D, resulting in higher mass.43,44 Another explanation could be that when fibrinogen undergoes conformation change, hydrophilic amino acids are exposed on the surface of the fibrinogen molecule. (40) Wertz, C. F.; Santore, M. M. Langmuir 1999, 15, 8884–8894. (41) Tzoneva, R.; Heuchel, M.; Groth, T.; Altankov, G.; Albrecht, W.; Paul, D. J. Biomater. Sci., Polym. Ed. 2002, 13, 1033–1050. (42) Lu, D. R.; Park, K. J. Colloid. Interface. Sci. 1991, 144, 271–281. (43) McHale, G.; Newton, M. I. J. Appl. Phys. 2004, 95, 373–380. (44) Macakova, L.; Blomberg, E.; Claesson, P. M. Langmuir 2007, 23, 12436–12444.

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This will increase the hydration water content. In both proposed adsorption models, the conformational change results in large incorporation of water into the film. On PLMA, on the other hand, the fibrinogen protein layer is more tightly packed and contains lower amounts of water. One feasible explanation could be that the very flexible PLMA surface could adjust to the new interface, that is polymerprotein, during the adsorption process. The first evidence of such polymer rearrangement due to protein adsorption was recently published using sum frequency generation vibrational spectroscopy by Clarke and Chen.45 It can be described as when fibrinogen adsorbs on the surface it exert some stress on the polymer chains. This stress could be due to increased interfacial tension as the water phase is replaced by the more hydrophobic regions of the protein or due to adhesion forces such as hydrogen or van deer Waals interactions between protein and polymer chains. This stress usually causes a conformational change in the protein. However, in the case of PLMA, in which the intra- and intermolecular forces are weak, and potentially lower than those for the peptide chains in fibrinogen, the polymer has to sacrifice its conformation. The high adaptability of PLMA to new interfacial properties is also reflected in the high water contact angle hysteresis (Table 1). PLMA contains both hydrophilic (ester) and hydrophobic (alkyl) domains, and it could be that the polymer is fine-tuning the interfacial properties to match the properties found on the surface of the fibrinogen molecule and by this reduce the interfacial tension between the two. Ultimately, this means that fibrinogen maintains a more native conformation on this surface, compared to the less flexible PIBMA surface. Decreasing the fibrinogen concentration 10-fold to 0.1 mg/mL results in comparable mass on PLMA (1620 ng/cm2 as determined with QCM-D). In comparison, on PIBMA, the mass decrease was significant (1110 ng/cm2), a result which again demonstrates the impact of molecular flexibility on the protein conformation and corresponding film structure. To fully understand such a large mass shift when decreasing the protein concentration, further investigations with, for example, TM-AFM, are needed. In addition to the P1 and P2 domains, fibrinogen contains a cell binding RGD motif near the C-terminus.46 It is likely that this cell binding motif will be exposed due to a conformation change. As has been indicated with QCM-D and SPR the conformation change seems to be more pronounced on the PIBMA surface. These small differences in fibrinogen structure could be one of the reasons for the differences in biological response upon implantation we have previously observed.35,36 In accordance with Wertz and Santore, the relaxation and conformational change is a time-dependent process and decreasing the concentration would allow the proteins to spread even more on the surface.40 At higher concentrations, neighboring fibrinogen molecules on the surface sterically restrict a full conformational change. Moreover, if the proteins assume a more flat configuration with stronger adhesion to the underlying substrate, one would assume that the dissipation would decrease. In Figure 5, the adsorbed mass/dissipation as a function of fibrinogen concentration is presented. The figure was generated from the end value, that is, after 30 min of adsorption. As can be seen, the amount of mass needed for one unit of dissipation increases as the (45) Clarke, M. L.; Chen, Z. Langmuir 2006, 22, 8627–8630. (46) Ugarova, T. P.; Budzynski, A. Z.; Shattil, S. J.; Ruggeri, Z. M.; Ginsberg, M. H.; Plow, E. F. J. Biol. Chem. 1993, 268, 21080–21087.

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Figure 5. Adsorbed mass/dissipation as a function of fibrinogen concentration on the three different model substrates, PIBMA, PBMA, and PLMA. The values were calculated from the QCM-D measurements and after 30 min of adsorption.

fibrinogen concentration decreases. This effect is more pronounced on the stiff PIBMA polymer compared with the flexible PLMA surface. If PIBMA is more likely to cause a conformational change, decreasing the concentration will even further accelerate the process, as is indeed indicated in Figure 5. In contrast, for PLMA, which is assumed to cause less conformational change, the adsorbed mass/dissipation is not so strongly affected by the decreased fibrinogen concentration. The process of polymer rearrangement upon adsorption could also be related to molecular imprinted polymers (MIPs). Molecular imprinting is a versatile technique providing functional materials able to recognize and in some cases respond to biological and chemical cues.47,48 The hypothesis is that the molecule of interest generates a pocket or a groove in the polymer. The polymer is then extensively cross-linked to keep the configuration, and the molecule is washed away. When the polymer is exposed to a mixture of molecules, the interaction is maximized with the molecule that fits in this “pocket” of the polymer. Applications in affinity separation, catalysis, organic synthesis, and chemical analysis have been discussed.49 Up to date, attempts to imprint proteins have met with partial success50 and the focus has been on small molecules (molecular mass < 1000 Da). Understanding both the potential protein conformational change and the mutual polymer rearrangement opens up new ways of increasing the interaction between protein and the MIP. The interplay of weak intra- and intermolecular forces is always present in all biological systems, and preventing conformational change by matching the protein surface properties via polymer rearrangements could be one way of increasing the efficacy of MIPs. Moreover, the protein of interest is more likely to keep its biological function if the conformational change could be minimized. It should be stressed that we do not directly probe the conformational change or the polymer chain rearrangement with (47) Wulff, G. Angew. Chem., Int. Ed. 1995, 34, 1812–1832. (48) Ye, L.; Mosbach, K. Chem. Mater. 2008, 20, 859–868. (49) Mayes, A. G.; Mosbach, K. TrAC, Trends Anal. Chem. 1997, 16, 321–332. (50) Shi, H. Q.; Tsai, W. B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature (London) 1999, 398, 593–597.

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our experimental setup, but it is a reasonable model which account for the observed responses. Other methods that could be used in combination with our experimental setup to further understand the process include atomic force microscopy,18,30,32,51 monoclonal antibodies against certain epitopes in the molecule that are exposed after conformational change,22,46 circular dichroism or infrared spectroscopy, which are able to detect changes in secondary structure,52,53 and mass spectrometric mapping.54 Even though these techniques are very powerful, they still lack the possibility to measure the timeresolved changes upon adsorption as is possible with QCM-D and SPR. Also worth remembering is that in our case we include the water in our models. As water is a crucial part of all biological systems, and could affect the biological response, it is important to include it in the discussions. As discussed above, only fibrinogen showed a clear trend between polymer flexibility and conformational change. Why is fibrinogen so remarkably different? It could be the biological function of the protein that dictates the response. Fibrinogen is considered to be a part of the “defense system” of the body, responsible for both the homeostasis, thrombotic events and immune response.24,25 It is in the nature of the molecule to be able to differentiate between foreign surfaces and “self”, that is, the body. If this statement holds, one can say that the soft, compliant PLMA polymer is more like “self”. Organs in the body are water filled and rather flexible structures. It could be that fibrinogen is a “mechanosensor” and could detect a difference in elastic modulus or stiffness between different materials. To increase the biocompatibility of implants, one should try to use as flexible or compliant materials as possible. It should be stressed that bone is certainly not a compliant material, so this discussion is for the moment limited to soft tissues. The compliance difference between PLMA and PIBMA is rather large (at least 3 orders of magnitude). In future studies, this range should be decreased. The conformational change is not restricted to solely take place in the polymer or in the protein. We also suggest that there is a zone of polymer flexibility in which both protein and polymer are adapting to the new interface via molecular rearrangements and by this reducing the interfacial tension between the two. This process of mutual molecular rearrangement is schematically illustrated in Figure 6. A suitable methodological approach to probe eventual mutual conformational changes (51) Choukourov, A.; Grinevich, A.; Saito, N.; Takai, O. Surf. Sci. 2007, 601, 3948–3951. (52) Maste, M. C. L.; Norde, W.; Visser, A. J. Colloid Interface Sci. 1997, 196, 224–230. (53) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 8168–8173. (54) Scott, E. A.; Elbert, D. L. Biomaterials 2007, 28, 3904–3917.

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Figure 6. Schematic of conformational change and incorporation of water after fibrinogen adsorption on polymers with variable flexibility (increased polymer chain flexibility from left to right). On rigid polymers, most of the conformational change occurs in the fibrinogen molecule. On the contrary, when the polymer chains are flexible, they adapt to the fibrinogen interface and most of the structural rearrangement occurs in the polymer. We also propose a range of polymer flexibility in which mutual fibrinogen and polymer rearrangement takes place. Note that the model is generalized and based on results generated in the flexibility range evaluated in this study.

could be the use of stiffness gradient surfaces.55-58 Gradient surfaces are currently being explored to understand complex biological phenomena.

Conclusions By combining the acoustic QCM-D technique with the optical SPR technique, we have shown significantly higher adsorbed organic mass, less water content, and less conformational change of fibrinogen on a flexible polymeric surface compared to a nonflexible surface. In comparison, no trend between polymer chain flexibility and protein film structure was found with albumin. With immunoglobulin G, the polymer chain flexibility had a minor effect as verified by the significant increase in dissipation as flexibility increased. This is to our knowledge the first report demonstrating the effect of polymer chain flexibility on protein adsorption and corresponding conformational change. As the conformation of fibrinogen on biomaterials has shown to be important for both acute immune response and inflammatory cell recruitment, the effect of polymer chain flexibility might be an important material parameter to consider when designing biomaterials with improved biocompatibility. (55) Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstrom, I. J. Colloid Interface Sci. 1987, 119, 203–210. (56) Ruardy, T. G.; Schakenraad, J. M.; vanderMei, H. C.; Busscher, H. J. Surf. Sci. Rep. 1997, 29, 3–30. (57) Kim, M. S.; Khang, G.; Lee, H. B. Prog. Polym. Sci. 2008, 33, 138–164. (58) Genzer, J.; Bhat, R. R. Langmuir 2008, 24, 2294–2317.

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