Fibronectin Conformation Switch Induced by Coadsorption with

Dec 9, 2010 - §Institute for the Chemistry of New Materials, Belarus National Academy of Sciences,. Staroborisovski Trakt 36, 220141 Mink, Belarus...
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Fibronectin Conformation Switch Induced by Coadsorption with Human Serum Albumin Nicoletta Giamblanco,† Mohammed Yaseen,‡ Genady Zhavnerko,§ Jian R. Lu,*,‡ and Giovanni Marletta*,† † Laboratory for Molecular Surfaces and Nanotechnology (LAMSUN), Dipartimento di Scienze Chimiche, Universit a di Catania and CSGI, Viale A. Doria 6, 95125 Catania, Italy, ‡Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, U.K., and § Institute for the Chemistry of New Materials, Belarus National Academy of Sciences, Staroborisovski Trakt 36, 220141 Mink, Belarus

Received October 13, 2010. Revised Manuscript Received November 15, 2010 The dynamic adsorption of human serum albumin (HSA) and plasma fibronectin (Fn) onto hydrophobic poly(hydroxymethylsiloxane) (PHMS) and the structures of adsorbed protein layers from single and binary protein solutions were studied. Spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation monitoring (QCM-D) together with atomic force microscopy (AFM) were used to measure the effective mass, thickness, viscoelastic properties, and morphology of the adsorbed protein films. Adsorbed HSA formed a rigid, tightly bound monolayer of deformed protein, and Fn adsorption yielded a thick, very viscoelastic layer that was firmly bound to the substrate. The mixed protein layers obtained from the coadsorption of binary equimolecular HSA-Fn solutions were found to be almost exclusively dominated by Fn molecules. Further sequential adsorption experiments showed little evidence of HSA adsorbed onto the predeposited Fn layer (denoted as Fn . HSA), and Fn was not adsorbed onto predeposited HSA (HSA . Fn). The conformational arrangement of the adsorbed Fn was analyzed in terms of the relative availability of two Fn domains. In particular, 4F1 3 5F1 binding domains in the Hep I fragment, close to the amino terminal of Fn, were targeted using a polyclonal antifibronectin antibody (anti-Fn), and the RGD sequence in the 10th segment, in the central region of the molecule, was tested by cell culture experiments. The results suggested that coadsorption with HSA induced the Fn switch from an open conformation, with the amino terminal subunit oriented toward the solution, to a close conformation, with the Fn central region oriented toward the solution.

1. Introduction Material biocompatibility is becoming increasingly understood in terms of the interplay between highly specific biological functions at biomaterial surfaces for the vast entourage of competing proteins, peptides, and cellular components. Accordingly, surface engineering strategies are addressing this requirement more and more with respect to functionalizing implant surfaces and tissue engineering scaffolds. In this context, a simple, direct strategy to enhance biocompatibility involves the preconditioning of biomaterials by protein adsorption.1 However, this strategy must cope with the well-known fact that the chemical structure of synthetic surfaces greatly affects the overall conformational state of the adsorbed proteins, both when a synthetic material is put into the body, thus interacting with the natural extracellular matrix (ECM) proteins, and when synthetic surfaces are preconditioned with specific proteins and/or peptides, including collagen, fibronectin, vitronectin, fibrinogen, and the derived peptide sequences.2 A large part of current research is dedicated to understanding and controlling the interplay between the surface chemical structure and the conformational state of proteins, which directly concern the mechanism of cell adhesion, proliferation, differentiation, and migration.3 Among the most important ECM proteins, fibronectin (Fn) is one of the well-studied ones. Its schematic molecular structure is *Corresponding authors. (J.R.L.) E-mail: [email protected]. Tel: 0044-161-3063926. (G.M.) E-mail: [email protected]. Tel: 0039-957-385130. (1) Dillow, A. K.; Tirrel, M. Curr. Opin. Solid State Mater. Sci. 1998, 3, 252–259. (2) Yamada, K. M. J. Biol. Chem. 1991, 266, 12809–12812. (3) Keselowsky, B. G.; Collard, D. M.; Garcı´ a, A. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5953–5957.

312 DOI: 10.1021/la104127q

shown in Figure 1 (in monomeric form). Fn is a multifunctional high-molecular-weight (450 KDa) dimeric glycoprotein that is present in the extracellular matrices of all connective tissues. It is known to play key roles in several fundamental cell functions including cell attachment and tissue repair.4 Fn exists in two forms: (i) as an insoluble glycoprotein dimer that serves as a linker in the ECM and (ii) as a soluble disulfide-linked dimer found in the plasma (plasma Fn). The two forms differ by the extra domains of A, B, and V (Figure 1). Indeed, the ECM form is synthesized by fibroblasts, chondrocytes, endothelial cells, macrophages, and certain epithelial cells and contains various combinations of extra domains A, B, and V. In contrast, the plasma form of Fn is synthesized by hepatocytes and contains only the V domain. Detailed structures and functions of Fn have been reported and extensively discussed in the literature.5-9 In particular, the two nearly homologous subunits of Fn are composed almost entirely of three different types of repeating motifs or modular tertiary structural units, in general indicated as type I, II, and III repeats. Fn has been shown to be able to mediate specific cell-surface interaction via either simple peptide sequences such (4) Pankov, R.; Yamada, K. M. J. Cell Sci. 2002, 115, 3861–3863. (5) Baugh, L.; Vogel, V. J. Biomed. Mater. Res. 2004, 69, 525–534. (6) Price, T. M.; Rudee, M. L.; Pierschbacher, M.; Ruoslahti, E. Eur. J. Biochem. 1982, 129, 359–363. (7) Bergkvist, M.; Carlsson, J.; Oscarsson, S. J. Biomed. Mater. Res. A 2003, 64, 349–356. (8) Velzenberger, E.; Pezron, I.; Legeay, G.; Nagel, M. D.; Kirat, K. E. Langmuir 2008, 24, 11734–11742. (9) Dolatshahi-Pirouz, A.; Jensen, T.; Kraft, D. C.; Foss, M.; Kingshott, P.; Hansen, J. L.; Larsen, A. N.; Chevallier, J.; Besenbacher, F. ACS Nano 2010, 4, 2874–2882.

Published on Web 12/09/2010

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Figure 1. Illustration of the organization of domains within an Fn monomer. The three types of Fn structural domains are represented by symbols: Fn type I (0), Fn type II (magenta )), and FN type III (Ο). The binding regions for Fn, fibrin, collagen, and heparin are indicated in green. The cell-binding site in the 10th type III Fn domain (dark blue O) is magnified to show the position of RGD in the β sandwich structure. Important synergy cell site PHSRN (light blue O) and spliced domains A, B, and V are also indicated. The anti-Fn used in this experiment binds to the 4F1 3 5F1 domains near the N terminal as shown.

as RGD and/or its synergistic site PHSRN through integrin receptors in the cell membrane or the interaction of the 4F1 3 5F1 segments, which are part of the heparin I (Hep I) and fibrin (Fbn) binding domains.10 RGD, located at the apex of the loop connecting the sixth and seventh β strands within the type III 10th unit (10FIII), is about 3.5 nm from the PHSRN synergy site located on the type III ninth unit. For such a protein, the crucial spatial distance of the correct peptide sequence and the appropriate structural conformation for its epitope “exposure” are basic requirements for maximizing the interaction with cells. Thus, the biological availability (i.e., the proper exposure of the two classes of epitopes for Fn in solution or onto surfaces) is a major issue in tuning the cell-adhesive function of Fn.11,12 Indeed, the cell-adhesive function of Fn could be tuned only if an optimal exposure of the relevant cell-binding motifs in Fn is achieved.12 However, the proper exposure of Fn binding motifs, as for any other protein, is critically conditioned by the simultaneous presence of other proteins in the biological medium and, in particular, in the ECM system. The interference is expected to increase as the interfering proteins become abundant. This is in particular the case for human serum albumin (HSA), the most abundant soluble blood protein whose lower molecular weight and much higher serum concentration should favor transport to synthetic surfaces with respect to larger proteins such as Fn.13 Accordingly, the competitive coadsorption of HSA with Fn has been studied in view of its possible effect on the adsorption and biological activity of Fn.14,15 Quite surprisingly, it has been reported that the coadsorption of HSA with Fn significantly affects the final density and surface orientation of the adsorbed Fn cell-binding (10) Hynes, R. O. Fibronectins; Springer-Verlag: New York, 1990. (11) Curtis, T. M.; McKeown-Longo, P. J.; Vincent, P. A.; Homan, S. M.; Wheatley, E. M.; Saba, T. M. Am. J. Physiol. Lung Cell. Mol. Physiol. 1995, 269, L248–L260. (12) Ugarova, P. T.; Zamarron, C.; Veklich, Y.; Bowditch, R. D.; Ginsberg., M. H.; Weisel, J. W.; Plow, E. F. Biochemistry 1995, 34, 4457–4466. (13) Putnam, F. W., Ed. The Plasma Proteins, 2nd ed.; Academic Press: New York, 1975; Vol. I, pp 133-181. (14) Sousa, S. R.; Bras, M. M.; Moradas-Ferreira, P.; Barbosa, M. A. Langmuir 2007, 23, 7046–7054. (15) Koenig, A. L.; Gambillara, V.; Grainger, D. W. J. Biomed. Mater. Res. A 2003, 64, 20–37. (16) Lewandowska, K.; Balachandar, N.; Sukenik, C. N.; Culp, L. A. J. Cell Physiol. 1989, 141, 334–345. (17) Renner, L.; Pompe, T.; Salchert, K.; Werner, C. Langmuir 2005, 21, 4571– 4577.

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modules, enhancing the availability of Fn cell-binding domains and in turn cell adhesion onto preconditioned biomaterial surfaces.16,17 Among the biomaterials, polysiloxanes are particularly interesting in view of their growing interest as highly biocompatible materials as supports for cell culturing in medical implants, as scaffolds for tissue regeneration, and for novel applications in the biosensor field.18-20 Accordingly, recent work on the surface conformation of adsorbed fibrinogen onto nanosiloxane biomaterials using a combination of AFM, immunochemical probing, and cell interactions has provided valuable evidence on how the protein structural conformation can improve biocompatibility.21 In particular, the adsorption and biological functionality of Fn onto poly(dimethylsiloxane) (PDMS) has been studied in view of the use of PDMS as a bioengineered compatible substrate, suggesting that, depending on whether hydrophilic or hydrophobic interactions dominate, the protein may be either denatured or stabilized on the surface of the material.22-24 In view of the above studies, this article aims to study the conformational state and the change induced in the Fn bioactivity by coadsorption and sequential adsorption with HSA onto poly(hydroxymethylsiloxane) (PHMS), which is representative of the large class of polysiloxanes. The surface structural conformation of Fn and its biological activity have therefore been examined to determine the availability of a specific Fn cell-binding domain (i.e., the 4F1 3 5F1 segments within heparin I (Hep I) and fibrin (Fbn) binding domains). Indeed, the position of these segments within the Fn chain makes them good markers of the exposure of the N terminus at the medium-substrate interface. At first, the 4F1 3 5F1 segments were targeted by using a polyclonal antifibronectin antibody as a probe, and the amount of adsorbed (18) (a) Satriano, C.; Conte, E.; Marletta, G. Langmuir 2001, 17, 2243–2250. (b) Satriano, C.; Carnazza, S.; Guglielmino, S.; Marletta, G. Langmuir 2002, 18, 9469– 9475. (c) Assero, G.; Satriano, C.; Lupo, G.; Anfuso, C. D.; Marletta, G.; Alberghina, M. Microvasc. Res. 2004, 68, 209–220. (19) Wu, M. H. Surf. Interface Anal. 2009, 41, 11–16. (20) Comelles, J.; Estevez, M.; Martı´ nez, E.; Samitier, J. Nanomedicine 2010, 6, 44–51. (21) Yaseen, M.; Zhao, X.; Freund, A.; Seifalian, A. M.; Lu, J. R. Biomaterials 2010, 31, 3781–3792. (22) Toworfe, G. K.; Composto, R. J.; Adams, C. S.; Shapiro, I. M.; Ducheyne, P. J. Biomed. Mater. Res. A 2004, 71, 449–461. (23) Abbasi, F.; Mirzadeh, H.; Katbab, A. A. Polym. Int. 2001, 50, 1279–1287. (24) Wittmer, C. R.; Phelps, J. A.; Saltzman, W. M.; Van Tassel, P. R. Biomaterials 2007, 28, 851–860.

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antibody was directly measured in situ for Fn, HSA, Fn coadsorbed with HSA, and various sequential adsorption processes using spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation monitoring (QCM-D). Atomic force microscopy (AFM) was employed to obtain information on the nanometric structure of the adsorbed protein layers. Furthermore, fibroblast adhesion and proliferation onto the surfaces preconditioned either with pure Fn or with Fn coadsorbed or sequentially adsorbed with HSA were determined to compare the relative efficiency of Fn adsorbed under different conditions in determining the cell-binding activity. The comparison of the data from antibody-selective binding and cell responses for the various adsorption conditions of Fn is expected to provide valuable insight into the relative exposure of the 4F1 3 5F1 segments, there by leading to a relationship between the Fn surface conformation and the cell-binding activity, which mainly seemed to involve the structural reorganization of Fn molecules from a “closed” to “open” conformation.10-12 Thus, the present study may shed light on the influence of coadsorption processes on Fn conformation, in view of the expected critical effect on cell response to surface preconditioning.

2. Materials and Methods 2.1. Substrates and Proteins. A concentrated solution of poly(hydroxymethylsiloxane) (PHMS) from Accuglass T-12B Honeywell International Inc. (Morristown, NJ) was diluted 12 and 30 times with a mixture of isopropanol/acetone/ethanol (35/20/45 v/v/v) (Sigma-Aldrich, Steinheim, Germany). The diluted solutions were spin coated onto freshly cleaned silicon wafers at 3000 rpm for 60 s. The coated wafers were then dried in vacuum for 1 h without any further curing. Each monomeric unit in PHMS contains one methyl group and one hydroxyl group bonded to the [Si-O-Si] backbone. The coated surface had a static water contact angle of θ = 90 ( 3°, consistent with the projection of the methyl group outward, rendering a hydrophobic substrate. PHMS films at two different thicknesses were deposited onto silicon wafers, and their swelling behavior and stability both in pure water and in PBS were assessed over a 50 min period using both SE and QCM-D. Human serum albumin (HSA) was obtained from SigmaAldrich (Steinheim, Germany, code A3782) and used as supplied. A stock solution of HSA was prepared in phosphate buffer with a concentration of 0.1 mg/mL (i.e., 1.4 μM). Human plasma fibronectin (Fn) was purchased from Sigma-Aldrich (code F2006) and used as supplied. A stock solution of Fn was prepared in phosphate buffer to a concentration of 0.1 mg/mL (0.2 μM). The dynamic processes of HSA and Fn adsorption on the PHMS films from 0.1 mg/mL protein solutions as well as from the binary solution adsorption were measured at an ambient temperature of 20-22 °C using the SE and QCM-D techniques. Coadsorption experiments were performed by using 1:1 binary solutions of Fn/HSA with a concentration of 0.1 mg/mL per protein. Polyclonal rabbit antifibronectin (anti-Fn) was obtained from SigmaAldrich (code F3648) and was employed as a solution in phosphate buffer at a final concentration of 0.042 μM. The employed anti-Fn belongs to the IgG1 subclass and interacts by its Fc portions with the 4F1 3 5F1 segments within Fn.25 Phosphate buffer solution (PBS) was prepared by dissolving 1 tablet (from SigmaAldrich) in 200 mL of ultrapure water (processed by a Millipore system with 18.2 MΩ resistivity), resulting in a solution ionic strength of 0.01 M for the phosphate salts, 0.0027 M for potassium chloride, and 0.137 M for sodium chloride (pH 7.4 at 25 °C). The same buffer was used for all of the adsorption experiments. 2.2. Cell Culture. The McCoy fibroblast cell line (from ATCC, no. CRL-1696) was used to test cell adhesion on the (25) Rostagno, A. A.; Gallo, G.; Gold, L. I. Mol. Immunol. 2001, 38, 1101–1111.

314 DOI: 10.1021/la104127q

preconditioned substrates. Cells were cultured in tissue culture polystyrene dishes using Dulbecco’s modified Eagle’s medium (DMEM EuroClone) supplemented with 10% (v/v) fetal bovine serum (FBS), L-glutamine (2 mM), and antibiotics (penicillin, 10 000 units; streptomycin, 10 mg/mL). Cultures were incubated at 37 °C under a humidified atmosphere in a CO2 (5%) incubator. Experiments were performed using cells in the fifth and ninth passages. Cells were detached from tissue culture polystyrene dishes using a trypsin-containing solution and were collected by centrifugation. Before cell seeding, the PHMS samples and controls were treated for 2 h with a solution containing 10 000 U/mL penicillin, 10 mg/mL streptomycin, and 25 mg/mL amphotericin and then washed twice in PBS. The cell suspension (containing 2.5  104 cell/dish) was added to various substrates including PHMS and protein molecular films of Fn, HSA, and a protein mixture (HSA þ Fn) adsorbed onto PHMS. Dishes were incubated at 37 °C in a 5% CO2 atmosphere. Each sample was made in triplicate. Cells were observed after the first, third, and fifth days to determine their attachment profiles. To evaluate the number of adhered cells, optical microscopy images were obtained at 10 magnification using a Leica DFC 320 camera (Leica HTML, Germany). The number of adhered cells was determined by counting cells on 1 mm2 fields under 10 magnification. Cell counts were expressed as the average number of five random fields of each sample. Statistical computation was performed with GraphPad Instat 3.00 software (GraphPad Software Inc., San Diego, CA), and the data were analyzed by a student’s t test. Values were considered to be significant at p < 0.05.

2.3. Techniques. 2.3.1. Spectroscopic Ellipsometry (SE). Using a Jobin-Yvon UVISEL spectroscopic ellipsometer, measurements were made over a wavelength range of 300-600 nm. A liquid cell was specially constructed to enable measurements at the solid/liquid interface at a fixed angle of incidence of 70° with respect to the sample surface. Results were analyzed using DeltaPsi I software developed by the Jobin-Yvon Company.26 For each spectroscopic ellipsometry measurement, the two optical angles, Ψ (measuring changes in the amplitude of light before and after reflection) and Δ (measuring changes in the phase of light before and after reflection), were recorded against wavelength of between 300 and 600 nm. Information about the amount of protein adsorbed was obtained by performing a uniform layer model fit to each pair of Ψ and Δ. The thicknesses of the coated polymer layers on which protein adsorption was undertaken were 8.5 and 18.7 nm. For such ultrathin interfacial layers, ellipsometry is incapable of separating the layer thickness from its volume fraction. In this study, the initial polymer layer thickness was fitted with a refractive index of 1.48. As for the HSA layer, it was initially adsorbed onto the bare silicon of a known oxide layer. Both the thickness and refractive index were fitted simultaneously for a particular concentration of the protein to give the best fit. From these results, the mean refractive index, n = 1.475, was obtained and fixed. This fixed n was then used to fit the thickness of the protein layer. A similar approach of fixing n to that of the pure protein has been shown to have little effect on the surface adsorbed amount or the surface excess.27 Following previous work by De Feitjer,28 we found that the surface excess (Γ in mg m2) can be estimated from the following equation Γ ¼

τðn - nb Þ 10a

ð1Þ

where τ is the protein layer thickness (in A˚), n is the corresponding refractive index of the layer, and nb is the refractive index of the aqueous phase or buffer. The value of a was related to the change (26) Provided by Jobin-Yvon Company: http://www.jobinyvon. (27) Tang, Y.; Lu, J. R.; Lewis, A. L; Vick, T. A; Stratford, P. W. Macromolecules 2001, 34, 8768–8776. (28) Feijter, J. A. D.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759– 1772.

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Giamblanco et al. in the solution’s refractive index with bulk concentration. To offset the unit conversion, the value of a was taken to be 1.88.29

2.3.2. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). The QCM-D technique (Q-Sense D300, Goteborg, Sweden) is based on an AT-cut piezoelectric quartz crystal with a gold film deposited on the electrode faces,9,14 allowing the simultaneous measurements of both frequency (f) and energy dissipation (D) of the sensor consisting of 5 MHz crystals (Q-Sense). The mass added or removed from surfaces induces a proportional decrease or increase in frequency. When the film does not slip on the electrodes and is thin and rigidly attached, it is possible to apply Sauerbrey’s equation.30 The PHMS polymer films were spin coated onto gold-coated crystals using the same conditions as discussed before and were stored in a vacuum heater for 1 h at 40 °C. The crystals were kept in air until the baseline was stabilized. The behavior of the polymer films was studied in phosphate buffer and in Millipore water. The QCM-D experiments for protein adsorption started with the sensors running in PBS buffer until the baseline was reached. The changes in D and f due to the addition of protein solution were monitored for both the fundamental frequency (n = 1 corresponding to f ≈ 5 MHz) and the first three overtones (n = 3, 5, and 7 corresponding to f ≈ 15, 25, and 35 MHz, respectively). When adsorption saturation was reached, a rinsing step was performed by exchanging protein solution with buffer in order to check possible desorption processes. 2.3.3. Atomic Force Microscopy (AFM). AFM analysis was performed for the PHMS films deposited on silicon or gold surfaces and after protein adsorption from solutions of 1.4 μM HSA, 0.2 μM Fn, and 1:1 HSA/Fn on PHMS substrates for 1 h. The samples were dried gently with a stream of N2. Topographical images were taken using a Digital Instruments (DI) Nanoscope IIIa under ambient conditions. The device was equipped with a calibrated scanner using the manufacturer’s grating. All samples were analyzed in tapping mode (TM) and in contact mode (CM). Nanoprobe cantilevers (100 mm and 200 mm standard spring constants ranging from 0.12 to 0.52 N/m) with oxide-sharpened Si3N4 integral tips (Veeco NanoProbe Tips NP-20) were used for the CM regime, and tapping silicon cantilevers with a resonance frequency of ∼260 kHz (Veeco NanoProbe Tips RTESP) were used for the TM regime. The applied force was varied over a wide range from several nanonewtons up to tens of nanonewtons in contact mode. The film thickness was estimated by measuring, at a scan speed of 5 Hz, the depth of an artificial hole that was scratched in the CM regime at a scan speed of 12 Hz. Image analysis was carried out using DI software, version 4.23r6. Height images were flattened to remove background slopes. The surface roughness was obtained from the 1  1 μm2 scanned areas from a minimum of three separate images obtained from different regions of each sample. The root-mean square roughness (Rrms) was used to characterize the material surfaces because it provides an indication of the deviation of height from the mean data plane.31

3. Results and Discussion 3.1. Polymer Film Characterization. SE measurements revealed an initial small thickness increase occurring in the first few minutes, but subsequently no further swelling of the polymer films occurred over 50 min with the final thicknesses constants at 8.5 ( 0.2 and 18.7 ( 0.5 nm (Figure SI1a). No difference was observed when films were immersed in pure water or buffer. Parallel QCM-D measurements, reported in Figure SI1b for the (29) J€onsson, U.; Malmqvist, M.; R€onnberg, I. J. Colloid Interface Sci. 1985, 103, 360–372. (30) Sauerbrey, G. Z. Phys. 1959, 155, 206–206. (31) Vilas, A. M.; Bruque, J. M.; Gonzalez-Martin, M. L. Ultramicroscopy 2007, 107, 617–625.

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thick film, also showed few changes against time. Specifically, the frequency shifts ΔF (upper trace in Figure SI1b) indicated that the uptake of water from the PBS solution, if any, was below the sensitivity of the technique (0.5 ng/cm2). The film exhibited few ΔD shifts (lower trace in Figure SI1b), suggesting that no changes occurred in the film’s viscoelastic behavior. The AFM topographic images for the films in air and under water are shown in Figure SI2 (roughness parameters of Rrms = 0.36 nm in air and 0.31 nm in buffer), with few variations between dry and wet surfaces. In summary, the deposited polymer films were very stable when immersed in pure water or buffer solution over the time required for experimental measurements. 3.2. Protein Adsorption. Typical mass uptake curves versus time from SE and QCM-D are shown in Figure 2a-c. The general features of the adsorption data were found to be independent of the PHMS film thickness. Specifically, the general shape and timescale of the adsorption curves are similar for both SE and QCM-D. The mass uptake calculated from QCM-D was found to be about 1.3 and 3.0 times higher than that found by SE for HSA and Fn, respectively. The differences, following the discussion in the literature, can be explained in terms of water bound to or hydrodynamically coupled to the adsorbed proteins, in particular, to Fn.32 The time-dependent protein adsorption as shown in Figure 2 shows the lower surface adsorbed amount (or surface excess) and the slower adsorption of HSA toward saturation, with the equilibrium plateau being reached after 30 min. In contrast, Fn adsorption on PHMS reaches saturation significantly faster, after only 10 min, and attains the highest adsorbed mass. Buffer rinsing after adsorption saturation resulted in no significant removal of adsorbed HSA or Fn molecules, showing that the adsorbed molecules were tightly bound. The slower kinetics of HSA adsorption on PHMS is likely to be associated with the structural reorientation and deformation of HSA prompting the irreversible adsorption, according to the current protein adsorption models.33 The saturated HSA adsorbed mass (1.50 mg/m2) as determined from SE would correspond to 70% of the full coverage, assuming a monolayer of side-on molecules with native state molecular dimensions of 4  4  14 nm3.34 However, SE revealed a thickness of about 2 nm, suggesting a monolayer of HSA that is thinner than its shortest axial dimension. A probable explanation is that the hydrophobic PHMS surface prompted HSA deformation and spreading, as driven by the hydrophobic interaction between the surface and hydrophobic HSA residues. The deformed HSA molecules would thus form a better covered monolayer, but the layer thickness would be less than the original axial dimension of HSA in the solution. In contrast, the faster Fn adsorption led to a plateau mass uptake of 4.0 mg/m2 as determined from SE. This surface adsorbed value would imply that the adsorbed Fn molecules had to adopt both side-on and end-on conformations to be fitted into the surface, given the approximate Fn molecular dimensions of 16.5  9.6  2.5 nm3.35 Fn is known to exist in a compact conformation at physiological pH and over low and medium ionic strength. Its compact form is stabilized by intersubunit ionic interactions between type III 2-3 and III 12-14 or type I 1-5 domains. The unravelling and extension of Fn may be triggered (32) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J. Colloids Surf., B 2002, 24, 155–170. (33) van der Veen, M.; Cohen, S M.; Norde, W. Colloids Surf., B 2007, 54, 136–142. (34) J. D. Andrade, V.; Hlady, A. N. Y. Acad. Sci. 1987, 516, 158–163. (35) MacDonald, D. E.; Markovic, B.; Allen, M.; Somasundaran, P.; Boskey, A. L. J. Biomed. Mater. Res. A 1998, 41, 120–130.

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Figure 2. QCM-D and SE mass uptake values (mg/m2) as a function of time (min) reported for HSA (a), Fn (b), and HSA þ Fn (c) adsorbed on PHMS (18.7 ( 0.5 nm) from 0.1 mg/mL protein solution. ΔD vs time for HSA (d), Fn (e), and HSA þ Fn (f) are also shown.

by an ionic strength increase or upon adsorption onto hydrophilic surfaces.7,36 The structural transition might allow the exposure of the hidden peptide epitope domains and an increased number of Fn-Fn interactions. Thus, the adsorption of Fn onto the PHMS surface resulted in fast but high surface mass adsorption with a thin, elongated Fn structure. Interestingly, the thickness of the adsorbed Fn layers was found to be 4.7 nm. This value is higher than the shortest axial length of the 3D structure but is well below the other two longer dimensions, showing that the Fn molecules must predominantly adopt a flat-on conformation with most domains in close contact with the surface. Some domains or fragments may be tilted away from the surface, contributing to the increase in layer thickness. Other conformations such as the sideon projection are also possible. Figure 2d,e shows the dissipation versus time curves for HSA and Fn adsorbed layers, respectively. In particular, the adsorbed Fn layer shows high-energy dissipation with ΔD/Δm ratios that are 1.7 higher than the values measured from the HSA layer. This is characteristic of the formation of a highly viscoelastic Fn layer with a large amount of trapped water, and HSA appears to be rather rigidly adsorbed on the PHMS surface. The dynamic adsorption from the binary HSA þ Fn solution on the PHMS surface is shown in Figure 2c,f. The adsorption profiles of the mixture monitored by SE and QCM-D methods are very similar to that of pure Fn. The mass uptake results are slightly lower than those obtained for Fn adsorbed from a single solution but are significantly higher than those obtained for HSA. Accordingly, the protein films obtained by HSA þ Fn coadsorption have a dissipation energy that is much higher than for HSA layers obtained from one-component HSA solution. Indeed, the ΔD/Δf ratios for the coadsorbed layer are around 5.6  10-8, (36) Johnson, K. J.; Sage, H.; Briscoe, G.; Erickson, H. P. J. Biol. Chem. 1999, 274, 15473–15479.

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which is slightly lower than those found for the pure Fn layer, ΔD/Δf = 6.2  10-8, suggesting that the coadsorbed layer predominantly consists of Fn with a minor amount of included HSA. According to the Vroman effect,37 smaller proteins such as HSA in this case would reach the surface faster but could subsequently be replaced by higher-molecular-weight protein Fn because Fn is larger and more surface-active. As shown in Figure 2c,f, both SE and QCM-D data indicate that the layer from the binary HSA þ Fn solution is adsorbed with the dominant feature of Fn adsorption, showing that Fn must be the main component in the adsorbed interfacial layer and that its adsorption occurred rather fast (over a period of minutes or shorter). This observation thus suggests that either Fn replaced most of the adsorbed HSA on a very short timescale or selective and immediate Fn adsorption occurred, preventing significant HSA adsorption. Finally, the effective thickness, deff, of protein layers adsorbed on the PHMS substrate was estimated using the following equation, which takes into account the mass uptake obtained from SE (MSE) and QCM-D (MQCM) measurements, the protein density (Fprot), and the solvent density (F): deff ¼ Fprot

MQCM " # MSE MSE þF 1MQCM MQCM

ð2Þ

.According to eq 2, the effective thickness values for adsorbed HSA and Fn are 1.6 and 10.0 nm, respectively. Because of the uncertainty in the real composition of the adsorbed layer, the evaluation of the effective thickness for the adsorbed film from the binary HSA þ Fn system was performed by assuming that the effective value of Fprot was 1.33 g/cm3 and that the density of (37) Vroman, L.; Adams, A. L. Surf. Sci. 1969, 16, 438–446.

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Table 1. Protein Layer Thickness (nm) and Roughness Values of the Root-Mean Square Roughness (Rrms, nm) Obtained from AFM-Scanned Areas of 1.0  1.0 μm2 on Dried HSA, Fn, and Binary HSA þ Fn Films on the PHMS Surface

HSA Fn HSA/Fn

thickness (nm)

Rrms (nm)

2.1 ( 0.2 4.8 ( 1.2 4.3 ( 0.5

0.60 ( 0.02 0.51 ( 0.03 0.45 ( 0.02

the PBS buffer was F = 1.00 g/cm3.32,38 Under these conditions, the effective thickness of the coadsorbed layer was deff = 9.85 nm, which is very close to the value obtained for the pure Fn layer. Representative AFM height, phase images, and z profiles for the dry HSA, Fn, and HSA þ Fn layers adsorbed on the PHMS surface are shown in Figure SI3, with the measured layer thicknesses and the related roughness parameter Rrms values listed in Table 1. Porous morphology was observed by AFM scanning for the adsorbed HSA layer (Figures SI3a and SI3d), in agreement with the SE and QCM-D data indicating only partial surface coverage. The Rrms value measured for this layer (Table 1) is accordingly high, reflecting the porous or uneven layer structure. Fn and HSA þ Fn adsorbed layers (Figure SI3b,c,e,f) showed both a smoother and rather uniform morphology, as confirmed by the corresponding Rrms values. Apart from morphological features, AFM measurements also allowed the thicknesses of the adsorbed layers to be estimated. In particular, for the HSA layer surface, the porous protein layer allowed the easy measurement of a layer thickness of 2.1 ( 0.2 nm, as compared to 2.0 and 1.6 nm obtained from SE and QCM-D measurements. In contrast, the Fn layer was rather uniform as explained earlier. Thus, the thickness was estimated by mechanically scratching the protein surface using the AFM tip. The value was found to be 4.8 ( 1.2 nm, which is close to that of 4.7 ( 0.2 nm as found by SE but markedly different from the value of 10.0 nm estimated by QCM-D. Finally, the same scratching method for the HSA þ Fn coadsorbed layer yielded a thickness of 4.3 ( 0.5 nm, which is closer to the 4.6 ( 0.4 nm estimated by SE but again lower than 9.85 nm as obtained from QCM-D. Thus, the above results show that the thickness values obtained from SE and AFM are in good agreement. The large discrepancy from the much higher thicknesses obtained by means of QCM-D in the case of the pure Fn and HSA þ Fn layers clearly arose from the large amount of water as already indicated.32 Given that the HSA layer did not undergo the same extent of water incorporation, the three techniques gave very similar thickness values for the HSA layer, further supporting the picture of stable HSA molecules firmly bound to the surfaces as a consequence of their irreversible denaturation. 3.3. Fn Orientation: Fn-Anti-Fn Recognition. The accessibility of the 4F1 3 5F1 segments in the heparin I (Hep I) and fibrin (Fbn) binding domains of Fn adsorbed onto the PHMS surface was tested by SE via the binding of the amount of an antifibronectin polyclonal antibody (anti-Fn) through the specific recognition of the site of interest.39 Figure 3 shows the SE measurements for the binding between anti-Fn and HSA, anti-Fn and Fn, and anti-Fn and HSA þ Fn layers, respectively. An interesting observation is that for the binding of anti-Fn onto preadsorbed Fn and HSA þ Fn layers the amount of bound antibody appeared to show linear growth with time over 50 min. In contrast, the (38) Tsai, J.; Taylor, R.; Chothia, C.; Gerstein, M. J. Mol. Biol. 1999, 290, 253–266. (39) Norde, W. Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; p 27.

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Figure 3. Adsorption of anti-Fn (2) onto preadsobed HSA (Δ), anti-Fn (b) onto preadsobed Fn (O), and anti-Fn (() onto a preadsobed 1:1 mixture of HSA þ Fn ()) on PHMS. The curves are shown to guide the reader.

Figure 4. Anti-Fn antibody binding to differently adsorbed Fn calculated on the basis of moles of antibody bound per mole of Fn adsorbed on different surfaces where HSA þ Fn is the 1:1 binary coadsorption mixture, the sequential adsorption of HSA onto preadsorbed Fn is abbreviated as Fn . HSA, and the sequential adsorption of Fn onto preadsorbed HSA represented as HSA . Fn.

binding of anti-Fn to the HSA layer surface did not show any noticeable time effect and the amount of anti-Fn bound was very low, consistent with the nonspecific adsorption. The level of antiFn bound to the two Fn surfaces was much greater, consistent with the specific antibody recognition. The amount of anti-Fn bound to 1 mol of Fn from each of the surfaces is shown in Figure 4, where the HSA surface clearly facilitated the least amount of antibody binding. The second smallest amount of anti-Fn binding occurred on the coadsorbed surface from the binary HSA þ Fn solution, and the highest amount of binding clearly occurred on the pure Fn adsorbed surface. However, it is interesting that whereas the SE and QCM-D data indicated that the adsorbed layers from HSA þ Fn coadsorption were almost exclusively composed of Fn, the lower binding of anti-Fn suggested that the Fn molecules from the 1:1 binary mixture were somewhat less available for anti-Fn binding. Therefore, because Fn/anti-Fn binding is due to the proper exposure of 4F1 3 5F1 domains, the reduced anti-Fn binding to the surface layer of HSA þ Fn coadsorption suggests that either a conformational change occurred for Fn, making the 4F1 3 5F1 domains unavailable, or coadsorbed HSA, however small the quantity, masked the binding domains. To discriminate between these two possibilities, sequential adsorption studies of HSA (0.1 mg/mL) on preadsorbed Fn (0.1 mg/mL) (i.e., Fn . HSA) and Fn (0.1 mg/mL) on preadsorbed HSA (0.1 mg/mL) (i.e., HSA . Fn) were performed. DOI: 10.1021/la104127q

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Figure 5. (Lower curve) Adsorption of Fn (2) onto preadsorbed HSA (Δ), followed by anti-Fn binding (). (Upper curve) Adsorption of HSA (b) onto preadsorbed Fn (O) followed by anti-Fn (*). 0 represents buffer washes. The coadsorption of the 1:1 mixture of HSA þ Fn ()) onto the PHMS surface is also shown, which closely follows the pure Fn (O) adsorption on the same surface. The curves are shown to guide the reader.

Figure 5 shows the time-dependent adsorption, with the relevant amount of anti-Fn binding to each single-component proteins shown in Figure 3. It can be seen that no significant Fn adsorption occurs on preadsorbed HSA under the studied conditions. Accordingly, the binding of anti-Fn with the adsorbed protein layer after sequential adsorption produced a mass increase almost identical to that found for pure HSA (i.e., Γ = 0.4 mg/m2), confirming that the exposed surface was predominantly HSA. Similarly, for the sequential adsorption of HSA onto preadsorbed Fn, no mass change was observed with respect to the initially adsorbed Fn (i.e., 3.9 mg/m2), with this value being close to the one measured from pure Fn adsorption at equilibrium. Moreover, a large amount of anti-Fn (i.e., around 1.0 mg/m2) was adsorbed onto the protein layer after the sequential adsorption, suggesting that the exposed surface in this case was predominantly composed of Fn. As already indicated, Fn has two subunits, each with 4F1 3 5F1 binding domains available for anti-Fn binding. Thus, the theoretical molar binding ratio is 2. For anti-Fn binding to pure Fn, the molar binding ratio is at most only about 1 from our results. It is noteworthy that anti-Fn binding onto preadsorbed Fn showed a steady increase over the period of the experiments, suggesting that the adsorbed Fn molecules continue to change their packing and structural conformation with time, an observation consistent with what was already reported in the literature.40,41 Although it is unlikely that a theoretical binding ratio of 2 would ever be reached because of the steric constraints at the interface, the mass adsorption results from the two sequential binding experiments suggest that no protein bilayers were formed, thus ruling out the possible HSA coadsorption masking the 4F1 3 5F1 binding domains. Accordingly, the reduced anti-Fn binding for the protein layer formed from HSA þ Fn coadsorption could arise from the conformational effects promoted by HSA coadsorption. The time-dependent anti-Fn binding from all Fn-containing surfaces adds strong support to the proposition of the structural reorganization of Fn molecules from a closed to an open conformation, leading to further exposure of the 4F1 3 5F1 binding domains. Co-adsorption of HSA could influence the initial adsorption and conformation of Fn molecules and also their subsequent structural adjustment or relaxation. (40) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313–328. (41) Xia, N.; May, C. J.; McArthur, S. L.; Castner, D. G. Langmuir 2002, 18, 4090–4097.

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Figure 6. Number of adhered McCoy cells, from suspensions of 2.5  104 cell/dish, on PHMS polymer (control) (B) and PHMS preadsorbed with HSA (E), Fn (C), and HSA þ Fn (D). The cells were cultured in a serum medium for T = 1, 3, and 5 days as shown. The reported data are representative of at least three separate experiments (*P b 0.05; **P b 0.01 compared to an untreated control by the student’s t test).

3.4. Fibroblast Adhesion and Proliferation. The Fn function of promoting cell attachment and proliferation largely arises from the induction of the RGD epitope, located in the 10th type III repeat within the central subunit. This is in contrast to the 4 F1 3 5F1 binding domains in Hep I, located near the N-terminus subunit that has been probed by the anti-Fn antibody binding.35 Accordingly, cell adhesion experiments could help discriminate the availability of the two different domains on Fn, bearing in mind that 4F1 3 5F1 binding domains in the coadsorbed layers from HSA þ Fn solutions were markedly less available with respect to the pure Fn layers, suggesting that a closed conformation masking 4F1 3 5F1 domains is preferentially adopted in coadsorption conditions. The cell adhesion experiments, using McCoy fibroblast lines, were performed on PHMS surfaces preadsorbed with HSA, Fn, and coadsorbed layer from HSA þ Fn 1:1 solution, respectively. Cell adhesion and proliferation were quantitatively assessed after 1, 3, and 5 days of incubation. The results reported in Figure 6 show the increase in the number of adhered cell with time on each surface from the representative microscopic images taken. From these results, we can see that at day 1 cell attachment is comparable for all of the bare and preconditioned surfaces, within small errors. At longer cell incubation times (i.e., at days 3 and 5), the cell population certainly grows on each surface. Obviously, as expected, cell adhesion and proliferation are significantly lower for HSA and bare PHMS surfaces but higher on Fn and HSA þ Fn surfaces, showing the clear effect of Fn adsorption on cell adhesion and proliferation. However, at day 3 the cell density on HSA þ Fn preconditioned surfaces is greater than on Fn surfaces, whereas at day 5 the cell density becomes almost identical on both surfaces. The difference observed at day 3 is a signature of the subtle effect of coadsorbed HSA on the conformation of surfaceimmobilized Fn. Indeed, the conformational arrangements of Fn molecules adsorbed from pure and 1:1 HSA þ Fn solution adsorption processes above were evaluated in terms of the different average exposure of the 4F1 3 5F1 binding domains close to the N termini of Fn. The cell adhesion data are instead representative of the availability of the RGD sequences located near the central region of the protein. Accordingly, the different availability of 4F1 3 5F1 and RGD segments is interpreted in terms of two predominant Fn conformations, the first with the N-terminal subunit (containing exposed 4F1 3 5F1) oriented toward the solution and the second Langmuir 2011, 27(1), 312–319

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ECM maturation cancel the conformational effects of preadsorbed Fn. Future study in this regard will need to examine cell growth rates on different protein surfaces within the first 3 to 4 days more systematically. Furthermore, whereas the difference in cell numbers at day 3 demonstrates the different availabilities of the RGD sequence, the current work could not identify the actual number of RGD epitopes available on the surfaces for the cells to access. It is thus not possible to assess the active surface-exposed RGD epitopes against the necessary surface density of RGD for maximal adhesion and cell growth. Nevertheless, the main outcome of this study concerning the different structural conformations of Fn with and without HSA coadsorption remains valid.

Figure 7. Schematic illustration of structural conformations of Fn (a) and HSA þ Fn (b) adsorbed on a PHMS surface, showing the effect of HSA coadsorption on the accessibility of 4F1 3 5F1 and RGD segments from the solution side.

exposing the central region of Fn (containing the RGD sequences) toward the solution.42,43 The two representative Fn conformations are schematically shown in Figure 7. As already indicated, the first type of binding domain was specifically targeted by using a polyclonal antifibronectin antibody (anti-Fn), and the exposure of the second has been determined by comparing the anti-Fn binding with the cell response data. The anti-Fn binding data indicated that the coadsorption of Fn and HSA strongly reduced the 4F1 3 5F1 domain availability with respect to the pure Fn surface, and the fibroblast cell responses to pure and coadsorbed Fn demonstrated that the RGD sequences remained available for both Fn surfaces (with slightly higher exposure of RGD for the HSA þ Fn surface), suggesting that coadsorption did not significantly affect the exposure of RGD domains as it did for the 4F1 3 5F1 domains. The different availability of 4F1 3 5F1 and RGD segments for pure and coadsorbed films suggests that Fn assumed different conformations depending on the fact that adsorption occurred with or without HSA. Note that for trypsinized cells, the growth after day 1 is still within the refractory period and this situation is consistent with the observation of similar cell densities (T1 in Figure 6). As to the levelling of the adhered cell density observed at day 5 for both Fn and HSA þ Fn preconditioned surfaces, it must be emphasized that this result is in good agreement with the recently demonstrated effects of ECM maturation.44 These effects respectively involve the cell-induced stretching of Fn fibrils, leading to partial unfolding of the secondary structure of individual protein modules45 and the expression at longer times of a cell’s self-made, thick, three-dimensional ECM microenvironment.44 In particular, for fibroblasts this last process has been shown to occur over the course of three days.44 Thus, our results allow us to complete the overall picture of the interaction of cells with preconditioned surfaces because the cell adhesion density observed at day 3 suggests that, under our conditions, this is indeed the temporal limit of action of the preconditioned surfaces, whereas at longer times (i.e., day 5) the processes of (42) Meadows, P. Y.; Walker, G. C. Langmuir 2005, 21, 4096–4107. (43) Koenig, A. L.; Gambillara, V.; Grainger, D. W. J. Biomed. Mater. Res. A 2003, 64, 20–37. (44) Baneyx, G.; Baugh, L.; Vogel, V. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14464–14468. (45) Antia, M.; Baneyx, G.; Kubow, K. E.; Vogel, V. Faraday Discuss. 2008, 139, 229–249.

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4. Conclusions The dynamic adsorption and the layer structure of Fn adsorbed onto the hydrophobic PHMS from single and binary solution with HSA were studied using in situ SE and QCM-D and ex situ AFM techniques. These studies together provided complementary measurements of the thickness, morphology, and effective mass of the adsorbed protein layers and also shed light on their viscoelastic properties related to the degree of surface binding and hydration. While the adsorption of HSA formed a tightly bound, thin monolayer of structurally destabilized protein, pure Fn adsorption yielded a relatively thick, viscoelastic molecular film. Although the Fn layer was also firmly bound to the surface, it was rather heavily hydrated. The protein layer obtained from the coadsorption of the binary HSA þ Fn solution was found to be predominantly composed of Fn, with a very low quantity of HSA present in the layer. The thickness and viscoelastic behavior indicated that the coadsorbed layer predominantly behaved as the pure Fn layer but the Fn had greater structural order within the coadsorbed layer. The coadsorption with HSA promoted the transition of the Fn conformation from open (in the pure Fn layer) to closed (in the coadsorbed layer), by analogy to the previously reported expanded-to-compact conformation transition induced by salt concentration and pH changes for Fn in solution36 or compactto-expanded ones onto liposomes.46 In this context, it must be stressed that the transition also seemed to occur if no significant amount of HSA was adsorbed within or onto the Fn layers. It is clear that the techniques employed to follow adsorption cannot provide the effective adsorbed layer composition and distribution and dynamic variations within the interfacial layer. Further experiments with high surface sensitivity are needed to clarify these crucial points addressing the detailed mechanistic processes associated with Fn conformational changes. Acknowledgment. G.M. and N.G. gratefully acknowledge the financial support of the Italian Ministry of University and Research (MIUR) under contract FIRB RBIP06KEWY (“Waterfall”). M.Y. and J.R.L. thank the U.K. Engineering and Physical Sciences Research Council (EPSRC) for supporting this work. Supporting Information Available: SE thickness for thin and thick films of PHMS in PBS solution measured as a function of time. QCM-D data for PHMS films in PBS solution. Tapping-mode AFM images and z profiles of PHMS films deposited on silicon and proteins adsorbed on hydrophobic PHMS. This material is available free of charge via the Internet at http://pubs.acs.org. (46) Halter, M.; Antia, M.; Vogel, V. J. Controlled Release 2005, 101, 209–222.

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