Elastic Repulsion from Polymer Brush Layers ... - ACS Publications

Jul 30, 2013 - Kyoko Fukazawa , Aiko Nakao , Mizuo Maeda , and Kazuhiko Ishihara. ACS Applied ... Kazuhiko Ishihara , Tomomi Kitagawa , and Yuuki Inou...
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Elastic Repulsion from Polymer Brush Layers Exhibiting High Protein Repellency Yuuki Inoue,†,§ Tomoaki Nakanishi,† and Kazuhiko Ishihara*,†,‡,§ †

Department of Materials Engineering and ‡Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan § Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 5 Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan ABSTRACT: Hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly(2-hydroxyethyl methacrylate) (PHEMA) brush layers with different thicknesses and graft densities were prepared to construct a model surface to elucidate protein−surface interactions. In particular, we focused on the steric repulsion of hydrophilic polymer layers as one of the surface properties that strongly influence protein adsorption and employed force-versus-distance (f−d) curve measurements obtained via atomic force microscopy to quantitatively evaluate the steric repulsion force, which is also referred to as the “elastic repulsion energy.” We also analyzed direct interactions between the surface and proteins via the f−d curve, because these interactions trigger the protein-adsorption phenomenon. Protein−surface interactions were extremely suppressed at surfaces with high elastic repulsion energies and highly dense polymer brush structures, which is in contrast to those at surfaces with low elastic repulsion energies and low density of the grafted polymer layers. These results indicate that the elastic repulsion from the grafted polymer layer at the surface is an important parameter for controlling protein−surface interactions and protein adsorption phenomenon.

1. INTRODUCTION Protein adsorption on the surfaces of materials triggers several undesirable biological reactions. Therefore, it becomes important to prevent protein adsorption when the materials are used in the biomedical field. To obtain nonbiofouling properties, it is necessary to understand the interactions between proteins and materials surfaces. Recently, much attention has been focused on arranging polymer chains with different chemical structures using surface-initiated living radical polymerization.1−4 In particular, the formation of a very dense polymer brush surface via surface-initiated atom transfer radical polymerization (SI-ATRP) enables the construction of a well-defined surface structure with wideranging monomer units, chain lengths, and chain density. Protein adsorption at polymer brush surfaces with different three-dimensional structures has been extensively studied using hydrophilic or zwitterionic polymers;5−11 for example, Brash and co-workers reported that oligo(ethylene glycol)- and phosphorylcholine-based polymer brush surfaces are extremely repellant toward protein adsorption (i.e., the amount of adsorbed proteins from a single or mixed protein solution is less than 5.0 ng/cm2), and the amount of adsorbed proteins is dependent on the layer thickness and density of the grafted polymer chains.12−15 However, the underlying mechanism for the dependence of protein adsorption on the surface structure has not yet been fully elucidated. The purpose of this study is to clarify the underlying mechanism for protein repellency of such hydrophilic polymer-chain−grafted surfaces from the perspec© XXXX American Chemical Society

tive of the repulsion energy of the surface. Polymer brush surfaces with precisely controlled chemical and topological structures are useful for investigating the effects of surface structure on protein-adsorption behaviors. Atomic force microscopy (AFM) is a powerful tool that not only can be used to image the surface topography but can also be used for the nanoanalysis of surface properties such as elasticity or softness and adhesion or interaction forces between two surfaces.16−19 Because the polymer brush surface consists of very dense polymer chains grafted perpendicular to the surface, the polymer chain mobility at the surface is considerably suppressed. This unique surface structure of the polymer brush layer enables the evaluation of the steric repulsion effect from the compression energy of the surface under aqueous conditions. Protein adsorption is triggered by the direct contact of the proteins with the surface through various intermolecular interactions. Accordingly, it is important to evaluate direct interactions between single-layered proteins and the surfaces in order to clarify the protein-adsorption behavior.20−23 However, there has been little research on the interactions between proteins and surfaces that do not adsorb proteins, such as the hydrophilic and dense polymer brush surfaces. This study provides a precise understanding of the protein-resistant properties of a hydrophilic polymer-chainReceived: June 7, 2013 Revised: July 28, 2013

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of polymer chains on the surface. Mn is assumed to be the same as the number-average molecular weight of each polymer in the polymerization solution, as calculated from the target DP; the conversion of each monomer to polymers was determined by 1H NMR spectroscopy and the molecular weight of each monomer.2,3 The static air contact angles in aqueous medium were measured at room temperature using the captive bubble method with a goniometer (CA-W, Kyowa Interface Science Co., Tokyo, Japan). The samples were immersed in water for 24 h prior to the measurements and then brought into contact with 5.0 μL of air bubbles during the measurements. All static air bubble contact angles were directly measured from photographic images. Data were collected at more than three positions for each sample. The elastic repulsion energy of the polymer brush surfaces in a phosphate-buffered saline (PBS, pH 7.4) was estimated by the forceversus-distance ( f−d) curve measurements with a silica-beadimmobilized cantilever. Figure 2 shows the typical approaching line

grafted surface from the perspective of steric repulsion between the polymer layers and proposes a novel concept for fabricating biomaterials that eliminate several undesired bioresponses.

2. EXPERIMENTAL SECTION 2.1. Materials. A phospholipid monomer, 2-methacryloyloxyethyl phosphorylcholine (MPC), was synthesized and purified according to a previously reported method.24 Silicon wafers were purchased from Furuuchi Chemical Co. (Tokyo, Japan); their surfaces were coated with ∼10-nm-thick SiO2 layers. Copper(I) bromide (CuBr), 2,2′bipyridyl (bpy), ethyl-2-bromoisobutyrate (EBIB), 2-hydroxyethyl methacrylate (HEMA), and fibrinogen from bovine plasma (Fraction I) were purchased from Sigma-Aldrich Co. (St. Louis, MO) and used as received. Triethylamine (TEA) was purchased from Kanto Chemical Co. (Tokyo, Japan) and used after atmospheric distillation at 95 °C. All other reagents and solvents were commercially obtained at extra-pure grade and were used as received. 2.2. Preparation of Well-Defined Polymer Brush Surfaces. Two types of hydrophilic polymer brush surfaces with grafted poly(MPC) (PMPC) and poly(HEMA) (PHEMA) chains were prepared on initiator-immobilized silicon wafers using SI-ATRP according to a previously reported method (Figure 1).25 Briefly, a

Figure 1. Chemical structures of polymer brush layers. surface-attachable initiator, 3-(2-bromoisobutyryl)decyl dimethylchlorosilane (BrC10DMCS), was synthesized as previously described1 and then immobilized on the silicon wafers at BrC10DMCS to decyl dimethylchlorosilane (CH3C11DMCS) molar feed ratios of 0.02, 0.1, and 1.0. Specific amounts of the monomers, CuBr and bpy, were dissolved in the degassed solvents; then the initiator-immobilized silicon wafers with different molar ratios of BrC10DMCS and EBIB, as free initiators, were simultaneously placed into the polymerization solution to initiate SI-ATRP. The polymerization solutions were stirred at room temperature for 24 h to complete the polymerization reaction. The target degrees of polymerization (DPs, [monomer]/ [initiator] ratio in feed) were set at 10, 20, 50, and 100. The conversion of each monomer to polymer and the molecular weights or polydispersities of the free polymers formed in the solutions were analyzed using proton nuclear magnetic resonance spectroscopy (1H NMR; α-300, JEOL, Tokyo, Japan) and gel permeation chromatography (GPC; JASCO, Tokyo, Japan), respectively. The target DPs and ratios of BrC10DMCS to CH3C11DMCS are indicated after the abbreviated polymer name, for example, PMPC10-1.0 and PHEMA100-0.02, when necessary. 2.3. Surface Characterization. The surface morphologies of the polymer brush layers under dry conditions were observed using a Nanoscope IIIa atomic force microscope (Bruker Japan Co., Ltd., Kanagawa, Japan) operated in the tapping mode. The measurement was performed using a commercially available cantilever (RTESP, Bruker Japan Co., Ltd.) at a scan rate of 1.0 Hz with a scan size of 1.0 μm × 1.0 μm. The root-mean-square (RMS) surface roughness values were calculated from the roughness profiles. The thicknesses of the PMPC and PHEMA brush layers under dry conditions were measured using spectroscopic ellipsometry (J. A. Woollam Co., Inc., Lincoln, NE). The graft density, σ, (chains/nm2) of the grafted polymer chains at the polymer brush surface was estimated using the following equation: σ = hρNA/Mn, where h (nm) is the ellipsometric layer thickness, ρ (g/cm3) is the density of the dry polymers (1.30 g/cm3 for PMPC5 and 1.15 g/cm3 for PHEMA6), NA is Avogadro’s number, and Mn is the number-average molecular weight

Figure 2. Typical force-versus-distance curve between polymer brush layer and silica-bead-immobilized cantilever. The elastic repulsion energy was defined as the region area enclosed by the obtained approaching line and two extended baselines. in the f−d curve measurements for the PMPC brush surface in the PBS. As shown in Figure 2, the elastic repulsion energy of the polymer brush layers was estimated from the region area enclosed by the obtained approaching line and two extended baselines in the f−d curve.16 The silica bead, with a diameter of 20 μm (Duke Scientific Co., Palo Alto, CA), was manually immobilized on the tip of a commercial probeless cantilever (NP-3 with an announced reported spring constant of 0.06 N/m, Bruker Japan Co., Ltd.). Using optical microscopy, we confirmed that the silica bead was successfully immobilized on the cantilever and that the immobilization did not change the spring constant of the cantilever. The silica-beadimmobilized cantilever approached the polymer brush layers in a PBS solution at a scan rate of 1.0 Hz with a load of ∼15 nN. 2.4. Adsorption Force of Fibrinogen against Polymer Brush Layers. Fibrinogen was covalently immobilized on a cantilever (OTR8 with an announced reported spring constant of 0.15 N/m, Bruker Japan Co., Ltd.) according to a previously reported method.25 Briefly, 3-nm-thick chromium and sequential 27-nm-thick gold films were sputtered onto a bare cantilever. The gold-sputtered cantilever was then immersed in a 1.0 mmol/L solution of 11-mercaptoundecanoic acid in ethanol for 24 h to form a carboxyl-terminated selfassembled monolayer (SAM) on the cantilever. The carboxyl groups on the prepared cantilever were activated using an aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.10 mol/L) and N-hydroxysuccinimide (0.05 mol/L). After incubating for B

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0.12 chains/nm2 for PHEMA-0.1, and 0.06 chains/nm2 for PHEMA-0.02. Because the graft density would be strongly influenced by the molecular size of the side chain in each monomer unit, it is important to determine the area occupied by the grafted chains at each polymer brush surface in order to clarify the dimensions of the grafted polymer chains at the surfaces.27,28 There was little difference between the occupied areas of the grafted polymer chains of the PMPC and PHEMA brush surfaces for the initiator-immobilized substrates with the highest (100%) and lowest (2%) densities of BrC10DMCS: The occupied areas of the grafted polymer chains of the PMPC-1.0 and PHEMA-1.0 brush surfaces were 0.39 and 0.44, respectively, while those for the PMPC-0.02 and PHEMA-0.02 brush surfaces were both 0.05. In contrast, the occupied areas of the PMPC-0.1 and PHEMA-0.1 brush surfaces were quite different at 0.17 and 0.09, respectively. These results show that the dimensions of the grafted polymer chains clearly differed among the polymer brush surfaces. In particular, the grafted polymer chains of PMPC-1.0, PMPC-0.1, and PHEMA-1.0 form high-density polymer brush structures, while those of PHEMA-0.1 form a diluted polymer brush structures and those of PMPC-0.02 and PHEMA-0.02 form mushroom structures.27 Previously, we obtained a smooth surface with very dense polymer brush layers using a 100% BrC10DMCS-immobilized substrate.25 As shown in Figure 4, the morphologies of the PMPC and PHEMA brush surfaces under dry conditions varied significantly with the BrC10DMCS ratio at the initiatorimmobilized surface: The surface roughness of the PMPC and PHEMA brush surfaces increased with decreasing BrC10DMCS ratio (RMS values of 0.3 nm for PMPC1001.0, 0.5 nm for PMPC100-0.1, and 1.6 nm for PMPC100-0.02; those of 0.5 nm for PHEMA100-1.0, 0.7 nm for PHEMA1000.1, and 1.1 nm for PHEMA100-0.02, respectively). These results also indicate that the conformation of the grafted polymer chains on the surface would differ at the polymer brush surface with controlled graft density. Figure 5 shows the elastic repulsion energy of the PMPC and PHEMA brush surfaces as a function of the molecular weight of the grafted polymer chain at the polymer brush surfaces with different graft densities. The elastic repulsion energy is the energy expended to achieve the completely compressed state from the equilibrated state of the water-swollen polymer chains grafted at the surface. Therefore, the elastic repulsion energy corresponds to the steric repulsion effect from the hydrophilic polymer chains in aqueous conditions. As evident from Figure 5, the elastic repulsion energy increased with increasing molecular weight and graft density, regardless of the chemical structure of the grafted polymer. Furthermore, the elastic repulsion energy of the polymer brush surfaces composed of the grafted polymer chains with same molecular weights increased with increasing graft density. These results indicate that an increase in the number of monomer units per defined area would increase the elastic repulsion energy of the polymer brush surface. On the other hand, the slope of the elastic repulsion energy versus the molecular weight of the grafted polymer chain differed among the polymer brush surfaces: The elastic repulsion energy of the PMPC-1.0, PMPC-0.1, and PHEMA-1.0 brush surfaces rapidly increased with increasing molecular weight of the grafted polymer chains, while that of the PMPC-0.02, PHEMA-0.1, and PHEMA-0.02 brush surfaces hardly increased. This is likely related to the fact that the PMPC-1.0, PMPC-0.1, and PHEMA-1.0 brush surfaces had very dense polymer brush structures, while the other polymer

30 min, the cantilever was washed with water and immediately placed in contact with a 1.0 mg/mL PBS solution of fibrinogen for 30 min at 37 °C. The desired fibrinogen-immobilized cantilever was obtained after washing with PBS and stored in PBS at 4 °C until used. The immobilization of fibrinogen on the cantilever was confirmed via quartz crystal microbalance with dissipation (QCM-D) and XPS measurements using a commercially available QCM-D gold sensor and gold-evaporated silicon wafer instead of the cantilever. The adsorption force of fibrinogen against the polymer brush layers was measured in PBS at room temperature using the f−d curve for the fibrinogenimmobilized cantilever and polymer brush layers. In the approaching and retracting traces of the f−d curve, the shift in the deflection value of the retracting trace from the bottom of the retrace line corresponds to the force between the proteins and surfaces. For each measurement, more than 100 approaching/retracting f−d curves were collected and the average measured force between the proteins and surfaces was defined as the adsorption force between fibrinogen and the polymer brush layers. There was little difference in the adsorption forces between the beginning and end of the f−d measurements, and they took a Gaussian distribution. These results indicate that the structure of the immobilized protein did not change during the measurement. All measurements were repeated at least three times.

3. RESULTS AND DISCUSSION 3.1. Surface Characterization. Previously, we confirmed that living radical polymerization of MPC and HEMA could be performed under the same conditions used in this study from an analysis of polymerization kinetics.26 Figure 3 shows the

Figure 3. Relationship between ellipsometric layer thickness and absolute molecular weight of polymer chains determined by 1H NMR. Circles: PMPC brush surface from an initiator-immobilized surface with (black) 100% BrC10DMCS (PMPC-1.0), (gray) 10% BrC10DMCS (PMPC-0.1), and (white) 2% BrC10DMCS (PMPC0.02). Squares: PHEMA brush surface from an initiator-immobilized surface with (black) 100% BrC10DMCS (PHEMA-1.0), (gray) 10% BrC10DMCS (PHEMA-0.1), and (white) 2% BrC10DMCS (PHEMA-0.02).

ellipsometric layer thickness of a polymer brush layer at the surface as a function of the molecular weight of free polymers in the polymerization solution. A linear relationship between the ellipsometric layer thickness and molecular weight was observed for each polymer, which indicates that the SI-ATRP of each monomer was well controlled. The calculated graft densities of the PMPC-grafted polymer chains were 0.26 chains/nm2 for PMPC-1.0, 0.11 chains/nm2 for PMPC-0.1, and 0.03 chains/nm2 for PMPC-0.02, and those of the PHEMAgrafted polymer chains were 0.59 chains/nm2 for PHEMA-1.0, C

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Figure 4. Images of heights of (a) PMPC100-1.0, (b) PMPC100-0.1, (c) PMPC100-0.02, (d) PHEMA100-1.0, (e) PHEMA100-0.1, and (f) PHEMA100-0.02 brush surfaces observed by atomic force microscopy (AFM) under dry conditions.

Figure 5. Relationship between repulsion energy of polymer brush surface under aqueous conditions and molecular weight of polymers. Circles: PMPC brush surface from an initiator-immobilized surface with (black) 100% BrC10DMCS (PMPC-1.0), (gray) 10% BrC10DMCS (PMPC-0.1), and (white) 2% BrC10DMCS (PMPC-0.02). Squares: PHEMA brush surface from an initiator-immobilized surface with (black) 100% BrC10DMCS (PHEMA-1.0), (gray) 10% BrC10DMCS (PHEMA-0.1), and (white) 2% BrC10DMCS (PHEMA-0.02).

polymer chains grafted at the surface. In this study, we focused on the direct interaction of proteins with the polymer brush surfaces, because the initial interaction between the proteins and surfaces is considered to be one of the important parameters that determine the protein adsorption onto the surface. Figure 6 shows the adsorption force of fibrinogen onto polymer brush surfaces with different graft densities as a function of the ellipsometric layer thickness. The adsorption force of fibrinogen was suppressed at polymer brush surfaces with a larger ellipsometric layer thickness. Moreover, the adsorption force of fibrinogen onto the PMPC brush surfaces was lower than that onto the PHEMA brush surfaces at the same ellipsometric layer thickness. These results are consistent with those of our previous study that showed that the amount of proteins adsorbed from 100% fetal bovine serum depended on the chemical structure and was suppressed at thicker

brush surfaces had a diluted polymer brush or mushroom structures. Tsujii et al. reported that the repulsive force originates from the steric interactions between the solventswollen polymer brush layer and the silica probe immobilized on the AFM cantilever.27 Furthermore, they reported that the repulsive force would increase with increasing equilibrium thickness of the solvent-swollen polymer brush layer in a good solvent, which increases as the graft density increases. These results indicate that a very dense polymer brush structure at the surface with a high elastic repulsion energy leads to the extremely high steric repulsion effect from the surface. 3.2. Adsorption Force of Proteins onto Polymer Brush Surfaces. As has been previously reported, the graft density at the hydrophilic polymer brush surface has a much greater influence on the amount of protein adsorbed than the thickness of the polymer brush layer.6,12,13 This is qualitatively considered to be because of the size-exclusion effect of the hydrophilic D

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Figure 6. Relationship between adsorption force of fibrinogen and ellipsometric layer thickness. Circles: PMPC brush surface from an initiatorimmobilized surface with (black) 100% BrC10DMCS (PMPC-1.0), (gray) 10% BrC10DMCS (PMPC-0.1), and (white) 2% BrC10DMCS (PMPC0.02). Squares: PHEMA brush surface from an initiator-immobilized surface with (black) 100% BrC10DMCS (PHEMA-1.0), (gray) 10% BrC10DMCS (PHEMA-0.1), and (white) 2% BrC10DMCS (PHEMA-0.02). Diamonds: Initiator-immobilized surface with 100% BrC10DMSC.

Figure 7. Relationship between adsorption force of fibrinogen and elastic repulsion energy of a polymer brush surface under aqueous conditions. Circles: PMPC brush surface from an initiator-immobilized surface with (black) 100% BrC10DMCS (PMPC-1.0), (gray) 10% BrC10DMCS (PMPC-0.1), and (white) 2% BrC10DMCS (PMPC-0.02). Squares: PHEMA brush surface from an initiator-immobilized surface with (black) 100% BrC10DMCS (PHEMA-1.0) and (gray) 10% BrC10DMCS (PHEMA-0.1).

polymer brush surfaces.26 This result demonstrates that the adsorption force of a protein at a surface with high repellency toward protein adsorption is strongly related to the amount of proteins adsorbed on the surface. On the other hand, the adsorption force of fibrinogen onto thinner polymer brush surfaces was affected by graft density, especially in the case of the PHEMA brush surfaces: The adsorption force of fibrinogen onto the polymer brush surfaces decreased as the graft density increased even when the surfaces had almost the same layer thicknesses. As mentioned above, there was a difference in the conformation of the grafted polymer chains among the polymer brush surfaces because of the graft density. Therefore, the adsorption force of fibrinogen toward the polymer brush surfaces is dependent on the conformation of the grafted polymer chains. The influence of the steric repulsion at the oligo(ethylene glycol)- or polyethylene oxide-grafted surfaces on protein adsorption has been extensively studied from several perspectives, including layer thickness, distance between the polymer terminal and surface, and concentration of polymers on the surface.29−36 In this study, in order to quantitatively evaluate the effect of polymer chain conformation on the adsorption force of fibrinogen, the relationship between the

adsorption force and elastic repulsion energy was plotted, as shown in Figure 7. It is evident from this plot that there is a stronger relationship between the adsorption force and elastic repulsion energy than between the adsorption force and thickness of the polymer brush layer. The adsorption force of fibrinogen toward the polymer brush layer monotonously decreased with increasing elastic repulsion energy of the surface. Proteins could not interact with surfaces that have high elastic repulsion energies, that is, the surfaces with very dense polymer layers, because of the strong repulsive force of the polymer layer. The surfaces with low elastic repulsion energies were mainly those with low graft densities; therefore, high mobility of the grafted polymer chains would be expected. However, proteins interacted with these surfaces, indicating that the steric repulsion from the grafted polymer layers has much greater influence on protein adsorption than the polymer chain mobility at the surface. These results clearly demonstrate that the adsorption force of proteins toward the hydrophilic polymer brush surface can be quantitatively explained via the elastic repulsion energy of the surface. From the perspective of the chemical structure of the polymer brush surface, the PMPC brush surfaces retained a small adsorption force of fibrinogen even onto surfaces with E

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polymerization. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 3350− 3359. (3) Husseman, M.; Malmstrom, E. E.; McNamara, M.; Mate, M.; Mecerreyes, D.; Benoit, D. G. Controlled synthesis of polymer brushes by “living” free radical polymerization techniques. Macromolecules 1999, 32, 1424−1431. (4) Kobayashi, M.; Mitamura, K.; Terada, M.; Yamada, N. L.; Takahara, A. Characterization of swollen states of polyelectrolyte brushes in salt solution by neutron reflectivity. J. Phys.: Conf. Ser. 2011, 272, 012019−012022. (5) Iwata, R.; Suk-In, P.; Hoven, V. P.; Takahara, A.; Akiyoshi, K.; Iwasaki, Y. Control of nanobiointerfaces generated from well-defined biomimetic polymer brushes for protein and cell manipulations. Biomacromolecules 2004, 5, 2308−2314. (6) Yoshikawa, C.; Goto, A.; Tsujii, Y.; Fukuda, T.; Kimura, T.; Yamamoto, K.; Kishida, A. Protein repellency of well-defined, concentrated poly(2-hydroxyethyl methacrylate) brushes by the sizeexclusion effect. Macromolecules 2006, 39, 2284−2290. (7) Yang, W.; Chen, S.; Cheng, G.; Vaisocherova, H.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Film thickness dependence of protein adsorption from blood serum and plasma onto poly(sulfobetaine)-grafted surfaces. Langmuir 2008, 24, 9211−9214. (8) Yang, W.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Pursuing “zero” protein adsorption of poly(carboxybetaine) from undiluted blood serum and plasma. Langmuir 2009, 25, 11911−11916. (9) Wu, Z.; Chen, H.; Liu, X.; Zhang, Y.; Li, D.; Huang, H. Protein adsorption on poly(N-vinylpyrrolidone)-modified silicon surfaces prepared by surface-initiated atom transfer radical polymerization. Langmuir 2009, 25, 2900−2906. (10) Gunkel, G.; Weinhart, M.; Becherer, T.; Haag, R.; Huck, W T. S. Effect of polymer brush architecture on antibiofouling properties. Biomacromolecules 2011, 12, 4169−4172. (11) Kitano, H.; Liu, Y.; Tokuwa, K.; Li, L.; Iwanaga, S.; Nakamura, M.; Kanayama, N.; Ohno, K.; Saruwatari, Y. Polymer brush with pendent glucosylurea groups constructed on a glass substrate by RAFT polymerization. Eur. Polym. J. 2012, 48, 1875−1882. (12) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Protein resistant surfaces: Comparison of acrylate graft polymers bearing oligo-ethylene oxide and phosphorylcholine side chains. Biointerphases 2006, 1, 50− 60. (13) Feng, W.; Gao, X.; McClung, G.; Zhu, S.; Ishihara, K.; Brash, J. L. Methacrylate polymer layers bearing poly(ethylene oxide) and phosphorylcholine side chains as non-fouling surfaces: In vitro interactions with plasma proteins and platelets. Acta Biomater. 2011, 7, 3692−3699. (14) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Adsorption of fibrinogen and lysozyme on silicon grafted with poly(2-methacryloyloxyethyl phosphorylcholine) via surface-initiated atom transfer radical polymerization. Langmuir 2005, 21, 5980−5987. (15) Jin, Z.; Feng, W.; Zhu, S.; Sheardown, H.; Brash, J. L. Proteinresistant materials via surface-initiated atom transfer radical polymerization of 2-methacryloyloxyethyl phosphorylcholine. J. Biomater. Sci. 2010, 21, 1331−1344. (16) Butt, H.-J.; Cappella, B.; Kappl, M. Force measurements with the atomic force microscope: Technique, interpretation and applications. Surf. Sci. Rep. 2005, 59, 1−152. (17) Hanley, W.; McCarty, O.; Jadhav, S.; Tseng, Y.; Wirtz, D.; Konstantopoulos, K. Single molecule characterization of P-selectin/ ligand binding. J. Biol. Chem. 2003, 278, 10556−10561. (18) Kienberger, F.; Kada, G.; Mueller, H.; Hinterdorfer, P. Single molecule studies of antibody−antigen interaction strength versus intramolecular antigen stability. J. Mol. Biol. 2005, 347, 597−606. (19) Han, X.; Qin, M.; Pan, H.; Cao, Y.; Wang, W. A versatile “multiple fishhooks” approach for the study of ligand−receptor interactions using single-molecule atomic force microscopy. Langmuir 2012, 28, 10020−10025. (20) Kidoaki, S.; Matsuda, T. Adhesion forces of the blood plasma proteins on self-assembled monolayer surfaces of alkanethiolates with

low elastic repulsion energy, as compared to the PHEMA brush surfaces. Protein adsorption would be influenced not only by the elastic repulsion energy of the surface but also by the surface wettability under wet conditions. The surface wettability, which results from the hydrophobic initiators or methyl groups at the surface and the characteristics of the grafted polymer chains, determines the extent of the hydrophobic interaction with the proteins under wet conditions. In this regard, there would be a big difference in the surface wettability of the PMPC and PHEMA brush surfaces, because these polymer chains have significantly different solubilities in water, wherein the static air contact angles of the highly watersoluble PMPC brush surfaces were always higher than those of the poorly water-soluble PHEMA brush surfaces with equivalent elastic repulsion energies. Therefore, the high wettability of the PMPC brush surfaces would lead to weaker hydrophobic interactions with fibrinogen than that between the PHEMA brush surfaces and fibrinogen. However, despite the difference in the static air contact angles of the PMPC and PHEMA brush surfaces with high elastic repulsion energies in water, there is only a small difference in the adsorption forces toward fibrinogen. This result indicates that the elastic repulsion energy of the hydrophilic polymer brush layer would dominate the inhibition of protein adsorption as compared to wettability under wet conditions.

4. CONCLUSIONS Well-defined hydrophilic polymer brush surfaces with severallayer thicknesses and graft densities were prepared as model surfaces in order to clarify the important parameters of the surface that suppress protein adsorption. In particular, we quantitatively analyzed the influence of the steric repulsion from the water-swollen polymer layer formed at the surface on the interactions between the protein and surfaces. The elastic repulsion energy of the surface, which is an indicator of the steric repulsion and is evaluated from the f−d curve measured using an AFM apparatus, could be controlled through the chemical structure and conformation of the grafted polymer chains. The adsorption force of protein toward the hydrophilic polymer brush surface decreased as the elastic repulsion energy increased. These results indicate that the elastic repulsion energy of the polymer-grafted surface is a crucial parameter for understanding the relationship between the proteins and surfaces that are highly repellant toward protein adsorption. A more precise understanding of the initial interactions between proteins and surfaces is progressing and would enable the design of novel materials that fully suppress protein adsorption.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-3-5841-7124. Fax: +81-3-5841-8647. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Ramakrishnan, A.; Dhamodharan, R.; Ruhe, J. Controlled growth of PMMA brushes on silicon surfaces at room temperature. Macromol. Rapid Commun. 2002, 23, 612−616. (2) Yamamoto, K.; Miwa, Y.; Tanaka, H.; Sakaguchi, M.; Shimada, S. Living radical graft polymerization of methyl methacrylate to polyethylene film with typical and reverse atom transfer radical F

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different functional groups measured by an atomic force microscope. Langmuir 1999, 15, 7639−7646. (21) Kidoaki, S.; Nakayama, Y.; Matsuda, T. Measurement of the interaction forces between proteins and iniferter-based graftpolymerized surfaces with an atomic force microscope in aqueous media. Langmuir 2001, 17, 1080−1087. (22) Sethuraman, A.; Han, M.; Kane, R. S.; Belfort, G. Effect of surface wettability on the adhesion of proteins. Langmuir 2004, 20, 7779−7788. (23) Cho, E. C.; Kong, H.; Oh, T. B.; Cho, K. Protein adhesion regulated by the nanoscale surface conformation. Soft Matter. 2012, 8, 11801−11808. (24) Ishihara, K.; Ueda, T.; Nakabayashi, N. Preparation of phospholipid polymers and their properties as polymer hydrogel membranes. Polym. J. 1990, 22, 355−360. (25) Inoue, Y.; Nakanishi, T.; Ishihara, K. Adhesion force of proteins against hydrophilic polymer brush surfaces. React. Funct. Polym. 2011, 71, 350−355. (26) Inoue, Y.; Ishihara, K. Reduction of protein adsorption on wellcharacterized polymer brush layers with varying chemical structures. Colloids Surf., B 2010, 81, 350−357. (27) Ejaz, M.; Ohno, K.; Tsujii, Y.; Fukuda, T. Controlled grafting of a well-defined glycopolymer on a solid surface by surface-initiated atom transfer radical polymerization. Macromolecules 2000, 33, 2870− 2874. (28) Tsujii, Y.; Ohno, K.; Yamamoto, S.; Goto, A.; Fukuda, T. Structure and properties of high-density polymer brushes prepared by surface-initiated living radical polymerization. Adv. Polym. Sci. 2006, 197, 1−45. (29) Lee, J. H.; Lee, H. B.; Andrade, J. D. Blood compatibility of polyethylene oxide surfaces. Prog. Polym. Sci. 1995, 20, 1043−1079. (30) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. A survey of structure-property relationships of surfaces that resist the adsorption of protein. Langmuir 2001, 17, 5605−5620. (31) Norde, W.; Gage, D. Interaction of bovine serum albumin and human blood plasma with PEO-tethered surfaces: Influence of PEO chain length, grafting density, and temperature. Langmuir 2004, 20, 4162−4167. (32) Kenausis, G. L.; Voros, J.; Elbert, D. L.; Huang, N.; Hofer, R.; Ruiz-Taylor, L.; Textor, M. J.; Hubbell, A.; Spencer, N. D. Poly(Llysine)-g-poly(ethylene glycol) layers on metal oxide surfaces: Attachment mechanism and effects of polymer architecture on resistance to protein adsorption. J. Phys. Chem. B 2000, 104, 3298− 3309. (33) Uchida, K.; Hoshino, Y.; Tamura, A.; Yoshimoto, K.; Kojima, S.; Yamashita, K.; Yamanaka, I.; Otsuka, H.; Kataoka, K.; Nagasaki, Y. Creation of a mixed polyethylene glycol tethered-chain surface for preventing the nonspecific adsorption of proteins and peptides. Biointerphases 2007, 2, 126−130. (34) Efremova, N. V.; Sheth, S. R.; Leckband, D. E. Protein-induced changes in poly(ethylene glycol) brushes: Molecular weight and temperature dependence. Langmuir 2001, 17, 7628−7636. (35) Halperin, A. Polymer brushes that resist adsorption of model proteins: Design parameters. Langmuir 1999, 15, 2525−2533. (36) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Prevention of protein adsorption by tethered poly(ethylene oxide) layers: Experiments and single-chain mean-field analysis. Langmuir 1998, 14, 176− 186.

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dx.doi.org/10.1021/la4021492 | Langmuir XXXX, XXX, XXX−XXX