Clarification of Protein Adsorption at Polymer Brush Surfaces Based

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Chapter 27

Clarification of Protein Adsorption at Polymer Brush Surfaces Based on Water Structure Surrounding the Surface Yuuki Inoue and Kazuhiko Ishihara* Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan *E-mail: [email protected]

Protein adsorption behavior at the biocompatible polymer brush surfaces was quantitatively investigated based on the water structure surrounding the surface. The high repellency of protein adsorption at the polymer brush surface was analyzed using both quartz crystal microbalance with dissipation method and force-distance curve measurement in atomic force microscopy. The diffusion coefficients of water molecules among micro-silica beads with polymer brush layers were analyzed using nuclear magnetic resonance spectroscopy. These results clearly demonstrated that the dynamics of water molecules determine the protein adsorption behavior at the biocompatible polymer brush surfaces. Thus, polymer brush surface with water molecules with high diffusion coefficients, such as a zwitterionic group-bearing polymer brush surface, was effective in inhibiting protein adsorption.

Biological reactions, such as thrombus formation, immunoresponses, and inflammatory responses, are induced at a material surface when the surface comes into contact with blood or tissues. In many cases, these reactions are initiated by protein adsorption on the surface (1). Biomaterial surfaces therefore require quite strong inhibition of protein adsorption. Several concepts for fabricating biomaterial surfaces have been proposed in order to reduce protein adsorption, such as the introduction of higher polymer chain mobility by poly(ethylene

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


oxide) (PEO) (2–4), the construction of artificial cell membrane structures by phospholipid polymers (5–8), and the fabrication of hydrophilic and highly dense polymer brush layers at the material surfaces (9–17). In particular, the hydrophilic polymer brush surfaces composed of zwitterionic monomer units have extremely higher resistance to protein adsorption than PEO-grafted surface. The underlying mechanism for the repellency of protein adsorption on the PEO-grafted surface has been theoretically and experimentally investigated (18–22). On the other hand, the comparable intermolecular interactions at the several kinds of hydrophilic polymer brush surfaces make it difficult to explain their high repellency of protein adsorption precisely. Water structure surrounding the hydrophilic polymer brush surfaces therefore should be considered in order to understand protein adsorption at a surface and obtain surfaces that do not adsorb proteins. Computer simulation (23, 24) or a lot of experimental data (6, 25–28) have indicated that protein adsorption would be strongly influenced by the hydration state around the surface. For example, Ishihara et al. clearly demonstrated that phospholipid polymer surfaces with a high fraction of free water reduced protein adsorption (6). Tanaka et al. reported that platelet adhesion was suppressed on the surface of poly(2-methoxyethyl acrylate) with bound freezing water, whose mobility is between that of nonfreezing water and that of free water (27). However, the role of water molecules at the protein–material interface is only assumed from information on static and bulk water molecules around the polymer chain. In addition, these studies involved the use of polymer-coated surfaces, which made it difficult to evaluate the water structure because of entanglement of the polymer chains or physical entrapment of water molecules in the polymer network. From these points of view, both surface-specific analysis of the water structure and a well-defined surface structure would be necessary to clarify the relationship between the protein adsorption behavior and water structure at the surface with extremely high biocompatibility. The objective of this study is to quantitatively evaluate the relationship between protein adsorption behavior and water structure at biocompatible materials surfaces. Five kinds of polymer brush layers were prepared on several kinds of material surfaces using surface-initiated atom transfer radical polymerization (SI-ATRP). The surface structure and representative surface properties of the polymer brush layer were characterized well enough to regard the polymer brush structure as a model surface for polymeric biomaterials. The ultralow protein adsorption mass was analyzed using quartz crystal microbalance with dissipation (QCM-D) method to show the high protein adsorption repellency of the polymer brush surfaces. Furthermore, the interaction forces between proteins and polymer brush surfaces were quantitatively analyzed using force–distance curves obtained from atomic force microscopy (AFM) to more clarify the protein adsorption behavior at the polymer brush surfaces. The diffusion coefficients of water molecules among micro-silica beads with polymer brush layers were evaluated using proton nuclear magnetic resonance (1H-NMR) measurements for the surface-specific analysis of the water structure (29, 30). Our study indicated that a surface with water molecules of high mobility would reduce the direct interactions with proteins, leading to extremely high inhibition of protein adsorption. 606 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Experiments


Materials 2-Methacryloyloxyethyl phosphorylcholine (MPC) was synthesized and purified using a previously reported method (5). [2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl) ammonium hydroxide (SBMA) and 2-hydroxyethyl methacrylate (HEMA) were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA). N-Methacryloyloxyethyl-N,N-dimethylammonium α-N-methyl carboxylate (CBMA) was obtained from Osaka Organic Chemical Industry, Ltd. (Osaka, Japan). Trimethyl-2-methacroyloxyethylammonium chloride (TMAEMA) was purchased from the Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Copper(I) bromide (CuBr), 2,2′-bipyridyl (bpy), and ethyl 2-bromoisobutyrate (EBIB) were purchased from the Sigma-Aldrich Co. and were used as received. Silicon wafers were purchased from the Furuuchi Chemical Co. (Tokyo, Japan). Gold sensor substrates for QCM-D measurement were purchased from Q-Sense (Gothenburg, Sweden). Micro-silica beads of diameter 10 μm were purchased from Fuji Silysia Chemicals, Ltd., Aichi, Japan.

Preparation of Initiator-Immobilized Substrates or Silica Beads The SiO2- and gold-attachable initiators for SI-ATRP, 11-(2-bromo-2methylpropionyloxy)undecyltrichlorosilane (BrC10TCS) and 11-(2-bromo-2methylpropionyloxy)undecylmercaptan (BUM), respectively, were synthesized using previously described methods (31, 32). The initiators were immobilized at the surface in a 5.0 mmol/L solution of BrC10TCS in toluene in the case of the silicon wafers and silica beads, and in a 2.5 mmol/L solution of BUM in ethanol in the case of the gold sensor substrates, for 24 h (15). The initiator-immobilized substrates and silica beads were removed from the solution, rinsed with solvents, and dried in a dry box under reduced pressure.

Preparation of Polymer Brush Layers on Initiator-Immobilized Substrates or Silica Beads MPC, SBMA, CBMA, HEMA, and TMAEMA were graft polymerized from the initiator-immobilized substrates or silica beads using SI-ATRP as follows (15). CuBr, bpy, and each monomer, in a specific molar ratio, were placed in a glass tube, and dehydrated and degassed solvents were added to the glass tube. Argon was bubbled into each monomer solution at room temperature for 10 min. The initiator-immobilized substrates or silica beads were then immersed in the solution, and EBIB was simultaneously added as the free-radical initiator at a [monomer]/ [EBIB] ratio (which means the target polymerization degree (DP)) ranging from 10 to 200. After the glass tubes were sealed, polymerization was performed at 20 °C with stirring. After 24 h, the obtained substrates or silica beads were rinsed with solvents, and dried in a dry box under reduced pressure. The structure of the polymer brush surface is shown in Figure 1. 607 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Chemical structures of the polymer brush surface fabricated in this study.

Physicochemical Surface Characterization The elemental composition of the polymer brush surfaces was determined using X-ray photoelectron spectroscopy (XPS; AXIS-Hsi, Shimadzu/Kratos, Kyoto, Japan) with a magnesium anode nonmonochromatic source. The polymer brush layers were prepared on the BrC10TCS-immobilized silicon wafers. High-resolution scans for C1s, N1s, P2p, S2p, and Br3d were acquired at a take-off angle of 90° for the photoelectrons. All the binding energies were referred to the C1s peak at 285.0 eV. The thickness of the grafted polymer layer under a dry condition was determined using a spectroscopic ellipsometer at an incident angle of 70° in the visible region (J. A. Woollam Co., Inc., Tokyo, Japan). The polymer brush layers were prepared on the BrC10TCS-immobilized silicon wafers. The thickness of the grafted polymer layer was estimated using the Cauchy layer model with an assumed refractive index of 1.49 at 632.8 nm. The graft density of the polymer chain in the polymer brush layer [σ (chains/nm2)] was calculated from the equation σ = hρNA/Mn. Here, h is the ellipsometric layer thickness (nm); ρ, the density of each dry polymer [1.30 g/cm3 for poly(MPC) (33), poly(CBMA), and poly(SBMA); 1.15 g/cm3 for poly(HEMA) (9) and poly(TMAEMA)]; NA, Avogadro’s number; and Mn, the absolute molecular weight of the polymer chains 608 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


on the surface. It is reported that the molecular weight of the grafted polymer chains was equal to that of the polymer chains formed in each polymerization solution (34, 35). In this study, the Mn was determined from the following equation, Mn = "Target DP" × "Conversion (%) / 100" × "Molecular weight of each monomer", where, the conversion value was determined from 1H-NMR of each polymerization solution. The surface coverage with each polymer chain was briefly estimated from the calculated graft density and cross-sectional area of each monomer unit. When we estimate the cross-sectional area, we consider a polymer chain as a cylinder and the contour length per monomer unit is set equal to the length of the C-C-C bond (0.25 nm) with the bulk density assumed to be unity (36). The surface morphologies of the polymer brush surfaces were observed using AFM (Nanoscope IIIa, Bruker Japan Co., Ltd., Kanagawa, Japan) operated in the tapping mode. The polymer brush layers were prepared on the BrC10TCS-immobilized silicon wafers. The measurements were performed under a dry condition with a standard cantilever at a scan rate of 1.0 Hz. The root-mean-square (RMS) value of the surface roughness was calculated from the roughness profiles. The Wilhelmy plate method was used to measure the dynamic contact angles in water for the polymer brush surfaces (DCA-100, Orientec Co., Ltd., Tokyo, Japan). Polymer brush layers at the approximate thickness of 5 nm were prepared on BrC10TCS-immobilized glass plates of dimensions 40 × 10 × 1.0 mm3. A value of 72.8 dyn/cm was used as the surface tension of pure water and the moving rate of the crosshead was 3.0 mm/min. The measurement was repeated for 5 cycles for each sample. The ζ-potential measurement was performed using an ELS-800 electrophoretic light-scattering spectrophotometer (Otsuka Electronics, Osaka, Japan) equipped with a plate sample cell to measure the surface potential in water containing 10 mmol/L sodium chloride. The polymer brush layers at the approximate thickness of 5 nm were prepared on the BrC10TCS-immobilized silicon wafer. The ζ-potential measurement for the polymer brush surfaces was performed at room temperature. The measurement was repeated at least three times.

Analysis of Water Structure Around Polymer Brush Surfaces Silica beads with polymer brush layers at the approximate thickness of 5 nm (500 mg) were packed into NMR tubing (ɸ = 10 mm, JEOL, Tokyo, Japan), and distilled water (500 μL) was added. The interspaces among the silica beads are extremely small, therefore we could investigate the water structure in the vicinity of the polymer brush surface (Figure 2) (29, 30). The diffusion coefficients of water molecules around the polymer brush layers were measured at 37 °C by 1HNMR spectroscopy (MU-25, JEOL, Tokyo, Japan) using the pulsed-field gradient method. The diffusion coefficient is assumed to be an indication of the mobility of the water molecules. 609 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2. Schematic representation of water among the silica beads with polymer brush layers for the analysis of the water structure using 1H-NMR.

Biological Analysis on Polymer Brush Surfaces The adsorbed amounts of proteins from 100% fetal bovine serum (FBS) on the polymer brush surfaces were quantified using QCM-D (15, 37). We prepared polymer brush layers on BUM-immobilized QCM gold sensors. The prepared QCM gold sensor was first exposed to phosphate-buffered saline (PBS, pH 7.4) at 37 °C until a stable baseline was established. Thereafter, the QCM sensors were exposed to FBS for 30 min, followed by PBS for an additional 10 min to replace FBS and to wash off the weakly adsorbed FBS from the surface. The change in the oscillator frequency was used to estimate the amount of adsorbed protein, using Sauerbrey’s equation, as follows (38): Amount of adsorbed protein (ng/cm2) = 17.7 × Frequency change at the seventh overtone (Hz). The adsorption force of bovine serum albumin (BSA, Sigma-Aldrich) on the polymer brush layers was estimated using force–distance (f–d) curve measurements in an AFM apparatus. The polymer brush layers at the approximate thickness of 5 nm were prepared on BrC10TCS-immobilized silicon wafers. BSA was covalently immobilized on the cantilever (OTR8 with a spring constant of 0.15 N/m, Bruker Japan Co., Ltd.) through the condensation reaction between the amino groups in the protein and the carboxyl groups on gold-evaporated cantilever (39). The immobilization of BSA on the carboxyl group-terminated self-assembled monolayer on the gold-evaporated surface was confirmed from QCM-D and XPS measurements. The adsorption force of BSA on the polymer brush was evaluated in PBS at room temperature by the deflection shift value from the baseline at the retract trace of the f–d curve. At each measurement, more than 100 approaching/retracting f–d curves were collected, and the average value of the measured forces was defined as the adsorption force of BSA on the surfaces. All measurements were repeated at least three times.

610 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Results and Discussion


Physicochemical Surface Properties In this study, five kinds of polymer brush layers, poly(MPC), poly(SBMA), poly(CBMA), poly(HEMA), and poly(TMAEMA), were prepared on several kinds of materials, such as silicon wafers, glass substrates, gold substrates, and micro-silica beads, using SI-ATRP with a free-radical initiator. The surface elements of the polymer brush surfaces were analyzed using XPS spectra. Specific peaks in the carbon (C1s), nitrogen (N1s), phosphorus (P2p), and sulfur (S2p) atom regions were detected at each polymer brush surface. Thus, XPS analysis confirmed the identities of each polymer chain at all the polymer brush surfaces. The ellipsometric layer thickness obtained for the grafted polymer layers under a dry condition were plotted against the absolute molecular weights of the polymer chains (Figure 3). The thickness of the grafted polymer layer could be linearly controlled in the range 1–20 nm by the molecular weight of the grafted polymer chains. We calculated the graft density of each polymer chain in the polymer brush layers using the slope of the line shown in Figure 3 and the equation described in the Experiments section. The graft densities of the polymer chains in the poly(MPC), poly(SBMA), poly(CBMA), poly(HEMA), and poly(TMAEMA) layers were 0.26, 0.48, 0.67, 0.79, and 0.31 chains/nm2, respectively. The well-defined structure of the polymer brush surface makes it possible to estimate the surface coverage with the grafted polymer chain at the surface using the method mentioned in the Experiments section. The calculated coverage with the grafted poly(MPC), poly(SBMA), poly(CBMA), poly(HEMA), and poly(TMAEMA) chains were 43%, 76%, 82%, 66%, and 37%, respectively. The graft density and coverage with the grafted polymer chains for all the polymer chains were greater than 0.1 chains/nm2 and 30%, respectively, which indicated that highly dense polymer brush surfaces were formed using the SI-ATRP method (40). The surface topology of the polymer brush surface and the RMS value as an indicator of the surface roughness were examined using AFM in a dry condition (12, 15, 39). Each polymer brush surface exhibited a slightly irregular structure, however the RMS value for the polymer brush surface was at most 1.0 nm. This RMS values were consistent with those reported in previous studies (12), indicating that the grafted polymer layers prepared by the SI-ATRP method would be considerably homogeneous. In particular, there was little difference among the RMS values of polymer brush surfaces with nearly equivalent thickness. This result indicated that the differences in surface properties among the polymer brush layers would mainly depend on the characteristics of the grafted polymer chains, not on the surface topology. The wettability of the polymer brush surface were evaluated using dynamic contact angle measurement. The receding contact angle would be more important than the advancing contact angle since biological reactions occur under aqueous conditions. The advancing and receding contact angles for zwitterionic polymer brush surfaces were around and below 20°, respectively, which indicated a high wettability of these surfaces in dry and aqueous conditions (41). The cationic poly(TMAEMA) brush surface also had a high wettability in an aqueous condition 611 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


(the receding contact angle was also below 20°), but the advancing contact angle for the surface was more than 60°. In contrast, the receding contact angle for the nonionic poly(HEMA) brush surface was more than 20°, which indicated that the poly(HEMA) brush surface had slightly low wettability in an aqueous condition. The hydrophilic hydroxyl group is smaller than the other hydrophilic functional groups and the poly(HEMA) synthesized in the polymerization solution was difficult to dissolve in water. This result suggested that the poly(HEMA) brush layer would contain very few water molecules. As a result, the wettability of the poly(HEMA) brush surface would be slightly lower than those of the other polymer brush surfaces in an aqueous condition.

Figure 3. Relationship between the ellipsometric layer thickness at polymer brush surface and molecular weight of free polymer. Open circles; poly(MPC), filled triangles; poly(SBMA), open squares; poly(CBMA), filled diamonds; poly(HEMA), and open triangles; poly(TMAEMA) (Average ± standard error of the mean (SEM), n = 5).

The surface potential of the polymer brush surfaces was evaluated using the ζ-potential measurement in water containing 10 mmol/L sodium chloride at room temperature. The ζ-potentials of zwitterionic and nonionic polymer brush surfaces were slightly negative from -1.8 mV (poly(CBMA) brush surface) to -7.8 mV (poly(SBMA) brush surface). On the other hand, the ζ-potentials of cationic poly(TMAEMA) brush surfaces was 45.0 mV. The surface potentials of the polymer brush surfaces corresponded to the charge property of each grafted polymer chain and there was little difference among the zwitterionic or nonionic polymer brush surfaces. 612 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Protein Adsorption Behavior on Polymer Brush Surfaces

The amounts of proteins adsorbed on the polymer brush surfaces from 100% FBS were quantified using QCM-D. It is known that the amount of adsorbed proteins on the polymer brush surface would decrease with the increasing layer thickness and graft density of polymer brush layer. That is, in the case that the polymer brush surface would have sufficient layer thickness and graft density, protein adsorption would be determined by the properties of grafted polymer chains, such as the charge condition or polymer chain-water interaction in a buffer solution. In this study, while five kinds of polymer brush surfaces have different graft densities according to the molecular size of each monomer unit, all the coverage with the grafted polymer chain was more than 40%. Therefore, it is considered that the there would be little influence of the different graft densities among the polymer brush surfaces on the protein adsorption behavior. The relationship between the amount of adsorbed proteins and the ellipsometric layer thickness is shown in Figure 4. There were large differences in this relationship, depending on the chemical structure of the grafted polymer chain. The amounts of adsorbed proteins on the zwitterionic polymer brush surfaces drastically decreased at thicknesses up to 5 nm, and those on the surfaces with an approximate layer thickness of 10 nm were 17 ng/cm2 for the poly(MPC) brush layer, 31 ng/cm2 for the poly(SBMA) brush layer, and 79 ng/cm2 for the poly(CBMA) brush layer. These results showed that the zwitterionic polymer brush surfaces would have excellent repellency of protein adsorption. In contrast, the amount of adsorbed proteins on the poly(HEMA) brush surface gradually decreased with increasing ellipsometric layer thickness, and that on the surface with an approximate layer thickness of 15 nm was 180 ng/cm2. The amount of adsorbed proteins on the cationic poly(TMAEMA) brush surface increased with increasing ellipsometric layer thickness. These results demonstrated that protein adsorption would depend not only on the layer thickness, but also on the characteristics of the grafted polymer chains. It is considered that the main driving force for protein adsorption on the cationic poly(TMAEMA) brush surface would be the electrostatic interaction between the poly(TMAEMA) brush surface and negatively-charged proteins at a physiological condition existing in FBS. And the density of positive charge at the poly(TMAEMA) brush surface would increase with the increasing ellipsometric layer thickness. Therefore, more proteins adsorbed on the thicker poly(TMAEMA) brush surface. To evaluate the direct interaction between negatively-charged proteins and polymer brush surfaces with different characteristics, the adsorption force of BSA was measured on the polymer brush layers with approximate thickness of 5 nm (Figure 5). The polymer brush layers with approximate thickness of 5 nm have the similar surface properties and the different amounts of adsorbed proteins, therefore, a clear relationship between the protein adsorption behaviors and water structure around the surfaces is expected. The maximum adsorption force of BSA was detected on the cationic poly(TMAEMA) brush surface, which means the adsorbed proteins via the strong electrostatic interaction hardly detach from the surface. This result also suggested that some negatively-charged proteins existing 613 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


in FBS would be denatured at the cationic poly(TMAEMA) brush surface. Additionally, the adsorption force of BSA on the poly(TMAEMA) brush surface was not so large compared to the amount of adsorbed proteins, that is, more proteins adsorbed on the poly(TMAEMA) brush surface than that expected from the adsorption force. On the other hand, the order of the adsorption forces on the other polymer brush surfaces was similar to the adsorbed amount of proteins from 100% FBS. The amounts of proteins adsorbed on the surfaces (poly(TMAEMA): > 1000 ng/cm2, other polymers: < 200 ng/cm2, shown in Figure 4) indicated that multilayer adsorption would occur at the cationic poly(TMAEMA) brush surface, whereas the proteins adsorbed on the other polymer brush surfaces would be below monolayer adsorption (42). This result indicated that there would be a positive relationship between the amount and force of protein adsorption at the surface where proteins adsorb below a monolayer. In particular, this result also indicated that the high repellency of protein adsorption on the zwitterionic polymer brush layers would arise from the extremely low interaction of the surfaces with proteins.

Figure 4. Relationship between frequency shift value (adsorbed amount of proteins) and the ellipsometric layer thickness at the different polymer brush surfaces. Filled circles; initiator-immobilized substrate, open circles; poly(MPC), filled triangles; poly(SBMA), open squares; poly(CBMA), filled diamonds; poly(HEMA), and open triangles; poly(TMAEMA). (Average ± SEM, n = 3).



Figure 5. Adsorption force of bovine serum albumin (BSA) against the different polymer brush surfaces with 5-nm-thick (Average ± SEM, n = 3). Water Structure Around the Polymer Brush Surface Figure 6 shows the diffusion coefficients of water molecules among micro-silica beads with various polymer brush layers. The diffusion coefficient of pure water is known to be 3.0 × 10-5 cm2/sec at 37 °C. On the other hand, the diffusion coefficient of water molecules among the bare micro-silica beads was measured to be 7.5 × 10-5 cm2/sec. We could not apparently explain the reason why the diffusion coefficient of water molecules among bare or polymer brush layer-modified micro-silica beads increased compared to that of pure water, it might be related that water molecules in extremely small space have different characteristics from those in bulk state, such as a higher viscosity and a lower dielectric constant (43). Here, we discuss the diffusion coefficients separately, according to the solubility of the polymer chain in water, because the water structure around the polymer brush layer would differ depending on the polymer chain solubility. Among the polymer brush surfaces with water-soluble polymer chains, the water molecules at zwitterionic polymer brush surfaces had higher diffusion coefficients than those at the cationic polymer brush surface. The water molecules at the poly(MPC) brush surface had a particularly large diffusion coefficient. This result indicated that water molecules near the poly(MPC) brush surface would have high mobility. In contrast, the mobility of water molecules near the cationic poly(TMAEMA) brush surface was restrained. Recently, Takahara et al. reported the solubilizing states and dimensions of polymer chains 615 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


in several polymer brush layers in aqueous media. In this report, the poly(MPC) chains expanded well in the aqueous media and the dimensions of the poly(MPC) chains were not changed by the high ionic strength of the aqueous medium (44). The relatively high diffusion coefficients of the water molecules and well-expanded structure of the poly(MPC) chains in ionic media indicated that water molecules may be bound quite weakly to the poly(MPC) chains. The water molecules at the poly(HEMA) brush surface, which had water-insoluble polymer chains and a relatively low wettability under aqueous conditions, showed higher diffusion coefficients than those at the other polymer brush layers. It is suggested that there would be a few water molecules having small diffusion coefficients as a result of interacting directly with the polymer chains. It was considered that the diffusion coefficients of the water molecules around the poly(HEMA) brush layer would mostly be high.

Figure 6. Diffusion coefficient of water molecules around the different polymer brush surfaces with 5-nm-thick (Average ± SEM, n = 3). To quantitatively analyze the relationship between the protein adsorption behavior and water structure at the polymer brush surface, the adsorbed amounts of proteins and adsorption force of BSA were plotted as a function of the diffusion coefficients of the water molecules, as shown in Figure 7. The adsorption force of BSA (Figure 7b) gradually decreased with increasing diffusion coefficient of the water molecules around the polymer brush surfaces with water-soluble polymer chains, in contrast to the rapid decrease shown in the adsorbed amount of proteins (Figure 7a). Two kinds of molecular interactions, the electrostatic interaction and water structure, would influence on the large adsorption force of BSA at the poly(TMAEMA) brush surface. On the other hand, this result clearly indicated that the high mobility of water molecules around the zwitterionic polymer brush surfaces would result in small interaction with proteins. Proteins are surrounded by hydrating water molecules, and the conformations and activities of the 616 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


proteins are maintained by molecular interactions through these hydrating water molecules. If the network structure of hydrating water molecules around the protein changes, the protein conformation is destroyed. In this regard, a surface with high-mobility water molecules would prevent proteins from detaching from the hydrating water molecules and changing the higher-order structure. As is also seen in Figure 7, a small difference in the diffusion coefficient resulted in a large difference in the protein adsorption mass. We hypothesize that this phenomenon is related to the diffusion range of the hydrating layer at the polymer brush surface, and studies of this are now progressing. The poly(HEMA) brush surface, which would have a different hydration state from that of the other polymer brush surfaces as mentioned above, was not in line with the other results. In the present study, we could not estimate the diffusion coefficient of the water molecules around the poly(HEMA) brush surface accurately. In addition, we could not explain the protein adsorption behavior at three kinds of zwitterionic polymer brush surfaces from the water structure surrounding the surfaces. Further analysis of the water structure around the polymer brush surface is needed to clarify the relationship between protein adsorption behavior and the water structure around the surface.

Figure 7. Relationship between (a) amount of adsorbed proteins and (b) adsorption force of bovine serum albumin (BSA) and diffusion coefficient of water at the different polymer brush surfaces (Average ± SEM, n = 3).

Conclusions We intensively researched protein adsorption behavior, based on the water structures at well-defined and biocompatible polymer brush surfaces. First, we successfully fabricated polymer brush layers with varying chemical structures on several kinds of material surfaces. The well-characterized structures and properties of the polymer brush surfaces enabled us to regard them as model surfaces. The interactions between proteins and polymer brush surfaces were quantitatively analyzed using AFM; the amounts of proteins adsorbed on the surfaces were also determined. The surface-specific water structures were evaluated using the diffusion coefficients of water molecules enclosed 617 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


among micro-silica beads with polymer brush layers. It was clarified that zwitterionic polymer brush surfaces, especially the polymer brush surface with phosphorylcholine groups, had little interaction with proteins, and the water molecules around these surfaces had higher mobility than those at the other water-soluble polymer brush surfaces. These results indicated that the active exchange of water molecules around the surface would reduce direct interactions with proteins, leading to extremely high repellence of protein adsorption. The control of surface polymer brush layers, focusing on the dynamics of water molecules, will lead to zero-protein-adsorption surfaces.

Acknowledgments This study was partially supported by a Core Research for Evolutional Science and Technology (CREST) grant from the Science and Technology Agency, Japan.

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