Mechanism of Resistance to Protein Adsorption on the MPC Polymer Surface

To reduce protein adsorption on polymer surfaces, surface modifications that induce repulsive interactions or reduce attractive interactions have been conducted. It has been known for quite some time that in general, as a surface becomes more hydrophobic, the extent of protein adsorption increases (Elwing et al. 1987; Golander and Pitt 1990; Golander et al. 1990; Lee and Lee 1993). The unusually strong attraction between proteins and hydrophobic surfaces in water has been described by a hydrophobic interaction, which arises primarily from the structural rearrangement of water molecules in the overlapping solvation zones as proteins adsorb to the surface. Thus, the hydrophobic interaction is primarily an entropic phenomenon without any specific associated bond. The orientation of water molecules adjacent to hydrophobic surfaces is entropically unfavorable. Upon adsorption of proteins to hydrophobic surfaces, the entropically unfavorable water is released in bulk, thereby reducing the total free energy. The hydrophobic interaction then results in the strong attraction of proteins toward the hydrophobic surface. To weaken the hydrophobic interaction, hydrophilic polymers [e.g., poly(2-hydroxyethyl methacrylate) (HEMA), poly(acryl amide), poly(vinylpyrrolidone)] have been applied for surface modification. While these hydrophilic polymers could relatively reduce protein adsorption, their use precludes the accurate control of protein adsorption.

Recently, it has been reported that the structure of water absorbed in the polymer materials influences protein adsorption on their surfaces. When protein molecules adsorb to a polymer surface, water molecules between the protein and the surface must be displaced, as shown in Fig. 4 (Lu et al. 1991). A repulsive solvation interaction arises whenever water molecules are associated with a surface that contains hydrophilic groups. Its strength depends on the energy necessary to disrupt the ordered water structure and ultimately dehydrate the surface. Even on a hydrophilic surface, the water structure has a major effect on protein adsorption and subsequent conformational change.

Tsuruta (1996) reported that the random networks of water molecules on the material surface are very important in explaining protein adsorption. Protein adsorption processes are considered to start with protein trapping by the random networks of water molecules on the material surface. The material surface, which cannot undergo hydrogen bonding with water, will then reduce protein adsorption. Table 1 lists the free water concentration in the hydrated polymer membrane with a 0.36 water fraction determined

ApproactVCorrtact -Adsorption -*< Irreversible adsorption

Diffusion Dehydration Denaturetion

Protein

ApproactVCorrtact -Adsorption -*< Irreversible adsorption

Diffusion Dehydration Denaturetion

Protein

Fig. 4. Schematic description of protein adsorption on the polymer surface. Water molecules are moved from contact sites between amino acid residues and the polymer surface. A change in the conformation of the adsorbed protein then occurs and the protein binds to the surface tightly. AF Free energy, AH enthalpy, T absolute temperature, AS entropy

Fig. 4. Schematic description of protein adsorption on the polymer surface. Water molecules are moved from contact sites between amino acid residues and the polymer surface. A change in the conformation of the adsorbed protein then occurs and the protein binds to the surface tightly. AF Free energy, AH enthalpy, T absolute temperature, AS entropy

Table 1. Characteristics of hydration state of polymer. Heq = (weight of water in the polymer membrane)/(weight of polymer membrane saturated with water) at 25 ° C, Poly(HEMA) poly(2-hydroxyethyl methacrylate), PMB poly(MPC-co-n-butyl methacrylate), Number after PMB is molar percent of MPC in the coplymer

Table 1. Characteristics of hydration state of polymer. Heq = (weight of water in the polymer membrane)/(weight of polymer membrane saturated with water) at 25 ° C, Poly(HEMA) poly(2-hydroxyethyl methacrylate), PMB poly(MPC-co-n-butyl methacrylate), Number after PMB is molar percent of MPC in the coplymer

Poly(HEMA)

PMB 10 30

Heq

0.40

0.23 0.84

Free water fraction at Heq

0.34

0.25 0.84

At H = 0.36

0.28

- 0.69

with differential scanning calorimetry (Ishihara et al. 1998). The fraction of free water (not bound water) in the MPC polymer was 0.65, which was found to be significantly higher than that in poly(HEMA), which was 0.28. In addition, the structure and hydrogen bonding of water in the vicinity of poly(MPC-co-butyl methacrylate) (PMB) were analyzed in their aqueous solutions and thin films with contours of O-H stretching of the Raman and attenuated total reflection infrared (ATR-IR) spectra, respectively (Kitano et al. 2000, 2003).

Figure 5 shows the O-H stretching Raman band of water in various polymer solutions recorded in the region between 2500 and 4000/cm by using the polarization method. For the polarization geometries X(ZZ)Y (parallel position, I//) andX(ZX) Y (perpendicular position, I±), a polarizer plate was rotated exactly 90 °C in front of the slit, where X and Y are the directions of the laser beam and observation, respectively.

A depolarization ratio, r, is an indicator of symmetry of the vibration mode, and is expressed by r = I///I±, where I// and I± are the intensities of the spectra observed with the polarizer oriented parallel and perpendicular to the incident laser beam, respectively. The component of the O-H

4000 3500 3000 2500

Wavenumber (cm"1)

4000 3500 3000 2500

Fig. 5. Raman shifts of O-H stretching region. a I// and I± spectra of pure water at 25 °C, where I// and I± are the intensities of the spectra observed with the polarizer oriented parallel and perpendicular to the incident laser beam, respectively. The I±/p spectrum (where p is the depolarization ratio) is shown with a dotted line. b The collective band of water. Reprinted with permission from Kitano et al. (2000), copyright (2000) American Chemical Society

4000 3500 3000 2500

Wavenumber (cm"1)

Fig. 5. Raman shifts of O-H stretching region. a I// and I± spectra of pure water at 25 °C, where I// and I± are the intensities of the spectra observed with the polarizer oriented parallel and perpendicular to the incident laser beam, respectively. The I±/p spectrum (where p is the depolarization ratio) is shown with a dotted line. b The collective band of water. Reprinted with permission from Kitano et al. (2000), copyright (2000) American Chemical Society stretching band of water centered at 3250/cm was highly polarized and diminished in the spectra at the perpendicular position. The polarized O-H stretching band of water, which is called a collective band, is ascribed to an H2O molecule executing u vibrations all in phase with each other but with a vibrational amplitude varying from molecule to molecule in water clusters, which are strongly hydrogen-bonded. Theoretical calculations of a random network model, which is characterized by fluctuating defects in water-water hydrogen bonds in a distorted tetrahedral network, support the interpretation.

To clarify the effect of polymers on the structure of water, the intensity of the collective band (Ic) observed at around 3250/cm was separated from the spectra using Eq. 1 (Fig. 5):

Since the intensities of Raman spectra are not absolute, the area of Ic was normalized as Eq. 2:

where w is the Raman shift in cm-1.

Figure 6 shows the effect of the molecular weight (Mw)of different kinds of polymers on the relative intensities of the collective bands (C) at a constant molar fraction of monomer units (px = 0.01 or 0.05). In the case of aqueous sodium poly(styrenesulfonate) solution, a very strong fluorescence background made the Raman spectroscopic measurements impossible. The values of C for other polymer solutions were almost constant in the region of relatively low Mw, and decreased with an increase in Mw in the region where the Mw values were larger than some critical values. In general, the whole concentration region of polymer solutions can be divided into three parts: dilute, semidilute, and concentrated. The macroscopic properties of polymer solutions in the different concentration regions are

Fig.6. Plots of values of the relative intensities of the collective bands (C) for various aqueous polymer solutions at molar fraction (px) = 0.01 and 25 °C versus molecular weight (Mw). Sodium poly(styrenesulfonate) (open triangles), poly-L-lysine hydrobromide (open circles),poly(2-methacryloyloxyethylphosphorylcholine) [poly(MPC);filledsquares], poly(N-vinylpyrrolidone) (filled triangles), and poly(ethylene glycol) (filled circles; px = 0.05). Reprinted with permission from Kitano et al. (2000), copyright (2000) American Chemical Society

Fig.6. Plots of values of the relative intensities of the collective bands (C) for various aqueous polymer solutions at molar fraction (px) = 0.01 and 25 °C versus molecular weight (Mw). Sodium poly(styrenesulfonate) (open triangles), poly-L-lysine hydrobromide (open circles),poly(2-methacryloyloxyethylphosphorylcholine) [poly(MPC);filledsquares], poly(N-vinylpyrrolidone) (filled triangles), and poly(ethylene glycol) (filled circles; px = 0.05). Reprinted with permission from Kitano et al. (2000), copyright (2000) American Chemical Society significantly altered. The figure seems to show that the structure of water in a dilute polymer solution and in a semidilute polymer solution are different, as discussed below.

The C value for the aqueous solution of poly(MPC)s was very close to that for pure water, as shown in Fig. 6, which is in contrast with the smaller C value in the aqueous solution of ordinary polyelectrolytes. A similar tendency was also observed on hydrated thin polymer films. These results suggest that the PMB does not significantly disturb the hydrogen bonding between the water molecules in either the aqueous solution or the thin film systems.

The equilibrium amount of the proteins bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) adsorbed on the polymer surface was measured and represented with the free water fraction in the hydrated polymers, as shown in Fig. 7 (Ishihara 2000). The amounts of both proteins adsorbed on poly(HEMA), poly(acryl amide (AAm)-co-n-butyl methacrylate) (BMA), and poly(N-vinylpyrrolidone (VPy)-co-BMA) were larger than were those on poly(MPC-co-dodecyl methacrylate) (PMD) and PMB. It was reported that the theoretical amount of BSA and BPF adsorbed on the surface in a monolayer state were 0.9 and 1.7^g/cm, respectively. On the surface of the MPC polymers, the amount of adsorbed proteins was less than these theoretical values.

Fig. 7. Relationship between free water fraction in hydrated polymer membrane and amount of proteins adsorbed on the polymer. Concentration of bovine serum albumin ([BSA]) in phosphate-buffered saline (PBS) = 0.45 g/dl (squares); concentration of bovine plasma fibrinogen ([BPF]) = 0.30 g/dl (circles). Poly(HEMA) Poly(2-hydroxyethyl methacrylate), Poly(AAm-co-BMA) poly(acryl amide-co-«-butyl methacrylate), Poly(VPy-c-BMA) poly(N-vinylpyrrolidone-co-butyl methacrylate), PMD poly(MPC-co-dodecyl methacrylate), PMB poly(MPC-co-butyl methacrylate)

Fig. 7. Relationship between free water fraction in hydrated polymer membrane and amount of proteins adsorbed on the polymer. Concentration of bovine serum albumin ([BSA]) in phosphate-buffered saline (PBS) = 0.45 g/dl (squares); concentration of bovine plasma fibrinogen ([BPF]) = 0.30 g/dl (circles). Poly(HEMA) Poly(2-hydroxyethyl methacrylate), Poly(AAm-co-BMA) poly(acryl amide-co-«-butyl methacrylate), Poly(VPy-c-BMA) poly(N-vinylpyrrolidone-co-butyl methacrylate), PMD poly(MPC-co-dodecyl methacrylate), PMB poly(MPC-co-butyl methacrylate)

Electrostatic interactions between proteins and solid surfaces have also been discussed. Almost all proteins in plasma are negatively charged under physiological conditions because their isoelectric points are below pH 7.4. Therefore, plasma proteins adsorbed favorably onto positively charged surfaces. Holmlin and coworkers (2001) compared surface ability to resist nonspecific protein adsorption on zwitterionic SAMs, referring both to SAMs formed from a 1:1 mixture of positively and negatively charged thiols and thiols combined in a positively and negatively charged moiety in the same molecule. As shown in Fig. 8, the amount of adsorbed protein on zwit-terionic SAMs was much less than that on positively charged or negatively charged SAMs. Moreover, single-component SAMs formed from thiols ter-

Fig. 8. Plots of the change in response unit (ARU) or irreversible adsorption of fibrinogen (a) and lysozyme (b) to different self-assembled monolayers (SAMs) as a function of the ionic strength of the buffer dissolving the protein. The buffer was 4.4 mM phosphate (pH 7.4, ionic strength 10 mM); the ionic strength was adjusted by dissolving NaCl in the appropriate concentrations. The symbols corresponding to the different functional groups presented at the SAM-buffer interface are defined above the plots. Reprinted with permission from Holmlin et al. (2001), copyright (2001) American Chemical Society

Fig. 8. Plots of the change in response unit (ARU) or irreversible adsorption of fibrinogen (a) and lysozyme (b) to different self-assembled monolayers (SAMs) as a function of the ionic strength of the buffer dissolving the protein. The buffer was 4.4 mM phosphate (pH 7.4, ionic strength 10 mM); the ionic strength was adjusted by dissolving NaCl in the appropriate concentrations. The symbols corresponding to the different functional groups presented at the SAM-buffer interface are defined above the plots. Reprinted with permission from Holmlin et al. (2001), copyright (2001) American Chemical Society minating in groups combining a positively charged moiety and a negatively charged moiety were capable of resisting the adsorption of proteins.

Poly(ethylene oxide) (PEO) is one of the most widely used hydrophilic polymers for surface modification of biomaterials (Harris 1992). The high water solubility of PEO is the result of a good structural fit between water molecules and the polymer. Steric repulsion and molecular flexibility are believed to be the dominant factors for reduction protein adsorption on PEO-immobilized surfaces. However, surface modification with few highly dense ethylene oxide oligomers is also effective in reducing protein adsorption (Johnston et al. 2005; Wu et al. 2000). The length of such oligomers is on the order of 1 nm. These results suggest that steric repulsion is not the dominating factor in the prevention of protein adsorption by surface modification. A similar behavior can be seen on a phosphorylcholine surface. In previous literature on MPC copolymer systems, chain mobility was thought to be the dominant factor in resisting protein adsorption onto a surface. Conversely, high-density MPC polymer brushes also reduced protein adsorption. The molecular mobility of a polymer brush, which is defined by the hysteresis between the contact angles of advancing and receding water, was much lower than that of the MPC copolymer.

The surface characteristics of a phosphorylcholine polymer surface for the reduction of protein adsorption are summarized in Fig. 9. The phos-

OCH2CH2OPOCH2CH2N+(CH3) Ó-

OCH2CH2OPOCH2CH2N+(CH3) Ó-

Phosphorylcholine

Neutral charge

Fig. 9. Possible factors for the nonfouling property of phosphorylcholine-bearing surfaces. Z-potential Charge potential phorylcholine group is very hydrophilic, which means it is unfavorable for attracting hydrophobic interactions with proteins. Moreover, the phospho-rylcholine polymer does not disintegrate the water structure around the polymer. This is a unique property of phosphorylcholine polymers in comparison with conventional hydrophilic polymers. In addition, electrostatic interactions between a phosphorylcholine polymer surface and proteins are weak because the charge potential (^-potential) of phosphorylcholine is neutral (Ishihara et al. 1994a). Therefore, phosphorylcholine polymer has several factors for reducing nonspecific protein adsorption. This polymer is one of the best polymer materials for making a nonbiofouling surface.

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