The Reduced Adsorption of Proteins at the Phosphoryl Choline

Hai Xu, Xiubo Zhao, Colin Grant, and Jian R. Lu , David E. Williams , Jeff Penfold .... Stuart Clarke, Martyn C. Davies, Clive J. Roberts, Saul J. B. ...
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Langmuir 1999, 15, 1313-1322

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The Reduced Adsorption of Proteins at the Phosphoryl Choline Incorporated Polymer-Water Interface E. F. Murphy and J. R. Lu* Department of Chemistry, University of Surrey, Guildford GU2 5XH, U.K.

J. Brewer and J. Russell Biocompatibles Ltd, Farnham Business Park, Weydon Lane, Farnham, GU9 8QL, U.K.

J. Penfold ISIS Neutron Facility, Rutherford Appleton Laboratory, CLRC, Chilton, Didcot OX11 0QX, U.K. Received September 28, 1998. In Final Form: November 23, 1998 The adsorption of lysozyme and bovine serum albumin (BSA) onto the surface of a hydrogel polymer has been characterized by neutron reflection and spectroscopic ellipsometry. The polymeric hydrogel was synthesized by copolymerizing methacrylate monomers bearing dodecyl chains and phosphorylcholine (PC) groups. The polymer surface was formed by dip-coating a thin layer of the polymer onto the polished surface of silicon oxide. While ellipsometric measurements at the solid-water interface showed that the polymer film could be represented by a uniform layer of 55 ( 5 Å, the subsequent measurements from neutron reflection suggested that the structural profile of the polymeric film was better described by a three layer model: the inner layer of 30 ( 3 Å mixed with less than 20% water, the middle layer of 20 ( 5 Å mixed with approximately 40% water, and the outermost layer of 25 ( 3 Å mixed with approximately 85% water. The neutron results thus suggest the uneven swelling of the polymer film along the direction normal to the interface. The adsorption of lysozyme and BSA onto the polymer surface was measured over a wide protein concentration range. It was found that the amount of proteins adsorbed on the coated polymer surface was substantially lower than that at the bare silica-water interface under the same solution condition. Thus, the amount of BSA adsorbed onto the polymer-coated surface at pH 5 and at 0.05 g dm-3 was found to be less than 0.5 mg m-2, as compared with 2 mg m-2 obtained at the bare silica-water interface. In addition, the amount of lysozyme adsorbed onto the coated surface at pH 7 and at 1 g dm-3 was also found to be about 0.5 mg m-2, as compared with 3.5 mg m-2 at the silica-water interface. The reduction in protein adsorption was attributed to the presence of a PC layer preferably formed on the outer surface of the coated polymer film.

Introduction Adsorption of proteins from bodily fluids onto medical devices is a well-known interfacial phenomenon. The deposition of blood proteins usually stimulates the attachment of fibrous and antibiotic moieties, leading to subsequent biological responses. Further examples of protein adsorption are the build-up of foulant layers onto food containers and the blockage of filtration membranes in bioseparation.1 The extent of protein adsorption is determined by the delicate balance of the interaction between the protein molecules and the solid substrate. Although hydrophobic and electrostatic interactions are often regarded as the most important driving forces for surface adsorption, the relative significance of these interactions in a given system depends on the details of the protein structure and the particular surface involved. The variety of the interactions and the difference in size, shape, and flexibility of protein molecules have made it difficult to rationalize the behavior of proteins at interfaces. The nature of the solid surface, including its hydrophobicity, charge type, and density, * All correspondence should be addressed to Dr. J. R. Lu, Department of Chemistry, University of Surrey, Guildford GU2 5XH, U.K. (1) Horbett, T. A.; Brash, J. L. Protein at Interfaces II; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995.

also has a strong influence on the structure and conformation of the protein layer. For example, a hydrophobic surface often induces exposure of the hydrophobic fragments within a globular protein, leading to irreversible adsorption.2-5 Even at a hydrophilic surface, where the interaction is entropically driven, the adsorption may also be irreversible because the accumulated number of direct contacts between protein fragments and the surface may be too large to allow desorption.1 It has been known for many years that coating surfaces with hydrophilic polymers can reduce protein adsorption.4,6 Various polymers including poly(vinyl chloride), poly(ethylene oxide), poly(etherurethane), poly(dimethylsiloxane), and poly(tetrafluoroethylene) were found to render varying degrees of reduction in protein adsorption. However, early work has shown that most of these polymers have poor blood compatibility and anti-thrombogenicity. The extent of their effectiveness in reducing protein deposition is therefore very limited. Various new polymeric materials offering improved blood biocompat(2) Haynes, C. A.; Norde, W. Colloids Surf., B: Biointerfaces 1994, 2, 517. (3) Prime, K.; Whitesides, G. M Science 1991, 252, 1164. (4) Iwasaki, Y.; Fujike, A.; Kurita, K.; Ishihara, K.; Nakabayashi, N. J. Biomater. Sci., Polym. Ed. 1996, 8, 91. (5) Feng, L.; Andrade, J. D. J. Biomed. Mater. Res. 1994, 28, 735. (6) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers; Plenum: New York, 1988; Vol. 2.

10.1021/la9813580 CCC: $18.00 © 1999 American Chemical Society Published on Web 01/22/1999

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ibility have recently been developed.4,7,8 One such polymer is that used in this study, termed PC100A. This polymer is mainly made up of a polymethacrylate backbone with pendant alkyl chains and phosphorylcholine (PC) groups. Phosphorylcholine is the headgroup of phosphatidylcholine, the major component of the extracellular side of cell membranes. The concept of surface biocompatibility has evolved from the study of cell membranes. Cell walls are composed of various amphiphilic molecules. Proteins in the bloodstream do not foul onto the surface of cells, suggesting that their outer surface is biocompatible. At the early stage, it was thought that biocompatibility was provided by the whole phospholipid molecules in the extracellular side of cell membranes. A great deal of effort was thereby made to immobilize phospholipid molecules onto solid substrates to reproduce the effect of biocompatibility.9 It was later suggested by Hayward et al.7 that it was the phosphorylcholine headgroup, instead of the whole molecule of phosphatidylcholine, that was responsible for the blood compatibility. This improved understanding led to the elevated activities in utilizing this concept in creating biocompatible surfaces. A significant amount of work has recently focused on the development of polymers incorporating PC groups, although some studies have also been made on the coating of self-assembled monolayers containing PC groups on the outer surfaces.7,10-12 Despite the rapid advances in the synthesis and application of PC-containing polymers, few direct studies have been made to understand the nature of the interaction between proteins and the coated surfaces. Several in vitro studies have been made to test the response of blood samples when in contact with a PC polymer coated surface, but the complexity of blood samples has made it impossible to extract any reliable information about the fate of proteins on the surface.4,6 In this work we use specular neutron reflection and ellipsometry to examine the adsorption of two model proteins, lysozyme and bovine serum albumin (BSA), onto the PC polymer surface. Since the adsorption of the two proteins at several model solidwater interfaces has already been investigated, the results from this work will enable a direct comparison to be made. Experimental Section Ellipsometric measurements were made on the Woollam Variable Angle Spectroscopic Ellipsometer 32 (WVASE32) manufactured by JA Woollam Co, Inc. Measurements were made over a wavelength range of 350-800 nm. At the air-solid interface, measurements were made at the incidence angles of 70° and 75°, and at the solid-water interface, measurements were made at 75° only. The incoming ellipsoidal beam entered the water solution through a glass window and was reflected from the solidwater interface and exited from the opposite end of the glass window. Thin glass slides were used as windows for the solid-liquid experiments, and the effects on the incoming and exiting beams were calibrated at the beginning of the measurements. (7) Hayward, J.; Chapman, D. Biomaterials 1984, 5, 135. (8) Yianni, Y. P. In Structural and Dynamic Properties of Lipids and Membranes; Quinn, P. J., Cherry, R. J., Eds.; Portland Press: New York, 1992. (9) Albrecht, O.; Johnston, D. S.; Villaverde, C.; Chapman, D. Biochim. Biophys. Acta 1982, 687, 165. (10) Hayward, J. A.; Durrani, A. A.; Shelton, C.; Lee, D. C.; Chapman, D. Biomaterials 1986, 7, 126. (11) Jia, C.; Haines, A. H. J. Chem. Soc., Perkin Trans. 1 1993, 2521. (12) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.; Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600.

Murphy et al.

Neutron reflection measurements were made on the white beam reflectometer CRISP at the RutherfordAppleton Laboratory13 using neutrons with wavelengths from 0.5 to 6.5 Å. The sample cell used was almost identical to that depicted by Fragneto et al. in ref 14, with the aqueous solution contained in a Teflon trough clamped against a silicon block of dimensions 12.5 × 5 × 2.5 cm3. The collimated beam entered the end of the silicon block at a fixed angle, was reflected at a glancing angle from the solid-water interface, and exited from the opposite end of the silicon block. Each reflectivity profile was measured at three different glancing angles, 0.35°, 0.8°, and 1.8°, and the results were combined. The beam intensity was calibrated with respect to the intensity below the critical angle for total reflection at the silicon-D2O interface. A flat background determined by extrapolation to high values of momentum transfer, κ (κ ) 4π sin θ)/λ) where λ is the wavelength and θ is the glancing angle of incidence, was subtracted. For all of the measurements, the reflectivity profiles were essentially flat at κ > 0.2 Å-1, although the limiting signal at this point was dependent on the H2O/D2O ratio. The typical background for D2O runs was found to be 2 × 10-6 and that for H2O to be 3.5 × 10-6 (measured in terms of the reflectivity). The large (111) face of the silicon block was polished using an Engis polishing machine. The block was lapped on a copper plate with 3 µm diamond polishing fluid and on a pad with 1 µm diamond fluid followed by 0.1 µm alumina fluid. The freshly polished surface was immersed in neutral Decon solution (5%) and ultrasonically cleaned for 30 min, and this was followed by a further 30 min of ultrasonic cleaning in water. The block was then copiously rinsed and soaked in acid peroxide solution (600 mL of 98% H2SO4 in 100 mL of 25% H2O2) for 6 min at 120 °C.15 The block was then thoroughly rinsed with ultrapure water to remove acid. It was then exposed to UV/ozone for 30 min to remove any further traces of organic impurities,16 and afterward, the surface was rehydrated by immersing in ultrapure water for at least 24 h. This procedure was found to produce smooth surfaces with the thickness of the oxide layer around 20 ( 5 Å, which was completely wetted by water. Silicon wafers (111) were also used in the ellipsometric measurements. They were cleaned in the same manner as the large block polished by ourselves. The final surface hydrophilicity is identical to the large neutron block as indicated by the amount of lysozyme adsorbed at 1 g dm-3. The thin polymer film was coated onto the silicon oxide surface by dipping the freshly cleaned block or wafer into a polymer solution. To reduce the possible effect of mechanical vibration, the whole dipping device was set on an anti-vibration table. The silicon block was attached in a vertical position to the end of the rod on the pulley system, and the coating was carried out by dipping the block into the polymer solution and then lifting it out. Parameters such as the motor speed, solvent composition, and polymer concentration were found to affect the smoothness and thickness of the coated film.17 The film (13) Penfold, J.; Richardson, R. M.; Zarbakhsh, A.; Webster, J. W. P.; Bucknall, D. G.; Rennie, A. R.; Jones, R. A. L.; Cosgrove, T.; Thomas, R. K.; Higgins, J. S.; Fletcher, P. D. I.; Dickinson, E. J.; Roser, S. J.; McLure, I. A.; Hillman, A. R.; Richards, R. W.; Staples, E. J.; Burgess, A. N.; Simister, E. A.; White, J. W. J. Chem. Soc. Far. Trans. 1997, 93, 3899. (14) Fragneto, G.; Lu, J. R.; McDermott, D. C.; Thomas, R. K.; Rennie, A. R.; Gallagher, P. D.; Satija, S. K. Langmuir 1996, 12, 477. (15) Brzoska, J. B.; Shahidzadeh, N.; Rondelez, F. Nature 1992, 360, 719. (16) Vig, J. R. J. Vac. Sci. Technol. 1985, A3, 1027. (17) Murphy, E. F.; Lu, J. R.; Keddie, J.; Brewer, J.; Russell, J. C. Biomaterials, submitted.

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detergent (Decon 90) followed by repeated washing in UHQ water. Theory of Ellipsometry Ellipsometry is an optical technique which measures the change in the polarization of light due to reflection from a thin film or substance on the reflecting surface. The change in amplitude and phase of polarization of the light upon reflection offers information about the structural composition of the interface and is determined in two components: in the plane of the reflection (the “p” plane) and perpendicular to this plane (the “s” plane). The ratio of the sample’s Fresnel reflection coefficients, Rp and Rs (corresponding to the p and s planes, respectively) defines the ellipticity, σ (a complex number). This ratio is related to the two ellipsometric angles, Ψ and ∆, by the following equation:19

Figure 1. Molecular structure of the copolymer.

used in this work was coated at 0.1 wt % polymer in the mixed solvent of ethanol and hexane (50 vol %) and the motor speed was controlled at 5 mm/s. These combined coating conditions resulted in a film thickness of 50 ( 10 Å. The coated surface was subsequently annealed at 160 °C under vacuum for 3 h. Because the glass transition of the polymer was around 130 °C, annealing was found to improve the smoothness of the coated film. The established procedure offered reproducible films on the solid surface, and, for the conditions described above, the difference in layer thickness was usually within 5 Å. The polymer used in this work was a methacryloylphosphorylcholine (MPC)/lauryl methacrylate (LM) copolymer (PC100A). The polymer was synthesized by Biocompatibles Ltd, and the synthetic procedure was outlined in ref 18. The final structure of the polymer was in the form of a methacrylate backbone grafted with dodecyl chains and phosphorylcholine headgroups. Figure 1 depicts the molecular structure of the polymer. The inclusion of a certain volume fraction of dodecyl side chains provided strong texture to the coated film. For the polymer used in this work m:n ) 1:2. Lysozyme from chicken egg white (Sigma, 99%+, Cat. No. L6876) was used as supplied. Its isoelectric point was around 11. The globular structure of lysozyme is ellipsoidal and has an approximate dimension of 30 × 30 × 45 Å3. Fatty acid free BSA (Sigma, Cat. No. A0281) was also used as supplied, and its isoelectric point was about 4.8. The globular dimension is about 40 × 40 × 140 Å3. Adsorption measurements were made at pH 7 for lysozyme and at pH 5 for BSA, all at 298 K. The solution pH was adjusted using phosphate buffer (Na2HPO4 and NaH2PO4), and the total ionic strength at each pH was fixed at 0.02 M. There were small differences in the pH between H2O and D2O solutions, but these were adjusted to be the same within an accuracy of 0.2 pH units. High purity Elgastat water (UHQ) was used throughout the work, and its surface tension was typically over 71 mN m-1 at 298 K, indicating the absence of any surface-active impurity. D2O was purchased from Florochem and was used as supplied. Its surface tension at 298 K was constant at 71.5 mN m-1. The glassware and Teflon troughs for the reflection measurements were cleaned using alkaline (18) Campbell, E. J.; O’Byrne, V.; Stratford, P. W.; Quirk, I.; Vick, T. A.; Wiles, M. C.; Yianni, Y. P. ASAIO J. 1994, 40 (3), M853.

σ)

Rp ) tan Ψ exp(i∆) Rs

(1)

In a spectroscopic experiment, the data is usually reported in terms of the parameters of Ψ and ∆ as a function of wavelength at a given incidence angle. Structural information with respect to surface normal direction can be obtained by fitting refractive index profiles to Ψ and ∆ simultaneously using the optical matrix method. In this work, the data was analyzed using the WVASE data fitting program operated in Windows 95. Neutron Reflection In a neutron reflection measurement, the intensities of the incoming and exiting beams are measured and the ratio gives the “reflectivity”. Neutron reflectivity is usually expressed as a function of scattering length density, F(z), which is related to the refractive index, n, by20

n2 ) 1 -

λ2 F π

(2)

For thermal neutrons, the refractive index differs from unity only by parts per million, and, as a consequence, neutron reflectivity is only measurable at the low angles of grazing incidence. The scattering length density is related to the chemical composition through the equation

F)

∑mibi

(3)

where mi is the number density of element i and bi is its scattering amplitude (scattering length). Because values of bi vary from isotope to isotope, isotopic substitution can be used to produce different reflectivities from a given chemical structure, and this can be a great help in revealing the structural details of an interface. This is particularly the case for systems containing hydrogen atoms. For example, the scattering lengths of D and H are of opposite sign, and, hence, the scattering length density of water can be varied over a wide range, which can be used to highlight an adsorbed protein layer in different ways. This technique is commonly called contrast variation. Thus, when the solution is made from water consisting of approximately 1 mol of D2O and 2 mol of H2O, which has a scattering length density close to that of silicon, the specular reflectivity is largely caused by the (19) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarised Light; North-Holland: Amsterdam, 1977. (20) Penfold, J.; Thomas, R. K. J. Phys. Condens. Matter. 1990, 2, 1369.

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coated polymer layer. Under this circumstance, neglecting the small contribution from the oxide layer on the silicon and assuming a model of a uniform layer distribution for the coated film, the amount of polymer in the layer (surface excess), Γ, can be deduced directly from the derived scattering length density F and thickness of the layer τ using

Γ)

Fτ pibi

∑

NA

(4)

where NA is Avogadro’s constant, pi is the number of ith atom, and pi and bi depend on the known formula of the polymer. The volume fraction of the polymer in the layer can be calculated using the following equation:

F ) Fpxp + Fw(1 - xp)

(5)

where Fp and Fw are the scattering length densities for the polymer and water and xp is the volume fraction of the polymer in the layer. Although in principle Fourier transformation could be used to determine the interfacial scattering length density profile, in practice, the procedure for extracting structural information from experimental profiles is usually done by fitting models to the data based on the optical matrix method. A structural model is assumed, the corresponding reflectivity is calculated using the optical matrix formula,21 the calculated reflectivity is compared with the measured one, and the structural parameters are modified in a leastsquares iteration. The parameters used in the calculation are the thicknesses of the layers, τi, and the corresponding scattering length densities, Fi. Since the scattering length density of a given layer varies with isotopic composition, the fitting of a set of isotopic compositions to a single structural model greatly reduces the possibility of ambiguity in the interpretation, although it adds to the complexity of the fitting procedure. Results and Discussion (A) Ellipsometric Measurements. Although the native oxide layer present on the surface of silicon block is very thin, it contributes to the signal of ellipsometric measurements. The thickness of the oxide layer was characterized by ellipsometry before the polymeric thin layer was coated. The measurements were made at the air-solid interface over the wavelength range of 350800 nm at the incidence angles of 70° and 75°. The simultaneous fitting to the profiles of Ψ and ∆ at the two incidence angles using a uniform layer model gave a thickness of 24 ( 5 Å. The refractive index for the oxide layer was taken to be the same as the bulk value, an assumption which will be justified later. Repeated measurements were performed on five other different spots on the same oxide surface, which produced the same layer thickness within the quoted error, suggesting that the bare oxide surface is smooth. No roughness was used in the fitting. A similar measuring procedure was subsequently used to determine the thickness of the coated polymer layer. Figure 2 shows the comparison of Ψ and ∆, measured at the air-solid interface, before and after coating the polymer layer. The data were plotted as a function of wavelength at the incidence angle of 75°. It can be seen that the coating of the polymer layer has resulted in the increase in the level of Ψ and ∆ over a large wavelength (21) Born, M.; Wolf, E. Principles of Optics; Pergamon: Oxford, 1970.

Figure 2. Plots of Ψ (a) and ∆ (b) as a function of λ at the PC polymer coated solid-air interface before (4) and after (O) coating a layer of the polymeric film. The measurements are shown for the incidence angle of 75° with respect to the surface normal and at 25 °C. The continuous lines were calculated using the optical matrix formula assuming an oxide layer thickness of 24 ( 3 Å and a polymeric film of 53 ( 5 Å.

range. The continuous lines through the profiles in Figure 2 were calculated using a thickness of 24 ( 5 Å for the oxide layer and 53 ( 5 Å for the polymer film. The uniformity of the coated film was characterized by repeating the same measurements over six different spots on the same surface over an area of 3 × 8 cm2 in the central area of the coated surface. It was found that the thickness of the polymer layer was constant at 53 ( 5 Å, suggesting that the film was reasonably uniform. It should be noted that, in fitting the measured profiles, the refractive indices for the oxide layer and the thin polymer film were taken to be the same as their bulk values; that is, no voids or cavities within the layers. It is difficult to justify this assumption from the elliptical data alone because when the layer is so thin, the thickness and refractive index are interrelated. If both thickness and refractive index were allowed to vary simultaneously in the fitting, unreasonable values of refractive indices were obtained. If, however, one of the parameters was fixed based on prior knowledge, the fitting led to a more reliable measurement of the other. PC100A contains a significant fraction of PC and pendant lauryl groups. It is thus expected to have the typical behavior of swelling with time like many other hydrogel polymers reported in the literature. The swelling is therefore a direct consequence of water penetration into the thin film. The expansion of the layer with time was monitored by following the possible change in elliptical signal with time. The measurements were made over the wavelength range of 300-700 nm, and each single scan was done in less than 2 min. A slight change in elliptical signal was observed within the first 30 min, which corresponded to a thickness increment of approximately 3-4 Å, which was nevertheless within the quoted error range. All of the subsequent ellipsometric measurements involving proteins were made after the coated surface was equilibrated with an aqueous solution to avoid the possible complication from the slight swelling of the coated film. The ellipsometric profiles of Ψ and ∆ at the polymer-

Reduced Adsorption of Proteins

Figure 3. Plots of Ψ (a) and ∆ (b) as a function of λ at the PC polymer coated solid-water interface before (4) and after (O) coating a layer of the polymeric film. The measurements were made at the incidence angle of 75° with respect to the surface normal and at 25 °C. The continuous lines were calculated using the optical matrix formula assuming an oxide layer thickness of 25 ( 3 Å and the thickness of polymeric film of 55 ( 5 Å.

water interface are shown in Figure 3, and the profiles corresponding to the bare oxide-water interface are also given for comparison. The difference between Ψ and ∆ in the presence and absence of the polymeric film is again quite large, indicating that the elliptical signal at the solid-water interface is also very sensitive to the structural dimension of the coated polymeric film. Quantitative information about the thickness of the film can be obtained from model fitting as described previously for the airsolid interface. The coupling between the thickness and refractive index was also found to be substantial at the solid-water interface, and we have taken the refractive index for the layer to be the same as the corresponding bulk polymer, that is, n ) 1.5. If the whole polymeric film is taken to be uniform, its thickness is calculated to be 55 ( 5 Å. If, however, the refractive index is taken to be 1.45, an equally good fit can be obtained, and the thickness of the polymer layer is subsequently 70 ( 5 Å. This observation suggests that, based on the elliptical results alone, layer thickness and composition cannot be disentangled reliably. Despite this, the good fits to Ψ and ∆ shown in Figure 3 suggest that the polymer layer can be approximated to a uniform layer distribution. Although the coating of the polymer layer has resulted in the increase in the level of Ψ, the extent of increase varies drastically with wavelength, with the largest increase occurring around 430 nm. A similar trend of increase in ∆ can also be seen in Figure 3 over most of the wavelength range, but some extent of decrease is also noticeable below 450 nm. This observation suggests that, for the silicon substrate, the sensitive variation in elliptical signal for the measurements at the silica-water interface occurs between 350 and 500 nm. It should be noted that at the wavelength of 632 nm, which is the wavelength of the laser source for most conventional ellipsometers, the difference in the signal becomes relatively small. Since the appropriate range in wavelength and angle tends to vary with the type of substrates, spectroscopic ellipsometry

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Figure 4. Plots of Ψ (a) and ∆ (b) as a function of λ at the PC polymer coated solid-water interface after adsorption of lysozyme onto the polymer film at 4 g dm-3 (O). The profiles obtained at the coated polymer-solution interface (4) are shown for comparison. The elliptical signals from 1 g dm-3 almost overlap with those at the coated polymer-water interface, suggesting that there is negligible lysozyme adsorption. The fitting to the profiles at 4 g dm-3 produces a surface excess of 1.3 ( 0.2 mg m-2. The total ionic strength of the phosphate buffer was fixed at 0.02 M. The measurements were made at the incidence angle of 75° with respect to the surface normal and at 25 °C.

has the advantageous flexibility in performing the measurements in the sensitive wavelength and angle regions for different solid-solution interfaces. The level of protein adsorption on the biocompatible polymer surface can be easily detected by comparing the elliptical profiles in the presence and absence of protein. Figure 4 shows the adsorption of lysozyme at pH 7 onto the coated polymer surface at the bulk concentration of 4 g dm-3, and the results are again presented in terms of Ψ versus λ (Figure 4a) and ∆ versus λ (Figure 4b). It should be noted that adsorption at 1 g dm-3 was also measured, but as there was a very small change in the profile from that in pure buffer, hence a very small amount of adsorption, the curve was not shown for clarity. The surface excess in this case was just above the range of error and was not expected to be greater than 0.5 mg m-2. It can be seen from Figure 4 that adsorption of lysozyme results in a slight increase in Ψ and ∆ from those corresponding to the pure D2O; however, the level of increment is wavelength-dependent and the overall change is small. Model fitting was made by assuming that the structural profiles for the oxide and polymer layers were the same as before protein adsorption. To reduce the coupling effect between thickness and refractive index, the thickness of the lysozyme layer was taken to be the same as one of its axial lengths. For example, if the lysozyme layer was assumed to be 30 Å thick at 4 g dm-3, equivalent to the sideways-on adsorption, the refractive index was fitted to be 1.41. The quality of the fits can be assessed by comparing the calculated and measured results shown in Figure 4. Alternatively, it could be argued that the thickness of the adsorbed protein layer was equivalent to its long axial length of 45 Å because of either its preferred orientation or the unevenness of the outer polymer surface. The corresponding refractive index was

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Figure 5. Neutron reflectivity measured from the PC polymer coated solid-D2O interface. The continuous line through neutron reflectivity was fitted with a three-layer model, and the corresponding polymer volume fraction distribution is inserted in the diagram. The dotted reflectivity profile was calculated using the uniform layer model of 55 Å obtained from the fitting to the ellipsometric data at the same interface.

then calculated to be 1.38. It is interesting to comment that an increment in layer thickness results in a small decrease in n, and the product of the two is virtually constant. Indeed, the amount of the adsorbed lysozyme calculated from both sets of data was found to be 1.3 ( 0.3 mg m-2. That the calculated Ψ and ∆ profiles are virtually identical at the two different layer thicknesses suggests that the measurements are insensitive to the layer thickness but are sensitive enough to detect the adsorbed amount. The adsorption of BSA to the polymer surface was also measured by ellipsometry at pH 5, close to its isoelectric point. At 0.05 g dm-3 the reflectivity profile did not show much change from that of the buffered water, indicating negligible adsorption. However, at a bulk concentration of 2 g dm-3, the profiles did change by a small amount, suggesting a slightly increased surface excess. If the BSA layer was taken to be 50 Å thick, the refractive index was fitted to be 1.36. The amount of adsorbed BSA was then calculated to be 1.0 ( 0.3 mg m-2. It was also found that, although the thickness could be varied to some extent, the total surface excess for BSA was within the quoted error unchanged. (B) Neutron Measurements. The structural composition of the bare and coated surfaces was also characterized by neutron reflection at the solid-water interface before any adsorption measurement was made. Neutron measurements were first done to determine the structure of the native oxide layer and its degree of hydration. The use of H2O and D2O can vary the scattering length density of the layer and thus serves to highlight the interfacial composition. All the reflectivity profiles under D2O, CM4 (D2O:H2O = 2:1), and CMSi (D2O:H2O = 1:2) were fitted using a thickness of 20 ( 3 Å and scattering length of 3.4 × 10-6 Å-2 for the oxide layer, suggesting that there is a negligible amount of water in the oxide layer. The thickness obtained is reasonably consistent with that of 24 Å from ellipsometric measurements. The coated surface was also measured under different water contrasts: D2O, H2O, and CMSi (F) 2.07 × 10-6 Å-2), and the resultant reflectivity profile in D2O is shown in Figure 5. The straightforward procedure for fitting these reflectivity profiles is to use uniform layer models similar

Murphy et al.

Figure 6. Neutron reflectivity measured from the PC polymer coated solid-D2O interface after adsorption of lysozyme onto the polymer surface at 1 g dm-3 (+) and 4 (O) g dm-3. The continuous line through the measured reflectivity is the fit to the profile at 4 g dm-3, giving a surface excess of 1.9 ( 0.2 mg m-2. The profile at 1 g dm-3 is close to that from the polymer coated solid-D2O interface (dashed line), suggesting that the adsorbed amount is small. The volume fraction distributions for the polymer film (solid line) and lysozyme layer at 4 g dm-3 (dashed line) are shown in the inserted diagram.

to that described from the ellipsometric measurements, allowing for an appropriate variation in water contrast. The dashed line in Figure 5 is the calculated reflectivity profile in D2O using a thickness of 55 Å for the polymer layer, as obtained from the ellipsometric measurement. The mismatch between the measured and calculated curves shows that the actual distribution of the polymer film cannot be modeled into a single uniform layer. A twolayer model was then tested with the inner layer rich in polymer and the outer layer lean in polymer. It was found that a two-layer model could not fit the measured profile well without incorporating roughnesses between the interfaces, particularly at the outer surface of the polymer film. A three-layer model was subsequently proposed: the dense inner layer has a small amount of water penetration, the middle layer has about twice as much water penetration, and the very diffuse outer layer contains a large fraction of water. The best fit is shown in Figure 5 as a continuous line and was calculated assuming the inner layer to be 30 ( 3 Å thick with 19% water, the middle layer to be 20 ( 3 Å with 44% water, and the outer layer to be 25 ( 5 Å thick with approximately 86% water. Although it is difficult to justify the reliability of the fit, the main feature out of the fitting procedure is that the unsymmetrical mixing of water into the coated film is substantial. Comparison of the results from the two experimental techniques appears to suggest that the outer polymer layer has been largely neglected in the ellipsometric measurement. This is likely to be caused by the heavy mixing of water into the outer polymer layer, resulting in a refractive index for the outer polymer layer close to that of pure water. In contrast, the use of D2O in neutron reflection helps to highlight the boundary region between the polymer film and water, providing more reliable information about the uneven distribution of the outer polymer surface. The adsorption of lysozyme onto the polymer surface at varying lysozyme concentrations was measured in the buffered D2O at pH 7, and the reflectivity profiles are shown in Figure 6. The extent of adsorption can be

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visualized by comparing the reflectivity profiles in the presence of lysozyme with that in the buffered D2O. At 1 g dm-3, the reflectivity profile changes very little from that of pure D2O, suggesting that there is very little lysozyme adsorption. The surface excess was found to be only 0.7 ( 0.2 mg m-2. The amount of lysozyme adsorbed at 4 g dm-3 was also obtained by fitting a non-uniform layer model to the measured reflectivity profile. The total surface excess at this high concentration was found to be 1.9 ( 0.2 mg m-2, in good agreement with the value of 1.3 ( 0.3 mg m-2 from the parallel ellipsometric work. The thickness of the layer was found to be 50 ( 10 Å, which is greater than the long axial length of lysozyme. In addition to this, there was a very low-density outer lysozyme layer of 35 ( 10 Å. The fitting also indicates that there was some lysozyme penetration into the outer diffuse polymer surface, as its scattering length density had decreased slightly from that at the polymer-water interface. The presence of lysozyme in all these layers was taken into account when calculating the surface excess above. The broadening of the distribution of the protein layer is clearly caused by the diffuse outer surface of the polymer film. A roughness of 15 ( 5 Å was required for both the polymer-lysozyme interface and the outer lysozyme-water interface. The broadening of the distribution of the layer and the roughness are both clearly a result of the diffuseness of the outer polymer surface, into which the protein can pentetrate. All the neutron measurements were made after the coated surface had been completely equilibrated with appropriate solutions. The first measurement was made after the surface had been in contact with water for about 2 h. The repeated neutron reflectivity profiles at the coated solid-water interface were identical over a period of 20 h, suggesting that the coated polymer film was very stable. The reversibility of the adsorption at the PC polymer surface was examined by rinsing the polymer surface with buffered D2O. It was found that a large fraction of the adsorbed lysozyme was still left after rinsing, suggesting that adsorption is irreversible with respect to protein concentration. A similar trend was observed at the hydrophilic silicon oxide-water interface where the surface excess was greater under the same bulk protein concentration.22 The preadsorbed surface was subsequently rinsed with dodecyl trimethylammonium bromide (C12TAB) at its critical micellar concentration (cmc ) 14 × 10-3 M). The reflectivity measurement in the buffered D2O returned to the D2O profile obtained before protein adsorption was started, suggesting that the adsorbed lysozyme was completely removed. Figure 7 shows the comparison of the reflectivity profiles in the buffered D2O during the course of the adsorption experiment. While the observation indicates that the cationic surfactant is effective at cleaning the fouled hydrogel polymer surface, the main finding is that the structure of the polymer film remained intact during the whole period of the experiment. This result is of direct relevance to the processing of the thin polymer films in many practical applications. The removal of the adsorbed protein from the polymer surface resembles what was observed at the hydrophilic silicawater interface22 where a complete desorption of proteins after rinsing with ionic surfactants also occurred. This might imply that, like adsorption at the silica-water interface, proteins adsorbed at the hydrogel polymerwater interface were not denatured. Lysozyme adsorption at the hydrophobic solid-water interface has also been

studied by neutron reflection.23 It was found that, unlike adsorption at the hydrophilic solid-water interface, adsorption at the hydrophobic surface led to the breakdown of the globular structure. Subsequent addition of a cationic surfactant resulted in the binding of the surfactant to the preadsorbed protein fragments. The surface was eventually composed of the complex mixture of surfactants and protein residues. Adsorption of BSA onto the polymer surface at pH 5 was also studied by neutron reflection. Figure 8 shows the adsorption of BSA at 2 g dm-3 compared with the reflectivity from the buffered D2O. The level of BSA

(22) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438.

(23) Lu, J. R.; Su, T. J.; Thirtle, P. N.; Thomas, R. K.; Rennie, A. R. J. Colloid Interface Sci. 1998, 206, 212.

Figure 7. Comparison of the neutron reflectivity profiles before lysozyme adsorption (O), after lysozyme adsorption at 4 (∆) g dm-3, and then after rinsing the adsorbed surface with C12TAB at its cmc (+). That the reflectivity profile after C12TAB rinsing is identical to that before protein adsoption suggests that the polymer film is very robust and that the cationic surfactant is very effective at removing the adsorbed protein.

Figure 8. Neutron reflectivity plotted as a function of κ for the adsorption of BSA onto the PC polymer coated solid-D2O interface at 2 g dm-3 (O). That the profile is very close to that from the PC polymer coated solid-D2O interface (dashed line) indicates a weak adsorption. The continuous line through the measured reflectivity is the fit to the profile at 2 g dm-3, giving a surface excess of 1.2 ( 0.2 mg m-2. The volume fraction distributions for the polymer film (solid line) and BSA layer at 2 g dm-3 (dashed line) are shown in the inserted diagram.

1320 Langmuir, Vol. 15, No. 4, 1999

Murphy et al.

Table 1. Surface Excesses for the Adsorption of Proteins at the Phosphoryl Choline Incorporated Polymer-Water Interfacea Γ ( 0.3/mg m-2 protein

C/g dm-3

pH

ellipsometry

neutron

lysozyme

1 4 0.05 2

7 7 5 5

0.5 1.3 0.3 1

0.7 (3.5) 1.9 (4.8) 0.5 (2.0) 1.2 (2.8)

BSA

a Numbers in brackets are the surface excesses at the bare silicon oxide-water interface obtained from refs 24 and 25.

adsorption can be visualized by the small gap between the two reflectivity profiles. It should be noted that adsorption at 0.05 g dm-3 was also measured but, because this reflectivity profile was very close to that between the coated solid-D2O interface, it was not shown for clarity. The surface excess at 0.05 g dm-3 was estimated to be 0.5 ( 0.3 g dm-3. The surface excess at 2 g dm-3 was calculated to be 1.2 ( 0.2 mg m-2, and the thickness of the layer was found to be 50 ( 10 Å with an outer low-density layer of 30 ( 10 Å. A roughness of 15 ( 5 Å was needed for both layers. It has been shown previously that at such a low surface excess, the adsorption leads to the formation of a sideways-on monolayer of approximately 30 Å on the silicon oxide surface.22 The increased thickness and roughness of the layer at the hydrogel surface is again a consequence of the diffuse outer surface of the polymer film. A summary on the variation of protein adsorption at the PC polymer-water interface is given in Table 1. The extent of reduction in the adsorption of lysozyme and BSA at the PC polymer surface can be realized when comparison is made with the adsorption at different solid-water interfaces. Adsorption of lysozyme and BSA under similar solution conditions at the hydrophilic silicon oxide-water interface has been studied by neutron reflection,22,24,25 and the corresponding surface escesses are also given in Table 1. The surface excess of lysozyme at the hydrophilic silicon oxide-water interface has also been examined by other techniques. Several independent measurements using in situ ellipsometry have produced the surface excess of 2.53.5 mg m-2 at 1 g dm-3 and a total ionic strength below 0.05 M,26-28 in good agreement with that of 3.5 mg m-2 obtained from neutron reflection at the same concentration. The surface excess on the bare oxide surface is thus significantly higher than the amount of lysozyme adsorbed on the polymer-water interface, which was found to be below 0.7 mg m-2. Furthermore, neutron measurements gave a surface excess of 4.8 mg m-2 at 4 g dm-3 for lysozyme adsorbed at the bare silicon oxide-water interface, which was almost three times greater than the value obtained from the PC polymer-solution interface. Lysozyme adsorption on mica was also determined by ellipsometry and XPS,29,30 and the surface excess was found to be between 4 and 5 mg m-2 when the bulk lysozyme concentration was below 1 g dm-3, indicating that the surface excess on (24) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. J. Colloid Interface Sci. 1998, 203, 419. (25) Su, T. J.; Lu, J. R.; Cui, Z. F.; Thomas, R. K.; Penfold, J. J. Phys. Chem. B 1998, 102, 8100. (26) Whalgren, M.; Arnebrant, T.; Lundstrom, L. J. Colloid Interface Sci. 1995, 175, 506. (27) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (28) Malmsten, M. J. Colloid Interface Sci. 1994, 168, 247. (29) Blomberg, E.; Claesson, P. M.; Froberg, J. C.; Tilton, R. D. Langmuir 1994, 10, 2325. (30) Claesson, P. M.; Blomberg, E.; Froberg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161.

mica is even higher than on the hydrophilic silica. Adsorption of lysozyme onto the hydrophobed solid-water interface has recently been studied by neutron reflection, and the results show that the total amount of the denatured protein deposited onto the hydrophobic surface is heavily dependent on the solution pH and the surface excess can vary from 2 to 5 mg m-2. Adsorption of globular and fibrous proteins onto poly(alkyl methacrylate) polymers was found to produce a densely packed protein layer,31 and the amount of protein adsorbed was strongly related to the type of protein molecules. Measurements have also been made on the adsorption of lysozyme onto various polymer-water interfaces in the form of flat interfaces or particulate dispersions. For example, lysozyme adsorption onto neutral and charged polystyrene particles has been studied by Haynes et al.32 and Fair et al.33 The surface excess was found to be between 2 and 3 mg m-2 at a lysozyme concentration around 1 g dm-3. Similar studies have been made on the adsorption of BSA onto different substrates,29,30,32,33 and the surface excess was typically found to be between 2 and 3 mg m-2 over a wide pH and concentration range. Because the results described above clearly show that hydrophobic and methacrylate surfaces induce strong protein deposition, the reduced adsorption after PC polymer coating tends to suggest that the outer polymer surface is covered by PC groups, instead of the dodecyl chains and polymeric backbones. This also implies the formation of a lamellar structure inside the coated film with the hydrophobic fragments buried in the middle. Although detailed structure within the coated layer can be probed by neutron reflection with the help of partial deuterium labeling to the polymer, such information is inaccessible under the current experimental conditions where only fully hydrogenated polymer was used. The preferential segregation of the fragments within the film might occur during the annealing stage when the coated surface was heated to 160 °C, approximately 30 °C above the melting point for the dry polymer. Further information about the presence of a PC layer on the outer surface can be gained by comparing our results with the adsorption of proteins onto phospholipids. Adsorption of several model proteins onto PC containing phospholipid surfaces has recently been studied by Malmsten28,34 using ellipsometry. Phospholipid molecules were coated onto the bare hydrophilic and methylated silicon oxide surfaces by Langmuir-Blodgett deposition, and the coating procedure was designed such that the PC headgroups would end up on the outer film surface. The extent of reduction in surface excess for the globular proteins adsorbed onto the phospholipid-coated surfaces is broadly consistent with what has been observed at the PC polymer-solution interface. For example, the residual amount of immunoglobulin (IgG) adsorbed on the PC phospholipid coated surface was found to be 0.2 mg m-2 at 0.1 g dm-3 at pH 7, as compared with 1 mg m-2 at the hydrophilic silicon-water interface under the same solution condition. The extent of reduction is very much comparable to what was observed at the polymer-water interface. Further, Malmsten has examined the possible difference in the method of surface layer deposition by performing parallel measurements using spin-coated (31) Lindon, J. N.; McManama, G.; Kushner, L.; Merrill, E. W.; Salzman, E. W. Blood 1986, 68, 355. (32) Haynes, C. A.; Norde, W. J. Colloid Interface, Sci. 1995, 169, 313. (33) Fair, B. D.; Jamieson, A. M. J. Colloid Interface Sci. 1980, 77, 525. (34) Malmsten, M. J. Colloid Interface Sci. 1995, 172, 106.

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Langmuir, Vol. 15, No. 4, 1999 1321

that the PC dipole is predominantly oriented almost parallel to the anchored plane as a result of intermolecular electrostatic interactions between the positive N-methyl group’s, and the negative phosphates of neighboring molecules in the same surface plane. Further, neutron scattering work on phospholipid bilayer membranes tends to indicate that upon change in system conditions, e.g., temperature, the structure of hydrocarbon chains varies significantly while that for the PC headgroups changes very little. This again reinforces the suggestion that PC groups have a strong tendency to form a closely packed monolayer, as a result of electrostatic attraction between the adjacent PC groups within the layer. From a simple geometric argument, the almost parallel orientation of PC groups also helps to bring the oppositely charged groups close to each other, thus making the neutralization almost complete. This is in sharp contrast with the charge distribution on the surface of a globular protein, where the globular framework keeps the opposite charges apart. The attractive force for the adsorption of protein onto the zwitterionic surface is therefore of van der Waals origin, arising mainly from the dispersion force and dipole-dipole interaction. The repulsive force between the surface and protein molecules must be related to the strong hydration of the headgroups which creates a steric barrier prohibiting protein deposition. The requirement of a well-packed hydrophilic layer for reducing protein adsorption has also been emphasized by Jeon et al.39 in their recent theoretical work on the interpretation of the interaction between a poly(ethylene glycol) grafted surface and protein molecules. The strong hydration of PC groups has been supported by the simulation work of Sheng et al.40 who have shown the heavy, nonrandom association of H2O molecules with PC. It should be reminded that in the precedent discussion, the entropic effect arising from either dehydration of the surface and protein molecules or change in the conformation of protein molecules during adsorption has not been considered.

phospholipid substrates, and no observable difference in the amount of protein adsorbed was detected, suggesting that upon the formation of the phospholipid layer, PC groups were preferentially expressed at the outer surface. The quality of packing of the outer PC layer will affect the residual amount of protein adsorbed, and this will in return be affected by the structure of the film. A smooth outer surface on the polymer film can enable the formation of a well-packed PC layer, leaving no gaps or voids for the exposure of hydrophobic fragments. The chemical structure of the polymer has a drastic effect on the formation of the thin film, and much has been discussed by Iwasaki and Hayward4,7,10 in terms of the spacer length under PC headgroups and the ratio of the hydrophobic and hydrophilic moieties within the polymer. Although discussion on the synthesis of polymers is beyond the scope of this work, it is useful to point out that, for a given polymer and solid substrate, the smoothness of the outer surface on the polymer film can be significantly improved by tuning the coating conditions.17 That there is a measurable amount of proteins adsorbed at high protein concentrations may indicate that some defects still exist on the outer surface. This is consistent with the outer surface being very diffuse. The uneven structure will deteriorate the packing of the outer PC polymer layer. Improvement in the uniformity of the outer surface is therefore expected to reduce the level of adsorption. However, because the residual amount of proteins adsorbed is comparable to that obtained at the phospholipid-coated surfaces, it can be said that at least the PC polymer surface is as effective as the phospholipid-coated ones. Electrostatic attraction and hydrophobic interaction are the two major driving forces causing surface adsorption in many cases. The occurrence of a large surface excess is usually associated with the existence of at least one of the two, as can be seen from the results described previously on the adsorption onto different solid substrates. In a simulation study on the interaction of globular protein with a charged solid substrate, Roth and Lenhoff35 have demonstrated that, even when the charge density on the solid substrate is very low, the magnitude of electrostatic force is still much greater than the contribution from the van der Waals force. This is even so in the case of BSA adsorption onto the hydrophilic silicon oxide at pH 5. Although under this pH the net charge of BSA is close to zero, the local positive charges on BSA will drive the molecule to the weakly negatively charged oxide, leading to a large BSA surface excess. In the case of a zwitterionic interface, little direct experimental information is available on its interaction with protein, but a close look at the packing of PC headgroups may help to understand the reason why PC surface strongly deters protein deposition. The structural conformation of zwitterionic PC groups in a bilayer membrane has been determined by neutron scattering and X-ray crystallography.36,37 The PC headgroup conformation, in terms of the vector directed from the phosphorus atom to the nitrogen (denoted by PN), was found to stay parallel or almost parallel to the average plane of the bilayer. The projection of the PC headgroup onto the surface normal was about 5 Å. The orientations of the PN vectors were heavily tilted, making angles of approximately 15°-30° with respect to the plane of the bilayer. This observation was recently confirmed by the simulation work of Granfeldt et al.,38 which again shows

Coating of phosphorylcholine polymers onto solid substrates can significantly reduce protein deposition. This observation is supported by the parallel measurements from both spectroscopic ellipsometry and specular neutron reflection. Although spectroscopic ellipsometry appears to be less sensitive to the structural distribution of the outer surface of the coated polymer film, both techniques produced consistent results with regard to the amount of protein adsorbed at the polymer-solution interface. In comparison with the surface excess at the hydrophilic silicon oxide-water interface, the residual surface excess on the PC polymer surface was found to be reduced by a factor of 2-4. The extent of reduction at the low- and medium-protein concentrations was more substantial although accurate estimates at low surface excess were limited by the resolution of both methods. Because dodecyl chains and methacrylate groups in the polymer induce strong protein deposition, the reduction in the adsorbed amount suggests that the outer polymer surface must be covered by a layer of phosphorylcholine groups. This hypothesis is supported by the results from the phospholipid-coated surfaces which showed a comparable level of reduction in protein adsorption. The results from this work strongly suggest that future improvement

(35) Roth, C. M.; Lenhoff, A. M. Langmuir 1993, 9, 962. (36) Buldt, G.; Seelig, J. Biochemistry 1980, 19, 6170. (37) Pearson, R. H.; Pascher, I. Nature 1979, 281, 499. (38) Granfeldt, M. K.; Miklavic, S. J. J. Phys. Chem. 1991, 95, 6351.

(39) Jeon, S. I.; Lee J. H.; Andrade J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (40) Sheng, Q.; Schulten, K.; Pidgeon, C. J. Phys. Chem. 1995, 99, 11018.

Conclusions

1322 Langmuir, Vol. 15, No. 4, 1999

in the uniformity of the outer surface on the polymer film, through chemical modification to the structure of the polymer or tuning of coating conditions, will improve the packing density of the outer PC layer, leading to a further reduction in the residual protein deposition. The diffuse outer surface has prevented us from revealing the possible structural conformation of the two proteins adsorbed at the polymer-water interface at the relatively high protein concentrations although the high depth resolution of neutron reflection is capable of probing such structural information at this level of resolution. The adsorption of globular proteins can also be used as a means to probe the electroneutrality of the zwitterionic

Murphy et al.

surface. The reduced level of adsorption observed in this work tends to suggest that electrostatic attraction, which is a common driving force for surface adsorption, has been predominantly eliminated and the zwitterionic surface must be neutral.

Acknowledgment. We thank the Biotechnology and Biological Sciences Research Council for support. E.M. thanks Biocompatibles Ltd for a studentship. LA9813580