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Reduced Protein Adsorption on the Surface of a Chemically Grafted Phospholipid Monolayer Jian R. Lu,* Emma F. Murphy, and Tsueu J. Su Department of Chemistry, University of Surrey, Guildford GU2 5XH, United Kingdom
Andrew L. Lewis and Peter W. Stratford Biocompatibles Ltd., Farnham Business Park, Weydon Lane, Farnham, GU9 8QL, United Kingdom
Sushil K. Satija NIST Centre for Neutron Research, Gaithersburg, Maryland 20899 Received December 12, 2000. In Final Form: March 12, 2001 We have modified the surface of hydrophilic silicon oxide by chemically anchoring an organic monolayer bearing terminal phosphorylcholine (PC) groups and subsequently characterized the structure of the PC layers and their effectiveness in inhibiting the deposition of a range of model proteins. The PC compound was synthesized through coupling of 3-aminopropyl trimethoxysilane with acryloyloxyethylphosphorylcholine in 2-propanol. The presence of the labile hydrogen on the secondary amine group of the monomer allowed a subsequent coupling of two monomers with a bridging spacer such as a diisocyanate to form a dimer. The PC dimer was coated onto a silicon substrate via dip coating, and the chemical grafting with the substrate was strengthened by annealing the coated layers at 150 °C under vacuum. Neutron reflection measurements showed that upon the formation of a well-packed monolayer, the small PC molecular coatings were as effective as the PC polymer coatings in reducing protein adsorption in vitro.
Introduction Protein adsorption is the first step in many biofouling processes and is a widespread phenomenon afflicting a variety of industrial systems. Its occurrence consequently constrains the application of many smart technologies. In the bioseparation industry, porous ceramic filtration membranes are robust and can withstand harsh cleaning conditions. They therefore offer attractive potential over the more widely used polymeric membranes. However, the poor biocompatibility between ceramic materials and biological molecules has seriously hindered their application in bioseparation and biomedical engineering.1,2 Protein deposition onto exterior and interior pores of porous ceramic membranes causes a fast decline of permeate flux, and this blockage may lead the filtration to halt in minutes. Protein adsorption is also the initial step in the response of bodily fluids (e.g., tears and blood) to the exposure to medical devices such as contact lenses and cardiovascular implants. The strong interaction between the adsorbed protein and the substrate surface induces the deterioration of proteins and enzymes that may trigger subsequent responses from the immune system, resulting in more serious biological consequences. Furthermore, the nonspecific deposition of biological molecules onto foreign substrates is also a common restriction to the improvement of the sensitivity and selectivity of biosensors and diagnostic devices. * To whom correspondence should be addressed. Present address: Department of Physics, UMIST, P.O. Box 88, Manchester M60 1QD, United Kingdom. Email:
[email protected]. Fax: 44161-2003673. (1) Marshall, A. D.; Munro, P. A.; Tragardh, G. Desalination 1993, 65, 91. (2) Su, T. J.; Lu, J. R.; Cui, Z. F.; Bellhouse, B. J.; Thomas, R. K.; Heenan, R. K. J. Membr. Sci. 1999, 163, 265.
Many studies in the literature have shown that coatings of certain polymers can reduce nonspecific protein deposition.2-6 Among many polymer coatings so far studied, those containing phosphorylcholine (PC) groups are extremely effective at reducing protein deposition in vitro.3,7-9 Similar protein repelling effects of these PC polymers have also been observed in the work by Campbell et al.10 and in a number of other literature studies referred to in ref 11 when the polymers were exposed to blood. From a biophysical viewpoint, the advantage in using pure proteins instead of blood is that the extent of protein deposition can be related to the structure and composition of the coated layer and that complications relating to the unknown composition of blood are avoided. This approach enables some delicate physical characterization to be made toward understanding the nature of surface biocompatibility. The encouraging results from PC polymers have motivated us to explore whether a similar antifouling effect is rendered by a molecular monolayer of phospholipids chemically grafted onto a solid substrate. It has however been widely perceived that a chemically (3) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers; Plenum: New York, 1988; Vol. 2. (4) Feng, L.; Andrade, J. D. J. Biomed. Mater. Res. 1994, 28, 735. (5) Iwasaki, Y.; Fujike, A.; Kurita, K.; Ishihara, K.; Nakabayashi, N. J. Biomater. Sci., Polym. Ed. 1996, 8, 91. (6) Yianni, Y. P. In Structural and Dynamic Properties of Lipids and Membranes; Quinn, P. J., Cherry, R. J., Eds.; Portland Press: London, 1992. (7) Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell, J.; Stratford, P. Macromolecules 2000, 33, 4545. (8) Murphy, E. F.; Lu, J. R.; Brewer, J.; Russell, J.; Penfold, J. Langmuir 1999, 15, 1313. (9) Murphy, E. F.; Keddie, J. L.; Lu, J. R.; Brewer, J.; Russell, J. Biomaterials 1999, 20, 1501. (10) Campbell, E. J.; O’Byrne, V.; Stratford, P.; Quirk, I.; Vick, T. A.; Miles, M. C.; Yianni, Y. P. ASAIO J. 1994, 40, M853. (11) Lewis, A. L. Colloids Surf., B 2000, 18, 261.
10.1021/la0017429 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/03/2001
Protein Adsorption on a Phospholipid Monolayer
anchored phospholipid monolayer would not offer a high level of biocompatibility because the attachment of these molecules onto a solid substrate constrains their mobility. Self-assembly of phospholipid bilayers has been reported to show high efficiency in reducing protein adsorption.12-14 However, the main shortcoming with the strategy in mimicking the exposure of PC groups on a bilayer structure of the cellular membrane wall is the lack of stability stemming from the physical coating. In this work, we show that contrary to the common belief a chemically anchored organic monolayer with terminal PC groups can reduce protein adsorption. We have developed two different methods for the attachment of PC molecular layers onto a silicon oxide surface through covalent bonding. We will demonstrate in this work that the monolayer- and bilayer-coated surfaces under the two synthetic routes are as effective as the PC polymer coatings under comparable solution conditions.
Langmuir, Vol. 17, No. 11, 2001 3383 Scheme 1. Assembly of Chemical Fragments Leading to PC Dimers
Experimental Section Neutron reflection experiments used large silicon blocks, each of dimensions 12.5 × 5 × 12.5 cm3. The coating of the PC layer was made on the large (111) face of the silicon block which was polished and cleaned prior to the coating.8 The cleaning process of the freshly polished surface started with the immersion of the block in 5% neutral Decon solution; it was ultrasonically cleaned for 30 min, followed by a further 30 min of ultrasonic cleaning in water. The block was then copiously rinsed and soaked in an acidic peroxide solution containing 600 mL of H2SO4 (98%) and 100 mL of H2O2 (29%). The block was then rinsed thoroughly with Elgastat ultrapure water (UHQ) to remove acid and exposed to UV/ozone for 30 min to remove any trace of organic impurities. The surfaces treated by this procedure were then used directly for organic coating. The uniformity of the oxide surface on each block was checked by ellipsometry (Woollam Variable Angle Spectroscopic Ellipsometer 32 (WVASE32))15 over spots at different locations, and the variation in the thickness was found to be typically within 20 ( 4 Å if the refractive index of the layer was taken to be the same as the bulk oxide. The subsequent neutron reflection was made to obtain more reliable measurements of the oxide layers before the PC layers were coated. Two neutron blocks were coated at PC dimer concentrations of 0.4 and 0.8 g dm-3. Preliminary ellipsometry measurements showed a large extent of reduction in protein adsorption on both of the coated surfaces. The quantitative amount of residual protein adsorption on the two surfaces was determined by neutron reflection using reflectometers NG-7 at the National Institute of Standards and Technology (NIST), Gaithersburg, MD,16 and SURF at the ISIS Neutron Facility, U.K.17 NG-7 uses neutrons with a wavelength of 4.75 Å. Its range of momentum transfer, κ (κ ) (4πsin θ)/λ) where λ is the wavelength and θ is the glancing angle of incidence), was provided by the variation of θ. In contrast, SURF uses a white beam with wavelengths ranging from 0.5 to 6.5 Å. Its full range of momentum transfer was provided by a combination of reflection measurements at three glancing incidence angles of 0.35°, 0.8°, and 1.8°. On both instruments, the incoming beams were directed downward toward the sample and were reflected from the interface upward and into the detector. For the measurement at the solid/water interface, a large silicon block (12) Cuypers, P. A.; Corsel, J. W.; Janssen, M. P.; Kop, J. M. M.; Hermens, W. Th.; Hemker, H. C. J. Biol. Chem. 1983, 258, 2426. (13) Malmsten, M. J. Colloid Interface Sci. 1995, 172, 106. (14) Malmsten, M. J. Colloid Interface Sci. 1994, 168, 247. (15) Su, T. J.; Green, R. J.; Wang, Y.; Murphy, E. F.; Lu, J. R.; Ivkov, R.; Satija, S. K. Langmuir 2000, 16, 4999. (16) Fragneto, G.; Lu, J. R.; McDermott, D. C.; Thomas, R. K.; Rennie, A. R.; Gallagher, P. D.; Satija, S. K. Langmuir 1996, 12, 477. (17) Penfold, J.; Richardson, R. M.; Zarbakhsh, A.; Webster, J. R. 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.; Roser, S. J.; McLure, I. A.; Hillman, R. A.; Richards, R. W.; Staples, E. J.; Burgess, A. N.; Simister, E. A.; White, J. W. J. Chem. Soc., Faraday Trans. 1997, 93, 3899.
was used and its largest polished face was clamped against a Teflon cell. The beam was directed into the solid/water interface from the smallest face of the silicon block and exited from the opposite side of the smallest face into the detector. The dimension of the beam at a given incidence angle was defined by sets of vertical and horizontal cadmium slits. The vertical slits set the width of the beams, and in this work the beam width was set at 25 mm on NG-7 and 30 mm on SURF. At a given incidence angle, the height of the beam was always maximized to optimize the beam intensity, but caution was taken not to overilluminate the sample surface. The three proteins chosen represent a range of size and different stability of the three-dimensional structures. Chicken egg white lysozyme (Sigma, 99%+, catalog no. L6876) was used as supplied. Lysozyme has an isoelectric point around pH 11. Its globular structure is roughly ellipsoidal and has an approximate dimension of 30 × 30 × 45 Å3. Bovine serum albumin (BSA) (Sigma, catalog no. A0281) was fatty acid free and was also used as supplied. Its isoelectric point was about pH 4.8, and the globular dimension is about 40 × 40 × 140 Å3. Fibrinogen (Sigma, catalog no. F8630, from bovine plasma) is known as a bloodclotting agent and has an isoelectric point of 5.5. Adsorption measurements were made at pH 7 for lysozyme and fibrinogen 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 for lysozyme and BSA solutions. For fibrinogen solutions, the total ionic strength for phosphate buffer was controlled at 0.05 M. To aid the dissolution of fibrinogen, additional sodium chloride was added to a concentration level of 0.1 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. Highpurity Elgastat water (UHQ) was used throughout the work, and D2O was purchased from Sigma and was used as supplied. The glassware and Teflon troughs for the reflection measurements were cleaned using alkaline detergent (Decon 90) followed by repeated washing in UHQ water. Organic Synthesis and Coating. In contrast to the stepwise connection used in our previous work,18 we have recently developed a one-step coating scheme (Scheme 1). In this new method, the PC compounds are assembled in bulk solution and the surface layer is formed via dip coating. A typical monomer (18) Wang, Y.; Su, T. J.; Green, R. J.; Tang, Y.; Styrkas, D.; Danks, T. N.; Bolton, R.; Lu, J. R. Chem. Commun. 2000, 587.
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synthesized was APTMS-APC that was made by refluxing 3-aminopropyl trimethoxysilane (APTMS) (from Aldrich, 97%) with acryloyloxyethylphosphorylcholine (APC, synthesized using a procedure similar to the formation of methacrylate) in 2-propanol for 2 h.19 1H and 13C NMR showed conversion to the Michael adduct, and proton Cosy NMR was used to assign the peaks. Because the PC compound has a labile hydrogen on the secondary amine group, its dimer form can be obtained by coupling two monomers with a bridging spacer such as a diisocyanate. The coupling was again carried out in 2-propanol by refluxing the monomer and the spacer for a further 2 h. The reaction procedure outlined in Scheme 1 leads to the formation of a dimer using hexaethylene diisocyanate. FT-IR confirmed the reaction of the isocyanate by disappearance of the NdCdO stretch at ca. 2230 cm-1. 1H and 13C NMR also confirmed the formation of the APTMS-APC dimer. An obvious advantage of this reaction scheme is the direct control of the surface coverage of the PC groups by the length of the spacer. The PC monolayer and bilayer were coated using a device described in our previous work.9 The whole device was put inside a Perspex box, and the dipping rig was placed on an antivibration bench. We have previously shown that in the course of dip coating, parameters such as temperature, the chemical nature of the solvent and its vapor pressure, motor lifting speed, and the solution concentration tend to affect the thickness and coverage of the layers. In this work, the temperature inside the Perspex box was controlled to 23 °C, and the use of the vibration-free table reduced mechanical disturbance. Because the PC dimer was synthesized in 2-propanol, the same solvent was chosen to prepare the coating solution. Because previous work had shown that the uniformity of the coated film was determined by the interplay between the lifting speed, the draining rate, and solvent evaporation, these parameters were assessed at the beginning of this work. It was found that when the speed was around 5 mm s-1 the uniformity of the coated layers was at the optimal state. The extent of film uniformity was judged from the parallel ellipsometric measurements at three fixed positions (two ends and the center) on the coated surface. In the case of the large neutron block, the two points at each end were chosen about 1 cm from the edge. The coated films were left to dry at the ambient temperature for a few hours before they were subject to annealing at 70 °C under vacuum overnight (about 16 h). The coated surfaces were then subject to rinsing with UHQ water before they were characterized by spectroscopic ellipsometry to reveal the relative amount of protein adsorption. The main parameter that was systematically examined was the concentration of the PC dimer, as will be outlined later. Hayward et al.20 explored the concept of the chemical grafting of PC monolayers onto silicon oxide some twenty years ago, but in their case, the attached alkyl chain bearing terminal PC groups was unstable as a result of the presence of an oxygen between carbon and silicon, the C-O-Si connection. The resulting alkyl silicate structure readily hydrolyses, with loss of the alkyl chains and their PC functionality. Our approach is similar to that of Hayward et al., but we have sought direct chemical bonding between the carbon and silicon so that stable chemical grafting is obtained. In comparison with C-O-Si bonding, the formation of Si-O-Si connections at the end of the organic monolayer is much more stable. This part of the layer is identical to the underlying silica layer in chemical composition although their structures might be different.
Lu et al.
Figure 1. Neutron reflectivity measured at the PC monolayer coated silicon oxide/water interface with the water isotopic contrast varying from D2O (O), CM4 (D2O/H2O ≈ 2:1) (+), and CMSi (D2O/H2O ≈ 1:2) (4). The simultaneous fitting produced an oxide layer of 16 ( 2 Å (defect free) and a PC monolayer of 18 ( 2 Å with 35% water.
κ. Figure 1 shows a set of reflectivity profiles measured at the PC layer coated silicon oxide/water interface. In addition to the PC layer, there is also a native layer of SiO2 on the surface of silicon. The reflectivity is a function of scattering length density (F) perpendicular to the surface.21,22 The scattering length density is subsequently related to the chemical composition of the layer through the following equation
F)
∑mibi Aτ
(1)
(I) PC Layer Coated at the Low Dimer Concentration. The structure of the PC layer coated at 0.4 g dm-3 and the extent of protein adsorption have been determined using reflectometer NG-7. In a typical neutron reflection experiment, neutron reflectivity, defined as the ratio of the intensity of the specularly reflected beam to that of the incoming one, is usually plotted as function of
where ∑mibi is the total scattering length of the species within the volume Aτ (A is the cross-sectional area and τ is the thickness). Thus, change in the chemical composition of the interface across the different layers directly affects neutron reflectivity. An important characteristic of neutron reflection is the use of deuterium labeling to alter neutron reflectivity without affecting the chemical composition of the interface. In this work, different ratios of H2O and D2O were used to highlight the interfacial layers differently. Because the scattering length densities for H2O and D2O are of opposite signs (6.35 × 10-6 Å-2 for pure D2O and -0.56 × 10-6 Å-2 for pure H2O), variation in the ratio of H2O and D2O allows F for the solvent to be varied over a wide range. As water fills the defects in the oxide layer and penetrates into the gaps within the PC layer, the shift of F in each layer from its bulk value gives an indication of the extent of mixing, as will be explained later. The combined reflectivity measurements under different isotopic contrasts for a given interfacial system hence greatly improve the certainty of structure determination. The three reflectivity profiles shown in Figure 1 were measured in D2O, CM4 (D2O/H2O ≈ 2:1, F ) 4 × 10-6 Å-2), and CMSi (D2O/H2O ≈ 1:2, F ) 2.07 × 10-6 Å-2).
(19) Biocompatibles Ltd., patent pending. (20) Hayward, J. A.; Durrani, A. A.; Shelton, C.; Lee, D. C.; Chapman, D. Biomaterials 1986, 7, 126. Also: Hayward, J. A.; Durrani, A. A.; Lu, Y.; Clayton, C. R.; Chapman, D. Biomaterials 1986, 7, 252.
(21) Lu, J. R.; Lee, E. M.; Thomas, R. K. Acta Crystallogr. 1996, A52, 42. (22) Lu, J. R.; Thomas, R. K. J. Chem. Soc., Faraday Trans. 1998, 94, 995.
Results
Protein Adsorption on a Phospholipid Monolayer
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Information about the thickness and composition of the layers across the interface was obtained from model fitting using the optical formula.23 The fitting procedure typically starts with the assumption of a model structure for the interface, and the corresponding reflectivity is calculated. The calculated reflectivity is then compared with the measured one, and the model is iterated until a good agreement is obtained between two reflectivity profiles. For each layer, the parameters used in the fitting are τi and Fi. The volume fractions of water (φw) and the PC species (φPC) in a given layer are related to Fi through the following equation:
Fi ) φwFw + φpFPC
(2)
where FPC represents the scattering length density of the pure PC compound and φw + φPC ) 1. Equation 2 is also applicable to the oxide layer and protein layer. The continuous lines through the measured reflectivity profiles in Figure 1 represent the best fits assuming the oxide layer to be 16 ( 2 Å thick without any defects and the PC layer to be 18 ( 2 Å containing 35 ( 3% water. It should be noted that the structure of the bare oxide was determined by neutron reflection prior to the PC layer coating and its thickness was found to be 14 ( 3 Å. Given that the difference is only 2 Å, the agreement is good. This structural feature is in contrast to the situation observed in our previous work16 on the coating of octadecyl trichlorosilane (OTS) onto the polished silicon oxide surface, where it was found that some readjustment had to be made about the structure of the oxide layer after OTS coating as a result of the porous feature of the oxide layer. Such adjustment was found to be unnecessary here, indicating that the oxide surface used in this work is smoother. After the characterization of the PC layer, lysozyme solutions were introduced into the sample cell. Measurements were made at the lysozyme concentrations of 0.03, 1, and 4 g dm-3, all at pH 7 and in D2O. For clarity, only the measurements at the lowest and highest lysozyme concentrations are shown in Figure 2. The reflectivity profile from the coated solid/pure D2O interface is shown as a dashed line for comparison. Because the difference between reflectivity profiles is small, the scale range was expanded so that the change caused by lysozyme adsorption is more easily visible. The reflectivity profiles were analyzed by assuming that the adsorption formed an additional layer on the surface of the PC layer and that the adsorption did not cause any structural change to the oxide and PC layers. The optimally fitted layer thicknesses together with the surface excess (Γ) and other related parameters are given in Table 1. The surface excess (mol m-2) is related to area per molecule (A, Å2) through
1020 NaA
(3)
Vp bp ) τFpφp τφp
(4)
Γ) A)
where Na is Avogadro’s constant, bp is the scattering length of protein, Fp is its scattering length density, Vp is its molecular volume, and φp is its volume fraction in the layer. The main observation from Table 1 is that the adsorbed lysozyme layer is thick but very diffuse. At the lowest (23) Born, M.; Wolf, E. Principles of Optics; Pergamon: Oxford, 1970.
Figure 2. Neutron reflectivity measured at the PC monolayer coated silicon oxide/D2O interface with lysozyme concentration at 0 (dashed line), 0.03 g dm-3 (O), and 4 g dm-3 (4). The continuous lines were calculated by taking the surface excesses to be 0.9 and 1.8 mg m-2 and the lysozyme layer thicknesses to be 60 and 68 ( 5 Å at 0.03 and 4 g dm-3 lysozyme, respectively. The solution pH was controlled at 7, and the total ionic strength was controlled at 0.02 M. Table 1. Protein Adsorption onto the PC Monolayer Coated on Silicon Oxide Measured at the Solid/Water Interfacea
protein lysozyme BSA fibrinogen a
FPro × ΓPro FPCm2 × 10-6 10-6 C τPCm2 τPro (0.3/ g dm-3 pH (2/Å (5/Å (0.1/Å-2 (0.1/Å-2 mg m-2 0.03 1 4 0.05 2.0 0.1 1
7 7 7 5 5 7 7
18 18 18 18 18 18 18
60 60 68 70 80 80 80
2.9 2.9 2.8 2.8 2.7 2.8 2.8
6.1 6.0 5.9 6.0 6.0 6.2 6.2
0.9 1.3 1.8 1.0 1.2 0.7 0.7
The thickness of the underlying oxide layer was 16 ( 2 Å.
lysozyme concentration of 0.03 g dm-3, the layer is some 60 Å thick and the volume fraction of protein is 0.08. The corresponding surface excess is 0.8 mg m-2. At the highest concentration of 4 g dm-3, τ ) 68 Å, φp ) 0.17, and Γ ) 1.8 mg m-2. The feature of the formation of thick and diffuse protein layers is remarkably similar to the observation found on the PC polymer surfaces in our previous work7,8 but is very different from the structural conformations observed on the bare oxide and OTS surfaces, as will be discussed later. After each protein adsorption study, the solution was drained and the surface was cleaned with pure buffered solution to see if any adsorbed protein could be removed. It was found that rinsing with water or buffer solution could only result in partial protein removal. As in the previous adsorption work, a cationic surfactant, dodecyl trimethylammonium bromide (C12TAB), was then used and the solution was made at its critical micellar concentration (cmc). Rinsing with this solution was found to remove the residual adsorption completely, as evidenced by the overlap of the reflectivity profile to the one before the introduction of lysozyme solution (Figure 3). The complete removal was also found for BSA and fibrinogen, as will be discussed later. Thus, the PC surface allowed a systematic assessment of the adsorption from different proteins. The adsorption of BSA was measured at 0.05 and 2 g dm-3 and at pH 5. Figure 4 shows the change of reflectivity
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Figure 3. Neutron reflectivity measured at the PC monolayer coated silicon oxide/pure D2O interface before (O) and after (+) lysozyme adsorption. The overlap between the two measured reflectivity profiles indicates the complete removal of the adsorbed protein and the stability of the PC layer.
Figure 4. Neutron reflectivity measured at the PC monolayer coated silicon oxide/D2O interface with BSA concentration at 0 (dashed line), 0.05 g dm-3 (O), and 2 g dm-3 (+). The continuous lines were calculated by taking the surface excesses to be 1 and 1.2 mg m-2 and the BSA layers to be 70 and 80 ( 5 Å thick at 0.05 and 2 g dm-3 BSA, respectively. The solution pH was controlled at 5, and the total ionic strength was controlled at 0.02 M.
with the concentration of BSA. As can be seen from Figure 4, the first addition of BSA at 0.05 g dm-3 causes a relatively bigger shift in the reflectivity and the subsequent increase in BSA concentration to 2 g dm-3 has a much smaller effect. The structural parameters obtained from data modeling are listed in Table 1, which also showed the formation of a thick but diffuse BSA distribution. The layer thickness at both BSA concentrations was found to be 80 Å with φp ) 0.08 and Γ ) 1 mg m-2. A similar adsorption measurement was subsequently made using fibrinogen, at the concentrations of 0.1 and 1 g dm-3, all at pH 7 in D2O, and the resultant reflectivity profiles are shown in Figure 5. It can be seen from Figure 5 that the reflectivity changes very little after the first
Lu et al.
Figure 5. Neutron reflectivity measured at the PC monolayer coated silicon oxide/D2O interface with fibrinogen concentration at 0 (dashed line), 0.1 g dm-3 (O), and 1 g dm-3 (+). The continuous lines were calculated by taking the surface excesses to be 0.7 mg m-2 and the layers to be 80 ( 5 Å thick for both concentrations. The solution pH was controlled at 7, and the total ionic strength was controlled at 0.02 M.
addition of fibrinogen, again showing that there is little effect of protein concentration. The result thus suggests a low level of adsorption of fibrinogen on the PC surface. The data analysis shows that the loosely adsorbed protein could also be approximated to a diffuse layer of some 80 Å with φp ) 0.08 and Γ ) 1 mg m-2 (see Table 1 for details). (II) PC Layer Formed at the High Dimer Concentration. The structure of the PC layer coated at the high dimer concentration and the subsequent protein adsorption were characterized using reflectometer SURF. As in the case of the characterization of the PC layer coated at the low dimer concentration, the same three water contrasts were used, and the resultant reflectivity profiles are shown in Figure 6. The simultaneous fitting produced a two-layer model: an inner layer of 15 Å with φPC ) 0.65 and an outer layer of 20 Å with φPC ) 0.25. The volume fraction of the inner layer is the same as that found for the monolayer coating, thus suggesting that the inner layer has reached its saturated density and increase in the PC dimer concentration leads to the attachment of a loose outer PC layer. Because the outer PC layer could not be washed off, the PC compound must have formed chemical bonding through silyl groups with either the substrate or other silyl groups from the nearby molecules. These results indicate the existence of some degree of structural disorder within the PC layers. The attachment of the loose outer PC layer may result in the exposure of some hydrophobic defects, which may subsequently attract protein attachment. It could however be argued oppositely that these loose PC molecules may help to cover the defects left within the inner layer, which may reduce protein adsorption. It is therefore important to compare the adsorbed amount between the PC bilayer and the PC monolayer surfaces. The adsorption of all three proteins under the same solution conditions as described previously has been measured, and the results analyzed from model fitting are given in Table 2. The reflectivity profiles measured from lysozyme adsorption are shown in Figure 7 to exemplify the adsorption measurements obtained from the white beam neutron source. In com-
Protein Adsorption on a Phospholipid Monolayer
Figure 6. Neutron reflectivity measured at the PC bilayer coated silicon oxide/D2O interface with the water isotopic contrast varying from D2O (O), CM4 (D2O/H2O ≈ 2:1) (+), and CMSi (D2O/H2O ≈ 1:2) (4). The continuous lines were calculated by taking the oxide layer to be 12 ( 2 Å (defect free), the inner PC layer to be 16 ( 2 Å with 35% water, and the outer PC layer to be 20 ( 2 Å with 75% water.
parison with the reflectivity profiles shown in Figure 2, more data points were obtained in each spectroscopic scan from SURF, but because the change in reflectivity caused by protein adsorption is again small, the two sets of neutron data are of comparable quality. One of the consistent trends that can be seen from Table 2 is that the protein layers are shorter than those determined from the monolayer PC surface shown in Table 1. This is because of the mixing of part of the protein layers with the outer PC layer on the bilayer PC surface. The mixing is evidenced by the slight decrease of the scattering length densities in the outer PC layers after protein adsorption. The surface excesses have been calculated by taking into account the contributions from the mixing into the outer PC layers. The extent of removal of residual protein adsorbed on the PC bilayer surface has also been examined using a similar procedure as described previously. After studying adsorption from each protein solution, the surface was rinsed with C12TAB solution, followed by copious rinsing of UHQ water. Figure 8 compares the two reflectivity profiles in D2O measured at the beginning and the end of the protein adsorption. The good reproducibility indicates that the PC bilayer surface is robust and the adsorbed protein is completely rinsed off. Discussion The difference in the performance between the coatings of the PC monolayer and bilayer can be found by comparing the surface excesses listed in Tables 1 and 2. It can be seen that the PC bilayer coating has further reduced protein adsorption and is therefore more effective. The extent of reduction is noticeably significant for lysozyme because its absolute residual adsorption was the highest among the three proteins studied. These results suggest that the PC bilayer produces a better PC surface shielding than the monolayer coating. We have previously reported an alternative synthetic route using a stepwise connection to attach the PC monolayer onto SiO2.18 As already described previously, the whole PC compound is assembled in bulk solution in a two-step, one-pot scheme and the coating is administered
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via dip coating. In contrast, the stepwise scheme starts with the attachment of the underlying organic layer onto the substrate. This intermediate layer bears terminal Cd C double bonds which are then converted into primary alcohol by the exposure of the coated surface to BH3‚THF for 10 min, followed by the treatment of alkaline hydrogen peroxide for 1 h. Subsequent connection of PC groups is achieved by reaction of the bound organic hydroxy groups with POCl3 in the presence of triethylamine, followed by a reaction with HOCH2CH2NMe+3OAc-. The adsorption of the three proteins under the same solution conditions was also studied using neutron reflection, and the surface excesses (ΓPCm1) are compared with those from the two PC surfaces studied in this work in Table 3 (ΓPCm2 for the monolayer and ΓPCbi for the bilayer reported in this work). The volume fraction of the PC monolayer coated from the stepwise connection was 0.61, some 4% lower than the coverage obtained from the monolayer surface coated in this work. Despite this difference, the surface excesses obtained from the two PC monolayer surfaces are almost identical for each of the proteins studied, showing that the small difference in PC coverage is insignificant and that the different coating chemistry did not seem to affect the performance either. The adsorption of proteins onto PC polymer coated surfaces has also been studied. Two PC polymers, one containing dodecyl chains and PC headgroups only (PC 100A) and the other containing dodecyl chains, PC headgroups, 2-propanol groups, and silyl cross-linking groups (PC 100B), were coated as thin films onto silicon substrates, and the surface excesses were similarly assessed by neutron reflection.7,8 PC 100B was found to be more effective than PC 100A. As a result, the inclusion of the surface excesses from PC 100B (ΓPC100B) in Table 3 is sufficient for comparison. It can be seen from Table 3 that the residual amount of protein adsorption on the PC polymer surface is broadly comparable to that found on the surfaces of PC monolayers, showing that the monolayer coatings are as effective as the polymer coatings in reducing protein adsorption in this in vitro type study. In addition to the reduced adsorption, there are two common features between the polymeric and monomeric PC surfaces. First, the residual protein layers are very thick and very diffuse and are all between 60 and 100 Å. Second, the absolute level of residual protein adsorption decreases with increasing the size of proteins. In all cases, the residual amount of lysozyme adsorption is always the highest and that for fibrinogen is the lowest. The overall performance of the coatings of PC materials in reducing protein adsorption can be realized by comparing the surface excesses from the PC surfaces with those from the bare silicon oxide (ΓSiO2) and the hydrophobic OTS (ΓOTS) surfaces. For each protein at a given concentration, the surface excesses on the PC surfaces are about a factor of 2-4 smaller than the corresponding values on SiO2 and OTS surfaces. The two sets of data also clearly show that on SiO2 and OTS surfaces the surface excess increases with the size and instability of the proteins whereas the opposite trend is seen on the PC surfaces. This suggests that the PC surfaces are more effective at prohibiting the nonspecific protein deposition than all the other surfaces studied. A further characteristic difference is the structure of the adsorbed protein layers between these surfaces. As already mentioned, the residual adsorption on all PC surfaces tends to form a loose and diffuse layer. In contrast, globular proteins adsorbed on the bare silicon oxide/water interface retain their globular structures and the volume fractions of the protein within the layers are usually less than 60%. The thickness of the
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Lu et al.
Table 2. Protein Adsorption onto the PC Bilayer Coated on Silicon Oxide Measured at the Solid/Water Interfacea protein lysozyme BSA fibrinogen
C g dm-3
pH
τPC1 (2/Å
τPC2 (2/Å
τPro (5/Å
FPC1 × 10-6 (0.1/Å-2
FPC2 × 10-6 (0.1/Å-2
FPro × 10-6 (0.1/Å-2
ΓPro (0.3/mg m-2
0.03 1 4 0.05 2.0 0.1 1
7 7 7 5 5 7 7
16 16 16 16 16 16 16
20 20 20 20 20 20 20
45 45 45 30 50 70 70
3.0 3.0 3.0 3.0 3.0 3.0 3.0
4.9 4.7 4.7 5.0 4.7 4.7 4.7
6.1 6.0 6.0 6.1 6.0 6.2 6.2
0.6 0.8 0.8 0.4 0.7 0.4 0.5
a τ PC1 and τPC2 denote the thicknesses for the inner and outer PC layers, and τPro denotes the thickness of the protein layer. FPC1, FPC2, and FPro are their respective scattering length densities. ΓPro represents protein surface excess.
Figure 7. Neutron reflectivity measured at the PC bilayer coated silicon oxide/D2O interface with lysozyme concentration at 0 (dashed line), 0.03 g dm-3 (O), and 4 g dm-3 (4). The continuous lines were calculated by taking the oxide layer to be 12 ( 2 Å (defect free), the inner PC layer to be 16 ( 2 Å, the outer PC layer to be 20 ( 2 Å, and the lysozyme layer to be 45 ( 2 Å. The slightly decreased scattering length density in the outer PC layer indicated the mixing of lysozyme, and this observation is consistent with the apparently shorter lysozyme layer thickness.
adsorbed layers is strongly dependent on protein concentration and solution pH.23-27 In the case of adsorption onto the OTS/water interface,28 proteins are completely denatured to form fragmented polypeptide distributions. The volume fraction of the polypeptides within the interfacial region can be as high as 90%. These structural differences together with the variation in the adsorbed amount clearly indicate the physicochemical effect of the surfaces, though it is still difficult to produce a generalized trend for these interactions. The antifouling effect of pentadecyl-1-ol (C15OH) chemically grafted onto the SiO2 surface has also been examined in our recent work,15 and the surface excesses (ΓC15OH) are also included in Table 3 for comparison. The presence of the terminal organic hydroxy groups substantially reduces protein adsorption, and the extent of reduction can be justified by comparing the respective surface excesses on (24) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438. (25) Su, T. J.; Lu, J. R.; Cui, Z. F.; Thomas, R. K.; Penfold, J. J. Phys. Chem. B 1998, 102, 8100. (26) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F. J. Phys. Chem. B 1999, 103, 3727. (27) Murphy, E. F. Ph.D. Thesis, University of Surrey, Guildford, U.K., 1999. (28) Lu, J. R.; Su, T. J.; Thomas, R. K.; Rennie, A. R.; Cubit, R. J. Colloid Interface Sci. 1998, 206, 212.
Figure 8. Neutron reflectivity measured at the PC bilayer coated silicon oxide/pure D2O interface before (O) and after (+) protein adsorption. The overlap between the two measured reflectivity profiles indicates the complete removal of the adsorbed protein and the stability of the PC bilayer.
C15OH with those on OTS. Also, the values of ΓC15OH are very close to the surface excesses on PC polymer and monolayer surfaces. Such a large extent of protein reduction on the organic hydroxy surface suggests the importance of a balanced surface hydrophobicity in improving the antifouling effectiveness of the surface and points to the direction for theoretical consideration in understanding the reduction in protein adsorption. However, although the organic hydroxyl surface does not appear to induce the denaturation of globular proteins such as lysozyme it tends to elicit the activation of platelets and complements and is therefore not truly biocompatible. A number of literature studies have been devoted to the understanding of the antifouling performance of different interfaces.3,6,29-36 These studies have indicated the importance of surface hydrophobicity and surface charge, but the chemical nature of the surfaces has not been properly considered. Because of the interrelation between these parameters, it has been difficult to generate any (29) Horbett, T. A.; Brash, J. L. Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (30) Prime, K.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (31) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (32) Granfeldt, M. K.; Miklavic, S. J. J. Phys. Chem. 1991, 95, 6351. (33) Sheng, Q.; Schulten, K.; Pidgeon, C. J. Phys. Chem. 1995, 99, 11018. (34) Ruiz, L.; Hilborn, J. G.; Leonard, D.; Mathieu, H. J. Biomaterials 1998, 19, 987. (35) Lindon, J. N.; McManama, G.; Kushner, L.; Merrill, E. W.; Salzman, E. W. Blood 1986, 68, 355. (36) Roth, C. M.; Lenhoff, A. M. Langmuir 1993, 9, 962.
Protein Adsorption on a Phospholipid Monolayer
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Table 3. Comparison of Adsorption of the Three Model Proteins onto Different Substrates Measured at the Solid-Water Interfacea protein lysozyme BSA fibrinogen
C g dm-3
pH
ΓSiO2 mg m-2
ΓOTS mg m-2
ΓPC100B mg m-2
ΓC15OH mg m-2
ΓPCm1 mg m-2
ΓPCm2 mg m-2
ΓPCbi mg m-2
0.03 1 0.05 2 0.1 1
7 7 5 5 7 7
1.0 3.7 2.0 2.8 5.0 5.8
1.9 4.2 2.2 3.8 5.2 6.4
0.4 1.2 0.4 1.0 0.3 0.7
0.7 1.6 1.2 1.2 1.0 1.0
0.2 1.2 0.4 1.0 0.3 0.7
0.7 1.3 1.0 1.1 0.7 0.7
0.6 0.8 0.4 0.7 0.4 0.5
aΓ SiO2, ΓOTS, ΓPC100B, andΓPC15OH denote surface excesses adsorbed on the surfaces of bare silicon oxide (refs 24-27), octadecyl trichlorosilane (OTS) terminally grafted onto silicon oxide (ref 28), PC polymer with a methacrylate backbone bearing dodecyl chains, isopropyl groups, silyl cross-linkers, and PC groups (PC100B) (refs 7 and 8), and pentadecyl-1-ol terminally grafted on silicon oxide (ref 15). The surface excesses adsorbed on small PC molecule coated surfaces are denoted by ΓPCm1 (grafted by stepwise connection (ref 18)) and ΓPCm2 and ΓPCbi (monolayer and bilayer coatings formed by the one-step coating scheme using the APTMS-APC dimer). The experimental errors are about (0.3 mg m-2.
useful outline to describe the effect of surface properties on its potential in reducing protein adsorption. The extent of complexity is well exemplified by the results obtained so far from our own studies. For example, although it is commonly believed that the hydrophilic surface is most effective at reducing protein adsorption, we have found that the lowest surface excess tends to occur on the surface with intermediate contact angles. Under the conditions where the surface charge is insignificant, the hydrophilic silicon oxide surface induces as much adsorption as the hydrophobic OTS. The main difference is that whereas the proteins adsorbed on OTS are completely denatured those adsorbed on the hydrophilic surface retain their globular structures. The PC dimer is “H” shaped. Its fully extended height was estimated to be about 20 Å, with the fully extended spacing between the two main chains to be around 13 Å and the total volume to be 1240 Å3.37,38 At the low coating concentration, the PC layer was found to be 18 ( 2 Å, in good agreement with the formation of a PC monolayer. Given that the volume fraction of PC dimer (φPC) is 0.65, its area per molecule (APC) can be calculated from eq 4 and the value was found to be 106 ( 10 Å2. If the cross-sectional area of the dimer is assumed to take a circular projection with the radius of 6.5 Å, its limiting area is 133 Å2 and is clearly higher than the calculated value. An alternative assumption is the projection of the cross-sectional area in the form of a rectangle. The width of the rectangle is then likely to be determined by the bulky PC headgroup. Following one of our recent studies on the surface adsorption of dodecyl phosphocholine (C12PC) on the surface of water, the limiting area per molecule at the critical micellar concentration was found to be 48 ( 3 Å2. Taking the PC groups on the dimer to be the same as the C12PC surfactant, the radius of its PC headgroup is about 4 Å and the resultant limiting area for the rectangle is 104 Å2, close to the value of 106 Å2 determined from the coated PC monolayer. The above calculation is meant only to outline the main structural feature at the interface. The attachment of a second PC layer though chemical bonding onto the outer surface is a good indication that not all the silyl groups
on the dimers have been connected onto the substrate and some degree of structural disorder certainly exists within the interface. One might expect that the attachment of the outer PC layer leads to an increased adsorption as a result of the poor packing on the outer surface, but Table 3 shows that the residual protein adsorption on the PC bilayer surface is marginally lower than on the PC monolayer and PC polymer surfaces, thus implying an effectively better shielding on the bilayer surface. Detailed information about the conformational structure of the PC groups and the underlying layers can be obtained when partially labeled PC compounds are used in neutron reflection. This work is being planned. Conclusions The chemically grafted PC monolayer surfaces are as effective as PC polymers in inhibiting protein deposition in vitro. The single-step attachment described in this work allows a more direct control over the surface coverage of PC groups, thus enabling a more systematic experiment to be made in the future to establish the relationship between the PC coverage and the residual amount of protein adsorption. The more simplified synthetic route over the one reported previously indicates the attractive potential in making the monolayer coating as commercially competitive as a polymer coating. In a range of chemical and medical applications, the monolayer coating may have advantages. Examples are the surface modification for dialysis membranes and nanofiltration membranes where the size of membrane pores is comparable to that of polymers and the coating of the polymer onto the surface of the inner pores is prohibited by the size exclusion.39 Monolayer coating allows an easier modification of inner and outer pore surfaces without altering the pore size and the subsequent permeate flux significantly. Acknowledgment. We thank the Engineering and Physical Sciences Research Council (EPSRC) for support through a ROPA grant. We also thank Biocompatibles for funding. LA0017429
(37) Weast, R. C. Handbook of Chemistry and Physics; CRC: Cleveland, OH, 1971. (38) Tanford, C. J. Phys. Chem. 1972, 76, 3020.
(39) Palkar, S. A.; Lenhoff, A. M. Colloids Surf., A 1996, 110, 119.
