Direct Measurement of Protein Adhesion at Biomaterial Surfaces by

Technology (IBITECH), University of Gent, Krijgslaan 281 S4bis, B-9000 Gent, Belgium,. Laboratory of Biophysics and Surface Analysis, Department of ...
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Direct Measurement of Protein Adhesion at Biomaterial Surfaces by Scanning Force Microscopy S. O. Vansteenkiste,*,† S. I. Corneillie,† E. H. Schacht,† X. Chen,‡ M. C. Davies,‡ M. Moens,§ and L. Van Vaeck§ Polymer Materials Research Group, Department of Organic Chemistry, Institute Biomedical Technology (IBITECH), University of Gent, Krijgslaan 281 S4bis, B-9000 Gent, Belgium, Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, U.K., and Department of Chemistry, University of Antwerp (UIA), Universiteitsplein 1, B-2610 Wilrijk, Belgium Received December 18, 1998. In Final Form: November 10, 1999 The adsorption of human serum albumin (HSA) onto tailor-made polyurethane biomaterial coatings was measured in a liquid environment by scanning force microscopy (SFM). The adhesion of HSA onto polyurethane films was probed by adhesion force measurements with protein-coated SFM tips. Results reveal that HSA adsorbs readily onto hydrophobic polyurethane surfaces. Adsorption time profiles of the HSA deposition were elucidated by dynamic in situ force-distance measurements. The introduction of poly(ethylene glycol) (PEG) grafts, present at the biomaterial interface, inhibited all interactions with HSA. Latter results were explained by the combined effects of steric repulsion forces, minimal interfacial free energy, and high chain mobility of the hydrated PEG grafts. These phenomena were in excellent agreement with measurements obtained in surface plasmon resonance experiments.

Introduction invention,1

scanning force microscopy (SFM) Since its has been an efficient tool for the topographic investigation of surfaces with molecular or atomic resolution.2 In the past few years, applications have extended into the field of biology,3,4 (bio)medicine,5,6 biochemistry,7 and material engineering.8 The ability of SFM to investigate a wide range of surface characteristics by force-distance measurements, e.g., local mechanical properties such as friction and elasticity, adhesion, and various intermolecular forces including van der Waals, electrostatic, structural, hydration, and sterical forces, has recently been highlighted.9 Moreover, the high sensitivity of the SFM technique resulted in the detection, quantification, and mapping of extremely small forces.10 Specific binding events between individual molecules such as hydrogen bonding, avidin-biotin pairs, antibody-antigen recognition, and complementary DNA-strand complexation within a controlled environment, have recently been reported.11-14 †

University of Gent. University of Nottingham. § University of Antwerp. ‡

(1) Binnig, G.; Rohrer, H.; Gerber, C.; Weibel, E. Phys. Rev. Lett. 1982, 49, 57. (2) Bottomley, L. A.; Coury, J. E.; First, P. N. Anal. Chem. 1996, 68, 185R-230R. (3) Shao, Z.; Mou, J.; Czajkowsky, D. M.; Yang, J.; Yuan, J. Adv. Phys. 1996, 45, 1. (4) Ikai, A. Surf. Sci. Rep. 1996, 26, 261. (5) Vansteenkiste, S. O.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Prog. Surf. Sci. 1998, 57 (2), 95. (6) Arnsdorf, M. F.; Xu, S. J. Card. Electrophysiol. 1996, 7, 639. (7) Radmacher, M.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Science 1994, 265, 1577. (8) Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. In Biocompatibility: Assessment of Materials and Devices for Medical Applications; Braybook, J., Ed.; John Wiley & Sons: London, 1997; p 65. (9) Butt, H. J.; Jaschke, M.; Ducker, W. Bioelectrochem. Bioenerg. 1995, 38, 191. (10) Cappella, B.; Baschieri, P.; Frediani, C.; Miccoli, P.; Ascoli, C. IEEE Eng. Med. Biol. 1997, 16, 58. (11) Hoh, J. H.; Cleveland, J. P.; Prater, C. B.; Revel, J. P.; Hansma, P. K. J. Am. Chem. Soc. 1992, 114, 4917. (12) Moy, V. T.; Florin, E. L.; Gaub, H. E. Science 1994, 266, 257.

Polyurethanes (PUs) are a class of materials frequently used in a broad range of biomedical applications such as the artificial heart or catheters.15 On exposure to body fluids, protein adsorption often results in the activation of coagulation factors and subsequent thrombus formation. These interface phenomena are linked directly to surface morphology and energetics.16-18 In recognition of the importance of the interfacial chemistry, considerable effort has been made to tailor surface properties to optimize the in vivo performance while retaining favorable bulk properties (e.g., mechanical strength, flexibility, ...). Among a wide range of amphiphilic polymers which are frequently employed in the design of biocompatible interfaces, poly(ethylene glycol) (PEG) is recognized as particularly effective in obtaining “protein-resistant” or blood-compatible biomaterials, because of its unique solution and surface properties in physiological media.19 Recent progress in measuring and imaging “artificial” model surfaces, e.g., self-assembled monolayers (SAMs) or Langmuir-Blodgett films with modified SFM tips,20-22 suggested that the state of the art of detecting small interaction forces would be sufficient for sensing protein adhesion and/or resistance of candidate biomaterials. (13) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477. (14) Lee, U. G.; Chrisey, L. A.; Colton, R. J. Science 1994, 6, 771. (15) Joshi, R. R.; Underwood, T.; Frautschi, J. R.; Phillips, R. E.; Schoen, F. J.; Levy, R. J. J. Biomed. Mater. Res. 1996, 31, 201. (16) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers 1: Surface Chemistry and Physics; Plenum Press: New York, 1985. (17) Missirlis, Y. F.; Lemm, W. Modern Aspects of Protein Adsorption on Biomaterials; Kluwer-Academic Publishers: Dordrecht, The Netherlands, 1991. (18) Horbett, T. A.; Brash, J. L. Proteins at Interfaces II: Fundamentals and Applications; ACS Symposium Series 602; American Chemical Society, Washington, DC, 1995. (19) Lee, J. H.; Lee, H. B.; Andrade, J. A. Prog. Polym. Sci. 1995, 20, 1043. (20) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Davies, J.; Dawkes, A. C.; Edwards, J. C. Langmuir 1997, 3, 44106. (21) Ito, T.; Namba, M.; Bu¨hlmann, P.; Umezawa, Y. Langmuir 1997, 13, 4323. (22) Van der Vegte, E. W.; Hadziioannou, G. Langmuir 1997, 3, 44357.

10.1021/la981740c CCC: $19.00 © 2000 American Chemical Society Published on Web 03/07/2000

Protein Adhesion at Biomaterial Surfaces

Figure 1. Chemical structures (a) of poly(THFCD) and (b) of linear polyurethane PU-BD and PEG-grafted polyurethane PU-PEG750.

Hence, the methodology presented in this paper involves the use of protein-modified SFM tips to access in situ the protein adhesion onto PEG-grafted polyurethanes (PEGPUs). Latter materials are envisioned as biocompatibilising coatings for medical devices such as catheters or stents. Materials and Methods Materials. Hexamethylene diisocyanate (HDI; Fluka, Bornem, Belgium), 1,4-butanediol and ethylene glycol (Acros Chimica, Geel, Belgium), dibutyltin diacetate (DBTDAc; Aldrich, Bornem, Belgium), (3-aminopropyl)dimethylethoxysilane (APDES; Fluorochem. Ltd., Old Glossop, Derbyshire, U.K.), human serum albumin (HSA; Sigma Chemical Co., Poole, Dorset, U.K.), and polystyrene (PS; Aldrich, Bornem, Belgium; Mw ) 35 000 g mol-1, Mw/Mn ) 1.03) were all used as received. Poly(THFCD), a difunctional linear polyether-polycarbonate-diol (Mw ) 2000 g mol-1) (Figure 1a), was a kind gift of BASF (Ludwigshafen, Germany). R-Methoxy-ω-diethanolamine-functionalized PEG (Mw ) 750 g mol-1), a specialty chain extender, was synthesized using the method detailed in ref 23. Poly(THFCD)- and R-methoxy-ω-diethanolamine-functionalized chain extenders were dried over P2O5 under reduced pressure at 60 °C (4 h) immediately before use. Preparation and Characterization of Polyurethane Materials. PUs were prepared by a classical two-step solution polymerization.23 Briefly, poly(THFCD) was endcapped with hexamethylene diisocyanate (HDI) in the presence of DBTDAc. The resultant prepolymer was reacted with equivalent amounts of chain extender using 1,4-butanediol and R-methoxy-ω-diethanolamine-functionalized PEG (Mw ) 750 g mol-1) in the case of PU-BD and PU-PEG750, respectively (Figure 1b). 1H NMR spectroscopy was performed in deuterated dimethyl sulfoxide (DMSO-d6) using tetramethylsilane (TMS) as the internal standard using a Brucker (Karlsruhe, Germany) AM 360 MHz instrument to confirm the polymer structure. Gel permeation chromatography (GPC) was used to obtain the relative molecular weights and molecular weight distributions of all polymers. Analyses were performed using a 104-105 Å styragel column (Polymer Laboratories, Church Stretton, Shropshire, U.K.) equilibrated at 45 °C connected with a MELZ LCD212 refractive index detector. Polymers were analyzed as 1% (w/v) solutions in N-methylpyrrolidone using monodispersed polystyrene standards for calibration (Waters data module M730, Brussels, Belgium). The following data were obtained: PU-BD, Mw ) 105 400; Mw/Mn ) 1.4, Rg ) 16 nm; PU-PEG750, Mw ) 47 200, Mw/Mn ) 1.8, Rg ) 13 nm. (23) Corneillie, S.; Lan, P. N.; Schacht, E. H.; Davies, M.; Shard, A.; Green, R.; Denyer, S.; Wassall, M.; Whitfield, H.; Choong, S. Polym. Int. 1998, 46, 251.

Langmuir, Vol. 16, No. 7, 2000 3331 Preparation of Polymer Coatings. The silver-coated slides were cleaned in Neutracon (Decon Laboratories Ltd., Sussex, U.K.) and then rinsed exhaustively with deionized water, followed by rinsing with chloroform, propanol, and methanol in an ultrasonic water bath. Then 100 µL of a 0.5% w/v PS or PU solution in chloroform was dropped onto the slides spinning at a rate of 2000 rpm. AFM analysis revealed the continuity and the smoothness (RMS ( 2 nm) of the coatings. The slides were then glued with thermoplastic adhesive Tempfix (Agar Scientific Ltd., Stansted, Essex, U.K.) onto the bottom of a homemade glass flow-through liquid cell of 20 mm diameter, allowing an easy refreshment or exchange of the liquid in the cell. The liquid cell was fixed on the XY translation stage provided with the Explorer. Ellipsometry experiments were undertaken on the polymercoated slides using a Gaertner L116B auto gain ellipsometer (Gaertner Scientific Corp., Chicago, IL), employing a 70° angle from normal using a HeNe 633 nm laser. Typical film thicknesses of 34 ( 0.5 nm were obtained independently of the polymeric material. Surface Analyses. X-ray photoelectron spectroscopy (XPS) analysis was carried out on a VG Scientific ESCALAB Mk II (VG, East Grinstead, U.K.) instrument employing Mg KR X-rays (hν ) 1253.6 eV). Samples were mounted on a sample stub using double-sided sticky tape. For each sample a wide scan (0-1000 eV) was recorded, followed by six narrow scans in the C 1s, O 1s, N 1s, Sn 3d, Si 2p, and Ag 3d regions. The X-ray gun was operated at 12 kV and 20 mA, and the analyzer was operated in a fixed analyzer transmission (FAT) mode with a pass energy of 50 eV (wide scan) and 20 eV (narrow scans). Electron takeoff angles of 45° and 75° normal to the surface were utilized to give a variation in sampling depth (approximately 7 and 3 nm, respectively).24 Time-of-flight (TOF) spectra were obtained using a Cameca TOF-SIMS IV instrument (CAMECA Instruments, Inc., Trumbull, CT). Targets were bombarded by a 25 keV Ga+ pulsed primary ion beam (pulse < 1 ns, repetition rate 10 kHz, current 0.2 pA) rastered over an area of 100 × 100 µm. Secondary ions generated by the primary ion pulse on the target surface were extracted and accelerated to an energy of 2 keV. An einzel lens and reflectron optics were used for focusing the secondary ion beam and for energy compensation in the TOF tube. The ions were postaccelerated to 10 keV just before the channel plate detector. Timer-to-digital conversion was used for data collection up to m/z 800. The mass resolution defined as full width at halfmaximum (fwhm) was about 10 000 at m/z 30. All data were collected on samples exposed to a primary ion dose < 1013 ions cm-2 to remain within the static regime. Surface Modification of SFM Probes with HSA. The Si3N4 probes (TopoMetrix Co., Saffron Walden, Essex, U.K.) are covalently coated with HSA using a method similar to that of Vinckier et al.25 Briefly, probes were cleaned and oxidized with oxygen plasma for 10 s at 30 Pa and 10 W power immediately before being immersed into a 4% (v/v) toluene solution of APDES for 2 h. The silanized probes were then rinsed in methanol followed by a sodium phosphate buffer (10 mM, pH 7.0) before being immersed into a 2.5% (v/v) aqueous solution of gluteraldehyde for 30 min and then rinsed with an excess of water. Finally, each probe was brought into a 0.01% (w/v) HSA solution (10 mM sodium phosphate buffer, pH 7.0) for 1 h. Each new probe was freshly coated before use, rinsed, and kept in a buffer solution. Statistical results of the adhesion forces measured on -CH3, -COOH, and -NH2 C-12 SAMs on gold showed no significant deterioration of the HSA-coated tip within 8 h of use or 48 h of storage time.26 Adhesion Force Measurements. The SFM instrument employed was a TMX2000 Explorer (TopoMetrix Co., Saffron Walden, Essex, U.K.) using a 3 × 3 µm scanner tube operating at 20 °C under liquid. The force-displacement measurement is (24) Shard, A. G.; Davies, M. C.; Li, Y. X.; Volland, C.; Kissel, T. Macromolecules 1997, 30, 3051. (25) Vinckier, A.; Heyvaert, I.; D’Hoore, A.; McKittrick, T.; Van Haesendonck, C.; Engelborghs, Y.; Hellemans, I. Ultramicroscopy 1995, 57, 337. (26) Chen, X.; Patel, N.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Davies, J.; Dawkes, A. C.; Edwards, J. C. Appl. Phys. A: Mater. Sci. Prog. 1998, 66, S631.

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achieved by varying the voltage applied on the piezoelectrical crystal (z-piezo) which controls the SFM probe’s vertical displacement, while the lateral scanning is temporally suspended. When the z-piezo extends, the probe-sample separation decreases, until the probe is brought into contact with the sample up to a preset point of maximal load. The z-piezo is then retracted, and the probe is withdrawn from the sample surface. During a full z-piezo extension-retraction cycle, the recorded z-piezo voltage, which represents the relative probe-sample distance, and the deflection of the cantilever, which represents the probesample interaction, form a force-displacement curve. To convert the relative force scale (nA) recorded in the Explorer into the absolute force scale, two steps in calibration are performed. First, using the gradient of the contact region in the retract portion of the force-displacement curve, the signal (nA) is transferred into a corresponding probe vertical movement or, equivalently, the deflection of the cantilever in nanometers. The force applied on the cantilever in nanonewtons is then calculated from Hookes law with the known spring constant of the cantilever. The spring constant (0.032 nN/nm) provided by the probe manufacturer was employed in the present study. Although this value possesses a large error, only the relative force differences are important in the in situ comparison of protein adhesion. Dynamic Contact Angle Measurements. The surface free energies were investigated using a dynamic contact angle analyzer (DCA-322, Cahn Instruments, Inc., Cerritos, CA). The advancing and receding contact angles were obtained by dip coating carefully cleaned stainless steel plates (7 × 1 × 20 mm3) using a 0.5% (w/v) solution of the polyurethane materials in chloroform. All samples were analyzed after overnight drying in a dust-free chamber at room temperature. Contact angles and the corresponding hystereses were obtained at room temperature using distilled water and ethylene glycol as the probe liquid. Immersion cycles were recorded from 0 to 12 mm at an immersion speed of 39 µm/s. All results presented in this paper are the average of five experiments. Free surface energies were determined from the advancing contact angles of water and ethylene glycol according to the harmonic mean method of Wu.27 Surface Plasmon Resonance (SPR) Analysis. SPR analysis was performed on an instrument constructed by Ortho Clinical Diagnostics (Ortho Clinical Diagnostics Ltd., Chalfont St. Giles, Buckinghamshire, U.K.). It utilizes a monochromatic laser light source (780 nm) focused onto a glass slide coated with approximately 50 nm of silver. Optical coupling between the prism and the SPR slide was achieved with index matching oil. The totally internally reflected laser light intensity, which included the SPR signal, was analyzed by a two-dimensional charged coupled device (CCD) array. Three isolated flow channels were formed across the PU coatings by pressing a temperatureregulated copper block at 34 ( 0.1 °C against Ag-coated sensors. For monitoring the HSA adsorption, a HSA solution (0.05% w/v) was pumped (240 µL/s) over the polymer films followed by buffer washes (10 mM sodium phosphate, pH 7.0) to remove loosely bound material. The shift in the SPR angle was measured after the buffer washes in units of millidegree angles (mDA) where 1 mDA ) 0.001° (n ) 8).

Results and Discussion XPS Analysis. XPS analysis was undertaken on the native and polymer-coated silver slides. The wide-scan XPS spectrum of the native slide is dominated by two large peaks, originating from the Ag 3d electrons at 368 eV. The continuous polystyrene film gives a single C 1s peak at 285 eV. Both PU materials show the expected peaks for C 1s, N 1s, and O 1s at 290, 405, and 538 eV, respectively. The analysis indicated the presence of trace amounts of tin catalyst (Sn 3d5/2 at 486 eV) originating from the polyurethane synthesis. In all cases, the Ag 3d peak is undetectable in the spectra, confirming the film homogenicity. XPS has proved to be a most valuable tool for characterizing poly(ether urethane) surfaces. A number of reports (27) Wu, S. Polymer Interface and Adhesion; Marcel Dekker: New York, 1982; p 178.

Vansteenkiste et al. Table 1. Comparison between Bulk Elemental Analysis and XPS Data for Polyurethanes PU material PU-BD PU-PEG750

takeoff angle

O/C

N/C

bulk 75° 45° bulk 75° 45°

0.387 0.296 0.348 0.403 0.349 0.374

0.031 0.022 0.028 0.027 0.015 0.029

have shown that the surface of these domain-forming segmented materials can exhibit an enrichment of polyether compared to the bulk, especially under the highvacuum conditions applied during the analysis.28,29 Therefore, in the study presented here, the use of angulardependent XPS for nondestructive depth profiling of casted PU films was explored. A comparison between the XPSderived elemental ratios as a function of sample depth and those expected based upon the stoichiometry of the polyurethanes is shown in Table 1. The experimental data clearly indicate large differences between expected and XPS-measured elemental ratios, particularly the N/C ratio. For example, PU-BD and PUPEG750 O/C ratios approach 90% of the theoretical bulk values at a penetration depth of (7 nm, whereas closer to the surface these levels drop to 76% and 87%, respectively, the latter value being higher because of the incorporation of PEG grafts. A similar trend is echoed in the N/C data, where the magnitude of the discrepancy is even more dramatic, especially for the PEG-containing polymer. These observations suggest that the soft segment migrates directly (Tg ) -67 °C) to the PU surface to form an interface layer enriched with the polyether. This preferential orientation of the soft segment conforms to the thermodynamic requirement of lowest surface free energy. Moreover, angular-dependent XPS data indicate that the more polar hard segment domains are largely localized within the bulk of the material. These phenomena are consistent with earlier findings on the surface characterization of segmented polyurethanes reported by Ratner et al.30,31 TOF-SIMS Analysis. The positive ion spectrum for PU-BD is typical for a number of segmented polyurethane materials prepared with PTHFCD diol. A series of low mass peaks appearing at 55, 71, 73, 85, 127, and 145 D can be attributed directly to fragments of the polyether PTHF units (Table 2).31 In contrast with earlier studies reported by Hearn et al.32 investigating the surface structure of polyurethanes with both aromatic and fluorinated chain extenders, no characteristic fragments originating from hard segments could be detected. This observation is consistent with the compositional gradients existing within the surface region as indicated by the XPS data. Moreover, one should note that the SIMS sampling depth of (1 nm is only representative for one or two monolayers, emphasizing sample depth heterogenicity. The positive ion spectrum of PU-PEG750 shows, besides the major peaks typical for the PTHFCD soft segment, evidence of diagnositic m/z values originating from the speciality chain extender. For example, the peak at m/z (28) Graham, S. W.; Hercules, D. M. J. Biomed. Mater. Res. 1981, 15, 465. (29) Paynter, R. W.; Ratner, B. D.; Thomas, H. R. In Polymers as Biomaterials; Shalaby, S. W., Hoffman, A. S., Ratner, B. D., Horbett, T. A., Eds.; Plenum Press: New York, 1984; p 121. (30) Yoon, S. C.; Ratner, B. D. Macromolecules 1986, 19, 1068. (31) Hearn, M. J.; Ratner, B. D.; Briggs, D. Macromolecules 1988, 21, 2950. (32) Hearn, M. J.; Briggs, D.; Yoon, S. C.; Ratner, B. D. Surf. Interface Anal. 1987, 10, 384.

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Table 2. Possible Structures of Characteristic Positive Secondary Ions from PU Materials

) 45 D, but in particular the peaks at m/z ) 59 and 103 D ([nM + CH3]+ series), which derive from the end group cleavage of the R-methoxy chain extender, occur with relatively high intensity, indicating the presence of hydrophilic PEG grafts at the uppermost surface, even under the applied vacuum conditions. This observation is rather surprising but consistent with the corresponding XPS results, demonstrating considerably higher O/C ratios for the PEG-grafted material. This may be explained by the relatively high PEG content in the PU-PEG750 material, namely, 14.6 wt %, and/or by the polymer architecture. Recently, XPS and SSIMS analysis of solvent-casted films of ABA block copolymers of PEG and poly(lactic and glycolic acid) (PLGA) by Shard et al.24 revealed that the PLGA component resides almost exclusively at the copolymer surface, even at percentages of PEG (B block) up to 40 wt%. Therefore, the present study demonstrates that grafting is an effective method of introducing flexible PEG segments at the interface.

Adhesion Force Measurements. Typical forcedisplacement retraction curves obtained for PU-BD and PU-PEG750 coatings with an HSA-modified SFM tip are presented in Figure 2. The corresponding distributions of the observed pulloff forces are visualized in Figure 3. The observed hysteresis in the retracting forcedisplacement curve of PU-BD is gradual and continuous. The pulloff is a characteristic feature of nonspecific probesample adhesion phenomena.10 Therefore, the pullout measured is explained by attractive hydrophobic and/or van der Waals forces33,34 between the HSA-modified tip and the uncharged surface of PU-BD in an aqueous environment. In a control experiment conducted with the (33) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992. (34) Christenson, H. K. In The long-range Attraction between Macroscopic Hydrophobic Surfaces; Schrader, M. E., Loeb, G., Eds.; Plenum Press: New York, 1992.

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Vansteenkiste et al. Table 3. Surface Energies as Calculated from Dynamic Contact Angle Measurements (n g 5)

Figure 2. Typical force-displacement curves measured on PU-BD and PU-PEG750 surfaces in 10 mM sodium phosphate buffer, pH 7.0 using HSA-modified SFM probes. Consistent maximum loading forces of =3.0 nN and a scan rate of 400 nm/s for both the extending (not shown) and retracting curves were used. The spring force constant (0.032 nN/nm) provided by the manufacturer was used for calibration.

Figure 3. Histograms of the pulloff forces between HSAmodified SFM probes and (a) PU-BD and (b) PU-PEG750. The measurements were performed in 10 mM phosphate buffer (pH 7.00) at room temperature ((20 °C).

same HSA-coated tip on a PS surface, typical adhesion forces of 12.84 ( 0.87 nN were observed consistently. In contrast to PS surfaces, the observed pulloff force of PUBD (495 ( 118 pN; n ) 250) is at least 1 order of magnitude smaller (Figure 5). The hysteresis provides information about the amphiphilicity of the interface, i.e., the content of hydrocarbons versus the presence of polar groups on the polymer surface. It can therefore be considered as a measure for the surface free energy. Hence, the inter-

polymer material

θΑ (deg)

θR (deg)

γd (10-3 J/m2)

γp (10-3 J/m2)

xd (γd/γ)

PS PU-BD PU-PEG750

97 ( 1 86 ( 1 77 ( 3

65 ( 1 47 ( 1 35 ( 3

26.5 ( 2.8 17.4 ( 0.5 16.3 ( 2.0

4.4 ( 0.7 10.4 ( 4.5 17.5 ( 3.0

0.858 0.626 0.482

pretation of these differences in adhesion forces is possible on the basis of surface free energies as calculated from dynamic contact angle experiments (DCA). When the harmonic mean method of Wu,27 which is designed for calculating the interfacial tension in polymeric systems, is applied, the total surface free energy can be separated into dispersion (nonpolar) and polar components i.e., γ ) γd + γp, where γd is a dispersion component arising from dispersion-force interactions and γp the polar component arising from various dipolar and specific interactions. The dispersity factor xd can be defined as xd ) γd/γ. The results obtained from DCA experiments are summarized in Table 3. Despite the fact that PU-BD and PS are characterized by identical surface energies (γPU-BD ) 27.8 ( 0.2 10-3J/ m2 compared to γPS ) 30.89 ( 2.11 10-3J/m2), it was observed that xdPS ) 0.858 is much larger in comparison with xdPU-BD ) 0.626. Therefore, it is not surprising that the adhesion force between an HSA-modified tip and a PS surface is significantly higher compared with the PUBD interface. This result is in excellent agreement with earlier findings of Sagvolden et al.,35 demonstrating an increased adhesion force between BSA-modified glass beads and PS surfaces with hydrophobicity. The introduction of PEG side chains (PU-PEG750) decreased the adhesion forces to negligible levels (65 ( 132 pN, n ) 240) (Figure 5). Despite the fact that the exact nature or mechanisms of the passivating effect of PEG chains is still not fully understood,36 it is generally accepted that parameters such as high chain mobility, excluded volume, and hydration forces are dominant factors governing its protein resistivity. The disappearance of the hysteresis also suggests that the surface density of the grafted PEG chains is sufficiently high to mask the underlining hydrophobic surface and exert an effective repulsion force.37-39 Moreover, no electrostatic interaction between the positively charged PU-PEG750 and the HSAcoated tip was observed. This phenomenon can possibly be explained by the architecture of the PU material in which the center carrying the positive charge is also the grafting point of the PEG chains. Attempts to estimate directly the surface coverage by the PEG side chains and therefore their conformation at the PU-water interface were not straightforward. In contrast to smooth monolayers (i.e., SAMs) or PEG chains grafted on atomically flat substrates,40,41 the situation is complicated by both the topography and morphology of the polymer film as well as by the reorientation of the hydrophilic PEG chains toward the surface after submersion into a physiological medium. An estimation of the (35) Sagvolden, G.; Giaever, I.; Feder, J. Langmuir 1998, 14, 5984. (36) Israelachvili, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8378. (37) McPherson, T. B.; Lee, S. J.; Park, K. In Proteins at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (38) Nagaoka, S.; Mori, Y.; Takiuchi, H.; Yokota, K.; Tanzawa, H.; Nishumi, S. Polymers as Biomaterials; Shalaby, S. W., Hoffman, A. S., Ratner, B. D., Horbett, T. A., Eds.; Plenum Press: New York, 1984; p 361. (39) Leikin, S.; Parsegian, V. A.; Rau, D. C.; Rand, R. P. Annu. Rev. Phys. Chem. 1993, 44, 369.

Protein Adhesion at Biomaterial Surfaces

Figure 4. Typical force-displacement curves measured on PU-BD and PU-PEG750 surfaces in 10 mM sodium phosphate buffer, pH 7.0 using Si3N4 SFM probes. Consistent maximum loading forces of =3.0 nN and a scan rate of 400 nm/s for both the extending (not shown) and retracting curves were used. The spring force constant (0.032 nN/nm) provided by the manufacturer was used for calibration.

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Figure 6. SPR sensorgrams depicting HSA adsorption onto PU-BD and PU-PEG750 (HSA concentration of 0.05% w/v).

Figure 7. Dynamic observation of HSA adsorption onto a PUBD surface. Retract force-displacement curves were measured on a PU-BD spin-cast interface with a HSA-coated probe after the surfaces were exposed to a HSA solution (0.01% w/v, pH 7.0, 10 mM sodium phosphate buffer) for 0, 10, and 30 min.

Figure 5. Histograms of pulloff forces between Si3N4 tips and (a) PU-BD and (b) PU-PEG750. The measurements were performed in 10 mM phosphate buffer (pH 7.00) at room temperature ((20 °C).

PEG surface density of a polymer layer of thickness 〈x〉 ) (π/2)1/2N0.6a ≈ 2.5 nm for PEG750 (good solvent conditions) indicates that for a PEG content of 14.6 wt% (bulk stoichiometry) the available area is about 77% of the theoretically predicted area for a mushroom configuration (area per chain ≈ RF2 with RF ) Flory radius). DCA measurements, i.e., θA ) 77 ( 3° and xdPU-PEG750 ) 0.482, confirm the presence of hydrophilic PEG chains at the material surface. Moreover, both the large contact angle hysteresis θA - θR ) 42° and the low receding contact angle θR ) 35° suggest PEG segment reorientations toward (40) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Israelachvili, J. N.; Smith, G. S. J. Phys. Chem. B 1997, 101, 3122. (41) Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 4, 8399.

the surface to produce a minimal interfacial energy. This concept is well-known in surfactant and polymer literature.42,43 Furthermore, the force-displacement curve of the PU-PEG750 substrate revealed a curved gradient and no sharp point of contact. This observation is in good agreement with a nonlinear hydration process accompanied by a reorientation of the PEG chains in PU coatings resulting in a similar bending of the AFM cantilever.44 Therefore, taking into account the results of both the TOFSIMS and XPS analyses as well as the preferential orientation of PEG at the surface in a water environment, it is very likely that the grafted PEG segments are occurring in a polymer brush regime. As a control experiment, the pulloff forces between unmodified Si3N4 tips and PU coatings were assessed. Figure 4 shows a typical example of the observed forcedisplacement retraction curve. The corresponding histogram of the measured pulloff forces is given in Figure 5. Routinely, a rather large adhesion force of 3.77 ( 0.84 nN (n ) 240) was determined between unmodified tips and PU-BD substrates. This observation reflects the hydrophobic nature of untreated Si3N4 tips, θA ) 64.6 ( 1.3° in water.21 On the other hand, negligible adhesion forces (30 ( 70 pN, n ) 250) were detected for PU-PEG750 samples, evidencing once more the presence of hydrophilic PEG grafts at the polymer interface. Monitoring the HSA adsorption in evanescent wave techniques such as SPR supported and complemented the information obtained by force-displacement measure(42) Malmsten, M.; Claesson, P. M.; Perzon, E.; Perzon, I. Langmuir 1990, 6, 1572. (43) Grainger, D.; Okano, T.; Kim, S. W. In Advances in Biomedical Polymers; Gebelein, C. G., Ed.; Plenum Press: New York, 1987; p 229. (44) Green, R. J.; Corneillie, S.; Davies, M. C.; Roberts, C. J.; Schacht, E. H.; Tendler, S. J. B. Langmuir 1999, in preparation.

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Figure 8. Histograms of pulloff forces between HSA-modified SFM probes and PU-PEG750 after (a) 0, (b) 10, and (c) 30 min.

ments. Flowing a HSA solution (0.05% w/v) over a PUBD polymer film resulted in the deposition of a HSA monolayer (∆θ ) 110 ( 10 mDA; Figure 6). Earlier adsorption profiles of HSA on PS, obtained under identical flow and buffer conditions, reported by Green et al.45 showed that the level of adsorption increases to a solution concentration of 0.05% w/v, beyond which the angle shift values were not significantly different. Although the reported adsorption kinetics were slower, the observed plateau value is indicative for the much higher albumin content in blood (4% w/v). Moreover, these attained values compared well with corresponding AFM and ellipsometry data on protein monolayer packing and thickness, respectively.23,45,46 Surprisingly, SPR sensorgrams for PS coatings show identical saturation profiles,45 unable to differentiate between both PS and PU-BD polymeric substrates. In contrast, data obtained from force-displacement experiments clearly offer a better opportunity to relate surface physicochemistry to protein adsorption phenomena. PU materials containing PEG chains resisted HSA adsorption as expected from force-displacement measurements. Varying the chain length of the PEG segment from 350 to 1900 g mol-1 did not influence the protein resistivity of the polymer surface.47 The time evolution of force-displacement curves when a PU-BD film was exposed to a HSA solution is shown in Figure 7. Before the protein solution was introduced into the liquid cell, adhesion forces due to probe-surface interactions were observed (Figure 2). The adhesion force strength decreased to about half its original value (495 (118 pN; n ) 250) after 10 min. Prolonged incubation (30 min) resulted in a disappearance of adhesion forces (7 ( 23 pN; n ) 240; Figure 8). Because at pH 7.0 HSA is known to possess a net charge of -18 (IEP 5.3),48 the lack of adhesion forces suggests the presence of a repulsive electrostatic “double-layer” force at large separations. Therefore, this SFM experiment clearly demonstrates that under static conditions HSA is adsorbed onto the PU-BD coating, resulting in a net negative charge on both the HSA-covered PU-BD and the SFM tip surface. (45) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 1997, 18, 405. (46) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. J. Biomed. Mater. Res. 1998, 42 (2), 165. (47) Corneillie, S. I. Ph.D. Thesis, University of Gent, Belgium, 1998. (48) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153.

Conclusions In this paper we have used both XPS and TOF-SIMS to investigate the surface chemistry of solvent-cast films of polyurethane materials. Angular-dependent XPS analysis showed a preferential orientation of the more hydrophobic soft segment toward the material interface, conforming to the thermodynamic requirement of lowest surface free energy. TOF-SIMS of polyurethanes, characterized by a high PEG content, demonstrated the occurrence of PEG grafts at the surface even under the vacuum conditions applied. Dynamic contact angle measurements indicated the presence of hydrophilic PEG chains at the surface of PEGcontaining polyurethanes in an aqueous medium. Moreover, the large contact angle hysteresis suggested PEGsegment reorientations toward the surface to produce a minimal interfacial energy. Direct force-displacement measurements performed with both an untreated Si3N4 tip and a protein-modified SFM tip were capable of evaluating quantitatively the protein adhesion onto polyurethane surfaces. For hydrophobic PU materials, the protein monolayer formation was studied under static conditions as a function of time by monitoring the pulloff force between the HSA-modified tip and the polymer substrate. Introduction of PEG-grafted chains present at the PU-liquid interface resulted in a complete disappearance of the observed hysteresis. Results could be rationalized by the hydrophilic/hydrophobic balance of both tip and polymer surfaces. Results obtained in force-distance experiments were in excellent agreement with observations made in evanescent wave techniques such as SPR. Therefore, we propose in situ force-displacement measurements as a novel procedure for a rapid screening of a wide variety of candidate biomaterials (i.e., PEG-containing polyurethanes) for their protein resistivity within a controlled aqueous environment. Acknowledgment. S.O.V. thanks the EC for financial support by providing a Training and Mobility Research grant (ERB4001GT957062). The authors also thank the Flemish Institute for the promotion of Scientific-Technological Research in Industry (IWT), the Fund for Scientific Research-Vlaanderen (FWO), and the Belgian Ministry of Scientific Programming (IUAP/PAI-IV/11). LA981740C