Measurement of the Interaction Forces between Proteins and Iniferter

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Measurement of the Interaction Forces between Proteins and Iniferter-Based Graft-Polymerized Surfaces with an Atomic Force Microscope in Aqueous Media Satoru Kidoaki,†,‡ Yasuhide Nakayama,† and Takehisa Matsuda*,§ Department of Bioengineering, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan, and Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan Received January 4, 2000. In Final Form: June 28, 2000 To investigate the characteristics of interaction forces between proteins and end-grafted polymer surfaces, force-versus-distance curves (f-d curves) were measured between protein-fixed probe tips (albumin (Alb) and lysozyme (Lyso)) and surfaces graft-polymerized with N,N-dimethylacrylamide (DMAAm) or acrylic acid (AAc) in an aqueous solution, using an atomic force microscope. DMAAm graft-polymerized surfaces with different chain lengths and AAc graft-polymerized surface were prepared by photopolymerization on a dithiocarbamate (iniferter)-immobilized surface. The effects of grafted chain length, grafting density, and electrostatic property of the grafted chain segments on the interaction forces in the processes of protein adsorption onto and desorption from the graft-polymerized surfaces were analyzed from the approaching and retracting traces of the observed f-d curves, respectively. (1) In the Alb/poly(DMAAm) system, steric repulsion was observed, in which the interaction range and the compressive force of the poly(DMAAm) layer linearly increased with increasing chain length of poly(DMAAm) except for very short chain lengths. Adhesion force was observed only for the poly(DMAAm) layer with short chains. (2) In the Alb/poly(AAc) system, repulsive force due to steric and electrostatic interactions, and “tooth-like” adhesion forces were observed. (3) In the Lyso/poly(AAc) system, electrostatic attraction and adhesion forces were observed. From observation 1, the grafting density, the elastic modulus of the poly(DMAAm) layer, and the conformation of the grafted chain (“mushroom” or “brush”) were deduced and are discussed in relation to the characteristics of the interaction force with the proteins. From observations 2 and 3, it was found that a polyanionic surface can provide a significant adhesion force not only to positively charged proteins but also to negatively charged ones at physiological pH.

Introduction Minimal biocolloidal adsorption has often been required in various surface-dependent industrial segments such as cosmetics, food processing, shipbuilding, and biomedical device fabrication. Protein adsorption onto the surface of a foreign material is the initial event that occurs when a surface comes in contact with physiological fluids such as blood, which is then followed by a series of biological reactions, such as platelet and leucocyte adhesion, blood coagulation, fibrinolysis, and thrombus formation. The biocompatibility of a material is known to be conditioned by the layer of protein initially adsorbed onto its surface.1-3 Therefore, many studies on protein adsorption onto various artificial surfaces have been carried out in order to understand the adsorption mechanisms involved or to appropriately design biocompatible artificial surfaces.1-9 * To whom correspondence should be addressed: TEL: +81-92-642-6210. FAX: +81-92-642-6212. E-mail: matsuda@ med.kyushu-u.ac.jp. † National Cardiovascular Center Research Institute. ‡ Present address: Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan, and CREST, Japan Science and Technology Corp., Kawaguchi, Saitama 3320012, Japan. § Kyushu University. (1) Horbett, T. A. In Biomaterials: Interfacial Phenomena and Applications; Cooper, S. L., Peppas, N. A., Eds.; Advances in Chemistry Series No. 199; American Chemical Society: Washington, DC, 1982; pp 233-244. (2) Andrade, J. D., Ed. In Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985.; Vol. 2, pp 1-80. (3) Park, K.; Mosher, D. F.; Cooper, S. L. J. Biomed. Mater. Res. 1986, 20, 589-612. (4) Macritchie, F. Adv. Protein Chem. 1978, 32, 283-326.

As one of the flexible methods to design a well-defined biocompatible surface, surface modification by graft polymerization or graft reaction has been extensively investigated over the years (see reviews, 10-12). Much attention has been focused on the reduction of the degree of protein adsorption onto surfaces grafted with nonionic hydrophilic polymers, such as poly(ethylene glycol).13-24 (5) Norde, W. Adv. Colloids Interface Sci. 1986, 267-340. (6) Ivarsson, B.; Lundstrom, I. CRC Crit. Rev. Biocompat. 1986, 2, 1-96. (7) Lundstrom, I.; Ivarsson, B.; Jonsson, U.; Elwing, H. In Polymer Surfaces and Interfaces I; Feast, W. J., Munro, H. S., Eds.; John Wiley & Sons: New York, 1987; pp 201-230. (8) Tengvall, P.; Lundstrom, I.; Liedberg, B. Biomaterials 1998, 19, 407-422. (9) Elwing, H. Biomaterials 1998, 19, 397-406. (10) Ikada, Y. Biomaterials 1994, 15, 725-736. (11) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 1043-1079. (12) Elbert, D. L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26, 365-394. (13) Amiji, M.; Park, K. Biomaterials 1992, 13, 682-692. (14) Bergstro¨m, K.; Holmberg, K.; Safranj, A.; Hoffman, A. S.; Edgell, M. J., Kozlowski, A.; Hovanes, B. A.; Harris, J. M. J. Biomed. Mater. Res. 1992, 26, 779-790. (15) Gombotz, W. R.; Guanghui, W.; Horbett, T. A.; Hoffman, T. S. J. Biomed. Mater. Res. 1991, 25, 1547-1562. (16) Lee, J. H.; Kopecek, J.; Andrade, J. D. J. Biomed. Mater. Res. 1989, 23, 351-368. (17) Lin, Y. S.; Hlady, V.; Go¨lander, C.-G. Colloids Surf., B 1994, 3, 49-62. (18) Han, D. K.; Park, K. D.; Ryu, G. H.; Kim, U. Y.; Min, B. G.; Kim, Y. H. J. Biomed. Mater. Res. 1996, 30, 23-30. (19) Huang, S.-C.; Caldwell, K. D.; Lin, J.-N.; Wang, J.-K.; Herron, J. N. Langmuir 1996, 12, 4292-4298. (20) Malmsten, M.; Van Alstine, J. M. J. Colloid Interface Sci. 1996, 177, 502-512. (21) Amiji, M.; Park, K. J. Biomater. Sci., Polym. Ed. 1993, 3, 217234.

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This effect has been discussed from both thermodynamic and mechanical aspects, including the effect of minimal interfacial free energy, entropic repulsion of grafted polymer chains, and chain mobility. Many experimental as well as theoretical works25,26 have been conducted in order to understand the mechanisms involved in protein adsorption onto such surfaces. However, the magnitude or interaction range of forces between protein and surfaces remains poorly understood due to the technical difficulty in the direct measurement of the molecular interaction. Concerning the direct force measurement, recent developments in the technique of force measurement using an atomic force microscope (AFM) have provided an useful strategy for investigating the interaction between the probe tip and the polymer-adsorbed or -grafted surface in an aqueous solution; some measurements have been successfully performed using a raw Si3N4 probe tip.27-32 On the other hand, concerning force measurement involving protein molecules, techniques of protein fixation or adsorption onto the AFM probe tip surface are utilized, e.g., the measurement of single molecular forces between specific protein pairs such as avidin/biotin pairs33-36 or antigen/antibody pairs37-41 and of nonspecific adhesion forces between proteins and material surfaces such as polystyrene42,43 or glass.43 In our previous study, we measured the force existing between the protein-fixed tip and the self-assembled monolayer surface of long-chain alkanethiolates and discussed the adhesion forces between the proteins and the closely packed functional groups such as methyl, hydroxyl, amino, and carboxyl.44 As an extension of that study, we use AFM to measure the interaction force between proteins and polymer-grafted surfaces in the present study. In contrast to force measurement using a surface force apparatus (SFA), the (22) Irvine, D. J.; Mayes, A. M.; Satija, S. K. Barker, J. G. SofiaAllgor, S. J., Griffith, L. G. J. Biomed. Mater. Res. 1998, 32, 498-509. (23) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecule 1998, 21, 5059-5070. (24) Llanos, G. R.; Sefton, M. V. J. Biomed. Sci., Polym. Ed. 1993, 4, 381-400. (25) Szleifer, I. Biophys. J. 1997, 72, 595-612. (26) Halperin, A. Langmuir 1999, 15, 2525-2533. (27) Kelley, T. W.; Schorr, P. A.; Johnson, K. D.; Tirrell, M.; Frisbie, C. D. Macromolecules 1998, 31, 4297-4300. (28) Roters, A.; Schimmel, M.; Ru¨the, J.; Johannsmann, D. Langmuir 1998, 14, 3999-4004. (29) Bemis, J. E.; Akhremitchev, B. B.; Walker, G. C. Langmuir 1999, 15, 2799-2805. (30) Li, H.; Liu, B.; Zhang, Xi; Gao, C.; Shen, J. Zou, G. Langmuir 1999, 15, 2120-2124. (31) Ortiz, C.; Hadziioannou, G. Macromolecules 1999, 32, 780-787. (32) Butt, H.-J.; Kappl, M.; Muller, H.; Raiteri, R. Langmuir 1999, 15, 2559-2565. (33) Pierce, M.; Stuart, J.; Pungor, A.; Dryden, P.; Hlady, V. Langmuir 1994, 10, 3217-3221. (34) Lee, G. U.; Kidwell, D. A., Colton, R. J. Langmuir 1994, 10, 354-357. (35) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415417. (36) Moy V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257259. (37) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368-1374. (38) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477-3481. (39) Allen, S.; Chen, X.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Sefton, J.; Tender, S. J. B.; Williams, P. M. Biochemistry 1997, 36, 7457-7463. (40) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guntherodt, H.-J.; Misevic, G. N. Science 1995, 267, 1173-1175. (41) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Guntherodt, H.-J. Biophys. J. 1996, 70, 2437-2441. (42) 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, 13, 4106-4111. (43) Sagvolden, G.; Giaever, I.; Feder, J. Langmuir 1998, 14, 59845987. (44) Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646.

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Figure 1. (a) Scheme of the experimental setup. (b) Experimental setup for photograft polymerization of vinyl monomers on a dithiocarbamate-derivatized glass substrate.

AFM probe tip locally indents or partially penetrates the graft layer and interacts with polymer segments inside the layer. Therefore, from the measurement of force between the protein-fixed AFM probe tip and the graftpolymerized surface, it is expected that not only the interaction of a protein with the outermost surface of the graft layer but also the interaction inside the layer can be detected. This is an important factor in the protein adsorption process and is significantly affected by the grafting state, such as the length of the grafted chain, the surface grafting density, the spatial density of polymer segments inside the graft layer, and the electrostatic properties of segments of grafted chain. In this study, to investigate such details of a force profile between a protein and a relatively well-controlled graftpolymerized surface, the AFM force-versus-distance curve (f-d curve) was measured between protein-fixed probe tips (albumin (Alb, isoelectric point 4.7-4.9) and lysozyme (Lyso, isoelectric point 10.5-11.0)) and iniferter-based photograft-polymerized surfaces (poly(N,N-dimethylacrylamide) (PDMAAm) and poly(acrylic acid) (PAAc)) in an aqueous solution at physiological pH, as shown in Figure 1a. Since the iniferter, which is a molecule with a triple role as an initiator, a transfer agent, and a terminator in the polymerization process,45,46 can induce radical polymerization only during photoirradiation, the “graftingfrom” polymerization on the iniferter-derivatized substrate apparently proceeds in a quasi-living manner.47-50 Thus, (45) Otsu, T.; Yoshida, M. Makromol. Chem., Rapid Commun. 1982, 3, 127-132. (46) Otsu, T.; Matsumoto, A. Adv. Polym. Sci. 1998, 136, 75-137. (47) Nakayama, Y.; Matsuda, T. Macromolecules 1996, 29, 86228630. (48) Nakayama, Y.; Matsuda, T. Macromolecules 1999, 32, 54055410. (49) Lee, H. J.; Nakayama, Y.; Matsuda, T. Macromolecules 1999, 32, 6989-6995.

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a graft-polymerized surface with well-controlled chain length can be prepared by regulating the photoirradiation time. By use of iniferter chemistry, PDMAAm-grafted surfaces with different chain lengths and PAAc-grafted surfaces were prepared. The effects of grafted-chain length, grafting density, and electrostatic properties of graftedchain segments on the interaction forces produced during processes of protein adsorption onto and desorption from the graft-polymerized surfaces were analyzed from the approaching and retracting traces of the observed f-d curves, respectively. Experimental Section Materials. The following commercially available substances of reagent grade were used: bovine serum albumin (fraction V powder) (Alb, Sigma Chemical Co., St. Louis, MO), lysozyme (egg white) (Lyso, Funakoshi Co., Ltd., Tokyo, Japan), 10-carboxy1-dodecanethiol (Dojindo Laboratories, Kumamoto, Japan), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDPC, Sigma), N-hydroxylsuccinimide (NHS, Wako Pure Chemical Ind., Ltd., Osaka, Japan), (chloromethylphenyl)ethyl trichlorosilane (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan), sodium N,N-diethyldithiocarbamate trihydrate (Wako), N,Ndimethylacrylamide (DMAAm, Wako), and sodium acrylate (AAcNa, Aldrich Chemical Co., Inc., Milwaukee, WI). Solvents and other substances, all of which are also of reagent grade, were used after appropriate purification. Water was deionized using a Mill-Q reagent water system (Nippon Millipore, Ltd., Tokyo, Japan) to 18 MΩ cm resistivity (DI water). Preparation of Dithiocarbamate-Derivatized Glass Surface. Glass substrates (Matsunami Glass Ind., Ltd., Osaka, Japan) (∼120 µm thickness, 14.5 mm diameter) were thoroughly rinsed according to the following procedure: (1) sonication in an aqueous detergent and DI water, (2) immersion in 80 °C piranha solution (concentrated H2SO4:30% H2O2 ) 7:3) for 1 h, (3) rinsing with DI water, (4) rinsing with RCA-type solution (28% aqueous NH3:H2O:H2O2 ) 2.8:5:1), (5) rinsing with DI water and acetone, (6) rinsing with 1:1 solution of acetone and toluene, and (7) rinsing with toluene. Then, the cleaned glass substrates were shaken in 5% (v/v) toluene solution of (chloromethylphenyl)ethyl trichlorosilane for 18 h under argon atmosphere at room temperature and rinsed with acetone and toluene. These steps were repeated again. The obtained chloromethylated glass substrates were annealed at 115 °C for 10 min in air. Subsequently, the chloromethylated glass substrates were immersed in 5% ethanolic solution of sodium N,N-diethyldithiocarbamate trihydrate for 18 h at room temperature. After the solution was shaken, dithiocarbamatederivatized glass (DC-glass) substrates were obtained. The glass substrates were thoroughly rinsed with ethanol and DI water, air-dried, and stored in a dark desiccator. Surface Photograft Polymerization. Photograft polymerization of vinyl monomers (DMAAm and AAc) on the DC-glass surface was performed by UV irradiation of the DC-glass substrate immersed in each monomer solution, as shown in Figure 1b. After nitrogen was bubbled through a methanolic DMAAm solution (1.0 mol/dm3) and aqueous AAcNa solution (1.0 mol/ dm3), 100 µL of each monomer solution was dropped onto the DC-glass substrate placed in a glass vessel (27 mm diameter) which was then covered with sapphire glass (25 mm diameter) under a nitrogen atmosphere (∼100 µm solution thickness). UV light (5 mW/cm2 intensity, 200 W Hg-Xe lamp, L2859-01, Hamamatsu Photonics Ltd., Shizuoka, Japan) was irradiated to the DC-glass substrate at room temperature. After UV irradiation, the polymerized surfaces were repeatedly rinsed with ethanol and DI water and then nitrogen-dried. Preparation of Protein-Fixed AFM Probe Tips. Proteins were fixed on a commercially available Si3N4 AFM probe tip (DNP-S; 200-µm-long wide leg, Digital Instruments, Santa Barbara, CA) according to the following procedure: (1) The tips were vacuum-deposited with chromium (30 Å) and then gold (50) Higashi, J.; Nakayama, Y. Marchant, R. E.; Matsuda, T. Langmuir 1999, 15, 2080-2088.

Kidoaki et al. (500 Å) by thermal evaporation using the vacuum coater ULVAC VPC-260F (Shinku-kiko, Co., Ltd., Kanagawa, Japan). (2) The Au-coated tips were immersed in an ethanolic solution (1 mM) of 10-carboxy-1-dodecanethiol overnight at room temperature to prepare the self-assembled monolayer with closely packed carboxyl groups. (3) After being carefully rinsed with ethanol and nitrogen-dried, the carboxyl-functionalized tips were immersed in a 1:9 solution of EDPC (25 mg/mL in water) and NHS (2.0 mg/ml in 1,4-dioxane) with gentle shaking for 15 min at room temperature. After being thoroughly rinsed with DI water to remove any unreacted NHS, the tips were immersed in a protein solution (Alb or Lyso, 1.0 mg/mL in phosphate-buffered saline (PBS, pH 7.4), 137 mM NaCl, 2.68 mM KCl, 8.10 mM Na2HPO4, 1.47 mM KH2PO4) for 3 h at room temperature and then thoroughly rinsed with PBS in order to avoid multilayer adsorption of the protein. Concerning the characterization of the protein-immobilized AFM tip, we performed two types of analysis. One involves investigating the adsorption of proteins on the carboxylated SAM surface with sufficient area under conditions similar to those for the condensation reaction. We carried out this analysis by measuring the surface plasmon resonance (SPR670, Nippon Laser & Electronics Lab., Nagoya, Japan). As a result, we confirmed that under the reaction conditions described in the Experimental Section, the proteins adsorbed onto the carboxylated SAM surface and their kinetics showed a saturation curve (data not shown). The other method of analysis involves comparing the tip subjected to protein-immobilization reaction with the nontreated tip. We systematically performed such a comparison in our previous work.44 As a result, the treated tip gave a characteristic and reproducible adhesion profile which was clearly different from the profile obtained using the nontreated tip. The surface coverage of the proteins on the AFM tip was not determined. Surface Characterization. The chemical composition of the outermost layer of the graft-polymerized surfaces was determined by X-ray photoelectron spectroscopy (XPS) (ESCA-3400, Shimadzu Corporation, Kyoto, Japan) using a magnesium anode (Mg KR radiation) at room temperature under 5 × 10-6 Torr (10 kV, 20 mA) at an escape angle of 15°. The surface wettability was evaluated by measuring the static contact angles (advancing and receding) toward DI water using the sessile drop method with a contact angle meter (Kyowa Kaimen Kagaku Co., Ltd., Tokyo, Japan) at 25 °C. Force-versus-Distance Curve Measurement. Measurement of the f-d curve between the protein-fixed AFM probe tip and the graft-polymerized surface was performed with the Digital Instruments Nanoscope IIIa AFM (Dimension 3000, Digital Instruments), according to the method previously reported by us.44 Briefly, protein-fixed AFM probes were equipped with a commercial fluid cantilever folder (Digital Instruments), and immediately transferred into the custom-designed sample cell filled with PBS. Measurements were performed in PBS. Typically, more than 30 f-d curves were obtained at one location through repeated tip approaching/retracting cycles, and measurements were repeated at five locations on each sample. To confirm the reproducibility of the f-d curves, a series of experiments were performed using three newly prepared protein-fixed tips. The frequency of the approaching/retracting cycle was chosen to be 3.2 Hz so as to minimize fluctuation in a single f-d curve, and sequential f-d curves were collected at 1-s intervals using a computer software program. The spring constant (0.12 N/m) given by the probe manufacturer was used without further calibration in the present study.

Results Preparation of the Graft-Polymerized Surface. Surface photograft polymerization was carried out on the DC-glass substrate which was prepared by chloromethylation of a glass substrate and subsequent dithiocarbamation with sodium N,N-diethyldithiocarbamate trihydrate.49 After the DC-glass substrate was irradiated in methanolic DMAAm solution, the elemental ratios of C/Si, O/Si, and N/Si, which were determined from the XPS relative intensity, gradually increased with increasing photoir-

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Figure 3. Representative f-d curves measured between the Alb-fixed tip and the photograft-polymerized surfaces of DMAAm after photoirradiation for 10 s, 20 s, 1 min, 5 min, 10 min, and 20 min: dashed curves, approaching trace; solid curves, retracting trace. The zero position of the tip is expediently defined as the starting position of the linear part in the tipsurface contact region. Figure 2. Surface characterization of the photograft-polymerized surface of DMAAm. The elemental ratio as determined from the XPS relative intensity: (a) C/Si, O/Si, and N/Si; (b) O/C and N/C; (c) the water contact angle (advancing and receding).

radiation time (Figure 2a). The elemental ratios of O/C and N/C gradually approached the theoretical value for PDMAAm, 0.20 (Figure 2b). Water contact angles abruptly decreased at 20 s, and thereafter, a slight gradual decrease was observed (Figure 2c), indicating that the surface became hydrophilic due to the presence of hydrophilic PDMAAm chains. The abrupt decrease of the contact angle at 20 s is discussed later in relation to the f-d curve profile. Results of XPS and wettability studies verify the gradual increase in the amount of polymerization, which means especially for the iniferter-polymerization system that PDMAAm-grafted surfaces with different chain lengths are formed. The DC-glass substrate was also irradiated in aqueous AAcNa solution. Table 1 summarizes the results of elemental ratio and wettability measurements of the DCglass surface before and after photoirradiation (10 and 60 min). Irrespective of the irradiation time, the C/Si and

Table 1. Water Contact Angle and Elemental Ratio Determined from the XPS Relative Intensity on DC-glass and Photograft-polymerized Surfaces of AAc after 10 and 60min Photoirradiation water contact angle/deg irradiation time, min advancing 0 (DC-glass) 10 60

70.0 ( 0.7 35.2 ( 2.9 39.9 ( 2.5

receding

elemental ratio C/Si O/Si O/C

67.5 ( 1.0 4.40 1.63 0.37