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Combination of Electrografting and Atom-Transfer Radical Polymerization for Making the Stainless Steel Surface Antibacterial and Protein Antiadhesive Milena Ignatova,†,‡ Samuel Voccia,† Bernard Gilbert,§ Nadya Markova,| Damien Cossement,⊥ Rachel Gouttebaron,⊥ Robert Je´roˆme,*,† and Christine Je´roˆme† Center for Education and Research on Macromolecules (CERM), UniVersity of Lie` ge, Sart-Tilman, B6, B-4000 Lie` ge, Belgium, Laboratory of Analytical Chemistry and Electrochemistry, UniVersity of Lie` ge, Sart-Tilman, B6, B-4000 Lie` ge, Belgium, Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, and SerVice de Chimie Inorganique et Analytique LASSIE, UniVersity of Mons-Hainaut, AV. Nicolas Copernic, B-7000 Mons, Belgium ReceiVed July 20, 2005. In Final Form: October 20, 2005 A two-step “grafting from” method has been successfully carried out, which is based on the electrografting of polyacrylate chains containing an initiator for the atom transfer radical polymerization (ATRP) of 2-(tert-butylamino)ethyl methacrylate (TBAEMA) or copolymerization of TBAEMA with either monomethyl ether of poly(ethylene oxide) methacrylate (PEOMA) or acrylic acid (AA) or styrene. The chemisorption of this type of polymer brushes onto stainless steel surfaces has potential in orthopaedic surgery. These films have been characterized by ATR-FTIR, Raman spectroscopy, atomic force microscopy (AFM), and measurement of contact angles of water. The polymer formed in solution by ATRP and that one detached on purpose from the surface have been analyzed by size exclusion chromathography (SEC) and 1H NMR spectroscopy. The strong adherence of the films onto stainless steel has been assessed by peeling tests. AFM analysis has shown that addition of hydrophilic comonomers to the grafted chains decreases the surface roughness. According to dynamic quartz crystal microbalance experiments, proteins (e.g., fibrinogen) are more effectively repelled whenever copolymer brushes contain neutral hydrophilic (PEOMA) co-units rather than negatively charged groups (PAA salt). Moreover, a 2- to 3-fold decrease in the fibrinogen adsorption is observed when TBAEMA is copolymerized with either PEOMA or AA rather than homopolymerized or copolymerized with styrene. Compared to the bare stainless steel surface, brushes of polyTBAEMA, poly(TBAEMA-co-PEOMA) and poly(TBAEMA-co-AA) decrease the bacteria adhesion by 3 to 4 orders of magnitude as revealed by Gram-positive bacteria S. aureus adhesion tests.
Introduction In the recent past, an increasing number of experiments have focused on bioactive polymer coatings able to provide solid substrates with antibacterial and protein antiadhesive properties. One major limitation of stainless steel implants, extensively used in orthopedic surgery, is bioinertness in case of infection, which may require reoperation and result in amputation, osteomyelitis, or death.1 One way to tackle this problem is to coat the stainless steel surface with a polymer known for being protein antiadhesive. Surface properties (hydrophilicity and electrostatic charges) of both proteins and biomaterials have a decisive effect on the nonspecific adhesion of proteins to coated metal surfaces. Incorporation of negatively charged or neutral hydrophilic groups in the polymer coating is effective in reducing protein and bacteria adsorption because electrostatic attractive forces and hydrophobic interactions between the solid substrate and proteins have been supressed. The most promising protein inert surfaces have been prepared by coating solid surfaces (polyurethane, poly(tetrafluo* To whom correspondence should be addressed. Phone: (32)4-3663565. Fax: (32)4-3663497. E-mail:
[email protected]. † Center for Education and Research on Macromolecules (CERM), University of Lie`ge. ‡ Permanent address: Institute of Polymers, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. § Belgium, Laboratory of Analytical Chemistry and Electrochemistry, University of Lie`ge. | Bulgarian Academy of Sciences, Institute of Microbiology. ⊥ University of Mons-Hainaut. (1) Gristina, A. G.; Naylor, P. T. Implant Associated Infection. In Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, CA, 1996; p 205.
roethylene), poly(vinyl chloride), cellulose, gold, functionalized glass, etc.) with poly(ethylene glycol).2-8 Whenever inorganic surfaces are concerned, either monolayers of oligo(ethylene glycol) alkanethiolates have been chemisorbed on gold surfaces9-12 or silane coupling agents have been self-assembled on glass followed by covalent grafting of poly(ethylene glycol).13 There are also reports on the “grafting-from” polymerization of ionic and nonionic monomers in the case of poly(tetrafluoroethylene) fibers, which results in the desired decrease of protein adsorption.14 However, the aforementioned strategies that rely on antiadhesive (2) Sofia, S. J.; Merrill, E. W. Protein adsorption on poly(ethylene glycol)grafted silicon surfaces. In Poly(Ethylene Glycol): Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; American Chemical Society: Washington, DC, 1997; Chapter 22. (3) Gombotz, W. R.; Guanghui, W.; Horbett T. A.; Hoffman, A. S. Protein adsorption to and elution from polyether surfaces. In Poly(Ethylene Glycol) Chemistry Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum Press: New York, 1992; p 127. (4) Zhang, M.; Desai, T.; Ferrari, M. Biomaterials 1998, 19, 953. (5) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176. (6) Kiss, E.; Golander, C. G.; Eriksson, J. C. Prog. Colloid Polym. Sci. 1987, 74, 113. (7) Zou, X. P.; Kang, E. T.; Neoh, K. G. Plasmas Polym. 2002, 7, 151. (8) Gong, X.; Dai, L.; Griesser, H. J.; Mau, A. W. H. J. Polym. Sci., Part B: Polym. Phys. 2000, 38, 2323. (9) Mrksich, M.; Whitesides, G. M. Using self-assembled monolayers that present oligo(ethylene glycol) groups to control the interactions of proteins with surfaces. In Poly(Ethylene Glycol): Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; American Chemical Society: Washington, DC, 1997; Chapter 23. (10) Chapman, R. G.; Ostuni, E.; Liang, M. N.; Meluleni, G.; Kim, E.; Yan, L.; Pier, G.; Warren, H. S.; Whitesides, G. M. Langmuir 2001, 17, 1225. (11) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (12) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (13) Jo, S.; Park, K., Biomaterials 2000, 21, 605. (14) Kato, K.; Sano, S.; Ikada, Y. Colloids Surf. B 1995, 4, 221.
10.1021/la051954b CCC: $33.50 © 2006 American Chemical Society Published on Web 12/08/2005
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polymeric surfaces cannot totally inhibit bacteria attachment. Therefore, polymer coatings that combine antibacterial properties and protein antiadhesive characteristics might be a promising approach to overcome this limitation. In this respect, (co)polymers of 2-(tert-butylamino)ethyl methacrylate (TBAEMA) are known for antibacterial activity toward a broad range of microorganisms, although they have a low toxicity for humans.15-17 Therefore, polymer coatings in which poly(TBAEMA) and hydrophilic polymers (i.e., poly(acrylic acid) and PEO) are associated, could exhibit the antibacterial and protein antiadhesive properties required by a variety of applications. When polymerization of (meth)acrylates is initiated from a cathode surface under an appropriate potential in a solvent such as DMF, the chains are chemisorbed (electrografted) and rapidly passivate the electrode.18-20 If an initiator for the controlled radical polymerization of, e.g., styrene is part of the pendant ester groups, a brush of polystyrene can be formed, which is strongly adhering to the solid substrate.21,22 The thickness of the film formed in the second step is directly determined by the average molar mass of the tethered chains. This paper aims at reporting on the electrografting of polyacrylate chains onto stainless steel surfaces, which can serve as macroinitiators for the atom transfer radical polymerization (ATRP) of TBAEMA and mixtures of this monomer with acrylic acid (AA), R-methyl ether, β-methacrylate poly(ethylene oxide) (PEOMA), and St, respectively. The adhesion of the coatings to stainless steel has been assessed by peeling test. Effect of chemical composition, hydrophobicity, roughness, and homogeneity of the polymer coatings on protein adhesion (e.g., fibrinogen) has been studied with a dynamic quartz crystal microbalance. The adsorption of bacteria S. aureus has also been investigated as a criterion for biomedical applications. Experimental Section Materials. Styrene, acrylic acid, methacryloyl chloride, 2-hydroxyethyl acrylate, triethylamine, and TBAEMA were purchased from Aldrich, dried over calcium hydride, and distilled under reduced pressure. 2-(2-Chloropropionate)ethyl acrylate (cPEA) was dried over molecular sieves before electropolymerization. Phenylethyl bromide (PEBr) (Aldrich), Grubbs catalyst (Aldrich), ethylene oxide (Messer), triethylene glycol-monomethyl ether (Fluka), and potassium hydroxide (Fluka) were of analytical grade and used as received. Dimethylformamide (DMF) was dried for 5 days over phosphorus pentoxide, distilled at 70 °C under reduced pressure, and stored under dried nitrogen in a glovebox. Tetraethylammonium perchlorate (TEAP) was dried by overnight heating at 80 °C under vacuum prior to use. PEOMA was synthesized as follows. A dry toluene solution of methacryloyl chloride (2.4 mL, 24.52 mmol) was added dropwise to a precooled (0 °C) mixture of R-methyl ether, β-hydroxyl of poly(ethylene oxide) (CH3O-PEO-OH)23 (Mn ) 1030 determined by 1H NMR) (20.06 g, 22.29 mmol), 120 mL of dry toluene, and 3.4 mL of freshly dried and distilled triethylamine. This reaction mixture was stirred at room-temperature overnight under nitrogen. The solution was filtered, and the filtrate was evaporated to dryness. (15) Ottersbach, P.; Inhester, M. PCT Int. Appl., 2003, WO 03/068316 A1. (16) Ottersbach, P.; Kossmann, B. PCT Int. Appl., 2003, WO 03/033033 A2. (17) Ottersbach, P.; Kossmann, B. PCT Int. Appl., 2002, WO 02/048070 A1. (18) Yuan, W.; Iroh, J. O. Trends Polym. Sci. 1993, 1, 388. (19) Baute, N.; Martinot, L.; Je´roˆme, R. J. Electroanal. Chem. 1999, 472, 83. (20) Baute, N.; Je´roˆme, C.; Martinot, L.; Mertens, M.; Geskin, V. M.; Lazzaroni, R.; Bre´das, J. L.; Je´roˆme, R. Eur. J. Inorg. Chem. 2001, 5, 1097. (21) Voccia, S.; Je´roˆme, C.; Detrembleur, C.; Lecle`re, P.; Gouttebaron, R.; Hecq, M.; Gilbert, B.; Lazzaroni, R.; Je´roˆme, R. Chem. Mater. 2003, 15, 923. (22) Claes, M.; Voccia, S.; Detrembleur, C.; Je´roˆme, C.; Gilbert, B.; Lecle`re, Ph.: Geskin, V. M.; Gouttebaron, R.; Hecq, M.; Lazzaroni, R.; Je´roˆme, R. Macromolecules 2003, 36, 5926. (23) Vangeyte, P.; Je´roˆme, R. J. Polym. Sci., Polym. Ed., Part A 2003, submitted.
IgnatoVa et al. The residue was taken up with chloroform, and the PEOMA was isolated by precipitation in cold diethyl ether and dried in vacuo at room temperature. Yield: 85%. 1H NMR (CDCl3) of PEOMA, δ (ppm): 6.12 (1H, s, HCHdC(CH3)-), 5.57 (1H, s, HCHdC(CH3)), 4.28 (2H, dd, -COO-CH2-CH2-), 3.8-3.5 (94H, m, -OCH2CH2(OCH2CH2)n-1-), 3.37 (3H, s, (OCH2CH2)n-1-OCH3), 1.94 (3H, s, CH2dC(CH3)-). 95% of the hydroxyl end-groups of CH3OPEO-OH from the PEOMA were reacted, as determined by 1H NMR. cPEA was synthesized by reaction of 2-chloropropionyl chloride with 2-hydroxyethylacrylate in the presence of triethylamine, as reported elsewhere.22 Yield: 80%. Purity was determined by gas chromathography (97%), and the structure was confirmed by 1H NMR and IR spectroscopy. 1H NMR (CDCl ) of cPEA, δ (ppm): 6.38 (d, 1H, HCHdCHs), 3 6.05 (dd, 1H, HCHdCHs), 5.81 (d, 1H, HCHdCHs), 4.33 (m, 5H, -CH2CH2O-, -CH(CH3)Cl), 1.61 (d, 3H, -CH(CH3)Cl). IR (NaCl), cm-1: 2963 (aliphatic C-H stretching vibration), 1729 (ester CdO stretching vibration), 1450, 1410, 1379, 1340 (aliphatic C-H bending vibration), 1276, 1175, 1075 (C-O-C stretching vibration). Electrografting of cPEA. The electrografting of cPEA onto stainless steel and carbon fibers was carried out as previously reported.22 Briefly, it was performed in dry DMF (0.15-0.5 M) in the presence of TEAP (5 × 10-2 M) as a conducting salt. The water content of the solution was measured by the Karl Fisher method (Tacussel aquaprocessor) and found to be lower than 10 ppm. Before electropolymerization, the stainless steel electrodes were polished. The polished stainless steel electrodes and carbon fibers were washed with heptane and acetone under sonication, dried in vacuo at 150 °C and immediately transferred in the glovebox. All of the electrochemical experiments were conducted in the glovebox under inert and dry atmosphere (N2) at room temperature in a closed cell with platinum foils as counter-electrode and pseudo-reference electrode, respectivly. A PAR-EG&G (model 273A) potensiostat/ galvanostat was used for voltammetry. The grafted films were carefully washed with pure DMF and acetonitrile. Formation of Polymer Brushes from Surface Electrografted polycPEA Chains. TBAEMA Polymer Brushes. A mixture of TBAEMA (6.4 g, 34.5 mmol), PEBr (0.0202 g, 0.11 mmol), Grubbs catalyst (0.09 g, 0.11 mmol), and 1.1 mL of toluene was added into a reaction tube containing polycPEA grafted stainless steel plate was added. The tube was closed by a three-way stopcock and filled with nitrogen, heated at 100 °C for 24 h, cooled to RT, and the reaction mixture was dissolved in chloroform and twice precipitated in heptane. The structure of collected poly(TBAEMA) was confirmed by 1H NMR and IR spectroscopy. The stainless steel plate was continuously extracted with dichloromethane for 24 h and dried. The polymer brush was analyzed by contact angle measurements, IR, and Raman spectroscopies. TBAEMA-PAA Copolymer Brushes. The polycPEA grafted stainless steel electrode was placed into a tube containing the Grubbs catalyst (0.09 g, 0.11 mmol) and 5.2 mL (25.7 mmol) of TBAEMA, to which 1.8 mL (25.7 mmol) of AA, PEBr (0.02 g, 0.11 mmol) and 2.0 mL toluene were added with a syringe. The mixture was purged with nitrogen for 30 min. Copolymerization proceeded at 100 °C for 24h. The electrode was then continuously extracted with dichloromethane for 24 h in order to remove the ungrafted polymer. TBAEMA-PEOMA and TBAEMA-St copolymer brushes were similarly prepared, PEOMA and styrene, respectively, being the comonomer instead of AA. The copolymers were precipitated twice from chloroform into heptane at 10 °C. They were analyzed by IR, 1H NMR spectroscopy and size-exclusion chromatography (SEC). PolyTBAEMA: IR (film, cm-1): 2964, 2870 (alipatic C-H stretching vibration), 1729 (ester CdO stretching vibration), 1482, 1447, 1389 (aliphatic C-H bending vibration), 1362 (C-H wagging vibration), 1266, 1231, 1154 (C-O stretching vibration). 1H NMR (400 MHz, CDCl3), δ ppm: 0.88 (3H, s, -C(CH3)-CH2-), 1.061.26 (9H, m,(CH3)3C-NH-), 1.84 (2H, m, -CH2CH2-NH- from TBAEMA units), 4.03 (2H, m, -CH2-OCO-).
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Scheme 1. Grafting of Brushes of Poly(TBAEMA) and Copolymers of TBAEMA with AA, PEOMA, and St, Respectively, onto Stainless Steel Surfaces
Poly(TBAEMA-co-AA): IR (film, cm-1): 3400 (OH stretching vibration), 2979, 2788 (alipatic C-H stretching vibration), 1726 (ester CdO stretching vibration), 1640 (CdO stretching vibration), 1452, 1405, 1380 (aliphatic C-H bending vibration), 1356 (C-H wagging vibration), 1166 (C-O stretching vibration). 1H NMR (400 MHz, CD3OD), δ ppm: 1.05 (3H, s, -C(CH3)-CH2-), 1.26-1.40 (9H, m,(CH3)3C-NH-), 1.91 (4H, m,-CH2-CH-COOH from AA units, -CH2CH2-NH- from TBAEMA units), 2.51 (1H, m, -CH2CH-COOH), 3.25 (2H, m, -C(CH3)-CH2-), 4.37 (2H, m, -CH2COO-). Poly(TBAEMA-co-St): IR (film, cm-1): 3062 (aromatic C-H stretching vibration), 2944, 2964 (aliphatic C-H stretching vibration), 1726 (ester CdO stretching vibration), 1602 (C-C ring stretching vibration), 1494, 1454, 1388 (aliphatic C-H bending vibration), 1362 (C-H wagging vibration), 1230, 1180, 1129, 1075 (in-plane C-H bending vibrations of the aromatic ring and C-O stretching vibration), 757, 701 (in phase out of plane wagging vibration of the adjacent hydrogen atoms on the aromatic ring). 1H NMR (400 MHz, CDCl3), δ ppm: 0.88 (3H, s, -C(CH3)-CH2- from TBAEMA units), 1.05-1.26 (9H, m, (9H, s,(CH3)3C-NH-), from TBAEMA units), 1.57 (2H, m, -CH2-CH-C6H5), 1.95 (2H, m, -CH2CH2NH- from TBAEMA units), 2.43 (1H, m, -CH2-CH-C6H5), 2.74 (2H, m, -C(CH3)-CH2-), 4.0 (2H, m, -CH2-COO-), 6.7-7.13 (5H, m, -CH2-CH-C6H5). Poly(TBAEMA-co-PEOMA): IR (film, cm-1): 2960, 2871 (aliphatic C-H stretching vibration), 1728 (ester CdO stretching vibration), 1481, 1449, 1389 (aliphatic C-H bending vibration), 1361 (C-H wagging vibration), 1232, 1140, 1110 (C-O-C stretching vibration). 1H NMR (400 MHz, CDCl3), δ ppm: 0.87 (6H, s, -C(CH3)-CH2- from TBAEMA and from PEOMA units), 1.11-1.26 (9H, m, (CH3)3C-NH-), 1.94 (2H, m, -CH2CH2NH- from TBAEMA units), 2.80 (4H, m, -C(CH3)-CH2- from TBAEMA and from PEOMA units), 3.37 (3H, s, -(OCH2CH2)OCH3), 3.66 (94 H, s, -(OCH2CH2)n-), 4.04 (2H, m, -CH2COO- from TBAEMA units), 4.29 (2H, m, -O-CH2CH2-COOfrom PEOMA units).
Characterization. IR spectra of polymer films grown from electrografted stainless steel or Pt quartz crystal microbalance (QCM) electrodes were recorded by the reflection-absorption technique (FTIR-RAS) using a Brucker spectrophotometer. Raman diffusion spectroscopy was performed with a Dilor spectrometer (SuperLabram type), equipped with a microscope and with a 800-2000 CCD detector cooled by liquid nitrogen. The excitation laser beam was focused on the sample via the microscope. Contact angles were measured by the sessile drop method with a GBX Digidrop instrument. Angles from three different spots on each surface were measured 10 times and statistically compiled. Chemical composition of the surface-grafted polymers was determined by X-ray photoelectron spectroscopy (XPS, VG-ESCALAB 220iXL spectrometer) with a monochromatic A1 KR X-ray source (Eexc ) 1486.6 eV). Thermogravimetric analysis (TGA) was performed with a TA instrument Q500 thermogravimetric analyzer. A Nanoscope 3 Multimode Microscope from Veeco Inc. was operated in the tapping mode and equipped with the Extender, such that height and phase AFM images were recorded simultaneously. Polymers collected in solution were characterized by SEC with a Waters 600 liquid chromatograph, equipped with a 410 refractive index detector and Styragel HR columns (HR1 1000-500, HR2 500-20000, HR4 5000-600000) and eluted with Et3N containing THF at 40 °C. The column set was calibrated with Polystyrene standards for analysis of poly(TBAEMA-co-St) copolymers and with PMMA standards for the analysis of poly(TBAEMA) and copolymers of TBAEMA with PEOMA and AA, respectively. 1H NMR spectra were recorded in CDCl3 with a Brucker AM spectrometer (400 or 250 MHz) at 25 °C. Peeling tests were carried out with an Instron tensile tester at 180° and acrylic foam 4930 tape from 3M according to the ASTM standards D 3330M-90. Fibrinogen Adsorption. Fibrinogen adsorption was studied at 25 °C with an electrochemical quartz crystal microbalance (QCM),24,25 (24) Je´roˆme, C.; Gabriel, S.; Voccia, S.; Detrembleur, C.; Ignatova, M.; Gouttebaron, R.; Je´roˆme, R. Chem. Commun. 2003, 2500.
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Figure 1. Cyclic voltammograms for the reduction of cPEA (A) (0.15 M) at the surface of a stainless steel cathode in a 0.05 M TEAP solution in DMF (V ) 20 mV/s). The “grafting” peak (I), the first scan (a), and the second scan (b) are shown. Table 1. Molecular Characteristics of Poly(TBAEMA) and Copolymers of TBAEMA with AA, PEOMA, and St Prepared by ATRP polymer
feed composition
polymer compositiona
Mnb
Mw/Mnb
theoretical Mn (100% conversion)
[Grubbs catalyst]: [PEBr]:[TBAEMA]:[M]
conversion (%)c
poly(TBAEMA) poly(TBAEMA-co-AA) poly(TBAEMA-co-PEOMA) poly(TBAEMA-co-St)
50:50 91:9 50:50
47:53 94:6 40:60
38500 43500 38500 40500
1.7 1.4 1.8 1.9
57620 60250 62800 64000
1:1.1:314 1:1.1:234:234 1:1.2:220:22 1:1.3:221:221
65 85 70 65
a Determined by 1H NMR. b Determined by SEC analysis in THF/Et3N. Calibration with polystyrene standards for poly(TBAEMA-co-St) and with poly(methyl methacrylate) standards for the other polymers. c Determined by gravimetry.
equipped with a platinum coated AT-cut quartz crystal (0.2 cm2, Inficon) oscillating at 9 MHz. The lyophylized fibrinogen was purchased from Sigma (fraction I, pig plasma). The time dependence of the frequency change was monitored with the electrode dipped in a 1.1 × 10-7 M fibrinogen solution in a phosphate buffer (PBS, pH 7.4). The Pt QCM electrodes were coated under the same conditions as stainless steel. The chemical composition of the polymer deposited on these electrodes was analyzed by FTIR-ATR spectrometry. The frequency was converted into mass of adsorbed fibrinogen by the Sauerbrey equation:26 ∆m ) ∆f × A(µq × Fq)1/2/2 × fo2, where ∆m is the mass of fibrinogen adsorbed after 1320 s, ∆f is the experimental frequency shift at the same time, fo is the original frequency of the quartz crystal, A is the piezoelectrically active area (0.2 cm2), Fq is the density (2.648 g/cm3), and µq is the shear modulus of quartz (2.947 × 1011 g/cm‚s2). The reported ∆m values were the average of three measurements. In Vitro Adhesion Model for Staphylococcus aureus. In vitro adhesion of Staphylococcus aureus (from the National Bank of Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria) was investigated as previously reported.6 Functionalized stainless steel surfaces (2 cm2) were sterilized by UV treatment for 2 h and placed in sterile (100 × 15 mm) polystyrene dishes (Fisher). 100 µL of a 1.5 × 108 Staphylococcus aureus 749 bacteria/mL suspension was spread over the entire surface of the stainless steel substrate with a the sterile pipet tip. The samples were incubated at 37 °C for 1 h. The stainless steel substrate was removed from the medium, washed five times with sterile phosphate buffer solution and sonicated for 5 s in 5 mL of trypticase soy broth containing 0.05% Tween. This suspension was 10 times diluted before being plated on trypticase soy agar. The plates were incubated at 37 °C for 24 h. The number of colonies was counted to determine the density of colony-forming units (cfu’s) in the suspension formed by sonicating the coated stainless steel samples. Each sample was tested in triplicate.
1 consists of a peak at -1.8 V/Pt (peak I) assigned to the electrografting of polycPEA chains. The same observation was previously reported with steel instead of stainless steel as a cathode.22 When the electropolymerization is conducted in the potential range of peak I, only a residual current is observed upon repeating the potential scan, which is the signature of the cathode passivation by the insulating grafted chains (curve b, Figure 1). Thus after one scan, the cathode surface is quasi saturated by polycPEA chains, which are chemisorbed rather than being dissolved in a good solvent as DMF.19,27,28 In a previous study, some of us reported that approximately 10% of chlorine was lost during the electrografting of polycPEA onto steel, as result of the concomitant cleavage of the C-Cl bonds.22 In this study, the Cl 2p spectrum actually shows two components at 200.2 and 202 eV, respectively, with a 2:1 relative intensity, characteristic of chlorine bound to carbon atoms. The experimental ester carbon/chlorine atomic ratio is 2.1, thus close to the theoretical value of 2.0. From these figures, less than 5% of the chlorine atoms would have been lost during the monomer reduction onto the stainless steel surface. A transparent film is clearly seen by the naked eye at the surface of the stainless steel cathode after two cathodic scans until the maximum of peak I and washing with DMF (a good solvent for the polymer) and acetonitrile. This film has been analyzed by FTIR-RAS. The absorptions typical of the polycPEA chains are observed, i.e., absorptions by the CdO ester group at 1728 cm-1, by the aliphatic C-H (bending vibration) at 1455 and 1383 cm-1, by the C-O bond stretching vibration at 1151, 1072, and 1014 cm-1, and by the C-Cl bond at 755 and 693 cm-1, respectively. The contact angle of water is consistent with
Results and Discussion
(25) Tanaka, M.; Mochizuki, A.; Shiroya, T.; Motomura, T.; Shimura, K.; Onishi, M.; Okahata, Y. Colloids Surf. A: Physicochem. Eng. Aspects 2002, 203, 195. (26) Buttry, D. A.; Ward, M. D. Chem. ReV. 1992, 92, 1355. (27) Je´roˆme, R.; Mertens, M.; Martinot, L. AdV. Mater. 1995, 7, 807. (28) Mertens, M.; Calberg, C.; Martinot, L.; Je´roˆme, R. Macromolecules 1996, 29, 4910.
cPEA (0.15-0.5 M) was electropolymerized in DMF (a good solvent for polycPEA) added with a conducting salt (0.05 M TEAP) at the surface of cathodically polarized stainless steel commonly used in medical devices. The curve shown in Figure
Antibacterial and AntiadhesiVe Stainless Steel
the hydrophobicity of polycPEA (84°), although close to the contact value for naked stainless steel (86°). To increase both the thickness and hydrophilicity of the polymer coating, the controlled radical polymerization of polar (meth)acrylates has been initiated from the chemisorbed polycPEA, which is actually a potential macroinitiator for ATRP.22 Poly(cPEA) modified electrodes have been dipped into a toluene solution of TBAEMA or mixtures of TBAEMA with AA, PEOMA, and St, respectively, in the presence of the Grubbs catalyst and PEBr for 24h under nitrogen. As reported elsewhere,22 an initiator (PEBr) has to be added to the (co)monomers solution in order to control the growth of the chains involved in the active/ dormant species equilibrium. The grafted initiator has no significant influence on the course of the polymerization in solution because of a comparatively small content with respect to PEBr. In this study, the commercially available Grubbs catalyst (RuCl2(dCHPh)(PCy3)2) has been used for its capacity to control the radical polymerization of (meth)acrylates and styrene29-31 and for inertness toward stainless steel which was not the case for Cu-based catalyst and steel.22 After polymerization, the ungrafted polymer has been recovered and analyzed by SEC (Table 1). The molecular weight distribution lies between 1.5 and 1.9 and the molecular weight is close to expectation for controlled polymerization. After intensive washing with dichloromethane, a good solvent for poly(TBAEMA), poly(TBAEMA-co-AA), poly(TBAEMAco-PEOMA) and poly(TBAEMA-co-St), the modified surfaces have been analyzed by IR reflection-absorption and Raman spectroscopy. The Raman spectrum A in Figure 2 is characteristic of poly(TBAEMA) with the main bands at 2958 and 2926 cm-1 (aliphatic C-H stretching vibrations), 1733 cm-1 (CdO stretching vibration), 1451 and 1330 cm-1 (aliphatic C-H bending vibrations), and 1235 and 1129 cm-1 (C-O stretching vibration). According to the intensity of the Raman bands, the poly(TBAEMA) film is thick in contrast to the original polycPEA that was too thin for being recorded by Raman spectroscopy. Spectra B-D (Figure 2) mainly show the characteristic features of poly(TBAEMA), which indicates that this monomer dominates in the films. Spectra C and D (Figure 2) shows Raman bands characteristic of the PEOMA units at 2700 cm-1 (aliphatic C-H stretching vibration) and 1093-1036 cm-1(C-O stretching vibrations) and those typical of the PS units at 3055 cm-1 (aromatic C-H stretching vibration) and at 1031 and 1001 cm-1 (aromatic ring). Copolymerization of AA is however difficult to confirm, more likely because of the overlapping of the bands characteristic of the AA and TBAEMA comonomer units. As a rule, films of copolymers of TBAEMA with AA and PEOMA are thinner than films of poly(TBAEMA) as assessed by comparatively lower absorptions. This observation might indicate that the chain growth is slower and/or that chain termination occurs earlier when PEOMA and AA are added as comonomers. The contact angles of water at the surface of poly(TBAEMAco-PAA) (28°) and poly(TBAEMA-co-PEOMA) (57°) films are significantly lower compared to the original polycPEA coating (84°) as result of the higher hydrophilicity of the PAA and PEOMA units. Stainless steel grafted with homo polyTBAEMA (contact angle 75°) exhibits an intermediate hydrophilicity. The poly(TBAEMA-co-St) (83°) film deposited onto stainless steel does not change significantly the hydrophobicity of the poly(cPEA) film. Comparison of the reflection-absorption IR spectra (Figure 3) for the copolymer chains electrografted onto stainless (29) Simal, F.; Delaude, L.; Jan, D.; Demonceau, A.; Noels, A. F. Polym. Preprint 1999, 40, 336. (30) Simal, F.; Demonceau, A.; Noels, A. F. Tetrahedron Lett. 1999, 40, 5689. (31) Simal, F.; Demonceau, A.; Noels, A. F. Angew. Chem., Int. Ed. 1999, 38, 4.
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Figure 2. Raman spectra for stainless steel electrodes grafted with polyTBAEMA (A), poly(TBAEMA47-co-AA53) (B), poly(TBAEMA94-co-PEOMA6) (C), poly(TBAEMA38-co-St62) (D), (a) 2001800 cm-1 range, (b) 2400-3600 cm-1 range.
steel and the parent copolymer chains formed in solution and solvent cast onto stainless steel plates qualitatively confirms that
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Figure 3. IR reflection-absorption spectra for grafted polymer brushes (a) and reference solvent-cast films of polymers of the same composition (b), (A) poly(TBAEMA-co-AA), (B) poly(TBAEMA-co-St), (C) poly(TBAEMA-co-PEOMA), (D) polyTBAEMA. Table 2. Adhesion of the Grafted and Solvent-Cast Polymer Films Determined by the ASTM Peeling Test polymer coatingsa electrografted
cPEA adhesion (( 10% N/m) stainless steel after peeling
grafted-from
solvent-cast
poly poly poly poly poly poly neat poly (TBAEMA38 (TBAEMA47 (TBAEMA94poly (TBAEMA40 (TBAEMA47- (TBAEMA94- stainless (TBAEMA) co-AA53) co-PEOMA6) steel -co-St62) -co-AA53) co-PEOMA6) (TBAEMA) -co-St60)
2270
2510
2840
2690
2790
1860
2100
2080
1990
b
b
b
b
b
c
c
c
c
1880
a The subscripts refer to the content of TBAEMA, St, AA, and PEOMA in the copolymers (mol %), determined by 1H NMR. b Polymer remaining on stainless steel. c Polymer removed with the tape.
the molar composition of the two chain populations is quite similar (see the intensity ratio of the characteristic bands). Because the surface area of the stainless steel cathodes is too low for an accurate determination of the grafting density of the poly(TBAEMA), poly(TBAEMA-co-PEOMA), poly(TBAEMAco-PAA), and poly(TBAEMA-co-St) chains, the two-step “grafting from” method has been repeated with carbon fibers instead of stainless steel. The grafting density, Σ (molecules/ nm2), has been calculated as follows: Σ ) WNA/MnS, where W is the weight loss of the carbon fibers (mg) determined by TGA, NA is Avogadro’s number, Mn is the apparent number-average molecular weight determined by SEC for the chains collected in solution, and S is the surface area of the carbon fibers (18.85 cm2/cm fiber mesh). Because Mn’s of the chains are typically apparent values, calculated Σ are crude estimates. For the coatings under investigation in this study, Σ is found in the 1.7-1.9 molecules/nm2 range, which is consistent with polySt brushes (1.7 molecules/nm2) grown by nitroxide mediated polymerization from alkoxyamine containing polyacrylates electrografted onto carbon fibers.21
Copolymer modified stainless steel surfaces have been observed by atomic force microscopy (for pictures, see the Supporting Information). At least three spots of the same surface have been scanned, which has confirmed the homogeneity of the grafted (co)polymer coatings. The roughness of the polyTBAEMA films is approximately 24 nm for a (3 × 3) µm2 surface, which is an intermediate value compared to copolymers with St units (87 nm) and copolymers with AA units (5.6 nm) and PEOMA units (3.6 nm). Polar comonomers thus contribute to the smoothness of the film surface. This characteristic feature has an impact on the adsorption of bacteria and proteins, which actually increases with the surface roughness.32 It must however be pointed out that the AFM observations have been performed in contact with air and that the surface structure will change upon immersion in water. The adhesion of the polymer brushes onto stainless steel has been determined and compared to films of the same composition, solvent cast on the same substrate (stainless steel). Table 2 shows (32) An, Y. H.; Friedman, R. J. J. Biomed. Mater. Res. 1998, 43, 338.
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Figure 4. Time dependence of frequency changes typical of the fibrinogen adsorption onto polymer coated Pt-quartz microbalance crystal: (b) poly(TBAEMA94-co-PEOMA6), (c) poly(TBAEMA47-co-AA53), (d) poly(TBAEMA), (e) poly(TBAEMA38-co-St62), (f) polySt. Concentration of fibrinogen in PBS was 1.1 × 10-7 M. For sake of comparison the same plot has been reported for poly(TBAEMA) in PBS without fibrinogen (a).
that the solvent cast films are completely peeled off by the tape, such that the peeling strengths refer to the adhesion of these films to stainless steel. In contrast, when the grafted polymer brushes are concerned, the adhesion failure occurs between the tape and the organic coating, as assessed by FTIR reflectance analysis (ATR-FTIR) of the surface after peeling. These observations are consistent with the chemisorption of the chains prepared by electropolymerization and ATRP, to stainless steel. The actual adhesion is typically higher than the adhesion strengths reported in Table 2. The fibrinogen adhesion onto the grafted polymer coatings has been studied by using the dynamic quartz crystal microbalance, thus by recording the frequency change of a Pt coated electrode dipped in a dilute phosphate buffer solution of fibrinogen (1.1 × 10-7 M). The experiments have been carried out for 22 min, thus until saturation of the electrode surface. This type of experiment is important because it might be representative of what happens in the initial stage of bacteria adhesion. It is known indeed that the formation of a biofilm onto implanted biomaterials results from the adsorption of the serum constituents including fibronectin, fibrinogen, albumin, collagen, osteonectin, and vibronectin, which then promotes the bacteria adhesion.1,33 The QCM Pt electrode has been coated by the same two-step technique (combination of the electrografting of polycPEA and ATRP of TBAEMA and mixtures of TBAEMA with AA, PEOMA, and St, respectively) as reported for stainless steel. Successful formation of the polymer brushes onto the Pt electrode has been confirmed by the same experimental techniques as for stainless steel. A QCM Pt electrode coated by a PS film has been used as a reference, because it exhibits a larger frequency change than all the coatings of interest in this study. The larger amount of fibrinogen adsorbed onto PS more likely results from more hydrophobic interactions between these two components. Consistently, the fibrinogen adsorption is decreased upon copolym(33) Hendricks, S. K.; Kwok, C.; Shen, M.; Horbett, T. A.; Ratner, B. D.; Bryers, J. D. J. Biomed. Mater. Res. 2000, 50, 160.
Table 3. Adsorption of Fibrinogen on Polymer Surfaces polymer coatings
∆m (µg/cm2)a
contact angle of water
polySt poly(TBAEMA) poly(TBAEMA38-co-St62) poly(TBAEMA47-co-AA53) poly(TBAEMA94-co-PEOMA6)
0.72 ( 0.09 0.49 ( 0.01 0.53 ( 0.02 0.24 ( 0.01 0.14 ( 0.02
85° 75° 82° 28° 57°
a
∆m mass of adsorbed fibrinogen after 1320 s.
erization of styrene with a more polar monomer, TBAEMA, and formation of a more hydrophilic surface. This beneficial effect is amplified in case of poly(TBAEMA) films, which are still more hydrophilic as stated by the previously mentioned contact angle of water. Modification of the poly(TBAEMA) films by either acrylic acid units or water soluble PEO chains decreases further the fibrinogen adsorption (Figure 4, Table 3). PEO modified poly(TBAEMA) films are more effective in preventing fibrinogen from being adsorbed, in relation to higher hydrophilicity and mobility.34 The beneficial effect of the AA comonomer might result from electrostatic repulsion of negative charges in both the film and fibrinogen at pH 7.4 (isoelectric point of fibrinogen ) 5.8). The bacteria adhesion onto the coated stainless steel surfaces has been estimated by counting the viable bacteria that adhere to the surface after incubation in a bacteria suspension for 1h and washing. Gram-positive bacteria S. aureus has been used in the test, being the most common pathogen responsible for implant infection.1 A large number of bacteria (from 1.5 × 105 to 2.1 × 105 colony-forming units (cfu)/ml) adhere onto uncoated and PS coated (reference) stainless steel surfaces (Figure 5). Poly(34) Lee, J. H.; Kopecek, J.; Andrade, J. D. J. Biomed. Mater. Res. 1989, 23, 351. (35) Wan, S.; Mahmud, N.; Kushner, L.; Lindon, J. N.; Curme, J.; Merrill, E. W.; Salzman, E. W. Trans. Am. Soc. Artif. Intern. Organs 1982, 28, 482. (36) Coleman, D. L.; Gregonis, D. E.; Angrade, J. D. J. Biomed. Mater. Res. 1982, 16, 381. (37) Humphries, M.; Nemcek, J.; Cantwell, J. B.; Gerrard, J. J. FEMS Microbiol. Ecol. 1987, 45, 297.
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consists of the electrografting of polycPEA chains, which are used as macroinitiators for the ATRP of TBAEMA or mixtures of it with a series of comonomers. Poly(TBAEMA) brushes modified by either negatively charged carboxylate groups or flexible hydrophilic PEO grafts are very effective in restricting the protein (fibrinogen) adsorption and the number of adhering bacteria S. aureus compared to more hydrophobic poly(TBAEMA) and copolymers of TBAEMA with styrene. These characteristic features are of great interest for biomedical implants to be used in orthopaedic applications.
Figure 5. Adhesion of bacteria Staphylococcus aureus to uncoated and coated stainless steel surfaces. CFU stands for the colony-forming units, thus the number of bacteria bound to the substrate. The error bars are the standard deviations for triplicated experiments.
(TBAEMA-co-PEOMA), polyTBAEMA, and poly(TBAEMAco-AA) coatings are the more effective in decreasing the adhesion of S. aureus bacteria adhesion by 99.9% compared to PS coated or naked stainless steel control (Figure 5). The lower efficency of poly(TBAEMA-co-St) brushes compared to polyTBAEMA is thought to result from higher roughness and hydrophobicity. Once again, availability of negatively charged species or hydrophilic and flexible PEO grafts at the surface of poly(TBAEMA) coatings decreases further the adhesion of bacteria.
Conclusion Brushes of (co)polymer of TBAEMA have been successfully grafted onto stainless steel surfaces by a two-step technique that
Acknowledgment. M.I. gratefully acknowledges the “Fonds National de la Recherche Scientifique” (FNRS) for a post-doc fellowship at the University of Lie`ge. The authors are much indebted to the “Belgian Science Policy” for general support under the auspices of the “Interuniversity Attraction Poles Program (PAI V/03)”. C.J. is “Chercheur Qualifie´” by the FNRS. S.V. is grateful to the “Fonds pour la Formation a` la Recherche dans l’Industrie et dans l’Agriculture” (FRIA) for a fellowship. The authors are grateful to Catherine Vieujean (ULg) for FTIR reflectance measurements (FTIR-RAS), to Krasimira Dilova (Institute of Microbiology, BAS) for assistance in microbiological tests with bacteria S. aureus, to Stefan Lutz for technical assistance in Raman spectroscopy and to Dr. P. Vangeyte for the synthesis of CH3O-PEO-OH. Supporting Information Available: AFM micrographs and three-dimensional height AFM images. This material is available free of charge via the Internet at http://pubs.acs.org. LA051954B
