Langmuir 2008, 24, 8151-8157
8151
PEGMA/MMA Copolymer Graftings: Generation, Protein Resistance, and a Hydrophobic Domain Volker Stadler,*,† Robert Kirmse,‡ Mario Beyer,† Frank Breitling,† Thomas Ludwig,‡ and F. Ralf Bischoff† Research Groups “Chip-Based Peptide Libraries” and “MicroenVironment of Tumor Cell InVasion”, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany ReceiVed March 11, 2008. ReVised Manuscript ReceiVed May 8, 2008 We synthesized various graft copolymer films of poly(ethylene glycol) methacrylate (PEGMA) and methyl methacrylate (MMA) on silicon to examine the dependency of protein-surface interactions on grafting composition. We optimized atom transfer radical polymerizations to achieve film thicknesses from 25 to 100 nm depending on the monomer mole fractions, and analyzed the resulting surfaces by X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle measurements, and atomic force microscopy (AFM). As determined by XPS, the stoichiometric ratios of copolymer graftings correlated with the concentrations of provided monomer solutions. However, we found an unexpected and pronounced hydrophobic domain on copolymer films with a molar amount of 10-40% PEGMA, as indicated by advancing contact angles of up to 90°. Nevertheless, a breakdown of the protein-repelling character was only observed for a fraction of 15% PEGMA and lower, far in the hydrophobic domain. Investigation of the structural basis of this exceptional wettability by high-resolution AFM demonstrated the independence of this property from morphological features.
Introduction Atom transfer radical polymerization (ATRP) is a powerful tool for the generation of both homopolymers in solution and graft polymer films on surfaces.1 This living radical polymerization allows a wide range of functional groups to be present in the monomer, solvent, or initiator, and thus can be applied to a broad spectrum of polymeric systems.2–4 The “living” character is expressed by a remarkably fast rate of polymerization with unusually high monomer conversions, first-order monomer kinetics, and predetermined molecular weights with narrow molecular weight distributions.5 In graft polymerizations on surfaces, the formation of homopolymer is completely obviated due to immobilized initiators and very low concentrations of polymer radicals.1,6 Besides a high tolerance of impurities3 and structures,7 the mild reaction conditions of ATRP even enabled the functionalization of corrosion-sensitive CMOS microchips with grafted poly(ethylene glycol) methacrylate (PEGMA) films for biomedical applications.8 Such PEGMA films dispose of an additional * Corresponding author. Telephone: +49-6221-424744. Fax: +49-6221421744. E-mail:
[email protected]. † Research Group “Chip-Based Peptide Libraries”. ‡ Research Group “Microenvironment of Tumor Cell Invasion”. (1) (a) Wang, J. S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614– 5615. (b) Sawamoto, M.; Kamigaaito, M. Trends Polym. Sci. 1996, 4, 371–377. (2) (a) Wang, X.-S.; Armes, S. P. Macromolecules 2000, 33, 6640–6647. (b) Balachandra, A. M.; Baker, G. L.; Bruening, M. L. J. Membr. Sci. 2003, 227, 1–14. (3) (a) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921–2990. (b) Kamigaito, M.; Ando, T.; Sawamoto, M. Chem. ReV. 2001, 101, 3689–3746. (4) (a) Neugebauer, D.; Zhang, Y.; Pakula, T.; Matyjaszewski, K. Macromolecules 2005, 38, 8687–8693. (b) El Tahlawy, K.; Hudson, S. M. J. Appl. Polym. Sci. 2003, 89, 901–912. (5) Wang, X.-S.; Lascelles, S. F.; Jackson, R. A.; Armes, S. P. Chem. Commun. 1999, 1817–1818. (6) (a) Feng, W.; Chen, R.; Brash, J. L.; Zhu, S. Macromol. Rapid Commun. 2005, 26, 1383–1388. (b) Xu, D.; Yu, W. H.; Kang, E. T.; Neoh, K. G. J. Colloid Interface Sci. 2004, 279, 78–87. (7) Mulvihill, M. J.; Rupert, B. L.; He, R.; Hochbaum, A.; Arnold, J.; Yang, P. J. Am. Chem. Soc. 2005, 127, 16040–16041. (8) Stadler, V.; Beyer, M.; Ko¨nig, K.; Nesterov, A.; Torralba, G.; Lindenstruth, V.; Hausmann, M.; Bischoff, F. R.; Breitling, F. J. Proteome Res. 2007, 6, 3197– 3202.
blocking of nonspecific protein-surface interactions9 and significantly reduce the adsorption of cells,10 which makes them very useful as antifouling coatings of biomaterials11 or solid supports in array technologies.8,12 Based on this, it was recently demonstrated that peptide arrays can be stained specifically with primary and secondary antibodies without using any additional blocking agents.13 This biocompatibility results from a minimum interfacial free energy of the hydrated poly(ethylene glycol) side chains upon contact with water, combined with a high surface mobility, and steric repulsion effects.14,15 In particular, the hydrophilicity of PEG-based coatings diminishes the short-range strong hydrophobic attraction between a hydrophobic surface and corresponding patches on a protein. Here, we synthesized graft copolymer films of PEGMA and methyl methacrylate (MMA) on silicon wafers to examine the dependency of protein adsorption on the grafting composition and thus on the hydrophobicity of the resulting films. Starting from rather hydrophilic PEGMA graftings (advancing contact angles with water of ∼53°),16 we gradually increased the MMA mole fraction up to pure PMMA films with a low water wettability (9) (a) Mayes, A. M.; Allgor, S. J. S.; Fujii, J. T.; Griffith, L. G.; Ankner, J. F.; Kaiser, H.; Johansson, J.; Smith, G. D.; Barker, J. G.; Satija, S. K. Macromolecules 1997, 30, 6947–6956. (b) Zhang, F.; Kang, E. T.; Neoh, K. G.; Wang, P.; Tan, K. L. Biomaterials 2001, 22, 1541–1548. (10) (a) Zhang, F.; Kang, E. T.; Neoh, K. G.; Wang, P.; Tang, K. L. J. Biomed. Mater. Res. 2001, 56, 324–332. (b) Zhang, F.; Kang, E. T.; Neoh, K. G.; Huang, W. J. Biomater. Sci., Polym. Ed. 2001, 12, 515–531. (11) (a) Kim, M. K.; Park, I. S.; Park, H. D.; Wee, W. R.; Lee, J. H.; Park, K. D.; Kim, S. H.; Kim, Y. H. J. Cataract RefractiVe Surg. 2001, 27, 766–774. (b) Hester, J. F.; Mayes, A. M. J. Membr. Sci. 2002, 202, 119–135. (12) Beyer, M.; Felgenhauer, T.; Bischoff, F. R.; Breitling, F.; Stadler, V. Biomaterials 2006, 27, 3505–3514. (13) Beyer, M.; Nesterov, A.; Block, I.; Ko¨nig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V. ; Bischoff, F. R.; Stadler, V.; Breitling, F. Science 2007, 318, 1888. (14) (a) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149–158. (b) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159–166. (15) Szleifer, I. Biophys. J. 1997, 72, 595–612. (16) Qiu, Y. X.; Klee, D.; Pluster, W.; Severich, B.; Hocker, H. J. Appl. Polym. Sci. 1996, 61, 2373–2382.
10.1021/la800772m CCC: $40.75 2008 American Chemical Society Published on Web 07/08/2008
8152 Langmuir, Vol. 24, No. 15, 2008
(advancing contact angles of ∼72.5°).17 According to this, we also expected a slight increase of advancing contact angles with decreasing PEGMA content, which should provide a correlation between the protein-repelling properties of the graft copolymer films and their wettability. For the gradual dilution of PEGMA, MMA was chosen due to several reasons: Both monomers have similar reactivities,18 while the sterical influence of the methoxy side group is as small as possible compared with other methacrylates. Since protein-surface interactions also depend on polymer film densities,14 this sterical factor was of additional interest. The moderate hydrophobicity of MMA/PMMA should ensure a homogeneous monomer distribution in the copolymer; on the other hand, this should also avoid a jump up of contact angles. Moreover, PMMA itself is also widely used in the field of biomaterials, e.g., for microfluidic chips or laboratory-on-achip devices,19,20 as keratoprostheses,21 intraocular lenses,22 denture resins or dental fillings,23,24 and hemodialysis membranes.25 In these applications, biofouling due to protein or cell adhesion is a major problem, which is why such protein-repelling coatings or even corresponding novel PEGMA/MMA copolymers are of substantial interest. Suitable PEGMA/MMA copolymer films might also result in biocompatible coatings with improved stability in vivo or in vitro compared to pure poly(ethylene glycol)based films, which usually are prone to degrade enzymatically or by oxidation.26 PEGMA/MMA copolymer graftings eventually enable the investigation of the interfacial properties of such materials. First, the synthesis of PEGMA/MMA graft copolymer films on silicon wafers was optimized, and the resulting surfaces were investigated by X-ray photoelectron spectroscopy (XPS), ellipsometry, and contact angle measurements. We incubated coated wafers with several protein solutions (BSA, lysozyme, fibrinogen, γ-globulin, and diluted human serum) and analyzed the amount of nonspecific protein adsorption by supersensitive XPS. To our surprise, the hydrophobicity of grafted PEGMA/MMA copolymer films did not show a linear increase with increasing MMA mole fraction, but exhibited a hydrophobic domain around a PEGMA fraction of 25% with advancing contact angles of up to 90°. Nevertheless, a breakdown of biocompatibility was only observed on coatings with 15% PEGMA and lower, which is why copolymer films with a PEGMA fraction of 25% should be the most hydrophobic surfaces resisting nonspecific protein adsorption known. This compares, for example, to advancing contact angles of 81° of protein-repelling mixed alkanethiol monolayers with methylated sorbitol endgroups.27 The origin of the hydrophobic domain was investigated by analyzing the morphologies of the copolymer films with atomic force microscopy (AFM). (17) Chai, J. N.; Lu, F. Z.; Li, B. M.; Kwok, D. Y. Langmuir 2004, 20, 10919– 10927. (18) Lee, J. H.; Oh, J. Y.; Kim, D. M. J. Mater. Sci.: Mater. Med. 1999, 10, 629–634. (19) (a) Bi, H.; Zhong, W.; Meng, S.; Kong, J.; Yang, P.; Liu, B. Anal. Chem. 2006, 78, 3399–3405. (b) Kitagawa, F.; Kubota, K.; Sueyoshi, K.; Otsuka, K. Sci. Technol. AdV. Mater. 2006, 7, 558–565. (c) Liu, J. K.; Pan, T.; Woolley, A. T.; Lee, M. L. Anal. Chem. 2004, 76, 6948–6955. (20) Becker, H.; Locascio, L. E. Talanta 2002, 56, 267–287. (21) Patel, S.; Thakar, R. G.; Wong, J.; McLeod, S. D.; Li, S. Biomaterials 2006, 27, 2890–2897. (22) Kurosaka, D.; Kato, K. J. Cataract RefractiVe Surg. 2001, 27, 1591– 1595. (23) Edgerton, M.; Raj, P. A.; Levine, M. J. J. Biomed. Mater. Res. 1995, 29, 1277–1286. (24) Kang, I. K.; Kwon, B. K.; Lee, J. H.; Lee, H. B. Biomaterials 1993, 14, 787–792. (25) Parzer, S.; Balcke, P.; Mannhalter, C. J. Mater. Sci.: Mater. Med. 1993, 4, 12–16. (26) Branch, D. W.; Wheeler, B. C.; Brewer, G. J.; Leckband, D. E. IEEE Trans. Biomed. Eng. 2000, 47, 290–300. (27) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. J. Am. Chem. Soc. 2000, 122, 8303–8304.
Stadler et al.
Interestingly, we observed significant differences between the surfaces at the nanoscale, which, however, were independent of the aforementioned phenomenon.
Experimental Section Materials. Graft copolymer synthesis and analysis were performed on polished single crystal silicon (100) wafers (Silicon Sense, Nashua, NH). PEGMA (Mn ∼ 360), copper(I) chloride (CuCl, p.a.), copper(I) bromide (CuBr, p.a.), 2,2′-bipyridyl (bipy, g98.0%), methanol (MeOH, p.a.), tetrahydrofuran (THF, p.a.), bovine serum albumin (BSA, Cohn fraction V, g96%), fibrinogen (from human plasma, clottable protein ∼95%), γ-globulin (from human blood, 99%), and lysozyme (from chicken egg white, lyophilized powder, ∼95%) were purchased from Sigma-Aldrich (Taufkirchen, Germany), and phosphate-buffered saline (PBS) tablets were purchased from Fluka (Buchs, Switzerland). MMA (p.a.) and (-)-sparteine (99%) were received from VWR International (Darmstadt, Germany), acetonitrile (p.a.) was obtained from AppliChem (Darmstadt, Germany), dimethylformamide (DMF, peptide grade) was obtained from Biosolve BV (Valkenswaard, Netherlands), and tri(ethylene glycol) monomethyl ether (TEGMME, p.a.) was obtained from Th. Geyer (Renningen, Germany). Anisole (99%) and 1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA, 99+%) were obtained from Acros Organics (Geel, Belgium). All chemicals were used as received with no further purification. Graft Polymerization. Surface pretreatment and silanization of silicon wafers with 2-bromo-2-methyl-N-propyltriethoxysilyl isobutyramide as ATRP initiator were described elsewhere.8 Graft polymerization of PEGMA was typically carried out with 5 mL of monomer (15.3 mmol) in 10 mL of a water/methanol mixture (1:1). Then, 64 mg of CuBr (0.45 mmol) and 141 mg of bipy (0.90 mmol) were added as ATRP catalyst. The brown reaction mixture was immediately degassed and sonicated under nitrogen atmosphere to dissolve the CuBr, before the silanized wafers were immersed. Polymerization took place under nitrogen atmosphere for at least 24 h. Afterwards the slides were extensively rinsed with distilled water, blown dry in a stream of nitrogen, and stored under nitrogen atmosphere at -20 °C. Graft polymer films of about 80 nm thickness and more were indicated by a blue staining of silicon substrates. Graft polymerizations of PEGMA/MMA mixtures and MMA were optimized according to a procedure described by Kimani and Moratti.28 With an amount of 50% PEGMA and above, 69.4 mmol of the monomer mixture itself was used as solvent with 34.6 mg of CuCl (0.35 mmol) and 146.3 µL of PMDETA (0.70 mmol) as ATRP catalyst. Otherwise, we added 5 mL of TEGMME as solvent and adjusted the monomer amount to 30.6 mmol. In contrast to CuBr/ bipy in MeOH/H2O, the reaction mixtures were colored deep blue. The graft polymerizations were processed as described for pure PEGMA films, unless we rinsed with TEGMME and distilled water. Incubation with Proteins. Initially, samples were hydrated with PBS buffer for 10 min. The buffer was removed and the wafers were immersed into 10 mL of the respective protein solution (PBS buffer, 2 mg/mL) in a Petri dish. Human serum was diluted 1:240 in PBS buffer to adjust the final protein concentration to approximately 2 mg/mL. After 60 min, the protein solutions were diluted continuously withPBSbuffertoremovetheproteinsandtoavoidLangmuir-Blodgettlike protein transfer onto the surface upon dehumidification of the sample. Finally, the slides were rinsed with Millipore water to remove residual PBS, blown dry with nitrogen, and stored at -20 °C under nitrogen. Surface Analysis. X-ray photoelectron spectra and ellipsometric data were recorded as described elsewhere.12 Advancing water contact angles (θa) with Millipore water were measured with a Kru¨ss Model G1 goniometer microscope under ambient conditions. Droplets were dispensed from a syringe with a micrometer gauge. The reported values are the average of five measurements taken for different samples with the tip contacting the drop. The instrument, however, (28) Kimani, S. M.; Moratti, S. C. Macromol. Rapid Commun. 2006, 27, 1887– 1893.
PEGMA/MMA Copolymer Graftings
Langmuir, Vol. 24, No. 15, 2008 8153
does not allow the measurement of receding contact angles. Atomic force microscopy was performed with a Nanowizard II AFM (JPK Instruments, Berlin, Germany). Coated silicon wafers were glued with double-sided adhesive tape to magnetic steel disks and mounted onto the sample holder. Imaging in air was performed in tapping mode with oxide-sharpened silicon nitride tips on 125 µm long cantilevers with a nominal spring constant of 40 N/m (type NCH, Nanoworld, Neuchaˆtel, Switzerland), a scanning speed of 1 Hz, and a cantilever drive frequency of approximately 250 kHz. Imaging in fluid was performed in contact mode with oxide-sharpened silicon nitride tips on 100 µm long cantilevers with a nominal spring constant of 0.38 N/m (type NP-S from Veeco Metrology Inc., Santa Barbara, CA). Images were processed using the Scanning Probe Image Processor (SPIP version 4.6.0, Image Metrology, Lyngsø, Denmark) and the JPK image processing software supplied with the instrument. Data analysis was done with SigmaStat 3.0 software (STATCON, Witzenhausen, Germany). The mean surface roughness (Ra) and the root mean square of the surface roughness (Rq) were calculated from (2 × 2) µm2 AFM images. With the SPIP software package, Ra is calculated as the average difference in height of surface variations according to M-1 N-1
∑ ∑ |z(xk, yl) - µ|,
1 Ra ) MN k)0
with µ )
l)0
M-1 N-1
∑ ∑ z(xk, yl) (1)
1 MN k)0
l)0
Here, µ is the mean height; M and N describe an M × N rectangular array of discrete points of the image. To obtain a statistical measure of the magnitude of height variations on individual copolymer surfaces, we also calculated the root mean square of the average surface roughness. Statistical significance was tested with a oneway ANOVA by the Holm-Sidak method for pairwise comparison.29 The threshold for significance was 0.05.
Results and Discussion Based on an inositol macroinitiator, Kimani and Moratti described the ATRP of star polymers of both MMA and PEGMA from TEGMME with CuCl/PMDETA as catalyst.28 They demonstrated that such ethylene glycol based solvents are most suited for ATRP with hydrophilic as well as hydrophobic monomers due to good solubility of both. Hence, we adapted this approach to PEGMA/MMA graft polymerization on silicon wafers: To optimize reaction conditions, we immobilized 2-bromo-2-methyl-N-propyltriethoxysilyl isobutyramide as initiator on silicon wafers, followed by immersion into an equimolar monomer mixture and CuCl/PMDETA in TEGMME. Based on previous experiments on the ATRP of PEGMA on identical surfaces,8 polymerization times of at least 20 h incubation were chosen. The main criterion of successful graft polymerization was film thickness, which was determined by ellipsometry: Basically, we succeeded with the catalyst/solvent system proposed, as indicated by films of several hundred angstroms thicknesses. On the basis of other reports, however, we also assayed other solvents and catalysts: In short, neither solvents like DMF or THF, nor a mixture of anisole/acetonitrile, worked at ambient conditions. CuBr/bipy as catalyst yielded no graft polymerization as well. This clearly indicated that the CuCl/ PMDETA/TEGMME system described was already adjusted to both, PEGMA and MMA, and also highlighted a low tolerance of ATRP reaction conditions with a particular monomer. With an equimolar PEGMA/MMA mixture and a fixed incubation time of 20 h, we initially observed a dependency of grafting thickness on the monomer concentration: with 15.3 mmol (29) Walker, G. A. Common Statistical Methods for Clinical Research with SAS Examples, 2nd ed.; SAS Publishing: Cary, NC, 2002.
Figure 1. Grafting thickness as a function of PEGMA mole fraction in the monomer mixture. Despite differing ATRP conditions, we observed a largely linear decrease in film thickness with a decreasing fraction of the more voluminous PEGMA monomer.
of the monomer mixture in 10 mL of TEGMME (1.53 M), we obtained films of (387.4 ( 31.9) Å on average; 24.5 mmol of monomers in 7 mL of TEGMME (3.5 M) resulted in films of (406.1 ( 49.8) Å; and 30.6 mmol in 5 mL of TEGMME (6.12 M) resulted in films of (453.2 ( 35.5) Å. This led us to the idea to carry out ATRP within a pure monomer solution of 11.32 mL of PEGMA and 3.68 mL of MMA (34.7 mmol each, in total 69.4 mmol), whereupon we obtained films of (677.7 ( 31.6) Å. Without TEGMME and with decreasing PEGMA fraction, however, the ethylene glycol effect described by Kimani and Moratti diminished.28 Accordingly, ATRP of pure MMA (7.36 mL, 69.4 mmol) without any solvent completely failed, which is why we had to dilute monomer mixtures with a PEGMA content of less than 50% with 5 mL of TEGMME. In doing so, we took two conflicting effects into account: A reduction of ethylene glycol units, either of the PEGMA monomer or of TEGMME as solvent, generally restrained ATRP, whereas increased monomer concentrations yielded thicker graft polymer films. This necessarily resulted in a decrease of total monomer amount from 69.4 mmol in pure monomer solutions (mixtures with 50% PEGMA and more) to 30.6 mmol in TEGMME diluted solutions (mixtures with less than 50% PEGMA), compared to only 15.3 mmol of pure PEGMA from MeOH/H2O. With these optimized but differing ATRP conditions, we observed a largely linear dependency of film thicknesses on the PEGMA fraction in the monomer mixture: From about 270 Å with pristine PMMA films, we obtained up to 1000 Å with PEGMA coatings after 20 h of incubation (Figure 1). This increase by a factor of 3.5 corresponded well with the higher molecular volume of PEGMA (0.543 nm3) compared to MMA (0.176 nm3), as calculated from the molar weight and density of the pure liquid components. The linear relationship observed seemed to contrast the correlation of film thickness with monomer concentration found with an equimolar mixture. Admittedly, the increase in film thickness from 30.6 mmol monomer amount to 69.4 mmol was comparatively small, which was presumably related to an excess of reactants. We hence supposed that the concentration effect might become blurred over the whole range of concentrations investigated here, with thicknesses depending more on a given PEGMA content than on monomer amount. The stoichiometry of PEGMA/MMA coatings was determined by XPS, which further allowed us to deduce the graft copolymer compositions. Although the attenuation lengths of core electrons emitted by X-rays is merely in the range of several nanometers,30 (30) Bain, C. D.; Whitesides, G. M. J. Phys. Chem. 1989, 93, 1670–1673.
8154 Langmuir, Vol. 24, No. 15, 2008
Stadler et al.
Figure 2. XP detail spectra of a PMMA coating with a C 1s ratio of approximately 3:1:1 (A) and an O 1s ratio of 1:1 (B). PEGMA films also revealed ratios close to the expected values of 3:10:1 for C 1s (C) and 6:1 for O 1s (D). Quantification was done by a peak-fitting routine with a Shirley background subtraction and Voigt profiles.
which corresponded only to the upper part of our graft polymer films, we assumed equivalent compositions of upper and lower film regions. According to this, XP spectra on silicon wafers featured only carbon (C 1s orbital) and oxygen (O 1s orbital) signals that related to the PEGMA/MMA films; Si 2p signals of the support were completely shielded. In all cases, a peak-fitting routine (Shirley background subtraction, Voigt profiles) revealed three individual C 1s peaks, which were assigned to the acrylate backbone (C-C) at 284.6 eV, the PEG or OMe side chains (C-O) at 286.1 eV, and the carboxyl group (CdO) at 288.4 eV (Figure 2A,C). The stochiometric ratios C-C:C-O:C)O obtained for pristine polymer films matched well with the expected theoretical values of 3:1:1 for PMMA and 3:10:1 for PEGMA (see inset in Figure 2). O 1s signals were divided into two peaks ascribed to the C-O and the C)O species with stoichiometric ratios of 1:1 for PMMA and 6:1 for PEGMA, respectively (Figure 2B,D). Based on numerical integration and the theoretical ratios expected, we deduced the composition of graft copolymer films from the ratios of C-O:C-C and C-O:CdO. These ratios should vary by a factor of up to 10 for the C 1s signals (10:3 for PEGMA vs 1:3 for PMMA) and up to 6 for the O 1s signals (6:1 for PEGMA vs 1:1 for PMMA) over the whole range of grafting mixtures realized. For each polymer grafting, we determined the stoichiometry by fitting and averaged the results over C 1s and O 1s signals as well as over at least five samples for each composition. As expected for similar monomer reactivities,18 we observed a linear dependency of the PEGMA fraction in the polymer on the mole fraction provided in the monomer solution (Figure 3). This result indicated a largely statistical distribution of PEGMA and MMA within the graft polymer chain, since otherwise a favored incorporation of one of the species would be accompanied by block copolymer formation: An initial
Figure 3. Correlation of grafting composition with PEGMA mole fraction in the monomer solution. Individual data points were obtained by numerical integration of C 1s and O 1s XP detail spectra, followed by averaging over both signals and at least five different samples.
preference for one monomer sort would cause its depletion in the ATRP solution, with an inverted monomer preference in the outer layer of the copolymer coatings due to concentration effects. This in turn should have been detectable by XPS, resulting in a nonlinear correlation of the monomer fractions of polymer and solution, respectively. The main emphasis of this work was placed on a correlation of grafting wettability and nonspecific protein adsorption. Accordingly, we measured advancing contact angles (θa) with water and verified the corresponding film composition for all different monomer mixtures employed. When plotted against the determined PEGMA fraction in the polymer, a gradual increase of θa from about (53.0 ( 2.0)° for pure PEGMA films16 to (72.5 ( 0.6)° for PMMA films was expected.17 Experimentally evaluated θa values for pure films were (56.3 ( 2.6)° for PEGMA
PEGMA/MMA Copolymer Graftings
Langmuir, Vol. 24, No. 15, 2008 8155
Figure 4. Advancing contact angles (θa) with water as a function of PEGMA mole fraction in the polymer (top). The hydrophobic domain is highlighted in gray. Amount of nonspecific protein adsorption referenced to the PEGMA mole fraction in the monomer solution (bottom); a collapse of biocompatibility was observed at 15% PEGMA and below, ranging far in the hydrophobic domain.
and (71.3 ( 1.4)° for PMMA, which agreed with the literature within standard deviations. The progression of θa with increasing MMA fraction, however, revealed an unexpected deviation from linearity in the range of mole fractions from 0.15 to 0.40 PEGMA (Figure 4, top). We observed a sudden gain in hydrophobicity with maximum values of up to 91° next to 25-30% PEGMA, significantly more than anticipated from the neat components. This surprising hydrophobic domain (Figure 4, gray area) was followed by a relapse of θa to “ordinary” values with 15% PEGMA and below. To correlate wettability with biocompatibility, we investigated the interaction of four different proteins (fibrinogen, bovine serum albumin, γ-globulin, and lysozyme) and diluted human serum with the various graft copolymer films. The extent of nonspecific protein adsorption was quantified by XPS, since N 1s signals were assigned exclusively to amide and amino groups of proteins. Under the experimental conditions (PBS buffer, pH 7.4), fibrinogen is a negatively charged, large, and sticky protein (340 kDa), which generally plays a key role in thrombus formation and blood clotting. In contrast, lysozyme is a small and positively charged protein (14 kDa).31 BSA and γ-globulin represent globular proteins with abundance in blood, whereas albumin, contrary to fibrinogen and γ-globulin, tends to adsorb to antithrombogenic surfaces.32 As already mentioned in the Introduction, pure PEGMA films effectively block nonspecific interactions of proteins even from diluted human serum,8 mainly due to their hydrophilic character and sterical effects.14,15 In (31) Colton, I. J.; Anderson, J. R.; Gao, J.; Chapman, R. G.; Isaacs, L.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 12701–12709. (32) 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.
Figure 5. Morphologies of PEGMA/PMMA films in air and in ultrapure water measured by AFM. Shown are the height images of PEGMA (A), PMMA (B), and copolymers with 25% (C), 50% (D), and 75% PEGMA (E) in tapping mode. PMMA (F) and a film with 25% PEGMA (G) were also imaged in ultrapure water (scale bar ) 500 nm).
accordance with that, we found no protein adsorption on pure PEGMA films (Figure 4, bottom). When we increased the MMA fraction up to 75%, nonspecific protein adsorption was still negligible or even completely blocked. This was a rather surprising finding, since the hydrophobic character observed in this molar range should act counterproductively on the protein-repelling nature of graft copolymer films. Presumably, sterical repulsion due to the high surface mobility of PEG chains still outbalanced the strong hydrophobic interactions between proteins and the surface. This mobility of PEG side chains might even be enhanced in PEGMA/MMA copolymer films, since the small methoxy side chains of MMA should reduce the polymer packing density compared to pure PEGMA films. Nevertheless, when the MMA fraction was continuously increased, we followed a collapse of biocompatibility with 15% PEGMA and below. Although θa then relapsed to less hydrophobic surfaces, the impact of PEG side chains was further weakened and finally completely inhibited. The observed dependency of protein-repelling properties on the grafting composition, however, clearly demonstrated that even minor PEGMA fractions of 20% were sufficient for blocking nonspecific adsorptions on PEGMA/MMA copolymer surfaces. This should be of importance for nonfouling coatings of PMMA devices for biomedical research, or might even give rise to novel polymers effectively combining different material properties,
8156 Langmuir, Vol. 24, No. 15, 2008
Stadler et al.
Figure 6. Roughness of copolymer surfaces calculated from AFM images. The plot shows the average roughness (Ra, light gray) and the root mean square of the average roughness (Rq, dark gray) in air and water. A significant change in surface roughness could only be detected on copolymers with PEGMA mole fractions of 0.75 and 0.5, but not in the hydrophobic domain.
e.g., the nonaging of weatherproof PMMA with biocompatibility of PEGMA. We hypothesized that the hydrophobic domain from 15-40% PEGMA could be attributed to one or both of two supposable origins: (1) The most hydrophobic component of PEGMA/MMA copolymers, the aliphatic backbone, was somehow presented preferentially at the outermost surface of the grafting. (2) In contact with water, the copolymer surface featured a specific topography in the micro- or nanometer range similar to the lotus effect.33 The latter hypothesis was tested by high resolution imaging by AFM in air and in contact with water. In air, we mapped the pure graft polymer films of PMMA and PEGMA as well as mixtures with 25%, 50%, and 75% PMMA, whereas the latter mixture represented the hydrophobic domain. (Figure 5A-E). On the face of it, only the 100% PMMA surface showed significantly different surface features. To gain a deeper insight, we analyzed and compared the mean roughness Ra of the various copolymer films (Figure 6, light gray bars). The results of these roughness analyses demonstrated that most of the individual copolymer surfaces exhibit a largely similar roughness, except for the copolymers with 50% and 75% PEGMA (Table 1). The statistical differences were studied using a one-way ANOVA with the Holm-Sidak method for pairwise comparison, whereas a significance level of 0.05 was used. The same is true for the root-mean-square average of the roughness Rq, which is sensitive for surface features: Besides copolymers with 50% and 75% PEGMA, the Rq values of individual copolymer surfaces were not significantly different in air as well (Figure 6, dark gray bars). In particular, we focused on copolymer films within the
hydrophobic domain in the range of 15-40% PEGMA, which is why we analyzed PMMA films as well as films with 25% PEGMA in water. By looking at the AFM images themselves (Figure 5F,G), we observed that these copolymer surfaces exhibited the same features as when scanned in air (Figure 5B,C). We found that even the surface roughness did not change substantially and was in the range of the surface roughness measured in air (Figure 6). Again, statistical differences were only observed in comparison with copolymer films with 50% and 75% PEGMA (Table 1). In summary, the results obtained from AFM measurements did not reveal any specific structural feature of copolymer surfaces located within the hydrophobic domain. We were yet not able to attribute the unexpected deviation of advancing contact angles with water to any morphological effect and can only speculate about this unknown phenomenon. It can be conceived that in some way the aliphatic backbone is preferentially presented in the outermost surface of films of the hydrophobic domain, which would result in an increased hydrophobicity and low wettability. Referring to this, the advancing contact angles of aliphatic polymers such as polypropylene and polyethylene with water are about 99° and 110°, respectively.34 At present, this hypothesis seems to be most evident, but in turn raises the question of how this can correlate with biocompatibility. First experiments with PEGMA/BuMA copolymer graftings yielded advancing contact angles of up to 110°: this might form a basis to track this phenomenon. In a forthcoming study, we are going to focus on the dependency of XPS data, film thicknesses, and particularly advancing as well as receding contact angles with water on the composition of different PEGMA copolymer systems. Besides PMMA, we are going to employ methacrylates with ethyl, butyl, and 2,2,3,3,3-pentafluoropropyl side chains. Combining a variety of different copolymers with a closer look at contact angles and contact angle hysteresis may provide additional information on the surface morphology of such PEGMA coatings.
Conclusions We optimized the synthesis of PEGMA/MMA graft copolymer films on silicon wafers with respect to film thickness and investigated the composition and water wettability of the resulting coatings. Although the ATRP conditions varied over the investigated range of copolymer mixtures, we observed a largely linear increase of film thickness with the PEGMA mole fraction in solution, which was attributed to the different molecular volumes of the monomers used here. We also found a linear correlation of the mole fraction provided in solution with the polymer composition, which was determined by numerical integration of XP spectra. The wettability of PEGMA/MMA films was examined by measurement of advancing contact angles with water: We revealed a hydrophobic domain in the range of 15-40% PEGMA with values of up to 90° significantly exceeding the contact angles of the pure components. Nevertheless, even the most hydrophobic copolymers blocked the nonspecific
Table 1. Comparison of the Statistical Differences of the Mean Roughness (Ra) between the Individual Surfacesa PEGMA mole fraction 1 (air) 0.75 (air) 0.50 (air) 0.25 (air) 0 (air) 0.25 (liquid) 0 (liquid) a
1 (air) ++ ++ -----
+ +, significant; - -, not significant.
0.75 (air)
0.50 (air)
0.25 (air)
0 (air)
0.25 (liquid)
0 (liquid)
++
++ ++
-++ ++
-++ ++ --
-++ ++ ---
--++ ----
++ ++ ++ ++ --
++ ++ ++ ++
----
---
--
PEGMA/MMA Copolymer Graftings
adsorption of proteins (fibrinogen, lysozyme, γ-globulin, BSA, and diluted human serum), as shown by quantification of the XP spectra of N 1s regions. A breakdown of the protein-repelling properties was only observed for 15% PEGMA and lower, which indicated a prevalence of the sterical repulsion by PEG side chains over the attractive hydrophobic interaction of proteins with a surface. We hypothesized that either a specific morphological effect or a preferred presentation of hydrophobic modules at the polymer surface was responsible for the hydrophobic domain observed. Therefore, we utilized AFM to map the pure polymer films as well as copolymer films with 75%, 50%, and 25% PEGMA in air and in contact with water. Significant statistical differences in surface roughness were only observed on copolymer films with 50% and 75% PEGMA but not on films within the hydrophobic domain. At present, we cannot explain the deviations in advancing contact angles with water on the basis of surface morphology.
Langmuir, Vol. 24, No. 15, 2008 8157
In conclusion, it was proven that even minor PEGMA fractions were sufficient for blocking nonspecific protein interactions on PEGMA/MMA copolymer films. This might open up new perspectives for nonfouling coatings of PMMA devices in biomedical research, or even for novel polymers combining different material properties with the biocompatibility of PEGMA. Acknowledgment. The authors thank Reiner Dahint and Michael Grunze for providing surface analytical methods. This work was supported by the German Federal Ministry of Education and Research (Grant Nos. 03N8710 and 0313375A), the MaxBuchner-Forschungsstiftung at the DECHEMA, the Deutsche Forschungsgemeinschaft (LU 854/ 3-1), and the Fonds der Chemischen Industrie. LA800772M (33) Barthlott, W.; Neinhuis, C. Planta 1997, 202, 1–8. (34) Tretinnikov, O. N.; Ikada, Y. Langmuir 1994, 10, 1606–1614.
