Langmuir 2008, 24, 6649-6658
6649
Imparting Antifouling Properties of Poly(2-hydroxyethyl methacrylate) Hydrogels by Grafting Poly(oligoethylene glycol methyl ether acrylate) Dimitriya Bozukova,† Christophe Pagnoulle,| Marie-Claire De Pauw-Gillet,‡ Nadia Ruth,§ Robert Jérôme,† and Christine Jérôme*,† Center for Education and Research on Macromolecules (CERM), Laboratory of Histology and Cytology, and Center for Protein Engineering (CIP), UniVersity of Liege, B6 Sart-Tilman B-4000 Liege, Belgium, and PhysIOL SA, Parc Scientifique du Sart-Tilman, Allee des noisetiers 4, B-4031 Liege, Belgium ReceiVed October 30, 2007. ReVised Manuscript ReceiVed February 8, 2008 The antifouling properties of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) hydrogels were improved by the surface grafting of a brush of poly(oligoethylene glycol methyl ether acrylate) [poly(OEGA)]. The atom-transfer radical polymerization (ATRP) of OEGA (degree of polymerization ) 8) was initiated from the preactivated surface of the hydrogel under mild conditions, thus in water at 25 °C. The catalytic system was optimized on the basis of two ligands [1,1,4,7,10,10-hexamethyl-triethylenetetramine (HMTETA) or tris[2-(dimethylamino)ethyl]amine (Me6TREN)] and two copper salts (CuIBr or CuICl). Faster polymerization was observed for the Me6TREN/CuIBr combination. The chemical composition and morphology of the coated surface were analyzed by X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy, contact angle measurements by the water droplet and captive bubble methods, scanning electron microscopy, and environmental scanning electron microscopy. The hydrophilicity of the surface increased with the molar mass of the grafted poly(OEGA) chains, and the surface modifications were reported in parallel. The antifouling properties of the coatings were tested by in vitro protein adsorption and cell adhesion tests, with green fluorescent protein, β-lactamase, and lens epithelial cells, as model proteins and model cells, respectively. The grafted poly(OEGA) brush decreased the nonspecific protein adsorption and imparted high cell repellency to the hydrogel surface.
Introduction Hydrogels are hydrophilic polymeric networks that are able to absorb a large amount of water.1 They are soft and flexible, with physical characteristics reminiscent of soft tissues.2 Poly(2hydroxyethyl methacrylate) (polyHEMA) is a very well known component of hydrogels and has great potential in the design and production of uretenal stents and urethral catheters,1 implantable drug delivery systems,3 neural tissue devices,2 nerve guidance channels,4 and contact and intraocular lenses.5–8 Nonspecific protein adsorption occurs at the surface of a foreign material whenever it is inserted into a biological environment. The adsorption of plasma proteins is an initial step of the inflammatory cascade, which is usually followed by the adhesion of macrophages that secrete mediators of inflammation and tissue destruction.3,9,10 Foreign-body giant cells that serve as markers * Corresponding author. Tel: +32 4 3663491. Fax: +32 4 3663497. E-mail:
[email protected]. † Center for Education and Research on Macromolecules (CERM), University of Liege. ‡ Laboratory of Histology and Cytology, University of Liege. § Center for Protein Engineering (CIP), University of Liege. | PhysIOL SA. (1) Jones, D. S.; McLaughlin, D. W. J.; McCoy, C. P.; Gorman, S. P. Biomaterials 2005, 26, 1761–1770. (2) Flynn, L.; Dalton, P. D.; Shoichet, M. S. Biomaterials 2003, 24, 4265– 4272. (3) Dziubla, T. D.; Torjman, M. C.; Joseph, J. I.; Murphy-Tatum, M.; Lowman, A. M. Biomaterials 2001, 22, 2893–2899. (4) Dalton, P. D.; Flynn, L.; Shoichet, M. S. Biomaterials 2002, 23, 3843– 3851. (5) Kwon, O. H.; Nho, Y. C.; Lee, Y. M. J. Ind. Eng. Chem. 2003, 9, 138–145. (6) Lloyd, A. W.; Faragher, R. G. A.; Denyer, S. P. Biomaterials 2001, 22, 769–785. (7) Parson, C.; Jones, D. S.; Gorman, S. P. Expert ReV. Med. DeVices 2005, 2, 161–173. (8) Abraham, S.; Brahim, S.; Ishihara, K.; Guiseppi-Elie, A. Biomaterials 2005, 26, 4767–4778. (9) Lan, S.; Veiseh, M.; Zhang, M. Biosens.Biolelectron. 2005, 20, 1697– 1708.
for foreign-body response are then formed.9 Any time this biological response is undesired, the surface of the substrate must be modified to make it resistant to protein adsorption and cell adhesion. There is, thus, a need for coatings with antifouling properties that are able to improve the performances of implanted biomedical devices. In this respect, poly(ethylene oxide) (PEO) is a synthetic polymer that is very effective at preventing protein adsorption and cell adhesion as a result of its hydrophilicity, high surface mobility, and low interfacial free energy with water.10,11 The molecular architecture of PEO is also important, with nonlinear chains (star-shaped and brush-shaped) being more effective than the linear counterparts at preventing nonspecific protein adsorption.12–15 The reason has to be found in a larger surface area occupied per chain and a higher surface mobility of PEO. The great variety of techniques for attaching a polymer to the surface of a substrate might be divided in two main groups: “grafting to” and “grafting from” techniques.16 Grafting to relies on the chemical bonding of a preformed polymer by reaction with functional groups available at the surface. Although the polymer can be tailored and characterized before grafting, this method suffers the limitation of a low grafting density. Indeed, the earlier grafted chains rapidly hinder extra chains from diffusing to and reacting with the surface. In the grafting from method, (10) Lee, J. H.; Oh, S. H. J. Biomed. Mater. Res. 2002, 60, 44–52. (11) Pasche, S.; De Paul, S. M.; Vörös, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 19, 9216–9225. (12) Groll, J.; Ademovic, Z.; Ameringer, T.; Klee, D.; Moeller, M. Biomacromolecules 2005, 6, 956–962. (13) Irvine, D. J.; Mayes, A. M.; Griffith-Cima, L. Macromolecules 1996, 29, 6037–6043. (14) Irvine, D. J.; Mayes, A. M.; Satija, S. K.; Barker, J. G.; Sofia-Allgor, S. J.; Griffith, L. G. J. Biomed. Mater. Res 1998, 40, 498–509. (15) Gabriel, S.; Dubruel, P.; Schacht, E.; Jonas, A. M.; Gilbert, B.; Jérôme, R.; Jérôme, C. Angew. Chem., Int. Ed. 2005, 44, 5505–5509. (16) Feng, W.; Chen, R.; Brash, J. L.; Zhu, S. Macromol. Rapid Commun. 2005, 26, 1383–1388.
10.1021/la7033774 CCC: $40.75 2008 American Chemical Society Published on Web 05/27/2008
6650 Langmuir, Vol. 24, No. 13, 2008
a polymerization initiator is attached to the surface, possibly at high density, followed by chain growth from the surface. The characterization of grafted chains is usually a problem that can be alleviated whenever the polymerization is controlled and conducted simultaneously in solution and at the surface. Surface-initiated atom transfer radical polymerization (SIATRP) is a recent example of the grafting from technique,16–20 which is illustrated by the grafting of poly(N,N-dimethylacrylamide) on polystyrene latex21 and by the grafting of silicon wafers by a variety of polymers, including PEO,16,19,20 poly(2methacryloyloxyethyl phosphorylcholine),19,20 polystyrene-bpolymethyl acrylate-b-polystyrene,22 and recently by glycomonomer on polypropylene microporous membranes.23 In this article, we report on ATRP of oligo(ethylene glycol) methyl ether acrylate (OEGA) initiated from the surface of preactivated poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) [poly(HEMA-co-MMA)] hydrogels. Commercially available poly(HEMA-co-MMA) hydrogels, used for the fabrication of IOL, were the substrate of choice because one of our indirect purposes was to use a material with real industrial application as a substrate for the modification. IOL implantation is one of the most frequently implemented surgical interventions. Protein adsorption and lens epithelial cell (LEC) adhesion on the posterior side of an implanted IOL is an undesired postoperative complication that leads to secondary lens opacification, known as posterior capsular opacification (PCO). The only way to treat the latter is with Nd:YAG laser capsulotomy. Therefore, creating protein- and cell-repellent coating on the lens surface would be an efficient way to prevent protein adsorption and cell adhesion. For the SI-ATRP modification of these materials, several ligands and copper salts were tested to optimize the catalytic system. The chemical composition and morphology of the modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), contact angles measured by the water droplet (CAwd) and captive bubble (CAcb) techniques, scanning electron microscopy, and environmental scanning electron microscopy (ESEM). Because our goal is to elucidate the antifouling properties of the grafted surface, in vitro testing of protein adsorption and cell adhesion has been performed. Green fluorescent protein (GFP) and β-lactamase along with lens epithelial cells were used as model proteins and cells, respectively.
Experimental Section Materials. 2-Bromopropionyl bromide (BPB), anhydrous ethylene glycol (EG), the OEGA macromonomer containing eight ethylene oxide units (degree of polymerization, DP ) 8), and 1,1,4,7,10,10hexamethyl-triethylenetetramine (HMTETA) from Aldrich were analytical-grade products and used as received. Water was purified by a Milli-Q system from Millipore. Ten grams of CuIBr or CuICl (Aldrich), respectively, was dispersed within 100 mL of glacial acetic acid under stirring overnight and then filtered, washed with methanol, dried under reduced pressure, and stored under argon. (17) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921–2990. (18) Shen, Y.; Tang, H.; Ding, S. Prog. Polym. Sci. 2004, 29, 1053–1078. (19) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Biointerphases 2006, 1, 50–60. (20) Feng, W.; Brash, J. L.; Zhu, S. Biomaterials 2006, 27, 847–855. (21) Jayachandran, K. N.; Takacs-Cox, A.; Brooks, D. E. Macromolecules 2002, 35, 4247–4257. (22) Boyes, S. G.; Brittain, W. J.; Weng, X.; Cheng, S. Z. D. Macromolecules 2002, 35, 4960–4967. (23) Yang, Q.; Tian, J.; Hu, M. X.; Xu, Z. K. Langmuir 2007, 23, 6684–6690.
BozukoVa et al. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was synthesized from tris-(2-aminoethyl)-amine (Aldrich) as described elsewhere.24 1H NMR (CDCl , 25 °C, 250 MHz): δ 2.5–2.56 (-CH -N(CH ) ), 3 2 3 2 2.27–2.32 (-CH2-N(CH2)2-), 2.15 (-CH3). The relative intensities of the resonances of the -CH2- protons and the -CH3 protons confirmed the structure of Me6TREN. No other peaks have been observed, which might be relevant to a pure product. A purity of more than 98% has also been estimated by gas chromatography. Activation of Poly(HEMA-co-MMA) Hydrogels. Commercially available random poly(HEMA-co-MMA) hydrogels (BENZ FLEX 26 UV, equilibrium water content of 26 wt %) that are used for the fabrication of intraocular lenses were chosen as the substrate. They were cut out with a diamond-equipped tool to the desired size and thickness and purified by extraction with Milli-Q water for 5 days in order to eliminate any residual monomer and oligomer. They were dried in a vacuum oven at 50 °C for 2 days, just before use.25 The hydroxyl groups available on the surface were esterified with BPB for 30, 60, or 120 min at room temperature in the bulk and in an inert atmosphere. Nonreacted BPB was removed by washing the samples with acetonitrile, followed by drying under reduced pressure for 2 h. The samples were analyzed by ATR-IR, the contact angle (CA) of water, and XPS. If not used immediately, the samples were stored for less than 1 day. Synthesis of r-Hydroxyethyl-2-bromopropionate (HEBP). A flame was applied, under vacuum, to a glass reactor equipped with a Teflon stir-bar and a dropping funnel and then anhydrous EG (300 g, 4.84 mol, 75 mol equiv) was added under argon. After the reactor was cooled in an ice bath, BPB (14.01 g, 0.0649 mol, 1 mol equiv) was added dropwise through the dropping funnel within 20 min. The temperature was allowed to increase slowly to room temperature for the 4 h reaction. Then, 200 mL of Milli-Q water and 50 mL of a saturated NaHCO3 solution were added, and the reaction product was extracted twice with 200 mL of dichloromethane. The organic phase was washed twice with 150 mL of Milli-Q water, dried over magnesium sulfate for one night, and filtered. The reaction product was collected as a colorless liquid after the removal of the solvent, and vacuum distillation was carried out at 82 °C and 0.05 torr. Yield ≈25%. 1H NMR (CDCl3, 25 °C, 400 MHz): δ 2.37 (s, HO-), 3.82 (t, HO-CH2-), 4.26 (t, -CH2-OC(O)-), 4.4 (t, -CH-Br), 1.8 (d, -CH3). From the relative intensities of the resonances of the HO-CH2- and -CH3 protons, the structure of a monofunctional product was estimated, although the presence of a bifunctional one could not be excluded. (See the forthcoming discussion.) Surface-Initiated ATRP of OEGA. The reaction solvent, Milli-Q water, was degassed by bubbling argon for 1 h just before use. For each experiment, 4 mL of OEGA was degassed in a separate flask by three vacuum-argon cycles under stirring and added to 2 mL of water. In a typical ATRP grafting run, the HEBP initiator (1 mol equiv), the CuI catalyst (1.2 mol equiv), and the ligand (1.44 mol equiv) were added to a flask equipped with a Teflon stir-bar and stainless-steel pliers used to maintain a piece of preactivated dry hydrogel immersed in the final reaction mixture. The flask was purged by three cycles of freezing-vacuum-argon-melting, and 2 mL of water was added to dissolve the reactants. The OEGA solution was finally added, and the reaction was allowed to proceed at 25 °C for 20 h, unless otherwise noted. The hydrogel sample was then washed with water in a Soxhlet extractor for 5 days. It was either kept hydrated until analysis or dried in a vacuum oven at 50 °C for 2 days. Hydrated samples were observed by ESEM and used for biological tests and captive bubble contact angle measurements. Dry samples were analyzed by ATR-IR and water droplet contact angle measurements and were observed by SEM. Aliquots of the polymerization mixture were characterized by 1H NMR in order to determine the monomer conversion, if desired. The rest of the solution was purified by elution through a silica column with a tetrahydrofuran/methanol (70/30 v/v) solvent mixture. Solvents (24) Ciampolini, M.; Nardi, N. Inorg. Chem. 1966, 5, 41. (25) Bozukova, D.; Pagnoulle, C.; De Pauw-Gillet, M. C.; Ruth, N.; Jérôme, R.; Jérôme, C. Biomacromolecules 2007, 8, 2379–2387. (26) Luo, Y.; Dalton, P. D.; Shoichet, M. S. Chem. Mater. 2001, 13, 4087– 4093.
Imparting Antifouling Properties by Grafting
Langmuir, Vol. 24, No. 13, 2008 6651
Scheme 1. Reaction Scheme for the Grafting of OEGA from the Surface of Preactivated Poly(HEMA-co-MMA) Hydrogels by ATRP in Water
were eliminated in a rotating evaporator, and the polymer was analyzed by size exclusion chromatography (SEC, details given in a forthcoming section). Chemical Characterization. The chemical composition of the surface-modified samples was determined by ATR-IR with a Nicolet spectrophotometer (Ge crystal) in the 800–1800 nm range. In some of the spectra, a small peak for CO2 from the ambient atmosphere at about 2600 cm-1 was observed. XPS was performed with an SSX-100 spectrometer (Surface Science Instruments), equipped with a monochromatic Al KR source and a spherical sector analyzer with a multichannel detector. The energy resolution corresponds to the full width at half-maximum (fwhm) of 0.65 eV for the Ag 3d5/2 peak. All of the spectra were collected in the normal emission with an X-ray spot size of 1000 µm. Electrostatic charging was stabilized by mounting a nickel grid about 1 to 2 mm above the sample and by flooding the sample with a wide beam of low-energy electrons. Contact angles were measured with a DGD Fast/60 contact angle meter and WINDROP++ software. At least three measurements per sample were taken. Contact angles of the dry surface were measured by the water droplet (CAwd) technique at a fixed time of 30 s with an average deviation of (2°. The measurement is representative of the solid/liquid interface (θs1) of the dry surface. CAwd ≈ 0° stands for the absorption of the water droplet within less than 30 s. Once deposited on some surfaces, the water drop immediately spread on the surface of the hydrogel, preventing the measurement from being properly performed. Such a sample has been referred to as having a CAwd of 0°. Contact angles of the hydrated surface were measured by the captive bubble (CAcb) technique with an average deviation of (3°. The contact angle of the solid/gaseous interface (θb) was directly measured and was then transferred to the contact angle of the solid/liquid interface (θs2) by the equation θs2 ) 180° - θb in order to facilitate the comparison of the hydrophilicity of the dry surface and the hydrated surface. The surface morphology of the dry hydrogels before and after modification has been observed by SEM on a Jeol JSM 840A instrument. The surface of the hydrated hydrogels have been observed by ESEM (XL30 ESEM-FEG from Philips) under the conditions reported in ref 26. An accelerating voltage of 10.0 kV and 1000× magnification have been applied to limit both the heating effect and sample damage. In the sample chamber, the pressure was maintained at 4.4–5.3 Torr (see image captions), and the temperature was maintained at 1.5 ( 0.5 °C using a Peltier-cooled sample stage. The sample chamber was periodically flushed with water vapor to maintain a satisfactory partial pressure of water, preserving the hydrogels from dehydration. 1H NMR measurements have been performed on a Bruker AM 400 MHz in D2O. Gas chromatography was carried out on a Varian 3900 instrument equipped with a flame ionization detector and a WCOT fused silica column (stationary phase, CP-Sil 5CB; column length, 0.39 mm; film thickness, 0.25 µm).
SEC was carried out in dimethylformamide containing 25 mM LiBr (flow rate, 1 mL/min) at 55 °C with a Waters 600 liquid chromatrograph equipped with a 410 refractive index detector (four Waters styragel columns: HR 1 (100–5000), HR 3 (500–30 000), HR 4 (5000–500 000), and HR 5 (2000–4 000 000) (7.8 × 300 mm2)). PEO standards were used for calibration. Green Fluorescent Protein Adsorption (in Vitro Test). The GFP adsorption test was carried out as described elsewhere.25 The GFP was expressed in Escherichia coli strain BL21 (DE3) transformed with plasmid coding for a GFP mutant (GFP mut2). The production and purification were performed as described by Dreesen et al.27 The purity of the protein solution was estimated by SDS-PAGE (>96% purity). Briefly, the hydrogels were preconditioned in a phosphate buffer solution (pH 7) for 24 h. They were then immersed in a PBS solution of GFP (0.15 mg/mL) and maintained for 3 days in an incubator (37 °C, 5% CO2). They were gently rinsed with PBS just before observation. Fluorescence microscopy images of the surfaces were recorded with a cooled AxioCam MRm (Zeiss) mounted on a Zeiss Axio Imager.Z1 microscope through a EC Plan-NEOFLUAR 100 × 1.3 oil immersion objective with a 400 to 500 nm band-pass excitation filter and a 460 to 560 nm band-pass emission filter. AxioVision Rel 4.5 (Zeiss) software was used. β-Lactamase Adsorption (in Vitro Test). The β-lactamase adsorption test was performed as described in ref 28a,b. It consisted of incubation of unmodified and modified samples (according to the conditions from entry 3 in Table 2) in phosphate buffer solution containing 0.3 mg/mL β-lactamase BlaP at 37 °C for 150 min. Three washing steps with phosphate buffer solution were then carried out at room temperature to eliminate the loosely adsorbed protein prior to UV–vis quantification. The hydrogel samples were then dipped in an aqueous solution of nitrocefin (100 µM), and the absorbance was measured as a function of time. A calibration curve allowed the absorbance to be converted into the actual mass of adsorbed, active β-lactamase. The activity of protein adsorbed onto the surface of the samples was measured by UV–vis according to the procedure described in ref 28b. LEC Isolation and Culturing. Lens epithelial cells (LECs) were isolated and cultured from the lens crystalline capsule of pig eyes (Pietrain-Landrace pig, from the Abattoir communal de Liège) as (27) Dreesen, L.; Humbert, C.; Sartenaer, Y.; Caudano, Y.; Volcke, C.; Mani, A. A.; Peremans, A.; Thiry, P. A.; Hanique, S.; Frère; J, M. Langmuir 2004, 20, 7201–7207. (28) (a) Xu, F.; Zhen, G.; Yu, F.; Kuennemann, E.; Textor, M.; Knoll, W. J. Am. Chem. Soc. 2005, 127, 13084. (b) Gabriel, S.; Cécius, M.; Fleury-Frenette, K.; Cossement, D.; Hecq, M.; Ruth, N.; Jérôme, R.; Jérôme, C. Chem. Mater. 2007, 19, 2364–2371.
6652 Langmuir, Vol. 24, No. 13, 2008
BozukoVa et al.
Figure 1. ATR-IR spectra of (a) a nonactivated poly(HEMA-co-MMA) hydrogel and the same material activated by BPB for (b) 1 and (c) 2 h.
reported elsewhere.25,29,30 Cells after the fourth passage were used for testing. LEC Adhesion (in Vitro Test). The test was similar to the one reported in ref 25. The samples were preconditioned in a SERAG BSS physiological solution (pH 7.3) for 2 days and cut as disks with 6 mm diameter, thus fitting the test wells. They were washed and sterilized in physiological solution at 1.5 bar, 120 °C for 21 min. They were then fixed on the bottom of a 96-well plate and conditioned with culture medium (CM) in an incubator for 2 h. After the removal of the conditioning CM, a 5000 cells/100 µL suspension was added to each well, followed by 100 µL of CM. After 3 days of contact of the hydrogels and the LEC suspension in the incubator, 50 µL of CM was added. After 2 extra days of incubation, the culture medium was removed, followed by careful washing with 1 mL of PBS to eliminate the nonadhering and dead cells. The cells were fixed with 4% paraformaldehyde at 4 °C for 2 h, washed three times with PBS, and kept in PBS at 6 °C. The sample surface was observed with a Zeiss Axioplan optical microscope equipped with Epiplan-NEOFLUAR objectives (5× magnification) and a CCD camera (model ICD-42E). The images were visualized with XnView software. Each sample was analyzed in triplicate. Counting of the number of adhered cells on the samples surface has been performed with Dpx View Pro Image management software.
Results and Discussion Surface Preactivation. In the first step of the treatment, the hydroxyl groups available at the surface of poly(HEMA-coMMA) hydrogels were esterified by 2-bromo propionyl bromide (BPB), as demonstrated in Scheme 1, thus making activated bromides and well-known ATRP initiators17 grafted to the surface. The surface modification was monitored by ATR-IR and XPS versus time. After 30 min, no significant change in the ATR-IR spectra of the hydrogel surface was observed. The amount of grafted bromide, if any, is too low to be detected by this method, which is probing a thickness of about 1–3 µm. However, for longer reaction time, functionalization takes place not only on the surface but also in the upper layer of the substrate, and after 60 min of reaction time, the intensity of the IR absorption band characteristic of the OH groups from the neat hydrogel at 3420 (29) DumaineL. Correlation Entre les Proprietes Physico-Chimiques de Polymeres Permettant de Moduler la Proliferation Cellulaire et Celles Permettant de Favoriser le Glissement de Lentilles Intraoculaires. Thesis, Ecole Centrale de Lyon, 2004,79-85. (30) Linnola, R.; Salonen, J. I.; Happonen, R. P. J. Cataract Refract. Surg. 1999, 25, 1480–1485.
Table 1. XPS Analysis of the Surface of Poly(HEMA-co-MMA) Hydrogels Activated by BPB esterification time, min
C%
O%
Br%
30 60 120
68 64 67
29 33 30
3 3 3
Scheme 2. Comparison of the Chemical Structure of the Sacrificial and the Grafted ATRP Initiators Used in This Work
cm-1 dramatically decreased (Figure 1b to be compared to Figure 1a for the neat hydrogel) or even disappeared after 120 min of reaction time (Figure 1c). In parallel, absorption bands typical of the C-Br stretching were observed at 1225 and 1336 cm-1. Thus, the grafting reaction of BPB to the hydrogel did occur under the tested conditions, and the bromide is able to diffuse within the first micrometers of the top layer of the hydrogel. Nevertheless, ATRP-initiating moieties in the volume of the sample would be useless for the forthcoming surface grafting process, and modification of the bulk material should be avoided to preserve its properties. Therefore, analyzing the surface after modification at a shorter reaction time (30 min) was considered by XPS, a technique more sensitive to the top surface layer. By this method, bromine was clearly detected, and the C/O/Br atomic ratio was determined for the upper 10 nm layer (probing depth of XPS) from the sample (Table 1). This ratio remained constant while increasing the reaction time up to 60 min and even up to 120 min. Therefore, 30 min of esterification was enough for the hydrogels to reach the maximum ATRP-initiator grafting over a depth of 10 nm. These samples were selected for further modification.
Imparting Antifouling Properties by Grafting
Langmuir, Vol. 24, No. 13, 2008 6653
Table 2. Experimental Data for the ATRP of OEGA initiated by BPB-Activated Hydrogels
entry unmodified ATRP-initiated 30 min 1 2 3 4 5 6 7 8 9 10
OEGA/In (mol eq./ mol eq.)
100/1 100/1 100/1 10/1 7/1 5/1 100/1 100/1 100/1 100/1
catalyst (mol eq.)
CuIBr (1.2) CuIBr (1.2) CuIBr (1.2) CuIBr (1.2) CuIBr (1.2) CuIBr (1.2) CuICl (1.2) CuICl (1.2) CuICl (1.2) CuICl (1.2)
ligand (mol eq.)
HMTETA (1.44) HMTETA (1.44) Me6TREN (1.44) Me6TREN (1.44) Me6TREN (1.44) Me6TREN (1.44) Me6TREN (1.44) HMTETA (1.44) HMTETA (1.44) HMTETA (1.44)
time min
20 120 20 180 180 180 20 20 60 120
conversion (a) %
27 56 77 ∼100 ∼100 ∼100 59 13 16 –
Mnth (b) g/mol
12 000 25 500 34 500 4700 3400 2500 27 000 5700 7300 –
MnSEC (c) g/mol
4500 14 000 33 500 3200 2600 2050 24 000 6200 6500 16 000
Ip (c)
CAwd θs1 ( 2°
CAcb θs2 ( 3°
1.30 1.70 1.65 1.30 1.25 1.20 1.40 1.30 1.35 1.65
66° 65° 77° 53° ∼0° 76° 69° 68° ∼0° 78° 77° 42°
43° 40° 40° 35° 33° – – 37° 33° – 39° –
a In stands for initiator. b Calculated by 1H NMR in D2O. c Calculated as (n mols of monomer/n mols of ATRP initiator) × MM monomer x % conversion of the monomer. d Determined for the polymer in solution; (Ip ) Mw/Mn).
Synthesis of a sacrificial initiator: r-hydroxyethyl-2-bromopropionate (HEBP). For grafting from experiments to be controlled, a sacrificial initiator is needed such that enough deactivating species are available to impart control to ATRP as rapidly as possible.31 In the case of ATRP of MMA initiated from the activated surface of magnetite nanoparticles, it was shown that the activities of both the surface initiator and the sacrificial initiator were similar, which resulted in good control of the growth of the grafted chains and the chains in solution.32 This is the reason that the sacrificial initiator used in this work (i.e., HEBP) was selected for a structure reminiscent of that of the grafted initiating species. These chemical structures are compared in Scheme 2. The structure of a monofunctional product was estimated by the NMR analysis of HEBP. Moreover, a monomodal molar mass distribution was observed by SEC for the polymer formed in solution with this sacrificial initiator. (A detailed discussion is available in a forthcoming section.) Therefore, it might be assumed that a monofunctional initiator has been formed, but the presence of a nonestimated amount of a bifunctional one could not be excluded, although it was not detected. On the basis of the above discussion and the high structural similarity of the sacrificial initiator and the one grafted on the surface, the chains formed in solution are considered to be representative of the grafted chains. Surface-Initiated ATRP of OEGA. Because the materials envisioned in this work are intended to be in contact with biological tissues, special attention was paid to the choice of the polymerization solvent. Recently, Wang et al.33,34 reported on the well-controlled ATRP of methoxy-capped oligo(ethylene glycol) methacrylate (OEGMA) in water at 20 °C. The monomer conversion was close to completion within 0.5 to 2 h. The grafting from ATRP of OEGMA was accordingly conducted in water at 25 °C, which are nonharmful conditions for the hydrogel and homogeneous conditions for the polymerization. ATRP is faster when conducted in water rather than in apolar solvents.16,23,35,36 Although the reason for this kinetic effect is not yet fully understood, it is thought that modifications of the halogen substitution of the metal complex in the higher oxidation (31) Yamamoto, K.; Miwa, Y.; Tanaka, H.; Sakaguchi, M.; Shimada, S. J. Pol. Sci., Part A: Pol. Chem. 2002, 40, 3350–3359. (32) Muritani, E.; Yamamoto, S.; Ninjbadgar, T.; Tsujii, Y.; Fukuda, T.; Takano, M. Polymer 2004, 45, 2231–2235. (33) Wang, X. S.; Lascelles, S. F.; Jackson, R. A.; Armes, S. P. Chem. Commun. 1999, 18, 1817–1818. (34) Wang, X. S.; Armes, S. P. Macromolecules 2000, 33, 6640–6647. (35) Nanda, A. K.; Matyjaszewski, K. Macromolecules 2003, 36, 599–604. (36) Coullez, G.; Carlmark, A.; Malmström, E.; Jonsson, M. J. Phys. Chem. A 2004, 108, 7129–7131.
state might shift the equilibrium between active and dormant species toward the active ones.35–37 As a rule, a faster process is economically beneficial, which is an additional incentive for using water in ATRP of OEGMA. A polymerization temperature of 25 °C is quite convenient for keeping the hydrogel intact (no cracking, no syneresis, etc.) and for preventing parasitic reactions such as the nucleophilic attack of the brominated initiator by water.37 Where the catalytic system is concerned, the ligand and the halide that are used are known to influence the course of ATRP.17 Attention was thus paid to the ATRP of OEGMA in water initiated by the activated (brominated) hydrogel at a constant monomer/ initiator molar ratio (100/1) in the presence of CuICl or CuIBr as metal catalysts and HMTETA or Me6TREN as ligands (Table 2). Polymerization is definitely faster when Me6TREN is the ligand (Table 2, entries 3–7) rather than HMTETA (Table 2, entries 1–2 and 8–10). The substitution of CuIBr for CuICl in the presence of HMTETA, all the other conditions being the same, has a beneficial kinetic effect as supported by an increase in the monomer conversion from 13% (Table 2, entry 8) to 27% (Table 2, entry 1 after 20 min polymerization). The same conclusion holds when Me6TREN is the ligand, with the monomer conversion being higher in the presence of CuIBr (77%; Table 2, entry 3) compared to CuICl (59%; Table 2, entry 7). This observation is consistent with a faster halogen exchange between the initiator and the catalyst when CuIBr is involved rather than CuICl, as reported by Matyjaszewski et al.38 As expected, the catalytic complex CuIBr/Me6TREN, providing the fastest polymerization rates, led to almost 100% monomer conversion within 180 min in the case of entries 4–6, Table 2, for small monomer/initiator molar ratios. As a rule, the polydispersity index (Ip) of the polymer collected in solution increases with molecular weight whatever the catalytic complex. For instance, an increase in the PDI from 1.20 to 1.65 is observed for the CuIBr/Me6TREN catalytic complex when MnSEC is increased from 2050 to 33 500 (Table 2, entries 3–6), and a PDI increase from 1.30 to 1.65 is observed in the case of the CuICl/HMTETA for MnSEC from 6200 to 16 000 (Table 2, entries 8–10). Moreover, more polydisperse polymer was formed in solution in the presence of Me6TREN as the ligand and CuIBr as the catalyst, probably because of the very fast polymerization rates. (37) Qui, J.; Charleux, B.; Mayjaszewski, K. Prog. Polym. Sci. 2001, 26, 2083–2134. (38) Matyjaszewski, K.; Qin, S. Macromolecules 2003, 36, 1843–1849.
6654 Langmuir, Vol. 24, No. 13, 2008
BozukoVa et al.
Figure 2. ATR-IR spectra of the poly(HEMA-co-MMA) hydrogel (a) before and after grafting of poly(OEGA) in the presence of (b) HMTETA (Table 2, entry 1) or (c) Me6TREN (Table 2, entry 3).
Figure 3. SEM images of dry poly(HEMA-co-MMA) hydrogels grafted by poly(OEGA) under the conditions of (a, b) entry 1 in Table 2 and (c, d) entry 2 in Table 2. ESEM images of hydrated hydrogels from (e) entry 1 in Table 2 and (f) entry 2 in Table 2.
Besides, an increase in the polydispersity index has been observed in the line PDI(CuICl/HMTETA) ) PDI(CuIBr/HMTETA) < PDI(CuICl/Me6TREN) < PDI(CuIBr/Me6TREN) when all other conditions are the same, demonstrating the greater dependence of the polymerization on the ligand rather than on the catalyst. Because of the difference between the molecular architectures of poly(OEGA) and the linear PEO standard used for SEC measurements, a direct comparison between Mnth and MnSEC is not reasonable. However, increasing the monomer/ initiator ratio
clearly increases the hydrodynamic volume of the poly(OEGA) polymers, with the polydispersity index remaining low ( 20 000, which might be explained by the surface roughness and PEG content variations. The hydration of the coatings rendered all poly(OEGA)-grafted surface more hydrophilic than the unmodified one. Large core–shell self-assemblies were observed at the hydrated surface because of the tendency of the PEG segments to expose themselves to water whereas the hydrophobic backbone to which they are attached exhibits the opposite behavior. Even short poly(OEGA) brushes can impart antifouling properties to the hydrogel, as assessed by biological in vitro tests. The GFP and β-lactamase adsorption in vitro tests indeed confirmed the efficient protein repellency of the coatings. In vitro tests of LEC adhesion showed the same results when cells were concerned.
BozukoVa et al.
The strategy proposed in this work is thus very promising for the surface modification of “soft” biomaterials by a coating with antifouling properties. Acknowledgment. We are indebted to Professor M. Galleni and Dr. B. Wolf (and contract FRFC 9.4527.05-2.4546.05 (FNRS, Belgium)) of the Center for Protein Engineering (ULg, Belgium) for the gift of GFP and FM analysis. We are also grateful to the Walloon Region of Belgium for financial support in the framework of the First Europe (SURFOLIO) Program and the Belgian Science Policy for support in the framework of the Interuniversity Attraction Poles Programme (PAI 6/27) - Functional Supramolecular Systems. We also thank Dr. S. Yunus and Dr. L. Köhler (UCL, Belgium) for the XPS measurements. C. Je´roˆme is a Research Associate of the Fonds National de la Recherche Scientifique (Belgium). LA7033774
