Lateral Control of Protein Adsorption on Charged Polymer Gradients

Jan 5, 2009 - reactor with a moving shutter. A homogeneous layer of cationic poly(2-aminoethyl methacrylate hydrochloride) was first formed, followed ...
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Langmuir 2009, 25, 3755-3762

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Lateral Control of Protein Adsorption on Charged Polymer Gradients Tobias Ekblad, Olof Andersson, Feng-I Tai, Thomas Ederth, and Bo Liedberg* DiVision of Molecular Physics, Department of Physics, Chemistry and Biology, Linko¨ping UniVersity, SE- 581 83 Linko¨ping, Sweden ReceiVed October 16, 2008. ReVised Manuscript ReceiVed December 5, 2008 This work describes the fabrication, characterization, and protein adsorption behavior of charged polymer gradients. The thin gradient films were fabricated by a two-step technique using UV-initiated free-radical polymerization in a reactor with a moving shutter. A homogeneous layer of cationic poly(2-aminoethyl methacrylate hydrochloride) was first formed, followed by a layer of oppositely charged poly(2-carboxyethyl acrylate) with a continuously increasing thickness. Adsorption from protein solutions as well as human blood plasma was investigated by imaging surface plasmon resonance and infrared microscopy. The results showed excessive protein adsorption in the areas where one of the polymers dominated the composition, while there was a clear minimum at an intermediate position of the gradient. The charge of the surface was estimated by direct force measurements and found to correlate well with the protein adsorption, showing the lowest net charge in the same area as the protein adsorption minimum. We therefore hypothesize that a combination of the charged polymers, in the right proportions, can result in a protein-resistant surface due to balanced charges.

Introduction The control and understanding of surface-protein interactions, often with the intention of minimizing nonspecific protein adsorption, is of great interest in biomedical and antibiofouling applications. With this aim, we have previously developed a method to reproducibly fabricate thin hydrogel films on a variety of substrates, using a quick and facile technique. The method, which has been described as self-initiated photografting and photopolymerization (SIPGP),1 is based on UV-initiated grafting of acrylate or methacrylate monomers,2,3 and has been demonstrated to be a viable technique for fabrication of surfacebound polymer films on several organic substrates. We have so far mainly utilized it to produce poly(ethylene glycol) (PEG)containing protein-resistant coatings for biosensor and microarray applications.4-6 Although shown to be highly effective in repelling protein adsorption as well as the settlement of biofouling organisms,7 the PEG portion of the hydrogel could be susceptible to oxidative and enzymatic degradation, and this concern has led us to investigate other routes to produce surfaces with low protein adsorption. One option is charge-balanced surfaces, previously demonstrated in formats such as thiolatebased self-assembled monolayers (SAMs),8,9 zwitterionic polymers,10-16 or amphoteric copolymers.17-19 Such materials gen* Corresponding author. E-mail: [email protected]. (1) Steenackers, M.; Lud, S. Q.; Niedermeier, M.; Bruno, P.; Gruen, D. M.; Feulner, P.; Stutzmann, M.; Garrido, J. A.; Jordan, R. J. Am. Chem. Soc. 2007, 129, 15655–15661. (2) Wang, H. L.; Brown, H. R. Macromol. Rapid Commun. 2004, 25, 1095– 1099. (3) Deng, J. P.; Yang, W. T.; Rånby, B. Macromol. Rapid Commun. 2001, 22, 535–538. (4) Larsson, A.; Ekblad, T.; Andersson, O.; Liedberg, B. Biomacromolecules 2007, 8, 287–295. (5) Larsson, A.; Du, C.; Liedberg, B. Biomacromolecules 2007, 8, 3511–3518. (6) Larsson, A.; Liedberg, B. Langmuir 2007, 23, 11319–11325. (7) Ekblad, T.; Bergstro¨m, G.; Ederth, T.; Conlan, S. L.; Mutton, R.; Clare, A. S.; Wang, S.; Liu, Y.; Zhao, Q.; D’Souza, F.; Donnelly, G. T.; Willemsen, P. R.; Pettitt, M. E.; Callow, M. E.; Callow, J. A.; Liedberg, B. Biomacromolecules 2008, 9, 2775–2783. (8) Holmlin, R. E.; Chen, X. X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841–2850. (9) Chen, S.; Yu, F.; Yu, Q.; He, Y.; Jiang, S. Langmuir 2006, 22, 8186–8191. (10) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355–360. (11) Lewis, A. L. Colloid Surf. B: Biointerfaces 2000, 18, 261–275.

erally lack the inherent instability of PEG and have repeatedly been shown to be highly protein resistant. Additionally, the charged groups may facilitate functionalization with proteins such as antibodies, a useful aspect in biomedical applications.20 It has been suggested that the biocompatibility of charge-balanced amphoteric and zwitterionic polymers is a result of high hydrophilicity combined with unusually weak disruptive effects on the hydrogen bond structure of water.17,21 Surface-bound gradients can offer advantages over homogeneous surfaces, for instance, in experiments aiming to find favorable surface properties for protein adsorption or cell adhesion. The developments in this field, both in terms of fabrication and applications, have recently been comprehensively reviewed.22-24 While certain properties, most notably wettability,25-27 have been studied extensively using the gradient format, relatively few examples of well-defined charge gradients can be found in the literature. A SAM-based approach with varying ratios of amine and carboxylic acid moieties was developed in our laboratory,28 but the self-assembly process strongly favors 1:1 composition of differently charged (12) Feng, W.; Zhu, S. P.; Ishihara, K.; Brash, J. L. Langmuir 2005, 21, 5980– 5987. (13) Zhang, J.; Yuan, Y. L.; Wu, K. H.; Shen, J.; Lin, S. C. Colloid Surf. B: Biointerfaces 2003, 28, 1–9. (14) West, S. L.; Salvage, J. P.; Lobb, E. J.; Armes, S. P.; Billingham, N. C.; Lewis, A. L.; Hanlon, G. W.; Lloyd, A. W. Biomaterials 2004, 25, 1195–1204. (15) Chang, Y.; Chen, S. F.; Zhang, Z.; Jiang, S. Y. Langmuir 2006, 22, 2222–2226. (16) Ishii, T.; Wada, A.; Tsuzuki, S.; Casolaro, M.; Ito, Y. Biomacromolecules 2007, 8, 3340–3344. (17) Kitano, H.; Nagaoka, K.; Tada, S.; Gemmei-Ide, M.; Tanaka, M. Macromol. Biosci. 2008, 8, 77–85. (18) Bernards, M. T.; Cheng, G.; Zhang, Z.; Chen, S. F.; Jiang, S. Y. Macromolecules 2008, 41, 4216–4219. (19) Chen, S. F.; Jiang, S. Y. AdV. Mater. 2008, 20, 335–338. (20) Zhang, Z.; Chen, S.; Jiang, S. Biomacromolecules 2006, 7, 3311–3315. (21) Kitano, H.; Tada, S.; Mori, T.; Takaha, K.; Gemmei-Ide, M.; Tanaka, M.; Fukuda, M.; Yokoyama, Y. Langmuir 2005, 21, 11932–11940. (22) Morgenthaler, S.; Zink, C.; Spencer, N. D. Soft Matter 2008, 4, 419–434. (23) Genzer, J.; Bhat, R. R. Langmuir 2008, 24, 2294–2317. (24) Kim, M. S.; Khang, G.; Lee, H. B. Prog. Polym. Sci. 2008, 33, 138–164. (25) Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstro¨m, I. J. Colloid Interface Sci. 1987, 119, 203–210. (26) Go¨lander, C. G.; Lin, Y. S.; Hlady, V.; Andrade, J. D. Colloids Surf. 1990, 49, 289–302. (27) Chaudhury, M. K.; Whitesides, G. M. Science 1992, 256, 1539–1541. ¨ stblom, M.; Lundstro¨m, I.; Svensson, S. C.; Denier van der (28) Riepl, M.; O Gon, A. W.; Schäferling, M.; Liedberg, B. Langmuir 2005, 21, 1042–1050.

10.1021/la803443d CCC: $40.75  2009 American Chemical Society Published on Web 01/05/2009

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thiols in the gradient,8,9 making it difficult to achieve controlled gradual variation. Polyelectrolyte-based gradients have also been prepared, most often composed of either polycations or polyanions. Lee et al. used a corona discharge process to create initiation sites for polymerization of the charged monomers acrylic acid, sodium p-styrene sulfonate, and N,N-dimethyl aminopropyl acrylamide. Gradients were formed by varying the power of the corona discharge. These surfaces were then tested in respect to blood platelet adhesion29 and adhesion and growth of cultured cells.30 These properties were shown to correlate with the content of grafted polyelectrolyte along the gradient, although the behavior was quite complex. Atom transfer radical polymerization (ATRP) has also been used to create polyelectrolyte gradients. For instance, Wu et al. fabricated gradients of the polyanion poly(acrylic acid) (PAA) using gradually varying surface concentrations of immobilized ATRP initiators.31 Polyelectrolyte gradients incorporating both negative and positive charges have also been reported from the same research group.32 This was achieved by block copolymerization of PAA with a poly(dimethyl aminoethyl methacrylate) brush of gradually varying molecular weight. No protein adsorption results were reported from these experiments. An alternative approach, using a “grafting-onto” method with sequential and heat-controlled immobilization of two preformed polyelectrolytes, poly(2-vinylpyridine) and PAA, on the same silicon wafer has also been reported.33,34 Although no protein adsorption experiments were performed on these gradients either, similar surfaces in a nongradient format showed significant protein adsorption, albeit highly controlled by the solution pH and ionic strength. The protein adsorption was not tested at the isoelectric point of the surface, and hence the charges were not balanced in any of the measurements.35 It is also worth mentioning that so-called immobilized pH gradients (IPGs) have been commercially available for decades and are used for isoelectric focusing of proteins. These materials are formed by mixing acidic and basic monomers with acrylamide and N-N′-methylene bisacrylamide and carefully distributing the mixtures in a mold. After polymerization, the acids and bases act as immobilized buffers within the gel, affecting the local pH, which in turn determines the net charge of proteins.36 In this work, we have used an alternative route to prepare a mixed polyelectrolyte gradient, employing free-radical grafting to deposit the anionic polyelectrolyte poly(2-carboxyethyl acrylate) (PCEA) within and on top of an cationic polyelectrolyte layer consisting of poly(2-aminoethyl methacrylate) (PAEMA). The gradient fabrication setup is illustrated in Figure 1 and is based on a method described previously.6 The gradient format was used in order to conveniently achieve varying ratios of the polyelectrolytes on a single sample. The surfaces were characterized along the gradient with respect to chemical composition, thickness, surface charge and protein adsorption, using null ellipsometry, infrared (IR) microscopy, atomic force microscopy (AFM), and imaging surface plasmon (29) Lee, J. H.; Khang, G.; Lee, J. W.; Lee, H. B. J. Biomed. Mater. Res. 1998, 40, 180–186. (30) Lee, J. H.; Lee, J. W.; Khang, G.; Lee, H. B. Biomaterials 1997, 18, 351–358. (31) Wu, T.; Gong, P.; Szleifer, I.; Vlcek, P.; Subr, V.; Genzer, J. Macromolecules 2007, 40, 8756–8764. (32) Bhat, R. R.; Tomlinson, M. R.; Wu, T.; Genzer, J. AdV. Polym. Sci. 2006, 198, 51–124. (33) Houbenov, N.; Minko, S.; Stamm, M. Macromolecules 2003, 36, 5897– 5901. (34) Ionov, L.; Houbenov, N.; Sidorenko, A.; Stamm, M.; Luzinov, I.; Minko, S. Langmuir 2004, 20, 9916–9919. (35) Uhlmann, P.; Houbenov, N.; Brenner, N.; Grundke, K.; Burkert, S.; Stamm, M. Langmuir 2007, 23, 57–64. (36) Righetti, P. G. In Laboratory Techniques in Biochemistry and Molecular Biology; Burdon, R. H., van Knippenberg, P. H., Eds.; Elsevier: Amsterdam, 1990; Vol. 20, Chapter 1.

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Figure 1. Cartoon of the polymerization setup used for gradient fabrication. PAEMA is marked blue, and PCEA is marked orange. The exposure was varied by moving the shutter, gradually exposing more and more of the surface to the UV light. The preceding step was carried out with AEMA solution and no shutter. The illustration is not drawn to scale, L ≈ 50 µm and d ≈ 40 nm in reality.

resonance (iSPR). The main objective of this study was to investigate whether, and under which conditions, a partly interpenetrating semilayered network of oppositely charged polyelectrolytes can compare with zwitterionic homopolymers or amphoteric copolymers in terms of protein resistance. A better understanding of this effect would be beneficial since chargebalanced surface chemistries show great potential for many applications where low protein adsorption is desired.

Materials and Methods Materials. The monomers 2-aminoethyl methacrylate hydrochloride (AEMA) and 2-carboxyethyl acrylate (CEA) were purchased from Sigma-Aldrich Sweden AB and used without purification. Both monomers were polymerized in phosphate-buffered saline buffer (PBS) (10 mM sodium hydrogen phosphate, 10 mM potassium dihydrogen phosphate, 150 mM NaCl), adjusted to pH 6.0 with HCl. The monomer concentrations were 15% (w/v) for AEMA and 1% (v/v) for CEA. The AEMA monomer solution was used immediately after preparation because of the possibility of acyl migration.37 Pepsin (from porcine gastric mucosa, MW ) 35 kDa), lysozyme (from chicken egg white, MW ) 15 kDa), human serum albumin (HSA, MW ) 67 kDa), and 11-mercaptoundecanoic acid were purchased from Sigma-Aldrich. Human citrated plasma was kindly provided by Lars Faxa¨lv, Department of Clinical Chemistry, Linko¨ping University. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acidbuffered saline (10 mM HEPES, 150 mM NaCl, pH 7.4, hereafter called HBS-N) was attained from GE Healthcare. The PEG-thiol HS(CH2)11CONH(C2H4O)11CH3 (hereafter called mPEG) was purchased from Polypure AS, Norway. Gradient Fabrication. The substrates used in the iSPR experiments consisted of 12 × 12 mm2 glass slides coated with a 45 nm thick gold layer (kindly provided by GE Healthcare, Uppsala, Sweden). For the IR, ellipsometry, and AFM experiments, silicon (110) (2 nm native oxide, Topsil Semiconductor Materials A/S, Denmark) was coated with 2.5 nm of chromium (99.9% Balzers, Liechtenstein) followed by 200 nm of gold (99.99% Nordic High Vacuum AB, Sweden) in a custom-made resistive evaporation system. As a first step in the gradient fabrication process, the gold-coated substrates were cleaned in a 5:1:1 mixture of Milli-Q water (MilliQ, Millipore), 30% hydrogen peroxide (Merck KGaA, Germany), and 25% ammonia (Merck KGaA) for 5 min at 85 °C. The iSPR substrates were then incubated in a 100 µM solution of mPEG in ethanol for at least 24 h. The silicon/gold substrates were instead coated with polystyrene (PS). For this process, a solution of 0.25% PS in xylene was spin coated at 1500 rpm for 30 s using a WS400B-6NPP/Lite spin coater (Laurell Technologies Corp.). The approach of using different substrates for the preparation of grafted (37) Smith, D. A.; Cunningham, R. H.; Coulter, B. J. Polym. Sci., Part A-1: Polym. Chem. 1970, 8, 783–784.

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Figure 2. Stacked IR spectra measured along the same line of a gradient surface before (A,B) and after (C,D) incubation in lysozyme/pepsin mixture (0.5 mg/mL of both in HBS-N, pH 7.4). The assignments for the peaks marked in A and C are found in Table 1. These spectra are respectively plotted against position along the gradient (x) in the 1800-1500 cm-1 region in B and D. Note the gradually increasing carbonyl peak at 1735 cm-1 and the COO- peak at 1587 cm-1. After protein incubation, two new peaks appeared at 1664 cm-1 and 1545 cm-1, respectively, both typical for vibrations in the amide bond of proteins.

polymers has been used previously with good results,5 and the mPEG SAM was chosen since it appears to closely match spin-coated PS, in terms of chemical composition and thickness of the grafted film. Irrespective of substrate type, the polyelectrolyte gradient surfaces were prepared by SIPGP in two steps, closely resembling the method described by Larsson et al.5 The setup is illustrated in Figure 1. A homogeneous layer of PAEMA was first fabricated on the substrate, followed by the PCEA gradient. A UV lamp (Philips TUV PL-L, 18 W) with a main emission peak at 254 nm was used to initiate the polymerization reaction in both cases. The irradiation time for the PAEMA layer was 4 min in all cases. For the PCEA step, a moving shutter produced gradually varying UV exposure times along the surface. The shutter setup has been described previously.6 The shutter moved with a set velocity of either 2.3 mm/min (3 min max. exposure time) or 3.0 mm/min (4 min max. exposure time). The latter setting was used for the preparation of the samples for the combined IR/AFM experiment. After the coating process, the surfaces were ultrasonicated in 50% ethanol for 30 s, followed by thorough rinsing with Milli-Q water. To further remove any physisorbed polymer, the samples were kept in buffer or Milli-Q water for at least 12 h before performing any experiments. Imaging Null Ellipsometry. An imaging null ellipsometer (EP,3 Nanofilm Technologie, Germany) was used to measure the thickness profile of the gradient surfaces. The ellipsometer was operated at a wavelength of 831 nm with the angle of incidence set at 60°. Measurements were carried out in the dry state, and the refractive index of the grafted polymer was assumed to be 1.5. The refractive index of the underlying gold substrate was obtained from previous measurements of the refractive index of clean gold substrates. The thickness of the spin-coated PS film was determined beforehand assuming a refractive index of 1.6.38 IR Microscopy. The IR microscopy measurements largely agree with those described by Larsson et al.6 In short: A Bruker Hyperion 3000 IR microscope connected to a Tensor 27 IR spectrometer and equipped with a computerized sample stage was used for the measurements. The objective utilized double surface reflections with (38) Nikolov, I. D.; Ivanov, C. D. Appl. Opt. 2000, 39, 2067–2070.

angles ranging between 52° and 83° relative to the surface normal. A nitrogen-cooled single element mercury-cadmium-telluride (MCT) detector collected the spectra with a resolution of 4 cm-1, and 200 interferograms were recorded to obtain a spectrum from each measurement point. A three-term Blackman-Harris apodization function was applied to the interferograms before Fourier transformation. All spectra were baseline corrected using a concave rubber band method with 64 baseline points. A clean gold surface was used to collect the background spectrum, and the measurement chamber and instrument were continuously purged with dry N2. The chemical composition of the sample was mapped by collecting several spectra along the gradient (parallel with the direction of increasing UV exposure time in the previous fabrication stage). Each measurement was made with an aperture of 150 × 200 µm, with the longer side perpendicular to the gradient direction. The distance between each measurement point was 250 µm and typically 33 spectra were taken, spanning a distance of 8 mm. In cases when the sample was moved between measurements, for instance for protein incubations, care was taken to perform all successive measurement series with exactly the same starting position and along the same line. Slightly different incubation protocols were used for the experiments presented in Figures 2, 3, and 5. For the experiment combining IR with ellipsometry (Figures 2 and 3), three IR measurements were performed, each following different incubations. The sequence of incubations was (1) 50 mM NaOH (5 min); (2) HBS-N buffer (40 min); and (3) a mixture of lysozyme and pepsin (0.5 mg/mL for both) in HBS-N buffer (20 min, followed by 10 min in pure HBS-N). For the experiment combining IR with AFM (Figure 5), the NaOH stage was omitted, and the proteins were not mixed. Instead, the first measurement was performed after HBS-N soaking, and the surface was then incubated first in HSA for 20 min and then in lysozyme for 20 min (0.5 mg/mL for both proteins, in HBS-N) and finally in pure HBS-N for 10 min, followed by the second measurement. In all cases, the sample was rinsed quickly with Milli-Q water and dried with a stream of N2 before it was placed in the IR microscope sample chamber.

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Figure 3. Plot of the integrated area of the ester carbonyl peak (circles) and the ellipsometric thickness (solid line), as a function of position. The same sample as in Figure 2 was used for the measurement.

Imaging Surface Plasmon Resonance. The iSPR instrumentation employed in this work was based on spectral interrogation. Surface plasmons were excited by means of a 25 mm equilateral prism (BK7 glass, Melles Griot) onto which the substrates were optically coupled using a small drop of refractive index matching oil (Cargille-Sacher Laboratories, Inc.). Light from a monochromator (SpectraPro 300i, Acton Research Corp.) was collimated and polarized before being guided toward the prism by means of a mirror configuration. A charge-coupled device (CCD) detector (Retiga Exi, Qimaging Corp., 12bit 1MP without IR-filter) with imaging optics was used to measure the intensity of the reflected light. The angle of incidence was kept fixed at 72.5°. At this angle, one pixel of the CCD detector depicted a 2.2 × 7.0 µm2 area of the substrate. A small-volume poly(methyl methacrylate) (PMMA) flow cell was tightly sealed to the substrate, and protein solutions were injected over the surface at a flow rate of 30 µL/min by means of a syringe pump. The protein solutions had concentrations of 1 mg/mL in HBS-N, except human plasma which was diluted to 5% in HBS-N. Between protein injections, a continuous flow of HBS-N buffer was maintained. SPR images were acquired by scanning the wavelength in the range 600-800 nm and recording the reflectivity spectra of s- and p-polarized light from each individual pixel. The spectra were subsequently normalized, and the wavelength at which there was a minimum in reflectance (the SPR wavelength, λSPR) was determined for each pixel of the image. The magnitude of the difference in λSPR before and after sample injection reflects the amount of adsorbed biomolecules. Force Measurements by AFM. The force measurements were performed with a NanoScope IVa Dimension 3100 SPM (Veeco Instruments, Inc., USA) equipped with a liquid compartment. Polyelectrolyte gradients were prepared as described above. Silicon nitride probes (DNP probes, cantilever C, normal spring constant 0.32N/m, Veeco Instruments, Inc.) were used to determine the surface charge along the gradient. The probes were first affirmed to be negatively charged by recording the force against a gold surface coated with a SAM of 11-mercaptoundecanoic acid in 100 mM bicarbonate solution. HBS-N buffer was used for the force measurements on the gradient surfaces, and the measurements were carried out by approaching the surface with the probe and recording the cantilever deflection as a function of the displacement. The ramp size of the probe was between 200 and 350 nm. The deflectionposition curves were converted to deflection-separation data assuming a hard-wall contact (zero separation) in the constant compliance region. Force measurements were recorded with a spacing of 250 µm along the gradient through the area exhibiting a “neutral” charge.

Results and Discussion Composition and Steepness of the Gradient. IR microscopy was used to characterize the chemical composition and steepness

Figure 4. (A) SPR wavelengths (λSPR) measured over a 2 × 0.5 mm2 section of a gradient before exposure to protein. The PCEA content varied from predominating (left; higher λSPR) to minor (right; lower λSPR). (B) Shift in λSPR upon exposure to solutions of pepsin (upper) and then lysozyme (lower). (C) Data of the same type as in B, but from a second experiment in which the pepsin was replaced with diluted human plasma. Both experiments show the same trends, with adsorption of oppositely charged proteins to the surface. Notably, human plasma, which contains a mixture of proteins, exhibits adsorption to the PAEMAdominated region, but also to a minor extent to the PCEA. In both experiments, it was only in the intermediate gradient region that protein adsorption was completely suppressed, as signified by the very small shift in λSPR (