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Langmuir 2009, 25, 3610-3617
Polyelectrolyte Multilayers Capped with Polyelectrolytes Bearing Phosphorylcholine and Triethylene Glycol Groups: Parameters Influencing Antifouling Properties Andreas Reisch,† Jean-Claude Voegel,‡,§ Eric Gonthier,† Gero Decher,† Bernard Senger,‡,§ Pierre Schaaf,*,† and Philippe J. Me´sini*,† Institut Charles Sadron, CNRS, 23 rue du Loess, BP 84047, 67037 Strasbourg Cedex 2, France, Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 977, 11 rue Humann, 67085 Strasbourg Cedex, France, and UniVersite´ de Strasbourg, Faculte´ de Chirurgie Dentaire, 1 place de l’Hoˆpital, 67000 Strasbourg, France ReceiVed NoVember 14, 2008. ReVised Manuscript ReceiVed December 22, 2008 In this paper, we investigate the design of antifouling surfaces by the deposition of polyelectrolytes modified by grafting of antifouling groups onto a (PSS/PAH)n precursor multilayer film [PSS, poly(styrenesulfonate); PAH, poly(allylamine)]. Different polyelectrolytes and different antifouling moieties are investigated, in particular, (EO)3 and (EO)3PC moieties (EO, ethylene oxide; PC, phosphorylcholine group). We find that protein adsorption can strongly be reduced and even practically suppressed through the deposition of only one layer of polyelectrolyte modified with PC and/or (EO)3 groups. We discuss the influence of various parameters such as the nature of the polyelectrolyte backbone, the nature of the antifouling moiety, and the grafting ratio on the reduction of protein adsorption. We find in particular that (EO)3 and (EO)3PC moieties grafted on poly(acrylic acid) (PAA) totally prevent protein adsorption for grafting ratios of 25% or more, at least within the detection limits of the used quartz crystal microbalance and optical waveguide light mode spectroscopy devices. The mechanism that leads to the antifouling property is discussed and compared to that leading to the antifouling properties of ethylene oxide self-assembled monolayers. Finally, by incorporating biotin on top of the precursor film, we show that one layer of PAA-(EO)3PC is not sufficient to prevent interaction with streptavidin but a PAA-(EO)3PC/PAH/PAA-(EO)3PC multilayer largely protects the biotin from interacting with streptavidin.
Introduction Nonfouling surfaces find numerous applications as bloodcompatible materials and as biosensor coatings. Extensive research has thus been carried out in this field. As a general observation, hydrophilic surfaces appear to reduce adsorption of proteins, but this property is often not sufficient to totally inhibit their adsorption and to prevent cell or bacteria adhesion. In the late 1980s, a major step forward resulted from the discovery that poly(ethylene glycol) (PEG) moieties strongly resist protein adsorption. This led to the important development of PEGmodified surfaces with polymers, copolymers, brush polymers, or self-assembled monolayers (SAMs).1-4 These different functionalization strategies can be roughly classified in two categories: those with PEG chains of molecular weights typically of 2000 or larger and those with oligo(ethylene glycol) moieties deposited on gold or silver substrates in the form of self-assembled monolayers. It was suggested that two different mechanisms are responsible for the protein repellency. The protein resistance of “high” molecular weight PEG is well-explained by “steric repulsion”: confinement of PEG chains due to protein molecules approaching the surface results in a free energy increase due to * To whom correspondence should be addressed. Tel: +33(0)388414012. Fax: +33(0)388414099. E-mail:
[email protected] (P.S.), mesini@ ics.u-strasbg.fr (P.J.M.). † Institut Charles Sadron. ‡ Institut National de la Sante´ et de la Recherche Me´dicale. § Universite´ de Strasbourg.
(1) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 1043. (2) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (3) Kenausis, G. L.; Vo¨ro¨s, J.; Elbert, D. L.; Huang, N. P.; Hofer, R.; RuizTaylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 104, 3298. (4) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164.
dehydration and conformational entropy decrease. On the other hand, protein resistance of self-assembled oligo(ethylene glycol) monolayers appears to be related to the ability of the polyethers constituting the SAMs to coordinate water both in their interior and on their surface. This enhanced water binding aptitude leads to an overall increase of the protein resistance.5 The problem of nonadhesive surfaces has, in fact, already been solved by nature. In a seminal paper, Zwaal et al. showed that the phospholipids of the outer leaflet of the erythrocyte membrane that contains predominantly phosphorylcholine (PC) moieties as polar groups are responsible for their nonthrombogenic character.6 The nonfouling property of PC groups originates from their zwitterionic character, and it was found that other zwitterionic groups such as sulfobetaine or carboxybetaine lead to a similar antifouling character on surfaces.7 In order to mimic nature, different ways to functionalize surfaces with zwitterionic groups have been investigated. Direct surface grafting of such groups onto various substrates either by free radical polymerization8 or by atom transfer radical polymerization (ATRP) has been carried out,9,10 self-assembled monolayers on gold or silicon surfaces have been generated,11,12 and polymers bearing zwitterionic side chains have been used to coat various substrates.13,14 (5) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359. (6) Zwaal, R. F. A.; Comfurius, P.; Van Deenen, L. L. M. Nature 1977, 268, 358. (7) Zhang, Z.; Chao, T.; Chen, S.; Jiang, S. Langmuir 2006, 22, 10072. (8) Ishihara, K.; Iwasaki, Y.; Ebihara, S.; Shindo, Y.; Nakabayashi, N. Colloids Surf., B 2000, 18, 3259. (9) Feng, W.; Zhu, S. P.; Ishihara, K.; Brash, J. L. Biointerphases 2006, 1, 50. (10) Feng, W.; Zhu, S. P.; Ishihara, K.; Brash, J. L. Langmuir 2005, 21, 5980. (11) Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841. (12) Chen, S.; Zheng, J.; Li, L.; Jiang, S. J. Am. Chem. Soc. 2005, 127, 14473.
10.1021/la8037846 CCC: $40.75 2009 American Chemical Society Published on Web 02/12/2009
Multilayers Capped with Polyelectrolytes
All these functionalization techniques possess advantages and drawbacks. In particular, they are all highly dependent upon the nature or topology of the substrate and must thus be adapted to each specific application. This can be circumvented by the use of polyelectrolyte multilayers, a coating method that is universal and simple.15 It consists of the alternate deposition of polyanions and polycations onto almost any kind of surface, whatever its nature and topology. This can be achieved either by dipping or spraying the different constituents. However, polyelectrolyte multilayers are usually rather prone to protein adsorption, and such architectures must thus be further functionalized in order to render them protein and/or cell repellent. This can be achieved by the use of polyelectrolytes with zwitterionic grafted side chains.16,17 Chitosans modified with phosphorylcholine groups have also been integrated into polyelectrolyte multilayers; however, the influence on protein adsorption was not investigated.18 We recently showed that PSS/PAH multilayers [PSS, poly(styrenesulfonate); PAH, poly(allylamine hydrochloride)] coated with a single layer of PAA-(EO)3PC chains [PAA(EO)3PC, poly(acrylic acid) modified by grafting (EO)3PC side chains; EO, ethylene oxide; PC, phosphorylcholine group] can almost totally prevent serum protein adsorption. Moreover, when deposited on silicone sheets, these layers proved to keep their protein repellency, even under stretching of the substrates, at least up to a stretching degree of 150%.19 In this paper, we investigate in more detail the surfaces obtained by the adsorption of polyelectrolytes bearing antifouling moieties onto PSS/PAH polyelectrolyte multilayer films and especially their nonfouling properties. We selected PSS/PAH as underlying precursor film for two main reasons: first, it is one of the most robust and characterized multilayer films and it can be built easily on any kind of surface; second, this multilayer film appears to be well-adapted for tissue engineering applications, because it induces very strong cell adhesion, at least partially due to its glasslike mechanical properties, thus favoring cell attachment, proliferation, and differentiation.20 If such films could be rendered nonadsorptive to proteins or nonadhesive to cells by a simple treatment, the micropatterning of this functionalization could become an ideal tool for the development of patterned coatings that would specifically interact with given proteins or cells. Moreover, precursor multilayers allow us to be fairly independent of the substrate, whatever its nature or geometry, and hence also to compare results from different methods. In our previous work, we obtained nonfouling properties by using -(EO)3PC side chains grafted on a PAA backbone. However, both (EO)3 and PC groups possess, independently, nonfouling properties. We thus want to investigate precisely the effect of different moieties on the nonfouling character of the multilayer film. Moreover, we tried to evaluate if a synergetic effect between the two groups exists, when associated in the same side chain. Finally, the influence of the chemical nature of the polyelectrolyte backbone on the antifouling character has also been investigated. (13) Iwasaki, Y.; Ishihara, K. Anal. Bioanal. Chem. 2005, 381, 534. (14) Lewis, A. L. Colloids Surf., B 2000, 18, 261. (15) Decher, G. Science 1997, 277, 1232. (16) Olenych, S. G.; Moussallem, M. D.; Salloum, D. S.; Schlenoff, J. B.; Keller, T. C. S. Biomacromolecules 2005, 6, 3252. (17) Salloum, D. S.; Olenych, S. G.; Keller, T. C. S.; Schlenoff, J. B. Biomacromolecules 2005, 6, 161. (18) Kujawa, P.; Schmauch, G.; Viitala, T.; Badia, A.; Winnik, F. M. Biomacromolecules 2007, 8, 3169. (19) Reisch, A.; Hemmerle´, J.; Voegel, J.-C.; Gonthier, E.; Decher, G.; Benkirane-Jessel, N.; Chassepot, A.; Mertz, D.; Lavalle, P.; Me´sini, P.; Schaaf, P. J. Mater. Chem. 2008, 18, 4242. (20) Boura, C.; Menu, P.; Payan, E.; Picart, C.; Voegel, J.-C.; Muller, S.; Stoltz, J.-F. Biomaterials 2003, 24, 3521.
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Materials and Methods 1. Materials. Poly(ethylene imine) (PEI, Sigma, MW 750 000), poly(allylamine hydrochloride) (PAH, Aldrich, MW 70 000), poly(sodium 4-styrenesulfonate) (PSS, Aldrich, MW 70 000), poly(4styrenesulfonate-co-maleic acid) (PSSMA, Aldrich, MW 20 000, styrenesulfonate to maleic acid ratio ) 1: 1), poly(acrylic acid) (PAA, Aldrich, 35 wt % in water, MW 100 000), Trizma base (Sigma, g99%), Trizma hydrochloride (Sigma, g99%), and NaCl (Sigma, g99.5%) were used as received. Fetal bovine serum (FBS, Gibco), bovine serum albumin (Sigma, g99%), chicken egg white lysozyme (Sigma, L6876), human plasma fibrinogen (Sigma, F 4883), and streptavidin (Sigma, S4762) were used for the adsorption tests. Millipore water was used for all the experiments. The different compounds used during this study are represented in Scheme 1. Synthesis and characterization of the modified polyelectrolytes are detailed in the Supporting Information and in ref 21. The grafting ratio (GR) is defined as the percentage of carboxylate or amine functions on the polyelectrolyte modified by (EO)3PC, (CH2)6PC, or (EO)3 groups. For PAA and PAH this corresponds to the percentage of monomeric units that are modified. In the case of PSSMA, it corresponds to the percentage of carboxylate groups of the maleic acid that are modified with (EO)3PC or (CH2)6PC and does not include the styrenesulfonate units. The GR was determined by NMR spectroscopy and verified by IR spectroscopy. It should be noted that the GR was termed DS, degree of substitution, in our prior publications on these polymers.19,21 2. QCM Measurements. Quartz crystal microbalance (QCM) measurements were performed on a QCM-D D300 device (Q-sense, Sweden), using gold-coated crystals at room temperature. The fundamental resonance frequency was on the order of 5 MHz. The changes in frequency and dissipation of the third, fifth, and seventh harmonics were used for analysis. The thickness of the polyelectrolyte layers and the amount of adsorbed proteins were evaluated using a viscoelastic model.22 The corresponding masses per unit area were obtained supposing a density of 1 g/cm3. 3. OWLS Measurements. Optical waveguide light mode spectroscopy (OWLS) was performed on an OWLS 120 (Microvacuum, Budapest, Hungary) using OW 2400 waveguides made of Si0.8Ti0.2O2 (Microvacuum, Budapest, Hungary). The technique has been extensively described elsewhere23 and experimentally applied to polyelectrolyte multilayers24 and to protein adsorption.25 In brief, a laser beam is shined on a grating imprinted in the waveguide and one determines the incoupling angles for both the transverse electric (TE) and the transverse magnetic (TM) waves into the guide. To each incoupling angle there corresponds an effective refractive index, respectively N(TE) and N(TM). From these effective refractive indices the optical thickness, d, and refractive index, n, of an adsorbed layer can be obtained with the homogeneous and isotropic monolayer model. The adsorbed masses of polyelectrolytes were calculated using the refractive index increments determined in the same buffer (see Table 1 for the used dn/dc values and Supporting Information for experimental details on their determination). For the calculation of the mass of adsorbed proteins, a refractive index increment of 0.18 mL/g was used.26 4. Multilayer Assembly. Adsorption of proteins and serum was evaluated on a PEI(PSS/PAH)5 multilayer on which the polyanion to test was adsorbed or on a PEI(PSS/PAH)4PAH multilayer if a modified PAH was tested. The multilayers were assembled in situ in the measurement cell of the QCM or the OWLS. (21) Reisch, A.; Voegel, J.-C.; Decher, G.; Schaaf, P.; Me´sini, P. J. Macromol. Rapid Commun. 2007, 28, 2217. (22) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scripta 1999, 59, 391. (23) Vo¨ro¨s, J.; Ramsden, J. J.; Csu´cs, G.; Szendro, I.; De Paul, S. M.; Textor, M.; Spencer, N. D. Biomaterials 2002, 23, 3699. (24) Picart, C.; Ladam, G.; Senger, B.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J. G.; Gergely, C. J. Chem. Phys. 2001, 115, 1086. (25) Ho¨o¨k, F.; Vo¨ro¨s, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf., B 2002, 24, 155. (26) Ball, V.; Ramsden, J. J. Biopolymers 1998, 46, 489.
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Scheme 1. Structures of Poly(acrylic acid) (PAA), Poly(styrenesulfonate) (PSS), Poly(styrenesulfonate-co-maleic acid) (PSSMA), Poly(allylamine hydrochloride) (PAH), and the Corresponding Modified Polyelectrolytes
Filtered solutions of 1 mg/mL of PEI, PSS, and PAH and of 0.5 mg/mL of PAA or modified PAA in Tris buffer (20 mM, NaCl 150 mM) at pH 7.4 were used for the construction of the multilayers. The multilayers were assembled by bringing the substrate (QCM crystal or waveguide) in contact with the polyelectrolyte solutions for 6 min in the case of PEI, PSS, and PAH and for 12 min in the case of PAA, PSSMA, and modified polyelectrolytes followed by rinsing twice with the buffer solution during 4 min each. After deposition of the last layer and rinsing, the multilayer was left to stabilize in contact with the buffer solution for at least 30 min prior to adsorption experiments with the different proteins or serum.
5. Adsorption of Serum. Serum adsorption was determined by QCM-D and OWLS experiments. Solutions of FBS were obtained by dilution of pure FBS with Tris buffer (20 mM, NaCl 150 mM) at pH 7.4, followed by filtration, yielding concentrations of 1-20 vol % of FBS, corresponding to total protein concentrations of 0.44-8.78 mg/mL. FBS solutions were used within 3 h after preparation. The adsorption of serum was studied by successive injections of six FBS solutions, starting with the lowest concentration, without intermediate rinsing, into the QCM cell after construction of the multilayer in situ. Each solution was left in contact with the multilayer for 26 min. Measurements were taken after 22 min, which was generally sufficient to obtain a stable signal (see Figure S5a in
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Table 1. Refractive Index Increments dn/dc for the Polyelectrolytes Used in This Study polyelectrolyte PAA PAA-(EO)3PC PAA-(CH2)6PC PAA-(CH2)11PC PAA-(EO)3
a
dn/dc GRa (mL/g) 9 18 25 12 23 33 14 20 24 33 42
0.172 0.150 0.168 0.153 0.148 0.165 0.171 0.152 0.166 0.165 0.167 0.166
polyelectrolyte PSSMA PSSMA-(EO)3PC PSSMA-(CH2)6PC PAH PAH-(EO)3PC
PAH-(CH2)6PC
dn/dc GR (mL/g) 25 25 17 30 54 80 50
0.176 0.164 0.165 0.217 0.156 0.168 0.163 0.154 0.153
Grafting ratio.
the Supporting Information). Four more minutes of contact before injection of the next solution were necessary due to experimental constraints. Adsorption was determined relative to the last deposited layer. Only the serum solution with a total protein concentration of 4.4 mg/mL was used for the OWLS experiments. Adsorption was measured after rinsing with buffer and stabilization of the signal. 6. Adsorption of Proteins. Protein adsorption on PAA and modified PAA was tested by QCM-D. Solutions of 0.2 mg/mL of albumin, lysozyme, and fibrinogen in Tris buffer (20 mM, NaCl 150 mM) at pH 7.4 were used. Albumin and lysozyme solutions were used as made, and fibrinogen solutions were centrifuged and only the supernatant was used within 15 min after preparation. Protein concentration was controlled by UV-vis spectroscopy at 280 nm. Adsorption of albumin, lysozyme, and fibrinogen on PAA was tested by bringing the protein solutions for 1 h in contact with the multilayer. One hour was sufficient for obtaining stable QCM signals for all three proteins and in particular for fibrinogen (see Figure S5a in the Supporting Information). This was followed by rinsing and contact with the buffer solution for 1 h. In the case of modified PAA (GR 25%), the same multilayer was brought in contact successively with albumin, lysozyme, and fibrinogen solutions with intermediate rinsing. Solutions of 0.1 mg/mL of streptavidin in the same buffer were brought in contact with the different surfaces for 10 min before rinsing. In the case of streptavidin, 10 min was generally sufficient for obtaining stable QCM signals with time (see Figure S5b in the Supporting Information). 7. ζ-Potential Measurements. For the determination of the ζ-potential of the functionalized surfaces, colloidal particles (polystyrene latexes, diameter 1 µm) were coated with a PEI(PSS/ PAH)2 multilayer capped with the polyanion under consideration. Multilayer assembly was performed by bringing a diluted dispersion of particles in contact with the polyelectrolyte solution followed by centrifugation. The particles were redispersed in buffer solution, and the buffer was removed by centrifugation twice. After redispersion, the following layer was deposited. A low level of aggregate formation was controlled by optical microscopy. The ζ-potential of the particles was then determined with a Zetasizer 3000 HS within the buffer solution.
Results and Discussion The goal of the present study was to investigate how nonfouling surfaces can be obtained by the deposition of one or a small number of layers of polyelectrolytes, modified with moieties known for their nonadsorptive character with respect to proteins, onto PSS/PAH multilayers used as precursor films and to elucidate which are the most important parameters involved in protein resistance of such surfaces. We used (EO)3, (EO)3PC, and (CH2)6PC as antifouling moieties and varied both the grafting ratio (GR) and the polyelectrolyte backbone, using PAA, PAH, and PSSMA. The moieties and polyelectrolytes were selected for their following properties: (i) PC and (EO)3 were chosen
because of their known antifouling character; (ii) the comparison between (EO)3 and (EO)3PC moieties should highlight a possible synergetic effect between (EO)3 and PC; (iii) the (CH2)6 spacer in (CH2)6PC possesses the same number of carbon atoms as (EO)3 but is more hydrophobic; (iv) PAA has already proven its nonfouling character once modified by (EO)3PC; (v) PAH permits us to check the influence of the charge of the polyelectrolyte backbone, and (vi) PSSMA was chosen because it bears strong acid groups and has a more hydrophobic character compared to PAA. 1. Characterization of the Antifouling Layers. Both the antifouling layers, which were constituted of modified polyelectrolytes deposited on a (PSS/PAH)n precursor multilayer, and their antifouling character were investigated by quartz crystal microbalance (QCM) and by optical waveguide lightmode spectroscopy (OWLS). QCM is a technique sensitive to the deposited mass, including the bound water, and thus effectively gives access to the hydrodynamic mass. OWLS is an optical technique and as such is sensitive to the difference of the refractive indexes of the adsorbed layer and the solution. It thus allows the mass of adsorbed polyelectrolytes to be determined. The comparison between the two masses furnishes an estimate of the degree of hydration of the adsorbed layer, usually regarded as a very important characteristic for antifouling surfaces. As a reference, we determined the water content of a PEI(PSS/PAH)3 multilayered film by this method. Using QCM, one finds a total mass of 1340 ng/cm2, whereas a mass of 1112 ng/cm2 was measured by OWLS (assuming a refractive index increment, dn/dc, of 0.2 mL/g). The water content is thus estimated to be on the order of 20%. The mass of the adsorbed polyelectrolyte, as determined by OWLS, gives furthermore the possibility to estimate the number n of antifouling side groups per square nanometer for known GR of the side groups on the polymer: n ) GR × (number of groups per repeat unit) × (adsorbed mass of polyelectrolyte per area)/(mean molecular weight per repeat unit). It is possible that not all of these groups are exposed at the surface. However, the surface density of antifouling groups calculated in this way should give a good measure of their presence in the surface layer. 2. Protein Adsorption onto Functionalized Multilayers. Once constructed, the films were brought in contact with FBS solutions and adsorption isotherms were determined. We first investigated protein adsorption from serum because it contains numerous proteins (albumin, fibrinogen, vitronectin, IgG, etc.) and other molecules found in blood. Thus, it constitutes a good model system to study the behavior of the films in contact with a biological fluid. The isotherms were determined in a sequential manner by bringing a given surface in contact with serum solutions of increasing total protein concentration, varying from 0.4 to 9 mg/mL. The adsorbed amounts were determined by QCM. The adsorbed masses increased with protein concentration until a plateau is reached (Figure 1). We also determined the amount of adsorbed proteins by OWLS but only for the 4.4 mg/mL serum solution. This concentration was chosen because it is close to the isotherm plateau but still low enough to avoid protein aggregation in the bulk solutions. For the films that showed the strongest antifouling potentiality, we also determined, by QCM, the adsorption of single proteins: solutions of bovine serum albumin, chicken egg white lysozyme, and human plasma fibrinogen. Bovine serum albumin was chosen because it is the most abundant protein in blood with a characteristic size of about (3 × 3 × 8 nm3),27 whereas chicken egg white lysozyme is a (27) Ferrer, M. L.; Duchowicz, R.; Carrasco, B.; de la Torre, J. G.; Acuna, A. U. Biophys. J. 2001, 80, 2422.
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Figure 1. Adsorption isotherms of proteins from fetal bovine serum on (filled square) PAA, (filled star) PAA-(EO)3PC, (half-filled diamond) PAA-(CH2)6PC, and (open circle) PAA-(EO)3 at grafting ratios of about 25% deposited on (PSS/PAH)5 multilayers as measured by QCM. The inset shows a magnification of the zone of low serum adsorption.
small and positively charged protein (size, 3.6 × 4.5 × 4.2 nm3)28 and fibrinogen (size, 6 × 9 × 45 nm3)29 constitutes one of the major actors in the coagulation cascade and is known to adsorb strongly on most surfaces. The results are summarized in Table 2, which will be used as a guide for discussion. The serum adsorption isotherms on PAA and PAA modified at a grafting ratio (GR) of about 25% with (EO)3PC, (CH2)6PC, and (EO)3 are given in Figure 1. Those on the other surfaces are given in the Supporting Information. The major trends that emerge from Table 2 (see in particular columns 8 and 9) are the following: (i) PC groups present an antifouling character whatever the polyelectrolyte backbone and the nature of the linker of the PC groups, i.e., [(EO)3 or (CH2)6]. (ii) For a given antifouling moiety, the nature of the polyelectrolyte backbone is of prone importance to reduce protein adsorption. (iii) One observes that (EO)3 and (EO)3PC are more efficient in suppressing protein adsorption than (CH2)6PC. (iv) If a synergetic antifouling effect between the (EO)3 and (EO)3PC groups exists, it must be small. (v) Finally, using PAA as the polyelectrolyte backbone and (EO)3 or (EO)3PC as antifouling moieties with grafting ratio of 25% or above renders the surfaces practically resistant to protein adsorption (within detection limit). We will now discuss more precisely these data. 3. Influence of the Nature of the Polyelectrolyte Outer Layer on Protein Adsorption. a. Role of the Nature of the Polyelectrolyte Backbone. The adsorption of unmodified PAA on a (PSS/PAH)n precursor multilayer film leads to a layer whose water content is much larger (85%) than that of a PSSMA layer (50%) deposited on a similar precursor film, or with PAH as terminating layer (20%) (see column 7, Table 2). This result was expected, since PAA is a more hydrophilic polyelectrolyte known for its strong interaction with water. The amounts of serum proteins adsorbed for these three polyelectrolyte types used as terminating layer in their unmodified form lead to plateau values in the adsorption isotherm of 1800 ng/cm2 for PAA, 1600 ng/ cm2 for PSSMA, and close to 2500 ng/cm2 for PAH (measured by QCM; see column 9, Table 2). Thus, the hydrophilicity of the used polyelectrolytes is not the major factor driving protein adsorption on these modified surfaces. The surface charge seems to play a more determinant role. The highest adsorbed amount (28) Imoto, T. In Encyclopedia of Life Sciences; John Wiley & Sons: New York, 2001. (29) Herrick, S.; Blanc-Brude, O.; Gray, A.; Laurent, G. Int. J. Biochem. Cell Biol. 1999, 31, 741.
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is found on PAH, which is positively charged, whereas the two negatively charged surfaces, which have similar ζ-potential (-39 mV), lead to comparable amounts of adsorbed proteins (see column 9, Table 2). This corroborates previous data showing that protein adsorption on polyelectrolyte multilayers is largely driven by electrostatic interactions.30 Moreover, due to the fact that most proteins are negatively charged, positively charged surfaces are usually more prone to adsorb proteins than negatively charged surfaces. Let us now analyze the polyelectrolytes modified with antifouling moieties. Whereas for a given grafting ratio of (EO)3PC, PAH-(EO)3PC always leads to the largest amount of adsorbed proteins, PAA-(EO)3PC much more efficiently prevents protein adsorption than does PSSMA-(EO)3PC. For example, at a GR of 25%, protein adsorption is on the order of the detection limit with PAA-(EO)3PC, whereas with PSSMA-(EO)3PC the adsorbed amount is on the order of 600 ng/cm2, as determined by QCM, and 40 ng/cm2, as determined by OWLS. Similarly, PAA-(CH2)6PC is much more prone to reduce protein adsorption than PSSMA-(CH2)6PC [140 (11) ng/cm2 compared to 1000 (53) ng/cm2 as determined by QCM (OWLS)]. PSSMA-(EO)3PC functionalized at 25% has a higher ζ-potential than PAA-(EO)3PC (-33 mV compared to -24 mV), but both are negatively charged, and the relatively small difference in ζ-potential cannot explain the great difference in adsorption behavior (see column 10, Table 2). One thus rather expects that the surface density of the antifouling moieties should constitute a more determinant parameter for protein adsorption. The surface density should depend on various parameters, in particular, the nature of the polyelectrolyte backbone, and this is indeed observed. For a given functionalization degree, the side-chain surface density is quite more important for PAA than for PSSMA-type backbones. For the (EO)3PC side groups and a GR of 25%, the surface density of functional groups is 2.0 nm-2 for PSSMA compared to 2.9 nm-2 for PAA (see column 6, Table 2). A similar trend is found for the (CH2)6PC moieties, but to a smaller extent. Such observations could result from structural differences between the PAA-X and PSSMA-X layers deposited on the precursor adlayer (X being the antifouling moiety). The differences in the layer structures become particularly apparent when the film thicknesses measured by QCM and OWLS were compared. The ratio between the QCM and OWLS thicknesses for PAA-(EO)3PC and PSSMA-(EO)3PC at the same GR of 25% are equal to 6.0 and 1.9, respectively. OWLS is mainly sensitive to the zone of the layer that possesses a high refractive index and is thus rich in organic material, whereas QCM is more sensitive to the hydrated layer and thus to the hydrodynamic thickness; therefore, PAA-(EO)3PC layers are clearly less dense, more diffuse, and more hydrated than the PSSMA-(EO)3PC. Another way to express these results is to use the water content that is estimated to be on the order of 90% for PAA-(EO)3PC layers and on the order of 45% for PSSMA-(EO)3PC ones (see column 7, Table 2). One can also compare the thicknesses measured by QCM to the length of the moieties grafted on the polyelectrolyte backbones. The QCM thickness of the PAA-(EO)3PC layer for a grafting ratio of 25% is 24 nm, whereas that of a PSSMA-(EO)3PC layer is 4 nm compared to the 2 nm of the -(EO)3PC moiety (see column 4, Table 2). This indicates that the PSSMA-(EO)3PC chains are deposited in a more stretched and flat way compared to the PAA-(EO)3PC chains, the latter being in a more loopy and loose conformation. On the basis of these results, the structures of the PSSMA-(EO)3PC and (30) Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel, J.-C. Langmuir 2001, 17, 878.
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Table 2. Summary of the Results Obtained for the Adsorption of One Layer of PAA, Modified PAA, PSSMA, or Modified PSSMA on a PEI(PSS/PAH)5 Precursor Layer and of PAH or Modified PAH on a PEI(PSS/PAH)4PSS Precursor Layer, and the Adsorption of Serum Proteins on These Surfaces -2
GR/% column no. PAA PAA-(EO)3PC PAA-(CH2)6PC PAA-(CH2)11PC PAA-(EO)3
PAH PAH-(EO)3PC
PAH-(CH2)6PC PSSMA PSSMA-(EO)3PC PSSMA-(CH2)6PC
1 0 9 18 25 12 23 33 14 20 24 33 42 0 17 30 54 80e 50 0 25 25
mass/ng cm (OWLS)a 2 103 207 221 285 207 222 267 230 217 249 229 276 114 176 162 194 197 226 159 225 244
thickness d/nmb OWLS
QCM
3 1.8 3.5 3.6 4.0 3.5 3.5 3.3 2.9 3.4 4.2 3.9 3.6 1.2 1.8 2.1 2.7 2.5 2.5 1.4 2.1 2.3
4 7 18 19 24 18 19 14 16 17 19 18 18 1 4 8 12 5 9 3 4 4
thickness ratio, number of side % water in dQCM/dOWLS groups per nm2 last layer 5 3.9 5.1 5.3 6.0 5.1 5.4 4.2 5.5 5.0 4.5 4.6 5.0 0.8 2.2 3.8 4.4 2.0 3.6 2.1 1.9 1.8
6 0 1.15 1.92 2.95 1.46 2.33 2.91 2.17 2.68 3.51 3.97 5.52 0 1.46 1.71 2.36 2.60 2.93 0 1.98 2.10
7 85 89 88 88 88 88 81 86 87 87 87 85 20 55 79 83 60 75 50 44 42
serum adsorption/ ng cm-2 c,d OWLS
QCM
ζ-potential/mV
8 297 64 15 1 42 11 81 51 18 1 1 1 385 302 165 47 22 126 205 40 53
9 1820 250 160 6 340 140 1320 170 55 8 17 20 2480 2300 1930 800 2200 1120 1590 610 960
10 -38.6 -24.1 -28.0 -24.2
+23.3 +8.8 +4.4 +11.1 -38.9 -33 -31.5
a Experiments were reproducible within (10 ng/cm2. b Thickness values obtained by OWLS were found to be reproducible within (0.2 nm. QCM results were reproducible within (10%. c For serum solutions having a total protein concentration of 4.4 mg/mL. d The experimental error was about (10%. Values below 20 ng/cm2 were reproducible within (2 ng/cm2 in OWLS experiments. Adsorption values below 50 ng/cm2 measured by QCM were found to be reproducible within about (5 ng/cm2. e The results given are only indicative and are not very reproducible, probably because the PAH-(EO)3PC layer corresponding to a GR of 80% is not adsorbed in a stable way.
Figure 2. Schematic view of the structure of PAA-(EO)3PC (a) and PSSMA-(EO)3PC (b) layers deposited on a (PSS/PAH)n adlayer and comparison with the size of albumin (Alb), streptavidin (Strep), lysozyme (Lys), and fibrinogen (Fib) molecules.
PAA-(EO)3PC adlayers are schematically represented in Figure 2. b. Influence of the Grafting Ratio (GR) and Role of the Nature of the Antifouling Group. For all the investigated systems, one observes that the antifouling group density on the surface increases when the grafting ratio (GR) increases (see Table 2, column 6 and Figure 3). This is not an obvious result, since the increase of GR reduces the polyelectrolyte charge and introduces lateral moieties, which may then influence the interactions of the functionalized polyelectrolyte with the precursor multilayer. For the adsorption of PAH-(EO)3PC-type polycations, the density of fixed antifouling groups reaches a plateau value for a GR value of 50%. Adsorption of PAH-(EO)3PC with a GR of 80% did not lead to stable and reproducible layers, probably due to
the too important decrease of the charge of the backbone. Moreover, one can notice that the smallest protein adsorption is found when the ratio between the thicknesses measured by QCM and OWLS is maximum, which corresponds also to the most extended and most loopy layer. As far as the PAA-based layers are concerned, the density of antifouling groups increases steadily when the functionalization degree increases (see Figure 3 and Table 2, column 6). For a GR of 25% with (EO)3PC and (EO)3 moieties grafted on PAA, the films become resistant to protein adsorption in the limits of the methods used here for its detection. In the case of (EO)3, higher GR values (33 and 44%) were investigated. No protein adsorption could be detected on these surfaces by OWLS. By QCM we found a small increase of the film mass after contact with the serum solution. This could
3616 Langmuir, Vol. 25, No. 6, 2009
Figure 3. Number of antifouling side groups on the multilayer surfaces per square nanometer as a function of the grafting ratio (GR) for modified PAA.
Figure 4. Influence of the surface density of the antifouling groups on the adsorption of proteins from serum as measured by QCM. Adsorption from a serum solution with a total protein concentration of 4.4 mg/mL is given.
be due to a very low amount of proteins that still is adsorbed. The amount could furthermore be overestimated due to the fact that QCM gives access to a hydrodynamic mass including bound water. In any case, if protein adsorption takes place at a GR of 33 and 44% for (EO)3 moieties, it is extremely weak. However, a small increase of protein adsorption relative to a GR of 25% could be possible. This might be attributed to a decrease of the electrostatic interactions between the PAA-(EO)3 and the precursor multilayer, as described above for PAH-(EO)3PC with a GR of 80%. Since for PAA the water content remained unchanged with the GR (about 90%) and the ratios between the thicknesses determined by QCM and OWLS are, for all GR values, quite large (close to 5 or above), the antifouling behavior seemed to be connected here to the surface density of antifouling groups. In the case of -(EO)3PC groups, the critical density, which leads to a total antifouling effect, is 2.9, and it is 3.5 for (EO)3 (column 6, Table 2 and Figure 4). It thus appears that, for a given surface density of antifouling groups, (EO)3PC appears to be slightly more efficient than (EO)3, even if, in both cases, the critical density is reached for the same GR of 25%. This is due to the interplay of structure of adlayer and the nature of side groups and how both influence the antifouling behavior. The former leads to a higher density of (EO)3 groups compared to (EO)3PC groups for a similar GR. One notices also that, despite
Reisch et al.
the large thickness ratios of QCM to OWLS thickness, this ratio is always slightly larger for (EO)3PC-modified PAA compared to (EO)3-modified PAA. (CH2)6PC groups are less efficient in reducing the adsorption of proteins than -(EO)3PC or -(EO)3 moieties for a given surface density and GR, although the water content of the outer layer and the ratio of the QCM/OWLS thicknesses are comparable. In this case, the less hydrophilic character of the side groups seems to be the cause for this less strong antifouling character. 4. Films Terminated with PAA-(EO)3PC or PAA-(EO)3 Having a GR of 25%. As noticed before, the multilayers terminated with PAA-(EO)3PC and PAA-(EO)3 layers largely prevent protein adsorption for a GR of 25%. In a more detailed investigation of these architectures, we determined by QCM the adsorption of three different proteins: albumin, lysozyme, and fibrinogen. The adsorbed amounts are presented in Table 3. For both modified polyelectrolytes, the adsorbed amounts of albumin and fibrinogen before the rinsing step were close to the detection limit of QCM measurements, but the adsorbed amount of lysozyme exceeded this limit, especially in the case of the PAA(EO)3-terminated architecture, where it reaches 180 ng/ cm2. This may be due to the fact that the outer layer is very loose, forming many loops and tails, as represented in Figure 2. This results in large density fluctuations within such layers, allowing for the penetration of lysozyme, which is a relatively small protein. When larger proteins such as albumin or fibrinogen are put in contact, the characteristic size of the density fluctuations is much smaller than the molecular size, and the outer layer thus acts as a repulsive, steric barrier. This also seems to indicate that the density fluctuations for the architectures ending with PAA-(EO)3PC or PAA-(EO)3 are typically of the size of the lysozyme molecule, that is, 3 nm. Moreover, the positive charge of lysozyme also favors its penetration in the negatively charged outer layer. Despite this penetration, lysozyme is not firmly attached to the layer and it is totally released during rinsing. This adsorption should thus not affect the film response to cell adhesion. After the rinsing step, the adsorbed amounts drop close or below the detection limit for all three investigated proteins, which confirms the results obtained for serum. In the case of (EO)3PC groups, protein adsorption after leaving the multilayer for 72 h in contact with the buffer solution gave the same results. Finally, one can still notice that, even though the amounts of proteins adsorbed were extremely small in both cases, (EO)3PC groups are found again to be slightly more efficient than (EO)3 groups in preventing protein adsorption. We found that the multilayers terminated by one PAA-(EO)3 layer become protein resistant when the (EO)3 group density is close to 2.8 nm-2. This density is comparable and even smaller than the value of 3.8 nm-2 found for (EO)3 self-assembled monolayers that fully prevent protein adsorption.5 Despite the important antifouling surface group density found here, the structure of the layer is totally different from that of a SAM, which is highly ordered. In the present case, as suggested in Figure 2, the antifouling effect seems to result mainly from steric hindrance and weak interactions of the proteins with the important hydration shell around the outer polyelectrolyte layer. 5. Deposition of Two Layers of Modified Polyelectrolytes and Interaction of Streptavidin with Underlying Biotin. We also investigated the antifouling behavior of PAA-(EO)3PC/ PAH/PAA-(EO)3PC layers, in which PAA-(EO)3PC with a GR of 25% was used, deposited on a PEI(PSS/PAH)3 precursor film. These films were brought in contact with serum. Similarly to what was found for a single PAA-(EO)3PC layer, the adsorbed amount of protein was on the order of the detection limit of the
Multilayers Capped with Polyelectrolytes
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Table 3. Adsorption of Albumin, Lysozyme, and Fibrinogen on PAA, PAA-(EO)3PC (GR 25%), and PAA-(EO)3 (GR 24%) Terminated Multilayers As Determined by QCM for Protein Concentrations of 0.2 mg/mL on PAA (ng cm-2) albumin lysozyme fibrinogen
on PAA-(EO)3PC (ng cm-2)
before rinsing
after rinsing
before rinsing
after rinsing
before rinsing
after rinsing
390 ( 38 220 ( 16 2010 ( 79
340 ( 33 44 ( 11 1640 ( 60
2(5 16 ( 5 8(5
0(5 7(5 3(5
0(5 180 ( 26 5(5
0(5 10 ( 6 5(5
Table 4. Adsorption of Streptavidin on Different Multilayers As Determined by QCM nature of the top layers [deposited on PEI(PSS/PAH)2PSS] PAH PAH/PAA-(EO)3PCa PAH-biotin PAH-biotin/PAA-(EO)3PC PAH-biotin/PAA-(EO)3PC/ PAH/PAA-(EO)3PC a
on PAA-(EO)3 (ng cm-2)
streptavidin adsorption before rinsing (ng cm-2)
after rinsing (ng cm-2)
110 ( 12 3(5 490 ( 22 100 ( 8 15 ( 7
65 ( 11 2(5 480 ( 16 100 ( 8 12 ( 7
PAA-(EO)3PC with a GR of 25% was used.
QCM device and was evaluated to 5 ng/cm2 for a 4.4 mg protein/ mL serum solution. This surface is thus as efficient as a single PAA-(EO)3PC layer at preventing protein adsorption. To further investigate the efficiency of PAA-(EO)3PC layers to protect against interaction of proteins with the underlying precursor film, we deposited PAH-biotin onto a PEI(PSS/ PAH)2PSS precursor film. Similar studies were carried out before with polyelectrolytes that did not have antifouling properties, and we show here that antifouling moieties represent a considerable advance over earlier studies,31 in which a larger number of polyelectrolyte layers were needed to prevent binding to the biotin ligands embedded in multilayer films. PAH-biotin is a PAH polyelectrolyte, which was functionalized with biotin moieties at a GR of 5%. This film, as made or covered with one or two layers of PAA-(EO)3PC, was brought in contact with a streptavidin solution at 0.1 mg/mL. The biotin-streptavidin interaction is known to be one of the strongest ligand/receptor interactions found in biology. The interaction has been followed by QCM and compared to films containing no biotin. The results are summarized in Table 4. Streptavidin adsorption on a surface covered with a PAH-biotin layer is much stronger than on a surface terminated with unmodified PAH. Moreover, the fraction of protein desorbed by rinsing is also much lower. These results show the difference between physicochemical interaction of streptavidin with the layer and specific ligand interactions. Streptavidin adsorption on films that do not contain biotin is totally prevented by one layer of PAA-(EO)3PC. When a PAH-biotin layer is covered by a single layer of PAA-(EO)3PC, the interaction with streptavidin is strongly reduced but not ruled out. We thus covered the PAH-biotin layer with a PAA-(EO)3PC/ PAH/PAA-(EO)3PC multilayer. Exposition to a streptavidin solution led to an adsorbed amount of 15 ng/cm2 on this multilayer (compared to the value of 490 ng/cm2 found on the PAH-biotinterminated film) and decreased to about 12 ng/cm2 upon rinsing, all these amounts being determined by QCM. This shows that PAA-(EO)3PC/PAH/PAA-(EO)3PC protects efficiently the precursor film from interacting with proteins of the size of streptavidin, even if strong, specific interactions are involved. Furthermore, these results seem to indicate that proteins can slightly penetrate the PAA-(EO)3PC layer but are not adsorbing unless a strong interaction is present. (31) Cassier, T.; Lowack, K.; Decher, G. Supramol. Sci. 1998, 5, 309.
Conclusion The present work dealt with the design of polyelectrolytebased coatings that are able to prevent or at least significantly reduce protein adsorption. We have shown that this goal can be achieved by depositing a single layer of polyelectrolytes modified with phosphorylcholine (PC) and/or ethylene oxide (EO)3 groups onto a (PSS/PAH)n precursor film. The extent to which the adsorption of proteins is reduced depends on the exact nature of the polyelectrolytes in the last layer, that is, the type of backbone, type of modification, and degree of modification (or grafting ratio GR). The nature of the polyelectrolyte determines the characteristics of the surface. Several main characteristics affecting the antifouling properties were identified. First, the charge of the polyelectrolyte and thus of the surface has an influence, with a positive charge leading to stronger adsorption than a negative charge. Generally, protein adsorption decreases with decreasing absolute charge, yet it is not necessary to have zero charge in order to obtain protein resistant surfaces. The second parameter is the structure of the surfaces, with strongly hydrated, loop-rich layers being most efficient against protein adsorption, as evidenced by the comparison between PAA-(EO)3PC and PSSMA-(EO)3PC, probably due to the formation of a steric barrier. However, this structure is only efficient if it bears a sufficient amount of antifouling groups per unit area. This minimum number density necessary to prevent totally the adsorption of proteins, as measured by QCM or OWLS, has been found to be 2.9 nm-2 for (EO)3PC and 3.5 nm-2 for (EO)3 groups and constitutes the third parameter that plays an important role in the antifouling properties of the coating. Furthermore, as local repulsion between the proteins and the antifouling layer is involved, the nature of these antifouling groups has also an influence on the protein resistance, with those being the most hydrated also being the more efficient. Only an interplay of these parameters leads to coatings that resist the adsorption of serum proteins including albumin, lysozyme, and fibrinogen. The most efficient coatings have been obtained with a PAA backbone modified by (EO)3PC and (EO)3 groups at grafting ratios of 25% or more. PSSMA and PAH modified with (EO)3PC were less efficient. (CH2)6PC groups were generally less efficient than (EO)3PC groups. Combining PC and (EO)3 groups in the same molecule leads to a small improvement of the antifouling properties. Finally, by implementing biotin at the surface of the precursor film, we could show that one layer of PAA-(EO)3PC is not sufficient to prevent interaction with streptavidin, but a PAA-(EO)3PC/PAH/PAA-(EO)3PC multilayer largely prevents the biotin from interacting with streptavidin. Acknowledgment. A.R. thanks the Re´gion Alsace for financial support. We also thank COST D 43 for supporting this research. Supporting Information Available: Details on the synthesis and the characterization of the compounds used in this work; the determination of the refractive index increments; serum adsorption isotherms on modified PAA, PAH, and PSSMA; QCM measurements of protein adsorption from serum on PAA-(EO)3PC and PAA-(EO)3; and kinetics of protein adsorption are given. This material is available free of charge via the Internet at http://pubs.acs.org. LA8037846
