Langmuir 2003, 19, 9873-9882
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Multilayered Polypeptide Films: Secondary Structures and Effect of Various Stresses F. Boulmedais,†,‡ M. Bozonnet,‡ P. Schwinte´,‡ J.-C. Voegel,*,‡ and P. Schaaf† Institut Charles Sadron (CNRS), 6, Rue Boussingault, 67083 Strasbourg Cedex, France, and INSERM Unite´ 595, Faculte´ de Chirurgie dentaire, 11 rue Humann, 67085 Strasbourg Cedex, France Received May 13, 2003. In Final Form: August 19, 2003
Polyelectrolyte multilayers constructed from polypeptides present secondary structures similar to those found in proteins (R-helices and β-sheets). These secondary structures are used as a tool to investigate the buildup and internal stability of multilayer films by means of Fourier transform infrared spectroscopy. Special attention is focused on the β-sheet contribution to the amide I band. Two main problems are addressed: (i) Does there exist a correlation between the local structure of the polypeptide multilayers and their corresponding polyanion/polycation complexes in solution? (ii) How stable is the local structure of these multilayers toward external stresses such as pH jumps, temperature rise, and changes of the nature of the outer layers of the film? Four different polypeptide multilayers, poly(L-glutamic acid)/poly(Llysine) (PGA/PLL), poly(L-aspartic acid)/poly(L-lysine), poly(L-glutamic acid)/poly(D-lysine), and poly(Lglutamic acid)/poly(L-ornithine), are studied. It is shown that the film secondary structures always closely resemble those of their corresponding complexes in solution. For example, the absence of β-sheet structures in the films correlates with their absence in solution. This shows the strong similarity between the physical processes leading to the formation of polypeptide complexes in solution and those involved in the multilayer formation. The secondary structures of (PGA/PLL)n films appear very stable against pH jumps for pH values ranging between 4 and 10.5. On the other hand, the sudden contact of a film constructed at pH 7.4 with a solution at pH 1.5 or 13.5 leads to a strong reduction of its β-sheet content together with a partial or total dissolution of the film. The structural response of a (PGA/PLL)n film to a temperature rise up to 89 °C depends on the way in which the temperature increase is performed: a slow temperature increase induces a reversible decrease of the β-sheet content at the expense of the R-helices. On the contrary, when the film is heated rapidly, the β-sheet content increases and a further increase is observed during cooling to room temperature. Finally, the deposition of poly(styrene sulfonate)/poly(allylamine) (PSS/PAH) bilayers on top of (PLL/PGA)n films leads to the total disappearance of the β-sheets. This seems to be related to the diffusion of PSS chains into the film during the first PSS deposition steps and an exchange of PGA molecules of the film by PSS ones deep in the architecture. Such an exchange process between two polyelectrolytes of different nature inside a multilayer architecture was, to the authors’ knowledge, never observed before.
I. Introduction When polyanion and polycation solutions are mixed, the two polyelectrolytes interact through electrostatic interactions and form complexes in solution. In a similar way, when a charged surface is alternately dipped in a polyanion and a polycation solution, the polycations (respectively polyanions) will interact with the previously deposited polyanions (respectively polycations). These polyelectrolytes adsorb on the surface forming an architecture whose mass and thickness increase with the number of deposition steps.1 Such an architecture is called a polyelectrolyte multilayer.2 The buildup of such films and their structure have received considerable attention during the past 10 years due to their large potential applications,3-6 and different behaviors have been observed depending among others on the nature of the polyelectrolytes and the buildup conditions. * Corresponding author. E-mail: Jean-Claude.Voegel@ medecine.u-strasbg.fr. † Institut Charles Sadron (CNRS). ‡ INSERM Unite ´ 595, Faculte´ de Chirurgie dentaire. (1) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Colloids Surf., A 1999, 146, 337-346. (2) Decher, G. Science 1997, 277, 1232-1237. (3) Kapnissi, C. P.; Akbay, C.; Schlenoff, J. B.; Warner, I. M. Anal. Chem. 2002, 74, 2328-2335. (4) Lvov, Y.; Caruso, F. Anal. Chem. 2001, 73, 4212-4217.
Roughly, two kinds of multilayers have been described: those whose mass increases linearly with the number of deposition steps7,8 and those whose mass increases superlinearly9 or exponentially.10,11 The first film type is mainly observed when the two polyelectrolytes are fully charged and the ionic strength of the polyelectrolyte solutions used for the film construction is small. The thickness increment per bilayer is then small; the built film is dense and presents a “periodic” structure where the interactions of each layer are constrained to its neighbors.2 For such systems, the polyelectrolytes from the solution interact only with the outer part of the multilayer and do not interact with polyelectrolyte layers more deeply embedded (5) Donath, E.; Moya, S.; Neu, B.; Sukhorukov, G. B.; Georgieva, R.; Voigt, A.; Baumler, H.; Kiesewetter, H.; Mo¨hwald, H. Chem.sEur. J. 2002, 8, 5481-5485. (6) Mamedov, A. A.; Kotov, N. A.; Prato, M.; Guldi, D. M.; Wicksted, J. P.; Hirsch, A. Nat. Mater. 2002, 1, 190-194. (7) Ruths, J.; Essler, F.; Decher, G.; Riegler, H. Langmuir 2000, 16, 8871-8878. (8) Advincula, R.; Aust, E.; Meyer, W.; Knoll, W. Langmuir 1996, 12, 3536-3540. (9) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Comm. 2000, 21, 319-348. (10) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J.-C.; Lavalle, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12531-12535. (11) Boulmedais, F.; Ball, V.; Schwinte´, P.; Frisch, B.; Schaaf, P.; Voegel, J.-C. Langmuir 2003, 19, 440-445.
10.1021/la0348259 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/30/2003
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in the film. This constitutes a marked difference with exponentially growing films. Such a growth results from the diffusion “in” and “out” through the whole film at each deposition cycle of at least one of the two constituting polyelectrolytes.10 Moreover, films made of two strong polyelectrolytes and those constituted of at least one weak polyelectrolyte12 can be also distinguished. The buildup of the former film type is fairly independent of the pH of the polyelectrolyte solutions used during the construction. The films grow linearly with a small bilayer thickness (typically a few nanometers). On the other hand, the construction of films constituted of at least one weak polyelectrolyte is pH sensitive.12 Under conditions where the weak polyelectrolyte is not fully charged, the thickness per bilayer becomes usually large (up to a few tens of nanometers). Under such conditions, hydrogen bonding or hydrophobic interactions can also become of importance.13,14 These films can grow linearly or exponentially. One can also distinguish between multilayers constituted of polyelectrolytes that form structureless complexes in solution and those whose complexes present a local structure.15-18 Except the alternating variation of the polyanion and polycation concentration perpendicular to the deposition surface, the first ones do not possess any local order. The second ones might present a local order fairly similar to that present in the complexes.18 Only very few results are available up to now in this field, and they are contradictory. Whereas a first study18 of the secondary structure of a polypeptide multilayer showed strong similarities between the structure of the complexes in solution and the corresponding multilayer, Arys et al.19 found no such correlation for films built by polycations belonging to the ionene family. A deeper understanding of the relation between the local structure of the complexes in solution and that of the multilayers should thus allow new insight into the multilayer buildup processes. Multilayers built from polyanions and polycations that form ordered complexes in solution are thus potentially of great interest not only for the properties that they can confer to the films but also from a fundamental point of view because they constitute an excellent tool to investigate the film buildup mechanisms. Films constituted of polypeptides belong to this category. Polypeptides are known to interact not only through electrostatic interactions but also through hydrogen bonding. Their chemical nature allows them to form secondary structures such as R-helices and β-sheets. The first structures are due to intramolecular hydrogen bonds, whereas the second ones can be due to intermolecular hydrogen bonds or to interactions between regions from the same chain that are far apart one from each other along the chain. These structures are at the origin of the tertiary protein structures and of their biological properties. We have already shown that (poly(L-glutamic acid)-poly(L-lysine))n (12) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 42134219. (13) Sukhishvili, S. A.; Granick, S. Macromolecules 2002, 35, 301310. (14) Choi, J. Y.; Rubner, M. F. J. Macromol. Sci., Pure Appl. Chem. 2001, 38, 1191-1206. (15) Cooper, T. M.; Campbell, A. L.; Crane, R. L. Langmuir 1995, 11, 2713-2718. (16) Cooper, T. M.; Campbell, A. L.; Noffsinger, C.; Gunther-Greer, J.; Crane, R. L.; Adams, W. Mater. Res. Soc. Symp. Proc. 1994, 351, 239-244. (17) Arys, X.; Laschewsky, A.; Jonas, A. M. Macromolecules 2001, 34, 3318-3330. (18) Boulmedais, F.; Schwinte´, P.; Gergely, C.; Voegel, J.-C.; Schaaf, P. Langmuir 2002, 18, 4523-4525. (19) Arys, X.; Fischer, P.; Jonas, A. M.; Koetse, M. M.; Laschewsky, A.; Legras, R.; Wischerhoff, E. J. Am. Chem. Soc. 2003, 125, 18591865.
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((PGA-PLL)n) multilayers constructed at pH 8.4 present secondary structures such as β-sheets and R-helices that are similar to those of the (PGA/PLL) complexes in solution.18 In this article, we present a more systematic study of the buildup and stability of multilayers constituted of polypeptides. We use the secondary structure as a tool to investigate in a deeper manner the relationship between the local structure of multilayers and their corresponding complexes in solution. We also investigate the stability of the local structure of such multilayer films toward external stresses such pH jumps, temperature rise, and changes of the chemical nature of the outer layers of the film. II. Experimental Section A. Materials. Poly(L-glutamic acid) sodium salt (PGA, P-4886, MW ) 50 300), poly(L-aspartic acid) sodium salt (PAA, P-6762, MW ) 33 400), poly(L-lysine) hydrobromide (PLL, P-2636, MW ) 32 600), poly(L-ornithine) hydrochloride (PLO, P-2533, MW ) 23 500), and poly(D-lysine) hydrobromide (PDL, P-4408, MW ) 27 200) were purchased from Sigma. Poly(ethyleneimine) (PEI, catalog no. 18,179-8, MW ) 750 000), poly(sodium 4-styrenesulfonate) (PSS, catalog no. 24,305-1, MW ) 70 000), and poly(allylamine hydrochloride) (PAH, catalog no. 28,322-3, MW ) 50 000-65 000) were purchased from Aldrich. Sodium chloride (purity g 99.5%) was purchased from Fluka, and 2-(N-morpholino)-ethanesulfonic acid (MES) and Tris-(hydroxymethylaminomethane) (Tris) from Sigma. B. Sample Preparation. The buffer media at pH 10.4, 11.3, and 13.3 were prepared from Tris (25 mM) and NaCl (100 mM) and adjusted by addition of NaOH. The buffer media at pH 7.4 and 8.4 were prepared from Tris (25 mM), MES (25 mM), and NaCl (100 mM) and adjusted by addition of NaOH or HCl, respectively. The buffer media at pH 4.4 and 1.5 were prepared from MES (25 mM) and NaCl (100 mM) and adjusted by HCl addition. Either Millipore filtered water (Milli Q-plus system) or deuterium oxide (99.9% D, catalog no. 15,188-2) from Aldrich was used as the solvent. All the polyelectrolytes were used without further purification. They were dissolved at a concentration of 5 mg/mL, except for the solutions of the polyanion/polycation complexes (at 2.5 mg/mL), obtained by mixing polycation (5 mg/ mL) and polyanion (5 mg/mL) solutions at a 1:1 mass ratio. C. Fourier Transform Infrared Spectroscopy. The “in situ” Fourier transform infrared (FTIR) experiments were performed on an Equinox 55 FTIR spectrometer (Brucker, Germany), using a liquid-nitrogen-cooled MCT detector. The experiments were all conducted in deuterated buffers. D2O was used as the solvent instead of water because the amide I band of the polypeptide is affected by the strong absorption of water around 1643 cm-1 (O-H bending), whereas the corresponding vibration in D2O is found at around 1209 cm-1.20 Spectra of polypeptide solutions were obtained by FTIR spectroscopy in transmission mode. Solutions of each polypeptide were prepared in deuterated buffer at pH 1.5, 4.4, 7.4, 8.4, and 10.4. Reference and sample spectra were taken by collecting 128 interferograms at 2 cm-1 resolution. For each spectrum of a polyanion/polycation polypeptide complex solution, we subtracted the spectrum of the corresponding buffer. The spectra relative to the polypeptide multilayers were determined in the attenuated total reflection (ATR) mode. Each polypeptide multilayer was built on a PEI(PSS-PAH)2 precursor film. A multilayer of the type PEI-(PSSPAH)2-(polyanion-polycation)n will be denoted as (polyanionpolycation)n, and (PGA-PLL)n stands for example for a PEI(PSS-PAH)2-(PGA-PLL)n architecture. Complexes formed in solution by PGA and PLL will be denoted PGA/PLL complexes. For each multilayer buildup, the different solutions (about 3 mL) were flown into an overhead in-compartment ATR cell (GRASEBY-SPECAC, U.K.), equipped with a top-plate (110 µL cell volume) fitted with a 45° trapezoidal ZnSe (internal reflection element) crystal (6 reflections, dimensions of 72 × 10 × 6 mm3). (20) Venyaminov, S.; Kalnin, N. N. Biopolymers 1990, 30, 12591271.
Multilayered Polypeptide Films The solutions were introduced into the cell by a peristaltic pump at a flow rate of approximately 0.25 mL/min. The multilayers were built up by an alternate deposition of polyanions and polycations on the ZnSe crystal. Each polyelectrolyte deposition was performed by flowing the solution for 12 min over the crystal, followed by an 8 min buffer rinse. After each polyelectrolyte deposition, at the end of the rinsing step, single-channel spectra from 512 interferograms were recorded between 400 and 4000 cm-1 with 2 cm-1 resolution, using Blackman-Harris three-term apodization and the standard Bruker OPUS/IR software (version 3.0.4). During the buildup, the film was continuously in contact with the buffer solution and never came into contact with air. For the analysis of the thermal stability of the films, we thermostated the experimental cell (Julabo, Germany) within 0.5 °C. Control experiments were performed on naked ZnSe crystals with the precursor film, PEI-(PSS-PAH)2. For all experiments with temperature changes, a continuous buffer flow of 0.25 mL/min was circulated through the thermostated cell. In the first type of experiments, the temperature was increased by 7 °C steps. A period of 5 min was then allowed for thermal equilibrium to be reached before interferograms were recorded during about 7 min. 512 interferograms were collected and used to calculate single-channel spectra. The absorbance of the multilayer was calculated as described by Schwinte´ et al.,21 by subtracting the sample spectrum at the actual temperature by the appropriate single-channel reference (the precursor film) spectrum taken at the same temperature. In the second type of experiments, the temperature was increased from 26 to 89 °C in 30 min and maintained constant during 8 h with the buffer continuously in contact with the film. Analysis of raw spectra obtained in the ATR mode was performed by subtracting the spectrum of the precursor PEI-(PSS-PAH)2 film from the spectrum of the actual polypeptide multilayer at the same temperature. Moreover, all raw infrared spectra were Fourier smoothed as described by Schwinte´ et al.21 The analysis was focused on the amide I band in the 1600-1700 cm-1 wavenumber range, except at pH 1.5 where the overlap of the COOH acid peak requires a study in the 1580-1750 cm-1 wavenumber range. The amide I band was decomposed into individual components which correspond to different secondary structures of the polypeptide. The decomposition was performed using the SPSERV software (version 3.20, BCS Software: Dr. Csaba Bagyinka, Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary). More details concerning the spectral decompositions can be found in ref 21. The frequencies of the different components forming the amide I band were first determined by means of the second derivative of the Fourier smoothed spectrum. There was thus no arbitrary decomposition into a preset number of bands. Once the number of component bands was determined, the amide I band was fitted by using the component frequency, width, and intensity (proportional to the area under the peak) as fitting parameters. All component bands were assumed to be Gaussian. The correspondence of each component band with a given secondary structure was established by comparing the frequency of its maximum to the value given in the literature.20,22,23 We defined the relative contribution of each component by the ratio of the area of each peak over the area of the total amide I band. D. Optical Waveguide Light Mode Spectroscopy (OWLS). We used OWLS to follow the film thickness and the mass of the films during their buildup. This optical technique allows for the determination of the optical thickness and the refractive index of films deposited on a Si0.8Ti0.2O2 waveguide. The technique has been extensively described elsewhere24 and experimentally applied to polyelectrolyte multilayers.25 It will only be briefly described here. A laser beam is shined on a grating imprinted (21) Schwinte´, P.; Voegel, J.-C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B 2001, 105, 11906-11916. (22) Rosenheck, K.; Doty, P. Proc. Natl. Acad. Sci. U.S.A. 1961, 47, 1775-1785. (23) Fasman, G. D. Handbook of Biochemistry and Molecular Biology; CRC Press: Boca Raton, FL, 1976. (24) Tiefenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B 1989, 6, 209220.
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Figure 1. Evolution of the multilayer thickness d during the buildup process of a PEI-(PSS-PAH)2-(PGA-PLL)7 multilayer followed by OWLS at different pHs: (O) pH 1.5, (b) pH 4.4, (4) pH 7.4, (2) pH 10.4, and (9) pH 11.3. Data for pH 8.4 are not represented due to poor experimental reproducibility. in the waveguide, and one determines the incoupling angles for both TE and TM waves into the guide. To each incoupling angle corresponds an effective refractive index, N(TE) and N(TM), respectively. The homogeneous and isotropic monolayer model is then applied to characterize the (optical) thickness d of the film. The OWLS experiments were conducted in H2O instead of D2O. All the polypeptide films were again built on a PEI-(PSSPAH)2 precursor film. The film deposited on the waveguide is sensed by an evanescent wave with a penetration depth typically of the order of 200 nm. When the film thickness largely exceeds this penetration length, the film behaves optically as an infinite medium.
III. Results and Discussion A. Buildup of (PGA/PLL) Multilayers as a Function of pH. (PGA-PLL)n multilayers were constructed at different pH values ranging from 1.5 to 11.3. At pH values exceeding 11.3, the films could no longer be constructed, in accordance with the absence of interaction between PLL and PGA above pH 11 found in solution.26 The film buildups were followed by OWLS. Figure 1 represents the evolution of the optical thickness d during their construction. The film thickness becomes largest when the multilayers are built under acidic (pH 1.5) or alkaline (pH 10.4) conditions (except for pH exceeding 10.4) and smallest when both polypeptides are fully charged (pH 4.4 and 7.4). Similar results were obtained by Rubner and collaborators for poly(allylamine)/poly(acrylic acid) multilayers built in the absence of salt.12 This effect is explained by the fact that when one of the polyelectrolytes is not fully charged, it interacts with the outer oppositely charged surface by forming loops that extend into the solution. This leads to larger film thicknesses and also usually to larger amounts of deposited material. From the pKa value of 4.5 reported for PGA, a larger film thickness at pH 4.4 was expected, but it is known that the pKa values of polyelectrolytes embedded in multilayers can be shifted.27-30 In Appendix A, we estimate the pKa value relative to PGA in (PGA-PLL)2 films to be of the order of 2.5. This explains the small value of the film (25) Picart, C.; Ladam, G.; Senger, B.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J. G.; Gergely, C. J. Chem. Phys. 2001, 115, 1086-1094. (26) Hammes, G. G.; Schullery, S. E. Biochemistry 1968, 7, 38823887. (27) Mendelsohn, J. D.; Barrett, C. J.; Chan, V. V.; Pal, A. J.; Mayes, A. M.; Rubner, M. F. Langmuir 2000, 16, 5017-5023. (28) Klitzing, R.; Mohwald, H. Langmuir 1995, 11, 3554-3559. (29) Rmaile, H. H.; Schlenoff, J. B. Langmuir 2002, 18, 8263-8265. (30) Burke, S. E.; Barrett, C. J. Langmuir 2003, 19, 3297-3303.
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thickness observed at pH 4.4. This shift of the pKa of PGA toward a smaller value originates from the stabilization of the negative charges distributed over the polyanion chains by their interactions with the positive charges carried by PLL. Moreover, at pH 11.3, a larger film thickness than at pH 10.4 was also expected. A careful analysis of the evolution of the film thickness at pH 11.3 reveals that d increases strongly during each PLL addition but decreases slightly after each PGA deposition. The large increase of d during the PLL deposition step is in accordance with the fact that at high pH, PLL is not longer fully charged and thus adopts loop and tail configurations on the surface.31 However, the reduction of the charge of the polycation chains also reduces their interactions with the PGA chains from the film. This smaller interaction between PGA and PLL chains at a high pH value is supported by the impossibility to build (PGA-PLL)n films at pH values exceeding 11.3. Indeed, when a film ending with PLL is further brought into contact with a PGA solution, some of the loose interactions between the PLL chains and the PGA ones in the film can be gradually replaced by interactions with PGA chains from the solution in a kind of exchange process. This could thus lead to a partial removal of PLL/PGA complexes from the surface. Such an effect may be similar to the partial film dissolution observed by Cohen Stuart and collaborators32,33 for multilayers in contact with polyelectrolyte solutions where soluble polyanion/polycations are formed. Finally, whereas the reproducibility of the film thickness was within 20% for all the reported pH values, it was rather poor (more than 100%) at pH 8.4 for a reason that remains unknown. A good reproducibility was, on the other hand, always found for FTIR-ATR experiments discussed below for all the investigated pH values, including 8.4. The evolution of the secondary structure of the (PGAPLL)n films during their buildup was then followed. We used FTIR in the ATR mode and focused on the amide I band. As already mentioned in the Experimental Section, the amide I band of each FTIR film spectrum was decomposed into individual bands. The number of bands was determined after Fourier smoothing by applying a second derivative on the spectra. Five (sometimes only three) bands were necessary to describe the whole amide I spectra. Three bands that were always present, centered at around 1620-1629, 1642-1646, and 1660-1665 cm-1, were attributed to R-helix, random, and turn structures, respectively.34-36 Two other bands that always appeared simultaneously and were centered at around 1610-1612 and 1677-1680 cm-1 were attributed to β-sheets. Their simultaneous appearance indicates that the β-sheets are antiparallel.26,36 Figure 2 represents the evolutions of the contributions of the different bands during the multilayer constructions. As a general trend, as the multilayers are constructed, an increase of the contributions of the β-sheet structures is observed at the expense of the random structures except at pH 1.5 where no β-sheet contribution is found. Above pH 8.4, after the deposition of 2 (PGAPLL) bilayers and 4-5 bilayers at pH 4.4 and 7.4, the secondary structures of the films become independent of (31) Hoogeveen, N. G.; Cohen Stuart, M. A.; Fleer, G. J.; Bohmer, M. R. Langmuir 1996, 12, 3675-3681. (32) Kovacevic, D.; van der Burgh, S.; de Keizer, A.; Cohen Stuart, M. A. Langmuir 2002, 18, 5607-5612. (33) Kovacevic, D.; van der Burgh, S.; de Keizer, A.; Cohen Stuart, M. A. J. Phys. Chem. B 2003, 107, 7998-8002. (34) Jackson, M.; Haris, P. I.; Chapman, D. Biochim. Biophys. Acta 1989, 998, 75-79. (35) Jackson, M.; Haris, P. I.; Chapman, D. J. Mol. Struct. 1989, 214, 329-355. (36) Susi, H.; Timasheff, S. N.; Stevens, L. J. Biol. Chem. 1967, 242, 5460-5466.
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Figure 2. Evolution of the relative contribution of the five components of the amide I band during the buildup of a (PGAPLL)7 multilayer film at different pHs: (9) pH 1.5, (O) pH 4.4, (b) pH 7.4, (4) pH 8.4, and (2) pH 10.4; (a) β-sheet 1, (b) R-helix, (c) random, (d) turn, and (e) β-sheet 2 contents. The x axis is valid for panels a-e.
the number of bilayers constituting the multilayer. Since the spectrum is relative to the whole film, these results indicate that the influence of the precursor film (PAHPSS)2 extends only over a few bilayers. At pH 4.4 and 7.4, the larger number of bilayers needed to reach a structure independent of the film thickness may be due to the fact that their thicknesses are smallest. Indeed, at these intermediate pH values the interactions between PGA and PLL are strongest and induce a small thickness increase per bilayer. In this case, the influence of each layer on the next deposited one may thus be stronger, so that a larger number of bilayers is required to vanish the influence of the substrate. B. Comparison between the Secondary Structure of Polypeptide Multilayers and the Corresponding Complexes in Solution. To compare the secondary structures of polypeptide polyanion/polycation complexes in solution to that of the corresponding polyelectrolyte multilayers, we performed two kinds of experiments. On one hand, we compared the secondary structures of (PGA/ PLL) complexes in solution, for various pH values, to that of the corresponding multilayers. On the other hand, at a given pH, the secondary structures of complexes and multilayers were studied for various polyanion/polycation systems differing from (PGA/PLL) by the chemical nature of the side chains of the polypeptides or by their chirality. We used transmission FTIR to investigate the structure of the complexes. The relative contributions of the different secondary structures to the amide I band of the (PGA/PLL) complexes in solution at various pHs and of their corresponding multilayers are gathered in Table 1. The contributions
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Table 1. Relative Contents of the Different Secondary Structure Contributions of the Amide I Band for PGA and PLL Solutions at Different pH Values, for PGA/PLL Complexes in Solution, and for (PGA-PLL)7 Multilayer Films Deposited on PEI-(PSS-PAH)2 Precursor Films Also at Different pHsa pH PLL
1.5
PLL
4.4
PLL
7.4
PLL
8.4
PLL
10.4
PGA
1.5
PGA
4.4
PGA
7.4
PGA
8.4
PGA
10.4
PGA/PLL
1.5
(PGA-PLL)7
1.5
PGA/PLL
4.4
(PGA-PLL)7
4.4
PGA/PLL
7.4
(PGA-PLL)7
7.4
PGA/PLL
8.4
(PGA-PLL)7
8.4
PGA/PLL
10.4
(PGA-PLL)7
10.4
β-sheet
1612 10%
1612 19% 1612 33% 1610 32% 1611 38% 1611 38% 1611 36% 1611 39% 1611 46%
R-helix
random
turn
1623 12% 1622 4% 1623 8% 1622 6% 1625 6% 1628 20% 1627 18% 1629 4%
1643 56% 1642 54% 1643 62% 1643 60% 1643 64% 1645 49% 1642 66% 1644 58% 1644 62% 1644 62% 1644 46% 1641 39% 1645 38% 1644 28% 1644 28% 1643 27% 1644 23% 1643 25% 1643 27% 1643 21%
1664 32% 1662 42% 1664 30% 1663 34% 1664 30% 1664 31% 1660 16% 1664 38% 1664 38% 1664 38% 1662 18% 1664 33% 1662 12% 1661 11% 1661 18% 1661 11% 1662 12% 1661 12% 1661 16% 1661 10%
1626 22% 1620 28% 1629 25% 1629 22% 1628 13% 1627 15% 1624 17% 1626 18% 1628 7% 1627 14%
β-sheet
Figure 3. Evolution of the multilayer thickness d during the buildup process of different polypeptide systems (A/C)7, (polyanion/polycation)7, at pH 7.4 with (b) A ) PGA/C ) PLL, (O) A ) PAA/C ) PLL, (9) A ) PGA/C ) PDL, and (0) A ) PGA/C ) PLO.
1680 4%
1679 6% 1678 6% 1677 9% 1677 9% 1678 10% 1678 9% 1678 11% 1677 9%
a
The first line for each polyelectrolyte, complex, or film architecture corresponds to the maximum frequency in cm-1 at which the component band appears. The second line corresponds to the value of its relative contribution to the amide I band (see text for definition).
relative to the spectra of PGA and PLL solutions at the same pH values are also given. As already reported in the literature, the interactions between PGA and PLL lead to the formation of β-sheet structures both in solution37 and in the multilayers.15,16,18 Table 1 shows that the secondary structures of the complexes and of the multilayers evolve similarly with pH: the contributions of the β-sheet band increase as the pH is increased. Correlatively, the R-helix contribution has a tendency to decrease as the solutions become more alkaline. The larger scatter in the data of the R-helix contributions may be due to the large overlap of the three bands at around 1628, 1644, and 1662 cm-1, which renders the decomposition more difficult. We also changed the chemical nature of the polypeptides and investigated poly(L-aspartic acid)/PLL (PAA/PLL) and PGA/poly(L-ornithine) (PGA/PLO) multilayers at pH 7.4. PAA and PLO differ from PGA and PLL, respectively, by the reduction of the side chains by one methylene group. (37) Jhon, M. S.; Jung, J. C. In Polymeric Materials Encyclopedia; Salamone, J. C., Ed.; CRC Press: New York, 1996; Vol. 8, pp 64666472.
Table 2. Relative Contents of the Different Secondary Structure Contributions in the Amide I Band of PDL, PAA, and PLO Solutions, of PGA/PDL, PAA/PLL, and PGA/PLO Complexes in Solution, and of (PGA-PDL)7, (PGA-PDL + PLL)7, (PAA-PLL)7, and (PGA-PLO)7 Multilayer Films Deposited on a PEI-(PSS-PAH)2 Precursor Film at pH 7.4a pH β-sheet R-helix random turn β-sheet PLO
7.4
PGA/PLO
7.4
(PGA-PLO)7
7.4
PAA
7.4
PAA/PLL
7.4
(PAA-PLL)7
7.4
PDL
7.4
PGA/PDL
7.4
(PGA/PDL)7
7.4
PDL + PLL
7.4
PGA/PDL + PLL
7.4
(PGA-PDL + PLL)7 7.4
1611 37% 1611 36%
1612 36% 1612 34%
1611 34% 1611 36%
1629 11% 1628 16% 1628 14% 1625 24% 1629 28% 1629 37% 1629 10% 1626 21% 1625 26% 1629 12% 1626 18% 1626 22%
1646 53% 1644 21% 1643 24% 1644 49% 1645 48% 1645 43% 1644 58% 1645 21% 1645 25% 1644 58% 1644 24% 1644 23%
1665 36% 1661 14% 1660 15% 1664 27% 1663 24% 1666 20% 1664 32% 1662 13% 1663 8% 1664 31% 1662 15% 1663 11%
1679 12% 1678 11%
1679 9% 1679 7%
1678 9% 1679 8%
a The first line for each polyelectrolyte, complex, or film architecture corresponds to the maximum frequency in cm-1 at which the component band appears. The second line corresponds to the value of its relative contribution to the amide I band (see text for definition).
We first followed the evolution of the OWLS signal during the film buildup and found that the (PAA-PLL)n and (PGA-PLO)n multilayers grow steadily (Figure 3). We then analyzed the secondary structures of these films and their corresponding complexes (Table 2). Here too, we found qualitatively a similar evolution for the secondary structure of the complexes in solution and of the corresponding multilayers: when β-sheets are absent in the complexes, they are absent in the film too, and vice versa. More precisely, the structures of the (PGA-PLO)n multilayers and of (PGA/PLO) complexes are close to those of their (PGA,PLL) counterpart, characterized by an important contribution of the β-sheets. The contributions
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of the β-sheets are even slightly more important for the (PGA/PLO) complexes than for the (PGA/PLL) ones (49% vs 41%). This result is in opposition with the findings of Hammes and Schullery,26 who reported that the interaction of PLO with PGA does not lead to the formation of β-sheets. On the other hand, in agreement with these authors, we found no β-sheets in the amide I band of PAA/ PLL complexes in solution. Similarly, no β-sheets are present in (PAA-PLL)n films. Finally, we also analyzed the influence of the chirality of the polypeptides on the film secondary structure by replacing PLL by PDL. (PGA-PDL)n films were thus constructed at pH 7.4 under conditions similar to those for (PGA-PLL)n multilayers. As can be seen in Table 2, the secondary structure of the (PGA-PDL)n films is quite similar to that of (PGA-PLL)n. It is also very similar to its complex counterpart in solution. On the other hand, the evolution of the thickness and mass of the (PGAPDL)n films with n is significantly slower than for (PGAPLL)n (Figure 3), showing that the buildup processes are not exactly similar in both cases. We then also constructed films by using a mixture of 2.5 mg/mL of PLL and 2.5 mg/mL of PDL at pH 7.4 as the polycation solution. The (PGA-(PDL + PLL))n multilayer films, with a 44% contribution in β-sheets, are structurally equivalent to their complex counterpart in solution and to the (PGAPLL)n and (PGA-PDL)n films. From these results, one can conclude that the secondary structure of the multilayers is qualitatively very similar to their complex analogues in solution. The small quantitative differences that are observed may be due to slightly different polyanion/polycation proportions in the films compared to the complexes. This is however difficult to verify experimentally because a change of the proportion of both polyelectrolytes in the solution may lead to solutions composed of complexes and of free polyelectrolytes without the possibility to determine the contribution of both entities to the spectra. C. Nature of the Polyanion-Polycation Interactions Leading to β-Sheet Structures. β-Sheet structures are formed of β-strands aligned adjacent to each other, which interact through hydrogen bonds between CO groups of one strand and NH groups of the other strand and vice versa. The side chains point alternatively above and below the β-sheet. Due to electrostatic interactions between the side chains, it is very difficult to form β-sheets in polyanion or polycation solutions. Among the acidic and basic polypeptides, only PLL and PGA are known to form β-sheets. PLL forms β-sheets in solution and only under alkaline conditions when heated above 50 °C or in ethylene glycol/water mixtures.38 PGA forms β-sheets in solution at low pH where it is insoluble39 and in a gel state by heating.40 Under such conditions, the electrostatic interactions become weak and hydrophobic interactions between adjacent lysine or glutamic residues stabilize the β-sheets. When polyanions and polycations are mixed, electrostatic interactions no longer only destabilize the β-sheets but the interactions between opposite charges of the side chains of both polypeptides can also stabilize them. With the exception of PLL/PAA, we indeed found β-sheets in all the investigated polypeptide polyanion/polycation complexes and multilayers. In such complexes, the β-sheets can be built in two different manners: The polyanions and polycations can form β-sheets separately (38) Davidson, B.; Fasman, G. D. Biochemistry 1967, 6, 1616-1629. (39) Lenormant, H.; Baudras, A.; Blout, E. R. J. Am. Chem. Soc. 1958, 80, 6191-6195. (40) Itoh, K.; Foxman, B. M.; Fasman, G. D. Biopolymers 1976, 15, 419-455.
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Figure 4. Schematic representation of antiparallel β-sheet conformations adopted by polyanion/polycation complexes of polypeptides (a) in the case of only positive β-strands, (b) in the case of negatively and positively charged β-strands with L-polycation and L-polyanion, and (c) in the case of negatively and positively charged β-strands with D-polycation and Lpolyanion. Lateral chains are represented by a line ending with a circle indicating their respective charges.
which stabilize each other as seen schematically in Figure 4a. Such an interaction scheme has already been proposed by Domard and Rinaudo.41 But β-sheets can also be formed by the direct interaction of polyanion β-strands with polycation ones through hydrogen bonds. The stabilization then takes place through the electrostatic interaction between adjacent side chains (Figure 4b). Whereas the first mechanism allows for the formation of β-sheets with a mixture of L-polyanions and D-polycations, the second does not. Indeed, if, in the second mechanism, β-strands of L and D chains would interact through the CO‚‚‚NH hydrogen bonds, their side chains could not all point perpendicularly to the β-sheet structure and thus be parallel to each other. The electrostatic interaction stabilization between the positive charges from the polycation and the negative ones from the polyanion would be lost. The first mechanism must thus be responsible for the β-sheet content of the PGA/PDL complexes and multilayers. However, PGA does not interact exactly in the same way with PLL and PDL as supported by the different evolution of the deposited mass during the (PGAPLL)n and (PGA-PDL)n film constructions (Figure 3). This suggests that both β-sheet formation mechanisms may be involved in (PGA/PLL) complexes and multilayers even (41) Domard, A.; Rinaudo, M. Macromolecules 1980, 13, 898-904.
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Figure 5. Encapsulation of (PGA-PLL)8 by (PSS-PAH) bilayers. (a) Evolution of the relative contribution of the β-sheet 1 component of the amide I band for a (PSS-PAH)2-(PGAPLL)8-(PSS-PAH)2 multilayer film built at pH 7.4. (b) Evolution of thickness d during the buildup process of the (PSSPAH)2-(PGA-PLL)8-(PSS-PAH)2 multilayer film at pH 7.4.
if the first mechanism is expected to dominate as suggested by the similar β-sheet contents for (PGA/PLL) and (PGA/ PDL) complexes or multilayers. The electrostatic interactions can, however, not be solely responsible for the β-sheet stabilization in complexes and multilayers; otherwise β-sheets would also be observed in (PAA,PLL) complexes and multilayers. As suggested in the literature,26,42 hydrophobic interactions between side chains must also play a stabilization role. PAA having a small side chain, this effect could be too weak to allow the formation of β-sheets in the (PAA,PLL) systems. D. Structural Response of the Multilayers to Various Stresses. a. Change of the Nature of the Polyelectrolyte in the Outer Layer. We deposited (PSSPAH) bilayers on top of (PGA-PLL)n films. The experiments were conducted at pH 7.4. Figure 5a shows the evolution of the contribution of the β-sheets (component band at 1610 cm-1) to the amide I band for n ) 8. Similar results were obtained for n ) 2 and 4. Clearly, the addition of two (PSS-PAH) bilayers on top of the (PGA-PLL)n architectures totally destroys the β-sheets over the entire (PGA-PLL)n film. Whereas the addition of (PSS-PAH) bilayers on top of the (PGA-PLL)8 multilayer is accompanied by an increase of the thickness and mass of the entire film (Figure 5b). A close analysis of the evolution of the IR spectra (Figure 6) reveals that at each adsorption step of PSS the characteristic bands of PSS (1007 and 1035 cm-1)43-45 increase while the COO- peaks of PGA (1561 cm-1)20,34 decrease. When the film is then further brought into (42) Mita, K.; Ichimura, S.; Zama, M. Biopolymers 1978, 17, 27832798. (43) Yang, J. C.; Jablonsky, M. J.; Mays, J. W. Polymer 2002, 43, 5125-5132. (44) Zundel, G. Hydration and intermolecular interaction; Academic Press: New York, 1969. (45) Orler, E. B.; Yontz, D. J.; Moore, R. B. Macromolecules 1993, 26, 5157-5160.
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Figure 6. Evolution of infrared spectra of a multilayer during its buildup process with (- -) (PGA/PLL)8, (-O-) (PGA/PLL)8PSS, (s) (PGA/PLL)8-PSS-PAH, (-4-) (PGA/PLL)8-(PSSPAH)-PSS, and (‚‚‚) (PGA/PLL)8-(PSS-PAH)2-PSS. (The spectrum of (PGA/PLL)8-(PSS-PAH)2 is not represented because it is too close to that of (PGA/PLL)8-(PSS-PAH)PSS.)
contact with the polycation solution (PAH in our case), the COO- band decreases even more strongly than after the first PSS deposition while the intensity of the PSS bands remains almost constant. During the second PSS deposition step, the intensities of the PSS peaks increase again strongly while the COO- band is no longer greatly affected (the slight increase may be due to slight rearrangements of the chains affecting their ionization degree). Additional depositions of PSS on top of the (PSSPAH)2 layers lead to a much smaller increase of the PSS bands while the intensity of the COO- band remains constant. Whereas the increase of the intensities of the bands attributed to PSS is clearly in direct relation with the increase of the amount of PSS present in the film, the changes in the intensity of the COO- band can, at first sight, have two different origins. Xie and Granick46 investigated the ionization of poly(methacrylic acid) (PMA), a weak polyelectrolyte, embedded in the bottom of a quaternized poly(vinylpyridine)/poly(styrene sulfonate) film. They found giant oscillations in the ionization of PMA directly correlated with the mass and nature of the polyelectrolyte deposited in the outermost layer of the film. These oscillations persisted with a decay length far exceeding the Debye length of the aqueous solution. The decrease of the intensity of the COO- peak consecutive to the first PSS adsorption on the top of our film could thus have a similar origin. However, if this were the case, one would anticipate an increase of the intensity of this peak when PAH is further deposited on the film. Moreover, oscillations of this peak intensity should be observed when new (PSS/PAH) bilayers are deposited. This is however not the case, the intensity of the COO- peak remaining constant after the deposition of two bilayers. The evolution of the FTIR-ATR spectra can have a second origin. During the first contact of the (PGA-PLL)n film with the PSS solution, PSS not only interacts with the outer layer but (46) Xie, A. F.; Granick, S. Macromolecules 2002, 35, 18051813.
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also diffuses into the film. These “free” PSS chains then exchange PGA ones that are part of the film architecture. The existence of such a diffusion mechanism followed by an exchange process could be proven for PLL chains in (hyaluronic acid-PLL)n10 and (PGA-PLL)n films.47 During the rinsing step, only a fraction of the free polyanions diffuse out of the film, a significant fraction of them remaining in the multilayer due to the existence of an electrostatic barrier. When the film is then further brought in contact with the polycation solution (PAH in our case), the remaining free polyanions diffuse out of it. Some of them interact with PAH to form the new outer layer, while others form complexes that escape into the solution. The exchange process of PGA by PSS thus reduces the number of COO- groups present in the film and thus also its corresponding absorbing band. During the next PSS deposition step, the bands of PSS increase again strongly while the COO- bands are no longer greatly affected (the slight increase may be due to slight rearrangements of the chains affecting their ionization degree). This suggests again the diffusion of PSS into the film but the absence of an exchange process between the remaining PGA chains in the film and the free PSS ones. A third deposition of PSS on top of the (PSS-PAH)2 layers leads to a much smaller increase of the PSS bands while the COO- bands remain unchanged. It thus seems that after the deposition of two (PSS-PAH) bilayers, PSS can no longer diffuse into the film. (PSS/PAH)2 thus forms a kind of barrier that becomes impenetrable for further chains. This effect is consistent with the fact that (PSSPAH)n films grow linearly with n. A similar “barrier” effect of two (PSS-PAH) bilayers also takes place on top of (PGA-PAH)n films (see Appendix B). Even if the evolution of the FTIR-ATR spectra does not constitute absolute proof of the exchange mechanism, it nevertheless strongly suggests it. To further prove the existence of the exchange mechanism, one should be able to directly prove the decrease of the amount of PGA in the film. This, however, is not possible with our system. Experiments on other polyelectrolyte systems are under preparation to specifically address this very general problem. One can thus conclude that the destruction of the β-sheets consecutive to the deposition of PSS and PAH layers seems to be due to the large exchange of PGA chains by PSS ones inside of the film. b. Stability of the Film Secondary Structure with Respect to pH Jumps. The secondary structures of the (PGAPLL)n multilayers change when the film buildup is performed at different pHs. Do the film secondary structures also vary when the pH is changed after its construction? To answer this question, we constructed (PGA-PLL)n films at various pHs and brought them into contact with solutions at other pHs. We first constructed the film at pH 4.4. It was then successively brought into contact with buffer solutions alternatively at pH 10.4 and 4.4 (9 h at each step). A slight increase of the β-sheet content is observed after the first two pH jumps (successively to pH 10.4 and 4.4) (Figure 7). Further contacts of the films with the buffer solutions at pH 4.4 and pH 10.4 no longer change their secondary structure. The β-sheet content is then slightly smaller than that obtained when the film is directly built at pH 10.4 (40% instead of 46%). When this film is finally brought into contact with a solution at pH 1.5, the β-sheet content strongly decreases during the last step (Figure 7). Correlatively, the 1700 cm-1 band attributed to COOH increases.34 This indicates (47) Jessel, N.; Atalar, F.; Lavalle, P.; Mutterer, J.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Ogier, J. Adv. Mater. 2003, 15, 692-695.
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Figure 7. Evolution of the relative contribution of the β-sheet 1 and R-helix components of the amide I band of a (PGA-PLL)7 multilayer film, built at pH 4.4, with alternative pH changes from 4.4 to 10.4 and finally to pH 1.5.
that PGA becomes uncharged. In addition, a strong reduction of the film absorbance is observed. This reduction is attributed to a partial “dissolution” of the film into the solution. This is confirmed by OWLS where an important decrease of the film thickness is observed. On the other hand, when a film prepared at pH 1.5 and thus having no β-sheet structure is put into contact with a solution at pH 7.4, the amide I band disappears totally. This indicates its total dissolution. Otherwise, when a film constructed at pH 10.4 is successively put into contact with solutions at pH 4.4 and 10.4 for at least 9 h, no significant changes in its secondary structure are observed. Finally, when a film constructed at pH 7.4 is brought into contact with solution at pH 13, it dissolves totally. To conclude, these results show that β-sheets stabilize the polypeptide multilayers and their secondary structures are very stable with respect to pH jumps as long as the stability of the film is not affected (between pH 4 and 10.5 for (PGA-PLL)n multilayers). Moreover, films constructed at pH 4.4 and subsequently put into contact with a solution at pH 10.4 do not reach their full equilibrium state. They seem to be frozen in an intermediate state between those prepared at pH 4.4 and those prepared at 10.4. When these multilayers are brought into contact with very acidic or very basic solutions, they are no longer stable. They lose both β-sheet content and mass. Moreover, (PGA-PLL)n films constructed at pH 1.5 are totally unstable with respect to pH jumps toward higher pH values. c. Stability of the Film Secondary Structure with Respect to Temperature Changes. We also investigated the effect of thermal stability of the polypeptide multilayer structure. Two types of experiments were performed. In both cases, (PGA-PLL)n films were first constructed at room temperature at pH 7.4. In the first type of experiment, the temperature was increased step by step from 26 to 89 °C by 7 °C increases in each step. The system remained 12 min at each temperature. After remaining at 89 °C for 12 min, it was rapidly cooled to room temperature in 1/2 h. One observes a slight but constant reduction (respectively increase) of the β-sheet (respectively R-helix) content of the multilayer (Figure 8a). The changes are fairly reversible upon cooling. Nevertheless, the β-sheet content is slightly higher after the thermal cycle, in accordance with the stabilization effect of β-sheets in multilayers. In the second type of experiment, the film was heated to 89 °C within 1/2 h and maintained at this temperature for 8 h before being cooled to room temperature within 1/2 h. Surprisingly, the β-sheet content increases rapidly while
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nection between the secondary structures of the multilayers and of their complex counterparts in solution and found a close similarity between both. We also studied their stability with respect to various external stresses: The secondary structures appear very stable with respect to pH jumps as long as they do not affect the stability of the film. Slight variations of the secondary structures are observed when the films are heated to 89 °C. The final film structure depends on the way in which the temperature rise is performed: a slow temperature increase leads to a slow but reversible decrease of the β-sheet content, while a rapid temperature rise leads to an irreversible increase of the β-sheet content. Finally, we also analyzed the effect of a change of the nature of the upper layers on the film secondary structure. When (PSS-PAH) bilayers are deposited on top of a (PGA-PLL)n film, its β-sheets are totally destroyed. This is explained by the diffusion of PSS chains into the (PGA-PLL)n film and the subsequent exchange of PGA chains from the film with free PSS ones. It is, to the authors’ knowledge, the first time that such an exchange process within a multilayer film has been observed.
Figure 8. Evolution of the relative contribution of the β-sheet 1 and R-helix components of the amide I band for a (PGAPLL)7 multilayer film built at pH 7.4 and (a) heated from 26 to 89 °C by steps of 12 min and (b) heated at 89 °C for approximately 8 h before a temperature decrease to 26 °C. Each x axis is valid for both graphs.
the R-helix content decreases during the first 20 min. The β-sheet content continues to increase slowly afterward before leveling off at around 48% (Figure 8b). When the film is cooled back to room temperature, a further increase (respectively decrease) of the β-sheet (respectively R-helix) content is observed. These results may be explained as follows: by increasing the temperature, one favors the formation of amide functions from the amine groups of PLL reacting with the carboxylic groups of PGA.48 The formation of such links between chains, even if their number is small, tends to stabilize the structure of the film. On the other hand, an increase in temperature tends to destabilize the film secondary structures. Such a destabilization takes place, however, only when the chains can move freely with respect to each other. In the case of a slow temperature rise, the destabilization process takes place before the reaction between some amine and carboxylic groups takes place. On the other hand, when the film is heated rapidly, some groups can react before the β-sheets are destabilized. The relative freedom of movement of the chains, gained by the temperature increase, only allows restructurations for local chains. This process allows the extension of the β-sheet domains but forbids their destruction (which would require larger movements). As the temperature is cooled, β-sheets become even more stable, thus leading to a further increase of their content. IV. Conclusions In this article, we investigated the secondary structures of polypeptide multilayers. We first analyzed the con(48) Harris, J. J.; DeRose, P. M.; Bruening, M. L. J. Am. Chem. Soc. 1999, 121, 1978-1979.
Acknowledgment. The work was supported by the programs Action Concerte´e Incitative 2002 “Technologie pour la sante´” No. 02C0170 and “Surfaces interfaces et conception de nouveaux mate´riaux” from the Ministe`re Franc¸ ais de la Recherche and by the Centre National de la Recherche Scientifique program “Physique et chimie du vivant”. We thank Dr. B. Szalontai for his help in the treatment of IR spectra. We also thank Dr. V. Ball and Professor G. Decher for fruitful discussions concerning polyelectrolyte multilayers. Appendix A For the pKa determination of PGA in (PGA/PLL)n multilayers, PEI-(PSS-PAH)2-(PGA/PLL)2 architectures were built at various pHs ranging between 2 and 10 in MES or/and Tris, NaCl buffer as described in the Experimental Section but prepared with water instead of deuterium oxide. The absorbance spectra of (PGA/PLL)2 were calculated using the single-beam spectrum (512 scans) of the underlying PEI-(PSS-PAH)2 as a reference. Absorbance spectra were Fourier smoothed, and the 13501750 cm-1 region was decomposed into Gaussian component bands as described in detail by Schwinte´ et al.21 In this region, the bands of interest for pKa determination are those of glutamate and glutamic acid. The intensities, corresponding to the areas under the peaks, of the symmetric stretching vibrations of the carboxylate anions νs(COO-) at 1405 cm-1 and symmetric stretching vibrations of the carbonyl group ν(CdO) at 1724 cm-1 were determined.20,34 The decomposition process led to the determination of bandwidth, position, and intensity, the intensity of the band being assumed proportional to the quantity of the corresponding functional group. We then plotted the relative percentage of each species versus pH as seen in Figure 9. The relative percentage of COOH species is calculated by ratio 1, where Ints represents the area of the corresponding peak:
% COOH )
Ints(COOH) Ints(COOH) + Ints(COO-)
(1)
The apparent pKa of PGA embedded in the multilayer corresponds to the pH at which 50% of the acidic groups are ionized and is equal to 2.5.
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Figure 9. The normalized intensity of (b) νs(COO-) at 1405 cm-1 and (O) ν(CdO) at 1720 cm-1 peaks of PGA embedded in PEI-(PSS/PAH)2-(PGA/PLL)2, calculated at different pHs by the following ratio, where Ints is the intensity of the peak (area): % COOH ) Ints(COOH)/[Ints(COOH) + Ints(COO-)].
Appendix B We prepared (PGA-PAH)n films in a manner similar to that for the (PGA-PLL)n films at pH 8.4. As expected, no β-sheets are present in these films but the PGA chains form R-helices (Figure 10a). As already reported,11 one observes that the R-helix content switches alternately from a value of around 20% when the film is in contact with a PAH solution to a higher value when it is in contact with the PGA solution. This effect was explained by the diffusion in and out of the film of PGA chains during each deposition step. Indeed, when the film is brought into contact with a PAH solution, no free PGA chains remain in the film and the R-helix content corresponds to that of PGA chains forming a network with PAH. On the other hand, when the film is brought into contact with a PGA solution, free PGA chains remain in the film even after the rinsing step. These chains seem to adopt conformations rich in R-helices. When two (PSS-PAH) bilayers are deposited on top of a (PGA-PAH)n film, the R-helix content of the film no longer changes (Figure 10a) in accordance with the fact that no free PGA chains diffuse within the film anymore. When a further (PGA-PAH)n film is grown on top of the (PSSPAH)2 bilayers, the R-helix content starts again to cycle with a high value that increases with the number of deposited bilayers. In addition, the value of the R-helix content after the first PGA deposition on top of the (PSSPAH)2 bilayers is significantly smaller than that observed before the deposition of the (PSS-PAH)2 bilayers. This clearly suggests that PGA no longer diffuses within the whole film but seems to diffuse only within the (PGAPAH)n film deposited on top of the (PSS-PAH)2 bilayers. A similar evolution is observed when a second (PSS-PAH)2 film is built on top of the previous architecture followed by a third subsequent (PGA-PAH)n film deposition.
Figure 10. (a) Evolution of the relative contribution of the R-helix component of the amide I band of PGA during the buildup of the (PGA/PAH)5-(PSS/PAH)2-(PGA/PAH)2-(PSS/ PAH)2-(PGA/PAH)5 multilayer film and (b) evolution of the effective refractive index N(TE), obtained by OWLS, during the buildup of the (PGA/PAH)7-(PSS/PAH)2-(PGA/PAH)2PGA multilayer film.
The barrier of two (PSS-PAH) bilayers is further supported by OWLS experiments. In this technique, one senses the film with an evanescent wave up to a thickness of the order of a few hundred nanometers. When the film becomes thicker, OWLS is only sensitive to refractive index changes in the zone of the film sensed by the evanescent wave, that is, near the deposition substrate. The diffusion of PGA in and out of (PGA-PAH)n films then leads to a cycling of the effective refractive indexes as shown in Figure 10b. When (PSS-PAH) bilayers are further deposited on such a film, the effective refractive index remains unchanged, showing that the refractive index of the film near the deposition substrate no longer changes.11 Further deposition of a (PGA-PAH)n film on top of the two (PSS-PAH) bilayers no longer changes the effective refractive indexes, clearly indicating that PGA can no longer diffuse up to the deposition substrate. This can only be because the two (PSS-PAH) bilayers act as impenetrable barriers. LA0348259
