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Langmuir 2008, 24, 7842-7847
Dynamics of Poly(L-lysine) in Hyaluronic Acid/Poly(L-lysine) Multilayer Films Studied by Fluorescence Recovery after Pattern Photobleaching Laurent Jourdainne,†,‡ Sigole`ne Lecuyer,§ Youri Arntz,†,‡ Catherine Picart,| Pierre Schaaf,§ Bernard Senger,†,‡ Jean-Claude Voegel,†,‡ Philippe Lavalle,*,†,‡,⊥ and Thierry Charitat§ Institut National de la Sante´ et de la Recherche Me´dicale, INSERM, Unite´ 595, 11 rue Humann, 67085 Strasbourg Cedex, France, UniVersite´ Louis Pasteur, Faculte´ de Chirurgie Dentaire, 1 place de l’Hoˆpital, 67000 Strasbourg, France, UniVersite´ Louis Pasteur, Institut Charles Sadron, CNRS UPR 22, 23 rue du loess, BP 84047, 67034 Strasbourg Cedex 2, France, UniVersite´ de Montpellier II, Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Centre National de la Recherche Scientifique, CNRS UMR 5235, Place Euge`ne Bataillon, 34095 Montpellier Cedex 5, France, and Hoˆpitaux UniVersitaires de Strasbourg, 1, Place de l’Hoˆpital, BP 426, 67091 Strasbourg Cedex, France ReceiVed December 22, 2007. ReVised Manuscript ReceiVed April 17, 2008 Poly(L-lysine) (PLL)/hyaluronic acid (HA) multilayers are films whose thickness increases exponentially with the number of deposition steps. Such a growth process was attributed to the diffusion, in and out of the whole film, of at least one of the polyelectrolytes constituting the film. In the case of PLL/HA, PLL is known to be the diffusing species. In order to better understand the growth mechanism of such films, it is of primary importance to well characterize the diffusion process of the polyelectrolytes in the multilayer. This process is studied here by fluorescence recovery after pattern photobleaching. We show that the diffusion behavior is different when we consider either PLL chains that are deposited on top of the film or PLL chains embedded in the film, even below only one HA layer. For chains that are embedded, we find two populations: a mobile one with a diffusion coefficient, D, of the order of 0.1 µm2 · s-1 and a population that appears immobile (D < 0.001 µm2 · s-1). For chains deposited on top of the multilayer, a third population appears which is rapidly diffusing (D = 1 µm2 · s-1). These results confirm the validity of the model generally accepted for the exponential growth process and in particular the existence of up to three subgroups of PLL chains from the point of view of their diffusion coefficient.
Introduction The alternate deposition of polyanions and polycations on solid surfaces leads to the formation of “polyelectrolyte multilayer films” (PEM films).1,2 The study of these films has received considerable attention during the last years due to widespread potential applications.3–11 Two types of polyelectrolyte multilayers can be distinguished: Those whose thickness and mass increase linearly with the number of deposition steps2 and those for which this growth is exponential.12 The present contribution * Corresponding author: phone, +33-(0)390-243061; fax, +33-(0)390243379; e-mail,
[email protected]. † Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 595. ‡ Universite´ Louis Pasteur, Faculte´ de Chirurgie Dentaire. § Universite´ Louis Pasteur, Institut Charles Sadron. | Universite´ de Montpellier II, Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques. ⊥ Hoˆpitaux Universitaires de Strasbourg.
(1) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831–835. (2) Decher, G. Science 1997, 277, 1232–1237. (3) Hiller, J.; Mendelsohn, J. D.; Rubner, M. F. Nat. Mater. 2002, 1, 59–63. (4) Vazquez, E.; Dewitt, D. M.; Hammond, P. T.; Lynn, D. M. J. Am. Chem. Soc. 2002, 124, 13992–13993. (5) Thierry, B.; Winnik, F. M.; Merhi, Y.; Tabrizian, M. J. Am. Chem. Soc. 2003, 125, 7494–7495. (6) Tang, Z. Y.; Kotov, N. A.; Magonov, S.; Ozturk, B. Nat. Mater. 2003, 2, 413–418. (7) Stanton, B. W.; Harris, J. J.; Miller, M. D.; Bruening, M. L. Langmuir 2003, 19, 7038–7042. (8) Rmaile, H. H.; Schlenoff, J. B. J. Am. Chem. Soc. 2003, 125, 6602–6603. (9) Schultz, P.; Vautier, D.; Richert, L.; Jessel, N.; Haikel, Y.; Schaaf, P.; Voegel, J.-C.; Ogier, J.; Debry, C. Biomaterials 2005, 26, 2621–2630. (10) Sukhishvili, S. A. Curr. Opin. Colloid Interface Sci. 2005, 10, 37–44. (11) Jessel, N.; Oulad-Abdeighani, M.; Meyer, F.; Lavalle, P.; Haikel, Y.; Schaaf, P.; Voegel, J.-C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 8618–8621. (12) Lavalle, P.; Gergely, C.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Picart, C. Macromolecules 2002, 35, 4458–4465.
focuses on this second type of films. One of the most investigated exponentially growing multilayers is the PLL/HA system (PLL, poly(L-lysine); HA, hyaluronic acid). Whereas for linearly growing films the polyelectrolytes from the solution always interact exclusively with the outer part of the multilayer during each deposition step, the exponential growth was attributed to the diffusion process “in” and “out” of the whole film during each bilayer deposition step of at least one of the polyelectrolytes constituting the PEM film. More precisely, let us assume that the polycation is the diffusing species. When the film ending by a polyanion layer is immersed in the polycation solution, the polycation chains first diffuse toward the film and strongly interact with the outer negative layer of the film, to form polyanion/ polycation complexes. However, in contrast to what happens for linearly growing films, polycationic chains diffuse into the film, over its whole thickness. These chains do not interact strongly with the polyanions inside the film and are called “free polycations”. During the rinsing step, a fraction of these free chains diffuses out of the film, but not all of them because of the presence of a positive electrostatic barrier at the film/solution interface. When the film is then brought in contact with a polyanion solution, the polyanions interact with the outer positive charges, reversing the sign of the outer excess charge of the film. This allows the remaining free polycation chains in the film to diffuse out of it. As soon as they reach the film/solution interface, they are complexed by the polyanions from the solution and these complexes form the new outer layer of the multilayer. Such a diffusion process was first demonstrated by confocal laser scanning microscopy where fluorescently labeled PLL chains were deposited on top of a (PLL/HA)n multilayer, rendering the
10.1021/la7040168 CCC: $40.75 2008 American Chemical Society Published on Web 06/26/2008
Poly(l-lysine) Dynamics in Polyelectrolyte Films
film totally fluorescent.13 However, the film remained totally fluorescent even after the following HA deposition step. This result is more surprising since it is expected that during the HA deposition step the PLL chains that have diffused during the previous step into the film should also diffuse out of it and be complexed by the HA chains. This discrepancy between the experimental observations and the model was explained by the exchange process between PLL chains that have diffused into the film during a deposition step and PLL chains constituting the film and that were incorporated during previous deposition steps. This exchange process should probably take place by keeping the density of “free chains” in the film constant. This model explaining the exponential growth was recently very nicely confirmed by Ko¨hler et al., who performed experiments on microcapsules with (PSS/PDADMA)n (PSS, poly(styrenesulfonate); PDADMA, poly(diallyldimethylammonium chloride)) multilayer walls.14 When such capsules are constructed under high ionic strength conditions, they grow exponentially. The authors found that when the walls were ended by PDADMA, the nitrogen/sulfur ratio in the walls was of the order of 1.1, whereas it was of the order of 0.7 when the walls were ended by PSS. Moreover, the swelling and shrinking behavior of these (PSS/ PDADMA)n microcapsules suggested that in the case of capsules ending by PDADMA, there was a large excess of PDADMA chains in the whole wall when compared to walls ending by PSS. Exponentially growing polyelectrolyte multilayers are of great interest because they can be used as reservoirs for active proteins, drugs, or peptides.15,16 Active peptides can also be covalently bound to the diffusing polyelectrolyte to render such films biologically active.17 Recently we also showed that two (PLL/ HA)n reservoirs can be separated by (PSS/PDADMA)m barriers which can be opened and closed reversibly by mechanical stretching.18 Once opened, PLL could diffuse from one compartment to the other. A better understanding and control of these processes as well as a better knowledge of the exponential growth process requires the investigation of the diffusion process of macromolecules in these films. In this paper, we focus on the diffusion of PLL in PLL/HA multilayers. We already undertook such a study by fluorescence recovery after photobleaching (FRAP), using confocal laser scanning microscopy to photobleach a circular area of several tens of micrometers on the film and recording the evolution of the fluorescence profile along a diameter of the bleached zone during a time lapse of about 1 h.19 On the basis of the assumption that the fluorescent molecules diffused according to Brownian motion, this technique allowed us to prove the existence of two populations of PLL chains: one population that appears immobile and another population that can diffuse within the multilayer with a diffusion coefficient of the order of 0.2 µm2 · s-1. However, this technique is not very sensitive and does not allow detecting different populations unless they are characterized by very different diffusion coefficients. Moreover, experimental uncertainties are important, so that it only indicates the order of magnitude of the diffusion coefficients. Diffusion (13) 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. (14) Ko¨hler, K.; Mo¨hwald, H. J. Phys. Chem. B 2006, 110, 24002–24010. (15) Benkirane-Jessel, N.; Lavalle, P.; Meyer, F.; Audouin, F.; Frisch, B.; Schaaf, P.; Ogier, J.; Decher, G.; Voegel, J.-C. AdV. Mater. 2004, 16, 1507–1511. (16) Vodouheˆ, C.; Le Guen, E.; Garza, J. M.; Francius, G.; Dejugnat, C.; Ogier, J.; Schaaf, P.; Voegel, J.-C.; Lavalle, P. Biomaterials 2006, 27, 4149– 4156. (17) Chluba, J.; Voegel, J.-C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001, 2, 800–805. (18) Mertz, D.; Hemmerle´, J.; Mutterer, J.; Ollivier, S.; Voegel, J.-C.; Schaaf, P.; Lavalle, P. Nano Lett. 2007, 7, 657–662. (19) Picart, C.; Mutterer, J.; Arntz, Y.; Voegel, J.-C.; Schaaf, P.; Senger, B. Microsc. Res. Tech. 2005, 66, 43–57.
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coefficients in thin films can be measured more accurately using fluorescence recovery after pattern photobleaching (FRAPP), in which optical fringes are employed to bleach the sample and to read the signals20 during fluorescence recovery toward a uniformly labeled substrate. This technique will allow us to (i) check that the recovery is, indeed, due to a Brownian diffusion process of the chains, (ii) detect the different populations of diffusing species that might exist, and (iii) determine their diffusion coefficients. This technique was indeed formerly used to study the diffusion coefficients of human serum albumin on and embedded in PSS/ PAH multilayers.21
Materials and Methods Chemicals. Poly(L-lysine) (PLL, Mw ) 5.89 × 104 Da) was purchased from Sigma (St. Quentin Fallavier, France). Hyaluronic acid (HA, Mw ) 4.0 × 105 Da) was obtained from BioIberica (Barcelona, Spain). Fluorescein isothiocyanate (FITC) labeled poly(Llysine) (PLLFITC, Mw ) 6.67 × 104 Da) was obtained from Sigma (St. Quentin Fallavier, France). Polyelectrolyte solutions (1 mg · mL-1) were prepared by dissolving the adequate amounts of polyelectrolytes in 0.15 M NaCl aqueous solution, pH 6.2. Buildup of Polyelectrolyte Multilayer Films. The PEM films were built up with an automatized dipping robot (Riegler & Kirstein GmbH, Potsdam, Germany) on glass slides (glass coverslips from Menzel GmbH & Co, Braunschweig, Germany). Before polyelectrolyte adsorption, the glass slides were cleaned with 0.1 M sodium dodecyl sulfate (dipping for 15 min), 0.1 M HCl (dipping for 15 min), and pure water. Then, the glass slides were first dipped in a polycation solution for 10 min. A rinsing step was performed by dipping the substrates for 10 min in 0.15 M NaCl solution (pH 6.2). The polyanions were then deposited in the same manner. The buildup process was pursued by alternated depositions of polycations and polyanions. After deposition of n bilayers, the film obtained is denoted (polycation/polyanion)n. Different films were built up for the present study: (PLL/HA)24-PLLFITC, (PLL/HA)48-PLLFITC, (PLL/ HA)24-PLLFITC-HA, (PLL/HA)24-PLLFITC-(HA/PLL)m with m ) 1, 6, or 24. Fluorescence Recovery Recording and Analysis. FRAPP was used to investigate the diffusion law of a labeled polyelectrolyte. It allows us to check the Brownian diffusion law at the micrometer scale. The experimental setup was similar to the one described by Davoust et al.,20 based on an intensity separation interferometer (Figure 1). The incident light beam of an etalon-stabilized monomode Ar laser (1 W at λ ) 488 nm) was split into two equivalent beams that crossed in the sample cell, providing an interference fringe pattern. The fringe spacing, i, is set by the crossing angle, θ, between the two beams: i ) λ/2 sin(θ/2). It defines the characteristic wave vector, q, of the fringe pattern: q ) 2π/i.20 Fluorescence bleaching of the labeled species in the illuminated fringes was obtained by producing a short (
