Hyaluronic Acid Films onto a

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Langmuir 2001, 17, 7414-7424

Buildup Mechanism for Poly(L-lysine)/Hyaluronic Acid Films onto a Solid Surface C. Picart,*,†,| Ph. Lavalle,† P. Hubert,‡ F. J. G. Cuisinier,† G. Decher,§ P. Schaaf,§,| and J.-C. Voegel† INSERM Unite´ 424, Fe´ de´ ration de Recherches “Odontologie”, Universite´ Louis Pasteur, 11 rue Humann, F-67085 Strasbourg Cedex, France; UMR 7568 CNRS-INPL and FR CNRS W0070, ENSIC-INPL, BP 451, F-54 001 Nancy, France; Institut Charles Sadron (CNRS-ULP), 6 rue Boussingault, F-67083 Strasbourg Cedex, France; and Ecole Europe´ enne de Chimie, Polyme` res et Mate´ riaux de Strasbourg, 25, rue Becquerel, F-67087 Strasbourg Cedex 2, France Received June 6, 2001. In Final Form: August 3, 2001 The formation of a new kind of biocompatible film based on poly(L-lysine) and hyaluronic acid (PLL/HA) by alternate deposition of PLL and HA was investigated. Optical waveguide lightmode spectroscopy, streaming potential measurements, atomic force microscopy, and quartz crystal microbalance (QCM) were used to analyze the different aspects of the buildup process such as the deposited mass after each new polyelectrolyte adsorption, the overall surface charge of the film, and its morphology. As for “conventional” polyelectrolyte multilayer systems, the driving force of the buildup process is the alternate overcompensation of the surface charge after each PLL and HA deposition. The construction of (PLL/HA)n films takes place over two buildup regimes. The first one is characterized by the formation of isolated islands that grow both by addition of new polyelectrolytes on their top and by mutual coalescence of the islands. The second regime sets in once a continuous film is formed after the eighth layer pair deposition in our working conditions and is characterized by an exponential increase of the mass. QCM measurements at different frequencies evidenced a viscoelastic behavior of the films with a shear viscosity on the order of 0.1 Pa‚s. This new kind of biocompatible film incorporating a natural polymer of the cartilage and a widely used polypeptide is of potential use for cell-targeted action.

Introduction The modification of biomaterial surfaces has become a major challenge over the past decade in particular to improve biocompatibility and to prepare surfaces that can resist or enhance cell adhesion by mimicking extracellular matrix components.1-3 Among the techniques enabling a high degree of control of both chemistry and spatial order on the surface, self-assembled monolayers (SAMs) and Langmuir-Blodgett techniques have been the most commonly used.4,5 More recently, the alternate deposition of polyanions and polycations has been proposed as a novel method for the buildup of multilayered polyelectrolyte films.6-8 This approach allows subnanometer control of positioning a large variety of materials in hybrid multilayers assemblies.9,10 In addition to integrating different nanosized objects (including a large number of polyelectrolytes, DNA, proteins, or colloids)11-13 in these ultrathin †

Universite´ Louis Pasteur. UMR 7568 CNRS-INPL and FR CNRS W0070, ENSIC-INPL. § Institut Charles Sadron (CNRS-ULP). | Ecole Europe ´ enne de Chimie, Polyme`res et Mate´riaux de Strasbourg. ‡

(1) Dillow, A. K.; Tirrell, M. Curr. Opin. Solid State Mater. Sci. 1998, 3, 252-259. (2) Healy, K. Curr. Opin. Solid State Mater. Sci. 1999, 4, 381-387. (3) Hubbell, J. Curr. Opin. Biotechnol. 1999, 10, 123-129. (4) Mrksich, M. Curr. Opin. Colloid Interface Sci. 1997, 2, 83-88. (5) Lo¨sche, M. Curr. Opin. Solid State Mater. Sci. 1997, 2, 546-556. (6) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831-835. (7) Decher, G. Science 1997. 277, 1232-1237. (8) Knoll, W. Curr. Opin. Colloid Interface Sci. 1996, 1, 137-143. (9) Hammond, P. T. Curr. Opin. Colloid Interface Sci. 2000, 4, 430442. (10) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid. Comm. 2000, 21, 319-348. (11) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995. 117, 6117-6123.

films in a programmed sequence, one can further engineer film properties by, for example, adjusting the ionic strength of the buffer in which the multilayers are built14 or by varying other deposition parameters such as time of adsorption, concentration of adsorbates, or intermediate drying. Last but not least, the technique is compatible with industrial production methods, and the first mass product containing this technology is expected to reach the market very soon. Up to now, the multilayer technique has been mainly applied to different types of synthetic polyelectrolyte systems. Several techniques have been used to investigate their buildup, including X-ray reflectometry, ellipsometry, and quartz microbalance.6,15,16 A linear increase in the thickness of the assembly with the addition of layers is usually observed for systems poly(styrene-sulfonate)/ poly(allylamine).17,16 One also finds a long-range order and stability of these films, although the layers highly interpenetrate at short range. Recently, the multilayer technique has also been applied to the natural biopolymer alginate and biocompatible poly(L-lysine)/alginate multilayers were constructed in order to build a nonadhesive barrier deposited on an adhesive collagen film.18 Measurements of the thickness of the dried film by ellipsometry (12) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427-3433. (13) Cassier, T.; Lowack, K.; Decher, G. Supramol. Sci. 1998, 5, 309315. (14) Ladam, G.; Schaad, P.; Voegel, J. C.; Schaaf, P.; Decher, G.; Cuisinier, F. J. G. Langmuir 2000, 16, 1249-1255. (15) Caruso, F.; Rinia, H. A.; Furlong, D. N. Langmuir 1996, 12, 2145-2152. (16) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422-3426. (17) Ramsden, J. J.; Lvov, Y. M.; Decher, G. Thin Solid Films 1995, 254, 246-251. (18) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355-5362.

10.1021/la010848g CCC: $20.00 © 2001 American Chemical Society Published on Web 10/12/2001

Poly(L-lysine)/Hyaluronic Acid Films

indicated that the deposited amount of polyelectrolytes growths exponentially with the number of deposited layers. These results are an example of overshooting layer growth which is not as frequently observed as quasi-linear growth.6,7 To explain these results, Elbert et al. evoked the possibility of the formation of a poly(L-lysine)/alginate complex coacervate, a kind of gel formed on the surface during the successive depositions.18 Another polysaccharide of great biological interest is hyaluronic acid (HA). HA is a linear polysaccharide chain with average molar masses ranging from about 105 to 107 Da that is found in the extracellular (ECM) matrix of mammalian connective tissues as sodium hyaluronate. This natural polyacid is the only nonsulfated glycosaminoglycan (GAG) and its primary structure consists of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine with β (1f4) interglycosidic linkage. HA participates in a hydrated network with collagen fibers (e.g., vitreous humor) in the ECM, where it acts as an organizing core. It also constitutes the structural backbone of the cartilage proteoglycans assembly. The physical properties and functions of HA are based on its ability to form viscoelastic aqueous solutions,19 the rheological behavior of which depends on the shear stress.20 It exerts lubrication functions in joints and is responsible for the viscoelasticity of the joint synovial fluid and eye vitreous humor,21 thus making it an ideal candidate for use in optical surgery and as a viscosupplementation agent in joint diseases.22 HA is known to be highly hydrophilic and is able to bind large amounts of water.23 Therefore, it participates in the control of tissue hydration and water transport, and due to these hydrating properties, it is also used in cosmetic formulation.19 To get a better insight into its hydration properties, X-ray diffraction studies,24-26 NMR experiments,27 fluorescence recovery experiments,28 and molecular simulations have been performed on its constituting disaccharide,29,30 trisaccharide, and tetrasaccharide forms.31 Several conformations were found indicating the presence of hydrogen bonds involving the ring oxygen and connecting the consecutive residues. Intrinsic viscosity measurements indicated that, in aqueous salt solutions, sodium hyaluronate behaves as a semirigid polymer.32 This stiffness has been proposed to arise partly from the presence of an extended network of intramolecular (19) Laurent, T. C. The chemistry, biology, and medical applications of hyaluronan and its derivatives; Wennner-gren international series; Cambridge University Press: Cambridge, U.K., 1998; Vol. 72. (20) Fouissac, E.; Milas, M.; Rinaudo, M. Macromolecules 1993, 26, 6945-6952. (21) Scott, D.; Coleman, P. J.; Mason, R. M.; Levick, J. R. Microvasc. Res. 2000, 59, 345-353. (22) Lapcik, L.; Lapcik, L.; De Smedt, J.; Demeester, J. Chem. Rev. 1998, 98, 2664-2684. (23) Whitson, K. B.; Lukan, A. M.; Marlowe, R. L.; Lee, S. A.; Anthony, L.; Rupprecht, A. Phys. Rev. E. 1998, 58, 2370-2377. (24) Atkins, E. D.; Sheehan, J. K. Science 1973, 179, 562-564. (25) Sheehan, J. K.; Atkins, E. D.; Nieduszynski, I. A. J. Mol. Biol. 1975, 91, 153-163. (26) Sheehan, J. K.; Atkins, E. D. Int. J. Biol. Macromol. 1983, 5, 215-221. (27) Scott, J. E.; Heatley, F. Proc. Natl. Acad. Sci. U.S.A.. 1999, 96, 4850-4855. (28) Gribbon, P.; Heng, B. C.; Hardingham, T. E. Biophys. J. 1999, 77, 2210-2216. (29) Scott, J. E.; Cummings, C.; Brass, A.; Chen, Y. Biochem. J. 1991, 274, 699-705. (30) Almond, A.; Brass, A.; Sheehan, J. K. J. Mol. Biol. 1988, 284, 1425-1437. (31) Haxaire, K.; Braccini, I.; Milas, M.; Rinaudo, M.; Perez, S. Glycobiology 2000, 10, 587-594. (32) Morris, E. R.; Rees, D. A.; Welsh, E. J. J. Mol. Biol. 1980, 138, 383-400.

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hydrogen bonds.33 Individual extended HA chains that self-interact have been visualized by atomic force microscopy for very dilute solutions.34 HA offers also the potentiality to be chemically modified in order to prepare cell-targeted polymeric prodrugs35 or even to be conjugated with PLL (poly(L-lysine)) for use as a DNA carrier.36,37 Alginate/HA associations have recently been proposed38 in order to prepare efficient biomaterials that combine the gel forming properties of alginate and the healing capacities of HA. Using HA of different molecular weights allowed a control of the size of the cavities formed in these porous gels. The presence of such cavities seems to favor cell invasion and bioactive substances entrapment. HA, in addition to be biocompatible is also nonimmunogenic and is involved in numerous biological processes such as cell adhesion migration and proliferation, both in the ECM and within the cell.39 It is also a ligand for different cell types such as chondrocytes through the CD44 and RHAMM receptors.40,39 HA appears thus to be an excellent candidate for incorporation into bioactive surface films aimed at improving compatibility and/or performance of medical devices in contact with tissue favoring tissue repair, cell specific adhesion, or drug delivery. The aim of the present work is to investigate the buildup of PLL/HA multilayers films obtained by alternate adsorption of cationic PLL and anionic HA. The counterpolycation PLL was chosen not only because it is biocompatible, but also because it offers the possibility of being easily conjugated with bioactive molecules.41,37 The adsorption process was monitored stepwise using several complementary in situ techniques: optical waveguide lightmode spectroscopy (OWLS), ζ potential measurements, atomic force microscopy (AFM), and dissipation enhanced quartz crystal microbalance (QCM-D). Because of the unique properties of both polyions (PLL and HA), multilayer films composed of these substances are expected to be interesting substrates for cell growth of numerous types of cell, such as chondrocytes or fibroblasts, HA being a natural and specific ligand. Another potential application lies in the facile incorporation of bioactive substances coupled to HA or PLL multilayer films designed for cell-targeted action. Materials and Methods Polyelectrolyte Solutions. HA samples in the sodium hyaluronate form were prepared by submitting high molecular weight parent HA (Acros) to controlled acidic hydrolysis (20 min, 4 °C in ethanol 70%/HCl) to afford samples of respective weightaverage molecular weights 2.4 × 105 Da (HCl 3 N) and 1.5 × 105 Da (HCl 12 N). Average molecular weights were determined by size exlusion chromatography-multi angle laser light scattering (SEC-MALLS). SEC was performed using a Waters HPLC pump with a serial set of SP 806, 805, and 804 OH Pack columns and SBP OH Pack as the guard column (Shodex, MerckEurolab, (33) Atkins, E. D.; Meader, D.; Scott, J. E. Int. J. Biol. Macromol. 1980, 2, 318-319. (34) Cowman, M.; Li, M.; Balazs, E. A. Biophys. J. 1998, 75, 20302037. (35) Luo, Y.; Ziebell, R.; Prestwich, G. D. Biomacromolecules 2000, 1, 208-218. (36) Asayama, S.; Nogowa, M.; Takei, Y.; Akaike, Y.; Maruyama, A. Bioconjugate Chem. 1998, 9, 476-481. (37) Takei, Y.; Maruyama, A.; Kawano, S.; Nishimura, Y.; Asayama, S.; Nogawa, M.; Ikejima, K.; Hori, M.; Akaike, T.; Lemasters, J. J.; Watanabe, S.; Sato, N. Transplant. Proc. 1999, 31, 790-791. (38) Oerther, S.; Maurin, A.-C.; Payan, E.; Hubert, P.; Lapicque, F.; Presle, N.; Dexheimer, J.; Netter, P.; Lapicque, F. Biopolymers 2000, 54, 273-281. (39) Lee, J. Y.; Spicer, A. Curr. Opin. Cell Biol. 2000, 12, 581-586. (40) Ishida, O.; Tanaka, Y.; Morimoto, I.; Takigawa, M.; S. Eto, J. Bone Mineral. Res. 1997, 12, 1657-1663. (41) Mezo, G.; Katjar, R.; Barna, K.; Gaal, D.; Hudecz, F. J. Controlled Release 2000, 63, 81-95.

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France). Elution (0.7 mL/min) was monitored both by MALLS detection (Mini Dawn, Wyatt) and differential refractometry (Waters 410, Waters). HA is a polyanionic (1 carboxylic acid group/ disaccharide unit) with a pKa ≈ 2.922 and hence a net charge of -1 at physiological pH. Tris (hydroxymethylaminomethane) (Tris), sodium dodecyl sulfate (SDS), and poly(L-lysine) (PLL) (3.26 × 104 Da) were purchased from Sigma (St Quentin Fallavier, France). PLL is a polycation (1 amino group/monomer repeat unit) in physiological conditions with a pKa ≈ 9.42 Sodium chloride (purity 99.5%) was obtained from Fluka (St Quentin Fallavier, France). All solutions were prepared using Ultrapure water (Milli Q-plus system, Millipore) with a resistivity of 18.2 MΩ.cm. Polyelectrolyte solutions were always prepared by direct dissolution of adequate weights of HA and PLL in 0.15 M NaCl, i.e., in a medium close to physiological conditions and containing only 2 types of ions (Na+ and Cl-) since the behavior of HA in solution is known to depend on its counterions.43 For streaming potential experiments, as the experimental device is extremely sensitive to very slight pH changes, polymer solutions were prepared in a Tris-HCl buffer (0.5 mM Tris, 0.15 M NaCl, pH 7.3). HA solutions (1 mg/mL) were in the dilute regime, as evidenced by a rapid calculation of its overlap concentration, C* ) 8 mg/mL (C* ) 3.8/[η], with intrinsic viscosity [η] ) 0.4 mL/g for HA ) 1.5 × 105 Da in 0.1 M NaCl, as reported by Fouissac et al.20). PLL solutions were prepared at two concentrations: 20 and 1 mg/mL. The pH of the water and of the prepared PLL and HA solutions was always in the range of 6.0-6.5. With regard to the respective pKa’s, the polyelectrolytes are fully dissociated at these pHs. Optical Waveguide Lightmode Spectroscopy. Optical waveguide lightmode spectroscopy (OWLS) has been extensively described44,45 and will only be briefly summarized here. The principle of OWLS is to incouple two waves (TE and TM) of a laser into a planar waveguide through an input grating coupler. The diffraction grating is incorporated in a planar optical waveguide of high refractive index (nF ≈ 1.77). To detect the refracted waves, the waveguide is mounted on a goniometer allowing to vary the incidence angle θi between the laser beam and the grating normal. At certain discrete values of the incidence angles θi the beam diffracted from the grating matches a possible guided mode (TE or TM) and incoupling takes place. To each of these incoupling angles there corresponds an effective refractive index N given by44

N ) sin θi + {lλ}/{Λ}

(1)

where l represents the diffraction order, λ corresponds to the wavelength of the light and Λ is the characteristic diffraction grating spacing; under our experimental conditions, l ) 1, λ ) 632.8 nm, and 1/Λ ) 2400 lines/mm. In our case, there is one incoupling angle for the TE (respectively TM) wave and the corresponding effective refractive index will be denoted by NTE (respectively NTM). The power coupled into the waveguide is measured with a photodiode situated on both ends of the waveguide. NTE and NTM are the solutions of the phase equation for both the transverse electric (TE) and the transverse magnetic (TM) polarizations:

2kz,FdF + ΦS,F + ΦF,A ) 2πm

(2)

where m is the order of the guide, all the quantities entering in this equation being functions of N. All the guides used in our experiments are monomode guides so that m can only take the value 0. ΦS,F and ΦF,A represent the phase shifts between the incident and the reflected beams relative to the substrate/ waveguide (S, F) interface and to the waveguide/film (F, A) interface, respectively. kz,F (kz,F ) k0(nF2 - N2)1/2) represents the (42) Fasman, G. D. Handbook of Biochemistry and Molecular Biology; CRC Press: Boca Raton, FL, 1976. (43) Lee, S. A.; Vansteenberg, M. L.; Lavalle, N.; Rupprecht, A.; Song, Z. Biophys. J. 1994, 24, 1543-1552. (44) Tiefenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B. 1989, 6, 209220. (45) Ramsden, J. J. J. Mol. Recogn. 1997, 10, 109-120.

component perpendicular to the interface of the wavevector in the guide. The values of NTE and NTM depend on the refractive index profile of the film deposited on the oxide layer. The method used for calculating these phase shifts is given in appendix A in ref 46. The presence of an adlayer deposited on the waveguide perturbs the evanescent field of the guided modes and leads to a highly sensitive change of the effective refractive indices NTE and NTM. Qualitatively, an increase of NTE and NTM corresponds to an increase of the optical mass of the adlayer. All experiments were performed on a home-built experimental setup with a He-Ne laser using ASI2400 waveguides made from Si0.8Ti0.2O2 (Artificial Sensing Instruments, Zu¨rich, Switzerland). The incidence angle θi is varied over a range of (7 deg with a precision of 3 × 10-5 degree by means of a translation stepped motor (Microcontroˆle, Evry, France). Each experiment is preceded by a cleaning procedure of the waveguides, first in a 0.01 M sodium dodecyl sulfate (SDS) solution for 15 min followed by 15 min in 0.1 N HCl, both in a boiling water bath. This cleaning procedure is followed by an extensive water rinse and drying of the guide under a stream of N2. The waveguide is introduced in its holder and connected to a three-hole sealed cover (injection port, buffer entrance port, and buffer exit port tubes). The measuring cell is tightly sealed to the chip by a circular perfluorinated O-ring (“Kalrez”, Dupont, Wilmington, DE) and has an internal volume of 37 µL. All the experiments were performed at a temperature of 24.5 ( 0.2 °C. Buffer is flushed through the cell at a constant flow rate (10 mL/h) with a syringe pusher. Measurement in buffer continues until constant values of the incoupling angles are reached (less that 10-5 absolute variation on the values of NTE and NTM). These values are used to calculate the waveguide refractive index nF and its thickness dF with the three layers mode equation,44 knowing the buffer refractive index (nC) which is measured for each buffer solution with an RFM 340 refractometer (Bellingham-Stanley, Kent, U.K.). The buildup of the (PLL/HA) pairs of layer is performed as follows: After the buffer flow is stopped, 100 µL of the PLL solution is manually injected in the cell through the injection port. NTE and NTM values increase and reach a plateau after about 10 min. When a stable adsorption signal is obtained, the buffer flow is restarted for about 15 min to rinse the excess material from the cell. The acquisition of one single data point takes approximately 100 s. In the same way, we continue with the alternate adsorption of HA and PLL on the waveguide. Thus, progressively PLL/HA, (PLL/HA)2, ..., (PLL/HA)n structures are deposited. After the nth (PLL/HA) deposition step, the film will be denoted as (PLL/HA)n. As will be discussed later on, in the case of HA paired with PLL, the deposition sequence may not conform to the actual film structure. Streaming Potential Measurements. Streaming potential measurements were carried out in order to determine the ζ potential of the (PLL/HA) multilayers. The experiments were performed on a homemade apparatus developed by Zembala and De´jardin.47 It is composed by a 20 cm long electrophoresis capillary made out of a fused silica tube with a radius of 530 ( 12 µm (SIN 2042-R10, Perichrom SARL, Saulx-les-Chartreux, France), connected to two flasks on each side of the capillary. Each flask contains an Ag/AgCl electrode used as a streaming potential sensor. The two electrodes are linked together through a voltmeter with a great internal impedance (Keithley 617 programmable electrometer). A pressure sensor calibrated in cm of water is also connected to both flasks. The pressure sensor and the two electrodes provide respectively the pressure and potential differences on both sides of the capillary. The streaming potential is due to the flux of the buffer solution through the capillary by applying an elevated N2 pressure to a flask directly connected to the capillary. In a typical experiment, the water pressure rises up to 200 cm of water. The pressure difference, the flow rate through the capillary, and the streaming potential are directly recorded on a microcomputer. The ζ potential is related to the (46) Picart, C.; Ladam, G.; Senger, B.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G.; Gergely, C. J. Chem. Phys. 2001, 115, 1086-1094. (47) Zembala, M.; De´jardin, P. Colloid Surf. B 1994, 3, 119-129.


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pressure difference ∆P and to the streaming potential ∆V (difference in the potential measured on the two Ag/AgCl electrodes) by the Smoluchovski relation:48

0πRcr2ζσ Lη

∆V ) ∆P

(3)

in which 0 is the permittivity of water (7.0 × 10-10 F/m at 20 °C), η represents the dynamic water viscosity (1.002 × 10-3 Pa‚s at 20 °C), L is the length of the capillary and r its radius, ζ is the zeta potential of the capillary surface, and Rc is the electrical resistance of the bare capillary filled with the buffer solution. In our experiments this resistance is typically of the order of 180 MΩ and is determined for each capillary. The experiments are realized as follows: (i) First, the capillary was filled and rinsed several times with water and then with the Tris-HCl buffer. This buffer is then maintened in the capillary for a whole night’s equilibrium. (ii) The streaming potential of the bare capillary filled with Tris-HCl buffer is measured. (iii) Then, 8 mL of the PLL solution is injected through the capillary with a syringe (1 mg/mL in 0.5 mM Tris containing 0.15 M NaCl). The solution is then kept in the capillary for 20 min in the absence of flow. This allows the PLL to adsorb on the capillary walls during this period of time. The capillary is then extensively rinsed and equilibrated by injection of 50 mL of TrisHCl buffer. The streaming potential is subsequently measured. (iii) HA (2.4 × 105 Da) is then brought in contact with the capillary in a similar way by using a HA solution (1 mg/mL in 0.5 mM Tris containing 0.15 M NaCl), and the streaming potential is measured by following the same procedure as described in step ii. (iv) PLL and HA adsorptions are performed in a similar way, and after each deposition step, the streaming potential is measured. As an adsorption step followed by rinsing and measurement takes almost 1 h, the experiment is performed over 3 days. Therefore, three freshly prepared solutions of HA and PLL are used. All the streaming potentials are determined in the same Tris-HCl buffer solution. Atomic Force Microscopy. Here, 12 mm diameter glass slides (Polylabo, Strasbourg, France) are used and cleaned with 0.01 M SDS and 0.1 N HCl and extensively rinsed with pure water and dried under nitrogen. A slide is glued onto a magnetic holder and is then deposited on the magnetic piezoelectric tube. A drop of 200 µL of a PLL solution (at 1 mg/mL in NaCl 0.15 M) is directly deposited over the slide and left for 15 min. The rinsing is achieved with 600 µL of 0.15 M NaCl solution. HA (MW 1.5 × 105 Da at 1 mg/mL in NaCl 0.15 M) is then deposited in the same manner. The surface is observed at different steps during the buildup, until the 10th PLL/HA layer pair. The AFM measurements are performed using a Nanoscope III (Digital Instruments, Santa Barbara, CA) equipped with silicon nitride cantilevers (model MLCT-AUHW Park Scientific, Sunnyvale, CA) having a spring constant of 0.03 N/m. The AFM is operated in constant force contact mode in liquid for each sample. Several scans are imaged over a given surface area in order to ascertain that there is no sample damage induced by the tip. Deflection and height mode images are scanned simultaneously at a fixed scan rate (between 2 and 4 Hz) with a resolution of 512 × 512 pixels. Quartz Crystal Microbalance. The measurements were performed using the dissipation enhanced QCM-D (Quartz Crystal Microbalance) system from Q-Sense (Go¨tenborg, Sweden). The QCM-D technique has been extensively described in details49 and will only briefly be summarized. It consists of measuring the changes in the resonance frequency f of a quartz crystal when material is adsorbed from solution.50 An AT-cut piezoelectric crystal coated on its two faces with gold electrodes is excited at its fundamental frequency (about 5 MHz), and observation takes place at the third, fifth and seventh overtones (corresponding to (48) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: New York, 1981. (49) Rodahl, M.; Kasemo, B. Sensors. Actuat. A 1996, 54, 448-456. (50) Ho¨o¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729-734.

15, 25, and 35 MHz, respectively). When a thin and rigid film of mass ∆m is attached to the electrodes, the resulting decrease ∆f in frequency is related to ∆m according to the Sauerbrey equation51

∆m ) -C∆f/ν

(4)

where ν is the overtone number and C the mass sensitivity constant (for the QCM-D system C≈17.7 ng‚cm-2‚Hz-1 at f ) 5 MHz in air) which depends on the properties of the crystal used. This equation holds for measurements in air. In solution, the density, the viscosity of the liquid and the viscoelastic properties of the film influence the frequency shift ∆f.15 The QCM-D instrument and data processing software allow one to simultaneously determine mass and viscoelastic properties of an adsorbed film. However, a decrease in the resonance frequency is usually associated, in a first approximation, to an increase of the mass coupled to the quartz and our discussion will remain essentially on a qualitative level. The adsorbing surface (electrodes) consists of an evaporated gold film. Before use, a cleaning process was applied by dipping the surface in a solution of 0.01 M SDS for 10 min followed by an extensive rinse with water. Using a similar protocol as for the other techniques, we first inject 0.5 mL of a 0.15 M NaCl solution into the measurement cell. After stabilization of the signals, 0.5 mL of a PLL solution at 1 mg/mL in 0.15 M NaCl is injected, left for 10 min and rinsed for 10 min with 0.15 M NaCl solution. During these time periods, the shifts in ∆f are continuously recorded. The same procedure is then used for the deposition of HA by introducing 0.5 mL of a HA solution (1.5 × 105 Da) at a concentration 1 mg/mL in 0.15 M NaCl. The buildup process is then continued by the alternated PLL and HA addition up to the 12th pair of layers. The signal is recorded every 12 s. Temperature of the solutions is stabilized at 22 ( 0.05 °C.

Results OWLS Measurements. The stepwise adsorption process was monitored by OWLS. A typical raw NTM signal obtained during the successive deposition of PLL (3.26 × 104 Da at 20 mg/mL) and HA (1.5 × 105 Da at 1 mg/mL) on a Si0.8Ti0.2O2 surface is given in Figure 1A. A similar evolution takes place for the effective refractive index NTE (data not shown). First, one observes, as expected, that the effective refractive index increases after each new PLL and HA deposition up to the sixth (PLL/HA) layer pair. During the rinsing steps, the signal remains almost stable when the outer layer is constituted of HA and decreases slightly for PLL. Then a second buildup regime sets in. It is characterized by a similar behavior for HA but by a decrease of the effective refractive indexes when PLL is added, followed by an index increase during rinsing. One can notice that after the rinsing of the PLL solution, the signal remains smaller than at the end of HA rinsing step. This indicates that, in the second regime, the total optical mass detected by OWLS decreases after each PLL addition. Moreover, after the deposition of the eighth layer pair the process becomes fully cyclic: NTE and NTM take similar values after each PLL addition, PLL rinsing, HA addition, and HA rinsing. By using a lower PLL concentration (1 mg/mL), one finds a comparable behavior with two characteristic regimes. This can be seen in Figure 1B where the evolution of NTM is represented during the film buildup process. A similar evolution is again found for NTE (data not shown). Although the shape of the curve is qualitatively the same as that found when the higher PLL concentration was used (Figure 1A), the transition between the two regimes appears smoother with still the gradual establishment of a cyclic evolution. The amplitude of this cyclic regime is also weaker. These signals were analyzed within the homogeneous and isotropic monolayer model.46 We found for the first PLL layer a thickness of 2.3 nm and an adsorbed amount

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Figure 2. Evolution of the ζ potential (mV) during the alternate deposition of PLL and HA layers as a function of the number of deposition cycles. PLL (MW 3.26 × 104 Da) and HA (MW 2.4 × 105 Da) were prepared at 1 mg/mL in a 0.5 mM Tris-HCl buffer (pH ) 7.4) containing 0.15 M NaCl. Values corresponding to PLL adsorption (O) and HA adsorption (9) are shown for each (PLL/HA) deposition cycle.

Figure 1. Raw NTM signal obtained during the alternate deposition of PLL and HA layers and measured by the OWLS technique, as a function of the time. PLL (MW 3.26 × 104 Da) and HA (MW 1.5 × 105 Da) were dissolved in 0.15 M NaCl at the following concentrations (A) PLL at 20 mg/mL and HA at 1 mg/mL and (B) PLL at 1 mg/mL and HA at 1 mg/mL. The alternating deposition was performed for 15 min adsorption and 15 min rinsing steps. The baselines (used for the calculation of the waveguide parameters) and the successive rinsing of the layers were achieved under flowing buffer at 10 mL/H, T ) 24.5 °C. Symbols indicate the value reached at the end of the (b) PLL deposition step, (O) PLL rinsing step with 0.15 M NaCl, (9) HA deposition step, and (0) HA rinsing step with 0.15 M NaCl. The number of cycles is also indicated on the figure.

of 0.25 µg/cm2. For a great number of experiments, especially for those performed at a PLL concentration of 20 mg/mL, neither the thickness nor the mass could be determined with the isotropic model over the whole buildup process. The solution of the equations gave negative values for the layer thickness. In the case where thicknesses could be determined after the deposition of 8-10 layers, their values were on the order of 100-200 nm. A rapid evaluation of the penetration depth corresponding to the characteristic decay length of the electromagnetic field gives ∆z ≈ 135-155 nm (according to Lukosz,52 ∆z ≈ 1/([ k0 (N2 - na2)1/2 ] with 1/k0 ) 100.66 nm for the He-Ne laser, nA≈ 1.4 is the refractive index of the adsorbed adlayer, N ≈ 1.55-1.59 for TM and TE waves respectively). Thus, the estimated thickness of the adsorbed multilayer is comparable to or even higher than the penetration depth of the evanescent wave propagating along the guide. This suggests that the film thickness might even be larger than ≈ 150 nm. In such a case, the optical signal variations would only be representative of (51) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (52) Lukosz, W. Biosens. Bioelectron. 1991, 6, 215-225.

the part of the film that is close to the waveguide surface and not to the whole film. Streaming Potential Data. The streaming potential technique was employed in order to determine the ζ potential of the film after each new polyelectrolyte deposition from a Tris-HCl buffer (containing 0.15 M NaCl) The evolution of the multilayer ζ potential during the buildup process is presented in Figure 2. The ζ potential of the bare capillary surface was approximately -100 mV under our conditions, which is in agreement with values currently reported in the literature for silica.47 After the deposition of the first PLL layer, the surface becomes positively charged (+70 mV). The deposition of the first HA layer again changes the ζ potential to a negative value (-55 mV). A charge reversal is observed after each new PLL and HA adsorption. This indicates that each newly deposited layer leads to an overcompensation of the previous charge excess as has been observed for other polyanion/polycation couples before.14,53,54 One can also notice that after the deposition of the ninth HA layer, the amplitudes of the ζ potential decrease and stabilize at about +50 mV for multilayers terminating with PLL and at about -30 mV for multilayers terminating with HA. It is important to note that there is no indication of the ζ potential going to zero, it continues to alternate after each new polyelectrolyte deposition although with slightly reduced amplitudes. AFM Investigations. The layer-by-layer deposition was also followed with the AFM until the buildup of the 10th layer pair. PLL deposited on a clean glass slide appears homogeneously distributed and exhibits a low surface roughness (RMS ≈ 0.35 nm) (Figure 3A). The visible dots whose lateral dimensions are on the order of 50 nm may correspond to individual molecules,55 although tip effects may lead to a certain overestimation of the sizes. The images do not allow concluding whether PLL is also adsorbed on the silica surface between the dots or if this corresponds to bare silica. The topography becomes completely different after the first HA deposition giving a (PLL/HA) film architecture (Figure 4A). Two kinds of structures are visible: large objects having typical sizes on the order of a few micrometers that we will call “islands” and smaller ones (53) Hoogeven, N. G.; Cohen Stuart, M. A.; Fleer, G.; Bo¨hmer, M. R. Langmuir 1996, 15, 3675-3681. (54) Caruso, F.; Mo¨hwald, H. J. Am. Chem. Soc. 1999, 121, 60396046. (55) Akari, S.; Schrepp, W.; Horn, D. Langmuir 1996, 12, 857-860.


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Figure 3. AFM image (height mode) obtained after deposition on a bare silica surface (A) of PLL (MW ) 3.26 × 104 Da) and (B) of HA (1.5 × 105 Da). For both images, the Z scale is 2 nm and image dimensions are 5 × 5 µm2. The height of the white dots is at least 2 nm.

“islets” whose characteristic size is on the order of 1 µm or even smaller. Even at this early stage of film deposition the height of individual islands is on the order of 1 µm. It is unlikely that the observed islets are composed of single HA molecules since the radius of gyration of HA was found to be on the order of 50 nm in 0.15 M NaCl aqueous solutions.56 Also, these islets and islands cannot be attributed to HA self-aggregates already formed in solution since images of HA deposited directly on a bare silica surface exhibit small globular structures of one hundred nanometers (Figure 3B). The islets and islands most probably result from the interaction of PLL with HA which may well be able to form complex coacervates on the surface, as was suggested for a somewhat related PLL/ alginate system,18 although no direct structural analysis was performed on this latter system. After the deposition of four layers (multilayer architecture (PLL/HA)2 (Figure 4B)), one notices that the structures have coalesced and formed larger sized islands whose heights remain nevertheless close to 1 µm. Moreover as for the (PLL/HA)1 case, many small islets cover the surface between the large ones. After the third and fourth PLL and HA additions, leading to (PLL/HA)4 films, the sizes of the islands increase whereas the smaller droplets become more rare (Figure 4C). One can notice that the PLL/HA multilayer buildup process seems to generate structures with shapes qualitatively resembling to those of so-called “breath figures”, which are observed during a continuous condensation of liquid on a surface from a supersaturated vapor.57 By looking at the time dependence on the buildup process, we found that the structures do not evolve as a function of the deposition time after a 15 min adsorption period, contrary to what was suggested for another polyelectrolyte system.58 Some images were even performed after a night left in the sample holder of the AFM without noticeable changes. From the sixth to the eighth (PLL/HA) layer pairs (Figure 4, parts D and E), neither individual islands nor islets are visible any more. All these structures seem to have coalesced, leading to the formation of an almost uniform film. This is even more noticeable after the deposition of the next PLL layer (film architecture (PLL/HA)8-PLL) (Figure 5F) where the surface becomes really homogeneous and flat (RMS ≈ 10 (56) Fouissac, E.; Milas, M.; Rinaudo, M.; Borsali, R. Macromolecules 1992, 25, 5613-5617. (57) Fritter, D.; Beysens, D.; Knobler, C. M. Phys. Rev. 1991, A43, 2858-2869. (58) Tsukruk, V. V.; Bliznyuk, V. N.; Visser, D.; Campbell, A. L.; Bunning, T. J.; Adams, W. W. Macromolecules. 1997, 30, 6615-6625.

nm). At this step, scanning a zone with applying a high set-point value allowed us to scratch the film. The material is partially displaced from the scratched zone toward the edges of the damaged area. Imaging after a zoom-out gives the indication that the film thickness remains to be about 850 nm (Figure 4G). Moreover, the smooth rounded shapes of the edges further indicate that the multilayer does not behave as a hard but rather a viscoelastic gellike material. One can also point out that the transition from “breath figure” type surfaces to continuous films takes place between the deposition of the sixth and the eighth (PLL/ HA) layer pairs. Therefore, the transition appears at the same buildup steps where one also observes changes in the evolution of the OWLS signals and just before a decrease of the ζ potential of the HA ending multilayers is noticeable. After the coalescence to homogeneous and flat film, additional deposition cycles lead to the presence of very small dots for films terminating with PLL (Figure 4G, near the scratched zone), and of larger dots for films terminating with HA (Figure 4H). Further imaging failed because the tip starts to become strongly attracted or repelled from the surface. This may result from higher local charge density fluctuations present on the (PLL/ HA)10 film than for the initial layers, even though the mean macroscopic charge density (ζ potential) appeared mainly constant from one deposition cycle to the following (see the section Streaming Potential). From cross-section images, it is also possible to estimate the contact angles of the islands on the surface during the buildup of the four first four layer pairs (Table 1). One can first notice that for a given film, the range of the measured angles is rather narrow ((5°) indicating that all the structures should have similar polyelectrolyte compositions and can thus be characterized by their contact angle. Moreover, the contact angles decrease as the number of deposited layers increases. This suggests that the polyelectrolyte composition of the islands evolves as the buildup process goes on. One also observes a flattening of the islands when new layers are added. As a control, the buildup of (PLL/HA) films on muscovite mica has also been investigated by AFM and similar topological evolutions have been observed. Thus, the filmforming process seems to be relatively independent of the nature of the substrate on which the film is being deposited. QCM Measurements. Figure 5A represents the evolution of the frequency shifts ∆f/ν during the film buildup

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Figure 4. AFM images obtained at the different steps of the alternate deposition of PLL and HA layers. The polyelectrolytes PLL (3.26 × 104 Da) and HA (1.5 × 105 Da) were used at a concentration 1 mg/mL in 0.15 M NaCl (same solutions as in Figure 1B). The surface is observed at different steps during the buildup process, corresponding to a given deposition cycle, until the 10th (PLL/HA) layer pair: (A) (PLL/HA)1, (B) (PLL/HA)2, (C) (PLL/HA)4, (D) (PLL/HA)6, (E) (PLL/HA)8, (F) (PLL/HA)8-PLL, and (G, right column) deflection image after a central scratch in the film (PLL/HA)8-PLL (H) (PLL/HA)10. In the left column of part G is plotted a cross section of the height image along a dashed line. Images dimensions are 50 × 50 µm2 and Z scales of height images (left column) are 1000 nm for images A-E, 300 nm for image F, and 30 nm for image H.

process corresponding to the first buildup regime up to the seventh (PLL/HA) layer pair and Figure 5B represents the evolution of ∆f/ν in the second buildup regime (layer pairs 9-12), ν being to the overtone number. Four different frequencies 5, 15, 25, and 35 MHz, corresponding to ν equal to 1, 3, 5, and 7, respectively, were recorded. In the case of films behaving as a rigid layer in which no dissipation takes place, ∆f/ν should, according to the Sauerbrey relation, be only proportional to the mass of the film and be independent of the frequency. This is, however, not the case for PLL/HA films. It should be noticed that data from the fundamental frequency (5 MHz) should be interpreted carefully due to its sensitivity to the mounting conditions of the quartz crystal in the cell.

In the first buildup regime, one observes qualitatively identical evolutions of the fundamental and the three harmonics with increasing numbers of adsorption steps. As expected, ∆f decreases with each PLL or HA injection, indicating that mass is added during each deposition step. When rinsing with buffer after PLL adsorption, an increase in ∆f/ν is observed probably due to the desorption of a small fraction of PLL, as measured with OWLS. The situation changes after the multilayer film becomes quasicontinuous, that is after the deposition of the eighth layer pair. Whereas the raw frequency signals show the same trend for each HA injection, i.e., ∆f/ν decreases for all the four investigated frequencies, each PLL addition leads to an unexpected increase of ∆f/ν at 15, 25 and 35 MHz.


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Figure 6. Increment in the QCM frequency shifts ∆(-∆f/ν) during the alternate deposition of PLL and HA layers (same polyelectrolyte solutions as for Figure 1B on gold electrodes, obtained as a function of the deposited layers at the 15 MHz frequency. Each ∆(-∆f/ν) value corresponds to the contribution of a given layer and is equal to the difference between the ∆f/ν at the beginning of the deposition step and ∆f/ν at the end of the rinsing step for HA (9) and PLL (O).

Figure 5. QCM frequency shifts (∆f/ν) during the alternate deposition of PLL and HA layers (same polyelectrolyte solutions as for Figure 1B on gold electrodes obtained at four different harmonics, (1) 5 MHz, (3) 15 MHz, (b) 25 MHz, and (O) 35 MHz, as a function of time: (A) from the first to the seventh pairs of layers; (B) from 9th to the 12th pairs of layers. The gray and black arrows correspond respectively to the beginning of the deposition of a new PLL or HA layer. The number of deposition cycles is also indicated on the figure. Only 10% of experimental data points have been represented with symbols for the sake of clarity. Table 1. Contact Angles (deg)a of the Islands on the Surface during the Alternate Deposition of the Four First Layer Pairs of PLL and HA Determined from AFM Images

(PLL/HA)1 (PLL/HA)2 (PLL/HA)3 (PLL/HA)4

contact angle

standard deviation

26.4 25.8 18.7 17.2

5.2 5.4 4.5 5.1

a These contact angles correspond to the average of at least 20 measurements.

Moreover, at these frequencies, another unexpected behavior is observed: during rinsing after PLL adsorption, ∆f/ν decreases. On the other hand, the qualitative behavior of the signal at 5 MHz after adsorption and after rinsing steps remains essentially unchanged in comparison with the first buildup regime. The effects observed at 15, 25, and 35 MHz become stronger as the buildup process progresses. When focusing on the amplitudes of the ∆f/ν variations for each (PLL/ HA) deposition step, one can also notice that the ∆f/ν decay is more pronounced for lower frequencies. For f ) 35 MHz, the absolute values of ∆f/ν no longer evolve as the deposition cycles are pursued. The existence of the two regimes shows up even more clearly when one plots the absolute increment in ∆f/ν relative to each polyelectrolyte deposition along the

Figure 7. Differences in the QCM frequency shifts -∆f/ν for each HA layer during the alternate deposition of PLL and HA (same solution as in Figure 1). (B) on gold electrodes; data are given for the four harmonics, (1) 5 MHz, (3) 15 MHz, (b) 25 MHz, and (O) 35 MHz.

buildup process. These variations, denoted as ∆(-∆f/ν), are represented in Figure 6 for the third harmonic (15 MHz) and correspond to the measurements after the rinsing step following each HA and each PLL addition. Up to the eighth layer pair, each polyelectrolyte adsorption leads to a slight increase in ∆(-∆f/ν). After (PLL/HA)9 one observes an exponential raise of ∆(-∆f/ν) relative to a HA deposition whereas a PLL deposition leads to a strong decrease, the reasons for which will be discussed further down. Finally, Figure 7 represents the evolution of -∆f/ν corresponding to each HA deposition and the consecutive rinsing step for all the harmonics. Whereas an exponentiallike raise is observed at 5 MHz and a similar trend is seen at 15 MHz although with a somewhat smaller amplitude, the signals appear to reach a plateau at 25 and 35 MHz. One can notice that these last curves resemble those obtained by OWLS, i.e., characterized by a first growth regime and a second cyclic regime. Figure 7 indicates that, after the deposition of the (PLL/HA)8 film, the buildup process still goes on. As we will see in the following section, the 5 MHz frequency is expected to reflect most closely the total mass coupled to the crystal. Therefore, our results suggest that the amount of deposited material increases in an exponential like manner with respect to the number of deposited layers as it has already been observed on the (PLL/alginate) system,18 in biotinylated PLL/streptavidine

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systems13 and in films composed of polyelectrolytes in which the charge is diluted along the main chain.59 Discussion The AFM experiments suggest that PLL adsorbs on silica by covering the whole substrate at the micrometer scale. A least some of the chains adopt coil configurations that are visible on the AFM images as small dots. It is however not clear whether a thin film of PLL covers the surface between these dots with PLL chains adopting more expected configurations with loops, trains and tails. The height of the dots ranges up to 2.5 nm and is consistent with the optical thickness found by OWLS. This layer leads to a charge reversal when compared to bare silica with a ζ potential on the order of +100 mV. When HA is brought in contact with this PLL layer, PLL/HA complexes must form. According to the AFM images, they reorganize into large sized islands and smaller islets. The islands have characteristic lateral dimensions on the order of several micrometers and a height on the order of 1 µm. The characteristic size of the islets is on the order of a few hundreds of nanometers. Because of the fact that the PLL/HA islands and islets form in the presence of the HA solution, HA adsorbs also on the surface of these structures rendering them negatively charged. This is in accordance with the charge reversal observed in the ζ potential measurements after HA deposition. One can expect that, at the end of the HA deposition process, there remain only a few polyelectrolyte molecules between the islands and islets. This allows the buildup process to continue in two ways when the surface is further brought in contact with a new PLL solution: PLL not only interacts with the negatively charged structures but also adsorbs directly on the depleted areas. When this film is again put in contact with a HA solution, new PLL/HA complexes form, giving rise to new islets and islands on the surface. These latter can coalesce with the structures already formed during the first (PLL/HA) deposition step. Such a coalescence process should lead to an increase of the characteristic sizes of the new islands as it is observed by AFM and generates again new areas depleted in polyelectrolytes. Moreover, coalescence should also restructure the material in the islands rendering them more or less homogeneous. It is then expected, in accordance with the experiments, that the islands are characterized by a well-defined contact angle with the surface. As HA also directly interacts with the positive islands already present rendering them negatively charged, the buildup process can evolve similarly with the addition of further PLL and HA. Such a process should then generate structures whose geometries qualitatively resemble the so-called “breath figures” even if differences exist with such condensation processes. Indeed, the polyelectrolytes from the solution can always interact with the surface through two different mechanisms, either by direct interaction with the silica (or PLL-covered silica areas) or by interaction with the droplets whereas breath figures are generated by a “simple” condensation process. In addition, the proportions of PLL/HA depositing on the surface by the two mechanisms are certainly different. It is thus expected that the PLL and HA composition of the droplets evolves during the buildup process. Measurements of the contact angles confirm such a continuous composition evolution toward smaller angles during the buildup process. If the contact angle would have remained constant along the buildup process, one would have (59) Kolarik, L.; Furlong, D. N.; Joy, H.; Struijk, C.; Rowe, R. Langmuir 1999, 15, 8265-8275.

Picart et al.

expected the surface coverage to remain constant, the augmentation of the deposited amount leading to an increase in the height of the islands.57 On the contrary, in our system, the lowering of the contact angle during the buildup induces a constant raise of the surface coverage. The penetration depth of the evanescent waves is on the order of 150 nm whereas the larger islands extend up to 1 µm. For this reason, the constant raise of the effective refractive indexes, NTE and NTM, observed by OWLS during the first regime, most probably reflects an augmentation of the surface coverage near to the silica/ islands interface rather than an increase of the total deposited amount. The constant decreases of the relative frequency shifts ∆f/ν measured by QCM at all the investigated frequencies during the first buildup regime reflect the constant increase in the total amount of deposited material on the quartz surface. The fact that ∆f/ν is not frequency independent shows the nonvalidity of the Sauerbrey relation. This can be due to various reasons: the surface is covered by islands and is not homogeneous; the deposited PLL/HA material does not behave as a rigid structure but may exhibit viscoelastic properties; the measurements are performed in water and not in air. At this stage it is unfortunately difficult to go beyond these observations in the analysis of the QCM signal. The evolutions of the QCM and the OWLS signals clearly indicate that, after eight (PLL/HA) deposition cycles, the buildup process enters into a new regime. The AFM images show that at this stage the whole surface is almost fully covered by islands that have coalesced, giving a uniform film that appears first homogeneous and smooth. Pursuing the buildup process, one observes an increase of the surface roughness with the formation of aggregates over the uniform (PLL/HA) film. This can explain the large charge fluctuations that are experienced along the film during the AFM imaging. The evolution of ∆f/ν for f ) 5MHz after the eighth deposition cycle confirms that new PLL and HA polyelectrolytes are deposited on top of the surface. Each new polyelectrolyte deposition leads again to a charge reversal that still should remain the motor of the buildup process. However, in this second buildup regime, the ∆f/ν changes at 15, 25, and 35 MHz show a different behavior as compared to the first regime: ∆f/ν increases (respectively decreases) when the films is brought in contact with the PLL solution (respectively during the rinsing step after the PLL deposition). The amplitudes of the ∆f/ν variations are strongly dependent on the excitation frequency, the variations being weaker when f increases. To explain the observed behaviors, one has to take into account the fact that HA presents strong viscoelastic properties.19 One can thus expect the (PLL/HA)n films to possess similar rheological properties. In such viscoelastic films, the propagation of shear waves is attenuated over a characteristic penetration length δ that varies, in first approximation, as

δ ) xη/πFf

(5)

where η corresponds to the shear viscosity and F is the density of the film. This shows that the penetration length decreases when the oscillating frequency increases as it is schematically depicted in Figure 8. Different frequencies are thus sensing different moments of the material distribution in the film. The increase of ∆f/ν for f ) 15-35 MHz and its decrease for f ) 5 MHz when the multilayer film is brought in contact with a PLL solution suggests a reduction of the coupled mass sensed by the shear waves over the smaller


Figure 8. Schematics of the spatial information gained from each experimental technique during the alternate deposition of PLL and HA layers. Because of the limited penetration length of the evanescent field, OWLS is sensitive to the inner part of the film, at the vicinity of the surface. Streaming potential measures the overall charge of the film that is mainly representative of the upper part of the film. The penetration length of the sound wave (which decreases with the harmonic number) allows QCM, with the four investigated harmonics, to cover the whole depth of the film, from the surface of the quartz electrodes to the outer side of the film. AFM gives an overview of the topography of the film.

penetration length (corresponding to the higher frequencies) and an increase of this mass over the penetration length corresponding to 5 MHz. The opposite behavior is expected during the PLL rinsing steps. Further information can be gained from the variations of the effective refractive indexes NTE and NTM. One must first keep in mind that the thickness of the film, as determined from AFM, is on the order of 1 µm. This is much larger than the penetration length of the optical waves (typically 150 nm). The changes in NTE and NTM observed during the polyelectrolyte injection and rinsing steps thus reflect changes that must occur in the close vicinity of the substrate and not near the outer part of the multilayer. Moreover, the polyelectrolyte film should behave, for the optical evanescent waves, as a semiinfinite medium characterized by a refractive index nA . Using the three layer mode equations44 corresponding to this situation, one then finds that the decrease (respectively increase) of NTE and NTM during the PLL adsorption (respectively rinsing) step reflects a decrease (respectively increase) of the refractive index nA attributed to the film (data not shown). A reduction of the refractive index is usually related to a decrease of the density of the material. The OWLS results are thus in accordance with the similar trend observed for the coupled mass determined by QCM at the higher frequencies sensing the smaller penetration lengths. These observations can be explained as follows: in the second buildup regime, after the deposition and rinsing steps of HA, all the PLL chains contained in the multilayer film are strongly bound to the HA chains. They can thus be considered as strongly anchored in the film. When such a film is brought in contact with a PLL solution, PLL chains can diffuse into the multilayer similarly to a diffusion in a porous material. The diffusion of PLL inside the films originates from the fact that almost no free PLL chains exist in the film which generates a great chemical

Langmuir, Vol. 17, No. 23, 2001 7423

potential difference of the PLL chains between the film and the solution. The interior of the film being neutral, it is expected that these chains do not form new strong complexes with the HA chains but rather interact weakly with them and in particular with the interfacial hydration layer of HA. HA is, indeed, known for its interaction with water, the interfacial hydration layer of HA being constituted of two types of water molecules that are closely associated with the biopolymer surface: the primary hydration shell involves strongly bound water molecules and the secondary hydration shell is constituted of more loosely bound ones.23,60 When PLL interacts with HA, it can then destructure the water organization around the HA chains and take the place of the water molecules in these hydration shells. Water molecules are then expelled from the interior of the film leading to a net outward mass flow. This destructuration of the water organization should also induce a density decrease of the film material as it is suggested by the OWLS and QCM experiments. One can also expect that free PLL chains that have diffused into the films can be exchanged by PLL molecules strongly interacting with HA. This would keep the concentration of the free PLL chains in the film unchanged. PLL chains do however not only diffuse into the interior of the film during this adsorption step but they also form new PLL/ HA complexes with the HA chains constituting the outer layer of the multilayer. The net mass balance of the whole film, sensed with the QCM at 5 MHz, can thus still be positive. During the rinsing step after the PLL deposition, the free PLL chains in the film can diffuse out of it with water molecules entering again into the film. The fact that the net coupled mass of the film remains smaller after the PLL rinsing step than before the previous PLL addition step indicates that only a fraction of the free PLL chains leave the film during the rinsing step. Once this film is brought in contact with a HA solution, HA chain interacts with the PLL chains lying at the outer part of the film. Moreover, the OWLS signal becoming perfectly cyclic in the second regime (see Figure 1), it is expected that all the free PLL chains that remain in the film at the end of the rinsing step, also diffuse toward the outer film surface that now acts as a perfect sink. As soon as these chains reach the surface, they interact with HA chains and thus also contribute to the buildup of the layer. One can assume that the amount of free PLL chains remaining in the film at the end of the rinsing step of the PLL solution by pure buffer is proportional to the film thickness. This would then lead to an exponential raise of the total mass of the film in the second buildup regime of the film. This is precisely what is seen from the evolution of -∆f/ν at 5 MHz with QCM. Finally these results also suggest that the penetration depth of the mechanical shear waves is smaller or on the order of the film thickness for f ) 15-35 MHz and is larger than the film thickness for f ) 5 MHz. By taking a film thickness of ≈1 µm and a film density F of 103 kg‚m-3, one obtains, from eq 5, a first rough estimate of the shear viscosity of the film material which is found to be on the order of 0.1 Pa‚s. This value is by a factor 10 smaller than the shear viscosity reported for hyaluronic acid solutions at 10 mg/mL but in a much smaller frequency domain.61 One can notice that all the results presented were obtained by building-up the films in solutions containing 0.15 M NaCl. Other buildup conditions could lead to different behaviors. For example, the ionic strength may (60) Middendorf, H. D. Physica B 1996, 226, 113-127. (61) Berriaud, N. Ph.D. Thesis, University Joseph Fourier, 1994, Grenoble, France.

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drastically influence the formation of the film by changing the polyelectrolyte charges, since electrostatic interactions are supposed to be the main basis of the construction.7,10 Further studies are thus necessary to get a full picture of the (PLL/HA) film buildup processes. Similarities with the poly(L-lysine)/alginate system, for which a rapid growth process was shown,18 could be suspected. Elbert el al.18 attributed this rapid growth process to the formation of complex gels or coacervates that could be correlated to a buildup mechanism greater than linear. However, no model could be advanced by these authors to explain the observed exponential growth mechanism. Conclusion The formation of a new kind of biocompatible film based on alternate adsorption of poly(L-lysine) and hyaluronic acid was investigated. The driving force of the buildup process appears, as for “conventional” polyelectrolyte multilayer systems, to be the alternate overcompensation of the surface charge after each PLL and HA deposition. It was shown that the (PLL/HA)n film construction takes place over two buildup regimes: The first regime is characterized by the formation of isolated islands and islets dispersed on the surface and which grow both by addition of new polyelectrolytes on their top and by mutual coalescence. The material constituting the islands appears structurally homogeneous as suggested by well-defined contact angle. The second regime sets in once a continuous film is formed which takes place in our buildup conditions after approximately the eighth (PLL/HA) deposition step. This continuous film seems to exhibit a viscoelastic behavior, as shown by QCM data at different frequencies with a shear viscosity that is on the order of 0.1 Pa‚s. During the second regime of the buildup process the overall

Picart et al.

mass of the film seems to increase in an exponential way. This exponential growth is explained by the diffusion of free PLL chains into the interior of the film when the film is in contact with a PLL solution and by the diffusion out of the film of a fraction of the free chain and their interaction with HA chains at the outer limit of the multilayer when the film is further brought in contact with a HA solution. The diffusion of free PLL chains into the film is also accompanied by an expulsion of water out of the film. These (PLL/HA) films will be used as model substrates for the design of bioactive films, HA being a natural extracellular matrix polysaccharide and a ligand for several cell types. In particular, this kind of film should enhance specific cell proliferation or adhesion. The incorporation of biologically active compounds in the films or their coupling to the PLL molecules is also foreseen. Indeed, investigation of chondrocytes interaction with these films, which are known to bind to HA through their CD44 receptors, will be investigated. Acknowledgment. The authors wish to thank M. Milas (CERMAV, Grenoble, France) and R. Richter from Q-Sense for fruitful discussion, and L. Szyk for assistance with the Streaming Potential measurements. Helpful discussions with C. M. Knobler about breath figures are also gratefully acknowledged. P.L. is indebted to the “Fondation pour la Recherche Me´dicale” for financial support. This work was supported by the program “Adhe´sion Cellules-Mate´riaux”, by the program CNRS “Physique et Chimie du Vivant”, and by the Association pour la Recherche sur le Cancer (Project No. 7597). LA010848G

Hyaluronic Acid Films onto a

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