Influence of Synthetic Polyelectrolytes on the Growth and Properties of

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Biomacromolecules 2009, 10, 294–301

Influence of Synthetic Polyelectrolytes on the Growth and Properties of Hyaluronan-Chitosan Multilayers Mikko Saloma¨ki* and Jouko Kankare Department of Chemistry, University of Turku, FIN-20014 Turku, Finland, and Turku University Centre for Materials and Surfaces (MatSurf), Turku, Finland Received September 11, 2008; Revised Manuscript Received November 7, 2008

Both hyaluronan (HA) and chitosan (CHI) are biocompatible polysaccharide electrolytes. The multilayers formed by these polyelectrolytes alone are known to be rather soft and strongly viscoelastic. In this work we study multilayers formed by incorporating synthetic nonsaccharide polyelectrolytes such as polyallylamine (PAH) and poly(acrylic acid) (PAA) in various proportions into the HA/CHI layers. The buildup was followed on a quartz crystal resonator. Surface acoustic impedance recorded in these measurements, in suitable conditions, gives a spiral when plotted in the complex plane. The shape of this spiral depends on the viscoelasticity of the layer material and regularity of the growth process. We found that poly(acrylic acid) destroys the soft diffuse matrix formed by hyaluronan. It forms diffusion barriers when deposited sparsely between the layers. If its proportion is higher, the film growth adopts a linear buildup in the layer-by-layer process. The linear buildup of CHI/PAA reveals that the buildup regime of a multilayer film does not determine the viscoelastic properties of the film. Linearly and exponentially growing films may have very similar mechanical properties. Polyacrylic acid forms a kind of scaffold inside the film giving the natively soft hyaluronan/chitosan film more mechanical strength. The optimal combination gave more than 100-fold increase in the shear modulus.

Introduction Polyelectrolyte multilayer films provide a simple and costeffective way to modify and to functionalize a variety of different surfaces.1 The possible applications are recognized in the fields of biomaterials,2 membranes,3 drug delivery,4 and so on. The naturally occurring polysaccharides such as hyaluronan, alginate, chitosan, and heparin provide valuable properties that are beneficial for biomaterials. These biocompatible, nontoxic, and highly hydrated polymers have many promising applications, for example, in tissue engineering.5 The layer-by-layer (LbL) method can be applied to these materials to construct stable but also preferentially decomposable polymer thin films.6 Fundamental studies have been carried out using polyelectrolyte multilayers as cell adhesion surfaces.7-11 The quality of a thin film on a cell-biomaterial interface is dependent on the chemical, topographical, and mechanical properties of the biomaterial. From the above-mentioned elements, the mechanical properties of thin films have only recently received higher attention.12 This is partly due to lack of available techniques that give rheological information in nano- to micrometer scale. With respect to the polyelectrolyte multilayer coatings, the stiffness of the film (usually addressed in terms of Young’s modulus) has been shown to play a crucial role in the cell adhesion. For example, native hyaluronan/chitosan multilayer film resembles a hydrogellike film and therefore resists chondrocyte adhesion.13 In general, it can be considered that there is a need for surfaces with the biocompatibility of polysaccharides combined with adjustable stiffness. The stiffness of a native multilayer film is related to the buildup regime in the LbL process. From the two different buildup regimes, linear and exponential, the exponentially growing films are generally considered to be softer than the * To whom correspondence should be addressed. E-mail: mikko.salomaki@ utu.fi.

films showing linear buildup.14 The linear buildup regime in the polyelectrolyte multilayer assembly is usually observed after the nonlinear initial buildup.15,16 According to the theory, every buildup process is inherently exponential, turning linear whenever the diffusion rate is not fast enough for distributing the mobile polyelectrolyte within the whole film.17 Also, slightly different suggestions on limiting factors have been given.18,19 The extended nonlinear buildup regime in the assembly is wellknown and it is often related to polyelectrolytes of biological origin.20,21 Highly hydrated natural polyelectrolytes seem to prefer the exponential buildup.13,22 There are multiple ways to modify the elastic properties of multilayer films. The stiffness of the LbL deposited film can be influenced by changing the conditions during the deposition or by a suitable post-treatment of the multilayer film. The factors affecting the stiffness of the films during the LbL procedure are, for example, temperature,17 the nature of the electrolyte,23 pH,24-26 and ionic strength.27,28 A particularly interesting and effective method is cross-linking between the charged units using an initiator or a linker molecule. With a linker molecule, such as glutaraldehyde, extremely stiff multilayer films can be produced.29 More than 10-fold stiffness increase has been observed by using chemical cross-linking in HA/poly(L-lysine) films.8 The cross-linking can also be done by post-treatment in an appropriate polyelectrolyte system by heating the film up to 130-215 °C.30 It is a very effective way to achieve almost total cross-linking of the polyion pairs in the film. However, the heating procedure cannot be applied to most biomacromolecules, because of polymer degradation at high temperatures. An alternative method to tailor film properties is to use blended polyelectrolyte solutions, that is, mixtures of polyanions or polycations where the components have different properties in the formation of multilayers.31-35 Although the viscoelasticity in these reports has not been directly estimated, in many cases, the growth regime could be changed from exponential to linear

10.1021/bm8010177 CCC: $40.75  2009 American Chemical Society Published on Web 01/02/2009

Hyaluronan-Chitosan Multilayers

by suitable blending, showing that most likely the viscoelasticity has undergone a considerable change. A different approach was taken in a recent study by Francius et al.36 The polyelectrolyte solutions used for the LbL procedure were not mixtures as in the previous methods, but the film buildup was done by first depositing several soft poly(L-lysine)/hyaluronan layers and capping them with hard poly(allyl amine)/poly(4-styrene sulfonate) layers. Considerable stiffening of the multilayer film was observed by using the AFM indentation technique and subsequent elegant mathematical modeling. The aim of this work is to take a typical polyelectrolyte multilayer with the exponential buildup regime and to study the influence of intervening polyelectrolyte layers of different buildup character. As is well-known, the exponential growth of the polyelectrolyte multilayers during the LbL process is assumed to be due to the fast diffusion of one of the electrolytes within the film. It can be anticipated that by adding a diffusion barrier into the film, the diffusion rate could be decelerated to such an extent that the growth would turn linear and the material stiffer. The diffusion barrier could be made of a polyelectrolyte which is known to sustain linear growth. Hyaluronan (HA) and chitosan (CHI) are polysaccharide electrolytes that are wellknown in forming multilayers with the exponential growth rate. On the other hand, synthetic polyelectrolytes poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) are known to form multilayers PAH/PAA, which reach a linear buildup almost at the beginning of the deposition.37 The buildup is dependent on pH of the solution and PAA as outermost layer improves rigidity of the film.38 Furthermore, these polyelectrolytes are suitable analogues for the polysaccharides HA and CHI bearing the same charged groups. Hence, these polymers are a natural choice for testing their capabilities as barrier layers. Simultaneous in situ monitoring both the mass and viscoelasticity of the layers during the growth process is possible by using a quartz crystal resonator (QCR). The information on the viscoelasticity is different from the AFM indentation measurements done, for example, in the work by Francius et al.36 In the QCR experiments dynamic shear parameters are measured at rather high frequencies, whereas the AFM indentation gives information on the tensile parameters at a considerably slower time scale. Also, the parameters from the QCR experiments correspond to the bulk material extending not far from the resonator surface whereas the results from the AFM indentation tally more or less with the stiff “crust” of the film. Still, the viscoelastic parameters from the QCR measurements are commonly used for estimating the relative stiffness of thin films.39 There are considerable ambiguities in the literature concerning the parameters coming out from the measurements on a QCR. Previously, the only parameter was the relative decrease in the oscillation frequency observed when the surface of the resonator is coated with some material. The well-known Sauerbrey46 equation tells that this relative decrease in frequency is in the first approximation equal to the relative increase in the mass of the resonator, provided that the mass increase is small enough. After it was observed that the resonator could be kept oscillating also in liquids, people paid attention to the energy needed to maintain oscillation with the resonator in contact with different materials. This energy is a new parameter and the main ambiguity is in the interpretation and presentation of this parameter. The first attempt into this direction was the Butterworth-Van Dyke (BVD) equivalent circuit where the electrical properties of QCR were described by a model composed of a series-connected inductance, capacitance, and resistance with an overall parallel-connected capacitance.40 Here

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the inductance and capacitance describe the resonant frequency and the resistance R the intrinsic losses and the losses induced by the material in contact with the resonator surface. In another presentation the losses are measured as the half-width of the impedance versus frequency curve.41 These models have been nearly completely superseded in the application literature by a so-called “dissipation model” or “QCM-D”, mainly due to the efficient marketing by the instrument manufacturer. Dissipation D is derived from the decay rate of the resonator oscillation when disconnected from the electrical circuit.42 However, dissipation D, BVD resistance R, and the half-width of the impedance curve are not the intrinsic properties of the material in contact with the resonator. All three parameters comprise also properties of the quartz resonator. The only natural parameter describing the quantity and properties of the coating material as well as the contacting medium is surface acoustic impedance, also called load impedance, acoustic load, or surface mechanical impedance, denoted here as ζ0.43 This is a complexvalued quantity comprising of a real part, mainly describing the viscoelastic properties of the material, and an imaginary part describing mainly the quantity of the material in thin layers but depending also on the viscoelastic properties in thicker layers. The main advantage of this parameter is that, as a complex number, any changes in the load such as growth, stratification, or softening can be described as mathematical transformations in the complex plane.28,52 Furthermore, as the layer on the resonator thickens, surface acoustic impedance converges to the bulk acoustic impedance of the layer material. For those preferring the expressions involving the relative frequency change ∆f/f and dissipation D, there is a simple relation of these quantities with the real and imaginary parts of surface acoustic impedance ζ0:

ζ0 ) ζ0 + jζ0 ; ∆f ⁄ f = -

2 1 ∆ζ ; D = ζ ωdqFq 0 ωdqFq 0

Here ω is the angular frequency ()2πf), dq is the thickness of the resonator, and Fq is the density of quartz. These equations are good approximations for thin layers. On the characterization of elastic properties of the films we have utilized a homemade quartz crystal impedance measurement system44 that is capable of providing reliable data even beyond the point of acoustic film resonance (AFR45). The AFR occurs when the film thickness reaches a critical thickness which equals to one-quarter of a wavelength of the acoustic shear wave in the material. Near to and after the AFR the classical Sauerbrey equation46 is no longer valid. It is often mentioned in the literature that a film with excessive damping generates a “viscoelastic effect” and, therefore, the QCM analysis is not reliable. However, as we have shown earlier, the valuable measurable information is unquestionably available also in the proximity of AFR.28 The complex acoustic impedance of the growing film on the surface of a thickness shear mode resonator (AT cut quartz crystal) forms eventually a spiral when represented in the complex plane (Argand diagram). At the pole of the spiral, the film on the surface has grown to the critical thickness where the acoustic shear wave generated by the resonator sees only the bulk of the film. This state is relatively easily reached in the case of exponentially growing films. At some conditions even less than 40 layers is enough.28 If the buildup is linear, the full spiral is very difficult to achieve because of the large number of layers needed. A shape or uniformity of the spiral describes the homogeneity of the material deposited on the surface of the resonator. Many of the films presented in this paper show acoustic film resonance within the layers deposited.

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Experimental Section Materials. Poly(allylamine hydrochloride) (PAH; Mw 65 kDa, Aldrich) and poly(acrylic acid) (PAA; Mw 100 kDa, Aldrich) were used as received. Sodium hyaluronate (HA; 1000 kDa, Acros Organics) was partially depolymerized by heating.47 The solution of HA (1 mg/ mL) in presence of NaCl was hydrolyzed thermally at 90 °C for 195 min. The molecular weight of the product was estimated to be about 530 kDa on the basis of the Mark-Houwink relation by measuring the intrinsic viscosity of the polymer solution. ([η] ) 0.0228 × M0.816).48 Chitosan (CHI; Aldrich, medium molecular weight, 93% deacetylated) was purified49 and the molecular weight was estimated as about 200 kDa on the basis of the Mark-Houwink relation.50 Chitosan (1 mg/ mL) was solubilized in 10 mM HCl. The amount of added Cl- was taken into account when the ionic strength of the solution was adjusted with NaCl. The concentrations of the coating solutions were 1 mg/mL for HA and CHI and 10 mM for PAH and PAA. The ionic strength of all solutions was adjusted to 0.15 M with NaCl, and 1 mM of acetic acid was added as a buffer. The pH of these solutions was adjusted to 4.5 with 1 M NaOH. All coating solutions were filtered with a 0.45 µm membrane filter because natural polyelectrolytes seemed to contain insoluble material. The viscosities and densities of the solutions were measured on an Anton Paar AMVn automated microviscometer and DMA45 density meter, respectively. Coating and Measurement. In this study the films are deposited by using a fully automated LbL machine. The instrumentation, coating procedure, and measurement have been described in detail in our previous publications.27,34 In short, the quartz crystal (10 MHz, 100 nm gold-plating with a chromium adhesion layer, Lap-Tech, Inc., South Bowmanville, Ontario) was plasma-cleaned, primed with a monolayer of 2-mercaptoethanesulfonic acid, mounted in the holder, measured without liquid loading, then as loaded with the supporting electrolyte and finally coated with polyelectrolytes in a fully automated system. At each step, the polyelectrolyte solution was allowed to stay in contact with the crystal exactly for 15 min and after rinsing with the supporting electrolyte the QCR measurement was done. The films were not dried during the measurement. These steps were repeated alternately with anionic and cationic polyelectrolytes until data for the wanted number of layers were collected. Coating and measurements were done at 25 ( 0.03 °C. A new unused crystal was each time taken for coating and measurements. Estimation of the values of the real and imaginary parts of surface acoustic impedance and their covariance matrices was done as described previously.44 Each film was deposited more than once to examine the reproducibility. Impedance Analysis. The impedance analysis of the sequentially growing films is based on the three layer model15,51 of polyelectrolyte multilayers (however, the zone I is considered negligible in thick films) and on the layer matrix representation described in our earlier papers.28,52 This particular calculation method gives the estimates of complex bulk acoustic impedance (Z) for two zones formed on the film if they can be distinguished from each other or from the solution (zone III, which is in contact with solution, and zone II, which is a bulk of the film). Bulk acoustic impedance is directly related to the complex shear modulus (G) of the material (Z ) FG), where F is the density of the material. The dependence between Young’s modulus (E) and the shear modulus for an elastomer is roughly estimated following G ) E/3. Also, the loss angle δ of the material can be estimated (tan δ ) G′′/G′).

Results and Discussion Two-Component Films. It was shown earlier that the native HA/CHI film undergoes an exponential buildup in the LbL process when deposited in 0.15 M NaCl. Furthermore, the growth exponent17 (β) can be fine-tuned by changing the ionic strength of the solution.13,28 According to Richert et. al, at a lower ionic strength the multilayer deposition will produce droplets instead of a uniform film,13 resulting in a linear buildup

Saloma¨ki and Kankare

pattern. The buildup study with two component films was conducted by using combinations of natural/natural and natural/ synthetic polyelectrolyte pairs in order to examine the effect of nature of the polyelectrolyte on the buildup process. Also the viscoelastic properties of the forming film are of the interest in this study. In our earlier study we showed that the growth of a CHI/HA film generates a well-shaped spiral in a complex impedance plot.28 Separate growth curves for polyanion and polycation terminated films can be observed, which was attributed to the deep internal diffusion of one of the polyelectrolytes. By replacing either of the natural polyelectrolytes in a CHI/HA film with the synthetic analogues, PAH or PAA, noticeable effects will occur. Surprisingly, by substituting CHI in the film with PAH resulted in only minimal changes in the film. No influence on the buildup regime was observed. The buildup in PAH/HA film is fully exponential, which has been observed also earlier,53 and it even has a higher growth exponent than the native CHI/HA film, 0.43 versus 0.25, respectively. This observation is an indication of the free diffusion inside the multilayer by at least one of the polyions. The buildup of PAH/HA shows a maximum in the imaginary part of acoustic impedance (corresponding to a minimum of the resonance frequency) earlier than CHI/HA multilayer by about 20 layers (Figure 1b). Also notable is that there is a clear separation of the values corresponding to the polyanion-capped and polycation-capped films near the maxima (Figure 1a and b). The impedance plots (Figure 1c and d) of CHI/HA and PAH/HA show common features. Both curves are divided into two separate pathways converging toward the same limiting values. The whole film is acoustically different after each polyanion deposition compared to the situation after the polycation deposition, which is an indication of deep diffusion. Convergence to the same limiting values can be explained by reaching the critical thickness of the bulk of the film. After a certain film thickness, the acoustic wave cannot penetrate to the top of the film anymore. Also, the polyion diffusion cannot reach the bottom of the film to be detected by the acoustic wave. At this point, the acoustic wave sees only the bulk of the film, that is, zone II, and further deposition outside the range of the acoustic wave is not detected. It is shown by Picart et al. that HA does not diffuse vertically through the film in HA/poly(L-lysine) system.21 Therefore, it is relatively safe to assume that at least PAH is capable of diffusing in PAH/HA film. The exponential buildup is attributed to hyaluronan, probably for the reason that it forms a diffuse matrix in which certain polycations can diffuse. Indirect evidence on the role of HA is obtained from the CHI/PAA deposition. The CHI/PAA film undergoes a linear buildup (ζ′′ indicates the mass) almost from the beginning of the deposition (Figure 1e). Therefore, it is assumed that there is no internal diffusion of significant extent inside this film. Despite having a linear buildup regime, this film also generates a maximum in acoustic impedance plot. There are no separate growth curves of polyanion and polycation terminated films. Also, the shear modulus values are quite close to the exponentially growing CHI/HA and PAH/HA films. Only the loss angle is somewhat lower, indicating slightly increased elasticity in this film. Nevertheless, it is shown here that the buildup regime does not determine the viscoelastic properties of the films, with viscoelasticity depending more on the individual properties of the polyelectrolytes and the complexes formed during the buildup process. Three-Component Films. The nature of the exponential buildup lies in the deep internal diffusion of either of the



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Figure 1. Impedance analysis of two component films. Real part (a) and imaginary part (b) of the local acoustic impedance of CHI/HA (squares) and PAH/HA (circles) multilayers. The impedance spirals of CHI/HA (c) and PAH/HA (d). Real and imaginary parts (e) of the local acoustic impedance of CHI/PAA multilayer. The impedance spiral of CHI/PAA (e) is shown in the inset. Lines indicate the fitting results of even layers (HA or PAA on top). Rayl ) kg m-2 s-1.

components participating in the buildup process. In order to study the ability to control the diffusion we have deposited CHI/ HA films containing various proportions of polyacrylate. The film with PAA as every fourth layer (CHI/HA/CHI/PAA)n shows strictly linear buildup with no evidence on the internal diffusion (Figure 2b and Figure 3). There are no separate growth curves and no maxima in the impedance plot. There is no impedance spiral formation. The resulting nearly straight line does not give additional information except the mass increase. Despite the fact that HA is present in the film, it is apparent that PAA layers form diffusion barriers in the film. Comparing this film with CHI/PAA film, it is noticed that the only common

feature shared by the films is the linear buildup. The (CHI/HA/ CHI/PAA)n film has a much higher mass increase per bilayer (Figure 3), and also the storage modulus (G′) is approximately 100 times higher (Table 1). If it is assumed that these films obey the Voigtian viscoelastic model, a commonly used but rather crude approximation, then also the Young’s modulus would be 100 times higher. If (CHI/HA)n and (CHI/PAA)n are hydrogel-like films, why is the combination (CHI/HA/CHI/PAA)n a stiff elastic film? The reason is most probably the same as in cross-linking. The mobility of individual chains is reduced and the whole internal

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Figure 2. Impedance analysis of three component films. Real part (a) and imaginary part (b) of the local acoustic impedance of CHI/HA/CHI/ PAA and (CHI/HA)3/CHI/PAA multilayers. The magnification of the first 60 layers (b) is shown in the inset. Real and imaginary parts (c) of the local acoustic impedance of (CHI/HA)7/CHI/PAA multilayer. The impedance spiral of (CHI/HA)7/CHI/PAA (c) is shown in the inset. Lines indicate the fitting results detailed in Table 1.

structure is immobilized. During the deposition, the incoming CHI is able to compensate the charges of HA to a very high extent, but at the same time, it complexes with PAA. The resulting film is composed of CHI-HA units, which favor the internal diffusion and exponential buildup, and also CHI-PAA units, which are diffusion-inhibiting and favor linear buildup. The high mass gain of CHI-HA complexes and the stable skeleton formed by CHI-PAA complexes will result in a film with all new qualities. If PAA is deposited as every eighth layer [(CHI/HA)3/CHI/ PAA]n, the film growth shows initially exponential buildup which turns linear after about 30 layers (Figure 2b, Figure 3). In this film the diffusion barriers are apparently farther away from each other but still the exponential buildup is inhibited by the PAA layer in eight layer periods. Therefore, the linear buildup occurs in a rather early stage. There are neither maxima nor separate growth curves detected in the plot within the number of layers deposited. However, the viscoelastic properties of this film are quite extraordinary. This film resembles neither an elastic film, like the (CHI/HA/CHI/PAA)n, nor a hydrogellike film, like (CHI/HA)n, but instead the bulk of the film contains material that resembles Newtonian fluid. Actually, the bulk is a very viscous fluid, like thick syrup, that gains mass and thickness during the growth process. This particular deposition and these calculations were conducted three times to ensure the absence of experimental artifacts, but the results were essentially similar. If PAA is deposited as every 16th layer [(CHI/HA)7/CHI/ PAA]n, the buildup is exponential in between the PAA steps,

but the buildup rate, indicated by ζ′′, starts over from almost zero after a PAA step (Figure 2c). This behavior is clearly visible between the layers 32 and 48. The diffusion barrier formation is most clearly shown in this film. The evidence of limited diffusion is also shown in the impedance plot (Figure 2c). There is no clear separation of the polyanion-capped and polycationcapped growth curves. The diffusion depth of a polycation is evidently limited by the distance to the last added PAA layer. The buildup of the multilayer shows a maximum later than in the native CHI/HA multilayer. The resulting peak in the imaginary part of the local acoustic impedance is rather broad which is probably due to the discontinuities brought by PAA in the deposition. Still, even when there are breaks in the buildup, the film is able to grow beyond the first resonance in a reasonable number of layers (ca. 250 layers). Question of Homogeneity. The two-zone model used in this work assumes homogeneity of the zones.28 In reality, the zones do not have a sharp boundary surface but a more or less broad interfacial region where the impedance varies smoothly from one zone to the other. In addition, the LbL method may create subzones where the impedance fluctuates between two values determined by the alternating polyelectrolytes. In this case we have to use the average impedance as discussed previously.44 Furthermore, the layer interpenetration usually exceeds four layers in multilayer films.54,55 Often the zone III in contact with the solution approaches values corresponding to a Newtonian fluid. This can be explained by the loops and tails of the polymer chains extending into the solution and forming a fuzzy interfacial structure containing water and small ions as charge compensation.



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Figure 3. Areal mass densities vs number of layers in the film. The results are obtained from the fitting detailed in Table 1. Zone II is indicated with dark gray and zone III, if detected, is indicated with light gray. A plateau in the mass indicates that the film has grown to a critical thickness, where the acoustic wave can not detect additional growth of the film. Table 1. Estimated Values of Acoustic Impedance, Loss Angle, and Storage Modulus of the Multilayer Films system

Z/zone II (kRayl)

δ/zone II (degrees)

PAH/HA;a even-numbered layers from 2 to 38 CHI/HA; even-numbered layers from 2 to 200 CHI/PAA;a even-numbered layers from 2 to 182 CHI/HA/CHI/PAA; every fourth layer from 2 to 278 (CHI/HA)3/CHI/PAA; every eighth layer from 2 to 282 (CHI/HA)7/CHI/PAA;b every 16th layer from 2 to 146

49 61 40 443 1640 57

59 62 36 18 90 14

Z/zone III (kRayl)

δ/zone III (degrees)

13

88

100 14 29

90 90 49

G′/zone II (MPa)c 1.0 1.4 1.1 156 Viscosity ca. 36 Pa.s 2.6

a One zone model gives best results for these films. The zone III cannot be distinguished from the zone II or from the solution. The values for the solution are about 7.5 kRayl and 90°. b The two zone model does not apparently fit in this film due to inhomogeneity. c Based on the density of 1200 kg/m3.

The (CHI/HA/CHI/PAA)n film seems to have a homogeneous structure with a linear buildup almost from the beginning of

the deposition. However, it is questionable whether the two other hybrid films have a sufficiently homogeneous structure. The

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[(CHI/HA)3/CHI/PAA]n film resulted in interesting properties of bulk of the film, resembling a very viscous Newtonian fluid. We have to point out that the measurement has been done at 10 MHz. It might well be that we are dealing with a Voigtian fluid with a very long relaxation time. Also inhomogeneity of the film may have its effect on the results. The film could actually have a number of different domains inside the bulk. The mutual effect of these domains would result in an extraordinary result. Additionally, as can be seen in the Figure 2a, the fit is not perfect, which supports the assumption of inhomogeneity. Also, an interesting feature is that every single deposition in an eight layer period seems to create its own pathway in the plot. The fitting results of each pathway gave similar results. However, experimentally, this film behaves like thick syrup and the best fit with the smallest number of adjustable parameters leads to the present result. The same question of homogeneity arises in the case of [(CHI/ HA)7/CHI/PAA]n, but this time apparently demanding more attention. The fitting of the impedance data is, actually, very complicated. A very likely explanation is that the film is divided into sequential soft and hard stratified zones. The soft zone consists only of HA and CHI having a hydrogel-like shear modulus values, referring to native CHI/HA film (Table 1). The hard zone consists of HA, CHI, and PAA forming much stiffer material, referring to (CHI/HA/CHI/PAA)n film (Table 1). Compared to the latter case, the distance between the PAA layers is apparently large enough to break the mechanical connection between the layers. So there will be no structure supporting skeleton formed by PAA, which would strengthen the material. The behavior of the acoustic wave in this kind of film is very difficult to analyze. The shear force generated by the acoustic wave probably induces strain in the soft zone, while the hard zones are moving with the soft zones, giving distorted picture of the elasticity in the film. On the basis of the impedance analysis, this film is only to some extent stiffer than the native CHI/HA film. However, the application of the impedance analysis assumes homogeneity of the zones, and in this case, the simple model is not apparently valid. Nevertheless, the real impedance values might be close to the obtained values, because the impedance values converge quite close to the values of CHI/ HA film.

Conclusions We have shown here that by hybridizing polysaccharide based polyelectrolyte multilayers with synthetic polyelectrolytes, the resulting films have new properties in terms of the LbL buildup regime and viscoelasticity. The buildup regime, whether being linear or exponential, does not alone determine the elastic properties of the film, as illustrated in CHI/HA and CHI/PAA multilayers. Hyaluronan is responsible for the exponential buildup of the films. However, the exponentially developing film can be tuned to linear by codepositing polyacrylate in the multilayers. Polyacrylate has apparently two main functions in the hyaluronan/chitosan film. Polyacrylate destroys the soft diffuse matrix formed by hyaluronan. It forms diffusion barriers when deposited sparsely between the layers. If its proportion is higher, the film adopts a linear buildup. Polyacrylate seems to form a kind of scaffold inside the film giving the natively soft hyaluronan/chitosan film more mechanical strength, but it will function only when all three components are present. Further experiments will show whether the mechanical properties of these films are improved by polyacrylate without impairing their biocompatibility.


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BM8010177

Influence of Synthetic Polyelectrolytes on the Growth and Properties of

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