Mechanical properties of model and natural gels have recently been demonstrated to play an important role in various cellular processes such as adhesion, proliferation, and differentiation, besides events triggered by chemical ligands. Understanding the biomaterial/cell interface is particularly important in many tissue engineering applications and in implant surgery. One of the final goals would be to control cellular processes precisely at the biomaterial surface and to guide tissue regeneration. In this work, we investigate the substrate mechanical effect on cell adhesion for thin polyelectrolyte multilayer (PEM) films, which can be easily deposited on any type of material. The films were cross linked by means of a water-soluble carbodiimide (EDC), and the film elastic modulus was determined using the AFM nanoindentation technique with a colloidal probe. The Young's modulus could be varied over 2 orders of magnitude (from 3 to 400 kPa) for wet poly(L-lysine)/hyaluronan (PLL/HA) films by changing the EDC concentration. The chemical changes upon cross linking were characterized by means of Fourier transform infrared spectroscopy (FTIR). We demonstrated that the adhesion and spreading of human chondrosarcoma cells directly depend on the Young's modulus. These data indicate that, besides the chemical properties of the polyelectrolytes, the substrate mechanics of PEM films is an important parameter influencing cell adhesion and that PEM offer a new way to prepare thin films of tunable mechanical properties with large potential biomedical applications including drug release.

The buildup and secondary structure of poly(L-glutamic acid)/poly(allylamine) (PGA/PAH) multilayer films were investigated by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, and Fourier transform infrared spectroscopy in attenuated total reflection mode. The thickness and the mass of these films grow exponentially with the number of deposited bilayers. Moreover, PGA undergoes a random/R-helix transition when interacting with PAH during the film buildup process. This structural transition leads to (PGA/PAH)i films with an R-helix content (contribution of the R-helices to the amide I band) that switches regularly between 30% and 40% during the film buildup, when the multilayer is alternatively brought into contact with the PAH and PGA solutions. The secondary structure of the film is thus entirely driven by the last deposited layer. The independence of the R-helix content with the number of deposited bilayers also strongly suggests that the film is structurally homogeneous over its whole thickness.

The structural changes in fibrinogen as a consequence of its adsorption onto the surface of or its embedding into the interior of poly(allylamine hydrochloride) (PAH) or poly(styrenesulfonate) (PSS) multilayers are investigated by means of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. It is found that both adsorption and embedding preserve the secondary structure of the fibrinogen molecules. Furthermore, the interactions of the polyelectrolytes with the protein molecules prevent their aggregation, especially in the embedded state, at room temperature. Thus, it seems that the structure and the biological activity of proteins adsorbed on or embedded in polyelectrolyte multilayers could largely be preserved, which opens up great perspectives in the design of new bioactive surfaces. The nature and the extent of the polyelectrolyte-protein interactions are further studied via analysis of the thermotropic responses of the different architectures. It is found that both PAH-and PSS-terminated polyelectrolyte multilayers can elevate the onset temperature of the structural changes in adsorbed/embedded fibrinogen molecules by about 5 °C as compared with that for fibrinogen in solution. These polyelectrolytes also broaden the thermally induced structural transitions in the adsorbed/embedded fibrinogen molecules. The magnitude of these thermally induced structural changes is polyelectrolyte-and architecture-dependent. Whereas multilayer PAH-fibrinogen and multilayer PSS-fibrinogen constructions exhibit roughly the same large-scale thermally induced structural changes, in all architectures where fibrinogen is embedded the scale of these structural changes is restricted. The restriction becomes stronger as the presence of PSS at the polyelectrolyte-fibrinogen interfaces increases (PAH-fib-PAH < PAH-fib-PSS ≈ PSS-fib-PAH < PSS-fib-PSS). In the PSS-fib-PSS arrangement, the secondary structure of fibrinogen as determined from its infrared spectrum changes only slightly up to 90 °C. The underlying processes of the thermally induced structural changes is, in addition, different for fibrinogen molecules adsorbed onto or embedded into PAH-terminated polyelectrolyte multilayers. A tentative model based on "encapsulation" of the embedded protein by the polyelectrolytes is proposed to explain the observed features.

The mechanism of aggregation of bovine serum albumin (BSA) by poly(allylamine) hydrochloride (PAH) is investigated as a function of the mixing ratio r defined as the ratio of the number of BSA molecules and PAH chains present in the solution, under pH conditions of strong binding between the two partners. It is found that as r increases the turbidity first increases, passes through a maximum at a value r max before decreasing again. For small and large values of r, one forms small aggregates in the 10 nanometer size range, whereas at r max , the size of the aggregates becomes of the order of micrometers. The structure of the aggregates appears to be independent of the history of the systems but depends only on the value of r despite the strong BSA/ PAH binding. The desaggregation of the large aggregates formed at r max by the addition of BSA or PAH is shown to be an isenthalpic process and is thus entirely entropically driven. Moreover, we prove that r max corresponds to the state of the system where both the PAH chains and the BSA molecules interact one with each other, both with their maximum number of interaction points per molecule. This explains the independence of r max on the BSA or PAH concentration in the solution and why it varies linearly with the molecular weight of the polyelectrolyte. Moreover, we show that at r max , all the BSA and PAH molecules present in the solution are involved in the aggregates. At small (respectively large) values of r, the aggregates appear positively (respectively negatively) charged, corresponding to a charge excess at the surface of the aggregates. Finally, it is found that the protein/polyelectrolyte interaction is endothermic, indicating that the BSA/PAH binding must thus be entropically driven. The binding enthalpy of BSA molecules with PAH chains for r < r max is of the order of 400 kJ‚mol -1 of BSA molecules for solutions containing less than 0.1M of NaCl. The effect of the salt concentration of the solution on the binding process is also briefly discussed.

Polyelectrolyte multilayers constructed from polypeptides present secondary structures similar to those found in proteins (R-helices and β-sheets). These secondary structures are used as a tool to investigate the buildup and internal stability of multilayer films by means of Fourier transform infrared spectroscopy. Special attention is focused on the β-sheet contribution to the amide I band. Two main problems are addressed: (i) Does there exist a correlation between the local structure of the polypeptide multilayers and their corresponding polyanion/polycation complexes in solution? (ii) How stable is the local structure of these multilayers toward external stresses such as pH jumps, temperature rise, and changes of the nature of the outer layers of the film? Four different polypeptide multilayers, poly(L-glutamic acid)/poly(Llysine) (PGA/PLL), poly(L-aspartic acid)/poly(L-lysine), poly(L-glutamic acid)/poly(D-lysine), and poly(Lglutamic acid)/poly(L-ornithine), are studied. It is shown that the film secondary structures always closely resemble those of their corresponding complexes in solution. For example, the absence of β-sheet structures in the films correlates with their absence in solution. This shows the strong similarity between the physical processes leading to the formation of polypeptide complexes in solution and those involved in the multilayer formation. The secondary structures of (PGA/PLL)n films appear very stable against pH jumps for pH values ranging between 4 and 10.5. On the other hand, the sudden contact of a film constructed at pH 7.4 with a solution at pH 1.5 or 13.5 leads to a strong reduction of its β-sheet content together with a partial or total dissolution of the film. The structural response of a (PGA/PLL) n film to a temperature rise up to 89 °C depends on the way in which the temperature increase is performed: a slow temperature increase induces a reversible decrease of the β-sheet content at the expense of the R-helices. On the contrary, when the film is heated rapidly, the β-sheet content increases and a further increase is observed during cooling to room temperature. Finally, the deposition of poly(styrene sulfonate)/poly(allylamine) (PSS/PAH) bilayers on top of (PLL/PGA) n films leads to the total disappearance of the β-sheets. This seems to be related to the diffusion of PSS chains into the film during the first PSS deposition steps and an exchange of PGA molecules of the film by PSS ones deep in the architecture. Such an exchange process between two polyelectrolytes of different nature inside a multilayer architecture was, to the authors' knowledge, never observed before.

The structural changes of bovine serum albumin (BSA) and hen egg white lysozyme (HEL) upon their adsorption onto the surface or their embedding into the interior of poly(allylamine hydrochloride)-(poly(styrenesulfonate) (PAH-PSS) multilayer architectures were investigated by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The presence of the polyelectrolytes seems, as previously observed for fibrinogen (J. Phys. Chem. B 2001, 105, 11906-11916), to prevent intermolecular interactions and, thus, protein aggregation at ambient temperature. The secondary structure of the proteins was somewhat altered upon adsorption onto the polyelectrolyte multilayers. The structural changes were larger when the charges of the multilayer outer layer and the protein were opposing. The adsorption of further polyelectrolyte layers onto protein-terminated architectures (i.e., embedding the proteins into a polyelectrolyte multilayer) did not cause considerable further changes in their secondary structures. The capacity of the polyelectrolyte architectures to delay the formation of intermolecular beta-sheets upon increasing temperatures was not uniform for the studied proteins. PSS in contact with HEL could largely prevent the heat-induced aggregation of HEL. In contrast, PAH had hardly any effect on the aggregation of BSA. The differences are explained on the basis of protein-polyelectrolyte interactions, affected mostly by the nature and the strength of the ionic interactions between the polyelectrolyte-protein contact surfaces.

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We describe the build‐up of biomaterial coatings based on polypeptide multilayers possessing anti‐inflammatory properties. Poly(L‐lysine) (PLL) and poly(L‐glutamic acid) (PGA) are used as polypeptides, and piroxicam (Px) is used as the anti‐inflammatory agent. In order to embed high enough amounts of Px, the drug is incorporated in the films in the form of complexes with a charged 6A‐carboxymethylthio‐β‐cyclodextrin (cCD). It is shown that this cyclodextrin can solubilize higher amounts of Px than the cyclodextrins used commercially. The anti‐inflammatory properties are evaluated by determining the inhibition of TNFα production by human monocytic THP‐1 cells stimulated with lipopolysaccharide (LPS) bacterial endotoxin. Using Fourier‐transform (FT) Raman spectroscopy, we show that Px is mainly in the neutral form in cCD–Px complexes in solution, and that it remains biologically active under this form, whereas up to now only the zwitterionic form was reported to possess anti‐inflammatory properties. When incorporated in PLL/PGA multilayers, Px in the cCD–Px complexes changes from the neutral to the zwitterionic form. It is shown that these films present anti‐inflammatory properties, which can be delayed, and whose duration can be tuned by changing the film architecture.