Catherine Picart

Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers

The structure of poly(l-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an “in” and “out” diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.

Multilayer Polyelectrolyte Films Functionalized by Insertion of Defensin: a New Approach to Protection of Implants from Bacterial Colonization

ABSTRACT Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic surgery. In the present study, we developed a new strategy based on the insertion of an antimicrobial peptide (defensin from Anopheles gambiae mosquitoes) into polyelectrolyte multilayer films built by the alternate deposition of polyanions and polycations. Quartz crystal microbalance and streaming potential measurements were used to follow step by step the construction of the multilayer films and embedding of the defensin within the films. Antimicrobial assays were performed with two strains: Micrococcus luteus (a gram-positive bacterium) and Escherichia coli D22 (a gram-negative bacterium). The inhibition of E. coli D22 growth at the surface of defensin-functionalized films was found to be 98% when 10 antimicrobial peptide layers were inserted in the film architecture. Noticeably, the biofunctionalization could be achieved only when positively charged poly(l-lysine) was the outermost layer of the film. On the basis of the results of bacterial adhesion experiments observed by confocal or electron microscopy, these observations could result from the close interaction of the bacteria with the positively charged ends of the films, which allows defensin to interact with the bacterial membrane structure. These results open new possibilities for the use of such easily built and functionalized architectures onto any type of implantable biomaterial. The modified surfaces are active against microbial infection and represent a novel means of local host protection.

Ion pairing and hydration in polyelectrolyte multilayer films containing polysaccharides.

Thin films constituted of poly(L-lysine) (PLL) as polycation and of the anionic polysaccharides hyaluronan (HA), chondroitin sulfate (CSA), and heparin (HEP) as polyanions with increasing sulfate contents have been investigated for their internal structure, including water content and ion pairing. Film buildup in physiological solutions was followed in situ by quartz crystal balance with dissipation monitoring (QCM-D) and attenuated total internal reflectance (ATR-FTIR), infrared spectroscopy (ATR-FTIR), which allows an unambiguous quantification of the groups (sulfate, carboxylate, ammonium) present on the side groups of the polyelectrolytes. HA- and CSA-based films were the most hydrated ones. The monomer ratio (disaccharide/lysine) was very similar for all the films, whatever the polyanion, and tended toward a plateau value at approximately 0.5, indicating that there are two lysine molecules per disaccharide monomer. Thanks to the possibility to selectively cross-link carboxylate and ammonium ions via carbodiimide chemistry, the COO-/NH3+ and SO3-/NH3+ ion pairing was determined. We found that 46% of NH3+ groups are unpaired (i.e., extrinsically compensated by counterions) in HA-based films, 21% in CSA-based films and none in HEP ones, which is indeed in agreement with fluorescence recovery after photobleaching (FRAP) measurements of fluorescently labeled PLL diffusion in the films. In addition, the ratio of SO3- versus COO- pairing with NH3+ groups was close to the stoechiometry of these groups in the dissacharide monomeric unit, that is, 2:1 for HEP-based films and 1:1 for CSA based films. Thus, hydration, ion pairing, and PLL diffusion in the films are interconnected properties that arise from the specific structures of the biomacromolecules constituting the films.

Determination of structural parameters characterizing thin films by optical methods: A comparison between scanning angle reflectometry and optical waveguide lightmode spectroscopy

We present a comparative study of the structural parameters characterizing thin macromolecular adsorbed films that are obtained from two optical techniques: optical waveguide lightmode spectroscopy (OWLS) and scanning angle reflectometry (SAR). We use polyelectrolyte multilayers and polyelectrolyte multilayers/protein films to perform this study. The comparison between the information obtained with the two methods is possible because the buildup of the polyelectrolyte multilayers is known to become substrate independent after the deposition of the first few polyelectrolyte layers. The analysis of the optical data requires usually to postulate a refractive index profile for the interface. Two profiles have been used: the homogeneous and isotropic monolayer and the bilayer profiles. When the refractive index profile of an adsorbed film is well approximated by a homogeneous and isotropic monolayer, as shown by using an analysis of the deposited films in terms of optical invariants, the two optical techniques...

Designing Hyaluronic Acid-Based Layer-by-Layer Capsules as a Carrier for Intracellular Drug Delivery

Polyelectrolyte microcapsules were prepared by the layer-by-layer assembly of hyaluronic acid (HA) and a polycationic polymer, poly(allylamine) (PAH) or poly(lysine) (PLL). The influence of the polycationic partner on the morphology, stability, permeability properties, and enzymatic degradation of microcapsules was thoroughly analyzed. It was found that these properties could be tuned by shell cross-linking. Confocal microscopy studies of cellular uptake of the capsules showed that the polyelectrolyte shells remain stable outside the cells but readily break open once internalized by cells, suggesting their potential as carrier for intracellular drug delivery.

Self Assembling and Crosslinking of Polyelectrolyte Multilayer Films of Chitosan and Alginate Studied by QCM and IR Spectroscopy

The formation of novel biocompatible multilayer films based on the alternate deposition of CHI and ALG was investigated for the first time by QCM-D and FTIR-ATR. A linear increase of the thickness was found during the film build-up. GLUT was used to crosslink the films terminated with either CHI or ALG. A change in the QCM-D signal was observed just in the first case, indicating that crosslinking only takes place in the top CHI layer. The evolution of the dissipation factor during crosslinking was modelled with a first-order kinetics; this reaction was found to be faster for chitosan terminated films with a lower number of multilayers. It was also found that more robust films could be produced by crosslinking the intermediate CHI layers during the build-up.

Human blood shear yield stress and its hematocrit dependence

Human blood cells build a percolating physical gel all over the sample when at rest. This gel is progressively broken when it is continuously sheared in the bulk. It can slip at the wall, a phenomenon that depends on the roughness of the surface. Hence, smooth and rough walls were used to investigate the rheometrical shear properties of blood. A Couette-type rheometer with cylindrical walls allowed the shear rate to be varied in the range 10−3–10 s−1 and the hematocrit in the range 0.53–0.95. Calibration was performed with standard silicon oil. The stress measured at low shear rates with rough walls seemed, indeed, to tend to a constant yield stress value. The value of the shear stress at the shear rate of 10−3 s−1 was taken as a realistic approximation of the yield stress of blood. This yield stress was measured for different values of cell concentration. Data were fitted and showed to be proportional to the cube of the concentration over the range of hematocrit studied. However, variability between the ...

Free-Standing Polyelectrolyte Membranes Made of Chitosan and Alginate

Free-standing films have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we prepared free-standing membranes by the layer-by-layer assembly of chitosan and alginate, two widely used biomaterials. Our aim was to produce a thick membrane and to study the permeation of model drugs and the adhesion of muscle cells. We first defined the optimal growth conditions in terms of pH and alginate concentration. The membranes could be easily detached from polystyrene or polypropylene substrate without any postprocessing step. The dry thickness was varied over a large range from 4 to 35 μm. A 2-fold swelling was observed by confocal microscopy when they were immersed in PBS. In addition, we quantified the permeation of model drugs (fluorescent dextrans) through the free-standing membrane, which depended on the dextran molecular weight. Finally, we showed that myoblast cells exhibited a preferential adhesion on the alginate-ending membrane as compared to the chitosan-ending membrane or to the substrate side.

Variation of Polyelectrolyte Film Stiffness by Photo-Cross-Linking: A New Way To Control Cell Adhesion

We report on the preparation of polyelectrolyte films based on biopolymers whose nanomechanical properties can be tuned by photo-cross-linking. Cationic poly(L-lysine) was layer-by-layer assembled with anionic hyaluronan (HA) derivatives modified by photoreactive vinylbenzyl (VB) groups. The study of the multilayer buildup by quartz crystal microbalance with dissipation monitoring showed that the presence of VB groups does not influence significantly the multilayer growth. Then the VB-modified HA incorporated into the films was cross-linked upon UV irradiation. UV spectroscopy measurements showed that the cross-linking rate of the multilayers increases with the amount of VB groups grafted onto HA chains. Force measurements performed by atomic force microscopy with a colloidal probe proved that the rigidity of the cross-linked films increases with the grafting degree of HA chains and consequently the number of cross-links. Cell culture assays performed on non-cross-linked and photo-cross-linked films with myoblast cells demonstrated that cell adhesion and proliferation are considerably improved with increasing film rigidity.