Microbial infections are a global problem. Due to the over and misuse of antibiotics, drug-resistant pathogens are becoming more common. It is imperative to explore broad spectrum antimicrobial approaches. In this work, we modified collagen/hyaluronic acid polyelectrolyte multilayers (PEMs) with the natural antimicrobial peptide, LL-37 to study hepatic infections. LL-37 was physisorbed and covalently linked to the surface of the PEMs. Escherichia coli DH10B were cultured in the presence of LL-37modified PEMs in bacterial adhesion and contact killing models. Physisorbed LL-37 PEMs prevented bacterial adhesion and could also kill pathogens in the surrounding environment due to the release of LL-37 from the film. Immobilized LL-37 PEMs resulted in less bacterial adhesion on the surface due to the presence of the peptide. Films were then placed in contact with primary rat hepatocytes as well as in hepatocyte/bacteria co-cultures. LL-37 input concentrations up to of 16μM did not exhibit cytotoxic effects on hepatocytes. The LL-37 modified PEMs exhibited a hepatoprotective effect on albumin and urea secretion functions in co-cultures. The hepatoprotective effects were dependent on the ratio of hepatocytes and bacteria as well as the concentration of LL-37. These findings are encouraging and demonstrate that LL-37 modified PEMs can be used to investigate hepatic infections caused by bacteria. / Master of Science

Layer-by-layer assembly of polyelectrolytes is a simple technique based on the self-assembly of polycations and polyanions mainly by electrostatic interactions, which has gained considerable scientific interest for its versatility of applications. Ease of fabrication process, inexpensive approach and use to coat surfaces with various geometries prompts the researchers to select this technique not only for the surface modification applications but also to study the processes which exploit the 3D matrix properties of polyelectrolyte multilayer films (PEMs). Recent advances have been made where PEMs coatings have been utilized for their bio-applications like drug delivery and in tissue engineering for modifying the biomaterial's surfaces. In the field of drug delivery and tissue engineering the location and availability of the constituent molecules is very important, which is defined by their ability to diffuse through the encapsulating material or reservoir. So the main objective of this thesis is to understand the transport of molecules in ultrathin Polyelectrolyte Multilayer Films in lateral as well as transverse direction to the substrate. To study this transport behaviour in PEMs, we have employed various strategies which can enhance or suppress the diffusivity across PEMs. Thus, understanding the diffusion at nanoscale resolution will lead us to design better host materials for loading of drugs and growth factors for various biomedical applications.

In this study we fabricated free standing multilayer films of polyelectrolyte complexes for potential use in tissue engineering of corneal stroma by using the layer-by-layer (LbL) approach. In the formation of these LbL films negatively charged, photocrosslinkable (methacrylated) hyaluronic acid (MA-HA) was used along with polycations polyethyleneimine (PEI) and poly(L-lysine) (PLL). Type I collagen (Col) was blended in with PLL for improving the water absorption and cell attachment properties of the films. It was shown that the LbL films could be easily peeled off from glass substrates due to the photocrosslinking of one of the LbL components, the hyaluronic acid. Film growth and composition were monitored with FTIR-ATR. Heights of peaks at 3383 cm-1, and 2958 cm-1increased along with the bilayer number confirming the polymer build-up. Film integrity and thickness were investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Films thicker than 5 bilayers (BLs) were found to be uniform in appearance and 10 BL (PEI/MeHA) films were calculated to be ca. 6 &mu / m thick. Atomic force microscopy (AFM) revealed that as the number of BLs increased, surface roughness decreased. Activity of methacrylated hyaluronic acid was shown by the increased resistance of photocrosslinked multilayer films against hydrolysis by hyaluronidase. Patterns could be created on the films by photocrosslinking further proving that the crosslinking step is successful. Since the ultimate goal was to construct a corneal stroma PEI/MA-HA films were tested with corneal stroma cells, keratocytes. Cell proliferation on PEI/MA-HA films was quite poor in comparison to TCPS. In order to improve the cell adhesion the tests were repeated with PLL/MA-HA. Collagen was added to decrease the hydrophilicity and introduce cell adhesion sequences (Arg-Gly-Asp, RGD) to improve cell proliferation on the films and thus PLL+Col/MA-HA films were also tested. Introduction of collagen to the PLL/MA-HA films was found to decrease water retention of the multilayer films and improve cell viability and proliferation. Col+PLL/MA-HA LbL thus appear to be a promising platform for tissue engineering, especially of corneal stroma.

Recent advances in multilayer self-assembly have enabled the precise construction of nanocomposite ultrathin films on a variety of substrates, from large-area planar surfaces to nanoparticles. As a result, a wide range of physico-chemical properties may be represented by selecting from an array of surface preparations, molecules, assembly conditions, and post-assembly treatments. Such multilayer nanofilm assemblies are particularly attractive for use as specialized membranes for selective transport, which have many applications for separations, sensors, and drug delivery systems. In this work, nanocomposite ultrathin films built with layer-by-layer (LbL) self-assembly methods have been applied to surface modification to control interfacial behavior, including diffusion, anti-fouling, and biomimetic membranes. Transport and interfacial properties of nanocomposite membranes constructed using LbL self-assembly with synthetic and/or bio-polymers were characterized, and permeability values of clinically relevant small molecules through the nanofilms were determined. Correlations between permeability and film properties were also examined. Nanofilm coatings around 100nm thickness decreased diffusion coefficients of glucose up to five orders of magnitude, and were found to greatly affect enzymatic glucose sensor responses. Surface modification on top of the nanofilms with poly(ethylene glycol) provided anti-fouling effects. However, weak-weak polyelectrolyte multilayers (PEMs) should not be used to control transport due to their susceptibility under normal physiological conditions. Natural/biological polymers also provided multilayer film structures at the specific conditions, but their transport-limiting properties were not significant compared to synthetic PEMs. Even when covalently crosslinked, biological PEMs did not reduce the permeability of a small molecule. Finally, the predicting model of projecting analyte permeation through multi-phase nanocomposite films comprised with known diffusion coefficients was theoretically and experimentally evaluated. The modeling was matched reasonably well to experimental data. The outcomes will be the key knowledge or engineering principles to support future efforts in research and development. It is anticipated that the system developed for determining transport properties will provide a general platform for assessing new candidate materials. The theory developed will be useful in estimating transport properties of novel nanocomposite materials that may be interesting in a broad array of chemical and biological systems, from analytical separations to implantable biomedical applications, and will provide useful design rules for materials and fabrication process selection.

Les films polyélectrolytes ont émergé comme un outil polyvalent dans le domaine desbiomatériaux et de l’ingénierie tissulaire. Dans cette étude, nous avons conçu des films à base debiopolymère, dont la rigidité peut être modulée par photo-réticulation. L’adhésion de bactéries etde cellules mammifères sur ces films a été étudiée. Une telle manipulation de rigidité superficielleconduit à une réponse différentielle des bactéries et des cellules mammifères. Les bactéries àGram négatif présentent une meilleure croissance sur des films nous alors que les cellulesmammifères préféraient les films plus rigides. Ces films ont été spatialement structurés à l’aided’un photomasque, permettant de créer des zones adjacentes de rigidité variable et de formecontrôlée. Les motifs photostructurés ont conduit les cellules à s’organiser préférentiellement surles zones les plus rigides. Une étude comparative a été réalisée avec des micropatronsbiochimiques. Les résultats ont montré des réponses similaires pour trois types cellulairesdifférents. Ces films offrent des perspectives intéressantes pour l’ingénierie tissulaire et pour letest de médicaments. / Polyelectrolyte multilayers have emerged as a versatile tool in the field of biomaterials and tissueengineering. In this study, photocrosslinkable polyelectrolyte films based on biopolymers whosestiffness can be easily tuned by UV irradiation were prepared. Then, they were tested againstbacteria and mammalian cells to address the influence of the film stiffness on cell behavior. Suchsuperficial stiffness manipulation resulted in differential response of bacteria and mammaliancells. Gram negative bacteria evidenced better growth on softer films while various mammaliancells preferred stiffer films. Stiffness patterns of various geometries and sizes were generated byexposing the films to the UV light through a photomask incorporated in transparent substrates.The patterned films composed of stiff motifs distributed in a soft background induced apreferential spatial organization, which depended on pattern shape and size. A comparative studywith commercial biochemical patterns revealed similar pattern fidelity for three differentmammalian cell types. Such mechanical patterns on a 2D film appear promising for futureapplications in tissue engineering or for drug screening.

No description available.

L'oxydation spontanée de la dopamine en solution légèrement basique a été étudiée sur la base de la publication de Lee [Science, 318:426-430, 2007], et le produit de la réaction a été identifié comme de la mélanine. La capacité de la mélanine de lier des groupements amines de façon covalente a été confirmée par la quantification des sites de liaison correspondants. En outre il est possible de rédisperser des agrégats de mélanine dans des solutions fortement basiques. Les grains de mélanine ainsi obtenus ont été utilisés pour construire des films multicouches avec le poly(diallyldimethyl ammonium) (PDADMA). Différentes méthodes d'oxydation de la dopamine pour former des films de mélanine à l'interface solide-liquide ont été développées. Toutes les méthodes mènent à la formation de films continus de mélanine ayant des morphologies de surface similaires. Elles deviennent imperméables à des sondes électrochimiques à partir d'une épaisseur de l'ordre de 10 nm. Une plus grande perméabilité à des sondes chargées positivement ou neutres qu'à des sondes négatives a été confirmée. L'adsorption de protéines à des revêtements de mélanine a été expliquée par une combinaison d'interactions électrostatiques et covalentes. Pour arriver à cette explication le potentiel zêta de dépôts de mélanine a été mesuré en fonction du pH. La formation de la mélanine dans des films multicouches de poly(L-lysine) (PLL) et de hyaluronate (HA) a été étudiée: la mélanine est capable de remplir des films (PLL-HA)n de manière homogène, et les composés ainsi obtenus peuvent être détachés de leurs substrats comme membranes autosupportées préparées par une méthode biomimétique sous conditions douces.

<p>Die vorliegende Arbeit beschäftigte sich mit zwei Themengebieten. Es ging zum einen um die mechanischen Eigenschaften von Polyelektrolythohlkapseln und zum anderen um die Adhäsion von Polyelektrolythohlkapseln.</p> <p>Die mechanischen Eigenschaften wurden mit der AFM „colloidal probe” Technik untersucht. Dabei zeigte sich, dass die Kraftdeformationskurven für kleine Deformationen den nach der Schalentheorie vorhergesagten linearen Verlauf haben. Ebenso wurde die quadratische Abhängigkeit der Federkonstanten von der Dicke bestätigt. Für PAH/PSS findet man einen E-Modul von 0.25 GPa. Zusammen mit der Tatsache, dass die Deformationskurven unabhängig von der Geschwindigkeit sind und praktisch keine Hysterese zeigen, sowie der Möglichkeit die Kapseln plastisch zu deformieren, kann man schließen, dass das System in einem glasartigen Zustand vorliegt.</p> <pt>Erwartungsgemäß zeigte der pH einen starken Einfluss auf die PEM. Während in einem pH-Bereich zwischen 2 und 11.5 keine morphologischen Änderungen festgestellt werden konnten, vergrößerte sich der Radius bei pH = 12 um bis zu 50 %. Diese Radienänderung war reversibel und ging einher mit einem sichtbaren Weicherwerden der Kapseln. Eine Abnahme des E-Moduls um mindestens drei Größenordungen wurde durch Kraftdeformationsmessungen bestätigt. Die Kraftdeformationskurven zeigen eine starke Hysterese. Das System befindet sich nun nicht mehr in einem glasartigen Zustand, sondern ist viskos bis gummiartig geworden.</p> <p>Messungen an Kapseln, die mit Glutardialdehyd behandelt wurden, zeigten, dass die Behandlung das pH-abhängige Verhalten verändert. Dies kann darauf zurückgeführt werden, dass das PAH durch den Glutardialdehyd quervernetzt wird. Bei einem hohen Quervernetzungsgrad, zeigen die Kapseln keine Änderung des mechanischen Verhaltens bei pH = 12. Schwach quervernetzte Kapseln werden immer noch signifikant weicher bei pH = 12, jedoch ändert sich der Radius nicht.</p> <p>Außerdem wurden Multilagenkapseln untersucht, deren Stabilität nicht auf elektrostatischen Wechselwirkungen sondern auf Wasserstoffbrückenbindungen beruhte. Diese Kapseln zeigten eine deutlich höhere Steifigkeit mit E-Modulen bis zu 1 GPa. Es wurde gefunden, dass auch dieses System für kleine Deformationen ein lineares Kraft-Deformationsverhalten zeigt, und dass die Federkonstante quadratisch von der Dicke abhängt. Die Kapseln lösen sich praktisch sofort bei pH = 6.5 auf. In der Nähe dieses pHs konnte das Abnehmen der Federkonstanten verfolgt werden.</p> <p>Außerdem wurde das Adhäsionsverhalten von PAH/PSS Kapseln auf mit PEI-beschichtetem Glas untersucht. Die Adhäsionsflächen waren zu einem großen Teil rund und ließen sich quantitativ auswerten. Der Adhäsionsradius nimmt mit dem Kapselradius zu und mit der Dicke ab. Das Verhalten konnte mit zwei Modellen, einem für die große und einem für die kleine Deformation beschrieben werden. Das große Deformationsmodell liefert um eine Größenordung niedrigere Adhäsionsenergien als das kleine Deformationsmodell, welches mit Werten von ‑0.2 mJ/m² Werte in einem plausiblen Bereich liefert. Es wurde gefunden, dass bei einem Verhältnis von Dicke zu Deformation von etwa eins "buckling" auftritt. Dieser Punkt markierte zugleich den Übergang von der großen zur kleinen Deformation.</p> / <p>This work had two objectives. The first was to study the mechanical properties of polyelectrolyte hollow capsules depending on the pH and the wall composition utilizing the AFM colloidal probe technique. The second objective was to study the adhesion of these capsules varying the radius and thickness.</p> <p>It was found that the AFM colloidal probe technique can be utilized to measure the shells spring constant. The acquired deformation curves for small deformations showed the linear force deformation relation predicted by the shell theory. The stiffness of capsules composed of polyallylamine and polystyrenesulfonate scales quadratically with the thickness giving Young’s modulus of 0.25 GPa.</p> <p>Changing the pH had no effect concerning the morphology and the elastic properties of the afore mentioned capsules up to pH = 11.5. At pH = 12 the capsules’ radius increased between 30 % and 50 % and the elastic modulus decreased by more than one order of magnitude. This pH-change can be viewed as the glass transition of the material, being in a glassy state below pH = 11.5 and becoming rubbery at pH = 12. Introducing crosslinking into the capsule wall inhibited any morphological changes at pH = 12, while leaving the capsule stiffness sensitive for small degrees of crosslinking.</p> <p>It was possible to prove the quadratic thickness dependency of the stiffness for a system where the internal forces keeping together the multilayer were hydrogen bonds (poly(methacrylic acid) / poly-4-vinylpyrrolidone). These apsules are significantly stiffer than the afore mentioned ones (Young’s modulus 0.6 to 1 GPa) and show a strong reaction towards increasing the pH by dissolving within seconds. Close to the dissolution pH these capsules also exhibit a transition from a glassy to a rubbery state.</p> <p>Capsules composed of polyallylamine / polystyrene sulfonate adhered to polyethylene imine coated glass. The radius of the adhesion disc increased with the radius of the capsule and decreased with increasing thickness. This behaviour could be semi quantitatively described with a strong and a weak deformation model giving a value for the adhesion energy of ‑0.02 mJ/m² for the small deformation model. At deformations of the order of the thickness, the theoretically predicted buckling was observed.</p>

Une analyse détaillée des propriétés physico-chimiques des nanoparticules (NP) anthropogéniques est nécessaire pour comprendre à un niveau mécanistique leurs interactions/toxicité potentielle avec/envers les composants biotiques des systèmes aquatiques naturels. Une telle analyse est également requise pour réaliser une évaluation complète et une optimisation de la performance des méthodes d’(ultra)filtration développées pour circonscrire le relargage des NPs dans les milieux aquatiques. Dans ce contexte, l'objectif de cette thèse de doctorat était de déchiffrer les processus physico-chimiques fondamentaux régissant la capture de nanodendrimères carboxylés (PAMAM-COOH) - utilisés fréquemment dans des applications biomédicales – par des films multicouches du type (poly(diallyldiméthylammonium)chlorure-poly(acide acrylique))n ((PDADMAC-PAA)n) assemblés par déposition séquentielle des composantes polymériques cationique et anionique. À cette fin, une étude systématique des propriétés électrohydrodynamiques des NPs PAMAM-COOH a d'abord été effectuée en fonction du pH et de la concentration en sel monovalent du milieu. Sur la base de la théorie électrocinétique de particules molles ayant une fonctionnalité zwitterionique, il est démontré que les caractéristiques électriques interfaciales des NPs considérées sont déterminées à la fois par des contributions électrostatiques de surface et volumique des nanoparticules, lesquelles dependent de l’extension intraparticulaire de la double couche électrique. L’existence de ces deux types de contributions conduit à un changement remarquable de signe de la mobilité des NPs en modifiant la concentration du sel monovalent en solution et à une dépendance prononcé du point de zéro mobilité des NPs avec la concentration de l’électrolyte. En outre, une confrontation quantitative entre résultats expérimentaux et théorie souligne comment les modifications structurales des NPs induites par des changements de pH et de salinité affectent les caractéristiques électrocinétiques des dendrimères. Dans une deuxième partie, la structure, la morphologie et les propriétés mécaniques des films PDADMAC-PAA et leur évolution temporelle dans des conditions de vieillissement naturel ou après traitement thermique ont été déterminées par microscopie à force atomique (AFM) et analyses microspectroscopie Raman. Les résultats démontrent que les films multicouches PDADMAC-PAA de type exponentiel présentent des caractéristiques mécaniques et structurelles typiques de films polyélectrolytes multicouches à croissance linéaire. En particulier, leur relaxation lente vers un état d’équilibre est accélérée après traitement thermique à 60°C et se révèle être intimement liée à l'instabilité de domaines de films riches en PDADMAC, épuisés en eau (faits confirmés par la théorie de la fonctionnelle de la densité) et marqués par la présence de structures caractéristiques en forme de ‘donuts’. Dans une dernière partie, des résultats préliminaires sont donnés pour la dépendance de l'élasticité des films multicouches PDADMAC-PAA avec la concentration en solution de nanodendrimères. Les résultats suggèrent que ces films multicouches complexes constituent une option prometteuse pour la capture et l'élimination de nanodendrimères carboxylés présents en milieux aqueux / A detailed analysis of the physicochemical properties of engineered nanoparticles (NPs) is required to understand on a mechanistic level their interactions/potential toxicity with/towards biotic components of fresh water systems. Such an analysis is further mandatory to achieve a comprehensive evaluation and optimisation of the performance of (ultra)filtration methods developed to prevent NPs release into aquatic media. Within this context, the aim of this PhD thesis was to decipher the basic physico-chemical processes governing the loading of carboxylated-poly(amidoamine) (PAMAM-COOH) nanodendrimers -commonly employed in biomedical applications- into layer-by-layer assembled (poly(diallyl dimethyl ammonium) chloride-poly(acrylic acid))n ((PDADMAC-PAA)n) multilayer films. For that purpose, a systematic investigation of the electrohydrodynamic properties of PAMAM-COOH NPs was first performed as a function of pH and monovalent salt concentration in solution. On the basis of advanced electrokinetic theory for soft particles with zwitterionic functionality, it is demonstrated that the interfacial electrostatic features of the considered NPs are determined both by surface and bulk particle contributions to an extent that depends on electrolyte concentration. This leads to a remarkable NPs mobility reversal with changing monovalent salt concentration and to a marked dependence of the point of zero NPs mobility on electrolyte content. In addition, confrontation between experiments and theory further highlights how pH- and salt-mediated modifications of the NP particle structure affect dendrimer electrokinetic features at large pH and/or low salt concentrations. In a second part, the structure, morphology and mechanical properties of PDADMAC-PAA films, and their evolution over time under natural aging conditions or after thermal treatment, were addressed from atomic force microscopy (AFM) and Raman microspectroscopy analyses. Results evidence that PDADMAC-PAA multilayer films of exponential type exhibit mechanical and structural features that are typical for polyelectrolyte multilayer films with linear growth. In particular, their slow relaxation to equilibrium is accelerated after heating treatment at 60°C and, in line with density functional theory computation, this relaxation dynamics is shown to be intimately connected to instability of film domains rich in PDADMAC, depleted in water and marked by the presence of characteristic donut-like structures. In a final part, the reported dependence of PDADMAC-PAA multilayer films elasticity on concentration of nanodendrimers in bulk solution suggests that these complex multilayer films constitute a promising option to be further investigated for the loading and removal of carboxylated nanodendrimers from aqueous environments

Polyelectrolyte complexation is a rapidly growing field with applications in functional multilayer (PEM) and nanoparticle (PEC) generation, where PEM films are deposited using Layer-by-Layer technique initiated by Decher and PECs are prepared using mixing-centrifugation technique initiated by our group. Its advantages (e.g. easy preparation) result in various applications in aqueous solution, especially in pharmaceutical and biomedical fields. Therefore, the objectives in this study are to explore interesting applications of polyelectrolyte complexation in the field of low molecular chiral compound and high molecular protein binding. Due to the rapidly growing demands for preparing optically pure compounds in the pharmaceutical field, herein, enantiospecific PEM and PEC were prepared using chiral polyelectrolytes (e.g. homo-polypeptide) and their ability of chiral recognition was investigated by ATR-FTIR, UV/Vis etc.. Chiral PEM and PEC showed pronounced enantiospecificity for both small (amino acids, vitamin) and large (protein) chiral compounds. This chiral recognition is performed by a diffusion process of chiral compounds into PEM based on the structures of chiral selector (PEM, PEC) and chiral probes (chiral compounds). However, the influences, e.g. pH value, ionic strength, surface orientation etc., were found to affect significantly the enantiospecificity. Beside planar substrates, porous membranes (e.g. PTFE) were modified using chiral PEM and successfully applied in enantiospecific permeation. Additionally, protein binding properties of PEC particle dispersions or PEC particle films were also studied. Due to the properties of polyelectrolytes used for PEC (e.g. molecular weight, charge density) and proteins (e.g. isoelectric point, size, hydrophobicity), PEC showed different uptake characteristics towards different proteins. Electrostatic and hydrophobic interaction as well as counterion release force were considered as possible driving forces for protein binding.