In vitro, la formation de structures de type tubulaire avec des cellules endothéliales de veine ombilicale humaine (HUVEC) a été étudiée en combinant la fonctionnalisation de la chimie des matériaux et le développement de la géométrie tridimensionnelle. Le polycarbonate (PC) a été utilisé comme modèle pour le développement de l'échafaud. Le film de polysaccharide naturel, basé sur un dépôt alternatif couche par couche (LbL) d’acide hyaluronique (HA) et de chitosane (CHI), a d’abord été appliqué sur une surface PC et caractérisé en termes de croissance d’épaisseur microscopie à balayage lascar (CLSM). Cette première fonctionnalisation se traduit par un revêtement complet de la couche PC. Une biofonctionnalisation supplémentaire avec un peptide adhésif (RGD) et deux peptides angiogénétiques (SVV et QK) a été étudiée, immobilisant ces peptides sur le groupe carboxylique de HA précédemment déposé, en utilisant la chimie bien connue du carbodiimide. La version marquée de chaque peptide a été utilisée pour caractériser l’immobilisation et la pénétration des peptides dans les couches de polyélectrolytes, aboutissant à une greffe réussie avec une pénétration complète dans toute l’épaisseur du LbL. Des tests in vitro ont été effectués à l'aide de cellules HUVEC pour évaluer leur efficacité d'adhésion et leur activité métabolique sur la LbL avec et sans immobilisation de peptides, ce qui a permis d'améliorer l'activité préliminaire lorsque des combinaisons de peptides sont utilisées. Enfin, les micro-canaux PC (μCh) ont été développés et caractérisés pour la première fois, et les autres expériences ont été réalisées sur un micromètre de 25 μm de largeur, fonctionnalisé avec une architecture (HA / CHI) 12,5 (PC-LbL) avec des peptides RGD et QK -RGD + QK) ou avec des peptides RGD et SVV (PC-RGD + SVV). Notre première expérience de tubulogénèse a montré de manière surprenante la formation de structures de type tubulaire déjà après 2h d'incubation en utilisant la combinaison double-peptides, mais uniquement avec PC-RGD + QK. Les tubes étaient également présents après 3 et 4 heures de culture. L'expérience de co-culture avec des péricytes humains dérivés du placenta (hPC-PL) montre comment la stabilisation des tubes a été améliorée après 3 et 4 heures également pour l'échantillon de PC-RGD + SVV. Globalement, notre matériel bio-fonctionnel avec les peptides PC-RGD + QK et PC-RGD + SVV permet la formation d'une structure de type tubulaire à la fois dans une expérience de monoculture et de co-culture. / In vitro, tubular-like structures formation with human umbilical vein endothelial cells (HUVECs) was investigated by combining material chemistry functionalization and three-dimensional geometry development. Polycarbonate (PC) was used as a template for the development of the scaffold. Natural polysaccharide’s film based on alternate layer-by-layer (LbL) deposition of hyaluronic acid (HA) and chitosan (CHI), was first applied to PC surface and characterized in terms of thickness growth both, in dry conditions using ellipsometry, and confocal lascar scanning microscopy (CLSM). This first functionalization results in a complete coating of the PC layer. Further biofunctionalization with one adhesive peptide (RGD) and two angiogenetic peptides (SVV and QK) was investigated, immobilizing those peptides on the carboxylic group of HA previously deposited, using the well-known carbodiimide chemistry. The labeled version of each peptide was used to characterize the peptides’ immobilization and penetration into the polyelectrolytes layers, resulting in a successful grafting with complete penetration through the entire thickness of the LbL. In vitro tests were performed using HUVECs to assess their adhesion efficiency and their metabolic activity on the LbL with and without peptide immobilization, resulting in a preliminary improved activity when peptide-combinations is used. Finally, PC micro-channels (μCh) were first developed and characterized, and the rest of the experiments were performed on μCh of 25μm width, functionalized with (HA/CHI)12.5 architecture (PC-LbL) with RGD and QK peptides (PC-RGD+QK) or with RGD and SVV peptides (PC-RGD+SVV). Our first tubulogenesis experiment surprisingly showed the formation of tubular-like structures already after 2h of incubation using the double-peptides combination but only using PC-RGD+QK the tubes were present also after 3 and 4 hours of culture. The co-culture experiment with human pericytes derived from placenta (hPC-PL) demonstrates how the stabilization of the tubes was improved after 3 and 4 hours also for the PC-RGD+SVV sample. Globally our bio-functional material with PC-RGD+QK and PC-RGD+SVV peptides allow the formation of tubular-like structure in both mono and co-culture experiment.

Entender os processos em superfície/interface de filmes e seus efeitos sobre as propriedades ópticas e elétricas de materiais orgânicos é de grande importância tecnológica. Esta pesquisa descreve a fabricação e caracterização de filmes poliméricos extremamente finos (espessura <10 nm) e homogêneos compostos por camadas de polímero/polieletrólitos e estruturas com modulação de energia ou poços quânticos. O objetivo principal foi o estudo dos processos de transferência de carga e energia em tais estruturas. Os polímeros luminescentes utilizados foram poli(9,9 dioctilfluoreno) (PFO) poli(p-fenileno vinileno (PPV). O PPV foi obtido a partir do precursor poli(cloreto de tetraidrotiofeno de xililideno) (PTHT). A técnica de deposição denominada deposição camada por camada assistida por spin (SA-LbL) foi utilizada para obtenção dos filmes. Medidas de absorbância confirmaram o crescimento linear das camadas para as interfaces polieletrólito/polieletrólito e polímero/polieletrólito. Com o objetivo de entender a transferência do elétron &pi; do polímero conjugado para o polieletrólito, as configurações das estruturas poliméricas foram alteradas através da deposição de diferentes monocamadas de polieletrólito sobre o filme polimérico. Observamos que os elétrons &pi; foram efetivamente transferidos para os polieletrólitos que possuem alta afinidades eletrônica. Este efeito interfere fortemente na absorção bem como nas características de condução do filme polimérico ultrafino. A absorção é restabelecida após a conversão de PTHT em PPV. Medidas de fotoluminescência (PL) em filmes PFO/PPV resultam em curvas de emissão com picos característicos de ambos os polímeros, o que confirma que a técnica SA-LbL permite a deposição de estruturas poliméricas multicamadas. As várias configurações de filmes obtidas elucidaram os processos de transferência que ocorrem em diferentes interfaces, tais como: mudança da sequencia de deposição do polieletrólito, número de camadas duplas PTHT/DBS e a introdução de camadas separadoras. Além disso, um único poço quântico, ou seja, estruturas formadas por uma camada PPV cercada por barreiras de PFO com 10 nm de espessura foram obtidas. Medidas de absorbâcia, PL e excitação mostraram uma eficiente migração estado excitado da barreira de PFO para o PPV (poço). A homogeneidade da imagem confocal, demonstrou um rigoroso controle da camada de cobertura ao nível de um única monocamada e sem contaminação pelos materiais depositados sequencialmente. A microscopia confocal de fluorescência (CFM) e espectroscopia de fluorescência resolvida no tempo (FLIM) foram utilizadas para caracterizar a dinâmica do exciton e o seu confinamento nos poços quânticos. As medidas de CFM demonstraram que excitons que são gerados na barreira de PFO são eficientemente transferidos para o PPV. Além disso, o tempo de decaimento da emissão PFO residual é fortemente reduzido devido a processos de migração concorrentes no poço. O tempo de decaimento de PPV diminui substancialmente para poços com espessuras abaixo de 5 nm como resultado da auto-aniquilação do exciton. Dessa forma, as estruturas de MQW obtidos pela técnica de SA-LbL podem ser usadas para estudar a transferência de energia, efeitos túneis e para a construção de novos dispositivos optoelectrónicos com maior eficiência. / The understanding of surface/interface processes and their effects on optical/electrical properties of organic materials is of strong technological importance. This research describes the fabrication and characterization of extremely thin (thickness <10 nm) and homogeneous multilayered polymeric structures including polymer/poly-electrolyte layers and structures with energy modulation such as quantum well. Our main purpose was the study of charge and energy transfer processes in such energy modulated structures. The luminescent polymers used were Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly(p-phenylenevinylene) (PPV). PPV has been obtained from the poly(xylyliden tetrahydrothiophenium chloride) (PTHT) precursor. The so-called Spin Self-Assembly Layer-by-Layer deposition method (SA-LbL) was utilized to obtain the films. Absorption measurements confirmed the linear growth of layers using for polyelectrolyte/polyelectrolyte and polymer/polyelectrolyte interfaces. In order to understand the &pi;-electron transfer from the conjugated polymer to charged states of the polyelectrolyte, the configurations of the polymeric structures were modified by depositing different polyelectrolyte monolayer on the polymer film. We observed that &pi;-electrons were effectively transferred to polyelectrolytes that have high electron affinities. This effect strongly affects both absorption and conduction features of such very thin polymeric film. The absorption is restored after the conversion of PTHT in PPV. Photoluminescence measurements on PFO/PPV films result in emission curves with characteristic peaks of both polymers, confirming that SA-LbL technique allows deposition of multilayer polymeric structures. The various film configurations elucidates the transfer processes occurring at different interfaces like: change of polyelectrolyte deposition order, number of PTHT/DBS bilayers and introduction of spacers. In addition, Single Quantum Well (SQW), i.e., structures consisted of PPV layer surrounded of 10 nm thick PFO barriers were obtained. Optical absorption, PL and excitation spectroscopy showed an efficient excited state migration from the PFO barrier to the PPV well. The confocal image homogeneity demonstrated the layer coverage control at a monolayer level and without layer intermixing of the sequentially deposited polymeric materials. High resolution Confocal Fluorescence Microscopy (CFM) and Fluorescence Life spectroscopy and Imaging (FLIM) were used to characterize the exciton dynamics and confinement in quantum well. The CFM measurements demonstrated that excitons generated at the PFO barrier are efficiently transferred to the PPV well. Furthermore, the decay time of the residual PFO emission is strongly reduced due to the competing migration process in the well. The decay time of PPV decreases substantially for well thicknesses below 5 nm as a result of exciton self-annihilation. Thus, the MQW structures obtained by SA-LbL technique can be used to study energy transfer, tunneling effects and to build up new optoelectronic devices with greater efficiency.

Entender os processos em superfície/interface de filmes e seus efeitos sobre as propriedades ópticas e elétricas de materiais orgânicos é de grande importância tecnológica. Esta pesquisa descreve a fabricação e caracterização de filmes poliméricos extremamente finos (espessura <10 nm) e homogêneos compostos por camadas de polímero/polieletrólitos e estruturas com modulação de energia ou poços quânticos. O objetivo principal foi o estudo dos processos de transferência de carga e energia em tais estruturas. Os polímeros luminescentes utilizados foram poli(9,9 dioctilfluoreno) (PFO) poli(p-fenileno vinileno (PPV). O PPV foi obtido a partir do precursor poli(cloreto de tetraidrotiofeno de xililideno) (PTHT). A técnica de deposição denominada deposição camada por camada assistida por spin (SA-LbL) foi utilizada para obtenção dos filmes. Medidas de absorbância confirmaram o crescimento linear das camadas para as interfaces polieletrólito/polieletrólito e polímero/polieletrólito. Com o objetivo de entender a transferência do elétron &pi; do polímero conjugado para o polieletrólito, as configurações das estruturas poliméricas foram alteradas através da deposição de diferentes monocamadas de polieletrólito sobre o filme polimérico. Observamos que os elétrons &pi; foram efetivamente transferidos para os polieletrólitos que possuem alta afinidades eletrônica. Este efeito interfere fortemente na absorção bem como nas características de condução do filme polimérico ultrafino. A absorção é restabelecida após a conversão de PTHT em PPV. Medidas de fotoluminescência (PL) em filmes PFO/PPV resultam em curvas de emissão com picos característicos de ambos os polímeros, o que confirma que a técnica SA-LbL permite a deposição de estruturas poliméricas multicamadas. As várias configurações de filmes obtidas elucidaram os processos de transferência que ocorrem em diferentes interfaces, tais como: mudança da sequencia de deposição do polieletrólito, número de camadas duplas PTHT/DBS e a introdução de camadas separadoras. Além disso, um único poço quântico, ou seja, estruturas formadas por uma camada PPV cercada por barreiras de PFO com 10 nm de espessura foram obtidas. Medidas de absorbâcia, PL e excitação mostraram uma eficiente migração estado excitado da barreira de PFO para o PPV (poço). A homogeneidade da imagem confocal, demonstrou um rigoroso controle da camada de cobertura ao nível de um única monocamada e sem contaminação pelos materiais depositados sequencialmente. A microscopia confocal de fluorescência (CFM) e espectroscopia de fluorescência resolvida no tempo (FLIM) foram utilizadas para caracterizar a dinâmica do exciton e o seu confinamento nos poços quânticos. As medidas de CFM demonstraram que excitons que são gerados na barreira de PFO são eficientemente transferidos para o PPV. Além disso, o tempo de decaimento da emissão PFO residual é fortemente reduzido devido a processos de migração concorrentes no poço. O tempo de decaimento de PPV diminui substancialmente para poços com espessuras abaixo de 5 nm como resultado da auto-aniquilação do exciton. Dessa forma, as estruturas de MQW obtidos pela técnica de SA-LbL podem ser usadas para estudar a transferência de energia, efeitos túneis e para a construção de novos dispositivos optoelectrónicos com maior eficiência. / The understanding of surface/interface processes and their effects on optical/electrical properties of organic materials is of strong technological importance. This research describes the fabrication and characterization of extremely thin (thickness <10 nm) and homogeneous multilayered polymeric structures including polymer/poly-electrolyte layers and structures with energy modulation such as quantum well. Our main purpose was the study of charge and energy transfer processes in such energy modulated structures. The luminescent polymers used were Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly(p-phenylenevinylene) (PPV). PPV has been obtained from the poly(xylyliden tetrahydrothiophenium chloride) (PTHT) precursor. The so-called Spin Self-Assembly Layer-by-Layer deposition method (SA-LbL) was utilized to obtain the films. Absorption measurements confirmed the linear growth of layers using for polyelectrolyte/polyelectrolyte and polymer/polyelectrolyte interfaces. In order to understand the &pi;-electron transfer from the conjugated polymer to charged states of the polyelectrolyte, the configurations of the polymeric structures were modified by depositing different polyelectrolyte monolayer on the polymer film. We observed that &pi;-electrons were effectively transferred to polyelectrolytes that have high electron affinities. This effect strongly affects both absorption and conduction features of such very thin polymeric film. The absorption is restored after the conversion of PTHT in PPV. Photoluminescence measurements on PFO/PPV films result in emission curves with characteristic peaks of both polymers, confirming that SA-LbL technique allows deposition of multilayer polymeric structures. The various film configurations elucidates the transfer processes occurring at different interfaces like: change of polyelectrolyte deposition order, number of PTHT/DBS bilayers and introduction of spacers. In addition, Single Quantum Well (SQW), i.e., structures consisted of PPV layer surrounded of 10 nm thick PFO barriers were obtained. Optical absorption, PL and excitation spectroscopy showed an efficient excited state migration from the PFO barrier to the PPV well. The confocal image homogeneity demonstrated the layer coverage control at a monolayer level and without layer intermixing of the sequentially deposited polymeric materials. High resolution Confocal Fluorescence Microscopy (CFM) and Fluorescence Life spectroscopy and Imaging (FLIM) were used to characterize the exciton dynamics and confinement in quantum well. The CFM measurements demonstrated that excitons generated at the PFO barrier are efficiently transferred to the PPV well. Furthermore, the decay time of the residual PFO emission is strongly reduced due to the competing migration process in the well. The decay time of PPV decreases substantially for well thicknesses below 5 nm as a result of exciton self-annihilation. Thus, the MQW structures obtained by SA-LbL technique can be used to study energy transfer, tunneling effects and to build up new optoelectronic devices with greater efficiency.

Macroporous ceramic materials have found widespread technological application ranging from particulate filters in diesel engines, tissue engineering scaffolds, and as support materials in carbon capture processes. This thesis demonstrates how the pore space of macroporous alumina can be manipulated, analysed in three-dimensions (3D) using visualisation techniques, and functionalised with a CO2-adsorbing material. A novel method was developed to produce macroporous alumina materials: by combining sacrificial templating with thermally expandable polymeric microspheres and gel-casting of an alumina suspension. This method offers a versatile production of macroporous ceramics in which the level of porosity and the pore size distribution can easily be altered by varying the amount and type of spheres. The permeability to fluid flow could be regulated by controlling the connectivity of the pore space and the size of the smallest constrictions between the pores. Sacrificial templating with particle-coated expandable spheres significantly increased the fraction of isolated pore clusters and reduced both the sizes and the numbers of connections between neighbouring pores, compared to templating with un-coated spheres. The macroporous alumina materials were characterised with X-ray micro-computed tomography (μ-CT). The 3D data-sets obtained by X-ray μ-CT were used to calculate the spatial variation in porosity, the throat and pore size distributions and to calculate the permeability to fluid flow. The throat and pore size distributions were also able to be accurately quantified in only one extrusion and intrusion cycle with water-based porosimetry; a relatively novel and simple characterisation technique. The pore walls of the macroporous alumina materials were also coated with zeolite films by a colloidal processing technique. The CO2-uptake of the coated alumina materials and of hierarchically porous monoliths of zeolites was evaluated and compared. / As the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Accepted. Paper 5: Manuscript. Paper 6: Submitted.

This thesis describes technical and scientific aspects of new types of composite films/coatings for corrosion protection of carbon steel, composite films with nanometer thickness consisting of mussel adhesive protein (Mefp‐1) and ceria nanoparticles, and polymeric composite coatings with micrometre thickness consisting of conducting polymer and ceria nanoparticles in a UV‐curing polyester acrylate (PEA) resin. The influence of microstructure on corrosion behaviour was studied for a Fe‐Cr‐V‐N alloy containing micro‐sized nitrides with different chemical composition spread in martensitic alloy matrix. The Volta potential mapping suggested higher relative nobility for the nitride particles than the alloy matrix, and the nitrides with higher amounts of nitrogen and vanadium exhibited higher nobility. Potentiodynamic polarization measurements in a 0.1 M NaCl solution at neutral pH and ambient temperature showed passivity breakdown with initiation of localized corrosion which started in the boundary region surrounding the nitride particles, especially the ones enriched in Cr and Mo. Mefp‐1/ceria nanocomposite films were formed on silica and metal substrates by layer‐by‐layer immersion deposition. The film formation process was studied in situ using a Quartz Crystal Microbalance with Dissipation (QCM‐D). The film grows linearly with increasing number of immersions. Increasing Mefp‐1 concentration or using Mefp‐1 with larger size leads to more Mefp‐1 being deposited. Peak Force Quantitative Nanomechanical Mapping (Peak Force QNM) of the composite films in air indicated that the elastic modulus of the film increased when the film deposited had a higher Mefp‐1 concentration. It was also noted that the nature of the outermost layer can affect bulk morphology and surface mechanical properties of the film. The QCM‐D study of Mefp‐1 on an iron substrate showed that Mefp‐1 adsorbs at a high rate and changes its conformation with increasing adsorption time. The QCM‐D and in situ Peak Force QNM measurements showed that the addition of Fe3+ ions causes a transition in the single Mefp‐1 layer from an extended and soft layer to a denser and stiffer layer. In situ ATR‐FTIR and Confocal Raman Microscopy (CRM) analyses revealed complex formation between Fe3+ and catechol groups in Mefp‐1. Moreover, optical microscopy, SEM and AFM characterization of the Mefp‐1/ceria composite film formed on carbon steel showed micron‐size aggregates rich in Mefp‐1 and ceria, and a nanostructure of well dispersed ceria particles in the film. The CRM analysis confirmed the presence of Mefp‐1/Fe complexes in the film. Electrochemical impedance microscopy and potentiodynamic polarization measurements showed that the Mefp‐1/ceria composite film can provide corrosion protection for carbon steel, and that the protection efficiency increases with exposure time. Composite coatings of 10 μm thickness composed of a UV‐curing PEA resin and a small amount of conductive polymer and ceria nanoparticles were coated on carbon steel. The conductive polymer (PAni) was synthesized with phosphoric acid (PA) as the dopant by chemical oxidative polymerization. The ATR‐FTIR and SEM analyses confirmed that the added particles were well dispersed in the coatings. Electrochemical measurements during long exposure in 0.1 M NaCl solution, including open circuit potential (OCP) and EIS, were performed to investigate the protective performance of the coatings. The results showed that adding ceria nanoparticles can improve the barrier properties of the coating, and adding PAni‐PA can lead to active protection of the coating. Adding PAni‐PA and ceria nanoparticles simultaneously in the coating can improve the protection and stability of the composite coating, providing excellent corrosion protection for carbon steel. / <p>QC 20131024</p>

It is widely known that surfaces play an important role in numerous biological processes and technological applications. Thus, being able to modify surface properties provides an opportunity to control many phenomena occurring at interfaces. One way of controlling surface properties is to adsorb a polymer film onto the surface, for example through layer-by-layer (LbL) deposition of polyelectrolytes. This simple but versatile technique enables various polymers, proteins, colloidal particles etc. to be incorporated into the film, resulting in a multifunctional coating. Due to recent legislations and a consumer demand for more environmentally friendly products, we have chosen to use natural polymers (biopolymers) from renewable resources. The focus of this thesis has been on the adsorption of biopolymers and their layer-by-layer formation at solid-liquid interfaces; these processes have been studied by a wide range of techniques. The main method was the quartz crystal microbalance with dissipation monitoring (QCM-D), which measures the adsorbed mass, including trapped solvent and the viscoelastic properties of an adsorbed film. This technique was often complemented with an optical method, such as ellipsometry or dual polarization interferometry (DPI), which provided information about the “dry” polymer or protein adsorbed mass. From this combination, the solvent content and density of the layers was evaluated. In addition, the surface force apparatus (SFA), X-ray photoelectron spectroscopy (XPS), total internal reflection fluorescence (TIRF), and fluorescence resonance energy transfer (FRET) were utilized, providing further information about the film structure, chemical composition, and polymer inter-layer diffusion. Adsorption studies of the glycoprotein mucin, which has a key role in the mucousal function, showed that despite the net negative charge of mucin, it adsorbed on negatively charged substrates. The adsorbed layer was highly hydrated and the segment density on the substrate was low. We showed the importance of characterizing the mucin used, since differences in purity, such as the presence of albumin, gave rise to different adsorption behaviours in terms of both adsorbed amount and structure. The adsorbed mucin layer was to a large extent desorbed upon exposure to the anionic surfactant sodium dodecyl sulfate (SDS). In order to prevent desorption, we demonstrated that a protective layer of the cationic polysaccharide chitosan could be adsorbed onto the mucin layer and that the mucin-chitosan complexes resisted the desorption normally induced by association with SDS. Moreover, the association between chitosan and SDS was examined at the solid-liquid interface, in the bulk, and at the air-water interface. In all these environments chitosan-SDS complexes were formed and a net charge reversal of the complexes from positive to negative was observed when the concentration of SDS was increased. Furthermore, the LbL deposition method could be used to form a multilayer-like film by alternate adsorption of mucin and chitosan on silica substrates. The LbL technique was also applied to two proteins, lysozyme and β-casein with the aim of building a multilayer film consisting entirely of proteins. These proteins formed complexes at the solid-liquid interface, resulting in a proteinaceous layer, but the build-up was highly irregular with an increase in adsorbed amount per protein deposition cycle that was far less than a monolayer.Continuing with chitosan, known to have antibacterial properties we assembled multilayers with an anti-adhesive biopolymer, heparin, to evaluate the potential of this system as a coating for medical implants. Multilayers were assembled under various solution deposition conditions and the film structure and dynamics were studied in detail. The chitosan-heparin film was highly hydrated, in the range 60-80 wt-% depending on the deposition conditions. The adsorbed amount and thickness of the film increased exponential-like with the number of deposition steps, which was explained by inter-diffusion of chitosan molecules in the film during the build-up. In a novel approach, we used the distant dependent FRET technique to prove the inter-layer diffusion of fluorescent-labelled chitosan molecules within the film. The diffusion coefficient was insignificantly dependent on the deposition pH and ionic strength, and hence on the film structure. With the use of a pH sensitive dye buried under seven chitosan-heparin bilayers, we showed that the dye remained highly sensitive to the charge of the outermost layer. From complementary QCM-D data, we suggested that an increase in the energy dissipation does not necessarily indicate that the layer structure becomes less rigid. / Det är välkänt att ytor spelar en viktig roll i många biologiska processer och tekniska tillämpningar. Att kunna modifiera en ytas egenskaper ger därför en möjlighet att kunna kontrollera många fenomen som sker på ytor. Ett sätt att kontrollera ytegenskaperna är genom att adsorbera en polymerfilm på ytan, till exempel genom att växelvis adsorbera olika polyelektrolyter (LbL-teknik). Denna enkla men mångsidiga teknik möjliggör att många olika material kan införlivas i filmen, vilket resulterar i en multifunktionell beläggning. På grund av dagens lagstiftning och konsumenters ökade efterfrågan på miljövänliga material beslutade vi oss för att använda biologiska polymerer (biopolymerer) i detta projekt. Fokus i den här avhandlingen har varit på adsorption av biopolymerer och deras LbL-formation på gränsytan vätska-fast fas, där adsorptionsförloppet och det adsorberade skiktet bestående av biopolymerer studerats med en mängd olika tekniker. Huvudtekniken var kvartskristallmikrovåg med energidissipations-registrering (QCM-D), som mäter massan inklusive inkorporerat vatten, samt de viskoelastiska egenskaperna hos ett adsorberat skikt. Som komplement till denna teknik användes ofta optiska metoder, till exempel ellipsometri och ”dubbel polarisationsinterferometri (DPI)”, två tekniker som endast mäter massan av de adsorberade biopolymererna. Genom denna kombination av metoder kunde massan av inkorporerat vatten i filmen och filmens densitet bestämmas. Dessutom användes ytkraftsapparaten (SFA), röntgenfotoelektronspektrometri (XPS), och fluorescens-spektroskopiteknikerna TIRF och FRET i några undersökningar för att erhålla information om skiktens struktur, kemiska sammansättning och polymerernas diffusion inom skiktet.Adsorptionsstudier av glycoproteinet mucin, som har en central roll i funktionen av slemhinnan, avslöjade att trots att mucinet har en negativ nettoladdning adsorberade det ändå på negativt laddade substrat. Det adsorberade lagret var väldigt hydratiserat och hade en låg andel mucin i direkt kontakt med ytan. Vi påvisade vikten av att noga undersöka mucinet som användes, eftersom olika renhet, till exempel i form av förekomsten av albumin gav upphov till olika adsorptionsbeteende gällande både adsorberad mängd och struktur. En stor andel av det adsorberade mucinlagret desorberade när det exponerades för den anjoniska tensiden natriumdodecylsulfat, SDS. Vi visade att ett skyddande lager av den katjoniska polysackariden chitosan kunde adsorberas på mucinet och att mucin-chitosan-komplexen inte desorberade när SDS tillsattes. Därtill studerades växelverkan mellan chitosan och SDS på gränsytan vätska-fast fas, i bulken och på luft-vattengränsytan. Komplex av chitosan-SDS bildades i samtliga miljöer och en nettoladdningsomsvängning från positiv till negativ observerades när koncentrationen av SDS ökades.Vidare kunde LbL-tekniken nyttjas för att skapa ett multilagerlikt skikt genom att alternerande adsorbera mucin och chitosan på kiseldioxidsubstrat. Denna teknik användes även med två proteiner, lysozym och β-kasein, med målet att skapa ett multilager bestående av endast proteiner. Dessa proteiner bildade komplex på gränsytan vätska-fast fas i form av ett blandat proteinlager, men uppbyggnaden var väldigt oregelbunden med en ökning i adsorberad mängd per proteindeponeringscykel som var avsevärt mindre än ett monolager.Inom området för biomaterial utgör de antibakteriella och antihäftande egenskaperna hos chitosan respektive heparin en lovande blandning för beläggningar av medicinska implantat. Baserat på detta konstruerade vi multilagerfilmer av chitosan och heparin med olika deponeringslösningar och undersökte dynamiken och filmens struktur i detalj. Chitosan-heparin-filmen var starkt hydratiserad, bestående av cirka 60-80 vikt-% vatten beroende på deponeringsbetingelserna. Den adsorberade mängden och tjockleken på filmen ökade nästan exponentiellt med antal deponeringar, vilket förklarades med chitosanets förmåga att diffundera genom filmen under uppbyggnaden. Med ett nytt angreppssätt använde vi FRET för att bevisa diffusionen av fluorescerande färgmärkt chitosan i filmen under uppbyggnaden. Diffusionskoefficienten var i princip oberoende av pH och jonstyrka under deponeringen och följaktligen av filmens struktur. Genom att använda ett pH-känsligt färgämne begravt under sju biskikt av chitosan-heparin visade vi att färgämnet i hög grad påverkades av laddningen på det yttersta lagret. Från QCM-D-data lade vi fram teorin om att en ökning av energidissipationen för ett lager inte nödvändigtvis indikerar att lagrets struktur har blivit mindre styvt. / QC 20100729

Les nanotechnologies sont en train de révolutionner le domaine biomédical et plus particulièrement l’ingénierie tissulaire. Elles permettent aujourd’hui, non seulement de réparer mais aussi de régénérer les tissus. Cette nanomédecine régénérative est particulièrement adaptée pour répondre aux besoins importants liés aux maladies dégénératives, au vieillissement et aux traumatismes.Mon travail de thèse s’inscrit dans ce contexte et concerne l’élaboration de biomatériaux nanofibreux et microporeux actifs pour la régénération osseuse. Notre objectif essentiel est de réaliser un implant biodégradable nanostructuré permettant d’accélérer la réparation du tissu osseux. Notre stratégie innovante repose non seulement sur la mise en oeuvre de membranes par électrospinning mais aussi sur leur fonctionnalisation par des facteurs de croissance. Cette fonctionnalisation originale a consisté à enrober ces principes actifs dans des nanoréservoirs en utilisant la technique multicouche de polyélectrolytes. Des membranes de polycaprolactone (PCL) nanofibreuses et microporeuses ont été obtenues par électrospinning puis les fibres ont été enrobées de réservoirs contenant le facteur ostéoinducteur, la protéine morphogénique osseuse 2 (BMP-2). L’induction osseuse engendrée par ces réservoirs actifs a été mise en évidence in vitro après culture d’ostéoblastes humains primaires. Des expérimentations in vivo chez la souris ont permis de confirmer l’accélération de la régénération osseuse grâce à ces nanoréservoirs.Cette même stratégie a été validée in vivo, chez la souris, en utilisant des membranes de collagène d’origine animal commerciales utilisées en clinique. L’activité de ces membranes fonctionnalisées par des nanoréservoirs de BMP-2 est en cours d’analyse dans le cadre de tests précliniques pour une application maxillofaciale et parodontale. / Nanobiotechnology enables the emergence of entirely new classes of bioactive devices intended for targeted intracellular delivery for more efficiency and less toxicities. Tissue engineering is an interdisciplinary field that has attempted to implement a variety of processing methods for synthetic and natural polymers to fabricate tissue and organ regeneration scaffolds.We report here the first demonstration of bone regeneration by using a strategy based on a synthetic nanostructured membrane. This electrospun membrane is manufactured by using a FDA approved polymer, PCL, (polycaprolactone), and functionalized with nanoreservoirs of a growth factor (BMP-2). Our expected outcomes are the development of clinical applications in the field of tissue engineering and nanomedecine and particularly in bone regeneration.We propose the development of smart nanostructured active implants for regenerative medicine. Our strategycombines a synthetic biodegradable electrospun nanofibrous membrane based on PCL and a bioactive growth factor (BMP-2) entrapped into polymer nanoreservoirs built atop the nanofibers according to the layer-by-layer technology. In this study, by using primary osteoblasts, we have shown the capacity of these sophisticated implants to promote and accelerate not only in vitro bone induction; but also, in vivo, bone formation (mouse model).We have also validated our strategy, in vivo (mouse model), by using an already used in the clinic collagen membrane (animal origin) to accelerate bone regeneration. This unique strategy is used to entrap, protect and stabilize the therapeutic agent into polymer coating acting as nanoreservoirs enrobing fibers of membranes.

The electrostatic layer-by-layer (LbL) self-assembly of polyelectrolyte’s has shown applications in thin film coatings, micro patterning, nano-bioreactors and capsules for drug delivery. The film architecture can be precisely designed and controlled to nanometer scale precision with a range from 5 nm to a few microns. Both in vitro and in vivo studies indicate potential applications in biology, pharmaceutics, medicine, and other biomedical areas. This thesis work focused on the design and development of protocols to fabricate polyelectrolyte multi-layer patterns on a variety of substrates such as glass, metals and plastics such as acrylic and polycarbonate. The micro-scale polyelectrolyte patterns have applications in the creation of DNA, protein or cell based microarrays. This work also demonstrated the use of polyelectrolyte multi-layers in the enhancement of fluorescence signals from fluorophore-tagged molecules captured within the multi-layers. In-situ measurements using Fiber Bragg Gratings were carried out to study the kinetics of adsorption and desorption of polyelectrolytes participating in the layer buildup process under different process environmental conditions.

Notre équipe a développé une stratégie innovante de fonctionnalisation d’implants nanofibreux synthétiques à base de nanoréservoirs actifs pour la médecine régénérative osseuse. Notre objectif essentiel est de proposer un implant synthétique, biodégradable, et nanostructuré permettant d’accélérer la réparation du tissu osseux. Ces nouveaux implants synthétiques représentent un choix alternatif aux membranes de collagène d’origine animale. Notre stratégie consiste à construire des nanoréservoirs de chitosane, contenant des facteurs ostéoinducteurs tels que la BMP-2 afin d’enrober les nanofibres de nos implants. L’implant synthétique et biomimétique a été conçu à partir du le poly(ε-caprolactone) (PCL),polymère biocompatible et biodégradable approuvé par la FDA, et élaboré grâce à la technique de l’electrospinning afin de mimer la matrice extracellulaire. L’optimisation de ce procédé a permis la mise en oeuvre d’implants d’épaisseurs différentes (jusqu’à 10mm). La double fonctionnalisation de l’implant a permis de le rendre bioactif et vivant en utilisant la combinaison de facteur de croissance et de cellules souches mésenchymateuses. L’efficacité de la double fonctionnalisation des implants de PCL a ainsi été mise en évidence par l’accélération de la régénération osseuse in vivo.L’activité de ces implants fonctionnalisés de nanoréservoirs bioactifs est en cours d’analyse dans le cadre de tests précliniques pour une application maxillo-faciale, parodontale et orthopédique en vu d’obtenir un marquage CE. De plus, une start-up (ARTiOS NanoMed) basée sur cette nanotechnologie a été crée. En conclusion, nous pensons que la technologie développée par notre laboratoire a permis une avancée dans le domaine de la régénération osseuse et que cette technologie présente un fort potentiel d’application en clinique. / Our team has developped a novel and unique strategy to functionnalize nanofibrous and synthetic implants based on active nanoreservoirs for bone regeneration. We propose a new synthetic biodegradable and nanostructured implant to accelarate restoration of bone tissue. These new implants could replace collagen membranes from animal origin. The nanoreservoirs are based on chitosan containing osteoinductive growth factors such as BMP-2. Poly(ε-caprolactone) (PCL) is a biodegradable and biocompatible polymer approved by FDA and has been used to produce the synthetic and biomimetic implants by electrospinning in order to mimic the bone extracellular matrix. Optimization of this process has allowed the elaboration of nanofibrous implants with different thicknesses reaching 10 mm. Using the combination of growth factors and mesenchymal stem cells in a double functionalization created a bioactive and living implant. This strategy has been validated in vitro and in vivo thanks to bone site implantation in murin model. Acceleration of bone regeneration in vivo has brought to light the efficiency of the double functionalization onto the PCL implants.The functionalized implants bioactivity is still currently in study for pre-clinical trials in order to obtain authorization for applications in maxillo-facial, parodontal, and orthopaedic fields. Moerover, astat-up (ARTiOS NanoMed) based on this nanotechnology has been founded.To conclude, we believe that our nanotechnology could lead to a new generation of engineered bone implants which has a great potential to be used in the clinic.

Ce travail de thèse a été consacré à l'élaboration d'un nouveau type de membranes de nanofiltration efficaces avec des propriétés améliorées (flux élevé et rétention élevée, et de bonnes propriétés mécaniques) en déposant un revêtement assemblé couche-par-couche (LbL) sur des supports poreux. Après avoir systématiquement étudié le mécanisme de croissance des films assemblés couche par couche des polyélectrolytes choisis et la relation entre les structures de ces films et les performances des membranes résultant, nous avons identifié avec succès les meilleures structures multicouches pour la construction de membranes de nanofiltration de référence avec des performances optimales. En outre, en prenant avantage de la technique LbL, nous avons introduit une couche de diffusion latérale assemblée soit de nanofibrilles de cellulose ou de nanotubes de carbone, qui permet d’augmenter le flux de 30% tout en conservant la même rétention par rapport à la membrane de référence. En plus, les films assemblés à base de chitosan et nanofibrils de cellulose ont montré une forte résistance à la traction allant jusqu’à 450 MPa et un module d’Young jusqu’à 50 GPa. / This thesis work was devoted to the development of a novel and efficient nanofiltration membrane with improved properties (high flux and high retention, good mechanical strength) by coating Layer-by-Layer (LbL) assembled films onto porous membrane support. After having systematically studied the growth mechanism of LbL-assembled films of chosen polyelectrolytes and the relationship between the structures of these films and the membrane performance of the resulting NF membranes, we successfully identified the best multilayer structures for constructing nanofiltration membranes (NF) of reference with optimal membrane performance. Furthermore, taking advantages of the LbL-assembly, we successfully introduced LbL-assembled lateral diffusion layer that is made of either cellulose nanofibrils or carbon nanotubes, which in turn led to membranes with 30% higher flux. In addition, the LbL-assembled films of chitosan and cellulose nanofibrils showed surprisingly strong tensile strength of up to 450 MPa and a high Young modulus of up to 50 GPa.