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.

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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

Orientador: Marisa Masumi Beppu / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-22T20:50:50Z (GMT). No. of bitstreams: 1 Taketa_ThiagoBezerra_M.pdf: 4178929 bytes, checksum: 10d5bd8df33f074970da2596a3ef43a0 (MD5) Previous issue date: 2013 / Resumo: A busca por materiais que possam constituir superfícies antimicrobianas é relevante uma vez que o contato com micro-organismos patogênicos representa um sério risco à saúde. Além das propriedades naturais biocidas de alguns materiais, a adição de compostos que também atuam como agentes antibióticos como íons metálicos ou peptídeos, podem potencializar essa funcionalidade. Existe também a alternativa de criar-se uma superfície antimicrobiana através da modificação da densidade e da mobilidade de cargas catiônicas dos materiais utilizados, assim como propriedades estruturais dos revestimentos visando dificultar a adesão ou crescimento de micro-organismos. Neste trabalho, os filmes foram construídos pela técnica layer-by-layer, na qual é feita a imersão de um substrato (lâmina de vidro) alternadamente em soluções que contêm cargas positivas ou negativas e, no intervalo entre cada submersão, o filme em formação passa por um processo de lavagem em água deionizada para a retirada do excesso de polieletrólitos. Especificamente, foram utilizados o biopolímero catiônico quitosana (CHI) e os polieletrólitos negativos alginato de sódio (ALG) e ácido hialurônico (HA). Os filmes constituídos de ALG/CHI e HA/CHI foram construídos em diferentes pH e força iônica das soluções polieletrolíticas. Por análises de perfilometria e microscopia de força atômica (AFM) foi possível monitorar mudanças de espessura e rugosidade dos recobrimentos e foi verificado que a presença de sal no meio de deposição faz com que os polímeros apresentem conformações com mais loops e tails e esse arranjo das cadeias em solução é transmitido para os filmes, que se tornam mais rugosos. Além disso, a espessura dos filmes pode ser controlada por meio do ajuste do pH do meio de deposição. A incorporação dos corantes rosa de bengala e azul de alciano, em conjunto com análises de FTIR possibilitou identificar os grupos funcionais responsáveis pela funcionalidade dos recobrimentos formados. Por fim, foram feitos testes dos recobrimentos, com e sem acetato de prata, frente à cepa Candida albicans e foi verificado que filmes contendo Ag+ foram capazes de inibir o crescimento do fungo. O uso sinérgico de polímeros naturais e da técnica LbL viabilizou, de uma maneira simples e versátil, a construção de filmes nanoestruturados com potencial para aplicações antimicóticas / Abstract: The search for materials that can constitute antimicrobial surfaces is relevant since the contact with pathogenic microorganisms is a serious health risk. Besides the natural biocidal properties of some materials, the addition of compounds that also act as antimicrobial agents, such as metal ions or peptides, may enhance this capability. There is also the alternative to create an antimicrobial surface by modifying the density and mobility of cationic charges of the materials used, as well as structural properties of the coatings in order to hinder the adhesion or proliferation of microorganisms. In this work, the nanostructured films were built by the layer-by-layer (LbL) technique, whose interaction among species with opposite electrostatic charges is, in most cases, the primary phenomenon responsible for the formation of multilayers. Specifically, we chose the cationic biopolymer chitosan (CHI) and the negative polyelectrolytes sodium alginate (ALG) and hyaluronic acid (HA). The aim of this study was to evaluate the effect of ionic strength (0 versus 200 mM) and pH (3 versus 5) on ALG/CHI and HA/CHI nanostructured multilayered thin films properties. From profilometry and atomic force microscopy (AFM) analysis, it was possible to monitor changes in thickness and roughness of the coatings and it was found that the presence of salt in polyelectrolyte solutions promoted polymer chains conformations with more loops and tails and this arrangement in solution is transmitted to films, resulting in rougher coatings. Furthermore, the film thickness can be precisely controlled by adjusting the pH of the polyelectrolyte solution. The incorporation of rose bengal and alcian blue dyes in conjunction with FTIR analysis allowed identifying the functional groups responsible for the functionality of the coatings formed. Finally, we evaluated the antifungal activity of the LbL coatings (with and without silver ions) against Candida albicans strain and we observed that films containing Ag+ were able to inhibit the growth of the fungus. The synergistic use of natural polymers and LbL technique made possible, in a simple and versatile way, the construction of nanostructured films with great potential for antifungal surfaces / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

Les systèmes plastiques à usage unique sont destinés aux industries biopharmaceutiques et biotechnologiques. Ils sont composés de polymères et sont appréciés pour leurs propriétés barrières au gaz et à la vapeur d’eau assurant une préservation des solutions contenues dans ces systèmes. Ces dispositifs présentent de nombreux avantages pour les industriels : ils permettent d’éviter un nettoyage des cuves en inox et ainsi de limiter des risques de contamination croisée. En vue de leur utilisation, ces dispositifs sont stérilisés par irradiation γ. Le but de cette étude est de mettre en évidence l'impact de la stérilisation γ sur certains polymères et sur certains antioxydants utilisés dans la fabrication des polymères. Plusieurs échantillons sont analysés avant et après la stérilisation en utilisant différentes doses de rayonnement γ et au cours du temps (vieillissements naturels). Les modifications de structure des polymères γ-irradiés (EVA, PE et EVOH) ont été caractérisées par diverses méthodes analytiques : IRTF, spectroscopie Raman, RPE, chromatographie… Les résultats sont traités en utilisant des méthodes chimiométriques, comme l’ACP (Analyse en Composantes Principales), l’AComDim (ANOVA Common Dimensions) et SIMPLISMA (Simple-to-use interactive self-modeling mixture analysis). L’ACP a permis de mettre en évidence l’impact de la dose d’irradiation sur le polymère. L’AComDim a conduit à identifier les facteurs responsables de l’évolution des structures (la dose γ, les temps et l’interaction dose γ × temps), alors que les différents lots n’induisent pas de déviation. Enfin, la méthode de résolution de courbe SIMPLISMA a été appliquée pour l’étude du cœur du matériau. / The single use plastic systems are designed for biopharmaceutical and biotechnology industries. They are principally made of polymers and they are very appreciated for their barrier properties to gas and water vapor ensuring preservation of the solutions contained in these single-use systems. These devices have many advantages for industrial: they avoid cleaning stainless steel tanks and thus limit the risk of cross-contamination. In view of their future use, these devices are sterilized by γ-irradiation. The purpose of this study is to highlight the impact of γ sterilization on some polymers and on some antioxidants used in the manufacture of polymers. The samples are analyzed before and after sterilization using different γ-doses and over time (natural ageing). The structural modifications of γ-irradiated polymers (EVA, EVOH and PE) were characterized by various analytical methods: FTIR, Raman spectroscopy, ESR, chromatography… Some results are analyzed using chemometric methods such as PCA (Principal Component Analysis), AComDim (ANOVA Common Dimensions), and SIMPLISMA (Simple-to-use interactive self-modeling mixture analysis). The PCA allowed to highlight the impact of the irradiation dose on the polymer. The AComDim method led to the identification of the factors contributing to the evolution of structures (γ-dose, ageing and γ × ageing interaction), whereas the several lots do not involve variation. Finally, the curve resolution method, SIMPLISMA, was applied to the study of the core of the material.

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The first thermal cycling experiments of ionic self-assembled multilayer (ISAM) films have been reported examining their survivability through repeated thermal cycles from -20° C to 120° C in ambient atmospheric conditions. The films were constructed from alternating layers of Nile Blue A and gold nanoparticles which provided a strong absorbance in the optical wavelength range. No degradation of the optical characteristics of the ISAM films was observed [1]. Techniques for measuring the capacitance and resistivity of various ISAM films have also been developed allowing for a more complete electrical characterization of ISAM films. Capacitance measurements enabled a calculation of the dielectric function and breakdown field strength of the ISAM films. The capacitance measurement technique was verified by measuring the dielectric function of a spin-coated thin film PMMA, which has a well characterized dielectric function [2]. Surface-enhanced Raman spectroscopy (SERS) has been studied as a possible detection method for malignant melanoma revealing spectral differences in blood sera from healthy horses and horses with malignant melanoma. A SERS microscope system was constructed with the capability of resolving the Raman signal from biologically important molecules such as beta-carotene and blood sera. The resulting Raman signals from sera collected from horses with malignant melanoma were found to have additional peaks not found in the Raman signals obtained from sera collected from healthy horses. A systematic analysis of the combination of absorbance and fluorescence signals of blood sera collected from populations of healthy dogs and dogs with cancer has resulted in a rapid and cost-effective method for monitoring protein concentrations that could possibly be used as part of a cancer screening process. This method was developed using the absorbance and fluorescence signals from known serum proteins, the combinations of which were used to match the absorbance and fluorescence signals of blood sera allowing for an accurate determination of protein concentrations in blood sera [3]. Finally, a novel method for measuring the melting point of DNA in solution using capacitance measurements is presented. This method allows for the determination of the melting temperature as well as the melting entropy and melting enthalpy of DNA strands. Two different short strands of DNA, 5'-CAAAATAGACGCTTACGCAACGAAAAC-3' along with its complement and 5'-GGAAGAGACGGAGGA-3' along with its complement were used to validate the technique as the characteristics of these strands could be modeled using theoretical methods. This experimental technique allows for the precise determination of the melting characteristics of DNA strands and can be used to evaluate the usefulness of theoretical models in calculating the melting point for particular strands of DNA. Additionally, a micro-fluidic device has been proposed that will allow for a rapid and cost-effective determination of the melting characteristics of DNA [4]. / Ph. D.

L'incidence des maladies cardiovasculaires d'origine athéromateuse constitue un problème majeur en santé publique et malgré le développement de techniques curatives endovasculaires, la chirurgie demeure nécessaire chez de nombreux patients. La faible disponibilité des vaisseaux naturels, autologues ou non, et les limites mécaniques et biologiques des substituts artificiels pour le remplacement des vaisseaux de petit calibre, imposent le recours à une nouvelle science : l'ingénierie vasculaire. Ce concept a émergé et évolué depuis quelques années. Il pourrait permettre de proposer de nouveaux types de substituts vasculaires synthétiques et/ou biologiques, en particulier grâce à l'utilisation de cellules souches, ouvrant d'intéressantes perspectives dans le domaine de l'ingénierie vasculaire. Le but de ce travail a été d'obtenir de manière fiable et reproductible des cellules à phénotype endothélial mature à partir de cellules souches mésenchymateuses (CSMs) issus de la gelée de Wharton (GW) du cordon ombilical et de la moelle osseuse (MO). Cependant, la différenciation de ces cellules nécessite une fonctionnalisation de la surface de culture, et notre groupe a démontré l'avantage des films multicouches de polyélectrolytes, constitués de PAH (hydrochlorure de poly(allylamine)) et de PSS (poly(styrène sulfonate)), sur l'adhésion, la prolifération et la différenciation cellulaire. Les cellules ont été cultivées sur films (PAH-PSS)4 ou sur collagène de type I (témoin), en présence de facteurs de croissance angiogéniques. La différenciation en cellules endothéliales (CEs) a été suivie par l'expression des marqueurs endothéliaux (PCR et western blot), et la fonctionnalité par leur capacité à incorporer les lipoprotéines acétylées (Ac-LDLs) ainsi que la capacité à produire du monoxyde d'azote et à exprimé le facteur von Willebrand (vWF). Après 14 jours de stimulation, seules les CSM-GWs étaient différenciées en CEs fonctionnelles démontrant l'intérêt de combiner l'utilisation des CSM-GWs et des films (PAH-PSS)4 dans le domaine de l'ingénierie vasculaire / The incidence of cardiovascular disease remains a major public health problem. Despite the development of endovascular therapies, surgical treatment is necessary for many patients. The low availability of natural vessels, autologous or not, and the mechanical and biological limits of artificial substitutes, led to the use of a new domain: vascular engineering. In recent years, the concept has emerged and evolved. He could afford to offer new types of synthetic vascular substitutes and / or biological, in particular through the use of stem cells, offering interesting perspectives in the field of vascular engineering. The purpose of this study was to obtain a reliable and reproducible protocol to generate functional endothelial cells (ECs) from mesenchymal stem cells (MSCs) derived from the umbilical cord Wharton's jelly (WJ) and bone marrow (BM). Nevertheless, their differentiation into vascular cells needs a culture surface functionalization; our group demonstrated the potential use of polyelectrolyte multilayer made of poly(allylamine hydrochloride): PAH, and poly(styrene sulfonate): PSS, in promoting cells adhesion, proliferation and differentiation. Cells were cultured on (PAH-PSS)4 films or collagen type I (used as control), in the presence of angiogenic growth factors. Cells differentiation into EC was followed through the expression of endothelial markers (PCR and western blot); cell functionality was checked through their ability to incorporate acetylated LDL (Low Density Lipoprotein), to produce NO (Nitric oxide) and to express the von Willebrand factor (vWF). After 14 days of stimulation, only WJ-MSCs were able to generate functional ECs demonstrating the potential of combining WJ-MSCs and (PAH-PSS)4 films in vascular tissue engineering field

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.