手術後の呼吸器からの空気漏出（エアリーク）と腹部及び胸部癒着はそれぞれ、未だに臨床にて大きな課題である。本研究では、安全性に優れた植物性多糖類のアルギン酸ナトリウムに着目し、ゲルやスポンジの材形に加工した。これをPGA不織布と併用して新規エアリーク防止材を開発した。この新規材料は、エアリーク防止だけでなく癒着防止に対しても優れた効果を発揮した。将来的に、この新規材料は、従来材料よりも優れた医療材料として臨床応用されることが期待できると考えられる。 / Sodium alginate is polysaccharide extracted from seaweed and used as a biomaterial clinically. The alginate in this study was used as gel- or sponge-formed and combined with a polyglycolic acid (PGA) mesh, a useful biomaterial clinically; namely this combination was the new sealing material. The purpose of this study was to prevent pulmonary air leak without inducing adhesion. This study was composed of the four animal experiments; the first half of them was about preventing air leak and the latter was about preventing adhesion. All experiments showed that new sealing material was superior to the conventional treatments. Therefore the new sealing material was expected to be applied clinically to a sealing material, which also has an anti-adhesive effect. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

Charles University, Faculty of Pharmacy in Hradci Králové Department of Pharmaceutical Technology Name: Martina Kišková Title of diploma thesis: Adhesive properties of matrix tablets Supervisor: PharmDr. Eva Šnejdrová, Ph.D. The diploma thesis deals with the evaluation of rheological and adhesive properties of the mucin, aqueous dispersions of polymeric carriers and matrix tablets based on chitosan and sodium alginate or iota-carrageenan loaded with the salicylic acid using absolute rotational rheometer. The theoretical part deals with the characterization and classification of matrix tablets, polymeric carriers (sodium alginate, chitosan and carrageenan) and with the principles of evaluation of rotational, adhesive and oscillational tests performed in the experimental part. The mucin from porcine gastric used as a model substrate for adhesion tests behaves as a viscoelastic solid and its adhesive strength decreases with increasing hydration. Significantly higher adhesive strength was found for chitosan at pH 1.2 and sodium alginate at pH 6.8 compared to the adhesive strength of iota-carrageenan. In terms of viscoelastic properties, chitosan and sodium alginate are viscoelastic fluids, but iota-carrageenan is a viscoelastic solid. Iota-carrageenan forms the stiffest gel after hydration at pH 6.8...

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Biological systems are composed of complex materials which are responsible for performing various functions, such as providing structural support, mobility, functional adaptation to the environment, damage repair, and self-healing. These complex materials display excellent mechanical properties and can rapidly adapt to external stimuli. Thus, nature inspires in terms of source materials, functions, and designs to develop new-generation structural and functional materials. Polymers (natural or synthetic) are excellent sources of developing materials to mimic the functions of soft segments in biological systems. This dissertation focuses on synthesizing and characterizing two different materials with tunable structures and properties: complexes from natural polysaccharides or polyelectrolytes and bioinspired hydrogels from synthetic polymers. Oppositely charged polyelectrolytes can form polyelectrolyte complexes (PECs) due to the electrostatic interactions. The structure and properties of PECs can be tuned by varying the salt concentration, as the addition of salt can facilitate associative phase separation. PECs were prepared from two biopolymers, positively charged chitosan and negatively charged alginate. Rheological experiments for the complexes displayed a tunable shear modulus with changing salt concentrations. The microstructural study conducted using small-angle X-ray scattering provided insights regarding the length scales of these complexes, and the results follow the observed rheological and phase behavior. Elastic biopolymers such as resilin display remarkable mechanical properties, including high stretchability and resilience, which many species exploit in nature for mechanical energy storage to facilitate their movement. Such properties of resilin have been attributed to the balanced combination of hydrophilic and hydrophobic segments present in the chain. In this work, we synthesized hydrogels with hydrophilic and hydrophobic components to mimic the properties of resilin. With this system, we determined the tensile, retraction (ability to revert to the original state after stretching), and swelling properties when (i) the concentration of the hydrophobic polymer was varied and (ii) additional hydrophobic components were included. The stretchability, stiffness, and strength of the gels varied as the compositions were altered. The fundamental understanding of the structure-property-function relationship for materials presented in this work provides insights into engineering materials for applications such as tissue engineering, drug delivery, wound healing, artificial muscles, soft robotics, and power amplification.

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<p>Hemophilia B patients may have frequent, spontaneous and life-threatening bleeds that are currently managed by an invasive and expensive treatment. Mesenchymal stem cells (MSCs) are increasingly being applied to clinically therapeutic strategies and lentiviral gene vectors have been shown to be safe and efficient tools for modifying stem cells for long-term expression of high levels of transgenes. In this study, MSCs were engineered with a lentivirus to express sustained and therapeutic levels of human FIX protein <em>in vitro </em>and in mice. The modified MSCs secreted human FIX protein at levels exceeding 4 μg/10⁶ MSCs/24 h with high FIX coagulant activity of greater than 2.5 mIU/10⁶ MSCs/24 h for 6 week <em>in vitro. </em>Functional FIX transgene was continually expressed by these cells when they were induced to differentiate into adipocyte, osteoblast and chondrocyte lineages <em>in vitro</em>. However, the modified MSCs transplanted via tail vein into NOD-SCID-γ mice expressed low levels of FIX <em>in vivo</em>. The transplantation procedure had an increased risk of death that was more pronounced in mice that received cell doses exceeding 2 million cells. Organ examinations suggested the deaths resulted from entrapment of MSCs in pulmonary capillaries. Modified MSCs encapsulated in alginate-PLL microcapsules and transplanted into the peritoneal cavity of both NOD-SCID-γ and hemophilia B mice at 9 million cells/mouse resulted in therapeutic expression around 100 ng of human FIX/mL of plasma only for a few days <em>in vivo</em> as human FIX expression quickly decreased to basal values by the end of the first week. Cultured <em>ex vivo</em>, human FIX expression by retrieved capsules indicated an innate immune response to the encapsulated cells prevented sustained expression of FIX. These investigations demonstrate that lentivirally modified MSCs have the potential to express therapeutic human FIX for sustained periods <em>in vitro</em>, even after their differentiation. However, they also highlight the challenges to overcome to optimize cell engraftment and survival following transplantation, and to minimize the immune responses associated with the xenogeneic translational<em> </em>models used.</p> / Doctor of Philosophy (PhD)

The development of structured macroporous hydrogels are of great interest in many industries due to their high permeabilities, large surface areas and large pore volumes. In drug delivery and wound healing applications, these macropores may theoretically be utilized as large drug reservoirs to deliver anti-inflammatory drugs to a wound site, while simultaneously absorbing exudate and maintaining a hydrated environment in which the wound may heal. However, current methods of generating macroporous structured hydrogels are low-throughput, expensive, and require the use of organic solvents, salts, and other additives that are difficult to remove from the crosslinked hydrogel scaffold. In contrast, the Pressurized Gas eXpanded liquid (PGX) processing technology, patented by the University of Alberta and licensed for all industrial applications by Ceapro Inc., has been shown to generate purified and exfoliated biopolymer scaffolds in a less expensive and more efficient way. Herein, the tunability of the PGX processing method was investigated in depth, varying solvent/anti-solvent ratios, nozzle mixing volume, polymer molecular weight, and polymer concentration to examine the resulting effects on produced polymer morphologies. PGX-processed chitosan and alginate scaffolds were stabilized as bulk hydrogels through post-processing crosslinking methods using anti-solvents, solid-state chemistries, and/or rapid gelation kinetics. The mechanical strength, swelling/degradation kinetics, affinity for protein uptake, and cytotoxicity of these stabilized scaffolds were subsequently examined and compared to hydrogels produced without the use of PGX processing. Furthermore, in situ crosslinking methods were explored, in which alginate and poly(oligoethylene glycol methacrylate) polymers were shown to form stable aerogels during the standard PGX processing method. Finally, the PGX apparatus was reconfigured to enable the impregnation of a model hydrophobic drug into pre-processed polymer scaffolds via circulation of supercritical CO2. The total loading was calculated and the release kinetics from loaded-scaffolds examined. In conclusion, this work outlines a novel method of creating structured macroporous hydrogels from PGX processed biopolymers with the potential to provide improved drug loadings and sustained release profiles. It is expected that this work will provide a basis for a great deal of research into the further stabilization of scaffolds for use in other applications, the investigation of a larger range of bioactive molecules for impregnation and release, and the exploration of PGX hydrogel scaffolds for in vivo wound healing. / Thesis / Master of Applied Science (MASc)

This work deals with the study of the drying-mediated assembly of colloidal gold nanoparticles (Au NPs) in mesoscale aggregation patterns and their manipulation by atomic force microscopy (AFM) on model surfaces. The assembly of NPs in mesoscale and complex aggregation patterns assisted by the wetting and the drying of complex fluids (suspensions of NPs, NPs/biopolymers mixed solutions) on homogeneous and heterogeneous molecular surfaces was studied. This issue is important, both for understanding fundamental processes of self-organization, and for generating new functional mesostructures. The drying of complex fluids often leads to the emergence of highly complex aggregation structures as shown and discussed in this work. The richness and the aesthetics of these complex structures generated by these interfacial phenomena reflect not only the bulk properties of fluids (different sizes and lengths- scales, kinetic changes in state), but more importantly, the coupling between the fluid properties and those of the substrate surface (wetting interactions, confinement, hydrodynamics). In the case of two important heterogeneous fluids which are Au NPs and polysaccharide solutions, these drying-mediated structure formation lead to the genesis of unusually large and highly ramified dendrites aggregation patterns. The growth mechanism and the critical parameters that control the morphogenesis of these complexes structures are addressed in this work. In addition, the aging mechanisms and kinetics of these structures that are metastable and evolve either through direct dislocation via clusters NPs mobility on the surface, or through undulation-induced roughning of the dendrite branches. To better understanding this NPs mobility and thus the dislocation mechanism of the aging, a detailed study based on the manipulation of NPs by atomic force microscopy in tapping mode (AFM) was developed. The threshold dissipated energy to manipulate (move) the NPs can be quantified according to the intrinsic parameters of the particle (size, shape, and chemical nature), the chemical nature and topography of the substrate, and finally the operating and environment conditions. This work enabled us to understand the mechanisms and characterize the critical parameters that may intervene in the dislocation (aging) of NPs-based functional structures, depending on the nature of the environment liquid and the substrate. Finally, this work proposes an approch of evaluation and of monitoring the stability and the aging of these aggregation structures, in particular, those formed from the drying of films and drops of nano-particles solutions (metal nanoparticles, blood: proteins, viruses ...).

Le papier bioactif est obtenu par la modification de substrat du papier avec des biomolécules et des réactifs. Ce type de papier est utilisé dans le développement de nouveaux biocapteurs qui sont portables, jetables et économiques visant à capturer, détecter et dans certains cas, désactiver les agents pathogènes. Généralement les papiers bioactifs sont fabriqués par l’incorporation de biomolécules telles que les enzymes et les anticorps sur la surface du papier. L’immobilisation de ces biomolécules sur les surfaces solides est largement utilisée pour différentes applications de diagnostic comme dans immunocapteurs et immunoessais mais en raison de la nature sensible des enzymes, leur intégration au papier à grande échelle a rencontré plusieurs difficultés surtout dans les conditions industrielles. Pendant ce temps, les microcapsules sont une plate-forme intéressante pour l’immobilisation des enzymes et aussi assez efficace pour permettre à la fonctionnalisation du papier à grande échelle car le papier peut être facilement recouvert avec une couche de telles microcapsules. Dans cette étude, nous avons développé une plate-forme générique utilisant des microcapsules à base d’alginate qui peuvent être appliquées aux procédés usuels de production de papier bioactif et antibactérien avec la capacité de capturer des pathogènes à sa surface et de les désactiver grâce à la production d’un réactif anti-pathogène. La conception de cette plate-forme antibactérienne est basée sur la production constante de peroxyde d’hydrogène en tant qu’agent antibactérien à l’intérieur des microcapsules d’alginate. Cette production de peroxyde d’hydrogène est obtenue par oxydation du glucose catalysée par la glucose oxydase encapsulée à l’intérieur des billes d’alginate. Les différentes étapes de cette étude comprennent le piégeage de la glucose oxydase à l’intérieur des microcapsules d’alginate, l’activation et le renforcement de la surface des microcapsules par ajout d’une couche supplémentaire de chitosan, la vérification de la possibilité d’immobilisation des anticorps (immunoglobulines G humaine comme une modèle d’anticorps) sur la surface des microcapsules et enfin, l’évaluation des propriétés antibactériennes de cette plate-forme vis-à-vis l’Escherichia coli K-12 (E. coli K-12) en tant qu’un représentant des agents pathogènes. Après avoir effectué chaque étape, certaines mesures et observations ont été faites en utilisant diverses méthodes et techniques analytiques telles que la méthode de Bradford pour dosage des protéines, l’électroanalyse d’oxygène, la microscopie optique et confocale à balayage laser (CLSM), la spectrométrie de masse avec désorption laser assistée par matrice- temps de vol (MALDI-TOF-MS), etc. Les essais appropriés ont été effectués pour valider la réussite de modification des microcapsules et pour confirmer à ce fait que la glucose oxydase est toujours active après chaque étape de modification. L’activité enzymatique spécifique de la glucose oxydase après l’encapsulation a été évaluée à 120±30 U/g. Aussi, des efforts ont été faits pour immobiliser la glucose oxydase sur des nanoparticules d’or avec deux tailles différentes de diamètre (10,9 nm et 50 nm) afin d’améliorer l’activité enzymatique et augmenter l’efficacité d’encapsulation. Les résultats obtenus lors de cette étude démontrent les modifications réussies sur les microcapsules d’alginate et aussi une réponse favorable de cette plate-forme antibactérienne concernant la désactivation de E. coli K-12. La concentration efficace de l’activité enzymatique afin de désactivation de cet agent pathogénique modèle a été déterminée à 1.3×10-2 U/ml pour une concentration de 6.7×108 cellules/ml de bactéries. D’autres études sont nécessaires pour évaluer l’efficacité de l’anticorps immobilisé dans la désactivation des agents pathogènes et également intégrer la plate-forme sur le papier et valider l’efficacité du système une fois qu’il est déposé sur papier. / Bioactive paper is obtained through the modification of paper substrate with biomolecules and reagents. It is used in the development of novel biosensors that are portable, disposable and inexpensive, aimed at capturing, detecting and in some cases deactivating pathogens. Generally bioactive papers are made by incorporating biomolecules such as enzymes and/or antibodies on to paper. The immobilization of such biomolecules on solid surfaces is widely used for different diagnostic applications such as in immunosensors and immunoassays but due to the sensitive nature of enzymes, their large scale incorporation into paper has faced several difficulties especially under industrial papermaking conditions. The functionalization of paper at large scale is possible because paper can be easily coated with a layer of microcapsules, which have proven to be an efficient immobilization platform for enzymes and to allow. In this study, we developed a generic alginate-based platform incorporating microcapsules that can be applied to current paper production processes to prepare antibacterial bioactive paper with the ability to capture pathogens on its surface and to deactivate them by producing an anti-pathogenic agent. The design of the antibacterial platform is based on constant production of hydrogen peroxide as the antibacterial agent inside the alginate microcapsules. Hydrogen peroxide production is achieved through oxidation of glucose, catalyzed by the enzyme glucose oxidase encapsulated inside the alginate beads. The different steps of development included the entrapment of glucose oxidase inside alginate microcapsules, the reinforcement and surface activation of microcapsules by adding an additional layer of chitosan, investigating the possibility of immobilization of antibodies (human immunoglobulin G as a model antibody) on the surface of microcapsules and, finally, verifying the antibacterial properties of the system against Escherichia coli K-12 (E. coli K-12) as a representative pathogen. During development, certain measurements and observations were made using various analytical methods and techniques such as Bradford protein assay, oxygen electroanalysis, optical and confocal laser canning microscopy (CLSM), matrix assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF-MS), etc. Appropriate tests were performed to validate the successful modification of microcapsules and to ensure that glucose oxidase is still active after each modification. It was found that the encapsulated glucose oxidase maintained the specific enzymatic activity of 120±30 U/g. Subsequent efforts were made to immobilize glucose oxidase on gold NPs of two different diameters (10.9 nm and 50 nm) to enhance the enzymatic activity and increase the encapsulation efficiency. The results obtained during this study demonstrate successful modifications on alginate microcapsules and also a successful response of such antibacterial platform regarding deactivation of the pathogen representative, E. coli K-12. The threshold for the enzymatic activity was found to be 1.3×10-2 U/ml for E. coli K-12 growth inhibition of 6.7×108 cells/ml. Further studies are needed to assess the efficiency of immobilized antibody in the capture of pathogens and also to incorporate the platform onto paper and to validate the efficiency of the system once it is coated on paper.