Dissertations / Theses on the topic 'Chitosan'

La quitina es un biopolímero muy abundante presente en el caparazón de crustáceos, insectos y en la pluma del calamar y la pota, entre otras fuentes. La desacetilación de la quitina forma la quitosana, un polisacárido más versátil por su solubilidad y mayor reactividad química. La quitosana es utilizada en aplicaciones médicas, farmacéuticas, cosméticas, tratamiento de aguas, agricultura e industria alimentaria.
Chitin is an abundant biopolymer that can be found in shells of crustaceans, insects and in squid and pota pen. Deacetylation of chitin produces chitosan, a more versatile polysaccharide due to its solubility and increased chemical reactivity. Chitosan is used in medicine, pharmaceutics, cosmetics, water treatment, agriculture and food industry.

Le chitosane présente des propriétés biologiques (biocompatibilité, biorésorbabilité, bioactivité) idéalement adaptées à des applications en ingénierie tissulaire. Dans cette étude partenariale (Programme ANR TECSAN 2010 ChitoArt), nous avons travaillé à l'élaboration d'hydrogels physiques de chitosane à propriétés physico-chimiques et biologiques variées et contrôlées, sans utilisation d'agents de réticulation externes. Ces hydrogels sont envisagés sous forme de tube mono ou pluri-membranaires pour une utilisation en tant que substituts vasculaires de petit diamètre (<6mm). En effet, l'ingénierie vasculaire présente, encore de nos jours, de nombreuses limitations lorsqu'il est question de vaisseaux de petits calibres. Notre démarche consiste en la modulation des paramètres structuraux (degré d'acétylation, masse molaire) et environnementaux (concentration du bain de gélification, du collodion) intervenants dans le procédé d'élaboration des hydrogels pour atteindre les critères physiques, biologiques et mécaniques compatibles avec cette application. L'étude morphologique des hydrogels par Cryo-Microscopie Électronique à Balayage (Cryo-MEB), via une méthode de préparation originale a permis une meilleure compréhension de l'organisation micro-structurale et multi-échelle des hydrogels de chitosane. Cette approche fondamentale a été couplée à une évaluation in vivo des propriétés biologiques des hydrogels ainsi qu'a des caractérisations mécaniques des substituts vasculaires. En particulier, l'évaluation de la suturabilité de nos substituts a mené au développement d'une formulation donnant lieu à des hydrogels physiques de chitosane suturables ayant fait l'objet d'un dépôt de brevet (N° de dépôt FR1363099). Le contrôle et la modulation des paramètres d'élaboration des hydrogels ont permis l'obtention de substituts vasculaire cellularisables et respectant les exigences (suture, compliance, résistance à l'éclatement) concernant leur implantation in vivo
Chitosan presents biological properties (biocompatibility, bioresorbability, bioactivity) ideally suited for tissue engineering. In this partnership study (ANR TECSAN 2010 ChitoArt program), we worked at the elaboration of physical chitosan hydrogels presenting various and controlled physicochemical and biological properties, without any external crosslinkers. These hydrogels are envisioned under mono- or poly-membranous tubes for small diameter vascular substitutes (<6mm) purposes. Indeed, vascular engineering presents, even today, numerous limitations for small calibre vessels. Our strategy consists in the modulation of both structural (degree of acetylation, molar mass) and environmental (neutralization bath and collodion composition and concentration) parameters involved in hydrogels elaboration process in order to reach physical, biological and mechanical requirements suitable for this application. The study of hydrogels morphology by Cryo-Scanning Electron Microscopy (Cryo-SEM), using an original sample preparation method led to a better comprehension of chitosan hydrogels fine structure and multi-scale organization. This fundamental approach was conducted through the in vivo biological evaluation of hydrogels but also to mechanical characterizations of vascular substitutes. In particular, our substitutes were evaluated in term of suture retention resulting in the development of a formulation that led to suturable physical chitosan hydrogels, which were protected by a patent (Deposit number: FR1363099). Hydrogels elaboration parameters control and modulation have resulted in the development of colonisable vascular substitutes matching their in vivo implantation requirements (suture retention, compliance, burst pressure)

Several approaches have been employed for directing the intracellular trafficking of DNA to the nucleus. Cationic polymers have been used to condense and deliver DNA and a few specific examples using chitosan as cationic polymer have been described. The concerted efforts in gene therapy to date have provided fruitful achievements toward a new era of curing human diseases. A number of obstacles, however, still must be surmounted for successful clinical applications. Therefore, chitosan-plasmid and quaternised chitosan-plasmid complexes (polyplexes) were investigated for their ability to transfect COS-1 cells and the results were compared with Transfectam/DNA lipoplexes for transfection efficiency. All of the chitoplexes utilised in this study proved to transfect COS-1 cells, however to a lesser extent than the Transfectam/DNA lipoplexes, which served as a positive control. Complexes formed with quaternised trimethyl and triethyl chitosan oligomers, specifically TMO L and TEO L, proved to be superior transfecting agents compared to other chitosans. The molecular mass of chitosan is considered to influence the stability of the chitosan/DNA polyplex, the efficiency of cell uptake and the dissociation of DNA from the complex after endocytosis. In literature it was shown that the toxicity of the chitosan1DNA polyplexes is relatively low compared to viral gene and lipid non-viral delivery vectors. This study showed that the percentage viable COS-1 cells when transfected with the chitosan polymers, oligomers, quaternised chitosan polymers and quaternised chitosan oligomers (chitoplexes) was higher than the percentage viable cells when transfected with lipoplexes prepared with Transfectam with the MTT assay. The Transfectam/DNA lipoplexes induced cell damage and a decreased viability of COS-1 cells were found. Chitosan/DNA and quaternised chitosan/DNA complexes did not affect the viability of the cell line. The degree of quaternisation of the polymers and oligomers and molecular size proved to be two important factors when considering effective non-viral gene delivery. It can be concluded that chitosan, especially quaternised oligomeric derivatives are polysaccharides that demonstrate much potential as a gene delivery system. The high solubility and low toxicity of chitosan allow its use in a wide variety of applications in the pharmaceutical industry and, as shown in this study, in gene delivery.
Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Les cultures céréalières sont sujettes aux invasions de champignons pathogènes, ce qui altère la qualité des grains et pose un problème de santé publique, en raison de mycotoxines potentiellement contenues dans ces grains. Face à la prise de conscience publique et politique de la nécessité d’inclure la composante environnementale dans nos modes de consommation et de production, les fongicides synthétiques traditionnels se voient petit à petit remplacés par des alternatives plus « vertes ». Dans ce contexte, ce travail de thèse a pour but de créer une formulation à base de produits renouvelables, pour contrôler le développement et la production de toxines d’espèces fongiques pathogènes. Ce travail exploite les propriétés remarquables de trois composés : le biopolymère de chitosane, dérivé de la chitine, la tétrahydrocurcumine (THC), un dérivé de curcumine, et des extraits de pins et de vigne. Les propriétés physico-chimiques des chitosanes ont tout d’abord été caractérisées. Ces solutions de chitosanes présentent des effets antifongiques prometteurs réduisant la croissance mycélienne du champignon modèle cible Fusarium graminearum et divisant sa production de mycotoxines de plus moitié. Un des principaux atouts de ce biopolymère réside dans le fait qu’il conserve ses propriétés antifongiques sous forme d’enduction. Des extraits végétaux possédant des activités antimicrobiennes ont ensuite été étudiés. Premièrement, la THC inhibe la production de toxines et est donc combinée avec du chitosane. Afin d’accroître la solubilité dans l’eau et l’efficacité de la THC, des complexes d’inclusion ont été formés avec des cyclodextrines. De même, cette THC a été encapsulée dans d’autres matrices de biopolymères variés, tels que l’amidon ou le chitosane. Deuxièmement, les extraits issus de ressources locales de pins maritimes et de vignes démontrent eux aussi des effets antifongiques et anti-mycotoxigéniques. En particulier, l’addition d’un de ces extraits, celui d’écorce de pin maritime, à une formulation de chitosane double l’efficacité de ce dernier contre le développement du mycélium
Cereals are subject to contamination by pathogenic fungi, which damage grains and threaten the public health with their mycotoxins. Recently, the raise of public and political awareness concerning environmental issues tend to limit the use of traditional fungicides against these pathogens in favour of eco-friendlier alternatives. In this framework, this thesis work aims to create a formulation based on renewable products in order to limit the fungal development and control the production of mycotoxins from cereal fungi. Our work exploits the remarkable properties of three compounds: the chitosan, a chitin derived biopolymer, the tetrahydrocurcumin (THC), a curcumin derivative, and plant extracts. In a first step, we studied and characterise the physicochemical properties of different chitosans. Chitosan solutions were shown to reduce the mycelial growth of a target model fungi Fusarium graminearum, and to divide by 2 the accumulation of mycotoxins. In addition, we showed that this biopolymer was able to maintain its antifungal properties as a form of a coating. In a second step, we focused on different plant extracts with antimicrobial activities. THC was able to inhibit the toxin production and a maritime pine by-product showed its potential to control the fungal growth. The combination of the THC or the wood extract with chitosan was then studied to increase the efficiency of the formulation. To this end, a significant work was made to increase the solubility of THC in water by forming an inclusion complex in cyclodextrins or by protecting it in starch or chitosan particles. In particular, we showed that the addition of pine extracts to a chitosan-based solution can double the effectiveness of the formulation

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

Le chitosane est un polymère naturel obtenu par deacétylation de la chitine. Ce polysaccharide est bien connu pour ses propriétés biologiques exceptionnelles : il est biocompatible et biorésorbable. Les fibres de chitosane peuvent être utilisées en chirurgie. L'objectif de cette thèse est d'étudier les phénomènes physico-chimiques mis en jeu, de développer un modèle du procédé, afin d'optimiser le procédé de filage mis au point au laboratoire.Après une revue de la littérature dans le premier chapitre, les techniques expérimentales d'obtention, de purification, et de caractérisation du chitosane sont décrits dans le deuxième chapitre. Une étude de la structure du chitosane obtenu est présentée. C'est l'un des résultats originaux de ce travail.Le principe du procédé étant par coagulation en solution, il est essentiel de déterminer dans quelle condition celle-ci s'effectue, et quel est le paramètre déterminant. Les études précédentes ont montré que celui-ci est le coefficient de diffusion de la soude dans le milieu. A cet effet, des mesures ont été effectuées, dans des géométries différentes. Cette étude constitue le travail présenté dans le chapitre trois.Dans le chapitre quatre est présentée une technique consistant à suivre au moyen d'un microscope l'avancée du front de coagulation. Cette technique a permis de déterminer précisément le coefficient de diffusion.Le dernier chapitre a consisté à élaborer des fibres au moyen d'un banc que possède le laboratoire (IMP Lyon 1). L'étape ultime de ce travail a été de modéliser le procédé, de prévoir les diamètres intérieur et extérieur des fibres obtenues, et de comparer le résultat de la modélisation aux résultats expérimentaux
Chitosan is a natural polymer obtained by deacetylation of chitin. This polysaccharide is well known for its exceptional biological properties: it is biocompatible and bio absorbable. Chitosan fibers can be used in surgery.The objective of this thesis is to study the physicochemical phenomena involved, to develop a process model, to optimize the filtering process in the laboratory.After a review of the literature in the first chapter, the experimental techniques for obtaining, purifying and characterizing chitosan are described in the second chapter. A study of the structure of the chitosan obtained is presented. This is one of the original results of this work.The principle of the coagulation method in solution, it is essential to determine in what condition it, and what is the determining parameter. Previous studies have shown that this is the diffusion coefficient of soda in the medium. One effect, measurements were made, in different geometries. This study constitutes the work presented in Chapter Three.In chapter four is presented a technique consisting in following by means of a microscope the advance of the coagulation front. This technique makes it possible to determine the diffusion coefficient.The last chapter consisted of developing fibers using a small scale plant existing in laboratory (IMP Lyon 1). The final element of this work consists of modelling the process, calculating the inside and outside diameters of the fibers obtained and comparing the result of the modelling with the experimental results

Le chitosane est un polysaccharide cationique biosourcé largement utilisé dans différents domaines tels que la santé, le traitement des eaux usées ou les compléments alimentaires. Un autre champ applicatif de ce polymère correspond au domaine des adhésifs. En effet, la demande actuelle visant à remplacer les polymères non naturels et toxiques par des polymères plus « verts » et plus sains est fortement présente dans le milieu des adhésifs et ouvre d'intéressantes opportunités au chitosane et à ses dérivés. Cependant, le chitosane est insoluble dans l'eau (à des pH > 7) et présente une résistance faible à l'eau lorsqu'il est utilisé comme adhésif. Cette constatation limite ses possibilités d'utilisation dans le domaine du collage.Des études ont été menées sur différents chitosanes industriels afin de sélectionner ceux présentant les caractéristiques physico-chimiques les plus attrayantes pour un usage en tant qu'adhésif. Ensuite, le chitosane a été chimiquement modifié par des réactions de réticulation covalente et plastifié afin d'améliorer les propriétés mécaniques de joints adhésifs à base de chitosane sur le bois en milieu humide. Un adhésif à base de chitosane présentant une résistance au cisaillement supérieure à 4 MPa sur éprouvettes humides a été obtenu avec l'ajout de glycérol diglycidyl éther avec un ratio massique glycérol diglycidyl éther/chitosane de 20 %
Chitosan is a cationic bio-sourced cationic polysaccharide widely used in different fields from health to wastewater treatment through food supplements. Another important use of this polymer is adhesion. Indeed, the current demand to replace non-natural and toxic polymers by greener ones is well present in the adhesive field and open good opportunities for chitosan and its derivatives. However, chitosan is not water soluble (at pH ≥ 7) and exhibits a poor water-resistance in the field of adhesion which reduces the possibilities of its utilization within the paste field.Studies have been carried out to identify commercial chitosans with the most promising characteristics as adhesive. Then, chitosan has been chemically modified by covalent crosslinking reaction and plasticizing to improve its mechanical resistance for wood bonding in presence of water. A chitosan-based adhesive with a shear resistance higher than 4 MPa on wet samples has been obtained by adding glycerol diglycidyl ether with a weight ratio glycerol diglycidyl ether/chitosan of 20 %

Les méthodes électrohydrodynamiques (EHD) permettent avec un dispositif peu coûteux de mettre en forme un polymère sous forme de film ou de particules par pulvérisation électrohydrodynamique (P-EHD) ou encore sous forme de fibres par électrofilage. Les structures obtenues peuvent être très régulières et avoir des dimensions micrométriques ou submicrométriques. En raison de ces avantages, ces méthodes sont étudiées pour la mise en forme du chitosan. Ce polymère biosourcé présente de nombreuses qualités telles que la biocompatibilité, la biodégradabilité, l’activité antibactérienne ou encore la muco-adhésion.Cette thèse aborde les méthodes EHD et principalement la P-EHD du chitosan, un sujet attractif dont les applications étudiées sont vastes : on peut notamment compter le médical (ingénierie tissulaire, vectorisation), l’alimentaire (additifs et films alimentaires), le traitement des effluents ou encore le textile. Cependant cette méthode possède certaines limitations. Tout d’abord, le procédé présente une grande variabilité selon le chitosan utilisé, la préparation des solutions et la configuration utilisée. Ensuite les particules obtenues dans la littérature sont inhomogènes en taille, ce qui limite leur application, notamment en vectorisation. La question est alors de savoir comment obtenir des particules de chitosan de tailles contrôlées et homogènes. Pour répondre à cette question, une analyse critique de la littérature a permis de préparer une démarche expérimentale menée en quatre étapes : (1) caractérisation des chitosans employés, (2) mesure des propriétés physiques des différentes solutions, (3) étude de la stabilité du procédé, (4) détermination de la morphologie et de la distribution en taille des particules obtenues. Cette étude a permis de mettre en avant des paramètres permettant une P-EHD stable, le contrôle de la taille et de l’homogénéité des particules. Enfin, dans une démarche biomimétique la mise en forme du chitosan a été étudiée pour tenter de reproduire les structures qui confèrent des propriétés remarquables comme l’hygrochromie ou l’hydrophobicité pour des insectes tels que les scarabées et les cigales
Electrohydrodynamic (EHD) methods enable to produce with one low cost set-up polymeric films/particles by electrospray or polymeric fibres by electrospinning. Particles or fibres produced by these methods can be very uniform in size. Therefore, electrospray and electrospinning of chitosan are appealing topics of re-search. Chitosan is a bio-based material possessing numerous qualities such as biocompati-bility, biodegradability, antibacterial activity and muco-adhesion. This thesis deals with EHD methods with a focus on chitosan electrospray whose applications are abundant in health (tissue engineering, drug delivery), food (nutrients en-capsulation, cling film), wastewater treatment and textiles. However electrospray has several limitations. First, effective electrospray depends of the grade of the chitosan, of the preparation of the solution and of used set-up. Second, particle-size distribution reported in literature are broad whereas applications such as drug delivery require monodisperse particle-size distributions. Then, the question is to know how to produce chitosan particles of monodisperse controlled size. To answer this question, a critical analysis of literature led to an experimental approach divided in four steps : (1) characterization of chitosan, (2) measurement of chitosan solution properties, (3) study of stability of electrospray process, (4) assessment of deposit morphology and particle-size distribution. Finally, as part of a biomimetic approach, imitation with chitosan of natural shapes has been studied. These shapes are part of structures that confer striking properties such as hygrochromic behavior and hydrophobicity to insects

Le traitement de la corrosion constitue un enjeu économique, environnemental et de sécurité sanitaire. Plus largement utilisée à l’échelle industrielle, la protection par revêtements consiste à isoler le métal du milieu agressif par une couche adhérente, continue et imperméable. Ils doivent répondre à trois propriétés majeures : 1) être fortement adhérent au substrat métallique, 2) posséder de bonnes propriétés barrière pour limiter la pénétration de l’eau et des espèces agressives et 3) apporter un rôle d’inhibition de la corrosion. Cependant, la protection des surfaces métalliques par les techniques actuelles génère une pollution notable liée à l’usage de chromates. L’utilisation de polymères biosourcés et solubles en milieu aqueux serait un challenge et contribuerait à préserver l’environnement. Les polysaccharides comme le chitosane sont des macromolécules biodégradables et respectueuses de l'environnement possédant des propriétés d’anticorrosion et sont donc des alternatives envisageables. Ces travaux de thèse portent sur le développement de revêtements anticorrosion à base de chitosane. Le chitosane possède deux points faibles pour être utilisé comme revêtement contre la corrosion : 1) une adhésion insuffisante sur la surface des matériaux et 2) un caractère hydrophile. En conséquence, le chitosane a été modifié chimiquement afin d’augmenter son adhésion et ses propriétés barrières. Afin d’améliorer son adhésion sur des substrats métalliques, des groupements de type acide phosphonique ont été ajoutés via la réaction de Kabachnik-Fields sur le chitosane. L’élaboration d’un chitosane possédant des fonctions catéchol a été également discutée. Dans un premier temps, le chitosane modifié a été testé et caractérisé par spectroscopie d’impédance électrochimique (SIE) en tant qu’inhibiteur de corrosion puis des revêtements à partir de ce même chitosane ont été réalisés et leurs protections contre la corrosion ont été évaluées. Deux approches de mise en forme des revêtements ont été testées : par dip-coating et par la technique Layer-by-Layer (LbL). Différentes voies de fonctionnalisation du chitosane ont également été présenté afin d’augmenter les propriétés barrière du revêtement. La chimie de phthaloylation du chitosane a été décrite puis le greffage de chaînes hydrophobes a été étudié
Corrosion treatment is an economic, environmental and health safety issue. More widely used on an industrial scale, coating protection consists in isolating the metal from the aggressive medium by an adherent, continuous and impermeable layer. They must have three major properties: 1) be strongly adherent to the metallic substrate, 2) have good barrier properties to limit the penetration of water and aggressive species and 3) provide a role in inhibiting corrosion. However, the protection of metal surfaces by current techniques generates significant pollution due to the use of chromates. The use of bio-based and soluble polymers in aqueous media would be a challenge and would contribute to preserving the environment. Polysaccharides such as chitosan are biodegradable and environmentally friendly macromolecules with anticorrosive properties and are therefore possible alternatives. These theses focus on the development of anticorrosion coatings based on chitosan. Chitosan has two weak points for use as a coating against corrosion: 1) insufficient adhesion on the surface of the materials and 2) hydrophilicity. As a result, chitosan has been chemically modified to increase its adhesion and barrier properties. In order to improve its adhesion on metal substrates, phosphonic acid groups have been added via the Kabachnik-Fields reaction on chitosan. The development of a chitosan with catechol functions was also discussed. Initially, the modified chitosan was tested and characterized by electrochemical impedance spectroscopy (EIS) as a corrosion inhibitor and coatings based on the same chitosan were made and their corrosion protection evaluated. Two approaches of coating elaborations were tested: dip-coating and Layer-by-Layer (LbL). Different ways of functionalizing chitosan have also been presented to increase the barrier properties of the coating. Phthaloylation chemistry of chitosan was described and hydrophobic chain grafting was studied

This thesis presents four different studis describing the characteristics and processing opportunities of two widely available biopolymers: chitosan and wheat gluten. The interest in these materials is mainly because they are bio-based and obtained as co- or by-products in the fuel and food sector In the first study, high solids content chitosan samples (60 wt.%) were successfully extruded. Chitosan extrusion has previously been reported but not chitosan extrusion with a high solids content, which decreases the drying time and increases the production volume. An orthogonal experimental design was used to assess the influence of formulation and processing conditions, and the optimal formulation and conditions were determined from the orthogonal experimental analysis and the qualities of the extrudates. The mechanical properties and processing-liquid mass loss of the optimized extrudates showed that the extrudates became stable within three days. The changes in the mechanical properties depended on the liquid mass loss. In a separate study, monocarboxylic (formic, acetic, propionic, and butyric) acid uptake and diffusion in chitosan films were investigated. It is of importance in order to be able to optimize the production of this material with the casting technique. The time of the equilibration uptake in the chitosan films exposed to propionic and butyric acid was nine months. This long equilibration time encouraged us study the exposed films further. The uptake and diffusivity of acid in the films decreased with increasing acid molecular size. A two-stage absorption curve was observed for the films exposed to propionic acid vapour. The films at the different stages showed different diffusivities. The acid transport was also affected by the structure of the chitosan films. X-ray diffraction suggested that the crystal structure of the original films disappeared after the films had been dried from their acid-swollen state, and that the microstructure of the dried films depended on the molecular size of the acid. Compared with the original films, the dried films retained their ductility, although a decrease in the molecular weight of the chitosan was detected. The water resistance of the acid-exposed films was increased, even though the crystallinity of these films was lower. The third study was devoted to chitosan/wheat gluten blend films cast from aqueous solutions. Different solvent types, additives and drying methods were used to examine their effects on the microstructures of the blended films. Chitosan and wheat gluten were immiscible in the aqueous blend, and the wheat gluten formed a discrete phase, and the homogeneity of the films was improved by using a reducing agent, compared with films prepared using only water/ethanol as cast media. Adding urea and surfactants resulted in a medium homogeneity of the films compared to those prepared with the reducing agents or with only water/ethanol. An elongated wheat gluten phase was observed in a film using glyoxal, in contrast to pure chitosan/wheat gluten blends. The opacity of the different films was studied. The mechanical properties and humidity uptake of the films increased with increasing chitosan content. The films containing 30 wt.% of wheat gluten showed the most promising mechanical properties, close to those of the pristine chitosan films. The final part describes the preparation and properties of a bio-foam composed of a blend of chitosan and wheat gluten. This foam was prepared without any porogen or frozen liquid phase to create porosity. A unique phase distribution of the chitosan and wheat gluten solutions formed without any agitation, and the foam was obtained when the liquid phase were withdrawn under vacuum. These foams showed high mass uptake of n-hexane and water in a short time due to their open pores and high porosity. The maximum uptake of n-hexane measured was 20 times the initial mass of the foam. The foams showed a high rebound resilience (94 % at 20 % compression strain) and they were not broken when subjected to bending.

QC 20150825

The aim of the study is to investigate the physicochemical, structural and sorption properties of the sorbent based on chitosan acetate, and the possibility of its use for the treatment of intoxication syndrome. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/31967

By mixing different stoichiometric ratios of chitosan with alginic acid (AA) and chitosan with p(toluenesulfonicacid) (PTSA), two new polymer networks were prepared. FT-IR spectroscopy results show the protonation of chitosan by AA and PTSA. Elemental analysis (EA) results show the composition of the networks. Thermal gravimetry analysis (TGA) and differential scanning calorimetry (DSC) results were used to characterize the thermal stability of the networks. Then, cholesterol oxidase (ChOx) enzyme were immobilized in these networks and checked for potential use of these enzyme entrapped polymer networks (EEPN) for enzyme immobilization. Additionally, the maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were evaluated for immobilized ChOx in these two polymer networks. Also, temperature and pH optimization, operational stability, shelf-life and the proton conductivity of these networks were investigated.

Ce projet vise à proposer des nouveaux candidats médicaments pour lutter contre les infections parasitaires cutanéo-muqueuses qui représentent un problème de santé majeur. C’est notamment le cas de la Trichomonose urogénitale et la leishmaniose cutanée.Malheureusement, l’administration systémique de première intention par le métronidazole (MTZ) pour traiter la trichomonose urogéntitale occasionne des problèmes de résistances et des effets secondaires indésirables. Ainsi, nous avons développé de nouvelles stratégies thérapeutiques en ciblant à la fois les mécanismes pharmacologiques et physiques de l’infection par Trichomonas vaginalis. Après avoir réussi à augmenter la solubilité apparente du MTZ dans l’eau en utilisant une beta-cyclodextrine méthylée, nous l’avons formulé dans un hydrogel thermosensible et mucoadhésif composé de pluronic® F127 et d’un biopolymère cationique et mucoadhésif, le chitosane. Cette formulation est spécifiquement adaptée à une application topique tout en offrant un contrôle de la libération du MTZ et une réduction de son passage systémique à travers la muqueuse vaginale. La viscosité élevée de l’hydrogel à température corporelle nous a conduit à étudier son effet sur la mobilité du protozoaire Trichomonas vaginalis. Il s’agit d’une stratégie physique d’immobilisation du parasite en parallèle à la chimiothérapie par le MTZ. Le suivi des trajectoires des parasites par vidéo-microscopie a montré la capacité de l’hydrogel seul ou en association avec le chitosane à immobiliser complètement T. vaginalis et à inhiber son attachement à la muqueuse. Ces évaluations ont été réalisées chez la souris. Cependant, le chitosane seul n’a pas permis d’immobilier les parasites et n’a pas montré une activité anti-T. vaginalis propre. Dans ce contexte, nous nous sommes inspirés des travaux antérieurs menés par notre équipe sur le développement de formulations à base de chitosane, et plus particulièrement des nanoparticules (NPs) composées de poly(isobutylcyanoacrylates) recouvertes de chitosane. Ces NPs ont une activité trichomonacide propre, même sans rajouter des substances actives, alors que des NPs sans chitosane étaient inactives. Nous avons étudié le mécanisme d’action et nous avons montré une meilleure internalisation des NPs lorsqu’elles étaient recouvertes de chitosane. Ces NPs ont provoqué des altérations morphologiques drastiques de la membrane du parasite. Cette activité pourrait être due en partie à l’interaction électrostatique entre la surface de T. vaginalis chargée négativement et les NPs recouvertes de chitosane cationique.Dans le but d’élargir le champ des applications de ces NPs à d’autres parasites, nous nous sommes intéressés à l’évaluation de leur effet anti-leishmanien vis-à-vis de Leishmania major. En effet, le chitosane connu pour ces propriétés cicatrisantes nous a paru particulièrement adapté pour cette pathologie. Nous avons ainsi montré in vitro et in vivo que les NPs recouvertes de chitosane avaient une activité anti-L. major propre, sans ajouter de substances actives. Dans un deuxième temps, nous avons décidé de nous orienter vers des particules de formes allongées et d’évaluer leur activité anti-leishmanienne. Ces particules appelées « plaquettes » sont constituées d’assemblages de chitosane hydrophobisé avec l’acide oléique et l’alpha-cyclodextrine dans l’eau. Cette stratégie nous a paru intéressante pour améliorer l’interaction des plaquettes avec la membrane de L. major, vue que ces parasites sont également de morphologie non-sphérique. Les résultats histologiques et immunohistochimiques des lésions cutanées ont montré une diminution significative du granulome inflammatoire et une réduction de la charge parasitaire par rapport à l'amphotéricine B seule utilisée comme référence.En conclusion, au cours de cette thèse, plusieurs formulations ont été développées et ont montré des efficacités biologiques en agissant sur des mécanismes pharmacologiques et/ou physiques des parasites
This project aims at developing new therapeutic strategies against parasitic muco-cutaneous infections such as urogenital trichomonosis and cutaneous leishmaniasis which still represents a major health problem worldwide.Unfortunately, metronidazole (MTZ) is a first-line systemic treatment for urogenital trichomoniasis that causes resistance and side effects. We have thus developed new strategies by acting on both the pharmacological and the physical mechanisms of Trichomonas vaginalis infection. After a successfull increase of the apparent solubility of MTZ in water using a methylated -cyclodextrin, we formulated it in a thermosensitive and mucoadhesive hydrogel composed of pluronic® F127 and a cationic and mucoadhesive biopolymer, chitosan. This formulation is specifically adapted for topical application providing a control of MTZ release and reduction of its systemic passage through the vaginal mucosa.Then, the ability of the high viscosity hydrogel to immobilize T. vaginalis was investigated by video-microscopy. Monitoring the trajectories of each parasite by multiple particle tracking showed the ability of the hydrogel alone or in combination with chitosan to completely immobilize T. vaginalis and to inhibit parasite attachment to the mucosa. These evaluations were performed on mice experimental model. However, chitosan alone did not allow parasite immobilization and did not show any anti-T. vaginalis activity. In this context, we were inspired by previous works conducted by our team on the development of formulations based on chitosan, and more particularly nanoparticles (NPs) composed of poly(isobutylcyanoacrylates) coated with chitosan. These NPs have their own trichomonacidal activity, even without adding active substances, while NPs without chitosan were inactive. Investigated of the mechanism of the activity showed better internalization of NPs when coated with chitosan. These NPs caused drastic morphological alterations on the parasite membrane. This activity could be due to the electrostatic interaction between negatively charged T. vaginalis surface and cationic chitosan coated NPs.In order to broaden the applications of these NPs to other parasites, we were interested in evaluating the anti-L. major activity of NPs coated or not with chitosan. Indeed, chitosan known for its healing properties could be particularly adapted for this pathology. We thus showed in vitro and in vivo that NPs coated with chitosan had intrinsic anti-L. major activity without adding any drug. In a second step, we decided to design chitosan elongated particles and to evaluate their anti-leishmanial activity. These particles called "platelets" are composed of chitosan hydrophobically-modified with oleic acid and cyclodextrin in water. This strategy could be interesting to improve the interaction of platelets with the L. major membrane, as these parasites had also non-spherical morphology. The histological and immunohistochemical results of skin lesions showed a significant decrease in inflammatory granuloma and a reduction in parasitic load compared with amphotericin B alone, used as a reference.In conclusion, during this thesis, several formulations were developed and showed biological activities by acting on pharmacological and/or physical mechanisms of the parasites

Wastewater from oil refineries is characterized mainly by the presence of hydrocarbons and, in particular, non-ionic volatile organic compounds such as benzene, toluene, ethyl benzene and xylene; lrnown as BTEX, The removal ofBTEX compounds is carried out by commercial activated carbon; however, the widespread use of this adsorbent is limited due to its high cost. As such, alternative novel adsorption techniques using non-conventional low-cost adsorbents are promising methods to remove BTEX compounds, The present study's main focus is to carry out a critical analysis on the removal efficiency of BTEX compounds from aqueous enviromnent in single and multi- component systems, investigate the advantages and limitations of each adsorbent, and evaluate the various adsorption mechanisms. Batch adsorption experiments ofBTEX-contaminated waters (5-200 mg/l) in single component systems were canied out by using chitin and chitosan as adsorbent materials, in order to evaluate the removal performance and to obtain the isotherm profiles. The effects of various parameters such as initial concentrations, adsorbent dose and contact time on the removal performance ofBTEX were investigated. The next part of this work examines the eqUilibrium sorption ofBTEX compounds; in multi-component system, from aqueous solutions by chemically modified chitosan. The enhancement of chitosan was carried out by crosslinking the chitosan with glutaraldehyde, and then grafting the poly(methaclylic acid) onto its backbone. Then, experimental work was carried out to study the adverse effects of major competing ions such as sulfate, phosphate and ammonium ions on the BTEX sorption isotherms and kinetics using chitosan and modified chitosan. The equilibrium data were analysed using Langmuir, Freundlich, RedlichPeterson, Temkin and Dubinin-Radushkevich isotherm models. The linear and non-linear regressions were carried out to determine the best fit model for each system. The linear correlation coefficient was found for each system and the Redlich-Peterson provided the best fit, over the concentration range studied. The non-linear regressions were carried out to evaluate the data by five error analysis methods; namely, the sum of the squares of the errors (ERRSQ), the hybrid fractional error function (HYBRID), Marquardt's percent standard deviation (MPSD), the average relative error (ARE), and the sum of the absolute errors (EABS). Overall, the values of error indicated that the Langmuir isotherm model provided the best quality of fit for the isotherm equilibrium data, for the selected adsorbents. The kinetic studies revealed that the adsorption followed the pseudo-second order rate model for the selected adsorbents, and the pore diffusion is not the only rate controlling step in the removal of BTEX compounds. In terms of added competing ions, it was found that the adsorption behaviour of BTEX compounds was insignificantly affected by the presence of ammonium, phosphate and sulphate ions. The major ions reduced the BTEX adsorption in order of HPO;- > SO;- > NH;J;. This study demonstrated that the chemically enhanced chitosan is a potential adsorbent for the removal of BTEX at concentrations as high as 200 mg/l.

Chitin is a natural polymer which is extracted from a number of biological sources. This polymer is deacetylated to form the related polymer chitosan. The design and preparation of chitosan derivatives has been investigated to tailor the physical and chemical properties towards specific applications. To this end, this thesis describes the synthesis and characterisation of a number of chitosan derivatives. The prepared polymers contained either naphthalimide groups, thiol groups, poly(methyl methacrylate) side chains or poly(oligoethylene glycol methyl ether methacrylate) side chains. Chitin was successfully extracted and purified from squid pens by literature methods to give polymers with DD values from 12 to 23%. The Kurita method was used to obtain chitosan samples with a range of DD values from 75 to 91%. The prepared polymers were characterised by microanalysis, IR, �H NMR and solid-state ��C NMR spectroscopy. The MW distributions of the chitosan samples were determined by GPC in 0.3 M AcOH/0.2 M AcONa(aq) solution with dextran standards. The N-(naphthalimide)-chitosan derivatives were fluorescent materials and were typically prepared from the reaction of 4-bromonaphthalic anhydride with chitosan. This reaction was thoroughly investigated to find the optimal reaction conditions. The bromo groups were subsequently displaced by one of four nucleophiles: dimethylamine, piperidine, methoxide or vinylferrocene. The polymers were characterised by microanalysis, GPC, IR, �H NMR, UV-vis and fluorescence spectroscopy. The excitation and emission wavelengths and Stokes shifts were dependent on the substituent present in the naphthalimide group. Chitin and chitosan were sequentially reacted with tosyl chloride, potassium thioacetate and sodium methoxide to form the thiolated derivatives. The success of the tosylation reaction was dependent on the synthetic route used and the DD of the polysaccharide. The thiolated chitin polymers were well defined although the samples were completely insoluble. The thiolated chitosan polymers were also characterised and were highly swollen in acetic acid solution. These thiolated polysaccharides were used as macroinitiators for the free-radical polymerisation of MMA. The products obtained from the reaction of 6-mercaptochitin in DMSO with MMA were typically prepared with low yields and low amounts of grafting. The products prepared from 6-mercaptochitosan under the same conditions contained larger amounts of the synthetic polymers although the products were highly variable. MMA was subsequently polymerised with 6-mercaptochitosan in acetic acid buffer to form highly grafted copolymers. Chitosan was reacted with the monomer OEGMA by ATRP to form chitosan-graft-poly(OEGMA) copolymers. Two synthetic routes were investigated. The "grafting-from" route involved. the formation of a chitosan macroinitiator and polymerisation of OEGMA with this polymer. The polymers contained a large amount of grafted side chains as estimated from the �H NMR spectra. However, the purification was not satisfactory as determined by the presence of two peaks in the GPC traces. The "grafting-to", route involved the formation of poly(OEGMA) by ATRP with activated initiators and subsequent attachment to chitosan. The prepared copolymers showed large differences in their appearance with even very low amounts of grafting. The purification of the polymers prepared by this method was successful with no detectable homopolymer as determined by GPC analysis.

Includes bibliographical references.
Water treatment has been an area of increasing concern over the last decade. This interest is due to exponential increase in demand of already limited water sources. Therefore the treatment of wastewater for re-use is a topic of great interest. The treatment applied depends on the source and quality of the water. Common water treatment options include filtration, flocculation, coagulation, lime softening, reverse osmosis and clarification to name a few. In addition, water should also be treated for the presence of harmful micro-organisms which is normally done using chlorine-based disinfection. Water purification filters which purify water by removal of impurities and micro-organisms are in great demand. Therefore the aim of this study was to develop ion exchange polymers and antimicrobial filters using ‘green’ materials.

Mestrado em Ciência e Engenharia de Materiais
Tissue engineering research attempts to satisfy the needs of support, reinforcement and in some cases organization of the regenerating tissue with a controlled supply of bioactive substances that might positively influence the behaviour of incorporated or ingrowing cells. As demonstrated by the recent advances on biomaterials, the ideal scaffold for tissue regeneration should offer a 3D interconnected porous structure behaving as a template to promote cells adhesion and proliferation and vascularisation as well thus stimulating the new tissue ingrowth. A special interest has been focused on chitosan (CH - the partially deacetylated derivative of chitin) scaffolds for bone regeneration due to its biological and physical properties, in spite of some drawbacks regarding its lack of mechanical strength and bioactivity. The incorporation of bioactive calcium phosphates materials in the polymer matrix is expected to reinforce chitosan scaffolds improving their mechanical performance and osteoconductivity. In the present work, chitosan based scaffolds were produced by freeze-drying CH solutions containing calcium phosphate (CaP) particles, either as fibers of hydroxyapatite (HA), platelets of monetite or a mixture of both. CaP particles were prepared by a wet precipitation method. The calcium phosphate precipitation was monitored by taking a number of samples during 3-days. Evolution of the morphology and crystal phase composition of the precipitated particles were followed by scanning electron microscopy (SEM), N2 adsorption using the BET isotherm (BET), and X-ray diffraction (XRD). It was observed that the increase of refluxing temperature allowed a faster transformation of octacalcium phosphate fibers into HA fibers, hence shortening the precipitation time required for obtaining HA fibers, Chitosan based scaffolds suspensions at two different pH values were frozen at three different temperatures before freeze-drying (thermally induced phase separation-TIPS). SEM, XRD, microcomputed tomography (μ-CT) and Fourier transformed infrared spectroscopy (FTIR) were used to analyze the physical and chemical properties of the composite scaffolds. Compressive mechanical tests were also undertaken to characterize the materials. Bioactivity studies were performed in simulated body fluid (SBF) solutions by monitoring the Ca and P concentration variations of SBF solutions. Highly interconnected macroporous scaffolds with a pore size ranging from of 50 to 250μm, interconnectivity around 91-98.5%, and porosity higher than 80% were obtained. The freezing temperature and the pH of chitosan solution/suspension revealed to play a significant influence in the pore structure. The higher pH (pH=5) and the higher freezing temperature (T=0ºC) were found as the most favourable conditions for ice crystal growth which resulted in larger pores. It was also observed that CaP particles incorporation in the CH matrix increased the scaffold mechanical strength which was also conditioned by the pore size and by the reinforcing particle morphology. The bioactivity studies revealed the CaP contribution for the scaffold bioactivity. The composite scaffolds having brushite and HA (obtained at pH=2) exhibited enhanced bioactivity as compared to composite CH/HA scaffolds based. CH based scaffolds were also prepared by incorporating HA granules loaded with dexamethasone (DEX), a drug model, in CH solution. The granules were obtained by spray drying HA nanosized particles suspended in DEX solution. The drug release profiles of DEX were determined in phosphate-buffered solution (PBS) by DEX concentration evaluation in the releasing medium by Ultraviolet (UV) spectroscopy at the wavelength of 242 nm. Among the different DEX release patterns corresponding to the various DEX loading methodologies which were tested, an adequate release profile could be selected: it showed that the release of 80% of the DEX loaded amount could be ensured during ~30 days, thus enabling a prolonged and slowest DEX release as compared to literature reports. It is thus found that the CH scaffolds engineered with a calcium phosphate based drug delivery system (DDS) provides the desirable association of a bioactive and osteoconductive matrix with an in situ controlled release of a therapeutic agent. These results point out an additional potential of the composite CH/HA scaffolds for behaving as a controlled drug release system (DDS).
A investigação em engenharia de tecidos (ET) tem procurado soluções para as necessidades de reforço e de regeneração dos tecidos recorrendo por vezes a substâncias bioactivas que podem favorecer a proliferação celular. Os avanços recentes em ET têm beneficiado da utilização de matrizes tridimensionais porosas (scaffolds) que permitem a adesão, proliferação e regeneração das células bem como a vascularização, estimulando a formação de novo tecido. A obtenção de scaffolds de quitosano (CH) para a regeneração óssea tem merecido especial interesse devido às suas propriedades biológicas e físicas, apresentando no entanto o inconveniente da falta de resistência mecânica e de bioatividade. A obtenção de scaffolds compósitos por incorporação na matriz polimérica de materiais bioactivos de fosfato de cálcio, permite reforçar o scaffold, melhorando o seu desempenho mecânico e a sua osteocondutividade. No presente trabalho, produziram-se scaffolds compósitos de quitosano/hidroxiapatite por processos de congelamento e liofilização de suspensões de fosfatos de cálcio (CaP) em soluções de CH. Utilizaramse CaP sintetizados laboratorialmente, quer na forma de fibras de hidroxiapatite (HA), quer de lamelas de monetite, quer de mistura dos dois. Os CaP foram sintetizados por um método de precipitação em meio aquoso, tendo-se monitorizado a precipitação de fosfato de cálcio durante 3 dias. Avaliou-se a evolução das fases cristalinas e da morfologia das partículas precipitadas por microscopia eletrónica de varrimento (SEM), difracção de raios X (XRD) e por adsorção de N2 usando a isotérmica de BET. Os resultados evidenciaram que o aumento da temperatura de refluxo acelera a transformação das fibras de octacalcium fosfato em fibras de HÁ, permitindo reduzir o tempo de precipitação total para obtenção de fibras de HA As soluções de quitosano e as suspensões de HAP em solução de CH, a dois valores de pH (pH=2 e pH= 5), foram congeladas a três temperaturas diferentes antes de serem liofilizadas. Caracterizaram-se os scaffolds por SEM, DRX, microtomografia computorizada (μ-CT) e espectroscopia de infravermelhos com transformada de Fourier (FTIR), tendo-se ainda avaliado o seu comportamento mecânico em compressão. Obtiveram-se scaffolds compósitos macroporosos com porosidade superior a 80%, tamanho de poro na gama 50-250μm e porosidade interconectada com grau de interconexão de 91-98.5%. Verificou-se que o tamanho e morfologia de poro dos scaffolds é condicionado pelo pH das suspensões e pela temperatura de congelamento. O valor de pH mais elevado (pH=5) e a temperatura de congelamento mais elevada (T=0ºC) são as condições que mais favorecem o crescimento de cristais de gelo e por conseguinte a formação de poros de maior dimensão. Verificou-se também que a incorporação de partículas de CaP na matriz polimérica de CH aumenta a resistência mecânica do scaffold, que é também condicionada pelo tamanho de poro e pela morfologia da partícula de CaP. O estudo do comportamento bioactivo dos scaffolds compósitos em soluções simuladoras do plasma humano (SBF), monitorizando a variação das concentrações de Ca e P na solução de SBF, evidenciou o contributo das partículas de CaP para a bioactividade do scaffold. Os scaffolds compósitos em que coexistem brushite e HA (preparados a pH=2) evidenciaram bioactividade superior á dos scaffolds compósitos CH/HA. Preparam-se também scaffolds incorporando grânulos de hidroxiapatite carregados com um fármaco modelo, a dexametasona (DEX), na solução inicial de CH. Os grânulos obtiveram-se por atomização de suspensões de HA nanométrica em solução de DEX. Construíram-se os perfis de libertação da DEX em solução tampão fosfato (PBS) por determinação da concentração de DEX por espectroscopia de ultravioleta (UV) ao comprimento de onda de 242 nm. Entre as várias curvas de libertação de DEX decorrentes das diferentes metodologias testadas para carregamento do fármaco, evidenciou-se um perfil de libertação de DEX segundo o qual cerca de 80% da DEX é libertado ao longo de ~30 dias, assegurando-se assim uma libertação mais lenta e prolongada do que as referidas na literatura para a DEX As características dos scaffolds compósitos preparados no presente trabalho apontam os materiais produzidos como promissores para aplicação em engenharia de tecidos, apresentando como potencial adicional a capacidade de se comportarem como sistemas de libertação controlada de fármacos.

A wet spinning procedure was developed for the production of chitosan fibres. The dope was prepared by dissolving 5% w/w medium grade chitosan in 2% aqueous acetic acid; after filtration and degassing, fibres were produced by extrusion of the dope into an aqueous NaOH bath and further drawing, washing, drying and winding. The effect of changes in the spinning conditions on the properties of chitosan fibres was studied; it was found that spinning variables such as jet stretch ratio, draw ratio and coagulation bath concentration had little effect on the fibre properties, though higher draw ratios would be obtained at lower jet stretch ratios and slightly improved tenacities were obtained using more dilute NaOH solutions as the coagulant. The drying methods, however, had a big effect on the fibre properties; the fibres obtained by air drying had a much higher extensibility than those dried by radiant heating. The addition of i-propanol to the dope gave much whiter fibres while the addition of Na2S04 to the coagulation bath produced the strongest fibres. Overall, the fibres produced in this work had tenacities between 0.61 and 2.48 g/dtex and extensibilities of 5.7 to 19.3%, with individual fibre decitex ranging from 2.5 to 7.5. The fibres had round cross-sections and a smooth surface when dried with heating or a rough surface when dried with acetone. The chelating properties of the chitosan fibres were studied; it was found that upto 8% Cu(II) and 5% Zn(II) can be absorbed into the fibres within 40 minutes. The Cu(II) and Zn(II) ions had strong effects on the fibre tensile properties; both dry and wet strengths were remarkably increased with the absorption of metal ions. In addition, the fibres chelated with ZnS04 had a LOI estimated to be 52%. The chitosan fibres were acetylated using acetic anhydride in methanol. It was found that 88% of the amine groups were acetylated within 30 minutes at 40 °C. The effects of temperature, time, ratio of anhydride to amine groups and the addition of water were studied; it was found that the addition of water to the methanol anhydride mixture greatly accelerated the reaction. Some O-acetylation was noticed in the reaction and this was removed by a treatment using 1 M aqueous NaOH. The changes in fibre properties after acetylation were also studied. It was found that after the acetylation process, the fibre had a better thermal stability and improved dry and wet strength. However, the fibre lost its chelating ability when substantially acetylated.

Ce travail est consacré à l'élaboration de nano-complexes polyélectrolytes (CPEs) ayant une stabilité améliorée en milieux physiologiques et à l'exemplification de leur fort potentiel d'application comme système de délivrance de (macro) molécules bioactives. Le chitosane comme polycation a été compléxé avec quatre polyanions naturels ayant différents densités de charges et groupements fonctionnels(-COO- et SO3-) à savoir l'acide hyaluronique (HYA), le chondroïtine sulfate (ChonS), le sulfate de dextrane (DS) et l'héparine (HEP). Les facteurs qui influent sur la formation et les propriétés physico-chimiques des nano-complexes chitosane-HYA ont été étudiés. Ces nanovecteurs perdent leur caractère colloïdal en milieux physiologiques. Pour améliorer leur stabilité dans ces conditions, une stratégie innovante qui implique l'ajout de zinc a été conçue. Cette stratégie de stabilisation a été démontrée comme étant polyvalente et a été étendue aux complexes polyélectrolytes (CPEs) chitosane-ChonS. Même si de cette manière une stabilité à long terme a été observée, cette stratégie reste uniquement applicable aux CPEs cationiques. Pour cette raison, une approche alternative permettant l'amélioration de la stabilité des colloïdes à charges positives ou négatives a été mise en oeuvre en concevant des nano-complexes de type coeur-couronne ternaires composés de polyacides forts c'est-à-dire de DS ou d'HEP associés au chitosane en coeur et un complexe chitosane-HYA en couronne. Tous les nano-complexes stables obtenus peuvent encapsuler le ténofovir, une molécule antirétrovirale et être fonctionnalisés par des IgAs de ciblage. En in vitro, ces nanovecteurs montrent une inhibition de l'infection des PBMC par le virus VIH-1 supérieure à l'antirétrovirale seule
This work is devoted to the elaboration of nano-polyelectrolyte complexes (PECs) systems with improved stability in physiological media and to the establishment of their high potential of applications as bioactive (macro) molecule delivery systems. Chitosan as polycation were complexed with four natural polyanions of different charged groups and densities (-COO- and SO3 - as negative charges), namely hyaluronan (HYA), chondroitin sulfate (ChonS), dextran sulfate (DS) and heparin (HEP). The factors impacting the formation and physical-chemical properties of chitosan-HYA nanocomplexes were investigated. However, these nanovectors lost their colloidal character in physiological media. To improve their colloidal stability in physiological conditions, an innovative stabilization strategy was designed, involving zinc ion. This stabilization strategy proved versatile and was extended to chitosan-ChonS PECs. Though a long-term stability was achieved, this strategy was only applicable to cationic PECs. Therefore, an alternate approach enabled the improvement of the colloidal stability in physiological media of both positive and negative colloids by designing core-shell ternary polyelectrolyte nanocomplexes composed of strong polyacid (DS or HEP)-chitosan PECs as core and a chitosan-HYA complex as shell. Furthermore, all of the stabilized nanocomplexes allowed the encapsulation of active molecules anti-retroviral drug tenofovir and surface functionalization with targeting IgAs. In vitro, these nanovectors exhibited an inhibition of infection of PBMCs by HIV-1 virus which could be superior to the free drug

Nicaragua is a country in which the toxic metal contamination of freshwater resources has become an increasingly important problem in certain regions posing a threat to the environment as well as to human health. Among the metals found in the waters of Nicaragua, arsenic is one of the most problematic since its long time consumption is connected to serious health problems such as cancer and neurological disorders. The arsenic contamination of water recourses in Nicaragua is mostly attributable natural factors, even though anthropogenic activities including gold mining may be a contributing factor.

In this work the biopolymer Chitosan was studied as a potential adsorption material for the removal of arsenic from aqueous solutions for water treatment design purposes.

The Chitosan used in this study was extracted from shrimp shells with an overall yield of 40% and a deacetylation grade of 59%. The maximum adsorption capacity was determined to 20.9 mg As/g at a controlled pH of 5.5 using the Langmuir isotherm. The adsorption was found to be strongly pH dependant with a fourfold increase in adsorption capacity when pH was well under the pKa of Chitosan. The pH dependence indicates that ionic exchange was the most important mechanism. No difference in adsorption capacity with respect to the initial pH of the solution was detected in the pH range 3-7. This was attributed to the ability of Chitosan to act as a weak base in water solutions.

The arsenic was desorbed from Chitosan using NaOH, (NH4) 2SO 4 and NaCl, with a 1M NaOH solution being the most efficient displaying a concentration ratio of 1.08. The NaOH and (NH4) 2SO 4 solutions displayed a steep desorption curvature with a large fraction of the arsenic being easily desorbed. The arsenic was, however, not completely desorbed from the Chitosan implying that the adsorption capacity would decrease for the coming cycles. Being a biopolymer the Chitosan is quite easily degraded in acid and alkali solutions, which might be a limiting step for the process applicability.

The absorption enhancing ability of chitosan, a linear polysaccharide, is mediated by protonated amino groups on the C-2 position of the molecules that induce interaction with the anionic sites on the cell membranes to subsequently alter tight junction integrity. In neutral and basic environments, such as those found in the small and large intestines, most chitosan molecules will lose their charge and precipitate from solution rendering it ineffective as an absorption enhancer. To increase the solubility of this polymer, methylation of the amino groups on the C- 2 position was proposed. A partially quaternised and water soluble derivative of chitosan, N-trimethyl chitosan chloride (TMC), which exhibits superior solubility in a basic environment compared with other chitosan salts was synthesised and included in a chitosan microbead solid drug delivery system. Two TMC derivatives were synthesised by reductive methylation from high and medium molecular weight Chitoclear™ chitosan respectively. The degree of quaternisation calculated from the 1H-NMR spectra for the medium molecular weight TMC (TMC-M) and the high molecular weight TMC (TMC-H) polymers were 74.7 % and 48.5 % respectively. The mean molecular weights of the synthesised TMC-M and TMC-H polymers were 64 100 g/mole and 233 700 g/mole respectively. The effect of different concentrations TMC-M and TMC-H on chitosan microbeads was studied with results obtained from scanning electron microscopy (SEM), TMC loading capacity and microbead swelling behaviour. After selection of the most suitable TMC concentration, the effect of varying concentration (0.1, 0.2 and 0.5 %) additives on TMC and ibuprofen release was studied. Commonly used modified cellulose gum (Ac-di-sol®(ADS)), sodium starch glycolate (Explotab®(EXP)) and ascorbic acid (AA) were added as disintegrants to different microbead formulations to promote release of both the ibuprofen as model drug and TMC from the beads. It was noticed that the loading (% drug loading capacity) of TMC-M was much lower than that obtained with TMC-H while the inclusion of different additives in varying concentrations did not seem to have a profound influence on the loading of either TMC-M or TMC-H. It was further noticed from the fit factors (f1 and f2) for dissolution profiles of eighteen chitosan microbead variations that the formulation containing TMC-H and 0.5% (w/v) ascorbic acid was the only formulation with a significantly higher ibuprofen and TMC-H release profile compared to all other formulations tested. The chitosan microbead formulation containing 2%(w/v) TMC-H and 0.5 % (w/v) ascorbic acid (H-AA-0.5) was used for in vitro absorption studies through rat intestine in Sweetana-Grass diffusion chambers. Chitosan containing TMC-H (no ascorbic acid) (CHIT-H) only and a plain chitosan microbead (CHIT) formulation was used as control formulations during the in vitro studies. Although the H-AA-0.5 formulation exhibited the highest transport rate for ibuprofen, the mean rate of transport (P app) obtained from the two formulations containing TMCH (CHIT-H and H-AA-0.5) showed no significant difference in the transport rate of ibuprofen. Compared to the CHlT formulation as control, both formulations containing TMC-H exhibited increased ibuprofen transport across in vitro rat jejunum. However, a statistical significant increase in transport was obtained only from the H-AA-0.5 formulation in comparison with the CHlT formulation. It can be concluded that the combination of high molecular weight TMC with a low degree of quaternisation and ascorbic acid (0.5% w/v) in a chitosan microbead lead to a statistical significant increase in the in vitro transport rate of ibuprofen through rat jejunum.
Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Philosophiae Doctor - PhD
Persistent root canal pathogens are one of the main causes of endodontic treatment failure. These pathogens are usually isolated in areas within the root canals that are inaccessible to mechanical instrumentation, chemical irrigants and medicaments resulting in incomplete sterilization of the root canal system. Furthermore, the development of resistant microbial species renders it difficult to disinfect the root canal system using commonly available root canal irrigants and intra-canal medicaments. Intra-canal medicaments are antimicrobial agents that are placed inside the root canal system in order to eliminate the remaining microorganisms that persist after mechanical instrumentation and irrigation. However, their antimicrobial efficacy is effective only against some of the root canal pathogens. Furthermore, the presence of tissue inhibitory factors such as dentine powder and serum albumine within the root canal system inhibits their antimicrobial activity. The use of nanoparticles as antimicrobial agents has recently attracted considerable attention especially in the medical field as a result of their unique antibacterial properties. These properties include their ability to use multiple mechanisms to eradicate microbial cells and their low potentiality to produce microbial resistance. Polymeric nanoparticles such as chitosan nanoparticles (Ch-Np) gained significant interest as a result of their biocompatible and antimicrobial properties. In medicine, several vehicles were designed to carry these antibacterial nanoparticles. Zeolites (Ze) are microporous crystalline hydrated sodium aluminosilicate material that is utilized in the chemical sciences as a carrier for various nanoparticles.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Nanoparticles (NPs) conquered an important role in many areas such as drug delivery. The number of studies that include them has grown in the last years. Nonetheless, the correlation between their properties and their effects on the immune system is poorly understood. Chitosan is a natural polymer that has shown interesting properties as biocompatibility and biodegradability. Considering the application of Chitosan NPs in the drug delivery field, the aim of this work was to study its immunotoxicity as a case study. On the one hand it was tried to clarify some controversial information that was found in the literature related with immunotoxicological properties of the chitosan NPs and on the other as a secondary objective to establish methods for testing immunotoxicity of the NPs that can be adapted to other nanomaterials which is important to their biomedical application and safe design. This work evaluated the Chitosan NPs toxicity in murine macrophages (RAW 264.7) and in human peripheral blood mononuclear cells (PBMCs). Other parameters important to the immunotoxicological assessment as the hemocompatibility were evaluated and was reported the relevance of the correct characterization of chitosan as their properties as deacetylation degree (DD) and molecular weight (MW). Chitosan NPs were produced by a coacervation method with different crosslinks: Method A with tripolyphosphate (ChitA NPs) and Method B with sodium sulphate (ChitB NPs). The average size of the ChitA NPs were 102.2 ± 8.7 nm, 133.1 ± 4.6 nm and 269.4 ± 38.5 nm with a chitosan DD of 80 %, 86 % and 93 %, respectively. With the method B, the average size of the ChiB NPs obtained were 351.7 ± 32.5 nm and 549.6 ± 12.4 nm with a chitosan DD of 80 % and 86 %, respectively. These results showed that the particle size increased with the chitosan DD and MW. Quantification studies showed that almost 100 % of chitosan was incorporated in the NPs. Stability tests were performed, and it was showed that ChitA NPs are stable at 4 ºC and 20 ºC at least for 5 weeks. The stability results in cell media showed a size increase after 24 h incubation with DMEM. In the incubation with RPMI the ChitA NPs 93 % also showed a size increase while the size of ChitA NPs decreased. Relatively to the immunotoxicological tests, the NPs showed to be more cytotoxic than the polymers in human PBMCs and in murine macrophages. An inhibitory effect of the NPs and polymers in LPS-induced NO production was observed which was more significant with the ChitA NPs with lower DD (80 %). However, without LPS, none of the concentrations of the NPs and polymers had a stimulatory effect in the NO production. The effect of chitosan in cytokine production was evaluated with two pro-inflammatory cytokines: TNF-α and IL-1β. None of the Chitosan NPs induced TNF-α, while the polymer with higher DD (93 %) showed to induce this cytokine production in a concentration-dependent manner. Both polymers and NPs were not able to stimulate the production of the IL-1β. The hemocompatibility of chitosan was also evaluated neither the NPs or polymers had hemolytic effect, but the ChitA NPs 80 % at 1mg/mL affected the plasma coagulation time by the intrinsic pathway. The effect on platelet aggregation wasn’t conclusive as during the assay, the interference of the NPs with the method was observed.These results show that the DD of chitosan and nanoparticle size can affect some immunotoxicological parameters. This work also highlights the importance of selecting appropriate methods and controls to avoid misinterpretations. These results together with further studies will contribute to develop a knowledge base and guidelines to implement the safe-by-design approach for nanobiomaterials, with focus on polymeric drug delivery systems.
As nanopartículas (NPs) conquistaram um papel importante em muitas áreas, como a entrega de fármacos. O número de estudos que as incluem tem crescido nos últimos anos. No entanto, a correlação entre suas propriedades e seus efeitos sobre o sistema imunitário é pouco compreendida. O quitosano é um polímero natural que tem mostrado propriedades interessantes tais como biocompatibilidade e biodegradabilidade. Considerando a aplicação de NPs de quitosano na área de entrega de fármacos, o objetivo deste trabalho foi estudar a sua imunotoxicidade como um caso de estudo. Por um lado, procurou-se esclarecer algumas informações controversas encontradas na literatura relacionadas às propriedades imunotoxicológicas das NPs de quitosano e, por outro, como objetivo secundário estabelecer métodos para testar a imunotoxicidade das NPs que podem ser adaptados a outros nanomateriais o que é importante para a sua aplicação biomédica e design seguro.Este trabalho avaliou a toxicidade de NPs de quitosano em macrófagos e murganho (RAW 264.7) e em células mononucleares do sangue periférico humano (PBMCs). Outros parâmetros importantes para a avaliação imunotoxicológica tais como a hemocompatibilidade foram avaliados e foi mostrada a relevância da correta caracterização do quitosano tal como suas propriedades como grau de desacetilação (DD) e peso molecular (PM). As NPs de quitosano foram produzidas por um método de coacervação com diferentes ligações cruzadas: Método A com tripolifosfato (ChitA NPs) e Método B com sulfato de sódio (ChitB NPs). O tamanho médio das NPs de ChitA foi de 102,2 ± 8,7 nm, 133,1 ± 4,6 nm e 269,4 ± 38,5 nm com um quitosano com DD de 80 %, 86 % e 93%, respetivamente. Com o método B, o tamanho médio das ChitB NPs obtidas foi de 351,7 ± 32,5 nm e 549,6 ± 12,4 nm com um quitosano com DD de 80 % e 86 %, respetivamente. Estes resultados mostraram que o tamanho das partículas aumentou com o DD e MW do quitosano. Estudos de quantificação mostraram que quase 100 % do quitosano foi incorporado nas NPs. Testes de estabilidade foram realizados, e foi demonstrado que as ChitA NPs são estáveis a 4 ºC e 20 ºC pelo menos por 5 semanas. Os resultados de estabilidade em meios celulares mostraram um aumento de tamanho após 24 h de incubação com DMEM. Na incubação com RPMI, as ChitA NPs 93 % também apresentaram um aumento de tamanho, enquanto o tamanho das ChitA NPs diminuiu. Relativamente aos testes imunotoxicológicos, as NPs mostraram ser mais citotóxicas do que os polímeros em PBMCs humanas e em macrófagos de murganho. Observou-se um efeito inibitório das NPs e polímeros na produção de NO induzida por LPS, que foi mais significativo com as ChitA NPs com menor DD (80 %). No entanto, sem o LPS, nenhuma das concentrações das NPs e polímeros teve efeito estimulante na produção do NO. O efeito do quitosano na produção de citocinas foi avaliado com duas citocinas pró-inflamatórias: TNF-α e IL-1β. Nenhuma das NPs de quitosano induziu TNF-α, enquanto o polímero com maior DD (93 %) mostrou induzir a produção de citocinas de maneira dependente da concentração. Ambos os polímeros e NPs não foram capazes de estimular a produção da IL-1β. A hemocompatibilidade do quitosano também foi avaliada, nem as NPs ou polímeros tiveram efeito hemolítico, mas as NPs de ChitA 80 % a 1mg/mL afetaram o tempo de coagulação plasmática pela via intrínseca. O efeito na agregação plaquetária não foi conclusivo, pois durante o ensaio, a interferência das NPs com o método foi observada.Esses resultados mostram que o DD do quitosano e o tamanho das nanopartículas pode afetar alguns parâmetros imunotoxicológicos. Este trabalho também destaca a importância de selecionar métodos e controlos apropriados para evitar interpretações erradas. Esses resultados, juntamente com outros estudos, contribuirão para o desenvolvimento de uma base de conhecimento e diretrizes para implementar a abordagem safe-by-design para nanobiomateriais, com foco em sistemas de entrega de fármacos poliméricos.
Outro - POCI-01-0145-FEDER-007440
Outro - PROSAFE/0001/2016

Tese de doutoramento em Engenharia Biomédica
Nanoparticles based on chitosan have been extensively studied for gene, drug and contrast agent delivery. The present work aimed to develop and characterize a glycol chitosan nanogel - also designated macromolecular micelles or hydrogel nanoparticles. The nanogel is obtained by chemically grafting hydrophobic chains on the hydrophilic glycol chitosan backbone, the amphiphilic structure obtained being capable of self-assembly in water, originating quite reproducible nanostructures with few hundred nanometers. The nanogel was decorated with folate conjugated polyethylene glycol, for active targeting purposes, as subsequently confirmed by in vitro assays. The positively charged nanogels exhibited colloidal stability up to at least four months. Considering their ability to complex siRNA, nanogels could represent promising vehicles for siRNA delivery. Moreover, their hydrophobic moieties could also carry hydrophobic drugs/imaging agents beyond nucleic acids, according to the theragnosis concept. The safety of the glycol chitosan nanogel as drug delivery system was comprehensively studied, since the biocompatibility of nanogels is still insufficiently reported. No cytotoxicity was detected in cell lines, RAW, 3T3, HMEC and HeLa, although a slight decrease on metabolic activity had been observed. Glycol chitosan nanogel didn’t induce cell membrane damage, cell death by apoptosis and/or necrosis, and cell cycling arresting (with exception to G1 arresting in RAW cells). Remarkably, glycol chitosan nanogel was poorly internalized by mouse bone marrow derived macrophages and does not trigger the activation of the complement system. Its blood compatibility was also confirmed through haemolysis and whole blood clotting time assays. The interaction of the nanogel with biological tissues, namely the endocytic mechanisms used by folate functionalized nanogels to entry HeLa cells, and the subsequent intracellular fate were studied using siRNA technology to deplete key proteins on regulation of each tested pathway. The nanogel cellular uptake is folate dependent, as expected since HeLa cells overexpress folate receptors. The internalization occurred mainly through clathrin and caveolin independent mechanisms, specifically by flotillin-1 and Cdc42-dependent endocytosis, as well as through micropinocytosis. Once internalized, after 7 h of incubation, approximately half of the nanogel was visualized in endolysosomal compartments, while the remaining was present in undefined regions of the cytoplasm. The biodistribution profile of the folate decorated glycol chitosan nanogel was assessed using in vivo near infrared fluorescence imaging as tool to track the nanogel over time in a mice model, after intravenous injection. Rapid nanogel whole body distribution was observed at early time points, and it is still detectable in a very wide distribution at least 6 h post-administration. The blood clearance occurred about 6 h post injection, with a blood half-life of approximately 2 h; surprisingly, the linear glycol chitosan seems to undergo a slower blood clearance. No accumulation in the organs was verified, the clearance from the body being observed apparently after a period of about 48h. In conclusion, the physicochemical features, the ability to complex nucleic acids and certain hydrophobic drugs, cell targeting ability, biocompatibility, internalization and intracellular trafficking, the fairly long blood circulation half-life and suitable body clearance, are pronounced hints of the engineered GC nanogel as a promising drug delivery system.
Nanopartículas à base de quitosano têm sido largamente estudadas para transportar ácidos nucléicos, fármacos e agentes de contraste. O presente trabalho tem por objectivo desenvolver e caracterizar um nanogel de glicol quitosano - também designado por micelas macromoleculares ou hidrogéis nanoparticulados. O nanogel é obtido através do enxerto químico de cadeias hidrofóbicas na estrutura hidrofílica do glicol quitosano, resultando uma estrutura anfifílica capaz de auto-organizar-se em água, originado nano-estruturas bastantes reprodutivéis com poucas centenas de nanómetros. O nanogel foi decorado com ácido fólico conjugado com polietilenoglicol para efeito de direccionamento controlado, tal como posteriormente confirmado em ensaios in vitro. O nanogel de natureza catiónica exibiu estabilidade coloidal pelo menos durante 4 meses. Considerando a sua habilidade para complexar siRNA, os nanogéis podem representar veículos promissores para transporte/entrega de siRNA. Além disso, os domínios hidrofóbicos podem também transportar fármacos hidrofóbicos/agentes de imagem para além de ácidos nucléicos, de acordo com o defendido no conceito de teragnóstico. A fiabilidade do nanogel de glicol quitosano como sistema de entrega de drogas foi estudada exaustivamente, uma vez que a biocompatibilidade dos nanogéis está ainda pouco reportada. Ausência de citotoxicidade foi observada nas linhas celulares RAW, 3T3, HMEC e HeLa, embora se tenha verificado uma ligeira diminuição na actividade metabólica das mesmas. O nanogel de glicol quitosano não danifica a membrana celular, assim como não induz morte celular por apoptose e/ou necrose, nem paragens no ciclo celular (com excepção para as células RAW onde se verifica paragem na fase G1). Surpreendentemente, o nanogel de glicol quitosano foi fracamente internalizado por macrófagos derivados de medula óssea de ratinho, para além de não desencadear a activação do sistema complemento. A sua hemocompatibilidade foi também confirmada através de ensaios de hemólise e de coagulação. A interacção do nanogel com os tecidos biológicos, nomeadamente os mecanismos de endocitose utilizados pelos nanogéis funcionalizados com ácido fólico para entrarem nas células HeLa e subsequente percurso intracelular foram estudados recorrendo à tecnologia de siRNA para silenciar proteínas chave na regulação de cada via analisada. A internalização do nanogel mostrou ser dependente do ácido fólico, tal como esperado uma vez que as células HeLa sobre-expressam receptores para ácido fólico. A internalização ocorreu principalmente através de mecanismos independentes de clatrina e caveolina, mais especificamente encocitose dependente de flotilina-1 e Cdc42, bem como macropinocitose. Uma vez internalizado, depois de 7 h de incubação, cerca de metade da população do nanogel foi visualizada em compartimentos endolisossomais enquanto que a porção restante se encontra em regiões indefinidas do citoplasma. O perfil de biodistribuição do nanogel de glicol quitosano decorado com ácido fólico foi avaliado num sistema de imagiologia in vivo de fluorescência no infravermelho próximo como ferramenta para monitorizar a distribuição do nanogel após injecção intravenosa ao longo do tempo, num modelo de ratinho. Uma rápida distribuição do nanogel por todo o corpo foi observada logo nos tempos mais precoces, sendo ainda detectável uma ampla distribuição até pelo menos 6 h após a administração. O desaparecimento do nanogel do sangue ocorreu cerca de 6 h após injecção, com um tempo de meia-vida de aproximandamente 2 h; surpreendentemente, o glicol quitosano de estrutura linear mostrou desaparecer mais lentamente do sangue. Não se verificou acumulação nos órgãos, e o desaparecimento do corpo acontece num período de aproximadamente 48h. Em conclusão, as características físico-químicas, a capacidade de complexar ácidos nucléicos e certamente fármacos hidrofóbicos, direcionamento celular, biocompatibilidade, internalização e percurso intracelular, período de semi-vida no sangue relativamente longo e razoável período de eliminação do corpo, são indícios de que o nanogel produzido poderá ser um sistema promissor de libertação controlada de drogas.
The grant SFRH/BD/64977/2009, SFRH/BD/27359/2006, SFRH/BD/27404 / 2006, from Fundação para a Ciência e Tecnologia (FCT), Portugal. This review was also supported by FCT through the project PTDC/BIO/67160/2006.

Este relatório pretende demonstrar o trabalho desenvolvido ao longo do último ano e meio, no que respeita à temática de Encapsulação de DNA, usando para o efeito Partículas de Gel e Nanopartículas. O trabalho a que se refere este relatório incidiu na criação de partículas de gel, preparadas por gelificação na interface água/água, através da interacção de dois polielectrólitos de carga oposta, o DNA e um oligossacarídeo de baixo peso molecular, o lactato de quitosano. É de destacar que as partículas foram obtidas sem utilizar agentes quelantes (cross-linkers), ou solventes orgânicos. Numa segunda parte do trabalho procedeu-se à experimentação de nanopartículas de Lactato de Quitosano contendo DNA no seu interior, usando para o efeito um nebulizador, que permitiu originar pequenas gotículas de DNA. De modo a caracterizar os sistemas obtidos, estes foram estudados através de uma análise de vários parâmetros físico-químicos: morfologia e estrutura das partículas assim como o grau de complexação sofrida pela gota de DNA, cinéticas de libertação de DNA por forma a comprovar a sua estabilidade, viscosidade dos polielectrólitos de forma a poder obter partículas de gel estáveis, tamanho e a carga das partículas, estudo o qual derivou na abordagem às Nanopartículas, uma vez que estas permitiriam uma transfecção celular mais eficaz. Foi também estudado, na Facultat de Farmacia em Barcelona, o efeito de tanto o Lactato de Quitosano como as partículas obtidas, na viabilidade celular através de ensaios com sal tetrazolium MTT em linhas celulares do tipo 3T3 fibroblast, bem como a interacção destes, com outros modelos celulares mais simples como os eritrócitos. Estes estudos permitiram verificar que o Lactato de Quitosano é um promissor agente no que respeita à transferência de genes.

碩士
中興大學
化學工程學系所
95
Chitosan nanocomposites were prepared by addition of 5 nm gold nanoparticles or silver nanoparticles in the chitosan-Au and chitosan-Ag matrix. The growth of human gingival fibroblasts on chitosan nanocomposites was enhanced. Chitosan-silver nanocomposites had the better antibacterial effect for Staphylococcus aureus. Results from Atomic force microscope showed that the surface crystalline domains of chitosan-Au or chitosan-Ag nanocomposites appeared larger at 60 or 120 ppm. At this time the mechanical properties and the glass transition temperature of the nanocomposites measured by the dynamic mechanic analyzer showed that chitosan-Au at 60 ppm and chitosan-Ag nanocomposites at 120 ppm were better. Transmission electron microscopy showed that aggregation of the nanoparticles at high concentrations may have destroyed the crystalline domains and reduced the physical properties. The free scavenging ability and hydrophilicity of the nanocomposites were better than those of the pure chitosan.

碩士
高雄醫學大學
醫藥暨應用化學研究所
97
Chitosan, CHI, is a natural cationic polysaccharide with good biocompatibility, biodegradability, bioactivity, low cytotoxicity and non- allergenicity. These properties make CHI a good candidate for multiple applications in industries such as a food additive agent or a fat blocker, cosmetics, textiles, agriculture, chemical additives, biomedical devices, biomedicine and biomaterials. Recently, CHI is also reported for its potential as both drug and gene carrier. Therefore, in developing a novel gene carrier, three CHI-succ(s) of different degrees of substitution were synthesized from a low molecular weight CHI prepared from the hydrolysis by NaBO3. CHI can be protonated in acidic condition (pH < 7) because its amino group has a pKa value of 6.5. This character facilitates CHI to form a complex with anionic DNA through electrostatic interactions, which is named as polyplex. From 1H-NMR spectrum, the degrees of deacetylation (D.D %) of hydrolyzed CHI(s) were around 86~89% while the degrees of substitution (D.S %) of succinic anhydride in three CHI-succ(s) were 5 %, 10 % and 20 %. After the substitution of succinic anhydrides, both COOH and amide absorption peak were observed in the IR spectra. The degree of crystallinity structure of CHI was decreased with increased succ substitution but the buffering capacity and pKa values were similar to CHI. Next, various weight ratios (w/w) of CHI / DNA and CHI-succ / DNA polyplexes were prepared for gel retardation, particle size, ζ-potential and TEM analysis. Results showed that the plasmid DNA is readily entrapped at w/w = 20, and the sizes and ζ-potentials were around 110~140 nm and ± 1~5 mV. Finally in the cellular studies, all polymers and polyplexes exhibited low cytotoxity in HEK 293T cell lines. Both CHI-succ 5% and CHI-succ 10% showed improved transfection efficiency compared to unmodified CHI, indicating a looser but proper binding ability for CHI-based nucleic acid delivery system.

博士
雲林科技大學
工程科技研究所博士班
96
There are four parts included in the work, concentration the preparation of various sulfonation degree sulfonated chitosan (SCS) and crosslinked sulfonated chitosan (GASCS) membranes first. Second part of this work was about introduced binary croslinked agents of hydrophilic chitosan network membranes (GASSACS). Third part of this work related to the synthesis Octa[3-propylglycidylether] dimethylsiloxy] petacycloctasilanae (OG) was synthesized as the crosslinked agent for the preparation of organic-inorganic hybrid chitosan network nanocomposite membranes. Eventually, the pervaporation separation performance for 90wt% ethanol aqueous dehydration of the above network membranes was studied. For Part I investigation, chitosan (CS) is modified by propane sultone via a sulfonation reaction to be subjected with various degrees of sulfonation. The sulfonated chitosan (SCS) has a pendant alkyl sulfate group dangling at the side chain, which can improve its hydrophilicity and water-solubility. Elemental analysis (EA), Fourier transform infrared spectroscopy (FT-IR) and 13C nuclear magnetic resonance (13C NMR) were adopted to identify the structure and determinine the distribution of the substituents of the final product. The degree of sulfonation in the SCS can be controlled and the hydrogen-bonding interaction can be reduced by varying the degree of sulfonation. A solubility test proved that solubility increased with degree of sulfonation at a wide range of pH values. X-ray diffraction patterns of sulfonated chitosan samples demonstrated that the crystallinity declined as the increasing degree of sulfonation. Thermogravimetric analysis (TGA) and modulated differential scanning calorimetry (MDSC) results indicated that thermal stability fell down but water absorbance increased with the degree of sulfonation. Better water soluble sulfonated chitosan is thus obtained. The controllable free pendant amino-groups of the chitosan polymer suggest new possibilities for the application of chitosan-based materials. Glutaraldehyde (GA) was used as a crosslinking agent for preparing crosslinking sulfonated chitosan membranes. The structures of crosslinking sulfonated chitosan membrane were elucidated using FT-IR and 13C NMR. The XRD results demonstrate that the degree of crystallinity of the GASCS membrane declines as the degree of crosslinking increase. An experiment on swelling and contact angle indicates that the degree of swelling and the contact angle of cross-linked chitosan membrane drop as the amount of glutaraldehyde increases; the hydrophilicity of GASCS decreases as the amount of glutaraldehyde increases. The pervaporation performance experiment indicates that the GASCS membrane separation is governed by diffusivity of the membrane. In Part II, chitosan was crosslinked using a sulfosuccinic acid (SSA) and glutaraldehyde mixture as the binary crosslinking agents to form hydrophilic chitosan network membranes. GA and SSA improve tensile strength and contribute to the hydrophilicity, respectively. The membranes that are produced by varying the crosslinking agent content are also characterized using FT-IR, X-ray diffraction and tensile testing, and by measuring their swelling ratio and thermal properties. Experimental results reveal that the contact angle of the membrane decreases from 84.54o to 69.83o and the maximum stress rises from 39.62 MPa to 133.66 MPa as the increase of the binary crosslinking agent content. These resultant membranes not only maintain the hydrophilicity but also enhance the mechanical strength. In Part III, chitosan was crosslinked using a synthesis Octa[3-propylglycidylether] dimethylsiloxy] petacycloctasilanae (OG) as the crosslinked agent to form a organic-inorganic hybrid chitosan network nanocomposite membranes(OGCS). The synthesis of OG was characterized using 1H-NMR and FT-IR. The OGCS was characterized by FT-IR. Contact angle measurement, swelling ratio, bulk density and fraction free volume all reveal that those properties increased at lower OG contents and then decreased with increasing OG contents. It was caused by the coagulation effect. The main factor of above results is the competition between crosslinking and coagulation. The degree of coagulation is enhanced with increasing OG contents in chitosan nanocomposite membranes. The OG coagulation behaves as the plasticizer and caused less improvement and the poorer properties. The tensile test, X-ray, TGA and MDSC analyses results show that the tensile properties and thermal properties slightly increased with increasing OG contents. This result originated from the OG regular cage-like structure and the X-ray pattern was demonstrated. Finally, the pervaporation separation performance for 90wt% ethanol aqueous dehydration of the above network membranes results show that a GA30SCS membrane at 60℃ has a permeation flux rate of 2532 g/m2-hr and the permeate contains 99.78% water.

博士
國立清華大學
化學工程學系
91
In this study, we make the favorable applications of chitosan in immobilization techniques. In enzymatic study, a method for immobilization of Candida rugosa lipase to two types of chitosan beads by activating the hydroxyl groups of chitosan using 1-Ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) coupling agent has been successfully developed. The ability of EDC to activate the hydroxyl groups of chitosan was confirmed using the electron spectroscopy for chemical analysis (ESCA) technique. The properties of lipase immobilized using dry and wet chitosan beads were also investigated and compared. Immobilization enhanced the enzyme stability against changes of pH and temperature. High storage stability of 30 days and an increased enzyme activity of 2,110% were observed in wet immobilized lipase, which indicated that the enzyme had a high affinity for chitosan supports. Immobilized lipase using dry and wet chitosan beads retained 78 and 85 % of its initial activity after 10 batch hydrolytic cycles. It was found that the swelling of dry chitosan beads in aqueous medium lead to a decrease in lipase loading as well as stability. The kinetic parameters such as Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined for the free and immobilized lipase. The activation energy (Ea) was found to decrease for immobilization of lipase on chitosan beads. On the other hand, immobilization of bata-cyclodextrin to chitosan (Ch-BCD) by chemical binding has been successfully developed and demonstrated that it possessed excellent capacity for cholesterol adsorption. The applicability of Langmuir and Freundlich equations to interpret the behavior of adsorption was studies. The experimental data on cholesterol adsorption fitted well in the Langmuir isotherm equation as compared with using Freundlich isotherm equation. The maximum amount of cholesterol adsorption determined using the Langmuir isotherm equation was 0.33 gram of cholesterol per gram of Ch-BCD which was the same as the experimental data. Cholesterol adsorption from yolk was also carried out with the Ch-BCD. Results indicated that 92% of cholesterol could be removed using 1% (w/v) Ch-BCD in 2 hours at 25oC. On the other hand, 96% of adsorbed cholesterol could be dissociated from the Ch-BCD by using 95% ethanol at 50oC. The adsorption of plasma cholesterol from human blood was also investigated. About 55% of total cholesterol from 20 ml of plasma could be removed in 30 minutes by using 0.5 gram of Ch-BCD.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
Cyclodextrins are oligosaccharides that have been studied for over 100 years and they have been used in numerous industries, ranging from food or cosmetic to pharmaceutical industry. However, some of them are still regarded as novel pharmaceutical excipients. Their characteristics make them particularly interesting since they can form complexes with poorly-water soluble lipophilic drugs, enhancing its water solubility. This strategy may improve the drugs oral bioavailability and their permeability through biological membranes. One of the interesting areas of research in drug delivery is the design of nanomedicines consisting of nanosystems that are able to deliver drugs to the right place, at appropriate time. Natural polysaccharides have received more and more attention in this field and they seem to be very promising materials in the preparation of nanometric carriers. The main goal of the present study was to develop dexamethasone nanoparticles using a polymer (chitosan) and an anionic cyclodextrin, sulfobutylether-β-cyclodextrin, and investigate its characteristics. The results obtained reveal that it is possible to form NPs consisting of those components, and they also show that the NPs present good proprieties such as stability at room temperature.
As Ciclodextrinas são oligossacáridos que têm vindo a ser estudadas há mais de 100 anos e são usadas em inúmeras indústrias, desde a indústria alimentar ou cosmética até à indústria farmacêutica. Contudo, muitas delas ainda são encaradas como excipientes farmacêuticos novos. As suas características tornam-nas particularmente interessantes, uma vez que podem formar complexos com fármacos lipofílicos, melhorando a sua solubilidade em água. E assim, melhoram a biodisponibilidade dos fármacos e influenciam a sua permeabilidade através das membranas biológicas. Uma das áreas de investigação na administração de fármacos é o design de nanomedicamentos que consistam em nanosistemas com capacidade para fazer o fármaco chegar ao local de acção no período adequado. Polissacáridos naturais têm recebido cada vez mais atenção nesta área e perecem ser materiais muito promissores na preparação de transportadores à escala nano. O objectivo principal deste estudo é desenvolver nanopartículas de dexametasona, usando um polímero (quitosano) e uma ciclodextrina aniónica, a sulfobutileter- β-ciclodextrina, e investigar as suas características. Os resultados demonstraram que é possível criar NPs que consistam nestes componentes, e ainda revelam que as NPs formadas apresentam boas propriedades, tal como a estabilidade à temperatura ambiente.

碩士
國立臺灣海洋大學
食品科學系
100
This study was investigated the effect of temperature and solution viscosity on MW of ultrasonic degraded of chitosan and effect of recovery method on DD of degraded chitosan. Chitosan was dissolved in acetic acid to produce 1% (w/v) chitosan solution, then ultrasonic degraded at 30, 40, 50℃ for 1-6 h respectively, and then the molecular weights and viscosities were detected. The results showed there is best ultrasonic degradation rate of chitosan at 30℃. The viscosity of chitosan solution decreased with the molecular weight decreasing. As the viscosity of chitosan solution below the specific viscosity range, the ultrasonic degradation rate was increased significantly. This lead to degradation rate of later period than degradation of previous period. To reduce the viscosity of the solution by lowering the concentration of chitosan solution, we found the lower concentration of chitosan solution have the higher degradation rate in the same degradation conditions. In addition, the predictive degradation curve of chitosan were calculated by the k of degradation formula. Comparison with the predictive and the experimental degradation curve, finding the predictive degradation curves of chitosan solution with lower viscosity were more closed to the experimental degradation curve. Therefore, the viscosity of chitosan solution is indeed an important factor for the ultrasonic degradation of chitosan. Additional, the probability of higher degree of deacetylation of chitosan was prepared will be high due to ultrasonic degradation time be longer.

Thesis (M.S.) -- University of Tennessee, Knoxville, 2005.
Title from title page screen January 30, 2006). Thesis advisor: John Mount. Document formatted into pages (x, 72 p. : ill. (some col.)). Vita. Includes bibliographical references (p. 50-60).