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Discharge of metal containing effluents into water has been a cause of major concern. Traditional treatment methods are proving to be ineffective and expensive. Chitosan was studied as a potential biosorbent due to its positive charge and relatively low cost. The study involves evaluating the metal binding performance of chitosan in a polymer enhanced diafiltration (PEDF) system which uses an ultrafiltration membrane to retain the chitosan which, in turn, binds the metal, thereby preventing passage into the permeate stream. Conditions for binding such as pH, concentration of polymer and chromium were studied. Optimal performance was obtained when the system was operated at pH values lower then the pKa of chitosan i.e. 6.3. Using 6 g/L chitosan at pH 4.0, chromium concentration was reduced to less than 1mg/L from a feed concentration of 20 mg/L. Equilibrium dialysis experiments were done to study the kinetics of binding and the uptake of metal per gram of polymer. Rheological measurements demonstrated that in the presence of 1-100 mM chromate, chitosan was found to be slightly shear-thickening at low concentrations such as 4 g/L and 6 g/L whereas it was slightly shear-thinning at higher concentrations like 12 g/L and 20 g/L This suggests that neutralization of chromium anions is due to the interaction of multiple chitosan molecules. This result is consistent with the relatively stiff nature of the polysaccharide. Overall, this study suggests that some modification of the native polymer would be required to improve uptake and make it an industrially workable process.

L’objectif du travail présenté dans cette thèse était de développer des nanoparticulesfonctionnelles ayant la capacité d’induire l’adhésion et la migration de kératinocyteshumains normaux. L’utilisation de systèmes particulaires pour favoriser l’adhésion etla migration cellulaire dans les processus de cicatrisation constitue une nouvelleapproche de l’ingéniérie tissulaire. Dans cette optique, un dérivé hydrosoluble du chitosan rendu fonctionnel par l’ajoutde peptides RGD a été développé. Les nanoparticules furent développées parcoacervation complexe entre le dérivé cationique du chitosan et le sulfate dechondroïtine anionique. La capacité du système particulaire à induire unchangement cellulaire phénotypique a été évaluée in vitro.Lors de l’évaluation de ce nouveau polymère, le succès de la synthèse a été montrépar l’absence de cytotoxicité et par la préservation de son activité biologique médiéepar les séquences RGD. Aussi bien les polymères que les nanoparticules ont induitl’adhésion et la mobilité de fibroblastes dermiques humains, confirmant le conceptde nanoparticules bio-actives. Cependant, concernant l’étude des interactions entreles nanoparticules et les kératinocytes, aucune conclusion n’a pu être tirée etd’autres travaux sont nécessaires. Pour résumer, un système particulaire bio-actif a été développé. Le choix despeptides RGD pour induire la migration des kératinocytes doit être réévalué, etl’utilisation de concentrations plus importantes, de mélange de peptides d’adhésionou l’utilisation de peptides d’adhésion différents doit être envisagée pour laréalisation d’études ultérieures. / The aim of the work presented in this thesis, was to develop functionalizednanoparticles with the ability to induce adhesion and migration in normal humankeratinocytes. Using particulate systems to promote and support cell adhesion andmigration in epidermal restoration is a novel approach of tissue engineering.In this view, a water-soluble chitosan derivative functionalized with RGD peptideswas developed. Nanoparticles were formed through complex coacervation betweenthe cationic chitosan derivative and the anionic chondroitin sulfate. The particulatesystem was evaluated in vitro for its ability to change phenotype in cells.In the evaluation of the novel hybrid polymer, the successful synthesis wasconfirmed by the absence of cytotoxicity and a preserved bioactivity specific to theRGD-moieties. Both the polymer and the particles formed thereof induced celladhesion and spreading in human dermal fibroblasts, proving the concept ofbioactive nanoparticles. However, when investigating the interaction between thenanoparticles and keratinocytes, no clear conclusion could be drawn and furtherassays are required. To summarize, a bioactive particulate system was developed. The choice of RGDpeptides to induce migration in keratinocytes needs to be re-evaluated and higherconcentrations, mixtures of adhesion peptides or other adhesion peptides might beconsidered for further investigations.

Chitosan is a biopolymer resulting from the deacetylation of chitin, the second most abundant biopolymer in nature. Chitosan has been successfully used in systems to remove metal ions and other pollutants from wastewater. Chitosan has shown promise as a sorbent for radionuclides in some aqueous waste streams. The sorption of these radionuclides by chitosan is studied to determine if chitosan could be used as a sorbent for aqueous waste streams containing these metals. The effect of various experimental conditions including sorbent particle size, agitation rate, hydration, temperature, pH, metal concentration and sorbent concentration are examined in this study. Results showed that sorption depends on the availability of access sites, controlled by the specific surface area of the sorbent. Sorption was observed to decrease with increasing temperature. The sorption isotherms and kinetics for Co(II), Eu(III) and U(VI) sorption onto chitosan were determined experimentally by batch sorption. Isotherms were fitted using the Langmuir and Freundlich models. Kinetics were modeled using the pseudo- first order, pseudo-second order, Elovich, and intraparticle diffusion models in order to determine possible rate-limiting steps. Most data were well described by the pseudo- second order and Elovich models. Multi-linearity was observed in the intraparticle diffusion model. The sorption capacity of the metals on chitosan was found to follow the order Co < Eu < U. / Graduation date: 2013

1. Die Deacetylierung von crabshell – Chitosan führte gleichzeitig zu einem drastischen Abfall der mittleren viscosimetrischen Molmasse ( Mv), insbesondere wenn die Temperatur und die Konzentration an NaOH erhöht werden. Diese Parameter beeinflussten jedoch nicht den Grad der Deacetylierung (DD).<br /> Wichtig ist jedoch die Quelle des Ausgangsmaterials: Chitin aus Pandalus borealis ist ein guter Rohstoff für die Herstellung von Chitosan mit niedrigem DD und gleichzeitig hoher mittlerer Mv, während Krill-Chitin (Euphausia superba) ein gutes Ausgangsmaterial zur Herstellung von Chitosan mit hohem DD und niedrigem Mv ist. Chitosan, das aus Insekten (Calliphora erythrocephala), unter milden Bedingungen (Temperatur: 100°C, NaOH-Konzentration: 40 %, Zeit: 1-2h ) hergestellt wurde, hatte die gleichen Eigenschaften hinsichtlich DD und Mv wie das aus Krill hergestellte Chitosan. Der Bedarf an Zeit, Energie und NaOH ist für die Herstellung von Insekten-Chitosan geringer als für crabshell-Chitosan vergleichbare Resultaten für DD und Mv.<br /> <br /> 2. Chitosan wurde durch den Schimmelpilz Aspergillus fumigatus zu Chitooligomeren fermentiert. Die Ausbeute beträgt 25%. Die Chitooligomere wurden mit Hilfe von HPLC und MALDI-TOF-Massenspektrmetrie identifiziert. Die Fermentationsmischung fördert die Immunität von Pflanzen gegen Bakterien und Virusinfektion. Die Zunahme der Immunität schwankt jedoch je nach System Pflanze-Pathogen. Die Fermentation von Chitosan durch Aspergillus fumigatus könnte eine schnelle und billige Methode zur Herstellung von Chitooligomeren mit guter Reinheit und Ausbeute sein. Eine partiell aufgereinigte Fermentationsmischung dieser Art könnte in der Landwirtschaft als Pathogeninhibitor genutzt werden. Durch kontrollierte Fermentation, die Chitooligomere in definierter Zusammensetzung (d.h. definierter Verteilung des Depolymerisationsgrades) liefert, könnte man zu Mischungen kommen, die für die jeweilige Anwendung eine optimale Bioaktivität besitzen.<br /> <br /> 3. Die aus Chitosan-Dispersionen hergestellten MCChB-Filme weisen bessere mechanische Eigenschaften (Bruchfestigkeit, Dehnung) und eine höhere Wasseraufnahmefähigkeit auf als Filme, die nach herkömmlichen Methoden aus sauerer Lösung hergestellt werden. Die Einführung von Proteinen ändert die mechanischen Eigenschaften der MCChB-Filme abhängig von der Art, der Proteine sowie des DD und der Mv des eingesetzte Chitosan. Die Zugabe von Protein beschleunigt den biologischen Abbau der MCChB-Filme. Aus den untersuchten MCChB-Filmen mit Proteinzusatz können leichte, reißfeste und dennoch elastische Materialen hergestellt werden.<br /> <br /> 4. Mit Hilfe von MCChB-Dispersion kann Papier modifiziert werden. Dadurch werden die mechanischen Eigenschaften verbessert und die Wasseraufnahme wird verringert. Die Zugabe von Proteinen verringert das Wasseraufnahmevermögen noch weiter. Ein geringes Wasseraufnahmevermögen ist der bedeutendste Faktor bei der Papierherstellung. Auch Papier, das mit einem MCChB-Protein-Komplexe modifiziert wurde, zeigt gute mechanische Eigenschaften.<br /> <br /> 5. Wird Chitosan durch unmittelbare Einführung von MCChB auf Cellulose-Fasern aufgebracht, so erhält man eine netzartige Struktur, während durch Ausfällung aufgebrachtes Chitosan eine dünne Schicht auf den Cellulose-Fasern bildet. Die netzartige Struktur erleichtert die Bioabbaubarkeit, während die Schichtstruktur diese erschwert.<br /> <br /> 6. Die guten mechanischen Eigenschaften, die geringe Wasseraufnahmefähigkeit und die mit Cellulose vergleichbare Bioabbaubarkeit von Papier, das mit MCChB modifiziert wurde, lassen MCChB für die Veredlung von Papier nützlich erscheinen. / 1. Deacetylation of the crustacean chitosan causes drastically decrease in the Mv with increasing reaction temperature and time as well as the concentration of sodium hydroxide. However, the DD are relatively less affected. Pandalus borealis is a good source for production of chitosan having high Mv and low DD, whereas chitosan of medium to low Mv can ideally be prepared using krill chitin.<br /> Insect chitosan is prepared under milder condition as compared with the crustacean chitosan, showed similar Mv and DD. Moreover, the consumption of time, energy and sodium hydroxide is much lower than for crustacean chitosan used. <br /> The properties of chitin (type of source, crystallinity, DD, Mv, swelling properties, particle size) affect the deacetylated polymer parameters.<br /> <br /> 2. Fermentation of chitosan using fungus Aspergillus fumigatus resulted in a composition of oligosaccharides with controlled molecular weight and yield at least 25 wt%. The product of fermentation effectively inhibited the viral and/or bacterial infection of the plant. This method can be an excellent, inexpensive system for preparation of bioactive agent.<br /> The preliminary purified fermentation mixture due to its antiviral and antibacterial behaviour is capable to be used as a natural, plant protection agent. The controlled degradation of chitosan connected with the production of various oligosaccharides having specified molecular weight allows obtaining the product with optimum bioactivity for suitable applications.<br /> <br /> 3. The films formed form microcrystalline chitosan (MCChB) gel-like dispersion demonstrate the better mechanical properties and higher swelling behaviour than typical films prepared using acidic solution of chitosan. The introduction of proteins significantly changes the mechanical strength and swelling behaviour. Addition of proteins causes the increase in their biodecomposition. The blended films containing proteins could be the base for formation of the resistant materials showed excellent elongation at break. <br /> <br /> 4. The application of MCChB in a paper formation as a modificator of the fibre-water interactions allows producing the paper sheets indicating the high increase in the mechanical properties and significant decrease in swelling properties. The introduction of MCChB with proteins causes a slight decrease in paper mechanical strength, if determined at low relative humidity. However, the mechanical strength measured at high relative humidity differ less than for paper sheet containing only MCChB.<br /> <br /> 5. Direct introduction of MCChB to a paper pulp forms the "web-like" structure of cellulose fibres and MCChB. <br /> The "web-like" structure of MCChB enables the faster biodecomposition of formed paper sheets. The precipitation of MCChB as wells as introduction of MCChB with proteins causes the "coat-like" structure. MCChB creates a thin layers coated the cellulose fibres lowering a biodecomposition rate.<br /> <br /> 6. The properties of paper sheets modified by MCChB such as: similar to cellulose biodegradation, excellent mechanical properties at rel. high humidity and the decrease in swelling properties as well as various possibilities to introduce MCChB allow to apple microcrystalline chitosan with or without proteins as the modificator of the fibre-water interactions in paper.

Taking inspiration from nature, where composite materials made of a polymer matrix and inorganic fillers are often found, e.g. bone, shell of crustaceans, shell of eggs, etc., the feasibility on making composite materials containing chitosan and nanosized hydroxyapatite were investigated. A new preparation approach based on a co-precipitation method has been developed. In its earlier stage of formation, the composite occurs as hydrogel as suspended in aqueous alkaline solution. In order to get solid composites various drying procedures including freeze-drying technique, air-drying at room temperature and at moderate temperatures, between 50oC and 100oC were used. Physicochemical studies showed that the composites exhibit different properties with respect to their structure and composition. IR and Raman spectroscopy probed the presence of both chitosan and hydroxyapatite in the composites. Hydroxyapatite as dispersed in the chitosan matrix was found to be in the nanosize range (15-50 nm) and occurs in a bimodal distribution with respect to its crystallite length. Two types of distribution domains of hydroxyapatite crystallites in the composite matrix such as cluster-like (200-400 nm) and scattered-like domains were identified by the transmission electron microscopy (TEM), X-ray diffraction (XRD) and by confocal scanning laser microscopy (CSLM) measurements. Relaxation NMR experiments on composite hydrogels showed the presence of two types of water sites in their gel networks, such as free and bound water. Mechanical tests showed that the mechanical properties of composites are one order of magnitude less than those of compact bone but comparable to those of porous bone. The enzymatic degradation rates of composites showed slow degradation processes. The yields of degradation were estimated to be less than 10% by loss of mass, after incubation with lysozyme, for a period of 50 days. Since the composite materials were found biocompatible by the in vivo tests, the simple mode of their fabrication and their properties recommend them as potential candidates for the non-load bearing bone substitute materials. / Inspiriert von Natur, bei der Kompositmaterialien aus Polymermatrices und anorganischen Füllstoffen z.B. in Knochen, Krustentieren und Eierschalen vorzufinden sind, wurde die Herstellungsmöglichkeit von Kompositmaterial aus Chitosan und Hydroxyapatitdispersionen untersucht. Basierend auf einem Kopräzipitationsverfahren wurde eine neue Herstellungsmethode entwickelt, die als flexibler Zugang zu einem Spektrum von Komposittypen führt. In den frühen Phasen der Kompositbildung entsteht ein in der wässrigen alkalischen Lösung suspendiertes Hydrogel, das durch Filtration und Zentrifugation isoliert werden kann. IR und Ramanspektroskopie klären das Vorhandensein von Chitosan und Hydroxyapatit im Kompositmaterial. Hydroxyapatit ist als Nanopartikel der Größe von 15-50 nm in bimodaler Verteilung in der Chitosanmatrix dispersiert, und in durch Transmissionselektronenmikroskopie (TEM), X-Ray Diffraction (XRD) und Konfokaler Laserscanmikroskopie (CSLM) nachweisbaren 200-400 nm großen Clustern assembliert. NMR-Relaxationsmessungen an Hydrogelkompositmaterial decken die Existenz zweier Klassen vorkommenden Wassers im Netzwerk auf, gebundenes und freies Wasser. Mechanische Tests zeigen, dass die mechanische Festigkeit etwa eine Größenordnung unter der von massivem Knochen liegt, der Festigkeit von porösem Knochen aber gleichkommt. Enzymatische Abbauraten des Kompostimaterials sind als langsam einzuschätzen. Eine 50-tägige Einwirkzeit von Lysozym führte zu einem Abbau von 10 % der Kompositmasse. Die sich durch in vivo Tests herausstellende Biokompatibilität, die einfachen Herstellungsmöglichkeiten und die physikochemischen Eigenschaften empfehlen dieses Material als vielversprechenden Kandidaten für Knochenersatzmaterial in mäßig belasteten Bereichen.

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.

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.

Thesis (M.S.) -- Mississippi State University. Department of Agricultural and Biological Engineering. / Title from title screen. Includes bibliographical references.

In search of better scaffolding materials for in vitro culture of chondrocytes, the combination of chitosan (similar to glycosoaminoglycans) and gelatin (denatured collagen) was tested due to its resemblance to cartilage extra-cellular matrix (ECM). Porous scaffolds were fabricated from chitosan gelatin blends (1:1, 2:1, and 3:1). The response of chondrocytes to them was evaluated from the amount of sulphated GAG and collagen type 2 secreted after 3 and 5 weeks. The effect due to static (transwell inserts) and dynamic (rotating bioreactor) culture methods was analyzed. Results indicate that 1:1 chitosan gelatin blends showed the best chondro-conductive potential. The rotating bioreactor facilitated better cell distribution across scaffold but did not show higher ECM secretion compared to transwell culture after 3 weeks. Gelatin leeched out by dissolution in culture media and left an open and interconnected chitosan network. Chitosan gelatin scaffolds show a potential for use in cartilage tissue engineering applications