Yes / In this study, we prepared chitosan (Cs_P) nanofibers (NFs) membrane by electrospinning. The Cs_P NFs membrane was then chemically functionalized (CsF) by a novel stepwise chemical process. The CsF NFs membrane was electrospray with TiO2 nanoparticles (NPs) to prepare the CsF_Coa NFs membrane. A second NFs membrane with embedded TiO2 NPs (Cs_Co) was also prepared by electrospinning. The TiO2 NPs, Cs_P, CsF s, CsF_Coa NFs, and Cs_Co NFs membranes were analyzed by standard spectroscopic, microscopic, X-ray, and thermal methods. Fourier transform infrared (FTIR) analysis confirmed the incorporation of the new functional group into the Cs structure. X-ray photoelectron spectroscopy (XPS) data confirmed the FTIR results and the fabrication of the CsF NFs membrane. Scanning electron microscope (SEM) micrographs showed a smooth morphology for the Cs_P NFs membrane and a denser morphology for the CsF NFs membrane (NFs swelled with functionalization). The SEM micrographs also showed a dense cloud of TiO2 NPs on the surface of the Cs_Coa NFs membrane. Transmission electron microscope (TEM) showed that the particle size of TiO2 NPs varied between 20 and 35 nm and tended to be spherical. The X-ray diffraction (XRD) pattern confirmed the existence of the anatase phase of the TiO2 NPs. The presence of TiO2 in the Cs_Coa and Cs_Co NFs membranes was also confirmed by energy-dispersive x-ray spectroscopy (EDX). Surface profilometry confirmed an increase in the surface roughness of the CsF and Cs_Coa NFs membranes. Brunauer–Emmett–Teller (BET) analysis revealed that the isotherms and hystereses for all NFs membranes were of the IV and H3 types, respectively, corresponding to mesopores and slit pores. The higher photocatalytic activity of the Cs_Coa NFs membrane (89%) compared to the Cs_Co NFs membrane (40%) was attributed to a balance between the short band gap, high surface roughness, and lower surface area.

Rupture of the anterior cruciate ligament (ACL) is one of the most common ligamentous injuries of the knee. Post rupture, the ACL does not heal on itself due to poor vasculature and hence surgical intervention is required to treat the ACL. Current surgical management of ACL rupture consists of reconstruction with autografts or allografts. However, the limitations associated with these grafts have prompted interest in tissue engineered solutions that combine cells, scaffolds and stimuli to facilitate ACL regeneration. This thesis describes a ligament tissue engineering strategy that involves incorporating biomolecules within fibers-based electrospun meshes which mimics the extra-cellular matrix microarchitecture of ligament. However, challenges exist with incorporation of biomolecules. Therefore, the goal of this research project was to develop two techniques to incorporate biomolecules within electrospun meshes: (1) co-axially electrospinning fibers that support surface-grafting of biomolecules, and (2) co-axially electrospinning fibers decorated with biomolecule-loaded microspheres. In the first approach, chitosan was co-axially electrospun on the shell side of poly caprolactone (PCL) and arginine-glycine-aspartate (RGD) was attached to the electrospun meshes. Bone marrow stromal cells (BMSCs) attached, spread and proliferated on these meshes. In the second approach, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded chitosan-alginate (CS-AL) microspheres were fabricated. The effects of cation to alginate ratio, type of alginate and concentration of CaCl2 on microsphere size, FITC-BSA loading and release were systematically evaluated. The CS-AL microspheres were then incorporated into the sheath phase of co-axially electrospun meshes to achieve microsphere-decorated fiber composite meshes. The results from these model study suggest that both approaches are tractable for incorporating biomolecules within fibers-based electrospun meshes. Both these approaches provide platform for future studies that can focus on ligament-relevant biomolecules such as FGF-2 and GDF-5. / Master of Science

A quitosana é um polímero natural obtido a partir da desacetilação química da quitina, sendo a quitina o segundo polissacarídeo mais abundante na natureza. O interesse em pesquisas por novas aplicações da quitosana vem aumentando muito em diversas áreas, como na indústria farmacêutica, na indústria de cosméticos e de alimentos. Isso se deve às importantes características biológicas e físico-químicas inerentes à quitosana, como: biocompatibilidade, biodegradabilidade, propriedade de formação de filmes e fibras, complexação de metais e distintas atividades biológicas. Além disso, a presença de grupos amino na molécula da quitosana permite modificações químicas das mais diversas. No entanto, esta funcionalidade tem mostrado ser dependente, não apenas da sua estrutura química, mas também do seu tamanho molecular. Pois muitas das propriedades físico-químicas, e funcionais de uma cadeia polimérica são definidas pela sua massa molecular. O presente trabalho objetivou a síntese, caracterização e estudo da atividade antibacteriana de derivados de quitosana: quitosanas de baixa massa molecular, utilizando-se o peróxido de hidrogênio como agente oxidante; N-succinilquitosana e 2-carboxibenzamido-quitosana, de alta e baixa massa molecular, em presença de anidridos cíclicos, anidrido succínico e anidrido ftálico, respectivamente. Foram realizadas análises de caracterização dos derivados sintetizados por espectroscopia Raman e Infravermelho e avaliação de propriedades físico-químicas, como viscosidade e solubilidade. A efetividade antimicrobiana da quitosana e de seus derivados sintetizados, de alta e baixa massa molecular, foi avaliada frente à concentração final de 106UFC/mL de Escherichia coli através da determinação da concentração mínima inibitória (CMI). Os derivados de baixa massa molecular se mostraram mais solúveis e com menor viscosidade em relação às amostras de alta massa molecular. Em relação à atividade antibacteriana, nenhuma das amostras testadas exibiu ação antibacteriana significativa. / Chitosan is a natural polymer derived from the chemical deacetylation of chitin. Chitin is the second most abundant polysaccharide in nature. The interest in research for new applications of chitosan has been increasing fast in many areas, such as pharmaceuticals, cosmetics and foods industries. This is due to important biological and physical-chemical properties inherent to chitosan, such as biocompatibility, biodegradability, film-forming properties and fiber, metal complexation and distinct biological activities. Moreover, the presence of amino groups in the molecule of chitosan allows a wide range of chemical modifications. However, this feature has shown to be dependent not only on its chemical structure but also on their molecular size. Because many of the physico-chemical and functional characteristics of a polymer chain are defined by their molecular weight. This paper aims at the synthesis, characterization and study of antibacterial activity of chitosan derivatives, low molecular weight chitosan, using hydrogen peroxide as an oxidizing agent, N-succinylchitosan and 2-carboxybenzamido-chitosan, high and low molecular weight in the presence of cyclic anhydrides, succinic anhydride and phthalic anhydride, respectively. Analyses were performed to characterize the derivatives synthesized by Raman and Infrared spectroscopy and assessment of physicochemical properties such as viscosity and solubility. The antimicrobial effectiveness of chitosan and its derivatives synthesized, high and low molecular weight, was evaluated against the final concentration of Escherichia coli 106 UFC/mL by determining the minimum inhibitory concentration (MIC). The low molecular weight derivatives were more soluble and lessviscous for samples of high molecular weight. Regarding the antibacterial activity, none of the samples tested exhibited significant antibacterial activity.

A quitosana é um polímero natural obtido a partir da desacetilação química da quitina, sendo a quitina o segundo polissacarídeo mais abundante na natureza. O interesse em pesquisas por novas aplicações da quitosana vem aumentando muito em diversas áreas, como na indústria farmacêutica, na indústria de cosméticos e de alimentos. Isso se deve às importantes características biológicas e físico-químicas inerentes à quitosana, como: biocompatibilidade, biodegradabilidade, propriedade de formação de filmes e fibras, complexação de metais e distintas atividades biológicas. Além disso, a presença de grupos amino na molécula da quitosana permite modificações químicas das mais diversas. No entanto, esta funcionalidade tem mostrado ser dependente, não apenas da sua estrutura química, mas também do seu tamanho molecular. Pois muitas das propriedades físico-químicas, e funcionais de uma cadeia polimérica são definidas pela sua massa molecular. O presente trabalho objetivou a síntese, caracterização e estudo da atividade antibacteriana de derivados de quitosana: quitosanas de baixa massa molecular, utilizando-se o peróxido de hidrogênio como agente oxidante; N-succinilquitosana e 2-carboxibenzamido-quitosana, de alta e baixa massa molecular, em presença de anidridos cíclicos, anidrido succínico e anidrido ftálico, respectivamente. Foram realizadas análises de caracterização dos derivados sintetizados por espectroscopia Raman e Infravermelho e avaliação de propriedades físico-químicas, como viscosidade e solubilidade. A efetividade antimicrobiana da quitosana e de seus derivados sintetizados, de alta e baixa massa molecular, foi avaliada frente à concentração final de 106UFC/mL de Escherichia coli através da determinação da concentração mínima inibitória (CMI). Os derivados de baixa massa molecular se mostraram mais solúveis e com menor viscosidade em relação às amostras de alta massa molecular. Em relação à atividade antibacteriana, nenhuma das amostras testadas exibiu ação antibacteriana significativa. / Chitosan is a natural polymer derived from the chemical deacetylation of chitin. Chitin is the second most abundant polysaccharide in nature. The interest in research for new applications of chitosan has been increasing fast in many areas, such as pharmaceuticals, cosmetics and foods industries. This is due to important biological and physical-chemical properties inherent to chitosan, such as biocompatibility, biodegradability, film-forming properties and fiber, metal complexation and distinct biological activities. Moreover, the presence of amino groups in the molecule of chitosan allows a wide range of chemical modifications. However, this feature has shown to be dependent not only on its chemical structure but also on their molecular size. Because many of the physico-chemical and functional characteristics of a polymer chain are defined by their molecular weight. This paper aims at the synthesis, characterization and study of antibacterial activity of chitosan derivatives, low molecular weight chitosan, using hydrogen peroxide as an oxidizing agent, N-succinylchitosan and 2-carboxybenzamido-chitosan, high and low molecular weight in the presence of cyclic anhydrides, succinic anhydride and phthalic anhydride, respectively. Analyses were performed to characterize the derivatives synthesized by Raman and Infrared spectroscopy and assessment of physicochemical properties such as viscosity and solubility. The antimicrobial effectiveness of chitosan and its derivatives synthesized, high and low molecular weight, was evaluated against the final concentration of Escherichia coli 106 UFC/mL by determining the minimum inhibitory concentration (MIC). The low molecular weight derivatives were more soluble and lessviscous for samples of high molecular weight. Regarding the antibacterial activity, none of the samples tested exhibited significant antibacterial activity.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

In oral sciences, chitosan application is of interest due to its antimicrobial and hemostatic activity. Chitosan coating of dentures and other intraoral devices could be beneficial for treatment of denture stomatitis or in the management of postoperative bleeding. Disinfection of dentures and prosthodontic materials is crucial before their use in patients. This study investigated the influence of chemical disinfectants on chitosan-coated surfaces. A total of 100 specimens were made: 50 of PMMA (polymethyl methacrylate), and 50 of PETG (polyethylene terephthalate glycol-modified) material and coated with 2% chitosan acetate solution. In each material, 5 groups (10 specimens each) were established and disinfected with Printosept-ID (L1), MD 520 (L2), Silosept (L3), or Dentavon (L4), or stored in distilled water (L0, control group). After disinfection, all specimens underwent abrasion tests (30,000 cycles in a tooth-brushing simulator). Areas without chitosan coating were measured by digital planimetry both before and after the disinfection/abrasion procedure and a damage-score was calculated. Regarding chitosan coating, the statistical analysis showed a significant influence of the disinfectants tested and significant differences between disinfectants (p < 0.05). Chitosan coating was most stable on PMMA and PETG after disinfection with MD 520 (L2). Otherwise, active oxygen containing disinfectants (L3, L4) led to the greatest alterations in the chitosan coating.

In oral sciences, chitosan application is of interest due to its antimicrobial and hemostatic activity. Chitosan coating of dentures and other intraoral devices could be beneficial for treatment of denture stomatitis or in the management of postoperative bleeding. Disinfection of dentures and prosthodontic materials is crucial before their use in patients. This study investigated the influence of chemical disinfectants on chitosan-coated surfaces. A total of 100 specimens were made: 50 of PMMA (polymethyl methacrylate), and 50 of PETG (polyethylene terephthalate glycol-modified) material and coated with 2% chitosan acetate solution. In each material, 5 groups (10 specimens each) were established and disinfected with Printosept-ID (L1), MD 520 (L2), Silosept (L3), or Dentavon (L4), or stored in distilled water (L0, control group). After disinfection, all specimens underwent abrasion tests (30,000 cycles in a tooth-brushing simulator). Areas without chitosan coating were measured by digital planimetry both before and after the disinfection/abrasion procedure and a damage-score was calculated. Regarding chitosan coating, the statistical analysis showed a significant influence of the disinfectants tested and significant differences between disinfectants (p < 0.05). Chitosan coating was most stable on PMMA and PETG after disinfection with MD 520 (L2). Otherwise, active oxygen containing disinfectants (L3, L4) led to the greatest alterations in the chitosan coating.

The present thesis presents new renewable, antimicrobial and biodegradable superabsorbent polymers (SAPs), produced from the cell wall of zygomycetes fungi. The cell wall was characterized and chitosan, being one of the most important ingredients, was extracted, purified, and converted to SAP for use in disposable personal care products designed for absorption of different body fluids. The cell wall of zygomycetes fungi was characterized by subsequent hydrolysis with sulfuric and nitrous acids and analyses of the products. The main ingredients of the cell wall were found to be polyphosphates (4-20%) and copolymers of glucosamine and N-acetyl glucosamine, i.e. chitin and chitosan (45-85%). The proportion of each of these components was significantly affected by the fungal strain and also the cultivation conditions. Moreover, dual functions of dilute sulfuric acid in relation to chitosan, i.e. dissolution at high temperatures and precipitation at lowered temperatures, were discovered and thus used as a basis for development of a new method for extraction and purification of the fungal chitosan. Treatment of the cell wall with dilute sulfuric acid at room temperature resulted in considerable dissolution of the cell wall polyphosphates, while chitosan and chitin remained intact in the cell wall residue. Further treatment of this cell wall residue, with fresh acid at 120°C, resulted in dissolution of chitosan and its separation from the remaining chitin/chitosan of the cell wall skeleton which was not soluble in hot acid. Finally, the purified fungal chitosan (0.34 g/g cell wall) was recovered by precipitation at lowered temperatures and pH 8-10. The purity and the yield of fungal chitosan in the new method were significantly higher than that were obtained in the traditional acetic acid extraction method. As a reference to pure chitosan, SAP from shellfish chitosan, was produced by conversion of this biopolymer into water soluble carboxymethyl chitosan (CMCS), gelation of CMCS with glutaraldehyde in aqueous solutions (1-2%), and drying the resultant gel. Effects of carboxymethylation, gelation and drying conditions on the water binding capacity (WBC) of the final products, were investigated. Finally, choosing the best condition, a biological superabsorbent was produced from zygomycetes chitosan. The CMCS-based SAPs were able to absorb up to 200 g water/g SAP. The WBC of the best SAP in urine and saline solutions was 40 and 32 g/g respectively, which is comparable to the WBC of commercially acceptable SAPs under identical conditions (34-57 and 30-37 g/g respectively). / <p>Disputationen sker fredagen den 1 oktober kl. 10.00 i KA-salen, Kemigården 4, Chalmers, Göteborg</p>

In this research, a series of synthetically engineered copolymers were synthesized containing polysaccharides (e.g., β-cyclodextrin and chitosan) to address the removal of trihalomethanes (THMs) from water environments. There are two main parts in this research thesis: i) the preparation and characterization of polysaccharide-based copolymers; ii) sorption studies of the copolymers with chloroform and total THMs (TTHMs) in aqueous solution. In the first part of this thesis, grafted polyester, polyester and grafted polyamide copolymers were prepared by cross-linking β-cyclodextrin (β-CD) and chitosan (CS) with various cross-linkers, including poly (acrylic acid) (PAA), terephthaloyl (TCl), and sebacoyl chloride (SCl), respectively. The synthesized copolymer materials were characterized by Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS), Scanning Electron Microscopy (SEM), Thermogravimetric analysis (TGA), Differential scanning calorimetry (DSC), elemental (C and H) analyses, and NMR spectroscopy. Nitrogen porosimetry was used to analyze the surface area and pore structure characteristics of the copolymers and starting materials in solid state. The sorption properties of the copolymers in aqueous solution were studied using different dye probes (e.g., p-nitrophenol and methylene blue) by UV–Vis spectrophotometry. The copolymers showed markedly varied interactions with dye probes in accordance with their composition, surface area, and pore structure characteristics. Diverse materials were afforded by variation of the synthetic conditions. The sorption isotherms were evaluated with various isotherm models (e.g., Langmuir, BET, Freundlich and Sips). The Sips isotherm showed the best overall agreement with the experimental results and the sorption parameters provided estimates of the sorbent surface area and the sorption capacity for various copolymers in aqueous solution. The copolymer sorbents display tunable physicochemical properties according to the synthetic conditions. In the second part of this thesis, the direct aqueous injection (DAI) method with gas chromatography (GC) with electron capture or electrolytic conductivity detectors (ECD) enabled quantitative detection of chloroform and TTHMs in water. A preliminary adsorption study and kinetic study of chloroform provided the information to establish the experimental protocol for the sorption study. The sorption parameters were evaluated using the Sips model. The sorption capacity (Qm) values of chloroform for these synthetically engineered copolymers at similar conditions ranged from 0.00335-1.70 mmol/g. The relative ordering of the Qm values was observed: β-CD/PAA 1:5 > SCl-5 > SCl-10 ~ CP-1 > β-CD/PAA 1:10 > CP-5 > AC > β-CD/PAA 1:5 at high mixing speed. An extension of the sorption study for copolymers toward the multi-component THMs in water was carried out. The copolymers showed distinct adsorption capacities to THMs: chloroform (0.0485-0.287 mmol/g); DBCM (0.0712-0.277 mmol/g); BDCM (0.0684-0.387mmol/g); and bromoform (0.0522-1.07 mmol/g). The copolymers exhibited relatively high selectivity toward individual components of THMs due to their variable molecular size and polarizability. The copolymers showed favorable adsorption (e.g., β-CD/PAA 1:5, CP-1) and each type of polysaccharide (e.g., β-CD and CS) copolymers displays great potential for the removal of halomethane-based contaminants.