Univ., Diss.--Würzburg, 1997.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2001. / Restricted until June 2002. Bibliography: leaves [145]-167.

Ph.D.

Chitosan (CS) is a man-made sugar based biopolymer derived from chitin, the second most abundant natural polymer after cellulose. Chitin is sourced from crustacean species such as shrimps and crabs. The chemical structure of chitin contains N-Acetyl D-glucosamine monomer units which forms CS upon deacetylation. In CS, ?-(1-4) linked D-glucosamine units are randomly distributed. Approximately 75% - 80% sugar units contains primary amine groups in commercially available low molecular weight CS. Biodegradability, low toxicity, mucoadhesive and transfecting properties of CS polymer are attractive for applications as oral and nasal drug delivery systems. Chitosan polymer is water insoluble at neutral pH. To solubilize CS, dilute mineral acid (such as hydrochloric acid and nitric acid) or organic acid (such as acetic acid) is often used. CS contains both hydroxyl and primary amine groups in its structure. In acidic solution, the amine functional groups become protonated (positively charged). Positively charged CS remains stable only in low pH condition due to electrostatic repulsion of charged polymer segments. Therefore, by using a suitable anionic (negatively charged) cross-linker, stable CS particles (such as nanoparticles and microspheres) can be prepared. This is popularly known as ionic gelation method. Extensive studies have been done on the synthesis of drug loaded CS particles where particle integrity is maintained by ionic gelation using tripolyphosphate (TPP, an anionic cross-linker). Drug encapsulated CS-TPP composite particles are shown to maintain biodegradability and biocompatibility. The CS-TPP composite particles exhibits very limited dispersibility at neutral pH conditions specifically in neutral buffered conditions. A number of biomedical applications (including systemic drug formulations) however demands buffer-stable CS composite particles for achieving optimal therapeutic outcome. To overcome the above dispersibility issues, CS polymer and CS particles units have been chemically modified using water soluble motifs (such as water soluble polymer or ligands). This approach is very cumbersome and usually involves multiple purification steps. Chemical modification of natural CS chain introduces risks of compromising biodegradability and biocompatibility. Therefore, there is a strong need for developing a straightforward method of making water soluble CS and CS particles. Chapter 1 of this dissertation presents an overview of the CS polymer, various applications of CS polymers, methods of making CS polymers and CS particles, current limitations of synthesis methods for preparing stable chitosan particles at neutral pH conditions and finally delineates the scope of the proposed research work. Chapter 2 describes development of chemo-hydrothermal synthesis method for producing water soluble CS polymer and water dispersible CS composite particles. In this method, a chemical (depolymerizing agent) is used to treat CS polymer in a hydrothermal (high temperature and high pressure) condition. Two types of depolymerizing agents have been used, an inorganic acid (e.g. hydrochloric acid, HCl) and a bicarboxylic organic acid (e.g. tartaric acid, TA). In both cases, 100% depolymerized CS polymer was obtained. Chemical characteristics of the depolymerized CS were comparable to acid solubilized CS. CS polymer exhibits weak fluorescence. Interestingly, hydrothermally depolymerized CS shows strong fluorescence properties irrespective of the nature of depolymerizing agent used. TA not only depolymerized CS but also formed CS-TA composite particulate structures in solution via self-assembly. The CS-TA composite particles are stable in a wide pH range from 5 to 11. Detailed spectroscopic and microscopic studies have been done to understand the basic mechanism of particle formation and increase in fluorescence properties (i.e. structure-property relationship). Usefulness of CS-TA in solubilizing water-insoluble cargos (such as fluorescein isothiocyanate, FITC) has been demonstrated. Chapter 3 is focused on hydrothermal synthesis of mixed-valence copper (Cu) oxide loaded CS-TA composite particles and their characterization. Crystalline Cu oxide nanoparticles were coated with the CS-TA layer. Water dispersibility of Cu oxide greatly improved upon coating with CS-TA material. To demonstrate catalytic activity of Cu-oxide loaded CS-TA film in sequestering carbon dioxide (CO2), an electrochemical setup was used. Electrochemical reduction of CO2 was successfully demonstrated. It was observed that CS-TA environment not only maintained catalytic properties of Cu oxide but also allowed solution processing of Cu-oxide film onto the electrode surface. Chapter 4 discusses a convenient method of making monodispersed water dispersible Cu loaded chitosan nanoparticles (Cu-CS) using HCl depolymerized CS polymer. The purpose of this study was to investigate if there was any improvement in antibacterial properties of Cu-CS nanoparticles prepared using hydrothermally treated CS polymer. Interestingly, it was observed that the antibacterial efficacy of Cu was not compromised in Cu-CS nanoparticles. Moreover, the materials exhibited improvement in antibacterial efficacy against both Gram-negative and Gram-positive bacteria species. A plausible mechanism has been proposed to explain antibacterial results. Chapter 5 summarizes major findings of this dissertation research and presents future research directions.

Over the past several decades, gene therapy technologies have been developed for a diverse number of applications ranging from DNA-based vaccines to gene silencing with RNAi. While all are powerful tools, a common limitation for these technologies is the need for effective and safe delivery to target sites within the body. Such delivery vectors are necessary for retention of bioactivity and stability, while also providing a method of cellular and tissue uptake and distribution, which may require endosomal escape. Although, viral and lipid-based technologies have shown promise as nucleic acid delivery vectors, both have inherent issues such as cytoxicity, oncogenicity, and immunogenicity. Thus, the development of polymer-based non-viral vectors has been an area of great focus over the past decade. While many polymeric vectors have been developed for plasmid DNA (pDNA) delivery, very few have shown effective delivery of short interfering RNA (siRNA), a powerful tool for gene silencing via the RNA interference mechanism. Furthermore, very few prospective delivery vectors have shown versatility for the administration of siRNA through multiple routes of administration. The overall goal of this research was to develop a biocompatible non-viral delivery system for the delivery of plasmid DNA, siRNA, and contrast agents through the modification of the natural biopolymer chitosan. We have synthesized an imidazole modified chitosan (chitosan-IAA) by conjugation of imidazole acetic acid to chitosan. Extensive evaluation and characterization of the modified polymer demonstrates enhanced solubility and buffering capacity within the physiological and endosomal pHs, thus providing enhanced endosomal escape by exploiting the "proton sponge" effect. We have demonstrated effective in vitro gene expression and gene silencing with chitosan-IAA mediated delivery of pDNA and siRNA, respectively. Furthermore, we have demonstrated in vivo gene silencing by delivery of siRNA through both intranasal and intravenous routes of delivery with chitosan-IAA/siRNA nanocomplexes. We have also demonstrated delivery of contrast agents up to 45 nm in size through mucosal tissue following treatment with chitosan and no contrast agent modification in both human and animal tissue. In conclusion, we have successfully developed a versatile and highly effective delivery vector for both nucleic acids and contrast agents. / text

Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Submitted in fulfillment of the requirements of the degree of Master of Engineering: Chemical Engineering, Durban University of Technology, Durban, South Africa, 2015. / The implementation of new water regulations from the local government has been a motivation for most industries to treat the effluent before disposal or reuse within the plant, in order to save costs and avoid sanctions. Tongaat-Huletts sugar refinery has therefore invested in this collaborative research with the Durban University of Technology in order to investigate new technologies for wastewater treatment and water recovery using an organic coagulant called chitosan. Chitosan is a natural non-toxic polymer extracted from the exoskeleton of crustaceans. Chitosan has gained extensive attention as a coagulant in the treatment of wastewaters from various industries. However, no attention has been given to the coagulation of effluents from the sugar industry using this polymer. In this work, chitosan coagulant (CCo) was prepared by dissolution of known amounts of chitosan powder in aqueous acid at 50℃. The solution was diluted to desired concentrations using distilled water at room temperature. The removal of impurities using chitosan was investigated for two effluent streams from the sugar refinery, namely the final effluent (FE) and the resin effluent (RE) by applying the one-factor-at-a-time (OFAT) method. The optimum chitosan loading was found to be 138 mg/l for the RE and 7.41 mg/l for the FE, beyond which the efficiency of the coagulant decreased. The coagulation of FE removed 97% of the total suspended solids (TSS), 61% colour and 35% chemical oxygen demand (COD). The treatment of RE resulted in the removal of 68% TSS, 30% colour and 15% COD due to its high content of impurities. Therefore, RE was not considered for statistical studies. The Box-Behnken (BBD) design, which is a statistical response surface methodology (RSM) model was used to study the simultaneous effect of pH, coagulant loading and settling time on the removal of the COD, TSS and colour, with the help of an overlay plot for the FE. The optimum values from the overlay plot were 92% for TSS, 83% for colour and 29% for COD. The model equations generated by the BBD for individual responses involved all the manipulated variables contrary to the OFAT which only considered one manipulated parameter per response. Moreover, the BBD allowed the simultaneous analysis of all the parameters and the identification of interactions which occur when the effect of one factor is dependent on the level of another. The most important interaction for the removal of TSS was the combination of the variation in pH and coagulant dosage. The COD removal was mostly affected by the interaction between the coagulant loading and the settling time. The colour removal increased with the simultaneous increase of the pH and the settling time. A comparative study between the wastewaters from the sugar industry, the brewery industry and milk processing industry revealed that the performance of the chitosan was also affected by the amount of total dissolved solids (TDS) in the wastewater. A model was developed relating the TSS, COD and TDS from all these wastewaters, and was used to predict the TSS removal for the effluent from the olive oil mills and the wastewater from the winery. Chitosan can be considered as a good alternative to inorganic and synthetic coagulants for the pre-treatment of the FE due to its ability to efficiently remove the levels of TSS and colour. Furthermore, the production of chitosan from crustacean shells is a good method of reducing pollution from the fishery industry. Chitosan can be produced locally at low cost due to both the abundance of crustacean shells in the coastal regions of South Africa and the simplicity of its preparation process. It is recommended that a mathematical model be developed to accurately predict the influence of chitosan on all types of effluent. Such a model will provide an indication of the performance of the chitosan and guide experimenters. It is further recommended that the effect of the use of organic coagulants on the destabilization of dissolved solids in wastewater be given greater attention.

Chitosan has been proposed as a "natural" coagulating agent to solve wastewater problems. The main hindrance in this commercial chitosan application has been its low cost effectiveness. The hypothesis in our research is that chitosan complexes with natural polyanions is more effective than chitosan alone, particularly in recovering low concentration proteins from food processing wastewater. Chitosan (Chi) was reacted with alginate (Alg), pectin (Pec) and carrageenan (Car) ex-situ to obtain chitosan-polyanion complexes (Chi-Pol). Analysis by Fourier Transform Infrared (FTER) spectroscopy confirmed electrostatic interactions as the mechanism for complex formation. Scanning electron microscopy revealed a tight, non-porous structure except for the porous Chi-Car complex. Tests with a bovine serum albumin solution revealed low adsorption rates with slightly higher values for Chi-Car suggesting the need for an improved complexation method. Chi-Pol complexes prepared in-situ at different monomeric weight ratios (MR) were evaluated using pH 6 adjusted Cheddar cheese whey and surimi wastewater (SWW). Complexes used at 30 mg complex/L whey showed higher turbidity reductions than at 10 mg/L. MR had no significant effect on turbidity reduction except for Chi-Alg at 30 mg/L; the value (72 %) at MR = 0.2 was higher than for MR = 0.8. UV-Vis spectroscopy confirmed in-situ complex formation with a preference for the adsorption of specific whey protein fractions. Complexes formed at 0.2 and 0.8 MR were evaluated at two concentrations for the treatment of SWW. Tests at 50 mg/L showed a turbidity reduction of up to 97 % at 24 h with a 81-90 % recovery of SWW proteins. At 150 mg/L, similar efficacy was achieved in only 1 h with turbidity reductions ranging 94-99 % and 78-94 % protein recovery. FTIR analyses confirmed the adsorption of proteins as indicated by similarities in the three amide bands for Chi-Alg recovered solids and untreated SWW. Differential scanning calorimetry (DSC) was employed to study interactions of SWW proteins and Chi-Alg complexes. Untreated and complex bound SWW proteins revealed single exothermic peaks at 23.3 and 38.0°C, respectively. This suggested Chi-Alg and SWW protein interactions increased the thermal stability of SWW proteins. However, further thermal analysis studies are needed to confirm this finding. / Graduation date: 2001

In some diseases it is preferable that the drugs used in their treatment are released in the colon. The colon is also suitable for systemic delivery of a variety of drugs. A variety of systems have been developed for the purpose of achieving colonic targeting. These approaches are either drug-specific (prodrugs) or formulation specific (coated or matrix preparations) and depends on the pH, transit time and pressure or bacteria in the colon. Different polymers, like chitosan, have been evaluated for their susceptibility to degradation by these bacterial enzymes. Chitosan is considered a good candidate for bacterial degradation and is widely available at low cost and has favourable biological properties. To investigate the influence of formulation factors on the properties of chitosan minitablets, it was necessary to ensure that the chitosan had satisfactory powder flow characteristics to ensure uniform compression in the tablet press and to prevent unacceptable variation in the tablet properties such as weight, thickness, disintegration and strength. Moisture content of the powder, particle size and the inclusion of glidants had an effect on the flowability and it could be improved from a composite flow index value of 32.7 to a value of 58.8. The compressibility of chitosan is very poor and different factors that might influence it, was investigated. Compression forces of between 15 and 20 bar resulted in tablets with acceptable physical characteristics. An increase in moisture content, using the powder fraction > 212 ym as well as a decrease in powder weight resulted in tablets with a higher tensile strength. Lower compression forces resulted in tablets that are extremely porous. This suggests that the chitosan can only be compressed at high compression forces which are difficult to obtain using a standard tablet press. The standard tablet press was therefore modified to fill more powder in the die and generate higher compression forces. Minitablets were compressed and the dissolution of isoniazide from these tablets was investigated. Varying the punch depth or the compaction of the powder did not result in the desired slower release of the drug as a result. The porosity of the tablets compressed at all the punch depth settings and compaction percentages was probably too high to have an effect on the wettablity of the tablets and as a result on the dissolution of the isoniazide from the tablets. The inclusion of excipients such as citric acid (an organic acid which would lower the pH in the tablet, allowing the chitosan to form a gel) and pectin (which would form an insoluble complex with the chitosan) into the formulation delayed the dissolution of the isoniazide from the minitablets. Coating of the minitablets with an enteric coating (Eudragit S ®) initially delayed the dissolution of the isoniazide and would protect the tablets from the harsh environment of the stomach so that the tablets will reach the colon and release the drug. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007