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.

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.

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.

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.