Over the past couple of years there has been rapid progress in the development and design of safe and effective delivery systems for the administration of protein and peptide drugs. The effective delivery of these type of drugs are not always as simple as one may think, due to various inherent characteristics of these compounds. Due to the hydrophilic nature and molecular size of peptide and protein drugs, such as recombinant human growth hormone, they are poorly absorbed across mucosal epithelia, both transcellularly and paracellularly. This problem can be overcome by the inclusion of absorption enhancers in peptide and protein drug formulations but this is not necessarily the best method to follow. This investigation focussed specifically on the evaluation of the ability of the PheroidTM carrier system to transport recombinant human growth hormone across mucosal epithelia especially when administered via the nasal cavity. The PheroidTM delivery system is a patented system consisting of a unique submicron emulsion type formulation. The PheroidTM delivery system, based on PheroidTM technology, will for ease of reading be called Pheroid(s) only throughout the rest of this dissertation. The Pheroid carrier system is a unique microcolloidal drug delivery system. A Pheroid is a stable structure within a novel therapeutic system which can be manipulated in terms of morphology, structure, size and function. Pheroids consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful substances to the desired site of action. The specific objectives of this study can be summarised as follows: a literature study on Pheroid technology; a literature study on chitosan and N-trimethyl chitosan chloride; a literature study on recombinant human growth hormone (somatropin); a literature study on nasal drug administration; formulation of a suitable Pheroid carrier; entrapment of somatropin in the Pheroid carrier, and in vivo evaluation of nasal absorption of somatropin in Sprague-Dawley rats. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

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.

This master´s thesis deals with the study of the incorporation of low and high molecular weight substances into liposomal systems. The aim of the work was to determine the encapsulation efficiency (EE) of the active substance and the influence of individual components of the liposomal system on EE. Liposomes were prepared from dipalmitoylphosphatidylcholine. They were stabilized by cholesteroland and phosphatidic acid was added to give a negative charge. Stealth properties gain the binding of polyethylene glycol and other trimethyl chitosan we enabled the entry of liposomes into the bloodstream by the paracellular pathway. Vitamin C and the enzyme bromelain were used for incorporation into liposomes. UV-VIS spectrophotometry was used to determine the encapsulation efficiency of liposomes prepared by combining the individual components. It has been suggested that vitamin C and the enzyme can be incorporated into liposomes, but an enzyme with a higher EE. Furthermore, phosphatidic acid and trimethyl chitosan have been found to affect EE, which increases the EE of vitamin C and decreases the EE of the enzyme.

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.

Research on nanoparticles for pharmaceutical applications has become increasingly popular in recent years. N-trimethyl chitosan chloride (TMC) is a cationic polymer that can enhance absorption across mucosal surfaces. It has been explored as a nanoparticulate drug delivery system for the delivery of vaccines, vitamins, insulin and cancer medication. It has special interest for intravenous use, as it is soluble over a wide range of pH values. However, polycationic nanoparticles run a great risk for intravenous toxicity, as the positive surface charge allows easy electrostatic interactions with negatively charged blood components, such as red blood cells and plasma proteins. Additionally, the small size of the nanoparticles permits the binding of more proteins per mass, than larger particles do. These interactions can lead to extensive hemolysis, cell aggregation, complement activation, inflammation and fast clearance of the particles from the circulation. A decrease in the surface charge density can ameliorate these toxic interactions. Such a decrease is achieved by adding poly(ethylene) glycol (PEG) to the particle’s formulation. PEG creates a steric shield around the particles, preventing a certain extent of interaction between the particles and the blood components. To be able to use TMC nanoparticles as a successful drug delivery system, the hemocompatibility must first be determined, which was the aim of this study. The influence of particle size, concentration and the addition of PEG were also examined. The extent of hemolysis and cell aggregation caused by the experimental groups (20% and 60% concentration small TMC nanoparticles, 20% larger TMC nanoparticles and 20% cross-linked PEGTMC nanoparticles) were determined by incubating the groups with whole blood and/or blood components. Complement activation was determined with a Complement C3 Human enzyme-linked immunosorbent assay (ELISA) and plasma protein interactions were quantified through rapid equilibrium dialysis and a colorimetric assay. It was determined that 60% concentration small TMC nanoparticles caused 49.08 ± 2.538% hemolysis at the end of a 12-hour incubation period, significantly more than any other experimental group. This group had also caused mild aggregation of the white blood cells and platelets. This was the greatest extent of cell aggregation seen in any of the groups. No significant complement activation was seen by any of the experimental groups. Because of the cationic nature of the particles, all groups had more than 50% of the initial particles in the sample bound to plasma proteins after a 4-hour incubation period. However, at 90.68 ± 0.828%, the 60% small TMC nanoparticles had had significantly more interaction with the plasma proteins than the other groups. Through the experimental measurements it was revealed that TMC nanoparticles had hemotoxic effects at high concentrations. The addition of PEG to the particle formulation stabilized the particles and decreased their zeta potential , but had no significant effect on improving hemocompatibility. It was concluded that although further tests are needed, TMC nanoparticles seem to have potential as a successful intravenous carrier for high molecular weight active pharmaceutical ingredients. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Research on nanoparticles for pharmaceutical applications has become increasingly popular in recent years. N-trimethyl chitosan chloride (TMC) is a cationic polymer that can enhance absorption across mucosal surfaces. It has been explored as a nanoparticulate drug delivery system for the delivery of vaccines, vitamins, insulin and cancer medication. It has special interest for intravenous use, as it is soluble over a wide range of pH values. However, polycationic nanoparticles run a great risk for intravenous toxicity, as the positive surface charge allows easy electrostatic interactions with negatively charged blood components, such as red blood cells and plasma proteins. Additionally, the small size of the nanoparticles permits the binding of more proteins per mass, than larger particles do. These interactions can lead to extensive hemolysis, cell aggregation, complement activation, inflammation and fast clearance of the particles from the circulation. A decrease in the surface charge density can ameliorate these toxic interactions. Such a decrease is achieved by adding poly(ethylene) glycol (PEG) to the particle’s formulation. PEG creates a steric shield around the particles, preventing a certain extent of interaction between the particles and the blood components. To be able to use TMC nanoparticles as a successful drug delivery system, the hemocompatibility must first be determined, which was the aim of this study. The influence of particle size, concentration and the addition of PEG were also examined. The extent of hemolysis and cell aggregation caused by the experimental groups (20% and 60% concentration small TMC nanoparticles, 20% larger TMC nanoparticles and 20% cross-linked PEGTMC nanoparticles) were determined by incubating the groups with whole blood and/or blood components. Complement activation was determined with a Complement C3 Human enzyme-linked immunosorbent assay (ELISA) and plasma protein interactions were quantified through rapid equilibrium dialysis and a colorimetric assay. It was determined that 60% concentration small TMC nanoparticles caused 49.08 ± 2.538% hemolysis at the end of a 12-hour incubation period, significantly more than any other experimental group. This group had also caused mild aggregation of the white blood cells and platelets. This was the greatest extent of cell aggregation seen in any of the groups. No significant complement activation was seen by any of the experimental groups. Because of the cationic nature of the particles, all groups had more than 50% of the initial particles in the sample bound to plasma proteins after a 4-hour incubation period. However, at 90.68 ± 0.828%, the 60% small TMC nanoparticles had had significantly more interaction with the plasma proteins than the other groups. Through the experimental measurements it was revealed that TMC nanoparticles had hemotoxic effects at high concentrations. The addition of PEG to the particle formulation stabilized the particles and decreased their zeta potential , but had no significant effect on improving hemocompatibility. It was concluded that although further tests are needed, TMC nanoparticles seem to have potential as a successful intravenous carrier for high molecular weight active pharmaceutical ingredients. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014