Malaria affects millions of people annually especially in third world countries. Increase in resistance and limited research being conducted adds to the global burden of malaria. Mefloquine, known for unwanted adverse reactions and neurotoxicity, is highly lipophilic and is still used as treatment and prophylaxis. Lipid drug delivery systems are commonly used to increase solubility and efficacy and decrease toxicity. The most generally used lipid drug delivery system is liposomes. The lipid bilayer structure varying in size from 25 nm to 100 μm can entrap both hydrophilic and lipophilic compounds. Similar in structure and size to liposomes, Pheroid™ technology consist of natural fatty acids and is also able to entrap lipophilic and hydrophilic compounds. The aim of this study was to formulate liposomes and Pheroid™ vesicles loaded with mefloquine and evaluate the physiochemical characteristic of the formulations followed by efficacy and toxicity studies. Pheroid™ vesicles and liposomes with and without mefloquine were evaluated in size, morphology, pH and entrapment efficacy during three month accelerated stability testing. Optimization of size determination by flow cytometry lead to accurate determination of size for both Pheroid™ vesicles and liposomes. During the three months stability testing, Pheroid™ vesicles showed a small change in size from 3.07 ± 0.01 μm to approximately 3 μm for all three temperatures. Confocal laser scanning microscopic evaluation of the liposomes showed structures uniform in spherical shape and size. No difference in size or structure between the Pheroid™ vesicles with and without mefloquine were obtained. Significant increase (p=0.027) in size from 6.46 ± 0.01 μm to above 10 μm was observed for liposomes at all the temperatures. Clearly formed lipid bilayer structures were observed on micrographs. With the addition of mefloquine to the liposome formulation, a decrease in the amount of bilayer structures and an increase in oil droplets were found. Entrapment efficacy was determined by firstly separating the entrapped drug from the unentrapped drug utilizing a Sephadex®G50 mini column. This was followed by spectrophotometric evaluation by UV-spectrophotometry at 283 nm. Initial entrapment efficacy of both Pheroid™ vesicles and liposomes was above 60%. An increase in entrapment efficacy was observed for Pheroid™ vesicles. The addition of mefloquine to already formulated Pheroid™ vesicles illustrated entrapment efficacy of 60.14 ± 5.59% after 14 days. Formulations loaded with mefloquine resulted in lower pH values as well as a decrease in pH over time. Optimization of efficacy studies utilizing propidium iodide was necessary due to the similarity in size and shape of the drug delivery systems to erythrocytes. A gating strategy was successfully implemented for the determination of the percentage parasitemia. Efficacy testing of mefloquine loaded in Pheroid™ vesicles and liposomes showed a 186% and 207% decrease in parasitemia levels compared to the control of mefloquine. Toxicity studies conducted include haemolysis and ROS (reactive oxygen species) analysis on erythrocytes as well as cell viability on mouse neuroblastoma cells. Pheroid™ vesicles with and without mefloquine resulted in a dose dependent increase in ROS and haemolysis over time. A dose dependent increase in ROS and haemolysis in both liposome formulations were observed, but to a lesser extent. Mefloquine proved to be neurotoxic with similar results obtained when mefloquine was entrapped in liposomes. Pheroid™ vesicles seem to have neuroprotective properties resulting in higher cell viability. Mefloquine could be entrapped successfully in Pheroid™ vesicles and less in liposomes. Pheroid™ vesicles was more stable over a three months accelerated stability testing with more favourable characteristics. The increase in ROS levels of Pheroid™ vesicles could be responsible for the higher efficacy and haemolytic activity. DL-α-Tocopherol in Pheroid™ vesicles possibly acted as a pro-oxidant due to the presence of iron in the erythrocytes. DL-α-Tocopherol showed possible antioxidant properties in the neurotoxicity evaluation resulting in higher cell viability. Even though liposomes illustrated higher efficacy and little haemolysis and ROS production, no difference in neurotoxicity was observed together with unfavourable properties during stability testing makes this drug delivery system less favourable in comparison to Pheroid™ vesicles. Mefloquine was successfully incorporated into Pheroid™ vesicles resulted in high efficacy and showed possible neuroprotection and therefore makes it an ideal system for treatment of malaria. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011

Malaria affects millions of people annually especially in third world countries. Increase in resistance and limited research being conducted adds to the global burden of malaria. Mefloquine, known for unwanted adverse reactions and neurotoxicity, is highly lipophilic and is still used as treatment and prophylaxis. Lipid drug delivery systems are commonly used to increase solubility and efficacy and decrease toxicity. The most generally used lipid drug delivery system is liposomes. The lipid bilayer structure varying in size from 25 nm to 100 μm can entrap both hydrophilic and lipophilic compounds. Similar in structure and size to liposomes, Pheroid™ technology consist of natural fatty acids and is also able to entrap lipophilic and hydrophilic compounds. The aim of this study was to formulate liposomes and Pheroid™ vesicles loaded with mefloquine and evaluate the physiochemical characteristic of the formulations followed by efficacy and toxicity studies. Pheroid™ vesicles and liposomes with and without mefloquine were evaluated in size, morphology, pH and entrapment efficacy during three month accelerated stability testing. Optimization of size determination by flow cytometry lead to accurate determination of size for both Pheroid™ vesicles and liposomes. During the three months stability testing, Pheroid™ vesicles showed a small change in size from 3.07 ± 0.01 μm to approximately 3 μm for all three temperatures. Confocal laser scanning microscopic evaluation of the liposomes showed structures uniform in spherical shape and size. No difference in size or structure between the Pheroid™ vesicles with and without mefloquine were obtained. Significant increase (p=0.027) in size from 6.46 ± 0.01 μm to above 10 μm was observed for liposomes at all the temperatures. Clearly formed lipid bilayer structures were observed on micrographs. With the addition of mefloquine to the liposome formulation, a decrease in the amount of bilayer structures and an increase in oil droplets were found. Entrapment efficacy was determined by firstly separating the entrapped drug from the unentrapped drug utilizing a Sephadex®G50 mini column. This was followed by spectrophotometric evaluation by UV-spectrophotometry at 283 nm. Initial entrapment efficacy of both Pheroid™ vesicles and liposomes was above 60%. An increase in entrapment efficacy was observed for Pheroid™ vesicles. The addition of mefloquine to already formulated Pheroid™ vesicles illustrated entrapment efficacy of 60.14 ± 5.59% after 14 days. Formulations loaded with mefloquine resulted in lower pH values as well as a decrease in pH over time. Optimization of efficacy studies utilizing propidium iodide was necessary due to the similarity in size and shape of the drug delivery systems to erythrocytes. A gating strategy was successfully implemented for the determination of the percentage parasitemia. Efficacy testing of mefloquine loaded in Pheroid™ vesicles and liposomes showed a 186% and 207% decrease in parasitemia levels compared to the control of mefloquine. Toxicity studies conducted include haemolysis and ROS (reactive oxygen species) analysis on erythrocytes as well as cell viability on mouse neuroblastoma cells. Pheroid™ vesicles with and without mefloquine resulted in a dose dependent increase in ROS and haemolysis over time. A dose dependent increase in ROS and haemolysis in both liposome formulations were observed, but to a lesser extent. Mefloquine proved to be neurotoxic with similar results obtained when mefloquine was entrapped in liposomes. Pheroid™ vesicles seem to have neuroprotective properties resulting in higher cell viability. Mefloquine could be entrapped successfully in Pheroid™ vesicles and less in liposomes. Pheroid™ vesicles was more stable over a three months accelerated stability testing with more favourable characteristics. The increase in ROS levels of Pheroid™ vesicles could be responsible for the higher efficacy and haemolytic activity. DL-α-Tocopherol in Pheroid™ vesicles possibly acted as a pro-oxidant due to the presence of iron in the erythrocytes. DL-α-Tocopherol showed possible antioxidant properties in the neurotoxicity evaluation resulting in higher cell viability. Even though liposomes illustrated higher efficacy and little haemolysis and ROS production, no difference in neurotoxicity was observed together with unfavourable properties during stability testing makes this drug delivery system less favourable in comparison to Pheroid™ vesicles. Mefloquine was successfully incorporated into Pheroid™ vesicles resulted in high efficacy and showed possible neuroprotection and therefore makes it an ideal system for treatment of malaria. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011