The available literature indicates that to date, few research has been performed on oil powders for topical delivery. The aim of this project was to investigate the release characteristics of oil powder formulations, as well as their dermal and transdermal delivery properties. Whey protein-stabilised emulsions were used to develop oil powders. Whey protein was used alone, or in combination with chitosan or carrageenan. Nine oil powders, with salicylic acid as the active ingredient, were formulated by using the layer-by-layer method. Three different pH values (pH 4, 5 and 6) were used to prepare the formulations, because of the different charges that polymeric emulsifiers may have. The characteristics of the prepared oil powders were determined, including their droplet sizes, particle size distributions, loss on drying, encapsulation efficiencies, oil leakage and water dispersibility. Release studies (membrane diffusion studies) were conducted by utilising cellulose acetate membranes (0.2 μm pore size) and Franz-type diffusion cells over a period of eight hours. The release of the active ingredient was determined for all nine powders, their respective template emulsions, as well as their respective oil powders redispersed in water. The release of salicylic acid from the respective redispersed oil powders was then further compared to its release from the template emulsions and from the oil powders. The effect of pH and different polymer types used in preparing the oil powders, their respective redispersed oil powders and the template emulsions were determined with regards to the release of the active ingredient from all these preparations. The effect of pH and different polymers used was furthermore determined on the oil powders and their respective redispersed oil powders, with regards to their dermal and transdermal deliveries. Transdermal delivery and skin uptake were investigated on specifically selected powders only, based on the outcomes of the oil powder characterisation and release data. The qualifying formulations were chitosan pH 4, 5 and 6, whey and carrageenan pH 6 oil powders, together with their respective redispersed oil powders in water. Human abdominal skin was dermatomed (thickness 400 μm) for use in the diffusion studies. Franz-type diffusion cells were used over a period of 24 hours. The results of the membrane release studies indicated that the oil powders had achieved a significantly higher release than their respective redispersed oil powders. The release of salicylic acid from the redispersed oil powders and from their respective emulsions was similar. The transdermal delivery test outcomes showed that the effect of pH could have been influenced by the degree of ionisation, resulting in a decrease in permeation with increasing ionisation of salicylic acid, in accordance with the pH partition hypothesis. Furthermore, biopolymers, such as chitosan had demonstrated a penetration enhancing effect, which had led to the enhanced dermal and transdermal delivery of salicylic acid. A correlation was also found between the powder particle size and transdermal delivery. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Motivation: C-reactive protein (hs-CRP) and other risk factors such as cortisol and obesity in the diagnosis of cardiovascular dysfunction (CVD) in African and Caucasian women has become increasingly imperative when one considers the escalation of hypertension among these groups. Recent studies have explored some aspects of these risk factors and the roles that they play within hypertension and possible future risk for cardiovascular disease. Hs-CRP has been associated with the increased prevalence of hypertension and obesity. Cortisol per se has also been linked with the development of both hypertension and the hypothalamic-pituitary adrenal cortex (HPA) response. Nevertheless, the exact mechanism remains rather uncertain due to conflicting outcomes of research within different ethnic groups. Several recent investigations have, however, linked hypocortisolism with both urbanisation and a subsequent increased likelihood of hypertension within African women as they have presented increased vascular blood pressure responses. Conversely, Caucasian women have displayed an increased central cardiac reactivity. The lack of data regarding the relationship between the above-mentioned parameters within both African and Caucasian women serves as the motivation for conducting this study. Objective: To investigate hs-CRP, cortisol and hypertension as contributors to the increased likelihood of cardiovascular dysfunction in both African and Caucasian women within South Africa. hs-CRP use this through whole document please Methodology: The manuscript presented in Chapter 2 has been compiled using data obtained from the POWIRS (Profiles of Obese Women with Insulin Resistance Syndrome) study. Apparently healthy African (N=102) and Caucasian (N=115) women, matched for age and body mass index, were recruited from the North-West Province of South Africa for participation within this study. Subjects were divided into normotensive (NT) and hypertensive (HT) groups according to the mean resting cardiovascular values that were taken using a Finometer device. High-sensitivity C-reactive protein (hs-CRP) and cortisol blood serum values were determined by immunochemistry and ELISA analyses. Significant differences within each ethnic group and between each of the groups (NT and HT) were determined by analysis of covariance (ANCOVA), for anthropometric, cardiovascular, hs-CRP and cortisol variables, while adjusting for cardiovascular covariates (age, smoking and alcohol consumption). Partial correlations analyses were used to examine the relationship between hs-CRP, cortisol, anthropometric and cardiovascular parameters adjusting for cardiovascular covariates. Logistic regression analyses was used within each ethnic group to determine the relationship between anthropometric, cardiovascular, hs-CRP and cortisol as independent variables and hypertension as dependent variable. This study was approved by the Ethics Committee of the North-West University and all subjects gave informed consent in writing. For a more detailed description of the subjects, study design and analytical procedures please refer to the Materials and Methods section within Chapter 2 of this dissertation. Results and Conclusion: Both ethnic groups presented higher hs-CRP and lower cortisol levels compared to normal values. Lower waist circumference (WC) and cortisol as well as higher blood pressure (BP) and vascular values were evident in Africans compared to Caucasians. Both HT ethnic groups were older and more visceral obese compared to their NT counterparts. HT Caucasians indicated higher central adrenergic responses whilst HT Africans showed vascular adrenergicresponses. Only NT Africans had lower cortisol values than NT Caucasians but the Africans (NT and HT) responded with higher diastolic blood pressure responses compared to their Caucasian counterparts. Moreover, hs-CRP within African women significantly correlated with all BP and obesity variables whilst hs-CRP only associated with stroke volume (SV) and compliance (Cw) within HT Caucasian women. Cortisol in both ethnic groups was strongly associated with vascular BP responses. Only BP contributed to the higher prevalence of HT in both ethnic groups. In conclusion, these results suggest the possible diverse roles of HPA axis dysregulation associated with higher inflammatory responses. This happens in conjunction with cardiac and vascular responses within more obese Caucasian and especially African women, respectively. / MSc (Physiology), North-West University, Potchefstroom Campus, 2009

The available literature indicates that to date, few research has been performed on oil powders for topical delivery. The aim of this project was to investigate the release characteristics of oil powder formulations, as well as their dermal and transdermal delivery properties. Whey protein-stabilised emulsions were used to develop oil powders. Whey protein was used alone, or in combination with chitosan or carrageenan. Nine oil powders, with salicylic acid as the active ingredient, were formulated by using the layer-by-layer method. Three different pH values (pH 4, 5 and 6) were used to prepare the formulations, because of the different charges that polymeric emulsifiers may have. The characteristics of the prepared oil powders were determined, including their droplet sizes, particle size distributions, loss on drying, encapsulation efficiencies, oil leakage and water dispersibility. Release studies (membrane diffusion studies) were conducted by utilising cellulose acetate membranes (0.2 μm pore size) and Franz-type diffusion cells over a period of eight hours. The release of the active ingredient was determined for all nine powders, their respective template emulsions, as well as their respective oil powders redispersed in water. The release of salicylic acid from the respective redispersed oil powders was then further compared to its release from the template emulsions and from the oil powders. The effect of pH and different polymer types used in preparing the oil powders, their respective redispersed oil powders and the template emulsions were determined with regards to the release of the active ingredient from all these preparations. The effect of pH and different polymers used was furthermore determined on the oil powders and their respective redispersed oil powders, with regards to their dermal and transdermal deliveries. Transdermal delivery and skin uptake were investigated on specifically selected powders only, based on the outcomes of the oil powder characterisation and release data. The qualifying formulations were chitosan pH 4, 5 and 6, whey and carrageenan pH 6 oil powders, together with their respective redispersed oil powders in water. Human abdominal skin was dermatomed (thickness 400 μm) for use in the diffusion studies. Franz-type diffusion cells were used over a period of 24 hours. The results of the membrane release studies indicated that the oil powders had achieved a significantly higher release than their respective redispersed oil powders. The release of salicylic acid from the redispersed oil powders and from their respective emulsions was similar. The transdermal delivery test outcomes showed that the effect of pH could have been influenced by the degree of ionisation, resulting in a decrease in permeation with increasing ionisation of salicylic acid, in accordance with the pH partition hypothesis. Furthermore, biopolymers, such as chitosan had demonstrated a penetration enhancing effect, which had led to the enhanced dermal and transdermal delivery of salicylic acid. A correlation was also found between the powder particle size and transdermal delivery. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Motivation: C-reactive protein (hs-CRP) and other risk factors such as cortisol and obesity in the diagnosis of cardiovascular dysfunction (CVD) in African and Caucasian women has become increasingly imperative when one considers the escalation of hypertension among these groups. Recent studies have explored some aspects of these risk factors and the roles that they play within hypertension and possible future risk for cardiovascular disease. Hs-CRP has been associated with the increased prevalence of hypertension and obesity. Cortisol per se has also been linked with the development of both hypertension and the hypothalamic-pituitary adrenal cortex (HPA) response. Nevertheless, the exact mechanism remains rather uncertain due to conflicting outcomes of research within different ethnic groups. Several recent investigations have, however, linked hypocortisolism with both urbanisation and a subsequent increased likelihood of hypertension within African women as they have presented increased vascular blood pressure responses. Conversely, Caucasian women have displayed an increased central cardiac reactivity. The lack of data regarding the relationship between the above-mentioned parameters within both African and Caucasian women serves as the motivation for conducting this study. Objective: To investigate hs-CRP, cortisol and hypertension as contributors to the increased likelihood of cardiovascular dysfunction in both African and Caucasian women within South Africa. hs-CRP use this through whole document please Methodology: The manuscript presented in Chapter 2 has been compiled using data obtained from the POWIRS (Profiles of Obese Women with Insulin Resistance Syndrome) study. Apparently healthy African (N=102) and Caucasian (N=115) women, matched for age and body mass index, were recruited from the North-West Province of South Africa for participation within this study. Subjects were divided into normotensive (NT) and hypertensive (HT) groups according to the mean resting cardiovascular values that were taken using a Finometer device. High-sensitivity C-reactive protein (hs-CRP) and cortisol blood serum values were determined by immunochemistry and ELISA analyses. Significant differences within each ethnic group and between each of the groups (NT and HT) were determined by analysis of covariance (ANCOVA), for anthropometric, cardiovascular, hs-CRP and cortisol variables, while adjusting for cardiovascular covariates (age, smoking and alcohol consumption). Partial correlations analyses were used to examine the relationship between hs-CRP, cortisol, anthropometric and cardiovascular parameters adjusting for cardiovascular covariates. Logistic regression analyses was used within each ethnic group to determine the relationship between anthropometric, cardiovascular, hs-CRP and cortisol as independent variables and hypertension as dependent variable. This study was approved by the Ethics Committee of the North-West University and all subjects gave informed consent in writing. For a more detailed description of the subjects, study design and analytical procedures please refer to the Materials and Methods section within Chapter 2 of this dissertation. Results and Conclusion: Both ethnic groups presented higher hs-CRP and lower cortisol levels compared to normal values. Lower waist circumference (WC) and cortisol as well as higher blood pressure (BP) and vascular values were evident in Africans compared to Caucasians. Both HT ethnic groups were older and more visceral obese compared to their NT counterparts. HT Caucasians indicated higher central adrenergic responses whilst HT Africans showed vascular adrenergicresponses. Only NT Africans had lower cortisol values than NT Caucasians but the Africans (NT and HT) responded with higher diastolic blood pressure responses compared to their Caucasian counterparts. Moreover, hs-CRP within African women significantly correlated with all BP and obesity variables whilst hs-CRP only associated with stroke volume (SV) and compliance (Cw) within HT Caucasian women. Cortisol in both ethnic groups was strongly associated with vascular BP responses. Only BP contributed to the higher prevalence of HT in both ethnic groups. In conclusion, these results suggest the possible diverse roles of HPA axis dysregulation associated with higher inflammatory responses. This happens in conjunction with cardiac and vascular responses within more obese Caucasian and especially African women, respectively. / MSc (Physiology), North-West University, Potchefstroom Campus, 2009

Introduction: In Africa, it is estimated that cardiovascular disease (CVD) will affect approximately 1.3 million people per annum over the following 20 years. C-reactive protein (CRP) is a predictor of CVD risk and certain CRP gene polymorphisms can result in altered CRP concentrations. The distribution of CRP gene polymorphisms is ethnic-specific and extrapolating information from other populations to the black South African population, reported to harbour considerable genetic variation, should be avoided. This highlights the fact that genetic research among black South Africans is necessary. Objectives: The main aim of this dissertation was to determine the association between various polymorphisms (reported and novel [single nucleotide polymorphisms (SNPs)] within the CRP gene with CRP concentrations [measured as high sensitivity (hs)-CRP concentrations] in a black South African population undergoing an epidemiological transition. Interactions between specific CRP polymorphisms and certain environmental factors on hs-CRP concentrations were also investigated. Methods: This cross-sectional study (n=1,588) was nested within the Prospective Urban and Rural Epidemiological (PURE) study. Genotyping was performed using Illumina VeraCode technology on the BeadXpress® platform. Hs-CRP concentrations were measured by the use of a sequential multiple analyser computer (SMAC) through a particle-enhanced immunoturbidometric assay. Results: All the SNPs adhered to the assumptions of Hardy-Weinberg equilibrium, although the distribution of several SNPs differed from that reported in other population groups. Three SNPs (rs3093058, rs3093062 and rs3093068) were associated with a significant (p ≤ 0.05) increase in CRP concentrations. Five SNPs (rs1205, rs1341665, rs2794520, rs7553007 and rs2027471) were associated with a significant (p ≤ 0.05) decrease in CRP concentrations. This difference in effect was most probably due to changes in gene function brought about by the localisation of these SNPs in the CRP gene. Men and urban individuals were more likely to present with significant associations between the SNPs investigated and CRP concentrations. The difference in the prevalence of the alleles associated with higher CRP concentrations in this population compared to non-African populations could possibly explain the increased CRP concentrations that are observed in the black South African population. Gene-gender (rs1205, rs1341665 and rs2027474) as well as gene-environmental (rs3093068) interactions were also observed. Conclusions: CRP concentrations are in themselves a complex trait and there are many factors at play that influence their expression. Numerous factors (both genetic and environmental) are involved and no single factor acting alone is likely to have enough of an influence to be used as a clinical diagnostic test of CRP concentrations. These results provide valuable information on the regulation of CRP in a black South African population as well as contribute to the literature of CRP on a global level. / Thesis (MSc (Nutrition))--North-West University, Potchefstroom Campus, 2013.

Introduction: In Africa, it is estimated that cardiovascular disease (CVD) will affect approximately 1.3 million people per annum over the following 20 years. C-reactive protein (CRP) is a predictor of CVD risk and certain CRP gene polymorphisms can result in altered CRP concentrations. The distribution of CRP gene polymorphisms is ethnic-specific and extrapolating information from other populations to the black South African population, reported to harbour considerable genetic variation, should be avoided. This highlights the fact that genetic research among black South Africans is necessary. Objectives: The main aim of this dissertation was to determine the association between various polymorphisms (reported and novel [single nucleotide polymorphisms (SNPs)] within the CRP gene with CRP concentrations [measured as high sensitivity (hs)-CRP concentrations] in a black South African population undergoing an epidemiological transition. Interactions between specific CRP polymorphisms and certain environmental factors on hs-CRP concentrations were also investigated. Methods: This cross-sectional study (n=1,588) was nested within the Prospective Urban and Rural Epidemiological (PURE) study. Genotyping was performed using Illumina VeraCode technology on the BeadXpress® platform. Hs-CRP concentrations were measured by the use of a sequential multiple analyser computer (SMAC) through a particle-enhanced immunoturbidometric assay. Results: All the SNPs adhered to the assumptions of Hardy-Weinberg equilibrium, although the distribution of several SNPs differed from that reported in other population groups. Three SNPs (rs3093058, rs3093062 and rs3093068) were associated with a significant (p ≤ 0.05) increase in CRP concentrations. Five SNPs (rs1205, rs1341665, rs2794520, rs7553007 and rs2027471) were associated with a significant (p ≤ 0.05) decrease in CRP concentrations. This difference in effect was most probably due to changes in gene function brought about by the localisation of these SNPs in the CRP gene. Men and urban individuals were more likely to present with significant associations between the SNPs investigated and CRP concentrations. The difference in the prevalence of the alleles associated with higher CRP concentrations in this population compared to non-African populations could possibly explain the increased CRP concentrations that are observed in the black South African population. Gene-gender (rs1205, rs1341665 and rs2027474) as well as gene-environmental (rs3093068) interactions were also observed. Conclusions: CRP concentrations are in themselves a complex trait and there are many factors at play that influence their expression. Numerous factors (both genetic and environmental) are involved and no single factor acting alone is likely to have enough of an influence to be used as a clinical diagnostic test of CRP concentrations. These results provide valuable information on the regulation of CRP in a black South African population as well as contribute to the literature of CRP on a global level. / Thesis (MSc (Nutrition))--North-West University, Potchefstroom Campus, 2013.

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