A viable <i>in vitro</i> excised human skin model was developed to accurately assess cutaneous delivery and metabolism of two ester type compounds; tetracaine (TC) and methyl salicylate (MS). This model could maintain the viability of fresh skin in diffusion cells for 24 hours. Skin viability was assessed using two methods; oxygen consumption measurement and confocal laser scanning microscopy. Two fluorescent probes, calcein AM and ethidium homodimer-1, were used as live and dead markers, respectively. General morphology and localization of nonspecific esterase activity in the skin samples from diffusion cell were checked histologically. Cutaneous delivery and metabolism of MS was evaluated with this viable skin model and compared to human skin homogenate model. A sensitive high performance liquid chromatography (HPLC) assay using reversed phase ion pair was developed/refined to simultaneously analyze TC and its metabolite (4-BABA). Several factors affecting this HPLC system were identified. The limit of detection for TC and 4-BABA was 0.3 ng and 0.5 ng, respectively. The limit of quantitation for TC and 4-BABA was 10 ng and 5 ng, respectively. Linearity was in the range of 10-120 ng for TC and 5-60 ng for 4-BABA. MS was hydrolyzed to salicylic acid (SA) during absorption through fall thickness human breast skin in diffusion cells. The extent of MS hydrolysis was significantly higher in viable skin than in non viable. The extent of absorption of SA through viable and non viable skins was similar. In human skin homogenate, MS was hydrolyzed at the rate of 72.31 nmol/h/[mu]g protein while the hydrolysis in phosphate buffered saline was very low. TC hydrolysis in human skin homogenate was not extensive due to substrate inhibition. From the kinetic study of TC hydrolysis in human skin homogenate, Km was in the 11-28 [mu]M range and Vmax was in the 2.0-2.8 [mu]mol/h/[mu]g protein range. Temperature over 60°C substantially reduced esterase activity in both models therefore caution must be taken during preparation and handling of tissue samples to preserve esterase activity. The viable <i>in vitro</i> excised skin model will provide more accurate quantitation of skin metabolism and absorption of xenobiotics.

This study was conducted to determine if electroporation could deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. The long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. The ability of electroporation to introduce biologically active molecules, e.g., plasmid DNA and siRNA, into these monolayers, to either express a protein of interest or modify cellular function, was also studied. Results showed that intracellular uptake of calcein, a small tracer molecule, and bovine serum albumin, a globular protein, occurred uniformly throughout the monolayers and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. Barrier function recovery depended on the electroporation conditions applied, with some monolayers recovering normal physiologic function within a day. Electroporation also increased the permeability of the monolayers to calcein and BSA, possibly through a combination of increased paracellular and transmonolayer transport. When compared to cationic lipid transfection (lipofection), transfection of intestinal epithelial monolayers with reporter plasmids by electroporation was more efficient in situations where high concentrations of DNA, and as a result, higher levels of expression were needed. Although uptake of DNA was high after electroporation and increased with increasing amounts of DNA, overall expreseion efficiency was still low (~3%). Electroporation-mediated transfection of intestinal epithelial monolayers with a plasmid that expressed inflammation inhibitor protein, IκВα was not always successful, probably because of low levels of protein expression. Introduction of the much smaller siRNA molecules into the monolayers by electroporation, on the other hand, was very successful at inhibiting the production of the nuclear envelope proteins lamin A and lamin C. The results of these experiments demonstrated that electroporation can introduce a wide variety of molecules intracellularly into model intestinal epithelia. These results should be useful to identify optimal electroporation conditions for transporting drugs, proteins, and genes into intestinal and, possibly, other epithelia for local drug and gene therapy, as well as for development of improved models of intestinal epithelium.

This dissertation focuses on utilization of poly(N-isopropylacylamide) (pNIPAm) based mirogels for regulated macromolecule drug delivery applications. There is particular emphasis on incorporation of stimuli responsive materials into multi-layer thin film constructs with the main goal being fabrication of highly functional materials with tunable release characteristics. Chapter 1 gives a broad overview of hydrogel and microgel materials focusing on fundamental properties of pNIPAm derived materials. Chapter 2 illustrates the progression of controlled macromolecule release from hydrogel and microgel materials and sets up the scope of this thesis work. Chapter 3 details studies on thermally modulated insulin release from microgel thin films where extended pulsatile release capabilities are shown. Chapters 4 and 5 focus on more fundamental synthesis and characterization studies of PEG and acrylic acid modified pNIPAm microgels that could ultimately lead to the design of protein loaded microgel films with tunable release characteristics. Chapter 6 illustrates fundamental macromolecule loading strategies, which could also prove useful in future protein drug delivery design using stimuli responsive networks. Chapter 7 concentrates on direct insulin release studies that probe the interaction between entrapped and freely diffusing protein and microgels. These model experiments could prove useful in design of tunable macromolecule drug release from functionally modified microgels and could aid in the tailored design of peptide-loaded microgel thin films. Chapter 8 discusses the future outlook of controlled macromolecule release from microgel based materials.

Most of the patterns of distribution channel of eggs traditional industry are through the middlemen. Therefore the distribution channel classifies multiple-steps distribution channel system. The price relationship between producers and sales works out by the middlemen for a long time and consequently cause many different viewpoints and conflicts about the price decision. In spite of the middlemen realize and tacitly agree the system of price establishment is not reasonable. They still can not change the actuality because it goes all the year round. Up to now, the producers and sales can not create a win-win situation. The change of marketing channels will reform structures of the production, sales and price forms. Therefore this study takes the home delivery as main body for the eggs marketing channels research, and adopts in-depth interview method to get data and information. At the same time this study penetrates the marketing strategy of egg industry , the business developments and characteristics of home deliveries. This study proceeds to interview the above information as SWOT analysis. To find out the feasibility of eggs home delivery and the point of strategy and problems. Finally, this study would offer some solution aspects and suggestions. The results showed that from the viewpoint of return, the possible pattern of eggs home delivery could create producers much mighty possibilities and big changes by a series of ways to make difference. It means that this study adopts the niche market to be the strategy of market segment and finds the target market and proper marketing mix, the package variation, functional egg developments, confidence-building with home deliveries and regulated contract of products quality. So that producers could develop the private channels and private label brand to change the price-decision system to reach the sustainable development.

The skin forms an attractive and accessible route for systemic delivery of drugs as alternative to other methods of administration, such as the oral and parental methods because of the problems associated with last mentioned methods. The lipophilic character of the stratum corneum, coupled with its intrinsic tortuosity, ensures that it almost always provides the principal barrier to the entry of drug molecules into the skin. Due to the fact that methotrexate (MTX) and acyclovir (ACV) have poor penetration properties through the skin, the aim of this study was to enhance the permeation of methotrexate and acyclovir with the use of two lamellar gel phase systems (LPGS) (Physiogel® NT and Physiogel® Dermaquadrille) and with Emzaloid® as transdermal drug delivery systems. Three different sets of experiments were done in this study: 1) the viscosity of the two Physiogel® creams was measured as an indication of stability and to determine whether the internal structure of the Physiogel® creams were affected by the investigated drugs; 2) the drug release rate from the three drug delivery vehicles was measured with a Vankel ® dissolution apparatus; 3) in vitro permeation studies were preformed using vertical Franz diffusion cells with human epidermal skin clamped between the donor and receptor compartments. The skin was hydrated with PBS buffer for one hour before 1% mixtures of the drugs in both the Physiogel® creams and Emzaloid® were applied to the donor chamber. Samples were taken at 2, 4, 6, 8, 10, 12 and 24 hours. It was then analysed by HPLC for methotrexate and acyclovir. The fluxes of drug permeation were determined. The viscosity measurements confirmed that the internal structure of the two Physiogel® creams was not influenced by the drugs. Acyclovir and methotrexate were both released from the delivery vehicles. There was an enhancement of acyclovir through the skin from one of the Physiogel® creams. The permeability of methotrexate in the presence of the two Physiogel® vehicles was not significantly enhanced. Emzaloid® as delivery vehicle increased the penetration of both drugs through the skin significantly. The lamellar gel phase system mimics the structure of the stratum corneum, but does not improve the drug permeation through the stratum corneum significantly. The utilisation of Emzaloid® as a drug delivery system could be advocated from these findings. As could be seen from the penetration profiles Emzaloid® was a superior delivery system for methotrexate and acyclovir compared to the lamellar gel phase systems. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Immuno-nanoparticles of poly(ᴅ,ʟ-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-g-poly(ethylene glycol) (poly(LA-co-TMCC)-g-PEG) have been used to target breast cancer cells through the specific binding of trastuzumab antibodies to over-expressed human epidermal growth factor receptor 2 (HER2). Small interfering RNA (siRNA) and antisense oligonucleotides (AONs) disrupt the synthesis of select proteins. It is hypothesized that oligonucleotides coupled to polymeric immuno-nanoparticles can be used for gene silencing and specifically to target luciferase. The first objective is to demonstrate the capacity to create dual functional micelles with antibodies and oligonucleotides. The second objective is in vitro testing of the nanoparticle for gene silencing activity. Oligonucleotides are conjugated to the nanoparticle by sequential click reactions of Diels Alder chemistry and copper catalyzed azide-alkyne cycloadditions, respectively. A luciferase assay is used to quantify knockdown of luciferase levels in SKOV-3luc cells (HER2+, luc+). When used in conjunction with a targeted drug delivery vehicle, the nanoparticles provide selective interactions with SKOV-3luc cells.

Immuno-nanoparticles of poly(ᴅ,ʟ-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-g-poly(ethylene glycol) (poly(LA-co-TMCC)-g-PEG) have been used to target breast cancer cells through the specific binding of trastuzumab antibodies to over-expressed human epidermal growth factor receptor 2 (HER2). Small interfering RNA (siRNA) and antisense oligonucleotides (AONs) disrupt the synthesis of select proteins. It is hypothesized that oligonucleotides coupled to polymeric immuno-nanoparticles can be used for gene silencing and specifically to target luciferase. The first objective is to demonstrate the capacity to create dual functional micelles with antibodies and oligonucleotides. The second objective is in vitro testing of the nanoparticle for gene silencing activity. Oligonucleotides are conjugated to the nanoparticle by sequential click reactions of Diels Alder chemistry and copper catalyzed azide-alkyne cycloadditions, respectively. A luciferase assay is used to quantify knockdown of luciferase levels in SKOV-3luc cells (HER2+, luc+). When used in conjunction with a targeted drug delivery vehicle, the nanoparticles provide selective interactions with SKOV-3luc cells.

Since the 1970s, the United States has encountered an increasing proportion of Cesarean deliveries (CS), surpassing the advised 10-15% maximum rate established in 1985 by the World Health Organization (World Health Organization, 1985). This increasing rate has fueled correlational and causational studies observing the impact of Cesarean delivery on several aspects of infant health. Previous studies on CS infants have observed a delay in gut colonization by beneficial bacteria – for instance Bifidobacteria – traditionally transmitted from the mother’s gut and vaginal microbiome as other environmental factors have influenced the initial microflora (Biasucci et al., 2010; Dong, Yang, & Wang, 2010; Penders et al., 2006). In addition, an increasing proportion of births are occurring within a home setting, providing an opportunity to study how these possible environmental factors may influence bacterial colonization. This initial gastrointestinal colonization is considered one of the most important factors towards immune system development and general health. This thesis proposes an examination of how the mode and setting of delivery influence the diversity of Bifidobacterium species in infants’ initial gut microbiomes. Additionally, while several studies have examined the impact of specific bacterial species on immune system development, this study will provide an approach to understanding how differences in the overall gastrointestinal (GI) ecologies of CS and vaginal delivery (VD) infants impact immune system development.

The skin forms an attractive and accessible route for systemic delivery of drugs as alternative to other methods of administration, such as the oral and parental methods because of the problems associated with last mentioned methods. The lipophilic character of the stratum corneum, coupled with its intrinsic tortuosity, ensures that it almost always provides the principal barrier to the entry of drug molecules into the skin. Due to the fact that methotrexate (MTX) and acyclovir (ACV) have poor penetration properties through the skin, the aim of this study was to enhance the permeation of methotrexate and acyclovir with the use of two lamellar gel phase systems (LPGS) (Physiogel® NT and Physiogel® Dermaquadrille) and with Emzaloid® as transdermal drug delivery systems. Three different sets of experiments were done in this study: 1) the viscosity of the two Physiogel® creams was measured as an indication of stability and to determine whether the internal structure of the Physiogel® creams were affected by the investigated drugs; 2) the drug release rate from the three drug delivery vehicles was measured with a Vankel ® dissolution apparatus; 3) in vitro permeation studies were preformed using vertical Franz diffusion cells with human epidermal skin clamped between the donor and receptor compartments. The skin was hydrated with PBS buffer for one hour before 1% mixtures of the drugs in both the Physiogel® creams and Emzaloid® were applied to the donor chamber. Samples were taken at 2, 4, 6, 8, 10, 12 and 24 hours. It was then analysed by HPLC for methotrexate and acyclovir. The fluxes of drug permeation were determined. The viscosity measurements confirmed that the internal structure of the two Physiogel® creams was not influenced by the drugs. Acyclovir and methotrexate were both released from the delivery vehicles. There was an enhancement of acyclovir through the skin from one of the Physiogel® creams. The permeability of methotrexate in the presence of the two Physiogel® vehicles was not significantly enhanced. Emzaloid® as delivery vehicle increased the penetration of both drugs through the skin significantly. The lamellar gel phase system mimics the structure of the stratum corneum, but does not improve the drug permeation through the stratum corneum significantly. The utilisation of Emzaloid® as a drug delivery system could be advocated from these findings. As could be seen from the penetration profiles Emzaloid® was a superior delivery system for methotrexate and acyclovir compared to the lamellar gel phase systems. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Thesis (Ph.D. (Pharmacy))--North-West University, Potchefstroom Campus, 2008.