The objective of this research is to investigate three classes of block copolymers, the vesicle structures they form, their response to stimuli in solution and their capabilities for use in biomimicry. The self-assembled structures of all classes of polymers will be used as a basis for templating hydrogel materials, in the interior of the vesicles, and the resulting particles will be designed to show the structural and mechanical properties similar to living cells. The synthetic block copolymers are a poly(ethylene glycol) and poly(butadiene) (PEO-b-PBd) copolymer, a poly(ethylene glycol) and a poly(dimethyl siloxane) (PEO-b-PDMS) copolymer and the polypeptide block copolymer is a lysine and glycine (K-b-G) copolymer. Investigation using the synthetic block copolymers will focus on whether the polymer can form vesicles, size limitations of vesicle structures, and the formation of internal polymer networks. Subsequent investigations will look at the needed steps for biomimicry. The PDMS copolymer is a novel entrant into amphiphilic block copolymers. Although characterization of the copolymer solution behavior is known, the mechanical properties of the polymer are not known. PDMS was investigated along with the PBd polymer due to the similar chemical structure and nature. The lysine-glycine copolymers are a new system of materials that form fluid vesicle structures. Therefore, characterization of how K-b-G assembly behavior and investigations of how K-b-G responds to solution conditions are needed before incorporating this copolymer into a cellular mimic. The size and mechanical behavior of the lysine-glycine vesicles are measured to compare and contrast to the synthetic systems. The goals in creating a biomimic are a hollow sphere structure with a fluid bilayer, a vesicle that has controllable mechanical properties, and with a controllable surface chemistry and density. Overall, these experiments were successful; the various properties are easily controllable: the size of vesicles created, the material properties of the vesicle interior and shell, as well as the surface chemistry of the vesicles. Investigations into the novel block copolymers were conducted, and the polypeptide block copolymer showed the ability to create vesicles that are responsive to changing salt and pH concentrations. The PDMS block copolymer system offers a new material system that will perform as well as the PBd system, but without some of the inherent drawbacks.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-118).

Approximately 350 million people worldwide suffer from diabetes mellitus (DM) and this number increases yearly. Since the discovery and clinical application of insulin in 1921, subcutaneous injections have been the standard treatment for DM. Because insulin is hydrophilic and has a high molecular weight and low bioavailability, this molecule is poorly absorbed if administered orally. The aim of this study is to evaluate nasal delivery systems for insulin, using Sprague Dawley rats as the nasal absorption model. Pheroid technology and N-trimethyl chitosan chloride (TMC) with different dosages of insulin (4, 8 and 12 IU/kg bodyweight insulin) was administered in the left nostril of the rat by using a micropipette. Pheroid technology is a patented (North-West University) carrier system consisting of a unique oil/water emulsion that actively transports drug actives through various physiological barriers. These formulations were administered nasally to rats in a volume of 100 p/kg bodyweight in different types of Pheroids (vesicles, with a size of 1.7 1 - 1.94 pm and microsponges, with a size of 5.7 1 - 8.25 pm). The systemic absorption of insulin was monitored by measuring arterial blood glucose levels over a period of 3 hours. The TMC formulation with 4 IU/kg insulin produced clinically relevant levels of insulin in the blood and as a result also the maximal hypoglycaemic effect. TMC is a quaternary derivative of chitosan and is able to enhance the absorption of various peptide drugs by opening tight junctions between epithelial cells. Pheroid formulations were also effective in lowering blood glucose levels but only at higher doses (8 and 12 IU/kg) of insulin. This study indicated that Pheroid rnicrosponges had a faster onset of action and a slightly better absorption of insulin when compared to Pheroid vesicles, but many more studies are needed in this field. Although the results of this study with absorption enhancers are encouraging, nasal insulin bioavailability is still very low, and the Pheroid formulations and long-term safety of nasal insulin therapy have yet to be investigated. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Advances in biotechnology and recombinant technologies have lead to the production of several classes of new drugs such as peptide and protein drugs. These compounds are mostly indicated for chronic use but their inherent characteristics such as size, polarity and stability prevent them from incorporation in novel dosage forms. The bioavailability of nearly all peptide drugs is very low due to poor absorption from the administration site. Several challenges confront the pharmaceutical scientist in developing effective and innovative dosage forms for these classes of drugs. A lot of attention has been given to the nasal route of drug administration for delivery of peptide drugs. The availability of several promising classes of absorption enhancers and new drug delivery technologies has also prompt scientists to develop new delivery systems for nasal administration of peptide drugs. It has been shown in recent years that N-trimethyl chitosan chloride (TMC), a quaternary derivative of chitosan, is effective in enhancing the absorption of several peptide drugs, both in the peroral route and in the nasal route of drug administration. Early indications are that new drug delivery technologies such as Pheroid technology will also be able to enhance peptide drug absorption in the nasal route. The aim of this study was to evaluate and compare the absorption enhancing abilities of TMC and Pheroid technology in the nasal delivery of calcitonin, a peptide hormone with low bioavailability. Pheroid vesicles and Pheroid microsponges were prepared and characterized for their morphology and size distribution. Calcitonin was entrapped into these vesicles and microsponges and TMC and TMO solutions (0.5 % w/v), containing calcitonin, was also prepared. These formulations were administered nasally to rats in a volume of 100 μl/kg body-weight to obtain a final concentration of 10 IU/kg body-weight of calcitonin. Plasma calcitonin and calcium levels were determined over a period of 3 hours. The results of this study clearly indicated that both Pheroid formulations and the TMC formulation increase the nasal absorption of calcitonin with a resulting decrease in plasma calcium levels, indicating an increased absorption of calcitonin. The highest increase in calcitonin absorption was obtained with the TMC formulation and this was explained by the difference in the mechanism of action in enhancing peptide absorption between TMC and Pheroid technology. It was concluded that Pheroid technology is also a potent system to enhance peptide drug delivery and that the exact mechanism of action should be investigated further. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

<p>The surface of cell membranes can display a high degree of lateral heterogeneity. This non-uniform distribution of constituents is characterized by mobile nanodomain clusters called rafts. Enriched by saturated phospholipids, cholesterol and proteins, rafts are considered to be vital for several important cellular functions such as signalling and trafficking, morphological transformations associated with exocytosis and endocytosis and even as sites for the replication of viruses. Understanding the evolving distribution of these domains can provide significant insight into the regulation of cell function. Giant vesicles are simple prototypes of cell membranes. Microdomains on vesicles can be considered as simple analogues of rafts on cell membranes and offer a means to study various features of cellular processes in isolation. </p><p>In this work, we employ a continuum approach to model the evolution of microdomains on the surface of Giant Unilamellar Vesicles (GUVs). The interplay of species transport on the vesicle surface and the mechanics of vesicle shape change is captured using a chemo-mechanical model. Specifically, the approach focuses on the regime of vesicle dynamics where shape change occurs on a much faster time scale in comparison to species transport, as has been observed in several experimental studies on GUVs. In this study, shape changes are assumed to be instantaneous, while species transport, which is modeled by phase separation and domain coarsening, follows a natural time scale described by the Cahn--Hilliard dynamics.</p><p>The curvature energy of the vesicle membrane is defined by the classical Canham--Helfrich--Evans model. Dependence of flexural rigidity and spontaneous curvature on the lipid species is built into the energy functional. The chemical energy is characterized by a Cahn--Hilliard type density function that intrinsically captures the line energy of interfaces between two phases. Both curvature and chemical contributions to the vesicle energetics are consistently non-dimensionalized.</p><p>The coupled model is cast in a diffuse-interface form using the phase-field framework. The phase-field form of the governing equations describing shape equilibrium and species transport are both fourth-order and nonlinear. The system of equations is discretized using the finite element method with a uniform cubic-spline basis that satisfies global higher-order continuity. For shape equilibrium, geometric constraints of constant internal volume and constant surface area of the vesicle are imposed weakly using the penalty approach. A time-stepping scheme based on the unconditionally gradient-stable convexity-splitting technique is employed for explicit time integration of nonlocal integrals arising from the geometric constraints.</p><p>Numerical examples of axisymmetric stationary shapes of uniform vesicles are presented. Further, two- and three-dimensional numerical examples of domain formation and growth coupled to vesicle shape changes are discussed. Simulations qualitatively depicting curvature-dependent domain sorting and shape changes to minimize line tension are presented. The effect of capturing the difference in time scales is also brought out in a few numerical simulations that predict a starkly different pathway to equilibrium.</p> / Dissertation

Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus. Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form. We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, the active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells. Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction.

No description available.

<p>Interfaces play a dominant role in governing the response of many biological systems and they pose many challenges to traditional finite element. For sharp-interface model, traditional finite element methods necessitate the finite element mesh to align with surfaces of discontinuities. Diffuse-interface model replaces the sharp interface with continuous variations of an order parameter resulting in significant computational effort. To overcome these difficulties, we focus on developing a computationally efficient spline-based finite element method for interface problems.</p><p>A key challenge while employing B-spline basis functions in finite-element methods is the robust imposition of Dirichlet boundary conditions. We begin by examining weak enforcement of such conditions for B-spline basis functions, with application to both second- and fourth-order problems based on Nitsche's approach. The use of spline-based finite elements is further examined along with a Nitsche technique for enforcing constraints on an embedded interface. We show that how the choice of weights and stabilization parameters in the Nitsche consistency terms has a great influence on the accuracy and robustness of the method. In the presence of curved interface, to obtain optimal rates of convergence we employ a hierarchical local refinement approach to improve the geometrical representation of interface. </p><p>In multiple dimensions, a spline basis is obtained as a tensor product of the one-dimensional basis. This necessitates a rectangular grid that cannot be refined locally in regions of embedded interfaces. To address this issue, we develop an adaptive spline-based finite element method that employs hierarchical refinement and coarsening techniques. The process of refinement and coarsening guarantees linear independence and remains the regularity of the basis functions. We further propose an efficient data transfer algorithm during both refinement and coarsening which yields to accurate results.</p><p>The adaptive approach is applied to vesicle modeling which allows three-dimensional simulation to proceed efficiently. In this work, we employ a continuum approach to model the evolution of microdomains on the surface of Giant Unilamellar Vesicles. The chemical energy is described by a Cahn-Hilliard type density functional that characterizes the line energy between domains of different species. The generalized Canham-Helfrich-Evans model provides a description of the mechanical energy of the vesicle membrane. This coupled model is cast in a diffuse-interface form using the phase-field framework. The effect of coupling is seen through several numerical examples of domain formation coupled to vesicle shape changes.</p> / Dissertation

No description available.

Approximately 350 million people worldwide suffer from diabetes mellitus (DM) and this number increases yearly. Since the discovery and clinical application of insulin in 1921, subcutaneous injections have been the standard treatment for DM. Because insulin is hydrophilic and has a high molecular weight and low bioavailability, this molecule is poorly absorbed if administered orally. The aim of this study is to evaluate nasal delivery systems for insulin, using Sprague Dawley rats as the nasal absorption model. Pheroid technology and N-trimethyl chitosan chloride (TMC) with different dosages of insulin (4, 8 and 12 IU/kg bodyweight insulin) was administered in the left nostril of the rat by using a micropipette. Pheroid technology is a patented (North-West University) carrier system consisting of a unique oil/water emulsion that actively transports drug actives through various physiological barriers. These formulations were administered nasally to rats in a volume of 100 p/kg bodyweight in different types of Pheroids (vesicles, with a size of 1.7 1 - 1.94 pm and microsponges, with a size of 5.7 1 - 8.25 pm). The systemic absorption of insulin was monitored by measuring arterial blood glucose levels over a period of 3 hours. The TMC formulation with 4 IU/kg insulin produced clinically relevant levels of insulin in the blood and as a result also the maximal hypoglycaemic effect. TMC is a quaternary derivative of chitosan and is able to enhance the absorption of various peptide drugs by opening tight junctions between epithelial cells. Pheroid formulations were also effective in lowering blood glucose levels but only at higher doses (8 and 12 IU/kg) of insulin. This study indicated that Pheroid rnicrosponges had a faster onset of action and a slightly better absorption of insulin when compared to Pheroid vesicles, but many more studies are needed in this field. Although the results of this study with absorption enhancers are encouraging, nasal insulin bioavailability is still very low, and the Pheroid formulations and long-term safety of nasal insulin therapy have yet to be investigated. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.