Even though the role of oxidative stress in a variety of disease states is known, strategies to alleviate this oxidative stress by antioxidants have not been able to achieve clinical success. Particularly, treatment of oxidative stress by small molecule antioxidants has not received due attention because of the challenges associated with its delivery. Antioxidant polymers, where small molecule antioxidants are incorporated into the polymer backbone, are an emerging class of materials that can address some of these challenges. In this work, biodegradable polymers incorporating phenolic antioxidants in the polymer backbone were synthesized. Antioxidant polymers were then characterized for their in vitro degradation, antioxidant release and their effect on oxidative stress levels (redox state) in the cells. Trolox, a water-soluble analogue of vitamin E, was polymerized to synthesize poly(trolox ester) with 100% antioxidant content which undergoes biodegradation to release trolox. Nanoparticles of poly(trolox ester) were able to suppress oxidative stress injury induced by metal nanoparticles in an in vitro cell injury model. In another study, we polymerized polyphenolic antioxidants (e.g. curcumin, quercetin) using a modified non-free-radical polymerization poly(β-amino ester) chemistry. This synthesis scheme can be extended to all polyphenolic antioxidants and allows tuning of polymer degradation rate by choosing appropriate co-monomers from a large library of monomers available for β-amino ester chemistry. Poly(antioxidant β-amino esters) (PABAE) were synthesized and characterized for their degradation, cytotoxicity and antioxidant activity. PABAE degradation products suppressed oxidative stress levels in the cells confirming antioxidant activity of degradation products.

Growth plate injuries account for 15-30% of long bone fractures in children. About 10% of these result in significant growth disturbances due to formation of a boney bar. If not treated correctly, this can lead to life-lasting consequences of limb length inequalities and angular deformities. Current treatments for growth plate injuries include removal of boney bar and insertion of fat, silicone, bone cement, etc.. This treatment y is inadequate, leaving almost half of these patients with continued deformities. This dissertation reports characterization of a DNA–containing porous poly(lactic-co-glycolic acid) (PLGA) scaffold system, chondrogenesis using insulin-like growth factor I (IGF-I) plasmid-releasing scaffolds in vitro, and in vivo testing of IGF-I plasmid-releasing scaffolds to regenerate growth plate . Controlled release of naked and DNA complexed with polyethylenimine (PEI) was achieved from porous PLGA scaffolds. PEI affected release of complexes from PLGA scaffolds, as PEI:DNA complexes were released at a lower rate compared to naked DNA encapsulated in low molecular weight (LMW) and high molecular weight PLGA scaffolds, as well as hydrophilic and hydrophobic PLGA scaffolds. Hydrophilicity and molecular weight of PLGA affected the release profiles of both naked DNA and PEI:DNA complexes from the scaffolds, as evidenced by later peak DNA and PEI:DNA release with increasing hydrophilicity and molecular weight. LMW hydrophilic PLGA scaffolds supported growth and chondrogenic differentiation of mesenchymal multipotent D1 cells, chondrocytes, and bone marrow cells (BMCs) in vitro. Culturing BMCs on IGF-I plasmid-encapsulated scaffolds resulted in elevated expression of IGF-I compared to blank scaffolds. Removal of boney bar and implantation of IGF-I plasmid-releasing LMW PLGA scaffolds in a rabbit model of growth plate injury resulted in some improvement of leg angular deformity compared to no scaffold implantation. Histological analysis of the newly developed cartilage showed growth plate-like columnar arrangement of chondrocytes in a defect that received IGF-I plasmid encapsulated scaffold, although the level of organization of newly formed cartilage was inferior to that of native growth plate. This appears to be the first report of the regeneration of growth plate-like structure without the use of stem cells in an animal model of physeal injury.

Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

Technological limitations have prevented interrogation and manipulation of many signaling pathways in model and living systems required for the development of diagnostic and therapeutic modalities in diseases, such as cancer. Liposome-supported plasmon resonant gold nanoshells are biologically inspired composite structures, in which the liposome allows for the encapsulation of substances, and the plasmon resonant structure facilitates rapid release of encapsulated contents upon laser light illumination. As shown in this work, we overcome current limitations in cellular manipulation using plasmon resonant gold-coated liposomes in conjunction with light-activated release to achieve accurate probing of complex cellular responses. Development toward this goal was demonstrated with four specific aims. The first specific aim was to develop a computational model of heat diffusion to investigate the light-induced heating of gold-coated liposomes. This model was used to optimize the photothermal process for release of an encapsulated payload. The second aim was to demonstrate encapsulation and on-demand release of molecules in a spectrally-controlled manner, where plasmon resonant nanoparticles only release content upon illumination with a wavelength of light matching their plasmon resonance band. The third specific aim was to demonstrate that this release mechanism can be used in a biological setting to deliver a peptide and extracellularly activate surface membrane receptors with single-cell spatial and high temporal resolution. The fourth specific aim further refined the level of spatial and temporal control of payload release using gold-coated liposomes with optical trapping to demonstrate mirco-manipulation of liposome movement and rapid content release to enable accurate perturbation of cellular functions in response to released compounds. Through this work, we have developed an experimental system with the potential for the delivery and localized release of an encapsulated agent with high spatial and temporal resolution. This on-demand release system is compatible with a broad range of molecules and uses biologically safe near-infrared light. In combination with the spectral tunability of these plasmon resonant nanoshells and spectrally-selective release, this technology may allow for interrogation of complex and diverse signaling pathways in living tissues or their models with unprecedented spatial and temporal control.

In 1982, several rectangular openings were cut in a 100 year old sub-alpine Norway spruce forest stand to initiate regeneration at the Lusiwald site at Davos, Switzerland. The openings on the steep, north-facing slope created rapid changes to the environment of the border trees. Growth reactions of these border trees were compared and analysed with reference trees from the adjacent closed canopy stand in 1997. The radial growth pattern of the two data sets differed within the 14-year period since the openings were cut; the border trees showed growth releases. The growth reaction at the stem base was larger than at breast height. Changes in wind exposure may have influenced border trees to adapt their root systems. Sub-alpine Norway spruce stands aged around 100 years, which are usually considered slow-growing on a north aspect, still seem capable of reacting to greater resource availability such as sudden light changes.

A model of the generation and release of fission gas, as well as the total swelling over time, was created. It uses an ideal spherical fuel grain with a time-dependent radius. UO2 and quasi-homogeneous SBR MOX fuels were simulated with this model, and the results were compared to a fixed grain radius model of gaseous swelling. Gaseous swelling and fission gas release were calculated for temperatures from 1600 K to 2200 K. The grain growth of UO2 was found to decrease the time needed to saturate the intergranular boundaries as compared to simple diffusion without grain growth. Small temperatures increased the time required for saturation, as did small rates of grain growth. Gaseous swelling was within the range of values found by experimental data.

The degree to which biotic interactions influence invasion success may partly depend on the evolutionary relationship between invaders and native species. In particular, since host-use by enemies such as invertebrate herbivores and fungal pathogens tends to be phylogenetically conserved, exotic plants that have close native relatives in the invaded range should be more likely to interact with enemies. In this thesis, I explore this idea using a series of experiments and field surveys at nested taxonomic levels. My results indicate that exotics from multiple plant families experience lower damage if their average phylogenetic distance from locally co-occurring native family members is higher. I then demonstrate that within the Asteraceae, foliar and capitular damage are lower on exotic compared to native species. Both damage types had a relatively large phylogenetic component, but did not decline with phylogenetic distance to native or exotic confamilials. Finally, I show that communities with versus without close relatives are unlikely to differ in resistance to the novel invader, Solidago virgaurea: biotic resistance imposed by competitors, generalist vertebrates, and specialist invertebrates resulted in similar patterns of damage and mortality regardless of the presence of congeneric natives. In some cases, effects of biota were positive: growth of S. virgaurea seedlings in soils collected near congeneric natives was enhanced more than in soils from communities where congenerics were absent. Overall, these results suggest that biotic interactions between exotic and native species can be phylogenetically structured, although trends based on distance measures tend to be weak. In some cases, damage does decline with phylogenetic distance to native species; however this trend is unlikely to be a strong force limiting invasion or structuring plant communities. These results have significant implications for current theories of invasion biology including the "Enemy Release Hypothesis" and "Darwin's Naturalization Hypothesis", as well as for community phylogenetics.

Device associated infections are a growing problem in the field of orthopaedics and dentistry. Bacteria adhering to implant surfaces and subsequent biofilm formation are challenging to treat with systemic administered antibiotics. Functionalization of implant surfaces with therapeutic coatings that are capable of inhibiting bacterial adhesion are therefore considered as a straight forward strategy to treat and prevent implant related infections. In this thesis, the use of crystalline, arc deposited TiO2 and biomimetic hydroxyapatite (HA) coatings were evaluated with respect to their potential as antibacterial surface modifications for bone-anchored implants. UV light induced photocatalysis of anatase dominated TiO2 coated surfaces was shown to provide a bactericidal effect against S. epidermidis under clinically relevant illumination times and doses. Major parts of the drug release work carried out was based on biomimetic HA (HA-B) coated fixation pins. The analysis of the coating characteristics revealed that the nanoporous structure of HA-B coatings in addition to the chemical composition and surface charge are essential parameters that influence the drug carrier performance. Loading by adsorption was demonstrated to be a feasible approach to quickly incorporate antibiotics. The controlled release of antibiotics was shown to facilitate bactericidal effects against S. aureus over application-relevant time periods, even when exposed to biomechanical forces during insertion into bone model materials. Antibiotic incorporation during coating growth was shown to promote somewhat longer drug release time periods than those obtained using adsorption loading. In summary, functionalization of implant surfaces with bioactive and biocompatible coatings is a promising concept to impact the clinical success for bone-anchored applications. The additional feature of optional, on-demand antibacterial properties of these coatings through either on-site drug release or photocatalytic antibacterial treatment is advantageous for the prevention and effective treatment of devices-associated infections. Both strategies provide an immediate response to the implant contamination by bacteria and are believed to contribute towards minimizing the origin of post-surgical infections, while at the same time improving the interfacial stability between implant and bone.

<p>Neuroinflammation associated with lumbar radiculopathy and peripheral nerve injury is characterized by locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF&alpha;). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in local delivery of anti-inflammatory drugs to treat this pathology, as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNF&alpha; in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. </p><p>ELPs are biopolymers capable of thermally-triggered in situ depot formation and have been successfully employed as drug carriers and biomaterials in several applications. A library of ELP-curcumin conjugates were synthesized and characterized. One lead conjugate was shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNF&alpha; and NF-&kappa;B with near-equivalent potency compared to free curcumin. When injected into the perineural space via intramuscular (i.m.) injection proximal to the sciatic nerve in mice, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4 days post-injection and decreased systemic exposure of curcumin 3-fold. </p><p>The results of this dissertation support the use of ELP as a drug carrier for local perineural drug delivery, and the strategy presented here for drug conjugate development and use of depot-forming ELP-curcumin conjugates represents a novel means of providing sustained treatment of neuroinflammation and pain associated with radiculopathy and peripheral nerve injury.</p> / Dissertation

Controlled release systems aim at achieving a predictable and reproducible drug release profile over a desired time period. These controlled release formulations offer many advantages over conventional dosage forms. These advantages include: reduced dosing intervals, constant drug levels in the blood, increased patient compliance and decreased adverse effects. Complex controlled release formulations such as those with sustained release properties, often require additional steps during the production phase. The cost and economic impact associated with these complex controlled release dosage formulations often outweigh the short term benefits. Thus the development of an economic method to produce controlled release particles is of great importance especially in third world countries. In controlled release formulations the drug is often equally dispersed throughout a polymer matrix. In the presence of a thermodynamically compatible solvent, swelling occurs and the polymer releases its content to the surrounding medium. The rate of drug release can be controlled by interfering with the amount of swelling and rate of diffusion by manipulating the viscosity of the polymer matrix. Chitosan is an ideal candidate for controlled drug delivery through matrix release systems. It is a biodegradable polymer with absorption-enhancing properties. Cross-linking chitosan with different cross-linking agents allow the preparation of beads. Beads are frequently used in controlled release dosage forms as they are very flexible in dosage form development and show various advantages over single unit dosage forms. Because beads disperse freely in the gastrointestinal tract they maximize drug absorption, reduce fluctuation in peak plasma, and minimize potential side effects without lowering drug bio-availability. Chitosan beads and excipient containing chitosan beads were prepared and investigated as possible controlled release formulations. Pyrazinamide was chosen as the model drug. Chitosan beads and excipient containing chitosan beads were prepared by ionotropic gelation in tripolyphosphate. In this study chitosan/pyrazinamide beads containing pharmaceutical excipients (Ascorbic acid, Explotab and Ac-Di-Sol) were produced. The excipients were added individually and in combinations to the chitosadpyrazinamide dispersion and the beads were characterized on the basis of their morphology, solubility, fiability, drug loading capacity and swelling behaviour, as well as drug release (dissolution properties). The drug loading of the pyrazinarnide loaded chitosan beads, was 52.26 % 0.57%. It was noted that the inclusion of excipients in the beads resulted in an increase in drug loading with the combination of Ascorbic acid and Ac-Di-Sol giving the highest drug loading of 67.09 ± 0.22%. It was expected that the addition of the pharmaceutical excipients would lead to a sustained release of pyrazinamide. Dissolutions studies, however, revealed a burst release in both phosphate buffer solution (PBS) pH 5.60 and 7.40 over the first 15 minutes and the curve reached a plateau after 30 minutes. Thus, apparently the inclusion of the pharmaceutical excipients did not contribute to a sustained release of pyrazinamide over the tested period of six hours. In future studies the dissolution time can possibly be extended to a period of 24 hours. It might be possible for the remaining drug (approximately 40%) in the beads to be released over the extended period. Other polymers can also be investigated to control the release of pyrazinamide. Further studies are, however, necessary to investigate this possibility in the future. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.