Polymeric nanoparticles were designed, synthesized, and loaded with metal ions to explore the therapeutic potential for transition metals other than platinum found in cisplatin. Nanoparticles were synthesized to show the potential for polymer based vectors. Metal loading and release were characterized via Inductively Coupled Plasma Mass Spectrometry (ICP MS), Energy Dispersive X-Ray Spectroscopy (EDX), X-Ray Photoelectron Spectroscopy (XPS), and Elemental Analysis. Targeting was attempted with the expectation of observed increased particle uptake by cancer cells with flow cytometry and fluorescence microscopy. Results demonstrated that a variety of metals could be loaded to the nano-sized carriers in an aqueous environment, and that the release was pH-dependent. Expected increased targeting was inconsistent. The toxicity of these particles was measured in cancer cells where significant toxicity was observed in vitro via dosing of high copper-loaded nanoparticles and slight toxicity was observed in ruthenium-loaded nanoparticles. No significant toxicity was observed in cells dosed with metal-free nanoparticles. Future research will focus on ruthenium loaded polymeric nanoparticles with different targeting ligands dosed to different cell lines for the aim of increased uptake and decreased cancer cell viability.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The primary aim of this study was to investigate the feasibility of the amphiphilic block copolymer poly((vinylpyrrolidone)-b-poly(vinyl acetate)) (PVP-b-PVAc) as a vehicle for hydrophobic anti-cancer drugs. PVP-b-PVAc block copolymers of constant hydrophilic PVP block length and varying hydrophobic PVAc block lengths were synthesized via xanthate-mediated controlled radical polymerization (CRP). The methodology consisted of growing the PVAc chain from a xanthate end-functional PVP. In an aqueous environment the amphiphilic block copolymers selfassembled into spherical vesicle-like structures consisting of a hydrophobic PVAc bilayer membrane, a hydrophilic PVP corona and an aqueous core. The self-assembly behaviour and the physicochemical properties of the self-assembled structures were investigated by 1H NMR spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic and static light scattering. Drug loading studies were performed using a model hydrophobic drug, clofazimine, and a common anti-cancer drug paclitaxel (PTX) to evaluate the potential of the PVP-b-PVAc block copolymers for drug delivery,. Clofazimine and PTX were physically entrapped into the hydrophobic domain of the self-assembled PVP-b-PVAc block copolymers via the dialysis method. The drug-loaded PVP-b-PVAc block copolymers were characterized regarding particle size, morphology, stability and drug loading capacity in order to assess their feasibility as a drug vehicle. The polymer vesicles had a relatively high drug loading capacity of 20 wt %. The effect of the hydrophobic PVAc block length on the drug loading capacity and encapsulation efficiency were also studied. Drug loading increased with increasing the hydrophobic PVAc block length. The effect of the drug feed ratio of clofazimine and PTX on the drug loading capacity and encapsulation efficiency were also investigated. The optimal formulation for the drug-loaded PVP-b-PVAc was determined and further investigated in vitro. The size stability of the drugloaded PVP-b-PVAc block copolymers was also assessed under physiological conditions (PBS, pH 7.4, 37 °C) and were stable in the absence and presence of serum. PVP-b-PVAc block copolymers were tested in vitro on MDA-MB-231 multi-drug-resistant human breast epithelial cancer cells and normal MCF12A breast epithelial cells to provide evidence of their antitumor efficacy. In vitro cell culture studies revealed that the PVP-b-PVAc drug carrier exhibited no cytotoxicity towards MDA-MB-231 and MCF12A cells, confirming the biocompatibility of the PVP-b-PVAc carrier. In vitro cytotoxicity assays using clofazimine-PVPb- PVAc formulations showed that when MDA-MB-231 cells were exposed to the formulations, an enhanced therapeutic effect was observed compared to the free drug. Cellular internalization of the PVP-b-PVAc drug carrier was demonstrated by fluorescent labeling of the PVP-b-PVAc carrier. Fluorescence microscopy results showed that the carrier was internalized by the MDAMB- 231 cells after 3 hours and localized in the cytoplasm and the perinuclear region. Overall, it was demonstrated that PVP-b-PVAc block copolymers appear to be promising candidates for the delivery of hydrophobic anti-cancer drugs. / AFRIKAANSE OPSOMMING: Die studie is gebaseer op die gebruik van amfifieliese blokkopolimere van poli((Nvinielpirolidoon)- b-poli(vinielasetaat)) (PVP-b-PVAc) as potensiële geneesmiddeldraers. PVP-b-PVAc blokkopolimere van konstante hydrofiliese bloklengte en verskillende hydrofobiese bloklengte is voorberei via die RAFT/MADIX-proses. Blokkopolimere met vinielasetaat is vanaf poli(N-vinielpirolidoon) met ‘n xantaatendfunksie voorberei. In ‘n wateromgewing vorm die PVP-b-PVAc blokkopolimere vesikel strukture met ‘n hydrofobiese membraan en ‘n hydrofiliese mantel. Die fisies-chemiese eienskappe van die PVP-b-PVAc blokkopolimere is gekarakteriseerd met gebruik van KMR spektroskopie, fluoresent spektroskopie, transmissie elektronmikroskopie (TEM) en dinamiese en statiese lig verstrooiing. Die potensiaal van PVP-b-PVAc as ‘n geneesmiddeldraer is ondersoek deur gebruik te maak van die hydrofobiese geneesmiddel, clofazimine, en ‘n anti-kanker geneesmiddel, paclitaxel. Clofazimine en paclitaxel is ge-inkapsuleer in die hydrofobiese gedeelte van die blokkopolimere via die dialise-metode. Clofazimine-PVP-b-PVAc en paclitaxel-PVPb- PVAc blokkopolimere is gekarakteriseerd met betrekking tot die partikel grootte, morfologie, stabiliteit en laai kapasitiet om die PVP-b-PVAc blokkopolimere as geneesmiddeldraers te evalueer. Die PVP-b-PVAc geneesmiddeldraer het ‘n relatiewe hoë laai kapsiteit van 20 gew % aangetoon. Die invloed van die bloklengte op die laai kapasitiet is ook ondersoek en beskryf. ‘n Toename in die laai kapasitiet is gesien met ‘n toename in die hydrofobiese bloklengte. Die invloed van die hoeveelheid geneesmiddel op die laai kapasitiet en die inkapsuleer doeltreffendheid is ook ondersoek. Die optimale formulasie is gevind en verder gebruik vir in vitro studies. Die stabiliteit van die geneesmiddeldraer in fisiologiese omstandighede (pH 7.4, 37 °C) is ook beskryf. Resultate toon aan dat die sisteem stabiel is onder hierdie omstandighede in die afwesigheid en aanwesigheid van serum. In vitro eksperimente is op MCF12A epiteel-borsselle en MDA-MB-231 epiteelborskankerselle getoets om die anti-tumoraktiwiteit te ondersoek. Resultate toon aan dat die PVP-b-PVAc geen sitotoxiese effek op die selle het nie, wat aandui dat die polimere bioverenigbaar is. Verder is dit bewys dat die PVP-b-PVAc geneesmiddel formualsie ’n hoër sitotoxisiteit besit as die vry-geneesmiddel. Fluoresent studies het aangetoon dat die geneesmiddeldraer na 3 uur opgeneen word deur MDA-MB231 selle en gelokaliseerd is in die sitoplasma en in die omgewing van die kern van die selle. In die algemeen is dit aangetoon dat PVP-b-PVAc blokkopolimere potensiële kandidate vir die lewering van hydrofobiese geneesmiddels is.

Aliphatic polyesters such as polycaprolactone and polylactide have received more attention in recent years for their use in biomedical applications because of their biodegradable nature. These polymers are often synthesized using homogeneous metal complexes. Unfortunately, using homogeneous metals as catalysts leads to metal contamination in the product polymer, a result which is highly undesirable in a polymer intended for biomedical use. More recent work has shown that these polymers can be synthesized using homogeneous metal-free complexes. These catatlysts are generally less active than metal catalysts, and although they do not contaminate the polymer with metal residue, they are still difficult to recover and hence recycle for further use. In this work, we attempted to create a metal-free, silica-supported catalyst for use in the synthesis of polycaprolactone or polylactide. Ultimately, n-propylsulfonic acid-functionalized porous and nonporous silica materials are evaluated in the ring-opening polymerization of epsilon-caprolactone. All catalysts allow for the controlled polymerization of the monomer, producing polymers with controlled molecular weights and narrow polydispersities. Polymerization rates are low, with site-time-yields generally one to three orders of magnitude lower than metal-based systems. The catalysts are easily recovered from the polymerization solution after use and are shown to contain significant residual adsorbed polymer. Solvent extraction techniques are useful for removing most of the polymer, although the extracted solids are not effective catalysts in recycle experiments. These new materials represent a green alternative to traditional metal-based catalysts, as they are recoverable and leave no metal residues in the polymer.

Novel surgical strategies for spinal disc repair are currently being developed that require materials that (1) possess the appropriate mechanical properties to mimic the tissue the material is replacing or repairing and (2) maintain their mechanical function for long durations without negatively affecting the tissue response of adjacent tissue (i.e. bone). Polymers formed through photopolymerization have emerged as candidate biomaterials for many biomedical applications, but these materials possess limited toughness in vivo due to the presence of water inherent in most tissues. Therefore, the overall objective of this research was to develop photopolymerizable (meth)acrylate networks that are both mechanically and biologically compatible under physiological conditions to be implemented in spinal repair procedures. The fundamental approach was to determine structure-property relationships between toughness and network structure in the presence of phosphate buffered saline (PBS) using several model copolymer networks in order to facilitate the design of photopolymerizable networks that are tough in physiological solution. It was demonstrated that networks toughness could be optimized in PBS by tailoring the Tg of the copolymer network close to body temperature and incorporating the appropriate "tough" chemical structures. The ability to maintain toughness up to 9 months in PBS was dependent upon the viscoelastic state and overall hydrophobicity of the network. In tandem, the effect of network chemistry and stiffness on the response of MG63 pre-osteoblast cells was assessed in vitro. The ability of MG63 cells to differentiate on (meth)acrylate network surfaces was found to be primarily dependent on surface chemistry with PEG-based materials promoting a more mature osteoblast phenotype than 2HEMA surfaces. Amongst each copolymer group, copolymer stiffness was found to regulate osteoblast differentiation in a manner dependent upon the surface chemistry. In general, photopolymerizable (meth)acrylate networks that were deemed "tough" were able to promote osteoblast differentiation in a manner comparable if not exceeding that on tissue culture polystyrene (TCPS). This research will impact the field of biomaterials by elucidating the interrelationships between materials science, mechanics, and biology.

In this study, we introduce novel, truly non-ionic hydrogen-bonded layer-by-layer (LbL) coatings for cell surface engineering capable of long-term support of cell function. Utilizing the LbL technique imparts the ability to tailor membrane permeability, which is of particular importance for encapsulation of living cells as cell viability critically depends on the diffusion of nutrients through the artificial polymer membrane. Ultrathin, permeable polymer membranes are constructed on living cells without a cationic pre-layer, which is usually employed to increase the stability of LbL coatings. In the absence of the cytotoxic PEI pre-layer, viability of encapsulated cells drastically increases to 94%, as compared to 20-50% in electrostatically-bonded shells. Engineering surfaces of living cells with natural or synthetic compounds can mediate intercellular communication, render the cells less sensitive to environmental changes, and provide a protective barrier from hostile agents. Surface engineered cells show great potential for biomedical applications, including biomimetics, biosensing, enhancing biocompatibility of implantable materials, and may represent an important step toward construction of an artificial cell.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Alternative materials have long been studied and developed to replace conventional skin dressings due to the emergence of new biopolymers and development of new polymeric film fabrication techniques. As a new material for polymeric dressings, films of poly (hydroxybutyrate) (PHB) blended with esterified alginate (ALG-e) and Poly (ethyleneglycol) were studied. The esterification of sodium alginate (ALG-e) generated a material with some amphiphilic characteristics and increased its compatibility with the PHB. PEG was added as plasticizer in PHB/ALG-e films, since PEG is often used in blends with PHB to improve its flexibility and reduce its hydrophobicity. At the amounts studied, it was found that both PEG and ALG-e increase the degree of crystallinity, but a decrease in the hydrophobic nature of PHB films was observed. At the maximum concentration of ALG-e and PEG used an increase in water vapor permeability and a decrease in tensile strength was reached due to the synergistic effect caused by better homogenization of PEG and ALG-e in the PHB matrix. / Materiais alternativos têm sido estudados e desenvolvidos para substituir curativos de pele convencionais devido ao surgimento de novos biopolímeros e o desenvolvimento de novas técnicas de fabricação de filmes poliméricos. Como um novo material para curativos poliméricos, filmes de poli (hidroxibutirato) (PHB) misturado com alginato esterificado (ALG-e) e poli (etilenoglicol) foram estudados. A esterificação de alginato de sódio (ALG-e) gerou um material com característica anfifílica e com uma compatibilidade maior com o PHB. PEG foi adicionado como plastificante ao sistema PHB/ALG-e, uma vez que PEG é muitas vezes utilizado em misturas com PHB para melhorar a flexibilidade e reduzir a hidrofobicidade. Nas quantidades estudadas, verificou-se que tanto PEG como ALG-e aumentaram o grau de cristalinidade, o entanto foi observada uma redução na natureza hidrofóbica dos filmes de PHB. Nos filmes com concentrações máximas de ALG-e e PEG um aumento na permeabilidade ao vapor de água e uma diminuição na resistência à tração foram alcançadas devido ao efeito sinérgico causado por uma melhor homogeneização de PEG e ALG-e na matriz de PHB.

Acute liver failure is a major cause of death in the world, and effective treatments are greatly needed. Liver macrophages (Kupffer cells) play a major role in the pathology of acute liver failure, and drug delivery vehicles that can target therapeutics to Kupffer cells have great therapeutic potential for treating acute liver failure. Microparticles, formulated from biodegradable polymers, are advantageous for treating acute liver failure because they can passively target therapeutics to Kupffer cells. However, existing biomaterials are not suitable for the treatment of acute liver failure because of their slow hydrolysis and acidic degradation products. In this dissertation, I present the development of a new class of biodegradable materials, termed aliphatic polyketals, which have considerable potential as drug delivery vehicles for the treatment of acute liver failure because of their neutral degradation products and tunable hydrolysis kinetics. The anti-inflammatory enzyme, superoxide dismutase (SOD), was delivered using polyketal microparticles to the liver for treating acute liver Failure. Our results demonstrated that polyketal microparticles significantly improved the efficacy of SOD in treating LPS-induced acute liver damage in vivo, as evidenced by decreased levels of serum alanine transaminase, which corresponds to the extent of damage in the liver, and serum level of tumor necrosis factor-alpha, which corresponds to the secretion of pro-inflammatory cytokines. The completion of this thesis research demonstrates the ability of polyketal-based drug delivery systems for treating acute inflammatory diseases and creates a potential therapy for enhancing the treatment of acute liver failure.

Amyotrophic lateral sclerosis (ALS) is a devastating disease. Currently, there is no cure for this disease, and effective treatment strategies are greatly needed. Calpain activation plays a major role in the motor neuron degeneration that causes ALS. Therefore, therapeutic strategies can inhibit calpain activity in the central nervous system (CNS) have great clinical potential. The calpain inhibitors AK295 and MDL-28170 have been demonstrated to be neuroprotective in animal models of neurological injury, and should have great potential to treat ALS; however delivery problems have hindered their clinical success. Therefore, development of a new strategy that can locally deliver the calpain inhibitors to the central nervous system could significantly improve the treatment of ALS. The objectives of my thesis research were (1) to develop high molecular weight polyketals that provide sustained release properties for hydrophobic molecules, (2) to formulate calpain inhibitor-encapsulated polyketal microparticles which have a release half life of one month in vitro, (3) and to evaluate the performance of polyketal microparticles for delivering calpain inhibitors to the spinal cord in vivo. In completing these specific aims, we have developed biodegradable polymeric microparticles for the delivery of calpain inhibitors, AK295 and MDL-28170 to treat ALS. The results of calpain assays showed that both AK-PKMs and MDL-PKMs maintained most of their inhibitory activities even after the robust emulsion process. The in vitro release profile of MDL-28170 in MDL-PKMs showed that 50 % of the drug was released in the first 30 days. Experiments using dye-encapsulated microparticles showed that polyketal microparticles (1-2 ìm) are not easily cleared in the neutral physiological environment and can have potential to continuously release drug from the injection sites in the spinal cord. The efficacy of calpain inhibitor-encapsulated PKMs were studied by evaluation the behavior and survival of SOD1G93A rats, a genetic rat model for ALS. We observed the trend toward improvements in grip strength and rotarod performance in the first two months from the AK-PKMs treated group, however, further improvements are needed to enhance their in vivo efficacy.

Biomimetic materials that recapitulate the complex mechanical and biochemical cues in load-bearing tissues are of significant interest in regenerative medicine and tissue engineering applications. Several investigators have endeavored to not only emulate the mechanical properties of the vasculature, but to also mimic the biologic responsiveness of the blood vessel in creating vascular substitutes. Previous studies in our lab generated the elastin-like protein polymer LysB10, which was designed with the capability of physical and chemical crosslinks, and was shown to display a range of elastomeric properties that more closely matched those of the native artery. While extensive validation of the mechanical properties of elastin-mimetic polymers has demonstrated their functionality in a number of tissue engineering applications, limited cell growth on the surfaces of the polymers has motivated further optimization for biological interaction. Recent biologically-inspired surface strategies have focused on functionalizing material surfaces with extracellular matrix molecules and bioactive motifs in order to encourage integrin-mediated cellular responses that trigger precise intracellular signaling processes, while limiting nonspecific biomaterial interactions. Consequently, this dissertation addresses three approaches to modulating cellular behavior on elastin-mimetic analogs with the goal of promoting vascular wall healing and tissue regeneration: genetic engineering of elastin-like protein polymers (ELPs) with cell-binding domains, biofunctionalization of elastin-like protein polymers via chemoselective ligation of bioactive ligands, and incorporation of matrix protein fibronectin for engineering of cell-seeded multilamellar collagen-reinforced elastin-like constructs. The synthesis of recombinant elastin-like protein polymers that integrate biologic functions of the extracellular matrix provides a novel design strategy for generating clinically durable vascular substitutes. Ultimately, the synthesis of model protein networks provides new insights into the relationship between molecular architecture, biomimetic ligand presentation, and associated cellular responses at the cell-material interface. Understanding how each of these design parameters affects cell response will contribute significantly to the rational engineering of bioactive materials. Potential applications for polymer blends with enhanced mechanical and biological properties include surface coatings on vascular grafts and stents, as well as composite materials for tissue engineered scaffolds and vascular substitutes.

Abdominal aortic aneurysms are a leading cause of death in the U.S. where 14,000 people die from aneurysm rupture and 178,000 are diagnosed each year. A novel alternative treatment for abdominal aortic aneurysms has been proposed, where a biodegradable polymer scaffold is photopolymerized in situ around the exterior of the aneurysm. This scaffold will mechanically constrain the aneurysm from further expansion, and will deliver a drug, doxycycline, to treat the underlying biological cause of the disease. In order for device development, a suitable polymer must be designed with appropriate mechanical properties, degradation rate, polymerization, and elution rate. Poly(β-amino ester) networks have been proposed as the material of choice; however, many of their structure-property relationships have yet to be determined. Therefore, the overall goal of this work is to determine the structure-property relationships of the poly(β-amino ester) networks in order to advance the design of the treatment, and has been divided into three objectives: (1) understand the structure-property relationships of poly(β-amino ester) networks, specifically the polymerization, degradation rate, and thermo-mechanical properties, (2) determine the impact of doxycycline incorporation on degradation rate and mechanical properties, (3) evaluate the effect of simulated physiological conditions on degradation rate and mechanical properties. In the initial chapters, the fundamental structure-property relationships are established between reactant chemical structure, step-growth polymerization, photopolymerization, thermo-mechanical properties, and degradation rate using a systematic approach of two homologous series of reactants. Further tailoring of degradation rate, water content, and modulus in vitro was performed by using a copolymer network. Doxycycline inhibited photopolymerization due to overlapping absorbance spectra with the photoinitiator, but full network formation occurred by increasing the photoinitiator concentration. Networks displayed varying controlled release rates, and the underlying release mechanism was determined for each network using established methods. In order to increase mechanical properties, a co-monomer, methyl methacrylate, was added to the network to increase the glass transition temperature, toughness, and deformation capacity. These co-networks displayed temporal-control of mechanical properties in simulated physiological conditions, since degradation caused a shift in the glass transition temperature, which changed the mechanical behavior of the network. The temporal-control of mechanical properties was further investigated under degradation conditions in vitro and in vivo. Due to the mechanically active loading environment in vivo, networks displayed a decrease in toughness, yet maintained mechanical properties similar to native biological tissues. These networks establish a multifunctional biomaterials platform with materials that can be easily synthesized, photopolymerized into various geometries, and sustain mechanical properties while undergoing degradation and therapeutic agent release.