The research presented in this thesis focuses on the synthesis of three amphiphilic block copolymer systems containing poly(ethylene oxide) (PEO) blocks. The polymer systems were developed for use in biomedical applications. The first of these is a series of poly(ethylene oxide-b-oxazoline) (PEO-b-POX) diblock copolymers for use in the progress towards novel non-viral gene transfer vectors. Poly(ethylene oxide-b-2-ethyl-2-oxazoline) (PEO-b-PEOX) and poly(ethylene oxide-b-2-methyl-2-oxazoline) (PEO-b-PMOX) were investigated. The PEOX block was hydrolyzed with acid to form linear polyethylenimine (L-PEI). The polycation L-PEI is well known for its DNA binding efficiency but the water solubility of the resulting DNA/polymer complex is limited. Addition of a PEO block is directed towards the formation of a water dispersible DNA/copolymer complex. Dynamic light scattering of the PEO-b-PEOX and PEO-b-PEI block copolymers indicated that both systems existed as single chains in aqueous solution at pH 7. PEO copolymers also play a significant role in the formation of magnetic magnetite nanoparticles, which are dispersible in water at biological pH (pH =7). There is significant interest in the design of magnetic nanoparticle fluids for biomedical applications including magnetic field-directed drug delivery, magnetic cell separations, and blood purification. For use in vivo, the magnetite nanoparticles must be coated with biocompatible materials. Such polymers render the nanoparticles dispersible in water. Harris1 et al. synthesized PEO based, polyurethane triblocks with pendant carboxylic acid groups for use in formation of stable aqueous magnetic fluids. Building from this work, two polyurethane and polyurethaneurea systems were synthesized with 1300 g/mol PEOX and 2500 g/mol and PEOX2070 g/mol poly(ethylene oxide-co-propylene oxide) tailblocks, respectively. The PEO/PPO random copolymer contained about 25 weight percent PPO, and this disrupted the capacity of the PEO to crystallize. The PEOX based urethane triblocks were synthesized through reacting the tailblocks with the monomers for the center block whereas the PEO/PPO based polyurethaneurea was synthesized through forming the central urethane block with pendant acid groups first and then terminating the copolymer with the monofunctional copolymer. Terminal amine groups on the PEO/PPO tailblock afforded a triblock linked with two urea groups. The new polyurethanes with the PEOX tailblocks and the new polyurethaneurea with the PEO/PPO tailblocks could be utilized to efficiently stabilize magnetite nanoparticles in water. / Master of Science

Les complexes métalliques des carbènes N-hétérocycliques (NHC) présentent un grand potentiel comme anticancéreux. En particulier, des études in vitro ont confirmés une cytotoxicité supérieure au cisplatine. Dans ce travail, nous avons introduit de la diversité moléculaire à de nouveaux complexes NHC-Pt par coordination de différents ligands NHC. Une deuxième stratégie, la post-fonctionnalisation de complexes de Pt a été étudié par : a) formation d’oxime, notamment avec une urée ciblant le PSMA, b) échange de ligand avec des polyamines hydrosolubles (PEI) ou des pnictogènes (phosphines, arsines, stibines), c) échange d’halogène avec des isotopes de l’iode. Les propriétés cytotoxiques de ces composés ont été évaluées in vitro. In vivo (souris), un complexe PEI-Pt montre une inhibition tumorale similaire à l’oxaliplatine. Néanmoins, aucun effet secondaire n’a été détecté contrairement à l’oxaliplatine (hématomes). Ces résultats ouvrent de nouvelles perspectives dans le domaine des anticancéreux sur la base de platine. / Metal N-Heterocyclic Carbene (NHC) complexes are of great potential for cancer therapy. In particular, in vitro studies confirmed their significantly higher cytotoxicity than cisplatin. In this work, we introduced molecular diversity on new NHC-Pt complexes by coordination of various NHC precursors to platinum. As a second strategy, post-synthetic functionalization of Pt complexes has been fully investigated by: a) oxime formation, e.g. with a PSMA targeting urea derivative, b) ligand exchange reaction with hydrosoluble polyamines (PEI) and pnictogen-based ligands (phosphines, arsines, stibines), c) halogen exchange with iodide isotopesCytotoxic properties of these new compounds were evaluated in vitro. Best candidate was selected for in vivo evaluation on mice model showing for PEI-Pt similar tumour inhibition as oxaliplatin. Besides, no “visual” side effects were detected in contrast to oxaliplatin (hematomas). These outstanding results opened up new perspectives in the field of platinum-based drugs.

A terapia gênica por RNA de interferência (RNAi) trata-se de um processo de silenciamento pós-transcricional capaz de suprimir a expressão de um determinado gene. No entanto, para se tornar eficaz, a terapia gênica utilizando siRNA (small interfering RNA - siRNA) é dependente de uma eficiente liberação e expressão do acido nucléico nas células-alvo dentro dos tecidos ou órgãos. A literatura atual mostra algumas propostas de sistemas de liberação tópica de siRNA, entretanto, o uso de nanopartículas híbridas polimérico-lipídicas para complexação e liberação de siRNA na pele para o tratamento tópico de doenças cutâneas, ainda é pouco explorado, o que configura a inovação deste trabalho. Neste contexto, o presente trabalho tem como objetivo o desenvolvimento de uma nanopartícula hibrida lipídica associada a um polímero catiônico (NLS-PEI) como sistema de liberação tópica para siRNA, visando a terapia gênica como nova abordagem no tratamento de patologias cutâneas. Primeiramente a composição e o método de produção das NLS-PEI foram otimizados. Como polímero catiônico, utilizou-se a polietilenoimina (PEI). As NLS-PEIs otimizadas, compostas pelo lipídeo Compritol® 888 ATO (2,0%), Poloxamer 188 (1,5%) e PEI (0,15 e 0,25%), apresentaram-se esféricas com superfície lisa e um tamanho médio de 165 nm, potencial zeta médio de +20 mV, concentração de partículas de 1013/mL e com alta estabilidade (90 dias a 4°C). Foi demonstrado, por DSC, que a PEI interage tanto com o tensoativo na superficie das nanoparticulas como também com o lipídeo. A avaliação de comportamento e ação in vitro das NLS-PEIs foi estudada em queratinócitos e fibroblastos, evidenciando que o tratamento não diminuiu a viabilidade celular e houve alta captação verificada por citometria de fluxo (acima de 80% de células transfectadas) e por microscopia confocal. No estudo in vitro de penetração das NLS-PEI complexado com siRNA-FAM em pele de orelha de porco, foi demonstrado a penetração cutânea aumentada de siRNA-FAM a partir das NLS-PEIs. Ensaio in vivo em modelo de inflamação cutânea aguda e crônica mostrou a eficácia das NLS-PEIs contendo siTNF-?, reduzindo a espessura da epiderme em comparação ao grupo controle, reduzindo o rubor, a descamação e os níveis da citocina pró-inflamatória TNF-? e seu RNAm correspondente. A NLS-PEI também promoveu a penetração cutânea das moléculas de siRNA nas camadas mais profunda da pele, in vivo. Diante dos resultados obtidos, podese concluir que as formulações desenvolvidas são sistemas de liberação promissores para administração tópica de siRNA para o tratamento de patologias cutâneas na terapia gênica. / Small-interfering RNA (siRNA) has a high application potential for therapeutic silencing of pathologic or drug-resistance genes. Nonetheless, free siRNA molecules are known to have poor transfection efficiency and biological stability, so a carrier use is necessary. Lipid-polymeric nanocarriers are the new generation of nanoparticulate that have been used as siRNA carrier system and are attracting attention as novel colloidal drug carrier for topical use. In this context, the aim of this study was to develop of hybrid lipidpolyethylenimine nanocarriers (SLN-PEI) as topical delivery systems for siRNA. First, the SLN-PEI composition and production method were optimized. Polyethyleneimine (PEI) was used as cationic polymer. The optimized SLN-PEIs, composed of the lipid Compritol® 888 ATO (2.0%), Poloxamer 188 (1.5%) and PEI (0.15 and 0.25%), were spherical with a smooth surface and average size of 200 nm, mean zeta potential of +20 mV, mean number of particles per mL in the 1013 frame and remained stable for 90 days at 4 °C. It has been demonstrated by DSC that PEI interacts with both the surface surfactant of the nanoparticles and with the lipid. The evaluation SLN-PEI\'s behavior and in vitro action was studied in keratinocytes and fibroblasts, evidencing that the nanoparticles did not decrease cell viability and there was high cellular uptake evidenced by flow cytometry (above 80% of transfected cells) and by confocal microscopy. In the permeation in vitro study, it was demonstrated that SLN-PEI increased cutaneous penetration of siRNA-FAM. In vivo assay, in model of acute and chronic skin inflammation, demonstrated the efficacy of SLN-PEIs carrying siTNF-?, reducing epidermal thickness compared to control group, reducing redness, desquamation and levels of the pro-inflammatory cytokine TNF- ? and its corresponding mRNA. SLN-PEI also promoted penetration of the siRNA molecules in the deeper layers of the skin, in vivo. In view of the results obtained, we can conclude that the formulations developed are promising delivery systems for topical administration of siRNA for the treatment of cutaneous pathologies in gene therapy.

There exists a dire need for improved therapeutics to achieve predictable and effective bone regeneration. Non-viral gene therapy is a safe method that can efficiently transfect target cells, therefore is a promising approach to overcoming the drawbacks of protein delivery of growth factors. The goal of this study was to employ cost-effective biomaterials to deliver genetic materials (DNA or RNA) in a controlled manner in order to address the high cost issues, safety concerns, and lower transfection efficiencies that exist with protein and gene therapeutic approaches. To achieve our goal, we set several aims: 1) To assess the bone regeneration capacity of polyethylenimine (PEI)-chemically modified ribonucleic acid (cmRNA) (encoding bone morphogenetic protein-2 (BMP-2)) activated matrices, compared to PEI-plasmid DNA (BMP-2)-activated matrices. 2) To explore the osteogenic potential of cmRNA-encoding BMP-9, in comparison to cmRNA-encoding BMP-2. 3) To use collagen membranes as integral components of a guided bone regeneration protocol and to enhance the bioactivity of collagen membranes by incorporating plasmid DNA (pDNA) or cmRNA encoding bone morphogenetic protein-9 (BMP-9). 4) To test whether the delivery of pDNA encoding BMP-2 (pBMP-2) and fibroblast growth factor-2 (pFGF-2) together can synergistically promote bone repair in a leporine model of diabetes mellitus, a condition that is known to be detrimental to union. 5) To investigated whether there is a synergistic effect on bone regeneration following delivery of pBMP-2 and pFGF-2, insulin and/or vitamin D. These investigations together provided new insights regarding the appropriate treatment methods for patients with fractures. Here we develop and test a non-viral gene delivery system for bone regeneration in challenging animal models utilizing a scaffold carrying PEI-nucleic acid complexes. We utilized three kinds of pDNA encoding either BMP-2, BMP-9 or FGF-2 as well as two kinds of cmRNA encoding either BMP-2 or BMP-9 formulated into PEI complexes. The fabricated nanoplexes were assessed for their size, charge, in vitro cytotoxicity, and capacity to transfect human bone marrow stromal cells (BMSCs). The in vivo functional potency of different nanoplexes embedded in scaffolds was evaluated using a calvarial bone defect model in rats, diaphyseal long bone radial defects in a diabetic rabbit model and intramuscular implantation in a diabetic rat. The results indicate that our non-viral gene delivery system induced migration and differentiation of resident cells to enhance bone regeneration. Together these findings suggest that scaffolds loaded with non-viral vectors harboring cmRNA or pDNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

<p>The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems <i>in vitro</i> and after lung administration <i>in vivo</i> were established for the first time.</p><p>Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells <i>in vitro</i> and after lung administration <i>in vivo</i>. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for <i>in vivo</i> gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI <i>in vitro</i> and <i>in vivo</i>. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for <i>in vivo</i> gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.</p>

The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems in vitro and after lung administration in vivo were established for the first time. Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells in vitro and after lung administration in vivo. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for in vivo gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI in vitro and in vivo. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for in vivo gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.

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Electrochemical synthesis of straight, separable, cylindrical nanowires for use as cantilevered mechanical resonators is the main focus of this dissertation. These types of nanowires are significant for many applications, but particularly so for chip-based sensor arrays made for ultrasensitive mass detection. Directed-assembly of nanowire-based devices has enabled the development of large-area fabrication of sensor devices with new functions such as cancer detection at early stage. Chemically stable noble metals gold and rhodium are interesting materials for making nanowire resonators. Gold makes a well-known, stable and strong bond with the thiol group, which enables a range of surface functionalization chemistries. Rhodium nanowires have desirable mechanical properties for resonant mass sensing as they can retain high quality factor (Q-factor) from high vacuum to near atmospheric pressures. As a versatile and inexpensive tool, electrodeposition provides the most suitable synthesis path for gold and rhodium resonator-grade nanowires in nanoporous templates. In this work, the structural characteristics of nanoporous membranes anodized aluminium oxide and track-etched polycarbonate was explored for use as electrodeposition template. New chemistries for making gold and rhodium nanowires are introduced. Although gold cyanide-based solutions work well for the electrochemical synthesis of separable nanowires, the toxicity of cyanide solutions makes non-cyanide alternatives desirable. However, electrochemical synthesis of gold nanowires in templates from non-cyanide solutions suffers from serious drawbacks. These include growth-arresting pellet formation, poor length control and defects such as inclusions. In this dissertation, the first electrochemical synthesis of straight, cylindrical, separable gold nanowires from a sulfite-based solution is presented. This work demonstrates a scheme that suppresses electroless particle growth in the weakly-complexed gold in solution by proper use of additives. The electrochemical nucleation and growth of rhodium nanowires from a sulphate-based solution is also discussed. The effect of pH on the length uniformity as well as the effect of EDTA and polyethylenimine as additives on the development of the wire nanostructure was studied. This study has shown that the control over hydrogen co-reduction on the electrode surface and its bubble transport rate allowed for tailoring the nanostructure of the grown nanowires. The control over electrochemical nucleation and growth of noble metal films for nanowire clamping has also been investigated in this work for making reliable defect-free clamps for nanoresonator measurements. Silver was introduced as a reliable replacement for gold for nanowire clamping. Resonance measurements of rhodium nanowires clamped with silver, confirmed a reliable and repeatable clamp with very small scatter in the plot of resonance frequency variation with appropriate geometric terms. In addition, we found that the elastic modulus of a set of rhodium nanowires synthesized and measured in this work, was 14% larger than in previous studies. / Graduate / 0794 / 0548 / mascotella@gmail.com