A cerebral aneurysm is a weakened area of an artery in the brain, creating an abnormal expansion. Recent research for treatment is utilizing a photopolymerizable hydrogel as a possible operation for injection in situ. This paper aimed to achieve bioconjugation of peptides on a PEGDMA polymer network (using the photoinitiator PEG-BAPO) to form a biocompatible photopolymerizable hydrogel, without compromise to any of its mechanical attributes. Achieving cell adhesion to the hydrogel surface is a critical requirement as that could drive the growth of endothelium between aneurysm and artery, to considerably enhance its sustainability and decrease the risk of inflammation. The hydrogel was synthesized by functionalizing RGD with a PEG-spacer and co-polymerize it with PEGDMA using UV-radiation to create an intertwined cross-linking network. Samples of various peptide concentrations were studied in cell culture to analyze cell adhesion, followed by mechanical tests to identify possible deviations. A subsequent study was established to create a dynamic prototype as a quantifiable replication of a hydrogel inside an aneurysm in vivo. The model was designed in SolidWorks and connected with an Ibidi sticky-Slide to roughly replicate a cerebral aneurysm connected to an artery with space to introduce a hydrogel sample.

The aortic valve maintains unidirectional blood flow between the left ventricle and the systemic circulation. When diseased, the valve is replaced either by a mechanical or a bioprosthetic heart valve, that carry issues such as thrombogenesis, long term structural failure, and calcification, necessitating the development of more structurally and biologically sufficient long-term replacements. Tissue engineering provides a possible avenue for development, combining cells, scaffolds, and biochemical factors to regenerate tissue. The overall goal of this dissertation was to create a foundation for the rational design of a tissue engineered aortic valve. The novel approach taken in this thesis research was to view each of the three leaflets as a laminate structure. The first three aims consider the leaflet as a laminate structure comprising of layers of collagen, elastin, and glycosaminoglycans (GAGs). In the first aim, the effect of GAGs on the tensile properties and stress relaxation in the leaflet was investigated, by removing GAGs through increasing amounts of hyaluronidase. A decrease in GAGs led to significantly higher elastic moduli, maximum stresses, and hysteresis in the leaflet. In the second aim, the 3D elastic fiber network of the leaflet was characterized using immunohistochemistry and scanning electron microscopy. This structure was found to have regionally varying thicknesses and patterns. In the third aim, a novel hydrogel-fiber composite design was proposed to match the anisotropy of the leaflet. This composite composed of aligned electrospun poly(ε-caprolactone) (PCL) within a poly(ethylene glycol) diacrylate (PEGDA) matrix. Surface modification and embedding of the PCL did not significantly alter the anisotropy or strength of the underlying PCL scaffold, providing the basis for an anisotropic, biocompatible scaffold. In the last aim, a novel co-culture model was designed using magnetic levitation as a layered structure of valvular endothelial cells and interstitial cells. This technique was used to create co-culture models within hours, while maintaining cell phenotype and function, and inducing extracellular matrix formation, as shown by immunohistochemical stains and their gene expression profiling. The overall result of this dissertation is a clearer understanding of the layered structure-function relationship of the aortic valve, and its application towards heart valve tissue engineering.

Cellulose nanofibrils (CNFs) are nanoscale fibers of high aspect ratio that can be isolated from a wide variety of cellulosic sources, including wood and bacterial cellulose. With high strength despite of their low density, CNFs are a promising renewable building block for the preparation of nanostructured materials and composites. To fabricate CNF-based materials with improved inherent rheological and mechanical properties and additional new functionalities, it is essential to tailor the surface properties of individual CNFs. The surface structures control the interactions between CNFs and ultimately dictate the structure and macroscale properties of the bulk material. In this thesis we have demonstrated different approaches, ranging from non-covalent adsorption and covalent chemical modification to modification of cellulose biosynthesis, to tailor the structure and surface functionalities of CNFs for the fabrication of advanced materials. These materials possess enhanced properties such as water-redispersibility, water absorbency, dye adsorption capacity, antibacterial activity, and mechanical properties. In Paper I, CNFs were modified via the irreversible adsorption of carboxymethyl cellulose (CMC). The adsorption of small amounts of CMC onto the surface of CNFs prevented agglomeration and co-crystallization of the nanofibrils upon drying, and allowed the recovery of rheological and mechanical properties after redispersion of dried CNF samples. In Paper II, CNFs bearing permanent cationic charges were prepared through quaternization of wood pulp fibers followed by mechanical disintegration. The activation of the hydroxyl groups on pulp fibers by alkaline treatment was optimized prior to quaternization. This optimization resulted in individual CNFs with uniform width and tunable cationic charge densities. These cationic CNFs demonstrated ultrahigh water absorbency and high adsorption capacity for anionic dyes. In Paper III, via a similar approach as in Paper II, CNFs bearing polyethylene glycol (PEG) were prepared by covalently grafting PEG to carboxylated pulp fibers prior to mechanical disintegration. CNFs with a high surface chain density of PEG and a uniform width were oriented to produce macroscopic ribbons simply by mechanical stretching of the CNF hydrogel network before drying. The uniform grafted thin monolayer of PEG on the surface of individual CNFs prevented the agglomeration of CNFs and facilitated their alignment upon mechanical stretching, thus resulted in ribbons with ultrahigh tensile strength and modulus. These optically transparent ribbons also demonstrated interesting biaxial light scattering behavior. In Paper IV, bacterial cellulose (BC) was modified by the addition of chitin nanocrystals (ChNCs) into the growing culture medium of the bacteria Acetobacter aceti which secretes cellulose in the form of entangled nanofibers. This led to the in situ incorporation of ChNCs into the BC nanofibers network and resulted in BC/ChNC nanocomposites exhibiting bactericidal activity. Further, blending of BC nanofibers with ChNCs produced nanocomposite films with relatively lower tensile strength and modulus compared to the in situ cultivated ones. The bactericidal activity increased significantly with increasing amount of ChNCs for nanocomposites prepared by direct mixing of BC nanofibers and ChNCs. In Paper V, CNFs were isolated from suspension-cultured wild-type (WT) and cellulose-binding module (CBM) transformed tobacco BY-2 (Nicotiana tabacum L. cv bright yellow) cells. Results from strong sulfuric acid hydrolysis indicated that CNFs from transgenic cells overexpressing CBM consisted of longer cellulose nanocrystals compared to CNFs from WT cells. Nanopapers prepared from CNFs of transgenic cells demonstrated significantly enhanced toughness compared to CNFs of WT cells. / <p>QC 20141103</p> / CARBOMAT

Environmentally benign alternatives for solvents and catalysts are essential for the development of sustainable chemical processes. Toward this end, we focused our research on the design of novel solvents and catalysts that reduce the environmental impact of these important materials. In this research, we develop switchable and tunable systems that couple reaction and separation to ease the processing requirements for product isolation and catalyst recovery. The switchable solvents use a ¡°switch¡± to transition from non-volatile, polar, aprotic solvents to volatile gases that can be easily separated. This allows us to facilitate reactions within the solvent and then enable easy separation through activation of the switch. We have used these materials for numerous reaction applications, including difficult reactions involving highly immiscible compounds. We also extended the work to acid-catalyzed reactions, in which we can avoid wasteful neutralization processes that are often associated with homogeneous acid catalysis. The tunable solvents use carbon dioxide pressure to ¡°tune¡± into desired solvent properties. We enable this through the dissolution of carbon dioxide into organic solvents, which generates gas-expanded liquids with solvent properties highly dependent on the carbon dioxide pressure. We can use this effect to couple homogeneous reaction with heterogeneous separation, allowing for recovery of expensive catalysts and ligands. In this work, we assess the possibilities of using liquid polyethylene glycol in the tunable systems, studying the phase behavior and industrial applications.

Optical assays to detect cancer-associated molecular biomarkers in biological substrates are commonly performed with antibody-targeted organic dye contrast agents but the potential for precise quantification, long-term imaging, and multiplexed readouts is limited by chemical and optical instability, non-optimal spectral characteristics, and complicated synthetic chemistry of the dyes. This dissertation tested the hypothesis that a novel class of optical contrast agents termed polymer-protected Raman-encoded nanoparticle tags (PRENTs) provides practical advantages over existing optical technologies for molecular diagnostic applications. First, PRENTs were developed through a modular design utilizing gold-nanoparticle-Raman reporter complexes protected and functionalized by polyethylene glycol derivatives. PRENTs produced optical readouts through surface enhanced Raman scattering (SERS) that were brighter and more photostable than the fluorescence of semiconductor quantum dots under identical experimental conditions. Unique spectral signatures were produced with a broader class of Raman reporters than is possible with silica coated Raman tags. Spectral signatures and colloidal stability of PRENTs were unaffected by harsh chemical conditions that cause spectral changes and aggregation of dyes, quantum dots, and protein coated Raman tags. Antibody-targeted PRENTs specifically tagged cell surface cancer biomarkers on living cells at reasonable integration times. PRENTs were non-toxic to cells under conditions exceeding those required for sensitive molecular detection. Next, PRENTs were efficiently optimized for excitation with near-infrared light through inclusion of near-infrared chromophores as Raman reporters and exploitation of the size-dependent optical enhancement of gold nanoparticles. Third, the development of a slide-based Raman-linked immunosorbent assay using antibody-conjugated PRENTs enabled quantification of protein biomarkers with a dynamic range of 3 to 4 logs. In summary, this dissertation establishes PRENTs as novel optical tags with unique features useful for biomedical applications and provides insights for further assay development.

Quantum dots (QDs) are potentially advantageous tools for both diagnostics and therapeutics due to their light emitting characteristics. The impact of QDs on biological systems however, is not fully understood. The aim of this project therefore, was to investigate the interaction of a series of different surface modifies QDs with macrophages and their subsequent toxicity. CdTe/CdSe (core), ZnS (shell) QDs with either an organic, COOH or NH2 polyethylene glycol (PEG) surface coatings were used. Fluorescent COOH polystyrene beads (PBs) at (Ø) 20nm and 200nm were also studied. J774.A1 murine ‘macrophage-like' cells were treated for two hours with QDs (40nM) of PBs ($50μg.ml^{-1}$) in the presence of 10% FCS prior to assessment of cellular uptake via confocal microscopy and flow cytometry. COOH and $NH_{2}$ (PEG) QDs, as well as 20nm and 200nm PBs entered macrophages within 30 minutes, and were found to locate within endosomes, lysosomes and the mitochondria. T.E.M. also illustrated particles, including organic QDs, to be present inside J774.A1 cells within membrane- bound vesicles at two hours. Organic QDs were unable to be visualised via fixed cell confocal microscopy. Live cell confocal microscopy (without 10% FCS) did suggest however, that organic QDs entered cells in low quantities up to 30 minutes, after which fluorescence declined. Particle toxicity was determined over 48 hours via the MTT, LDH and GSH assays, as well as via assessment of their potential to produce the pro-inflammatory cytokine (TNF-α) and effect cytosolic $Ca^{2+}$ signalling in the J774.A1 cells. Organic QDs were found to be highly toxic at all time points and concentrations used. Both COOH and $NH_{2 }$ (PEG) QDs induced significant (p<0.0001) cytotoxicity (MTT and LDH assays) at 80nM after 48 hours, as well as significant (p<0.01) GSH depletion over 24 hours at all doses, as well as increasing the level of cytosolic $Ca^{2+}$ at 40nM when assessed over 30 minutes. Organic and NH2 (PEG) QDs were found to significantly increase TNF-α production after 24 hours at 80nM. The findings of this study demonstrate that QDs differ in their uptake by macrophages according to their surface coating, with the organic surface coated QDs being the most toxic. At sub-lethal concentrations, in the presence of 10% FCS, the COOH and $NH_{2}$ (PEG) QDs are taken up resulting in GSH depletion and modulated $Ca^{2+}$ signalling, with $NH_{2}$ (PEG) QDs and organic QDs only eliciting limited TNF-α production. Interestingly however, despite these observations, QD surface coating does not affect the intracellular fate of these NPs, with all of the different surface coated QDs observed to be present in endosomes, lysosomes and the mitochondria within J774.A1 macrophage cells. Therefore, in conclusion, the surface coating of QDs plays a significant role in their interaction with macrophages, their uptake and their subsequent toxicity.

A epilepsia é normalmente a associação de pré-disposição genética e doença ou uma lesão cerebral. Aproximadamente 1 entre 50 a 100 pessoas apresentam essa pré-disposição à convulsões. Um dos fármacos mais prescritos e utilizados para o tratamento de convulsões é o ácido valpróico (AV), tornando-se a medicação de primeira escolha no tratamento da epilepsia infantil por apresentar um amplo espectro de ação, embora apresente efeitos colaterais bastante conhecidos como pancreatite e a hepatotoxicidade, que pode ser fatal. Sistemas nanoparticulados como nanocápsulas, obtidas a partir da utilização de polímeros biodegradáveis como o polietilenoglicol (PEG) e macromoléculas naturais como a quitosana (QS), que proporcionam hidrofilia e bioadesividade, têm sido estudadas com o objetivo de aumentar a penetração cerebral e reduzir a dosagem do fármaco. Nesse contexto, o presente trabalho teve como objetivo desenvolver e caracterizar físico-quimicamente nanocápsulas de poli(ε-caprolactona) contendo AV e revestidas com QS (NCQ) e/ou com PEG 6000 (NCP e NCQP), investigar a farmacocinética plasmática, a penetração do AV através da barreira hematoencefálica (BHE) por microdiálise e a hepatotoxicidade em ratos Wistar. Nanocápsulas revestidas com QS (NCQ) foram obtidas pelo método de nanoprecipitação do polímero pré-formado seguido do revestimento com adição de 5 mL de solução de QS a 1%. Para a preparação de NCP, foi utilizada metodologia similar adicionando 0,7% de PEG 6000 na fase aquosa. Para a preparação de NCQP, as nanocápsulas preparadas com a adição de 0,35% de PEG 6000 e revestidas posteriormente com solução de QS a 1% (2,5 mL). As formulações foram caracterizadas físico-quimicamente avaliando-se o tamanho das partículas, potencial zeta, pH e taxa de incorporação. As nanocápsulas foram visualizadas por MET e a estabilidade foi investigada por retroespalhamento de luz (Turbiscan Lab®). As formulações (AV 5 mg/mL) apresentaram um pequeno tamanho de partícula (144,2 ± 2,0 nm, 153,2 ± 1,8 nm, e 231,3 ± 15,6 nm, para NCQ, NCP e NCQP, respectivamente), com baixo índice de polidispersão, alta taxa de incorporação (95 a 98 %), pH ácido, potencial zeta positivo para NCQ (+8,7 ± 0,4 mV) e negativo para NCP (- 6,6 ± 0,8 mV) e NCQP (- 2,8 ± 1,3 mV). As fotomicrografias mostraram partículas de forma esférica e as formulações demonstraram boa estabilidade durante 24h de análise a 40°C. As concentrações plasmáticas foram investigadas em ratos Wistar (15 mg/kg via i.v. de AV) para todas as formulações e valproato sódico (grupo controle). A análise farmacocinética compartimental apresentou uma distribuição muito rápida para o AV em NCQ e a ASC0-∞ aproximadamente duas vezes menor em comparação à NCP, NCQP e o fármaco livre (3874 ± 1775; 8280 ± 2136; 7849 ± 1021 e 7978 ± 3622 μg/mL/min, respectivamente). O clearance do AV aumentou significativamente para NCQ (0,284 ± 0,156 L/h/kg) (α = 0,05%). A penetração do AV através da BHE foi realizada em ratos Wistar acordados por microdiálise (MD) cerebral, no córtex frontal utilizando sondas CMA/12 (3 mm). Os experimentos de MD mostraram um aumento de 5 vezes no fator de penetração cerebral após a administração de NCQ em comparação com o fármaco em solução (0,110 and 0,021, respectivamente), demonstrando a viabilidade da utilização de QS como polímero de revestimento objetivando a BHE. A NCP demonstrou um aumento de 1,7 vezes no fator de penetração cerebral e NCQP não demonstrou qualquer diferença na penetração. A investigação da hepatotoxicidade do AV foi realizada após cinco dias de tratamento (dose de 30 mg/kg q12h de AV) em solução ou em nanocápsulas (NCQ ou NCP) com grupo controle de solução salina. Os níveis séricos de asparto aminotransferase (AST), alanina aminotransferase (ALT), gama-glutamiltransferase (GGT), fosfatase alcalina (FAL), creatinina (CRE) e uréia foram determinados. Os resultados mostram a manutenção dos níveis normais de enzimas hepáticas como a ALT e FAL para NCQ (54,2 ± 11,2 UI/mL and 149 ± 26 UI/mL) demonstrando um efeito hepatoprotetor não observado para os outros grupos. Análises histológicas do fígado dos animais não apresentaram a formação de esteatose microvesicular para NCQ em comparação com a formação de esteatoses em todos os outros grupos, incluindo o grupo controle. Ao final, os resultados indicaram que NCQ possa ser uma formulação em potencial, necessitando ser investigada pelo aumento da penetração cerebral de AV e efeito hepatoprotetor observados. / Epilepsy is usually a combination of genetic pre-disposition and a disease or a brain damage. About 1 in 50 to 100 people has this genetic predisposition to seizures. One of the world’s most prescribed drugs to treat epileptic seizures is valproic acid (VA), which is the first choice drug to treat epilepsy in childhood due to its broad spectrum of action, although its well known side effects such as pancreatitis, hepatotoxicity can be fatal. Nanoparticulated systems such as nanocapsules, obtained from biodegradable polymers like polyethylene glycol and natural macromolecules like chitosan, who gives the system hidrophilicity and bioadhesivity, have been used to increase brain penetration and reduce drug doses. In this context, the present work aimed to develop and physicochemically characterize poly(ε-caprolactone) nanocapsules loaded with VA and coated with chitosan (NCQ) and/or polyethylene glycol (PEG) 6000 (NCP and NCQP), and to investigate their plasma pharmacokinetics, VA blood-brain barrier penetration (BBB) by microdialysis and hepatotoxicity in Wistar rats. Nanocapsules coated with chitosan were obtained by nanoprecipitation of preformed polymer followed by coating with 1% chitosan solution added prior to final adjustments at a volume of 5 mL. For NCP preparation, similar methodology was used adding 0.7% PEG 6000 in the aqueous phase. For NCQP preparation, the nanocapsules prepared with PEG 6000 (0.35% w/v) was further coated with chitosan 1% in solution adding 2.5 mL prior to the final adjustments. The formulations were physicochemical characterized by particle size, zeta potential, pH, incorporation efficiency. The particles were visualized by MET and the stability investigated by backscattering (Turbiscan Lab®). The formulations (VA 5 mg/mL) presented small particle sizes (144.2 ± 2.0 nm, 153.2 ± 1.8 nm, and 231.3 ± 15.6 nm, for NCQ, NCP and NCQP, respectively), with low polidispersion index, high incorporation efficiency (95 to 98 %) and acid pH. The zeta potential was positive for NCQ (+8.7 ± 0.4 mV) and negative for NCP (- 6.6 ± 0.8 mV) and NCQP (- 2.8 ± 1.3 mV). The photomicrography of all formulations showed spherically shaped particles. The formulations showed good stability during 24 hours investigation at 40 ºC. Plasma concentrations were investigated in Wisar rats after 15 mg/kg i.v. dosing of all formulations and sodium valproate solution (control group). The pharmacokinetic compartmental analysis showed a very rapid distribution of VA when incorporated in NCQ in comparison to the other formulations and the AUC0-∞ about two times lower in comparison to NCP, NCQP and the drug alone (3874 ± 1775; 8280 ±2136; 7849 ± 1021 and 7978 ± 3622 μg/mL/min, respectively). VA clearance was significantly increased after NCQ dosing (0.284 ± 0.156 L/h/kg) (α = 0.05 %). Drug penetration through BBB was performed in awaken Wistar rats by brain microdialysis at the frontal cortex using CMA/12 probes (3 mm). The microdialysis experiments showed a five times increase in VA brain penetration factor after NCQ administration in comparison to drug alone (0.110 and 0.021, respectively), demonstrating the viability of chitosan as coating polymer to aim the BBB. NCP showed only a 1.7 times increase in brain penetration factor and NCQP did not showed any difference in comparison to drug alone. The investigation of drug hepatotoxicity was conducted after 5 days i.v. dosing of VA 30 mg/kg q12h as solution or nanocapsules (NCQ or NCP). A saline control group was also investigated. Serum levels of asparte aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosfatase, creatinin and urea were determined. The results showed the maintenance of normal levels of hepatic enzymes such as alanine aminotransferase and alkaline phosfatase for NCQ (54.2 ± 11.2 IU/mL and 149 ± 26 IU/mL) showing a hepatoprotective effect not observed in the other groups investigated. Histological analysis of animals livers showed no microvesicular steatosis formation when NCQ was administered in comparison with the formation of steatosis in all other groups including control. Overall the results indicate that NCQ is a potential formulation to be investigated for the treatment of epilepsy due to its increase in VA brain penetration and hepatoprotective effect observed.

Biodesulfurization (BDS) is an alternative to hydrodesulfurization (HDS) as a method to remove sulfur from crude oil. Dibenzothiophene (DBT) was chosen as a model compound for the forms of thiophenic sulfur found in fossil fuels; up to 70% of the sulfur in petroleum is found as DBT and substituted DBTs; these compounds are however particularly recalcitrant to hydrodesulfurization, the current standard industrial method. My thesis deals with enhancing BDS through novel strains and through nanotechnology. Chapter highlights are: Chapter 2. My first aim was to isolate novel aerobic, mesophilic bacteria that can grow in mineral media at neutral pH value with DBT as the sole sulfur source. Different natural sites in Iran were sampled and I enriched, isolated and purified such bacteria. Twenty four isolates were obtained that could utilize sulfur compounds. Five of them were shown to convert DBT into HBP. After preliminary characterization, the five isolates were sent to the Durmishidze Institute of Biotechnology in Tbilisi for help with strain identification. Two isolates (F2 and F4) were identified as Pseudomonas strains, F1 was a Flavobacterium and F3 belonged to the strain of Rhodococcus. The definite identification of isolate F5 was not successful but with high probability it was a known strain. Since no new strains were apparently discovered, I did not work further in this direction. Chapter 3. In a second approach I studied the desulfurization ability of Shewanella putrefaciens strain NCIMB 8768, because in a previous investigation carried out at Cranfield University, it had been found that it reduced sulfur odour in clay. I compared its biodesulfurization activity profile with that of the widely studied Rhodococcus erythropolis strain IGTS8. However, S. putrefaciens was not as good as R. erythropolis. Chapter 4 and 5. I then turned to nanotechnology, which as a revolutionary new technological platform offers hope to solve many problems. There is currently a trend toward the increasing use of nanotechnology in industry because of its potentially revolutionary paths to innovation. I then asked how nanotechnology can contribute to enhancing the presently poor efficiency of biodesulfurization. Perhaps the most problematic difficulty is how to separate the microorganisms at the end of the desulfurization process. To make BDS more amenable, I explored the use of nanotechnology to magnetize biodesulfurizing bacteria. In other words, to render desulfurizing bacteria magnetic, I made them magnetic by decorating their outer surfaces with magnetic nanoparticles, allowing them to be separated using an external magnet. I used the best known desulfurizing bacterial strain, Rhodococcus erythropolis IGTS8. The decoration and magnetic separation worked very well. Unexpectedly, I found that the decorated cells had a 56% higher desulfurization activity compared to the nondecorated cells. I proposed that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product respectively. Supporting evidence for enhanced permeabilization was obtained by Dr Pavel Grigoriev, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino. In Chapter 6, to optimize attachment of the nanoparticles to the surface of the bacteria I created thin magnetic nanofilms from the nanoparticles and measured the attachment of the bacteria using a uniquely powerful noninvasive optical technique (Optical Waveguide Lightmode Spectroscopy, OWLS) to quantify the attachment and determine how the liquid medium and other factors influence the process.

A epilepsia é normalmente a associação de pré-disposição genética e doença ou uma lesão cerebral. Aproximadamente 1 entre 50 a 100 pessoas apresentam essa pré-disposição à convulsões. Um dos fármacos mais prescritos e utilizados para o tratamento de convulsões é o ácido valpróico (AV), tornando-se a medicação de primeira escolha no tratamento da epilepsia infantil por apresentar um amplo espectro de ação, embora apresente efeitos colaterais bastante conhecidos como pancreatite e a hepatotoxicidade, que pode ser fatal. Sistemas nanoparticulados como nanocápsulas, obtidas a partir da utilização de polímeros biodegradáveis como o polietilenoglicol (PEG) e macromoléculas naturais como a quitosana (QS), que proporcionam hidrofilia e bioadesividade, têm sido estudadas com o objetivo de aumentar a penetração cerebral e reduzir a dosagem do fármaco. Nesse contexto, o presente trabalho teve como objetivo desenvolver e caracterizar físico-quimicamente nanocápsulas de poli(ε-caprolactona) contendo AV e revestidas com QS (NCQ) e/ou com PEG 6000 (NCP e NCQP), investigar a farmacocinética plasmática, a penetração do AV através da barreira hematoencefálica (BHE) por microdiálise e a hepatotoxicidade em ratos Wistar. Nanocápsulas revestidas com QS (NCQ) foram obtidas pelo método de nanoprecipitação do polímero pré-formado seguido do revestimento com adição de 5 mL de solução de QS a 1%. Para a preparação de NCP, foi utilizada metodologia similar adicionando 0,7% de PEG 6000 na fase aquosa. Para a preparação de NCQP, as nanocápsulas preparadas com a adição de 0,35% de PEG 6000 e revestidas posteriormente com solução de QS a 1% (2,5 mL). As formulações foram caracterizadas físico-quimicamente avaliando-se o tamanho das partículas, potencial zeta, pH e taxa de incorporação. As nanocápsulas foram visualizadas por MET e a estabilidade foi investigada por retroespalhamento de luz (Turbiscan Lab®). As formulações (AV 5 mg/mL) apresentaram um pequeno tamanho de partícula (144,2 ± 2,0 nm, 153,2 ± 1,8 nm, e 231,3 ± 15,6 nm, para NCQ, NCP e NCQP, respectivamente), com baixo índice de polidispersão, alta taxa de incorporação (95 a 98 %), pH ácido, potencial zeta positivo para NCQ (+8,7 ± 0,4 mV) e negativo para NCP (- 6,6 ± 0,8 mV) e NCQP (- 2,8 ± 1,3 mV). As fotomicrografias mostraram partículas de forma esférica e as formulações demonstraram boa estabilidade durante 24h de análise a 40°C. As concentrações plasmáticas foram investigadas em ratos Wistar (15 mg/kg via i.v. de AV) para todas as formulações e valproato sódico (grupo controle). A análise farmacocinética compartimental apresentou uma distribuição muito rápida para o AV em NCQ e a ASC0-∞ aproximadamente duas vezes menor em comparação à NCP, NCQP e o fármaco livre (3874 ± 1775; 8280 ± 2136; 7849 ± 1021 e 7978 ± 3622 μg/mL/min, respectivamente). O clearance do AV aumentou significativamente para NCQ (0,284 ± 0,156 L/h/kg) (α = 0,05%). A penetração do AV através da BHE foi realizada em ratos Wistar acordados por microdiálise (MD) cerebral, no córtex frontal utilizando sondas CMA/12 (3 mm). Os experimentos de MD mostraram um aumento de 5 vezes no fator de penetração cerebral após a administração de NCQ em comparação com o fármaco em solução (0,110 and 0,021, respectivamente), demonstrando a viabilidade da utilização de QS como polímero de revestimento objetivando a BHE. A NCP demonstrou um aumento de 1,7 vezes no fator de penetração cerebral e NCQP não demonstrou qualquer diferença na penetração. A investigação da hepatotoxicidade do AV foi realizada após cinco dias de tratamento (dose de 30 mg/kg q12h de AV) em solução ou em nanocápsulas (NCQ ou NCP) com grupo controle de solução salina. Os níveis séricos de asparto aminotransferase (AST), alanina aminotransferase (ALT), gama-glutamiltransferase (GGT), fosfatase alcalina (FAL), creatinina (CRE) e uréia foram determinados. Os resultados mostram a manutenção dos níveis normais de enzimas hepáticas como a ALT e FAL para NCQ (54,2 ± 11,2 UI/mL and 149 ± 26 UI/mL) demonstrando um efeito hepatoprotetor não observado para os outros grupos. Análises histológicas do fígado dos animais não apresentaram a formação de esteatose microvesicular para NCQ em comparação com a formação de esteatoses em todos os outros grupos, incluindo o grupo controle. Ao final, os resultados indicaram que NCQ possa ser uma formulação em potencial, necessitando ser investigada pelo aumento da penetração cerebral de AV e efeito hepatoprotetor observados. / Epilepsy is usually a combination of genetic pre-disposition and a disease or a brain damage. About 1 in 50 to 100 people has this genetic predisposition to seizures. One of the world’s most prescribed drugs to treat epileptic seizures is valproic acid (VA), which is the first choice drug to treat epilepsy in childhood due to its broad spectrum of action, although its well known side effects such as pancreatitis, hepatotoxicity can be fatal. Nanoparticulated systems such as nanocapsules, obtained from biodegradable polymers like polyethylene glycol and natural macromolecules like chitosan, who gives the system hidrophilicity and bioadhesivity, have been used to increase brain penetration and reduce drug doses. In this context, the present work aimed to develop and physicochemically characterize poly(ε-caprolactone) nanocapsules loaded with VA and coated with chitosan (NCQ) and/or polyethylene glycol (PEG) 6000 (NCP and NCQP), and to investigate their plasma pharmacokinetics, VA blood-brain barrier penetration (BBB) by microdialysis and hepatotoxicity in Wistar rats. Nanocapsules coated with chitosan were obtained by nanoprecipitation of preformed polymer followed by coating with 1% chitosan solution added prior to final adjustments at a volume of 5 mL. For NCP preparation, similar methodology was used adding 0.7% PEG 6000 in the aqueous phase. For NCQP preparation, the nanocapsules prepared with PEG 6000 (0.35% w/v) was further coated with chitosan 1% in solution adding 2.5 mL prior to the final adjustments. The formulations were physicochemical characterized by particle size, zeta potential, pH, incorporation efficiency. The particles were visualized by MET and the stability investigated by backscattering (Turbiscan Lab®). The formulations (VA 5 mg/mL) presented small particle sizes (144.2 ± 2.0 nm, 153.2 ± 1.8 nm, and 231.3 ± 15.6 nm, for NCQ, NCP and NCQP, respectively), with low polidispersion index, high incorporation efficiency (95 to 98 %) and acid pH. The zeta potential was positive for NCQ (+8.7 ± 0.4 mV) and negative for NCP (- 6.6 ± 0.8 mV) and NCQP (- 2.8 ± 1.3 mV). The photomicrography of all formulations showed spherically shaped particles. The formulations showed good stability during 24 hours investigation at 40 ºC. Plasma concentrations were investigated in Wisar rats after 15 mg/kg i.v. dosing of all formulations and sodium valproate solution (control group). The pharmacokinetic compartmental analysis showed a very rapid distribution of VA when incorporated in NCQ in comparison to the other formulations and the AUC0-∞ about two times lower in comparison to NCP, NCQP and the drug alone (3874 ± 1775; 8280 ±2136; 7849 ± 1021 and 7978 ± 3622 μg/mL/min, respectively). VA clearance was significantly increased after NCQ dosing (0.284 ± 0.156 L/h/kg) (α = 0.05 %). Drug penetration through BBB was performed in awaken Wistar rats by brain microdialysis at the frontal cortex using CMA/12 probes (3 mm). The microdialysis experiments showed a five times increase in VA brain penetration factor after NCQ administration in comparison to drug alone (0.110 and 0.021, respectively), demonstrating the viability of chitosan as coating polymer to aim the BBB. NCP showed only a 1.7 times increase in brain penetration factor and NCQP did not showed any difference in comparison to drug alone. The investigation of drug hepatotoxicity was conducted after 5 days i.v. dosing of VA 30 mg/kg q12h as solution or nanocapsules (NCQ or NCP). A saline control group was also investigated. Serum levels of asparte aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosfatase, creatinin and urea were determined. The results showed the maintenance of normal levels of hepatic enzymes such as alanine aminotransferase and alkaline phosfatase for NCQ (54.2 ± 11.2 IU/mL and 149 ± 26 IU/mL) showing a hepatoprotective effect not observed in the other groups investigated. Histological analysis of animals livers showed no microvesicular steatosis formation when NCQ was administered in comparison with the formation of steatosis in all other groups including control. Overall the results indicate that NCQ is a potential formulation to be investigated for the treatment of epilepsy due to its increase in VA brain penetration and hepatoprotective effect observed.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-03T11:42:26Z No. of bitstreams: 2 Dissertação - Renata Klícia Mendes Caetano - 2018.pdf: 2847916 bytes, checksum: 2eaea78563b91986afd782253b20323a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-03T11:47:01Z (GMT) No. of bitstreams: 2 Dissertação - Renata Klícia Mendes Caetano - 2018.pdf: 2847916 bytes, checksum: 2eaea78563b91986afd782253b20323a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-10-03T11:47:01Z (GMT). No. of bitstreams: 2 Dissertação - Renata Klícia Mendes Caetano - 2018.pdf: 2847916 bytes, checksum: 2eaea78563b91986afd782253b20323a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-08-29 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The growing interest in bioactive compounds from renewable natural sources has stimulated more and more studies aimed at increasing the supply of this market. An example is phycobiliproteins, dyes used in various areas, such as food, pharmacological and cosmetic. The microalgae arthrospira platensis (Spirulina platensis) is one of the organisms that stands out most in the production of these dyes, producing allophycocyanin and phycocyanin; these two dyes are the ones that today have more demand in the market. These proteins have their application determined from their degree of purity obtained with the absorbance ratio read at 620 nm of the extract used for the absorbance read at 280 nm. For use of these dyes, in the food area, a purity value greater than 0.7 should be obtained, and for use in the analytical area, the purity should be greater than 4.0. In view of the importance of these dyes and their degree of purity, in order to develop means of obtaining phycobiliproteins in a purity satisfactory for use in food industries, phase separation techniques were used in aqueous biphasic systems (ABS) to evaluate the action of pH, phosphate salt concentration and the concentration of low chlorine content cationic polymer and polyethylene glycol 4000 (PEG 4000) on the efficiency of the extraction and purification process of Spirulina crude extract and also in the pre-purified extract of phycocyanin obtained by centrifugation. For this, a first experiment was carried out using a factorial design 2 4 to evaluate the variables that most influenced the extraction and purification of phycocyanin, allophycocyanin and purity of the extract. Values of 2.6 mg.mL -1 were reached for phycocyanin concentration, 1.4 mg.mL -1 for the allophycocyanin concentration and 1.6 for the purity of the extract. The significance of the PEG 4000 and PBC polymers was observed for extraction and purification of phycobiliproteins. Then, a second design was carried out, a Rotational Central Composite Design (RCCD) 2 2 , fixing the phosphate salt, pH and varying the PEG and low chlorine content cationic polymer concentrations. At the end of the extraction, it was possible to find concentrations of 2.7 mg.mL -1 for Phycocyanin, 1.3 mg.mL -1 of allophycocyanin and 2.5 for the purity of the extract, evidencing that the techniques used are effective for the extraction process and purification of phycobiliproteins, in which the levels achieved allow their use and application in food industry. / O crescente interesse por compostos bioativos de fontes naturais renováveis tem estimulado cada vez mais estudos que visam aumentar a oferta deste mercado. Um exemplo são as ficobiliproteínas, corantes utilizados em diversas áreas, como alimentícia, farmacológica e cosmética. A microalga Arthrospira platensis (Spirulina platensis) é um dos organismos que mais se destaca na produção destes corantes, produzindo a Aloficocianina e, principalmente, a Ficocianina, sendo estes dois corantes os que, hoje, mais têm demanda no mercado. Essas proteínas têm sua aplicação determinada a partir de seu grau de pureza, obtido com a relação da absorbância lida a 620 nm do extrato utilizado pela absorbância lida de 280 nm. Para utilização destes corantes na área alimentícia, deve-se obter valor de pureza maior que 0,7 e para sua utilização na área analítica, a pureza deve ser maior que 4,0. Tendo em vista a importância destes corantes e seu grau de pureza, a fim de se desenvolver meios de obtenção das ficobiliproteínas em um grau de pureza satisfatório para utilização em indústriasalimentícias, foram utilizadas técnicas de separação por fases em sistemas aquosos bifásicos (SAB) para avaliar a ação do pH, da concentração de sal fosfato, e da concentração dos polímeros catiônicos com baixo teor de cloro e polietilenoglicol 4000 (PEG 4000) na eficiência do processo de extração e purificação do extrato bruto da Spirulina, e também, em extrato pré-purificado de ficocianina obtido por processo de centrifugação. Para tal, realizou-se um primeiro experimento utilizando um planejamento fatorial 2 4 para a avaliação das variáveis que mais influenciaram na extração e purificação da ficocianina, da aloficocianina e da pureza do extrato. Foram atingidos valores de 2,6 mg.mL -1 para concentração de ficocianina, valor de 1,4 mg.mL -1 para concentração de aloficocianina e 1,6 mg.mL -1 para a pureza do extrato. Foi observada a significância dos polímeros PEG 4000 e polímeros catiônicos com baixo teor de cloro para extração e purificação das ficobiliproteínas. Então foi realizado um segundo delineamento, sendo um DCCR - Composto Central Rotacional 2 2 , fixando as variáveis de sal fosfato e pH; e variando as concentrações de PEG 4000 e polímeros catiônicos com baixo teor de cloro. Ao final da extração, foi possível encontrar concentrações de 2,7 mg.mL -1 para Ficocianina, 1,3 mg.mL -1 de Aloficocianina e 2,5 para pureza do extrato, evidenciando que as técnicas utilizadas são eficazes para o processo de extração e purificação das ficobiliproteínas;em que os níveis alcançados permitem sua utilização e aplicação em indústrias do setor alimentício.