Title: Polymer hydrogels: heterogeneous structure and deformation behavior Author: Evgeny Karpushkin Department: Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University in Prague Supervisor: Miroslava Dušková-Smrčková Ing. Dr., Institute of Macromolecular Chemistry AS CR, Heyrovského náměstí 2, 162 06 Prague 6 Abstract: Model series of crosslinked poly(2-hydroxyethyl methacrylate) hydrogels differing in morphology were prepared by polymerization-induced phase separation. Morphology, swelling and dynamic mechanical properties in broad frequency range of the model gels were tested. The vitrification of gels was found sensitive to microstructure of polymer network and to solvent content and to polymer-solvent interaction, but not to the macroscopic morphology of the gel. The low-frequency mechanical response was found sensitive to both polymer network properties and hydrogel morphology. Lightly crosslinked hydrogels showed relatively high mechanical losses in the rubbery plateau region, due to physical association of chains. Fused-particles type hydrogels revealed a weak secondary relaxation at low frequency, ascribed to motion of the dangling particles aggregates. These aggregates, elastically inactive, along with inhomogeneous stress field arisen in porous material, were...

abstract: Current treatment methods for cerebral aneurysms are providing life-saving measures for patients suffering from these blood vessel wall protrusions; however, the drawbacks present unfortunate circumstances in the invasive procedure or with efficient occlusion of the aneurysms. With the advancement of medical devices, liquid-to-solid gelling materials that could be delivered endovascularly have gained interest. The development of these systems stems from the need to circumvent surgical methods and the requirement for improved occlusion of aneurysms to prevent recanalization and potential complications. The work presented herein reports on a liquid-to-solid gelling material, which undergoes gelation via dual mechanisms. Using a temperature-responsive polymer, poly(N-isopropylacrylamide) (poly(NIPAAm), the gelling system can transition from a solution at low temperatures to a gel at body temperature (physical gelation). Additionally, by conjugating reactive functional groups onto the polymers, covalent cross-links can be formed via chemical reaction between the two moieties (chemical gelation). The advantage of this gelling system comprises of its water-based properties as well as the ability of the physical and chemical gelation to occur within physiological conditions. By developing the polymer gelling system in a ground-up approach via synthesis, its added benefit is the capability of modifying the properties of the system as needed for particular applications, in this case for embolization of cerebral aneurysms. The studies provided in this doctoral work highlight the synthesis, characterization and testing of these polymer gelling systems for occlusion of aneurysms. Conducted experiments include thermal, mechanical, structural and chemical characterization, as well as analysis of swelling, degradation, kinetics, cytotoxicity, in vitro glass models and in vivo swine study. Data on thermoresponsive poly(NIPAAm) indicated that the phase transition it undertakes comes as a result of the polymer chains associating as temperature is increased. Poly(NIPAAm) was functionalized with thiols and vinyls to provide for added chemical cross-linking. By combining both modes of gelation, physical and chemical, a gel with reduced creep flow and increased strength was developed. Being waterborne, the gels demonstrated excellent biocompatibility and were easily delivered via catheters and injected within aneurysms, without undergoing degradation. The dual gelling polymer systems demonstrated potential in use as embolic agents for cerebral aneurysm embolization. / Dissertation/Thesis / Ph.D. Bioengineering 2012

abstract: Aqueous solutions of temperature-responsive copolymers based on N-isopropylacrylamide (NIPAAm) hold promise for medical applications because they can be delivered as liquids and quickly form gels in the body without organic solvents or chemical reaction. However, their gelation is often followed by phase-separation and shrinking. Gel shrinking and water loss is a major limitation to using NIPAAm-based gels for nearly any biomedical application. In this work, a graft copolymer design was used to synthesize polymers which combine the convenient injectability of poly(NIPAAm) with gel water content controlled by hydrophilic side-chain grafts based on Jeffamine® M-1000 acrylamide (JAAm). The first segment of this work describes the synthesis and characterization of poly(NIPAAm-co-JAAm) copolymers which demonstrates controlled swelling that is nearly independent of LCST. The graft copolymer design was then used to produce a degradable antimicrobial-eluting gel for prevention of prosthetic joint infection. The resorbable graft copolymer gels were shown to have three unique characteristics which demonstrate their suitability for this application. First, antimicrobial release is sustained and complete within 1 week. Second, the gels behave like viscoelastic fluids, enabling complete surface coverage of an implant without disrupting fixation or movement. Finally, the gels degrade rapidly within 1-6 weeks, which may enable their use in interfaces where bone healing takes place. Graft copolymer hydrogels were also developed which undergo Michael addition in situ with poly(ethylene glycol) diacrylate to form elastic gels for endovascular embolization of saccular aneurysms. Inclusion of JAAm grafts led to weaker physical crosslinking and faster, more complete chemical crosslinking. JAAm grafts prolonged the delivery window of the system from 30 seconds to 220 seconds, provided improved gel swelling, and resulted in stronger, more elastic gels within 30 minutes after delivery. / Dissertation/Thesis / Ph.D. Bioengineering 2012

Doctor of Philosophy / Food Science Institute / Channa B. Rajashekar / Weiqun Wang / As health-promoting agents, phytochemicals are biosynthesized in the plants that typically respond to environmental stresses. This study focused on the analysis of phytochemical contents in vegetables in response to environmental changes of high tunnel and light spectra. A potential anticancer activity was further studied by developing a novel 3D cell culture model. Three specific studies were conducted as follows. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 μM. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 M. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application.

O óleo de copaíba é largamente utilizado na medicina popular da região amazônica, principalmente para tratar enfermidades relacionadas a inflamações. Estudos prévios demonstraram que o óleo extraído da espécie Copaifera multijuga Hayne tem uma promissora atividade anti-inflamatória, assim como seu componente majoritário, o β-cariofileno. Entretanto, o caráter untuoso deste óleo torna pouco aceitável sua aplicação direta na pele. Nosso grupo de pesquisa propôs a formulação de nanoemulsões contendo o óleo, uma vez que estes sistemas, além de hidrofílicos, possuem pequeno tamanho de gotícula e alta área de superfície, o que pode melhorar a penetração de substâncias através da pele. O único inconveniente destes é a sua baixa viscosidade, que pode ser contornado por sua incorporação em um hidrogel. No presente estudo, foi desenvolvida uma formulação de hidrogel contendo a nanoemulsão de óleo de copaíba (Copaifera multijuga Hayne) e avaliado seu perfil de permeação/retenção cutânea no modelo de pele de orelha suína. A viabilidade de incorporação da nanoemulsão em dois agentes geleificantes (Carbopol 980® e quitosana) foi testada. No período de sete dias, a formulação de quitosana, ao contrário da formulação de Carbopol 980® (CARB-NE) que permaneceu estável por 60 dias, apresentou visível instabilidade e aumento dos valores de tamanho de gotícula e índice de polidispersão. Devido a isto, a formulação CARB-NE foi escolhida para dar continuidade aos estudos de perfil reológico e de permeação/retenção cutânea. O perfil reológico de CARB-NE foi caracterizado como pseudoplástico, assim como o do hidrogel controle, o que indica que a nanoemulsão não interferiu na matriz polimérica do hidrogel. A formulação CARB-NE aumentou significativamente a penetração do componente β-cariofileno na derme comparada com a nanoemulsão não incorporada em hidrogel. Não houve um aumento da penetração na epiderme, e os valores não foram significativamente diferentes para as duas formulações. Portanto, o hidrogel de Carbopol 980® demonstrou ser o melhor agente geleificante para a nanoemulsão de copaíba, já que permaneceu estável durante o tempo testado e aumentou a penetração de β-cariofileno na pele, especialmente na derme, camada de interesse no tratamento da inflamação pela via tópica. / Copaiba oil is a natural product widely used in folk medicine of the Amazon region mostly to treat diseases related to inflammation. Previous studies have shown that the oil extracted from Copaifera multijuga Hayne has a promising anti-inflammatory activity, as well as its major component, β-caryophyllene. However, the unctuous nature of this oil makes it slightly acceptable to apply directly to the skin. Our research group has proposed the formulation of nanoemulsions containing copaiba oil, since these systems are hydrophilic, have a small droplet size and high surface area, which can improve the penetration of substances through the skin. The only disadvantage of these systems is their low viscosity that can be modified by incorporating it into a hydrogel. In the present study, we developed a hydrogel formulation containing the nanoemulsion of copaiba oil (Copaifera multijuga Hayne) and evaluated its skin permeation/retention profile in porcine ear skin model. The feasibility of incorporating the nanoemulsion into two gelling agents (Carbopol® 980 and chitosan) was assessed. Within seven days, chitosan formulation had increased values for droplet size and polydispersity index, in opposite to Carbopol® 980 formulation (CARB-NE), which continued stable for 60 days. Due to this, the formulation CARB-NE was chosen to continue studies of the rheological profile and skin permeation/retention profile. The rheological profile of CARB-NE was characterized as pseudoplastic, the same was observed for the control hydrogel, which indicates that the nanoemulsion had no effect on the hydrogel polymer matrix. The CARB-NE formulation significantly increased the penetration of β-caryophyllene in the dermis compared to the nanoemulsion not incorporated. There was no increase in the epidermis permeation, nor the values were significantly different for the two formulations. Therefore, the hydrogel Carbopol® 980 proved to be the best gelling agent for copaiba nanoemulsion since it remained stable over the tested time and increased β-caryophyllene penetration in the skin, especially in the dermis, the layer of interest in the treatment of topical inflammation.

Os extratos obtidos a partir da própolis vermelha brasileira (PVB) têm sido investigados devido às suas amplas atividades biológicas. Recentemente, em nosso grupo de pesquisa, demonstramos a viabilidade da incorporação de um extrato n-hexânico de PVB em nanoemulsões de uso tópico, bem como sua permeação/retenção em pele de orelha suína. No presente estudo, avaliamos as propriedades físico-químicas e reológicas de hidrogéis contendo essas nanoemulsões visando a obtenção de um produto semissólido adequado para aplicação tópica. Em uma primeira fase, foram preparadas nanoemulsões compostas de núcleo oleoso contendo extrato n–hexânico de PVB, miristato de isopropila, lecitina de ovo (NE) e DOTAP (NE/DT), e fase externa aquosa. O polímero gelificante hidroxietilcelulose foi incorporado às formulações após a sua obtenção por emulsificação espontânea (H-NE e H-NE/DT). As formulações apresentaram-se monodispersas com diâmetro médio na faixa de 200-300 nm, confirmado por microscopia eletrônica de transmissão. H-NE apresentou um potencial zeta negativo (-38mV), enquanto o mesmo parâmetro para H-NE/DT foi positivo (+36mV), devido à presença do lipídeo catiônico DOTAP na formulação. O teor de benzofenonas totais, determinado por cromatografia líquida de alta eficiência (CLAE), foi de cerca de 85 mg/g de extrato. Esses parâmetros mantiveram-se constantes durante 90 dias de armazenamento a 4C. As formulações H/NE e H-NE/DT apresentaram um comportamento não-Newtoniano pseudoplástico. Estudos de permeação/retenção das benzofenonas através da pele de orelha suína foram realizados utilizando células de difusão do tipo Franz. A maior retenção das benzofenonas na pele (18,11 μg/cm² após 8h) foi observada para a formulação H-NE/DT, demonstrando o efeito do lipídeo catiônico DOTAP nesse parâmetro. Em uma última etapa, investigou-se a capacidade das formulações de conferirem proteção à pele de orelha suína frente ao dano oxidativo gerado pela sua exposição à luz UVA/UVB. A proteção da pele de orelha suína foi evidenciada pelas técnicas de TBARS, carbonilação de proteínas e grupamentos tióis totais. Os resultados obtidos sugerem que os Hidrogéis contendo extrato de PVB apresentam propriedades físico-químicas e reológicas adequadas para serem utilizadas topicamente para a prevenção do dano oxidativo causado pela exposição à luz UVA/UVB. / Brazilian red propolis (BRP) extracts have been investigated due to their extensive biological activities. Recently, in our research group, we demonstrated the feasibility of incorporating an n-hexane extract of BRP into topical nanoemulsions, as well as the permeation/retention of these compounds in porcine ear skin. In the present study, we evaluated the physicochemical and rheological properties of hydrogels containing these nanoemulsions in order to obtain a semi-solid product suitable for topical application. In a first step, nanoemulsions composed of an oil nucleus containing BRP n-hexane extract, isopropyl myristate, egg lecithin (NE) and DOTAP (NE/DT), and an aqueous external phase were prepared. The hydroxyethylcellulose gelling polymer was incorporated into the formulations after being obtained by spontaneous emulsification (H-NE and H-NE/DT). The formulations were monodisperse exhibiting a mean diameter in the 200-300 nm range, confirmed by transmission electron microscopy. H-NE presented a negative zeta potential (-38mV), while H-NE/DT showed a positive value (+ 36mV) due to the presence of the cationic lipid DOTAP in the formulation. The total benzophenone content, determined by high performance liquid chromatography (HPLC), was about 85 mg/g extract. These parameters were maintained constant for 90 days of storage at 4°C. The formulations H/NE and H-NE/DT presented a non-Newtonian pseudoplastic behavior. Permeation/retention studies of benzophenones through porcine ear skin were performed using Franz type diffusion cells. The highest retention of benzophenones in the skin (18.11 μg/cm² after 8h) was observed for the H-NE/DT formulation, demonstrating the effect of the DOTAP cationic lipid on this parameter. In a last step, the ability of the formulations to confer protection of porcine ear skin against oxidative damage, generated by its exposure to UVA/UVB light, was investigated. The protection was evidenced by the TBARS, carbonylation of proteins, and total thiol groups techniques. The results obtained suggest that the hydrogels containing BRP have adequate physicochemical and rheological properties to be used topically for the prevention of oxidative damage caused by exposure to UVA/UVB light.

Esta dissertação teve como objetivo, a preparação de hidrogéis à base de alginato contendo argila e material magnético em sua estrutura. Foram analisadas as modificações nas características físico-químicas dos hidrogéis preparados com diferentes reticulantes (CaCl2 e FeCl3) e diferentes concentrações de material magnético (1 e 3 % m/m) e argila (1, 5 e 10 %). Após isso, os hidrogéis foram avaliados quanto à capacidade de remoção de íons Cu2+ e Zn2+ de soluções aquosas. As amostras foram caracterizadas quanto à composição química por espectroscopia na região do infravermelho (FTIR), quanto à morfologia por microscopia óptica e microscopia eletrônica de varredura (SEM) e quanto às propriedades magnéticas por magnetometria de amostra vibrante (VSM). Através da técnica de difratometria de raios-X (XRD), foi possível verificar a natureza do material magnético e a dispersão da argila nos hidrogéis. A estabilidade térmica das amostras foi analisada por análise termogravimétrica (TGA). Os resultados mostraram que tanto a argila como o material magnético ficaram bem dispersos nas amostras. De forma geral, foram preparados hidrogéis com morfologia esférica, sendo que os hidrogéis de alginato de cálcio tenderam a apresentar maior resistência térmica do que os hidrogéis de alginato de ferro. Todas as amostras magnéticas apresentaram comportamento superparamagnético, porém as amostras de alginato de ferro mostraram-se quebradiças após o intumescimento em água. O tempo médio de equilíbrio de intumescimento foi de 240 minutos. Os resultados de cinética de adsorção mostraram que os hidrogéis de alginato de cálcio preparados nas condições avaliadas nesta Dissertação foram eficientes na remoção dos íons Cu2+ e Zn2+, sendo que o cobre apresentou maior afinidade pelo hidrogel do que o zinco / The aim of this Dissertation was study the preparation of hydrogels based on alginate containing clay and magnetic material in its structure. The modifications on the physicochemical characteristics of hydrogels prepared with different crosslinking (CaCl2 and FeCl3), different concentrations of magnetic material (1 and 3 % w/w), and clay (1, 5 and 10 % w/w) were analyzed. After that, the removal capacity of Cu2+ and Zn2+ from aqueous solutions was evaluated. The samples were characterized according their chemical composition by infrared spectroscopy (FTIR), morphology by scanning electron microscopy (SEM) and optical microscopy, magnetic properties by vibrating sample magnetization (VSM). Through the X-rays diffraction (XRD) it was possible to ascertain the nature of the magnetic material and the clays dispersion into hydrogels. The thermal stability of the samples was analyzed by thermogravimetry analysis (TGA). The results showed that both clay and magnetic material were well dispersed in the samples. Spherical hydrogels were prepared and the calcium alginate hydrogels had tended to present higher thermal resistance than the iron alginate hydrogels. All the magnetic samples showed superparamagnetic behavior; however iron alginate samples had proved be brittle after swelling in water. The equilibrium time of swelling was 240 minutes. The adsorption kinetics results showed that calcium alginate hydrogels prepared under the conditions evaluated in this work were efficient in the removal of Cu2+ and Zn2+, and copper showed higher affinity than zinc for the hydrogel

Orientadora: Profa. Dra. Daniele Ribeiro de Araujo / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2015. / It is expected the improvement of BUD physicochemical properties through complexation with CD and the incorporation of BUD:HP-â-CD inclusion complex into PL-based hydrogels, contributing to a longer duration of anti-inflammatory effect associated with high bioadhesion in the treatment of ulcerative colitis. In this work, we propose the study of the system BUD:HP-â-CD inclusion complex incorporated into PL407 and PL407-PL403, since the micelles assembling to the thermoreversible hydrogels considering structural and thermodynamics parameters related to inclusion complex formation, micellization and sol-gel transition phase, as well as the interaction between BUD:HP-â-CD complex and PL. Complexation between BUD and HP-â-CD was confirmed by phase solubility studies (1:1 stoichiometry, Kc = 8662.8 M-1), DSC and microscopy analyzes. BUD solubility on upper and lower simulated colon fluids was improved in a dependence of both carriers association (HP-â-CD and PL407 or PL407-PL403). Micellar hydrodynamic diameter determination showed the interaction between HP-â-CD and PL blocks, as well as the reorganization of the micellar system in the presence of BUD. Micellization temperature (Tm) was not shifted, but in the presence of BUD or HP-â-CD, an increase on ÄH° values was observed for all formulations. Sol-gel phase transition studies showed that in the presence of BUD, HP-â-CD or BUD:HP-â-CD complex, the association PL407-PL403 favored the gel formation close to the physiological temperature. 1H-NMR studies revealed the interaction between PL407 and HP-â-CD and FTIR confirmed the effective incorporation of the drug and the inclusion complex in PL system. The in vitro drug release was sustained and the release rate was slowed by the addition of inclusion complex (under 38% of the BUD released after 2 days), suggesting a prolonged anti-inflammatory effect. Drug release of PL407 was best explained by Higuchi and Korsmeyer-Peppas (n < 0.5), indicating the Fickian diffusion mechanism. In the binary system with inclusion complex, drug release follows Hixson-Crowell cube root law, specially in lower colon fluid, indicating the erosion of polymer matrix as a mechanism that influences the BUD release profile. Sol/gel phase transition, solubility, turbidity, FTIR and in vitro release tests revealed no competitive displacement of BUD from the hydrophobic HP-â-CD cavity evoked by PL407 or PL407-PL403 addition. These promising findings point out the BUD-HP-â-CD in 20 % or 18/2 PL-based hydrogels as strategies for future in vivo investigations and also the development of new pharmaceutical formulations looking forward the treatment of ulcerative colitis. / O presente trabalho propoe a otimizacao das propriedades fisico-quimicas do glicocorticosteroide budesonida (BUD) para o tratamento da colite ulcerativa (COL), por meio da complexacao com ciclodextrinas (CD), bem como a incorporacao do complexo de inclusao farmaco:CD em hidrogeis a base de Poloxamer (PL), contribuindo para uma maior duracao do efeito antiinflamatorio associado a permanencia no local de aplicacao. Desta forma, para o complexo de inclusao BUD:HP-¿À-CD, como tambem para os hidrogeis termorreversiveis de PL407 e de PL407/PL403, foram realizados diferentes ensaios de caracterizacao fisico-quimica, abrangendo desde uma analise estrutural e morfologica ate ensaios de liberacao in vitro. A complexacao entre BUD e HP-¿À-CD foi confirmada por meio dos ensaios de solubilidade de fase (estequiometria 1:1), Calorimetria Diferencial Exploratoria (DSC), Microscopia Eleronica de Varredura (MEV), Ressonancia Magnetica Nuclear (RMN) e Espectroscopia no Infravermelho por Transformada de Fourier (FTIR), destacando a elevada afinidades entre a molecula do farmaco e a cavidade hidrofobica da HP- ¿À-CD (Kc = 8.662,8 M-1). A solubilidade da BUD em fluidos simulados do colon superior (FSCS) e inferior (FSCI) foi melhorada de forma dependente a ambos os carreadores (HP-¿À- CD e PL), sugerindo o efeito aditivo dos sistemas polimericos na solubilizacao do farmaco. Na presenca de BUD ou de BUD:HP-¿À-CD, a associacao PL407/PL403 favoreceu a formacao da estrutura gel proximo a temperatura fisiologica, otimizando a viscosidade da formulacao em relacao a forma comercial (enema). A analise termodinamica mostrou ocorrencia de um processo endotermico espontaneo, embora tenha sido observado um aumento nos valores de AH¿ para todas as formulacoes. Estudos de RMN-H1 revelaram a interacao entre PL407 e HP-¿À-CD, nao sendo o mesmo identificado para o PL403. Os resultados de FTIR comprovaram a efetiva incorporacao do farmaco e do complexo de inclusao nos sistemas PL, que sofreram uma desorganizacao na cadeia polimerica e a formacao de um novo arranjo como consequencia da interacao entre os aditivos e o PL. Ensaios de liberacao in vitro atestaram a efetividade dos sistemas polimericos associados ao complexo de inclusao BUD:HP-¿À-CD, diminuindo significativamente o fluxo e prolongando a liberacao, que alcancou menos de 38% de farmaco liberado apos 2 dias de experimentacao, nas formulacoes 18/2% PL407/PL403 e 20% PL407. O perfil cinetico dos sistemas isolados de PL407 foi melhor descrito por Higuchi e Korsmeyer-Peppas, com expoente de liberacao inferior a 0,5, indicando a difusao Fickiana como mecanismo predominante. A incorporacao do complexo de inclusao no sistema binario conferiu ao perfil de liberacao uma aderencia ao modelo de Hixson-Crowell, indicando a erosao da matriz polimerica como mecanismo que influencia a liberacao do farmaco. Esses resultados promissores atestam a efetividade dos sistemas hibridos propostos no presente trabalho, apontando o complexo de inclusao BUD:HP-¿À-CD em hidrogeis de 20% PL e 18/2% PL407/PL403 como estrategia para investigacoes futuras in vivo, visando otimizar o tratamento da COL.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this work was the development of nanostructured formulations containing clobetasol propionate. Initially, it was validated a chromatographic method to assay clobetasol propionate in nanocapsule suspensions. Clobetasol propionate-loaded nanocapsules and nanospheres of poly(ε-caprolactone) (PCL) and nanoemulsion (0.5 mg mL-1) were prepared by the interfacial deposition of preformed polymer method, nanoprecipitation and spontaneous emulsification, respectively. Formulations were characterized by means of drug content, encapsulation efficiency, pH, mean size, polydispersity index, zeta potential, morphology analysis, and stability under storage. The PCL nanocapsules showed the highest physicochemical stability, followed by the nanoemulsions and nanospheres. In the evaluation of in vitro release of clobetasol propionate, the nanocapsules showed a better control of drug release, according to the biexponential model. The photodegradation study of clobetasol propionate against UVA light showed the importance of the polymer and the oil in the nanoparticles to protect the drug from light. From these results, the nanocapsules were chosen for the study of the influence of the polymerid material on the physicochemical stability umder storage, photostability, release profile of the drug and its release mechanism. The nanocapsules prepared with poly(lactide) (PLA) showed a higher stability in comparison to the nanocapsules prepared with poly(lactide-co-glycolide) 50:50 and 85:15, although its stability was lower than nanocapsules prepared with PCL. Photodegradation studies demonstrated the pretection of the nanoencapsulated drug, regardless of the polymeric material of the nanocapsule s wall. The in vitro release study demonstrated the controlled release of the drug according to an anomalous transport. Due to these results, the nanocapsules prepared with PCL were selected for the development and preparation of hydrogels. Similar formulations containing nanospheres and nanoemulsion were used to evaluate the influence of polymer and oil on different properties of the hydrogels. These dosage forms were evaluated for drug content, pH, spreadability, rheology and in vitro drug release. All hydrogels presented properties compatible to the topical application. The presence of the drug-loaded nanoparticles in hydrogels led a slower drug release, especially for the formulation containing nanocapsules. The drug release profile was according to the Higuchi model. / Este trabalho teve como principal objetivo o desenvolvimento de formulações nanoestruturadas contendo propionato de clobetasol. Inicialmente, foi validado um método cromatográfico para análise do propionato de clobetasol em suspensões de nanocápsulas poliméricas. As nanocápsulas e nanoesferas de poli(ε-caprolactona) (PCL) e nanoemulsões contendo propionato de clobetasol (0,5 mg/mL) foram preparadas pelo método da deposição interfacial do polímero pré-formado, nanoprecipitação e emulsificação espontânea, respectivamente. Foram avaliados teores de fármaco, eficiências de incorporação, pHs, diâmetros de partícula, índices de polidispersão, potenciais zeta, características morfológicas e estabilidade frente ao armazenamento das diferentes formulações. As nanocápsulas apresentaram maior estabilidade físico-química, seguida pelas nanoemulsões e nanoesferas. Na avaliação da liberação in vitro do propionato de clobetasol, as nanocápsulas apresentaram o maior controle na liberação do fármaco, seguindo um modelo biexponencial. O estudo da fotodegradação do propionato de clobetasol frente à luz UVA demonstrou a importância da presença do polímero e do óleo para o aumento da fotoestabilidade. Diante destes resultados, as nanocápsulas foram selecionadas para o estudo da influência do material polimérico sobre as características físico-químicas, estabilidade frente ao armazenamento, fotoestabilidade, perfil de liberação do fármaco e seu mecanismo de liberação. As nanocápsulas preparadas com poli(ácido lactídeo) (PLA) apresentaram uma maior estabilidade frente ao armazenamento em comparação com as nanocápsulas preparadas com poli(ácido lactídeo-coglicolídeo) 50:50 e 85:15, embora sua estabilidade tenha sido inferior às nanocápsulas preparadas com PCL. O estudo da fotodegradação demonstrou a proteção do fármaco quando nanoencapsulado, independente do tipo de polímero empregado na sua preparação. A liberação in vitro demonstrou a liberação controlada do fármaco com transporte anômalo. Diante de todos esses resultados, as nanocápsulas preparadas com PCL foram selecionadas para o desenvolvimento de formas farmacêuticas semissólidas (hidrogéis). Formulações similares contendo nanoesferas e a nanoemulsão foram utilizadas para se avaliar a influência do polímero e do óleo sobre diferentes propriedades dos hidrogéis. Estas formas farmacêuticas foram avaliadas quanto ao teor de fármaco, pH, espalhabilidade, reologia e liberação in vitro do fármaco. Os hidrogéis apresentaram propriedades compatíveis com a aplicação tópica. A presença do fármaco nanoencapsulado nos hidrogéis proporcionou sua liberação controlada, principalmente para as formulações contendo as nanocápsulas. O perfil de liberação do fármaco a partir dos hidrogéis seguiu o modelo de Higuchi.

abstract: Traumatic brain injury (TBI) is a significant public health concern in the U.S., where approximately 1.7 million Americans sustain a TBI annually, an estimated 52,000 of which lead to death. Almost half (43%) of all TBI patients report experiencing long-term cognitive and/or motor dysfunction. These long-term deficits are largely due to the expansive biochemical injury that underlies the mechanical injury traditionally associated with TBI. Despite this, there are currently no clinically available therapies that directly address these underlying pathologies. Preclinical studies have looked at stem cell transplantation as a means to mitigate the effects of the biochemical injury with moderate success; however, transplants suffer very low retention and engraftment rates (2-4%). Therefore, transplants need better tools to dynamically respond to the injury microenvironment. One approach to develop new tools for stem cell transplants may be to look towards the endogenous repair response for inspiration. Specifically, activated cell types surrounding the injury secrete the chemokine stromal cell-derived factor-1α (SDF-1α), which has been shown to play a critical role in recruiting endogenous neural progenitor/stem cells (NPSCs) to the site of injury. Therefore, it was hypothesized that improving NPSC response to SDF-1α may be a viable mechanism for improving NPSC transplant retention and migration into the surrounding host tissue. To this end, work presented here has 1. identified critical extracellular signals that mediate the NPSC response to SDF-1α, 2. incorporated these findings into the development of a transplantation platform that increases NPSC responsiveness to SDF-1α and 3. observed increased NPSC responsiveness to local exogenous SDF-1α signaling following transplantation within our novel system. Future work will include studies investigating NSPC response to endogenous, injury-induced SDF-1α and the application of this work to understanding differences between stem cell sources and their implications in cell therapies. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2015