L’utilisation de nanovecteurs pour la livraison contrôlée de principes actifs est un concept commun de nous jours. Les systèmes de livraison actuels présentent encore cependant des limites au niveau du taux de relargage des principes actifs ainsi que de la stabilité des transporteurs. Les systèmes composés à la fois de nanovecteurs (liposomes, microgels et nanogels) et d’hydrogels peuvent cependant permettre de résoudre ces problèmes. Dans cette étude, nous avons développé un système de livraison contrôlé se basant sur l’incorporation d’un nanovecteur dans une matrice hydrogel dans le but de combler les lacunes des systèmes se basant sur un vecteur uniquement. Une telle combinaison pourrait permettre un contrôle accru du relargage par stabilisation réciproque. Plus spécifiquement, nous avons développé un hydrogel structuré intégrant des liposomes, microgels et nanogels séparément chargés en principes actifs modèles potentiellement relargués de manière contrôlé. Ce contrôle a été obtenu par la modification de différents paramètres tels que la température ainsi que la composition et la concentration en nanovecteurs. Nous avons comparé la capacité de chargement et la cinétique de relargage de la sulforhodamine B et de la rhodamine 6G en utilisant des liposomes de DOPC et DPPC à différents ratios, des nanogels de chitosan/acide hyaluronique et des microgels de N-isopropylacrylamide (NIPAM) à différents ratios d’acide méthacrylique, incorporés dans un hydrogel modèle d’acrylamide. Les liposomes présentaient des capacités de chargement modérés avec un relargage prolongé sur plus de dix jours alors que les nanogels présentaient des capacités de chargement plus élevées mais une cinétique de relargage plus rapide avec un épuisement de la cargaison en deux jours. Comparativement, les microgels relarguaient complétement leur contenu en un jour. Malgré une cinétique de relargage plus rapide, les microgels ont démontré la possibilité de contrôler finement le chargement en principe actif. Ce contrôle peut être atteint par la modification des propriétés structurelles ou en changeant le milieu d’incubation, comme l’a montré la corrélation avec les isothermes de Langmuir. Chaque système développé a démontré un potentiel contrôle du taux de relargage, ce qui en fait des candidats pour des investigations futures. / Controlled delivery of active compounds using nanoscale carriers is nowadays a common concept, but there are still limitations in current delivery systems related to active compound release rate and nanocarriers stability. To address these limitations, delivery systems can be made to incorporate both nanocarriers (liposomes, microgels and nanogels) and hydrogels. In this study, we have developed controlled delivery systems by combining different carriers in order to overcome deficiencies observed in systems using only one type of carrier. Such a combination could lead to an enhanced controlled release delivery system through synergistic stabilization. More specifically, we created a structured hydrogel embedded with either liposomes, microgels, or nanogels, each loaded with model active compounds that would be released in a controlled fashion by manipulating the temperature of release medium and nanocarriers composition and concentration. We compared drug loading and release kinetics of sulforhodamine B from liposomes (composed of DOPC and DPPC at different ratios) and nanogels (chitosan/hyaluronic acid) embedded in acrylamide hydrogels. We also compared drug loading and release kinetics of rhodamine 6G from microgels of N-isopropylacrylamide (NIPAM) with different ratios of methacrylic acid embedded in acrylamide hydrogel. Liposomes demonstrated a moderate drug loading capacity with sustained release for over ten days, while nanogels showed high drug loading but faster release kinetics, exhausting their contents within two days. Comparatively, microgels completely released their content within a day. Despite their faster release kinetics, microgels have shown the capacity to be finely tuned for efficient drug loading. The Langmuir isotherms indicated that it can be achieved by altering their structural properties or by changing their incubation medium. Each developed system has demonstrated a potential in controlling the release rate, which makes them candidates for further investigations in the future.

<p>Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior. </p><p>In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration. </p><p>Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines. </p><p>Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.</p> / Dissertation

A papaína é uma enzima proteolítica empregada no debridamento e cicatrização de feridas. Contudo, problemas de estabilidade na forma farmacêutica, bem como reações alérgicas reportadas por pacientes submetidos à tratamentos com a enzima, culminaram na restrição aos produtos contendo papaína para uso tópico por órgãos regulatórios internacionais. Este trabalho objetivou desenvolver hidrogel nanoestruturado contendo complexo de papaína e ciclodextrina visando obter forma farmacêutica estável e eficaz como curativo dérmico, com redução da resposta imunológica. A síntese do hidrogel foi realizada combinando fenômenos de cristalização e/ou reticulação e esterilização simultânea induzida por radiação gama, de modo a promover nanoestruturação adequada da membrana para veiculação da papaína nativa e do complexo. O complexo e o produto final tiveram suas propriedades biológicas e físico-químicas avaliadas. O hidrogel a base de PVA contendo complexo de papaína-ciclodextrina apresentou características adequadas para aplicação como curativo, além de apresentar indícios de redução na resposta imunológica e melhora na citocompatibilidade quando comparado à papaína nativa, isso devido ao encapsulamento molecular com a ciclodextrina e à alta retenção do complexo por parte da matriz. Por outro lado, a irradiação, não alterou o perfil citotóxico da enzima, mas acarretou leve diminuição em seu potencial imunogênico. O hidrogel se mostrou promissor para uso como curativo e demonstrou potencial redução nas reações adversas desencadeadas pelo uso da papaína. / Papain is a proteolytic enzyme applied for wound healing and debridement. However, stability issues as well as allergenic reactions reported by patients submitted to papain pharmaceutics led to restriction of papain containing products for topical use by international regulatory agencies. This work aimed the development of nanostructured hydrogel containing papain and cyclodextrin complex or papain in order to obtain stable and suitable pharmaceutical form as a wound dressing with reduced allergenic properties. The hydrogel synthesis was performed by combining freezing cycles and the simultaneous crosslinking and sterilization process promoted by gamma irradiation to achieve a nanostructured hydrogel for the loading of the papain and cyclodextrin complex. The biological and physical-chemical properties of the complex and the final product were assayed. The PVA based hydrogel containing cyclodextrin-papain complex presented desirable characteristics for wound dressing purposes. In addition, an in vitro shift in the immunological response and an increase in the cytocompatibility if compared to native papain were observed as a function of the molecular encapsulation with cyclodextrin. The process of irradiation was not capable of altering papain cytotoxicity, but conferred a slight decrease in the immunogenic properties. In conclusion, the developed hydrogel was promising as a novel papain containing dressing with potential reduced adverse reactions.

O objetivo principal desta tese foi desenvolver dispositivos microfluídicos do tipo língua eletrônica baseada em espectroscopia de impedância para análise de líquidos complexos. Dois tipos de língua eletrônica foram desenvolvidos. O primeiro com unidades sensoriais fabricadas com eletrodos interdigitados de ouro sobre vidro, selados com microcanal de PDMS, e recobertos com filmes automontados de polímeros condutores, materiais orgânicos e semicondutores. Essa língua eletrônica foi usada para diferenciar os sabores básicos, representados por soluções aquosas de NaCl (salgado), sacarose (doce), cafeína (amargo), HCl (azedo) e glutamato monosódico (umami), distinguir diferentes tipos de cafés, e detectar a presença de gliadina em soluções e alimentos. A distinção foi possível processando-se os dados de magnitude da capacitância com técnicas de projeção multidimensional IDMAP (Interactive Document Map) e análise de componentes principais (PCA). O segundo tipo de língua foi produzido com eletrodos interdigitados de carbono impressos sobre papel com canal hidrofílico e barreiras hidrofóbicas também impresso, modificado com hidrogel funcionalizado com ácido fenil-borônico (PBA) ou polilíquido iônico (PIL). Com dados de capacitância tratados com PCA e IDMAP, a língua foi usada para distinguir soluções dos açúcares glicose, frutose e sacarose, em diferentes concentrações, e diferentes marcas de suco de maçã. Nesta última língua, empregou-se a capacidade de intumescimento de hidrogéis contendo PBA na presença de açúcares, principalmente frutose, que também foi explorada na confecção de sensores com eletrodos de papel que puderam detectar concentrações baixas de glicose, próximas às encontrados no suor humano. Com a tecnologia de sensores em papel e microfluídica, podem-se conceber aplicações futuras, de baixo custo, em sensores na forma de emplastro para monitorar o nível de glicose no suor humano de maneira não invasiva e língua eletrônica para verificar a presença de glúten em alimentos. / The main aim of this thesis was to develop microfluidic devices of electronic tongue (e-tongue) type based on impedance spectroscopy to analyze complex liquids. Two types of e-tongue were developed. The first had sensing units fabricated with gold interdigitated electrodes onto glass with a microchannel sealed with PDMS and coated with layer-by-layer films of conducting polymers, organic and semiconductors. This e-tongue was used to distinguish the basic tastes, represented by aqueous solutions of NaCl (salty), sucrose (sweet), caffeine (bitter), HCl (sour) and monosodium glutamate (umami), to distinguish different types of coffees, and detect the presence of gliadin in solutions and food. The distinction was made possible by processing the capacitance data with the multidimensional projection techniques IDMAP (Interactive Document Map) and principal component analysis (PCA). The second type of e-tongue was produced with carbon interdigitated electrodes printed on paper with a hydrophilic channel with hydrophobic barriers, also printed, modified with functionalized hydrogel with phenylboronic acid (PBA) or poly(ionic liquid) (PIL). With capacitance data treated with PCA and IDMAP, the e-tongue was used to distinguish solutions at various concentrations of glucose, fructose and sucrose, in addition to different brands of apple juice. In this latter e-tongue, use was made of the ability of the hydrogel containing PBA to swell in the presence of sugars, mainly fructose, which was exploited in producing sensors with paper electrodes that could detect low concentrations of glucose, close to the values found in human sweat. With the technology of paper-based sensors and microfluidics, one may envisage future low cost applications, including patch sensors to monitor glucose in human sweat in a non-invasive manner and e-tongues to determine the presence of gluten in food.

O desenvolvimento de novos procedimentos e tecnologias tem proporcionado um grande avanço no tratamento de feridas e queimaduras, melhorando a qualidade de vida das vítimas e reduzindo as taxas de mortalidade de pessoas com queimaduras graves. Entretanto as complicações infecciosas continuam sendo um desafio e uma das principais causas de óbito de queimados. Por outro lado, em todo o mundo tem-se observado um crescente interesse no uso de hidrogéis para aplicação como curativos para queimaduras, ferimentos e úlceras de pele, tendo em vista que são capazes de absorver exsudatos, ajudam na cicatrização e proporcionam conforto ao paciente, uma vez que favorecem o alívio da dor. Além disso, os hidrogéis também podem ser aplicados como matrizes para sistemas de liberação controlada de princípios ativos e agentes antimicrobianos. O objetivo deste trabalho foi estudar as propriedades mecânicas e físico-químicas de hidrogéis compostos por blendas formadas por Poli(N-vinil-2-pirrolidona)/ Polietilenoglicol/ ágar (PVP/PEG/ágar), Poli(N-vinil-2-pirrolidona)/ glicerol/ ágar (PVP/glicerol/ágar); Poli(álcool vinílico)/ kappa-carragena/ ágar (PVA/KC/ágar), reticuladas por radiação ionizante. Para a preparação das blendas, utilizou-se planejamento de misturas como ferramenta, oferecendo assim subsídios a futuros desenvolvimentos de novos hidrogéis, que possam ser usados como curativos e como matrizes poliméricas para liberação de ativos hidrofílicos e lipofílicos. Foram otimizadas três formulações e sintetizadas seis, três das quais hidrogéis nanocompósitos de prata. As nanopartículas de prata (NPAg) foram sintetizadas, in situ, por radiação gama, sem uso de catalisadores ou outro reagente para obtenção de curativo mais puro e com ação antimicrobiana. A caracterização físico-química dos hidrogéis foi obtida por análises de fração gel, intumescimento, ensaios de tração e perfuração, microscopia eletrônica de varredura (MEV), espectroscopia na região do ultravioleta-visível (UV-vis). Também foi avaliada a atividade antimicrobiana dos hidrogéis e realizado estudo de citotoxicidade in vitro. O planejamento de misturas apresentou modelos confiáveis para os resultados de fração gel e intumescimento, porém, para as propriedades mecânicas, os resultados devem ser vistos com cautela. O uso da radiação se mostrou eficaz para a síntese de NPAg in situ nas blendas estudadas. Os resultados sugerem que os hidrogéis sintetizados não liberaram NPAg, porém os hidrogéis de PVP apresentaram atividade bactericida para S. aureus e P. Aeruginosa, enquanto que o hidrogel de PVA apresentou atividade bactericida para P. aeruginosa e atividade bacteriostática para S. aureus, sendo essa atividade pelo contato direto. / The development of new procedures and technologies have provided a major advance in the treatment of wounds and burns improving the quality of life of victims and reducing mortality rates of people with severe burns. However, infectious complications remain a challenge and a major cause death to burn. Furthermore, in the whole world, an increasing interest in the use of hydrogels as dressings for burns, wounds and skin ulcers has been observed, considering that they are capable of absorbing exudates, aid in healing and provide confort to the patient, since they favor the pain relief. In addition, hydrogels can also be applied as matrices for controlled release systems of active and antimicrobial agents. The aim of this study was to investigate the mechanical and physicochemical properties of hydrogels composed of blends formed by poly(N-vinyl-2-pyrrolidone)/ Polyethylene glycol/ agar (PVP/PEG/agar), poly(N-vinyl-2-pyrrolidone)/ glycerol/agar (PVP/ glycerol/agar) and poly(vinyl alcohol)/ kappa-carrageenan/ agar (PVA/KC/agar), crosslinked by ionizing radiation. In blends preparation, design of mixtures was utilized as a tool, thus offering information for future developments of new hydrogels, which can be used as dressings and as polymer matrices for the release of lipophilic and hydrophilic actives. Three formulations were optimized, and six synthesized, being three of these silver nanocomposite hydrogels. The silver nanoparticles (AgNPs) were synthesized, in situ, by gamma irradiation without using a catalyst or other reagent in order to obtain wound dressing with higher purity and antimicrobial activity. The physicochemical characterization of the hydrogels was obtained using assays of gel fraction, swelling, tensile and perforation, scanning electron microscopy (SEM), spectroscopy in the ultraviolet-visible (UV-vis) region. Antimicrobial activity of the hydrogels was evaluated, and cytotoxicity study in vitro also was performed. The mixture design offered reliable models for gel fraction and swelling, but for the mechanical properties the results must be seen with caution. The use of radiation has proven to be effective for synthesis of AgNPs in situ in the blends studied. The results suggest that hydrogels synthesized did not release AgNPs, but the PVP hydrogels showed bactericidal activity for S. aureus and P. aeruginosa, whereas the PVA hydrogel had bactericidal activity for P. Aeruginosa and bacteriostatic activity for S. aureus, being this activity by direct contact.

La cellulose est l’une des bio-ressources les plus abondantes sur terre ; elle forme des microfibrilles où alternent régions cristallines, de taille nanométrique, et régions amorphes. L’utilisation des nanocristallites de cellulose, appelés nanowhiskers, comme renforts dans des composites à matrice polymère thermoplastique a fait l’objet de nombreuses publications. En revanche, l’utilisation de nanowhiskers comme éléments structurants d’hydrogels n’a pas été vraiment explorée, en dépit d’un intérêt potentiel pour la formulation d’hydrogels verts innovants. L’objectif de la thèse était d’étudier les relations entre état structural à différentes échelles et propriétés rhéologiques de systèmes hydrocolloïdaux constitués d’hydrogels d’agarose chargés de nanowhiskers de cellulose issus de tunicier, un animal marin. Les travaux expérimentaux menés au cours de cette thèse ont montré que l’effet renfort apporté par les nanowhiskers à la matrice agarose pouvait être marqué, et qu’il pouvait être modulé en faisant varier la densité de charges électriques à la surface des nanowhiskers. Aux fractions volumiques étudiées, inférieures à 0,2%, les résultats ont montré que les nanowhiskers ne percolaient pas et l’effet renfort a été attribué à des modifications topologiques du réseau d’agarose, et à un transfert des contraintes efficace entre la matrice et les charges, qui interagissent via des liaisons hydrogène. Les résultats obtenus au cours de cette thèse permettent d’améliorer la connaissance des mécanismes qui gouvernent les propriétés renfort apportées par l’ajout de nanocharges cellulosiques à une matrice hydrogel, et donnent des pistes réalistes pour une formulation maîtrisée d’hydrogels verts innovants ayant de bonnes propriétés mécaniques. / Cellulose is the most abundant organic compound on Earth; it is composed of microfibrils, containing nanocrystalline regions, and amorphous regions. Cellulose nanocrystallites, called nanowhiskers, have been studied as reinforcement agents in polymer composites with thermoplastic matrix in numerous publications. However, the use of nanowhiskers to reinforce hydrogels has not really been explored up to now, despite potential interest in the formulation of green innovative hydrogels. The aim of this PhD thesis was to study the relationship between the structural state, on different length scales, and the rheological properties of hydrocolloid systems consisting of agarose hydrogels filled by cellulose nanowhiskers from tunicate, amarine animal. The experimental work performed in this thesis has shown that the reinforcing effect provided by the nanowhiskers could be marked, and could also be modulated by varying the density of electrical charges on the surface of nanowhiskers. Within the volume fraction range studied, that is less than 0.2%, the results have shown that the nanowhiskers could not percolate, and reinforcement effect was attributed to topological modifications of the agarose network, and to an efficient stress transfer between the matrix and the fillers, which interact via hydrogen bonds.The results obtained in this thesis improve the understanding of the mechanisms that govern there inforcement effect provided by the addition of cellulose nanofillers in a hydrogel matrix; they could also help to design innovative green hydrogels having good mechanical properties.

Two Systems were designed. The first permits tunable on-demand softening of a hydrogel network. The second permits reversible on demand ligand exchange within a hydrogel network. Both means were shown to be cytocompatible and their uses demonstrated in cell culture of mesenchymal stem cells and 3T3 fibroblast cells.

The extracellular matrix (ECM) governs many cellular processes through biochemical and mechanical cues. Particularly, the effect ECM mechanical properties on cells fate has been well established over the years. Many hydrogel systems have been used to mimic the dynamic stiffening processes occurring in ECM. However, changes in ECM stiffness does not fully recapitulate the mechanics of native ECM, as viscoelasticity is also a major factor contributing to ECM dynamic property. This thesis describes the design and characterization of an enzyme-crosslinked hydrogel system that is not only capable of being stiffened on demand, but also can be tuned to obtain viscoelasticity. The first objective of this thesis was to utilize horseradish peroxidase (HRP) to crosslink thiol-norbornene hydrogel and use mushroom tyrosinase (MT) to create secondary DOPA-dimer crosslinks that stiffened the hydrogel. The cytocompatibility of HRP-mediated thiol-norbornene gelation and the effect of stiffening on cell fate was evaluated. The second objective of this thesis represented the first step towards developing a hydrogel system whose viscoelasticity could be dynamically tuned. Thiol-norbornene hydrogel was designed to yield dynamically adaptable boronic ester bonds via partial enzymatic reaction. Thiol-norborne hydrogel was made to contain hydroxyl phenol as well as boronic acid residues within its network. MT, in this case was used to oxidize the hydroxy phenol moieties into DOPA, which then complexed with boronic acid, created dynamic bonds, introducing viscoelasticity to an initial elastic hydrogel.

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.

A papaína é uma enzima proteolítica empregada no debridamento e cicatrização de feridas. Contudo, problemas de estabilidade na forma farmacêutica, bem como reações alérgicas reportadas por pacientes submetidos à tratamentos com a enzima, culminaram na restrição aos produtos contendo papaína para uso tópico por órgãos regulatórios internacionais. Este trabalho objetivou desenvolver hidrogel nanoestruturado contendo complexo de papaína e ciclodextrina visando obter forma farmacêutica estável e eficaz como curativo dérmico, com redução da resposta imunológica. A síntese do hidrogel foi realizada combinando fenômenos de cristalização e/ou reticulação e esterilização simultânea induzida por radiação gama, de modo a promover nanoestruturação adequada da membrana para veiculação da papaína nativa e do complexo. O complexo e o produto final tiveram suas propriedades biológicas e físico-químicas avaliadas. O hidrogel a base de PVA contendo complexo de papaína-ciclodextrina apresentou características adequadas para aplicação como curativo, além de apresentar indícios de redução na resposta imunológica e melhora na citocompatibilidade quando comparado à papaína nativa, isso devido ao encapsulamento molecular com a ciclodextrina e à alta retenção do complexo por parte da matriz. Por outro lado, a irradiação, não alterou o perfil citotóxico da enzima, mas acarretou leve diminuição em seu potencial imunogênico. O hidrogel se mostrou promissor para uso como curativo e demonstrou potencial redução nas reações adversas desencadeadas pelo uso da papaína. / Papain is a proteolytic enzyme applied for wound healing and debridement. However, stability issues as well as allergenic reactions reported by patients submitted to papain pharmaceutics led to restriction of papain containing products for topical use by international regulatory agencies. This work aimed the development of nanostructured hydrogel containing papain and cyclodextrin complex or papain in order to obtain stable and suitable pharmaceutical form as a wound dressing with reduced allergenic properties. The hydrogel synthesis was performed by combining freezing cycles and the simultaneous crosslinking and sterilization process promoted by gamma irradiation to achieve a nanostructured hydrogel for the loading of the papain and cyclodextrin complex. The biological and physical-chemical properties of the complex and the final product were assayed. The PVA based hydrogel containing cyclodextrin-papain complex presented desirable characteristics for wound dressing purposes. In addition, an in vitro shift in the immunological response and an increase in the cytocompatibility if compared to native papain were observed as a function of the molecular encapsulation with cyclodextrin. The process of irradiation was not capable of altering papain cytotoxicity, but conferred a slight decrease in the immunogenic properties. In conclusion, the developed hydrogel was promising as a novel papain containing dressing with potential reduced adverse reactions.