The functional ingredient curcumin has a variety of biological and pharmacological actions, such as anti-tumor, anti-inflammatory, anti-virus, anti-oxidant, and anti-HIV properties coupled with low toxicity. However, curcumin possesses low bioavailability due to its poor solubility in water. The purpose of this study was to investigate the impact of different lipid-based formulations of curcumin on in vitro solubilization and bioaccessibility. Oils representing LCT, MCT, LCT:SCT mix and SCT were used to prepare O/W (nano)emulsions with droplet sizes as low as 174 nm. An in vitro digestion model simulating the small intestine milieu in the fasted and fed state was used to characterize rate, extent, and particle size associated with emulsion digestion. Rate and extent were oil dependent, but not particle size. SCT emulsions digested at the fastest initial rate, but MCT emulsions were digested to the largest extent. Bioaccessibility, a precursor to eventual bioavailability, was determined after digestion using a curcumin:lipid content dependent and independent method. MCT produced the highest bioaccessibility of curcumin for each method. Nanoemulsion digestion and bioaccessibility results were compared to conventional emulsions because an appropriate comparison was needed to determine the merits of the nanoemulsion delivery system. There was no significant difference in particle size and bioaccessibility between the conventional and nanoemulsions.

Yes / Extensive research has demonstrated the potential effectiveness of curcumin against various diseases, including asthma and cancers. However, few studies have used liquid-based vehicles in the preparation of curcumin formulations. Therefore, the current study proposed the use of nanoemulsion and microsuspension formulations to prepare nebulised curcuminoid for lung delivery. Furthermore, this work expressed a new approach to understanding the aerosol performance of nanoparticles compared to microsuspension formulations. The genotoxicity of the formulations was also assessed. Curcuminoid nanoemulsion formulations were prepared in three concentrations (100, 250 and 500 µg/ml) using limonene and oleic acid as oil phases, while microsuspension solutions were prepared by suspending curcuminoid particles in isotonic solution (saline solution) of 0.02% Tween 80. The average fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD) of the nebulised microsuspension formulations ranged from 26% and 7.1 µm to 40% and 5.7 µm, for 1000 µg/ml and 100 µg/ml respectively. In a comparison of the low and high drug concentrations of the nebulised nanoemulsion, the average FPF and MMAD of the nebulised nanoemulsion formulations prepared with limonene oil ranged from 50% and 4.6 µm to 45% and 5.6 µm, respectively; whereas the FPF and MMAD of the nebulised nanoemulsion prepared with oleic acid oil ranged from 46% and 4.9 µm to 44% and 5.6 µm, respectively. The aerosol performance of the microsuspension formulations were concentration dependent, while the nanoemulsion formulations did not appear to be dependent on the curcuminoids concentration. The performance and genotoxicity results of the formulations suggest the suitability of these preparations for further inhalation studies in animals.

Nanoemulsions are templates that have the potential to fill the gap between micellar systems and latex particles in the preparation of porous materials. A nanoemulsion can also be used as a carrier for uploading the desired materials inside the pore formed after the removal of the template. In this research, oil-in-water (O/W) nanoemulsions were prepared by means of a low-energy method based on a phase inversion composition (PIC) technique, using two nonionic surfactants (Tween 80 and Span 80), which can be mixed in order to adjust the hydrophilic-lipophilic balance (HLB). The influence of a number of parameters on the tunability and stability of such nanoemulsions was also studied. The effect of the simultaneous intercrossing of multifactors on droplet size was explored using a process- mixture design, and the size of the nanoemulsion oil droplets was measured by means of dynamic light scattering (DLS). The nanoemulsions were combined with sol-gel method in order to prepare porous silica with a macroporosity in the 50 nm to 400 nm range. The results demonstrate that a precise synergy between the silica source and the nanoemulsions is essential for effective interactions and homogeneous structures. Depending on the nature of such interactions, a variety of materials were observed, from hollow particles to continuous gels. Changing the size of the oil droplet and the volume of the nanoemulsions produced silica with differing pore sizes and varying total pore volumes. The obtained hierarchical porous silica (HPS) were characterized using mercury porosimetry, small angle X-ray scattering (SAXS), nitrogen isotherms, Fourier transform infrared (FTIR) analysis, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The parallel use of the oil vesicles as containers for the further synthesis of metal oxide is a novel method of internally functionalizing the silica. When hydrophobic metal precursors are dissolved into the oil phase before the preparation of the nanoemulsion, they are confined within the globular cavities of the silica. The thermal treatment applied to the material to burn the organics then leads to the final formation of metal oxide nanoparticles, which are larger than the porosity of the silica matrix but entrapped within the large cavities, producing a "rattle-like" structure. This method was demonstrated through the synthesis of Fe2O3, Fe3O4, and Co3O4 nanoparticles, and the results showed that a rather large amount of metal oxide (up to a 60 wt.% of metal oxide in nanocomposites) be generated while still maintaining the nanometric size observed at lower concentrations. This method allows control of the type of metal oxide, the concentration of the metal oxide, and the pore size, which enables the creation of different types of nanocomposites. Metal oxide hierarchical porous silica (MHPS) nanocomposites were characterized based on nitrogen isotherms, TEM and SEM observations, FTIR analysis, X-ray diffraction (XRD), and Mossbauer spectroscopy. Magnetic measurements were also taken. This new method, using the new templating objects, is a perfect illustration of the concept of "integrative synthesis,??? whereby the combination of building units and reactional mechanisms leads to complex structures as a result of true synergy among the elements during the reaction. In this case, the size of the nanoemulsion and the total water volume both contribute to the generation of distinctive architectures. In addition, the reaction of the metal oxide precursors within the cavities limits the extension of the final crystal size, but the surrounding solid matrix plays a role as well by keeping the particles apart. The final factor is that the reactive materials cannot leak from the silica because of the rattle-like structure, but the reagents can reach those particles through the porosity of the silica framework.

En cas de contamination cutanée radiologique par des composés d’uranium, les seuls traitements actuellement employés consistent en un rinçage de la zone contaminée par de l’eau et des détergents, ou par une solution de sel calcique de l’acide diéthylène-triamine-pentaacétique (Ca-DTPA). Ces derniers ne sont cependant pas efficaces vis-à-vis de l'uranium. De plus, en l'absence de traitement d'urgence, le passage transcutané de ce radionucléide est rapide, et induit une exposition interne après sa distribution dans l’organisme par le biais de la circulation sanguine. Une partie de l'uranium ainsi biodisponible est alors stockée dans les organes cibles que sont principalement les reins et le squelette, où ses effets toxiques se manifestent. C'est pourquoi une formulation topique consistant en une nanoémulsion huile dans eau, incorporant des molécules de calixarène tricarboxylique en tant qu'agent chélatant spécifique de l'uranium, a été initialement développée. Les travaux menés dans le cadre de cette thèse visent à évaluer l'efficacité de décontamination ex vivo et in vivo de ce nouveau traitement d'urgence à la fois sur peaux intactes et sur peaux lésées superficiellement. Pour cela, le modèle d'excoriation a été utilisé. Des modèles de lésions reproductibles ont également été mis en place afin de mimer des incisions par micro-piqûres et microcoupures. Ces études démontrent que la nanoémulsion de calixarène pourrait constituer un traitement de décontamination efficace, moins agressif que l’emploi de l’eau savonneuse actuellement employée. Sa potentielle toxicité cutanée a également été évaluée in vitro par l'utilisation d'épiderme humain reconstitué, combinée à trois différents tests de toxicité (MTT, LDH et IL-1-α). Dans le cadre de ces études, il a ainsi été démontré que la nanoémulsion de calixarène n’induit pas de toxicité cutanée, même après un temps de contact prolongé jusqu'à 24 h. / In case of radiological skin contamination by uranium compounds, the only treatments currently available consist in rinsing the contaminated skin area with water and detergent, or with a calcium salt of diethylene triamine pentaacetic acid (Ca-DTPA) solution. However, these procedures are not specific and no efficient treatment for cutaneous contamination due to uranium exists. In the absence of such treatments, uranium diffusion through the skin is fast, inducing an internal exposure after its distribution inside the body through the bloodstream. One part of the bioavalaible uranium is uptaken in target organs which are the kidneys and the skeleton, where its toxic effects occur. Therefore a topical formulation consisting of an oil-in-water nanoemulsion incorporating a tricarboxylic calixarene molecule, as a specific chelating agent for uranium, was previously developed. The work achieved in this thesis aimed at evaluating the ex vivo and in vivo decontamination efficiency of this new emergency treatment on intact and superficially wounded skin. For this purpose, skin excoriation model was used. Reproducible models of superficial wounds consisting of micro-cuts and micro-punctures were also developed in order to evaluate the efficiency of the nanoemulsion on physical wounds such as incisions. These studies showed that the calixarene nanoemulsion could be an efficient decontaminant treatment, less aggressive than using the current treatment: soaped water. Its potential cutaneous toxicity was evaluated on in vitro reconstructed human epidermis using three different toxicity tests (MTT, LDH and IL-1-α). These studies demonstrated that the calixarene nanoemulsion did not induce skin toxicity even after 24 h of exposure time.

Em estudos prévios, mostramos que uma nanoemulsão lipídica (LDE) é reconhecida e se liga aos receptores de LDL após sua injeção na corrente sanguínea. Como tais receptores estão superexpressos em células com altas taxas de proliferação, como ocorre no câncer e na aterosclerose, a LDE pode ser utilizada como veículo para direcionar fármacos a essas células, diminuindo sua toxicidade e aumentando sua eficácia terapêutica. Anteriormente, reportamos que o tratamento com um derivado do paclitaxel, o oleato de paclitaxel, associado à LDE (PTX-LDE), reduziu em 60% a área lesionada de aortas de coelhos submetidos à dieta aterogênica, comparados a animais não tratados. No presente trabalho, avaliamos o efeito da associação de sinvastatina, medicamento hipolipemiante, e PTX-LDE, sobre a aterosclerose induzida por dieta em coelhos. Trinta e seis coelhos machos da raça Nova Zelândia foram submetidos à dieta enriquecida com 1% de colesterol durante oito semanas. A partir da quinta semana, os animais foram divididos em quatro grupos, de acordo com o tratamento: controle (solução salina EV), sinvastatina (2mg/kg/dia, VO), paclitaxel (PTX-LDE, 4mg/Kg/semana, EV), ou combinação de sinvastatina (2mg/Kg/dia, VO) com paclitaxel (PTX-LDE, 4mg/Kg/semana, EV). Após oito semanas, os animais foram sacrificados para análise das aortas. Em comparação aos controles, a área lesionada das aortas foi em torno de 60% menor, tanto no grupo paclitaxel, quanto no grupo da combinação, e em torno de 40% menor no grupo sinvastatina (p<0,05). A razão entre as camadas íntima/média foi menor nos grupos tratados, em relação ao grupo controle (controles, 0,35±0,22, sinvastatina, 0,10±0,07, paclitaxel, 0,06±0,16 e combinação, 0,09±0,05, p<0,0001). Os grupos combinação e sinvastatina apresentaram um aumento da porcentagem de colágeno nas lesões (combinação, 20% e sinvastatina, 22%), em comparação aos controles (11%) e ao grupo paclitaxel (12%), (p<0,0001). Houve uma diminuição da porcentagem de macrófagos na lesão em todos os grupos tratados (paclitaxel, 11%, sinvastatina, 8% e combinação, 5%), comparados ao grupo controle (30%), (p<0,0001). O grupo paclitaxel apresentou menor porcentagem de células musculares lisas na lesão (20%) em relação aos controles (33%), (p<0,0001), já na combinação, houve aumento dessa porcentagem (44%), (p<0,0001). A combinação com sinvastatina não aumentou a eficácia do tratamento com PTX-LDE na redução da área de lesões ateroscleróticas, porém, os efeitos adicionais sobre o perfil lipídico e na composição das lesões, observadas com o uso da combinação, são achados importantes, que sugerem benefícios no sentido de aumentar a estabilidade das placas ateroscleróticas, o que nos abre um caminho de pesquisa muito promissor. / In previous studies we have shown that a lipid nanoemulsion (LDE) is recognized and binds to LDL receptors after injection into the bloodstream. As those receptors are upregulated in cells with higher proliferation rates, as occurs in cancer and atherosclerosis, LDE can be used as a vehicle to direct drugs to those cells, diminishing toxicity and increasing therapeutic efficacy. Previously, we reported that treatment with antiproliferative agent paclitaxel derivative, paclitaxel oleate, associated with LDE (PTX-LDE), reduced by 60% the injured area of the aorta of rabbits subjected to atherogenic diet compared to untreated animals. In the current study we aim to test the effect of a combination of lipid-lowering drug simvastatin with PTX-LDE on diet-induced atherosclerosis in rabbits. Thirty-six male New Zealand rabbits were fed a 1% cholesterol diet for 8 weeks. Starting from week 5, animals were divided into four groups, according to the following treatments: controls (I.V. saline solution injections), simvastatin P.O. (2mg/kg/day), paclitaxel (PTX-LDE I.V. injections, 4mg/Kg/week), or paclitaxel-simvastatin combination (PTX-LDE I.V., 4mg/Kg/week + simvastatin P.O., 2mg/Kg/day). After 8 weeks, the animals were sacrificed for aorta evaluation. Compared to controls, the injured area was reduced by 60% in both paclitaxel and combination groups, and by 40% in simvastatin group (p<0,05). The intima/media ratio was reduced in treated groups, compared to control group (controls, 0,35±0,22, simvastatin, 0,10±0,07, paclitaxel, 0,06±0,16 and combination, 0,09±0,05, p<0,0001). Simvastatin and combination groups showed increased collagen content within the lesions (simvastatin, 22% and combination 20%), compared to controls (11%) and to paclitaxel group (12%), (p <0.0001). Macrophage content within the lesions was reduced in all treated groups (paclitaxel, 11%, simvastatin, 8% e combination, 5%), compared to controls (30%), (p <0.0001). The percentage of smooth muscle cells in the lesions was diminished in paclitaxel group (20%) compared to control group (33%), while the combination group showed increased percentage (44%) of smooth muscle cells in the lesions (p<0,0001). The combination of simvastatin did not improve the efficacy of the treatment with PTXLDE in reducing the area of atherosclerotic lesions, but the additional effects on lipid profile and lesion composition observed with the use of the combination are important findings that suggest benefits in order to enhance the stability of atherosclerotic plaques, which may lead us to a very promising research path.

Uma nova alternativa para o tratamento do câncer foi proposta em estudos anteriores, consistindo no uso de uma nanoemulsão lipídica como transportadora de agentes quimioterápicos às células neoplásicas. A redução da toxicidade da quimioterapia promovida pelo direcionamento específico de quimioterápicos às células tumorais nos levou a testar o potencial de aplicação do sistema de nanopartículas lipídicas na terapêutica combinada do paclitaxel com a sinvastatina, um agente hipolipemiante que pode ser empregado como coadjuvante no tratamento do câncer. Nos dias 11, 14 e 19 após a inoculação de células de melanoma B16F10, camundongos C57BL/6J receberam pela via intraperitoneal soluções de oleato de paclitaxel associado à nanoemulsão lipídica 17,5&#181;mol/kg (Nano-paclitaxel), formulação comercial do paclitaxel 17,5&#181;mol/kg, nanoemulsão lipídica (Nanoemulsão) e solução salina (Controle). A sinvastatina 50mg/kg/dia foi administrada por gavagem do 11° ao 19° dia após a inoculação do tumor em um dos grupos de animais tratados com o Nano-paclitaxel (Nano-paclitaxel + Sinva), no grupo tratado com a formulação comercial do paclitaxel (Paclitaxel + Sinva) e como monoterapia (Sinva). Camundongos Balb-c saudáveis receberam os mesmos tratamentos para avaliação dos possíveis efeitos tóxicos dos diferentes tratamentos. A terapia combinada Nano-paclitaxel + Sinva apresentou toxicidade negligível em comparação com a terapia combinada Paclitaxel + Sinva que provocou perda de peso e mielossupressão nos animais. Nos animais portadores de tumor, o tratamento Nano-paclitaxel + Sinva inibiu 95% do crescimento tumoral, comparado à inibição de 44% promovida pelo tratamento Paclitaxel + Sinva. Além disso, apenas 37% dos animais portadores de melanoma submetidos ao tratamento com Nano-paclitaxel + Sinva apresentaram metástases, em contraste com 90% dos tratados com Paclitaxel + Sinva. A probabilidade de sobrevida também foi maior nos camundongos tratados com o Nano-paclitaxel + Sinva em comparação aos tratados com Paclitaxel + Sinva. A análise de amostras de tumores por citometria de fluxo mostrou que somente nos grupos de animais tratados com Sinva, Nano-paclitaxel ou com a combinação Nano-paclitaxel + Sinva houve aumento na expressão de p21 em comparação ao grupo Controle. Da mesma forma, apenas nos grupos Sinva e Nano-paclitaxel + Sinva houve redução na expressão de ciclina D1 em comparação ao grupo Controle. O teste de viabilidade celular com rodamina 123 mostrou despolarização da membrana mitocondrial com redução no número de células tumorais viáveis em todos os grupos de tratamentos em comparação aos grupos Nanoemulsão e Controle. A avaliação histológica dos tumores demonstrou que os grupos Nanoemulsão e Controle apresentaram alta densidade de células tumorais, diferentemente dos demais grupos de tratamento e que apenas os tumores do grupo Nano-paclitaxel + Sinva apresentaram aumento na presença de fibras de colágeno tipo I e III. Em comparação ao grupo Controle, os tumores dos grupos Sinva, Paclitaxel + Sinva, Nano-paclitaxel e Nano-paclitaxel + Sinva apresentaram redução na expressão imunohistoquímica de ICAM, MCP-1 e MMP-9 sendo que o grupo Nano-paclitaxel + Sinva apresentou a menor porcentagem de área marcada positivamente para a MMP-9. A terapia combinada com Nano-paclitaxel + Sinva é menos tóxica e mais efetiva na inibição do crescimento tumoral do que a mesma terapia com a formulação comercial do paclitaxel. / In previous studies we have proposed a novel approach for cancer treatment consisting of the use of a lipid nanoemulsion as a vehicle to direct chemotherapeutic agents to neoplastic cells. Reduction of chemotherapy toxicity promoted by specific targeting of antineoplastic agents to tumor cells led us to test the application of the lipidic nanoparticle system in combined treatment with paclitaxel and simvastatin, a cholesterol-lowering drug that can be used as coadjuvant in cancer treatment. On days 11, 14 and 19 after B16F10 melanoma cells inoculation, C57BL/6J mice were intraperitoneally injected with paclitaxel oleate associated to the lipidic nanoemulsion 17.5 &#181;mol/kg (Nano-paclitaxel), commercial formulation of paclitaxel 17.5 &#181;mol/kg, lipidic nanoemulsion (Nanoemulsion) or saline solution (Control). Simvastatin 50 mg/kg/day was administered by gavage from days 11 to 19 after tumor inoculation in one group of animals treated with Nano-paclitaxel (Nano-paclitaxel + Simva), in the group treated with commercial formulation of paclitaxel (Paclitaxel + Simva) and as monotherapy (Simva). Evaluation of possible toxic effects of the treatments was accessed in healthy Balb-c mice. Combined therapy with Nano-paclitaxel + Simva showed negligible toxicity as compared with the combination of Paclitaxel + Simva which resulted in animal weight loss and myelosuppression. In tumor-bearing animals, treatment with Nano-paclitaxel + Simva resulted in a remarkable tumor growth inhibition rate of 95%, compared to a 44% inhibition rate promoted by treatment with Paclitaxel + Simva. Moreover, only 37% of melanoma bearing animals treated with Nano-paclitaxel + Simva developed metastasis, in contrast to 90% of those treated with Paclitaxel + Simva. Survival rates were also higher in mice treated with Nano-paclitaxel + Simva in comparison to Paclitaxel + Simva treated animals. Analysis of tumor samples by flow cytometry showed that only animals treated with Simva, Nano-paclitaxel or Nano-paclitaxel + Simva increased the expression of p21 in comparison to Control group. Also, tumors from animals treated with Simva or Nano-paclitaxel + Simva presented a decrease in the expression of cyclin D1 in comparison to Control group. Cell viability test with rhodamine 123 showed mitochondrial membrane depolarization with reduction of tumor viable cells in all treatment groups in comparison to Nanoemulsion and Control groups. The histological study revealed that in contrast to drugs treated groups, tumors from Nanoemulsion and Control groups presented high tumor cell density and only Nano-paclitaxel + Simva treated animals presented tumors with increased presence of collagen fibers I and III. In comparison to Control group, tumors from groups Simva, Paclitaxel + Simva, Nano-paclitaxel and Nano-paclitaxel + Simva showed a reduction in immunohistochemical expression of ICAM, MCP-1 and MMP-9 and the group Nano-paclitaxel + Simva presented the lowest percentage of area positively stained for MMP-9. Combined therapy with Nano-paclitaxel + Simva was less toxic and more effective in promoting tumor growth inhibiton than the same combined therapy with the commercial formulation of paclitaxel.

A Terapia Fotodinâmica (TFD) é uma modalidade terapêutica que vem atuar no tratamento de diversos tipos de câncer, com interesse crescente nas últimas décadas. Atuando de forma pouco invasiva e complementar as terapias tradicionais, a TFD consiste basicamente na administração sistêmica ou tópica de um fármaco fotossensibilizador (FS) seguido pela exposição da lesão à luz visível resultando na regressão tumoral caracterizada por processos de morte celular, sendo que, o fármaco fotossensibilizador e a radiação luminosa separadamente não induzem nenhum tipo de dano aos tecidos. A radiação monocromática utilizada na maioria dos casos é do tipo LASER sendo direcionada sobre a lesão por um conjunto de fibras ópticas. A maioria dos estudos até o presente momento sugerem um mecanismo comum de ação para a TFD. Após a excitação do fotossensibilizador para os estados excitados singlete e triplete, e por transferência de energia para o oxigênio molecular (O2) leva a morte tumoral ocasionada por espécies reativas de oxigênio definidas como EROS (1O2, O2_ ,OH, H2O2) além de outras formas radicalares (CH3, NO2, R-CO2, etc.) que atacam centros específicos dentro dos sistemas celulares, desencadeando a morte de tais tecidos por processos de necrose e/ou apoptose celular. Neste trabalho de pesquisa avaliamos a interação do fármaco fotossensibilizador Foscan® com Nanoemulsões biodegradáveis como sistemas de liberação em TFD-tópica. Na primeira etapa do trabalho foram realizados estudos físico-químicos para avaliação da estabilidade termodinâmica e determinação de alguns parâmetros morfológicos. Consecutivamente, determinamos a caracterização fotofísica do fotossensibilizador em ambiente coloidal empregando-se técnicas de espectroscopia no estado estacionário e resolvida no tempo. Foi possível determinar importantes parâmetros que elucidaram o potencial fotodinâmico do sistema fármaco/veículo, confirmando sua viabilidade para aplicação em Terapia Fotodinâmica. A última parte do trabalho de dissertação consiste na determinação do perfil de interação cutânea do sistema a partir de estudos in vitro em modelo animal. Empregando-se protocolos de permeação e retenção cutânea, foram determinados os fluxos difusionais para, a taxa de retenção tecidual e o tempo de latência. Portanto, com estes estudos concluímos que o sistema Foscan®/NE apresenta caracterísiticas favoráveis que lhe conferem grande potencial para aplicação em TFD, dando margem para estudos in vivo e em fase clínica. / Photodynamic Therapy (PDT) is the therapeutics that comes to act in the treatment of several cancer types, with growing interest in the last decades. Acting in way a complemental the traditional therapies, PDT consists basically of the administration systemic or topical of a photosensitizer drug (FS) following for the exhibition of the lesion to the visible light resulting in the regression tumoral characterized by processes of cellular death, and, the photosensitizer drug and the luminous radiation separately don\'t induce any damage type to the lesion. The monochrome radiation used in most of the cases is of the type LASER being addressed on the lesion by a group of fiber optic. Most of the studies to the present moment they suggest a mechanism common of action for PDT. After the excitement of the PS for the states excited singlet and triplet, and for transfer of energy for the molecular oxygen (O2) it takes the death tumoral caused by species reactivate of oxygen defined like ROS (1O2, O2_ ,OH, H2O2) besides other forms radicalares (CH3, NO2, R-CO2, etc.) that attack specific centers inside of the cellular systems, unchaining the death of such woven by necrosis processes and/or cellular apoptosis. In this research work we evaluated the interaction of the photosensitizer drug Foscan® with biodegradable nanoemulsion (NE) as drug delivery system (DDS) in PDT-topical. In the first step of the work physiochemical studies were accomplished for evaluation of the thermodynamic stability and determination of some morphologic parameters. Consecutively, we determined the photophysical characterization of the drug in colloidal medium being used spectroscope techniques in the stationary state and in the time function. It was possible to determine important parameters that elucidated the photodynamic potential of the system (DDS), confirming its viability for application in PDT. The last step of the dissertation work consists of the determination of the profile of cutaneous interaction of the system starting from studies in vitro in animal model. Being used permeation protocols and cutaneous retention, they were certain the diffusion flux, the index of retention, skin up-take and the time-lag. Therefore, with these studies we ended that the system Foscan®/NE showed characteristics favorable and great potential for application in PDT-topical giving sequence to futures studies in vivo and in clinical phase.

O processo de cicatrização cutânea pode ser favorecido pela aplicação de laser de baixa potência, sendo a matriz de colágeno e elastina um substituto dérmico no tratamento de feridas descrita como um sistema adequado na engenharia tecidual em sistemas tridimensionais da pele. Com este intuito, foi elaborado um estudo utilizando o sistema nanoemulsão contendo cloro alumínio ftalocianina (ClAlPc) como o agente fotossensibilizante em biópsias de explants de pele de pacientes saudáveis irradiadas por luz proveniente de laser de baixa potência, visando estabelecer a melhor dose de luz para a bioestimulação de colágeno tipo I e elastina neste tecido. O sistema de liberação de fármacos foi sintetizado utilizando protocolos já conhecidos, cujas propriedades fotoquímicas e fotofísicas foram confirmadas por espectrofotometria de absorção e fluorescência, além de estudos de estabilidade medindo-se o tamanho das partículas, potencial Zeta e índice de polidispersão. Uma vez caracterizado este sistema e conhecendo-se a sua faixa de absorção, elaborou-se um protocolo para avaliar a ação do fármaco nos principais componentes da matriz extracelular, como colágeno e elastina, e a sua ação combinada com luz laser de baixa potência em três doses conhecidas distintas: 70, 140 e 700 mJ/cm2. Foi avaliada também a ação somente da irradiação sobre as biópsias, nas mesmas doses, podendo-se assim observar os seus efeitos. As estruturas morfológicas da pele foram estudadas por histologia, e posteriormente foram comparadas quantitativamente as porções de colágeno e elastina da pele tratada e irradiada com as amostras do controle, as quais não receberam nenhum tipo de tratamento. Tanto a análise para colágeno tipo I quanto a análise para elastina apontaram um aumento de quase 20% em relação às amostras não tratadas, utilizando a dose intermediária de 140 mJ/cm2 nas amostras tratadas com o fármaco de ftalocianina, durante um período de 14 dias após a irradiação. Este efeito foi bastante significativo, ao ser comparado com a ação somente da irradiação, que apresentou desempenho inferior. Outra técnica explorada neste trabalho e utilizada na detecção da expressão das enzimas MMP-2 e MMP-9, participantes do processo cicatricial, foi a zimografia de gelatina, utilizando o meio de cultivo de cada amostra. As bandas relacionadas à degradação da gelatina para cada amostra no zimograma foram quantificadas e os níveis de expressão de MMP-2 e MMP-9 comparados. Os resultados obtidos confirmaram a análise histológica, apontando um maior nível de expressão dessas enzimas nos grupos tratados com o fármaco fotossensibilizante e luz na dose intermediária (140 mJ/cm2) sendo esta combinação a mais promissora para um estudo mais aprofundado. / Wound healing process can be favored by Low Level Laser Therapy, with the collagen and elastin matrix a dermic substitute described as an appropriate system in tissue engineering and in tridimensional skin systems. With this purpose, a study was elaborated using the system nanoemulsion of aluminium-chloride phthalocyanine (ClAlPc) as photosensitizer in skin biopsies obtained after plastic surgery and irradiated by a low level laser, to establish the most appropriate dose of light for biostimulation of type I collagen and elastin fibers. The drug delivering system was synthesized using a well-known protocol and its photophysical and photochemical properties were confirmed by absorption and fluorescence spectrophotometry, besides stability studies measuring particle size, zeta potential and polidispersion index. Once characterized this system and known its absorption range in the UV-Vis region of light spectrum, a protocol was elaborated for evaluating the effect of the photosensitizer direct into extracellular matrix components, collagen and elastin, and the combined effect with low level laser therapy using three different doses: 70, 140 and 700 mJ/cm2. Also, only the effect of irradiation was evaluated using the same doses. Morphological structures of the skin were analyzed by histology, and portions of collagen and elastin of skin biopsies after the photosensitizer and light treatment were quantified and compared to the non-treated samples. The analysis for type I collagen and elastin pointed an increase of more than 20% compared to the non-treated samples, for the samples treated with the combination phosensitizer/light140 mJ/cm2 after 14 days of treatment. This effect was significant when compared to the effect of the irradiation only. Another technique was used in this work to detect the expression of MMP-2 and MMP-9, participant enzymes in various processes including tumoral progression and wound healing. Samples of biopsies culture medium were collected and analyzed by gelatin zymography, the bands related to gelatin degradation were quantified and MMP-2 and MMP-9 expression levels compared. The obtained results confirmed histological analysis, pointing a higher expression level of these enzymes for the group treated with the photosensitizer and the intermediate dose of light (140 mJ/cm2), leading to a promising combination of treatment for future studies.

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 pele é constituída por duas camadas: a epiderme e a derme sendo que a camada mais superficial da epiderme, o estrato córneo, camada mais superficial da pele é responsável pela sua função de barreira. Os hidratantes são formulações destinadas a manter o conteúdo de água na pele, uma vez que a hidratação é essencial para manter a função barreira íntegra. Assim, emulsões fluídas e semi-fluídas como as nanoemulsões, que apresentam glóbulos na faixa de 20-200 nm estão sendo amplamente utilizadas como veículos em produtos cosméticos por apresentarem vantagens em relação às macroemulsões, como melhor espalhabilidade e penetração, além de possuírem uma maior estabilidade cinética. O emprego de óleos vegetais como constituintes das nanoemulsões tem sido cada vez mais notório, uma vez que esses óleos são capazes de repor as frações lipídicas da pele hidratando-a. Desta forma, o presente trabalho tem como objetivo obter e caracterizar nanoemulsões O/A a base de óleo de framboesa, maracujá e pêssego e avaliar as propriedades cosméticas da formulação. A atividade antioxidante dos óleos foi avaliada por meio do método do DPPH. O EHL da fase oleosa foi então determinado utilizando-se os tensoativos lipofílicos monolaurato de sorbitano e mono-oleato de sorbitano e os tensoativos hidrofílicos polissorbato 20 , polissorbato 80 e PEG 36 castor oil combinados entre si de modo a originar 5 sistemas binários. A nanoemulsão foi obtida pela metodologia EPI (Emulsion phase inversion), utilizando-se os tensoativos lipofílicos monooleato de sorbitano e PEG-15 castor oil e os tensoativos hidrofílicos PEG-30, PEG-36, PEG-40 e PEG-54 castor oil combinados entre si, e as características físico-químicas da nanoemulsão foram determinadas. A nanoemulsão estável e com menor tamanho de glóbulo foi aditivada de (i) lanolina etoxilada e (ii) lanolina acetilada em diferentes concentrações e as alterações nas características físico-quimicas das nanoemulsões foram avaliadas. As nanoemulsões aditivadas de lanolina acetilada e lanolina etoxilada foram submetidas a testes de estabilidade preliminar e acelerada e a avaliação da influência dos derivados de lanolina na hidratação, oleosidade, valor de pH cutâneo e formação de eritema foram avaliados. A mistura dos óleos apresentou maior atividade antioxidante que os óleos separadamente. O EHL requerido para a fase oleosa da emulsão foi igual a 9 e nanoemulsões com menor tamanho de glóbulos foram obtidas com o par de tensoativos monooleato de sorbitano e PEG 36 castor oil. A lanolina tanto etoxilada quanto acetilada provocou alterações nas propriedades físico-químicas da nanoemulsão inicial. Frente aos testes de estabilidade, a formulação aditivada de lanolina etoxilada mostrou-se mais estável que a formulação aditivada de lanolina acetilada. As nanoemulsões aditivadas de derivados de lanolina provocaram maior hidratação da pele e aumento da oleosidade quando comparada a nanoemulsão não aditivada, não havendo alteração no valor de pH cutâneo e formação de eritema quando as formulações (tanto aditivadas quanto não aditivada) foram aplicadas. / The skin consists of two layers: the epidermis and dermis. The most superficial layer of the epidermis, the stratum corneum is responsible for barrier function, so moisturizing formulations are designed to maintain the water content in the skin, since hydration is essential for maintaining the barrier function intact. Thus, fluid and semi-fluid emulsions as the nanoemulsions that present droplets in the range of 20-200 nm are widely used in cosmetics as vehicles because they present advantages over macroemulsões as better spreadability and penetration, and they have greater stability kinetics. The use of vegetable oils as components of the nanoemulsion has been increasingly clear, since these oils are able to replace the lipid fractions of skin moisturizing it, and they have other activities of cosmetic concern. Thus, this study aims to obtain and characterize O/W nanoemulsions composed by raspberry, passion fruit and peach oils and evaluate the cosmetic properties of the formulation. The antioxidant activity of the oils was evaluated by the DPPH method. The HLB of the oil phase was then determined using the lipophilic surfactants sorbitan monolaurate and sorbitan monooleate and the hydrophilic surfactants polysorbate 20, polysorbate 80 and PEG 36 castor oil combined among themselves in order to create five binary systems. The nanoemulsion was obtained by the methodology EPI (Emulsion phase inversion), using the lipophilic surfactants sorbitan monooleate, PEG-15 castor oil and hydrophilic surfactants PEG-30, PEG-36, PEG-40 and PEG-54 castor oil combined among themselves, and the physicochemical characteristics of the nanoemulsion was determined. The stable nanoemulsion and with smaller droplet size was additivated (i) ethoxylated lanolin and (ii) acetylated lanolin in different concentrations and changes in physico-chemical properties of nano-emulsions were evaluated. Nanoemulsions were tested for primary and accelerated stability and the influence of derived from lanolin in hydration, skin pH, oily skin and training erythema were evaluated. The mixture of the oils showed higher antioxidant activity than oils separately. The required HLB for the oil phase of emulsion was equal to 9 and nanoemulsion with smaller droplet size were obtained with the pair of surfactants sorbitan monooleate, PEG 36 castor oil. Ethoxylated an acetylated lanolin caused changes in physico-chemical properties of nanoemulsions. The formulation additivated of ethoxylated lanolin was more stable than the formulation additivated of acetylated lanolin front accelerated stability testing. Nanoemulsions additivated of lanolin derivatives caused a higher skin hydration and increase oily skin when compared to the nanoemulsion without additives, with no change in the value of skin pH and erythema formation when the formulations (both with additives and without additives) were applied.