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Authors
Advisor(s)
Abstract(s)
O cancro da próstata manifesta-se a nível global como uma das principais causas de
óbito, resultando em milhões de fatalidades anualmente. Apesar da existência de uma
ampla gama de tratamentos considerados padrão, estes estão associados a baixas taxas
de sucesso e a variados efeitos colaterais debilitantes. desta forma, continua a ser de
elevada importância desenvolver abordagens que possam melhorar a eficácia terapêutica
e superar as limitações dos tratamentos atuais.
A sinvastatina, um fármaco originalmente desenvolvido para reduzir os níveis de
colesterol no sangue, tem demonstrado potencial para exercer efeitos além do seu uso
tradicional. Vários estudos têm sugerido que a sinvastatina tem propriedades que
poderiam ser exploradas no tratamento do cancro. Esses efeitos estão intrinsecamente
relacionados com a habilidade da sinvastatina de inibir a via de síntese do colesterol,
através da sua notável inibição da enzima hidroximetilglutaril-coenzima A redutase.
Além da modulação dos níveis de colesterol, a sinvastatina parece exibir outras
propriedades notáveis (efeitos pleiotrópicas) que demonstram potencial a nível
anticancerígeno. Por exemplo, a nível da inibição da via sintética que desempenha um
papel crucial na proliferação e na sinalização celular de células tumorais e não tumorais.
O foco central deste trabalho residiu no desenvolvimento, aprimoramento, e análise da
eficácia de micro e nanoemulsões na entrega da sinvastatina, com o intuito de aperfeiçoar
a sua eficácia terapêutica. De maneira mais precisa, esta pesquisa aborda o impacto da
sinvastatina no crescimento celular das células tumorais da próstata PC-3 e DU145. Para
tal, foi realizado um ensaio inicial do impacto da sinvastatina na ausência de
transportadores nas duas linhas celulares referenciadas anteriormente. Esse ensaio foi
crucial para estabelecer um padrão de comparação com o fármaco quando formulado.
Foi realizado posteriormente otimização e desenvolvimento de micro e nanoemulsões.
Um screening inicial da concentração de polietilenoglicol 4000 (0,75%, 1% ,2%) e das
condições de armazenamento (25 °C e 4 °C) foi realizado de forma a assegurar
propriedades como o tamanho hidrodinâmico e homogeneidade (PDI, do inglês
polydispersity index) de gotículas de forma desejável. Posteriormente foi feita uma
comparação das formulações desenvolvidas, tanto neutras como catiónicas.
Os perfis in vitro de libertação do fármaco foram caracterizados com recurso a câmaras
de Ussing horizontais, sendo posteriormente avaliados os respetivos perfis cinéticos.
Realizou-se um estudo sobre o impacto da adição de a-tocoferol na estabilidade química a uma temperatura de 40 °C durante 15 dias. As quantificações do fármaco nos ensaios
de libertação degradação foram realizadas por cromatografia líquida acoplada à deteção
UV, tendo ocorrido a sua validação previa. As micro e nanoemulsões com maior potencial
para administração intravenosa foram posteriormente testadas nas linhas celulares PC3 e DU145.
Tanto as nano como microemulsões neutras com fármaco demonstraram elevada
homogeneidade (PDI = 0,1). Nanoemulsões neutras com 5,55% de sinvastina exibiram
valores de PDI de 0,134 e 0,050, com tamanhos de gotícula em torno dos 160 nm e 130
nm, antes e após, respetivamente, da refrigeração das mesmas a 4 °C. Já as
microemulsões com 8,93% de sinvastatina exibiram um PDI de 0,034, e um tamanho de
31,2 nm.
As formulações catiónicas, exibiram tamanhos similares de 29 nm e 27 nm nas
microemulsões com 2,9% e 5,95% de fármaco, e 111 nm e 104 nm nas nanoemulsões com
1,85% e 3,7 % de fármaco. O PDI foi de 0,16 e 0,04 em nanoemulsões e 0,07 e 0,06 em
microemulsões. Inesperadamente não foi possível realizar a caracterização das maiores
concentração de cada formulação de 8,93% em microemulsão e 5,55% de nanoemulsão
devido a sua precipitação apos diluição.
Durante os ensaios de libertação in vitro, verificou-se uma libertação incompleta, tendo
sido obtidas percentagens totais de libertação ao fim de 6 horas de apenas 9,2%, 16%,
9,2% e 11,60% para a nanoemulsão catiónica, microemulsão catiónica, nanoemulsão
neutra e microemulsão neutra, respetivamente. Estas baixas taxas de libertação
resultaram, em parte, da alta adsorção da sinvastatina por parte da membrana/Câmaras
de Ussing. A qual demonstrou taxas muito superiores ao esperado, sendo exibido valores
de 37,48%, 76,24%, 46,26% e 45,03% para a nanoemulsão catiónica, microemulsão
catiónica, nanoemulsão neutra e microemulsão neutra, respetivamente. Demonstrando
que, o fármaco na sua maioria permanece retido ou adsorvido relativamente a pequena
percentagem que é libertada.
Usando como matéria-prima sinvastatina já adicionada de antioxidante, formulações,
com sem adição de DL-a-Tocoferol apresentaram estabilidade química pelo menos
durante 15 dias a 40 °C.
Foi demonstrada a promoção do efeito inibitório do crescimento celular da sinvastatina
com as novas formulações comparativamente ao fármaco livre, o que demonstra a sua
eficácia como veículos para administração de fármacos. No entanto, este efeito
promissor foi observado exclusivamente na linha PC-3 à concentração de 1-3 µM, alcançando-se valores de viabilidade de 31,7% nas microemulsões e 35,2% nas
nanoemulsões, o que não foi observado na linha DU145 mantendo valores de viabilidade
>85% nos dois tipos de emulsões. O uso de transportadores permitiu a inibição do
crescimento celular com concentrações inferiores de fármaco, tendo sido demonstrando
inibições de 30,1 % com as micro e 26,8% com as nanoemulsões a apenas 3 µM. Este
nível de inibição só foi observado com o fármaco livre a concentrações de 30 µM. Em
conclusão, por meio da administração da sinvastatina utilizando micro e nanoemulsões
neutras, foi viabilizada a otimização da dose citotóxica e a obtenção de elevada inibição
tumoral quando em comparação à resultante do fármaco livre. Esses resultados
destacam o potencial promissor das nano e microemulsões como uma abordagem de
entrega terapêutica.
Prostate cancer manifests globally as one of the leading causes of mortality, resulting in millions of fatalities annually. Despite the existence of a wide range of treatments considered standard, these are associated with low success rates and various debilitating side effects. Therefore, it remains of high importance to develop approaches that can improve therapeutic effectiveness and overcome the limitations of current treatments. Sinvastatin, a drug originally developed to reduce blood cholesterol levels, has shown potential to exert effects beyond its traditional use. Several studies have suggested that simvastatin has properties that could be explored in cancer treatment. These effects are intrinsically related to simvastatin's ability to inhibit the cholesterol synthesis pathway, through its notable inhibition of the enzyme hydroxymethylglutaryl-coenzyme A reductase. In addition to modulating cholesterol levels, simvastatin appears to exhibit other notable properties (pleiotropic effects) that demonstrate anticancer potential. For example, it inhibits the synthetic pathway that plays a crucial role in the proliferation and cell signaling of both tumor and non-tumor cells. The central focus of this work was on the development, enhancement, and analysis of the effectiveness of micro and nanoemulsions in delivering simvastatin to improve its therapeutic efficacy. More precisely, this research addresses the impact of simvastatin on the cellular growth of prostate tumor cells PC-3 and DU145. For this purpose, an initial assay of simvastatin's impact in the absence of transporters in the two aforementioned cell lines was conducted. This assay was crucial to establish a benchmark for comparison with the formulated drug. Subsequently, optimization and development of micro and nanoemulsions were carried out. An initial screening of polyethylene glycol 4000 concentration (0.75%, 1%, 2%) and storage conditions (25°C and 4°C) were performed to ensure properties such as hydrodynamic size and homogeneity (Polydispersity Index, PDI) of droplets met desirable standards. A comparison of the developed formulations, both neutral and cationic, was conducted. The in vitro drug release profiles were characterized using horizontal Ussing chambers, and their respective kinetic profiles were evaluated. A study on the impact of adding atocopherol on chemical stability at a temperature of 40 °C for 15 days was conducted. Drug quantifications in degradation release assays were performed using liquid chromatography coupled with UV detection, with prior validation. The micro and nanoemulsions with the highest potential for intravenous administration were subsequently tested on the PC-3 and DU145 cell lines. Both neutral nano and microemulsions with the drug demonstrated high homogeneity (PDI = 0.1). Neutral nanoemulsions with 5.55% simvastatin exhibited PDI values of 0.134 and 0.050, with droplet sizes around 160 nm and 130 nm before and after refrigeration at 4°C, respectively. Microemulsions with 8.93% simvastatin exhibited a PDI of 0.034 and a size of 31.2 nm. The cationic formulations showed similar sizes of 29 nm and 27 nm in microemulsions with 2.9% and 5.95% drug, and 111 nm and 104 nm in nanoemulsions with 1.85% and 3.7% drug. The PDI was 0.16 and 0.04 in nanoemulsions and 0.07 and 0.06 in microemulsions. Unexpectedly, it was not possible to characterize the highest concentration of each formulation, 8.93% in microemulsion and 5.55% in nanoemulsion, due to their precipitation after dilution. During in vitro release assays, incomplete release was observed, with total release percentages after 6 hours being only 9.2%, 16%, 9.2%, and 11.60% for cationic nanoemulsion, cationic microemulsion, neutral nanoemulsion, and neutral microemulsion, respectively. These low release rates resulted, in part, from the high adsorption of simvastatin by the membrane/Ussing chambers, which exhibited much higher rates than expected, with values of 37.48%, 76.24%, 46.26%, and 45.03% for cationic nanoemulsion, cationic microemulsion, neutral nanoemulsion, and neutral microemulsion, respectively. This demonstrates that the drug mostly remains retained or adsorbed rather than being released. Using simvastatin with added antioxidant as the raw material, formulations without the addition of DL-a-tocopherol showed chemical stability for at least 15 days at 40°C. The promotion of the inhibitory effect of simvastatin on cell growth was demonstrated with the new formulations compared to the free drug, indicating their efficacy as drug delivery vehicles. However, this promising effect was observed exclusively in the PC-3 cell line at concentrations of 1-3 µM, achieving viability values of 31.7% in microemulsions and 35.2% in nanoemulsions, which was not observed in the DU145 line, maintaining viability values >85% in both types of emulsions. The use of carriers allowed the inhibition of cell growth at lower drug, demonstrating inhibitions of 30.1% with microemulsions and 26.8% with nanoemulsions at only 3 µM. This level of inhibition was only observed with the free drug at the concentration of 30 µM. In conclusion, the administration of simvastatin using neutral micro and nanoemulsions allowed for the optimization of the cytotoxic dose and achieved high tumor inhibition compared to the result of the free drug. These results highlight the promising potential of nano and microemulsions as a therapeutic delivery approach.
Prostate cancer manifests globally as one of the leading causes of mortality, resulting in millions of fatalities annually. Despite the existence of a wide range of treatments considered standard, these are associated with low success rates and various debilitating side effects. Therefore, it remains of high importance to develop approaches that can improve therapeutic effectiveness and overcome the limitations of current treatments. Sinvastatin, a drug originally developed to reduce blood cholesterol levels, has shown potential to exert effects beyond its traditional use. Several studies have suggested that simvastatin has properties that could be explored in cancer treatment. These effects are intrinsically related to simvastatin's ability to inhibit the cholesterol synthesis pathway, through its notable inhibition of the enzyme hydroxymethylglutaryl-coenzyme A reductase. In addition to modulating cholesterol levels, simvastatin appears to exhibit other notable properties (pleiotropic effects) that demonstrate anticancer potential. For example, it inhibits the synthetic pathway that plays a crucial role in the proliferation and cell signaling of both tumor and non-tumor cells. The central focus of this work was on the development, enhancement, and analysis of the effectiveness of micro and nanoemulsions in delivering simvastatin to improve its therapeutic efficacy. More precisely, this research addresses the impact of simvastatin on the cellular growth of prostate tumor cells PC-3 and DU145. For this purpose, an initial assay of simvastatin's impact in the absence of transporters in the two aforementioned cell lines was conducted. This assay was crucial to establish a benchmark for comparison with the formulated drug. Subsequently, optimization and development of micro and nanoemulsions were carried out. An initial screening of polyethylene glycol 4000 concentration (0.75%, 1%, 2%) and storage conditions (25°C and 4°C) were performed to ensure properties such as hydrodynamic size and homogeneity (Polydispersity Index, PDI) of droplets met desirable standards. A comparison of the developed formulations, both neutral and cationic, was conducted. The in vitro drug release profiles were characterized using horizontal Ussing chambers, and their respective kinetic profiles were evaluated. A study on the impact of adding atocopherol on chemical stability at a temperature of 40 °C for 15 days was conducted. Drug quantifications in degradation release assays were performed using liquid chromatography coupled with UV detection, with prior validation. The micro and nanoemulsions with the highest potential for intravenous administration were subsequently tested on the PC-3 and DU145 cell lines. Both neutral nano and microemulsions with the drug demonstrated high homogeneity (PDI = 0.1). Neutral nanoemulsions with 5.55% simvastatin exhibited PDI values of 0.134 and 0.050, with droplet sizes around 160 nm and 130 nm before and after refrigeration at 4°C, respectively. Microemulsions with 8.93% simvastatin exhibited a PDI of 0.034 and a size of 31.2 nm. The cationic formulations showed similar sizes of 29 nm and 27 nm in microemulsions with 2.9% and 5.95% drug, and 111 nm and 104 nm in nanoemulsions with 1.85% and 3.7% drug. The PDI was 0.16 and 0.04 in nanoemulsions and 0.07 and 0.06 in microemulsions. Unexpectedly, it was not possible to characterize the highest concentration of each formulation, 8.93% in microemulsion and 5.55% in nanoemulsion, due to their precipitation after dilution. During in vitro release assays, incomplete release was observed, with total release percentages after 6 hours being only 9.2%, 16%, 9.2%, and 11.60% for cationic nanoemulsion, cationic microemulsion, neutral nanoemulsion, and neutral microemulsion, respectively. These low release rates resulted, in part, from the high adsorption of simvastatin by the membrane/Ussing chambers, which exhibited much higher rates than expected, with values of 37.48%, 76.24%, 46.26%, and 45.03% for cationic nanoemulsion, cationic microemulsion, neutral nanoemulsion, and neutral microemulsion, respectively. This demonstrates that the drug mostly remains retained or adsorbed rather than being released. Using simvastatin with added antioxidant as the raw material, formulations without the addition of DL-a-tocopherol showed chemical stability for at least 15 days at 40°C. The promotion of the inhibitory effect of simvastatin on cell growth was demonstrated with the new formulations compared to the free drug, indicating their efficacy as drug delivery vehicles. However, this promising effect was observed exclusively in the PC-3 cell line at concentrations of 1-3 µM, achieving viability values of 31.7% in microemulsions and 35.2% in nanoemulsions, which was not observed in the DU145 line, maintaining viability values >85% in both types of emulsions. The use of carriers allowed the inhibition of cell growth at lower drug, demonstrating inhibitions of 30.1% with microemulsions and 26.8% with nanoemulsions at only 3 µM. This level of inhibition was only observed with the free drug at the concentration of 30 µM. In conclusion, the administration of simvastatin using neutral micro and nanoemulsions allowed for the optimization of the cytotoxic dose and achieved high tumor inhibition compared to the result of the free drug. These results highlight the promising potential of nano and microemulsions as a therapeutic delivery approach.
Description
Keywords
Sinvastatina Agente Citotóxico Cancro da Próstata Entrega Microemulsão Nanoemulsão