Avaliação do potencial de carreadores lipídicos nanoestruturados funcionalizados com ácido fólico para incorporação de rapamicina no tratamento do câncer de mama
Carregando...
Data
2021-12-10
Autores
Rodero, Camila Fernanda
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O câncer de mama é o segundo tipo de câncer mais comum no mundo. Dentre os fármacos candidatos à utilização no tratamento deste tipo de câncer tem-se a rapamicina que é capaz de inibir a proteína mTOR, controlando seu crescimento e progressão no ciclo celular. No entanto, é um fármaco lipofílico, sofre sequestro por eritrócitos e dispõe de instabilidade química, fatores que comprometem sua biodisponibilidade. Desta maneira, sua incorporação em carreadores lipídicos nanoestruturados funcionalizados com ácido fólico pode ser uma estratégia interessante, pois este sistema pode proteger o fármaco de possíveis degradações, além de proporcionar ação mais lenta e sustentada. A funcionalização dos carreadores com ácido fólico possibilita a liberação do fármaco no local-alvo mediante ligação com receptores de folato que são expressos em grande quantidade por uma variedade de células tumorais, dentre elas as de mama. Frente ao exposto, o objetivo foi desenvolver e caracterizar carreadores funcionalizados com ácido fólico para incorporação de rapamicina e avaliar o seu potencial no tratamento do câncer de mama. Foram desenvolvidos seis diferentes carreadores, que apresentaram valores de diâmetro hidrodinâmico médio de 97,29±0,79 a 119,05±0,23 nm respectivamente, e valores de potencial zeta de -12,45±0,48 a -28,75±0,16 mV, respectivamente. Todas as formulações apresentaram morfologia esférica e em nanoescala, segundo análises de microscopia eletrônica de transmissão. As analises térmicas apresentam um evento de fusão em 50 ºC, o qual é endotérmico, mostrando um novo composto. A funcionalização do lipídio com ácido fólico foi confirmada por análises de espectrofotometria no ultravioleta visível, por ressonância magnética nuclear e espectroscopia vibracional na região do infravermelho. A quantificação da rapamicina foi realizada por cromatografia liquida de alta eficiência, mostrando estar dentro dos parâmetros requeridos e preconizados pela ANVISA e ICH, apresentando linearidade de R2=0,99, precisão e robustez. Os ensaios de eficiência de encapsulação evidenciaram que o carreador consegue encapsular cerca de 95% do fármaco. O ensaio de liberação evidenciou para os carreadores funcionalizados a liberação de 82% de rapamicina após 16 horas, já o carreador não funcionalizado liberou cerca de 45% do fármaco no mesmo período. Os ensaios in vitro de citotoxicidade evidenciaram que carreadores não apresentaram toxicidade frente às linhagens celulares LEWIS, MCF-7 e MCF10-A. Para o sistema, observou-se IC50 de 84, 176 e 110nM, respectivamente e para a rapamicina livre o IC50 frente às mesmas linhagens celulares foram 81,8, 141 e 95nM, respectivamente. A microscopia confocal evidenciou a internalização celular do sistema incorporada com DiO em linhagens MCF-7 e LEWIS. Os dados de citometria de fluxo corroboram os de microscopia confocal, sugerindo que a internalização da formulação. O modelo alternativo de Caenorhabditis elegans, demonstrou que a formulação não apresenta toxicidade. O modelo xenográfico in vivo de câncer de mama evidenciou que sistema, tanto administrado pela via intravenosa quanto intraperitoneal, no período de 18 dias de tratamento, levou a regressão do tumor, apresentando diferença estatística em relação aos controles. Os resultados sugerem que o carreador contendo rapamicina e funcionalizado com ácido fólico apresenta potencial promissor para o tratamento de câncer de mama.
Breast cancer is the second most common type of cancer in the world. Among the candidate drugs for use in the treatment of this type of cancer is rapamycin, which is capable of inhibiting the mTOR protein, controlling its growth and progression in the cell cycle. However, it is a lipophilic drug, undergoes sequestration by erythrocytes and has chemical instability, factors that compromise its bioavailability. Thus, its incorporation in nanostructured lipid carriers functionalized with folic acid can be an interesting strategy, as this system can protect the drug from possible degradation, in addition to providing slower and more sustained action. Functionalization of carriers with folic acid enables drug release at the target site by binding to folate receptors that are expressed in large amounts by a variety of tumor cells, including breast cells. Based on the above, the objective was to develop and characterize carriers functionalized with folic acid for incorporation of rapamycin and to evaluate its potential in the treatment of breast cancer. Six different carriers were developed, which presented mean hydrodynamic diameter values from 97.29±0.79 to 119.05±0.23 nm respectively, and zeta potential values from -12.45±0.48 to -28, 75±0.16 mV, respectively. All formulations showed spherical and nanoscale morphology, according to transmission electron microscopy analyses. Thermal analysis shows a fusion event at 50°C, which is endothermic, showing a new compound. The functionalization of the lipid with folic acid was confirmed by spectrophotometric analysis in the visible ultraviolet, by nuclear magnetic resonance and vibrational spectroscopy in the infrared region. Quantification of rapamycin was performed by high-performance liquid chromatography, showing that it was within the parameters required and recommended by ANVISA and ICH, with linearity of r2=0.99, precision and robustness. Encapsulation efficiency tests showed that the carrier can encapsulate about 95% of the drug. The release assay showed for the functionalized carriers the release of 82% of rapamycin after 16 hours, while the non-functionalized carrier released about 45% of the drug in the same period. In vitro cytotoxicity assays showed that carriers did not show toxicity against LEWIS, MCF-7 and MCF10-A cell lines. For the system, it was observed IC50 of 84, 176 and 110nM, respectively, and for free rapamycin the IC50 against the same cell lines were 81.8, 141 and 95nM, respectively. Confocal microscopy evidenced the cellular internalization of the system incorporated with DiO in MCF-7 and LEWIS lines. Flow cytometry data corroborate those from confocal microscopy, suggesting the internalization of the formulation. The alternative model of Caenorhabditis elegans demonstrated that the formulation does not present toxicity. The in vivo xenographic model of breast cancer showed that the system, both administered intravenously and intraperitoneally, within 18 days of treatment, led to tumor regression, showing a statistical difference in relation to controls. The results suggest that the carrier containing rapamycin and functionalized with folic acid has promising potential for the treatment of breast cancer.
Breast cancer is the second most common type of cancer in the world. Among the candidate drugs for use in the treatment of this type of cancer is rapamycin, which is capable of inhibiting the mTOR protein, controlling its growth and progression in the cell cycle. However, it is a lipophilic drug, undergoes sequestration by erythrocytes and has chemical instability, factors that compromise its bioavailability. Thus, its incorporation in nanostructured lipid carriers functionalized with folic acid can be an interesting strategy, as this system can protect the drug from possible degradation, in addition to providing slower and more sustained action. Functionalization of carriers with folic acid enables drug release at the target site by binding to folate receptors that are expressed in large amounts by a variety of tumor cells, including breast cells. Based on the above, the objective was to develop and characterize carriers functionalized with folic acid for incorporation of rapamycin and to evaluate its potential in the treatment of breast cancer. Six different carriers were developed, which presented mean hydrodynamic diameter values from 97.29±0.79 to 119.05±0.23 nm respectively, and zeta potential values from -12.45±0.48 to -28, 75±0.16 mV, respectively. All formulations showed spherical and nanoscale morphology, according to transmission electron microscopy analyses. Thermal analysis shows a fusion event at 50°C, which is endothermic, showing a new compound. The functionalization of the lipid with folic acid was confirmed by spectrophotometric analysis in the visible ultraviolet, by nuclear magnetic resonance and vibrational spectroscopy in the infrared region. Quantification of rapamycin was performed by high-performance liquid chromatography, showing that it was within the parameters required and recommended by ANVISA and ICH, with linearity of r2=0.99, precision and robustness. Encapsulation efficiency tests showed that the carrier can encapsulate about 95% of the drug. The release assay showed for the functionalized carriers the release of 82% of rapamycin after 16 hours, while the non-functionalized carrier released about 45% of the drug in the same period. In vitro cytotoxicity assays showed that carriers did not show toxicity against LEWIS, MCF-7 and MCF10-A cell lines. For the system, it was observed IC50 of 84, 176 and 110nM, respectively, and for free rapamycin the IC50 against the same cell lines were 81.8, 141 and 95nM, respectively. Confocal microscopy evidenced the cellular internalization of the system incorporated with DiO in MCF-7 and LEWIS lines. Flow cytometry data corroborate those from confocal microscopy, suggesting the internalization of the formulation. The alternative model of Caenorhabditis elegans demonstrated that the formulation does not present toxicity. The in vivo xenographic model of breast cancer showed that the system, both administered intravenously and intraperitoneally, within 18 days of treatment, led to tumor regression, showing a statistical difference in relation to controls. The results suggest that the carrier containing rapamycin and functionalized with folic acid has promising potential for the treatment of breast cancer.
Descrição
Palavras-chave
câncer de mama, carreadores lipídicos nanoestruturados, funcionalização, ácido fólico, rapamicina, breast cancer, nanostructured lipid carriers, rapamycin