Avaliação do potencial de lipossomas para co-delivery de rapamicina e trans-resveratrol na terapia do câncer de mama
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Data
2022-01-28
Autores
Reis, Leidiana Rocha dos
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Universidade Estadual Paulista (Unesp)
Resumo
Considerando os principais tipos de câncer diagnosticados com mais frequência está o câncer de mama, que afeta principalmente as mulheres. Foram estimados em 2020 no Brasil, 66.280 novas ocorrências de câncer de mama. A rapamicina (RAPA) é um fármaco que pode ser empregado na terapia do câncer de mama, como inibidor da rede de sinalização mecanística do mTOR membro da família da proteína quinase relacionada à fosfatidilinositol 3-quinase (PI3K), que é comumente desregulada no câncer de mama, especialmente em tumores ER-positivos. Apesar da efetividade, este fármaco é instável, possui baixa solubilidade aquosa, podendo promover resistência no tratamento mediante sua utilização. Uma estratégia a ser utilizada na tentativa de diminuir essa resistência ao tratamento é a associação com moléculas com potencial antitumoral que atuem de forma sinérgica com a RAPA, como o trans-resveratrol (RESV), que mantém a inibição da sinalização da proteína mTORC1, ao mesmo tempo que impede a regulação positiva da ativação de AKT e a autofagia, promovendo apoptose nas células cancerígenas. Embora o RES apresente bioatividade, sua biodisponibilidade é baixa. Desta forma, considerando as características de ambas as moléculas, a coencapsulação em sistemas nanoestruturados, como lipossomas, torna-se uma interessante ferramenta, pois, não há até o momento na literatura descrita a associação destas moléculas em sistemas nanoestruturados. Neste sentido, o objetivo deste trabalho foi desenvolver, caracterizar e avaliar o potencial in vitro de lipossomas contendo RAPA e RESV na terapia do câncer de mama. Utilizando a metodologia de homogeneização de alta pressão foi possível obter lipossomas acrescidos de RAPA e RESV, associados ou não, com diâmetro hidrodinâmico na faixa de 75-99 nm. Análises de NTA e microscopia eletrônica de transmissão sugerem tamanho aproximado de 135nm e 100nm, respetivamente, para as diferentes composições estudadas. A eficiência de encapsulação (EE%) das formulações obtidas foi de aproximadamente 63 a 76%. O índice de polidispersão das formulações foi menor que 0,250 e o potencial zeta na faixa de -15,40 a -18,66 mV. O método analítico desenvolvido possibilitou analisar simultaneamente a RAPA e o RESV, apresentando-se linear, seletivo, robusto e preciso. No ensaio de liberação in vitro, após 72h de ensaio, observou-se a liberação de 21,92 ± 1,87% e 30,99 ± 3,72% de RAPA e RESV, respectivamente, associado nos lipossomas. Os ensaios de viabilidade celular evidenciaram que a formulação de lipossomas vazios não apresentou citotoxicidade frente às células MCF7 e que os lipossomas contendo os fármacos co-encapsulados apresentaram citotoxicidade maior que para os fármacos livres. Os resultados de citometria de fluxo demonstraram a internalização de 34,2% de lipossomas nas células da linhagem MCF-7. Em conjunto, as abordagens desenvolvidas no presente estudo sugerem o potencial do sistema como terapia adjuvante no tratamento do câncer de mama. Ensaios in vivo serão necessários para melhor verificação da eficácia do sistema obtido.
Abstract: Among the main types of cancers most commonly diagnosed is breast cancer, covering mainly women. In 2020, 66,280 new cases of breast cancer were estimated in Brazil. Rapamycin (RAPA) is a drug that can be used in breast cancer therapy as an inhibitor of the mTOR mechanistic signaling network, a member of the phosphatidylinositol 3-kinase protein kinase family (PI3K), which is commonly deregulated in breast cancer, especially in ER-positive tumors. Despite the effectiveness of RAPA in breast cancer, this drug is unstable, has low aqueous solubility, and can promote resistance in the treatment through its use. One strategy to be used in an attempt to reduce this resistance to treatment is the association with molecules with antitumor potential that act synergistically with RAPA. Among these molecules, trans-resveratrol (RESV) stands out, which maintains the inhibition of mTORC1 protein signaling, while preventing the upregulation of AKT activation and autophagy, promoting apoptosis in cancer cells. Despite the bioactivity of RESV, its bioavailability is low. Thus, considering the characteristics of both molecules, coencapsulation in nanostructured systems, them now there is no literature associating these molecules in nanostructured systems such as liposomes, becomes an interesting tool in antitumor therapy. In this sense, the objective of this work was to develop, characterize and evaluate the in vitro potential of multifunctional liposomes containing RAPA and RESV in breast cancer therapy. Using the high-pressure homogenization methodology, it was possible to obtain liposomes added with RAPA and RESV, associated or not, with a hydrodynamic diameter in the range of 75-99 nm. NTA and transmission electron microscopy analyzes suggest an approximate size of 135nm and 100nm, respectively, for the different compositions studied. The encapsulation efficiency (EE%) of the formulations obtained was approximately 63 to 76%. The polydispersity index of the formulations was less than 0.250 and the zeta potential ranged from -15.40 to -18.66 mV. The analytical method developed made it possible to analyze the drug RAPA and RESV simultaneously, presenting themselves as linear, selective, robust, and accurate. In the in vitro release assay after 72 hours of testing, the release of 21.92 ± 1.87% and 30.99 ± 3.72% of RAPA and RESV associated in the liposomes. Cell viability assays against a formulation of empty liposomes did not show MCF7 cytotoxicity and the liposomes loaded with the drugs showed cytotoxicity similar to the solution containing both drugs and higher than for the free drugs. In addition, flow cytometry results demonstrated the cellular internalization of 34.2% of liposomes in MCF7 cells. Taken together, the approaches developed in the present study suggest the potential of the system as adjuvant therapy in the treatment of breast cancer. In vivo assays will be necessary to better verify the efficiency of the obtained system
Abstract: Among the main types of cancers most commonly diagnosed is breast cancer, covering mainly women. In 2020, 66,280 new cases of breast cancer were estimated in Brazil. Rapamycin (RAPA) is a drug that can be used in breast cancer therapy as an inhibitor of the mTOR mechanistic signaling network, a member of the phosphatidylinositol 3-kinase protein kinase family (PI3K), which is commonly deregulated in breast cancer, especially in ER-positive tumors. Despite the effectiveness of RAPA in breast cancer, this drug is unstable, has low aqueous solubility, and can promote resistance in the treatment through its use. One strategy to be used in an attempt to reduce this resistance to treatment is the association with molecules with antitumor potential that act synergistically with RAPA. Among these molecules, trans-resveratrol (RESV) stands out, which maintains the inhibition of mTORC1 protein signaling, while preventing the upregulation of AKT activation and autophagy, promoting apoptosis in cancer cells. Despite the bioactivity of RESV, its bioavailability is low. Thus, considering the characteristics of both molecules, coencapsulation in nanostructured systems, them now there is no literature associating these molecules in nanostructured systems such as liposomes, becomes an interesting tool in antitumor therapy. In this sense, the objective of this work was to develop, characterize and evaluate the in vitro potential of multifunctional liposomes containing RAPA and RESV in breast cancer therapy. Using the high-pressure homogenization methodology, it was possible to obtain liposomes added with RAPA and RESV, associated or not, with a hydrodynamic diameter in the range of 75-99 nm. NTA and transmission electron microscopy analyzes suggest an approximate size of 135nm and 100nm, respectively, for the different compositions studied. The encapsulation efficiency (EE%) of the formulations obtained was approximately 63 to 76%. The polydispersity index of the formulations was less than 0.250 and the zeta potential ranged from -15.40 to -18.66 mV. The analytical method developed made it possible to analyze the drug RAPA and RESV simultaneously, presenting themselves as linear, selective, robust, and accurate. In the in vitro release assay after 72 hours of testing, the release of 21.92 ± 1.87% and 30.99 ± 3.72% of RAPA and RESV associated in the liposomes. Cell viability assays against a formulation of empty liposomes did not show MCF7 cytotoxicity and the liposomes loaded with the drugs showed cytotoxicity similar to the solution containing both drugs and higher than for the free drugs. In addition, flow cytometry results demonstrated the cellular internalization of 34.2% of liposomes in MCF7 cells. Taken together, the approaches developed in the present study suggest the potential of the system as adjuvant therapy in the treatment of breast cancer. In vivo assays will be necessary to better verify the efficiency of the obtained system
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Palavras-chave
Rapamicina, Resveratrol, Lipossomas, Câncer de Mama