Lipossomas contendo moxifloxacino: citotoxicidade, permeabilidade em células intestinais, microscopia eletrônica e micronúcleo
Carregando...
Data
2022-05-20
Autores
Franzini, Maria Carolina
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A tuberculose (TB) é causada principalmente pelo Mycobacterium tuberculosis e continua
sendo uma preocupação de saúde pública em todo o mundo, principalmente devido ao
desenvolvimento de cepas resistentes aos antibióticos disponíveis e a patogênese particular da
doença. Diante desse contexto, usando a estratégia de nanoencapsular fármacos para
aprimorar sua performance biológica, foram desenvolvidos lipossomas contendo
moxifloxacino (MOX) para propor uma nova abordagem de “drug delivery” contra a TB.
Com base nisso, este trabalho teve o objetivo de avaliar a citotoxicidade destes lipossomas
com e sem moxifloxacino, LipMox e LipV, respectivamente, frente a quatro linhagens
celulares de mamíferos, sendo elas, J774.A1, MRC-5, HepG2 e Caco-2, a mutagenicidade
pelo método de micronúcleo (MN), a permeabilidade em células intestinais in vitro e a
interação dos lipossomas com osmacrófagos por meio da microscopia eletrônica de varredura
(MEV). O LipV apresentou IC50 maior que 19% (v/v) para as quatros linhagens celulares
analisadas. Já o LipMox apresentou IC50 entre 12 e 23% (v/v). Esses resultados mostram que
tanto o LipV quanto o LipMox são seguros frente às linhagens celulares analisadas. O ensaio
de MN foi realizado em células HepG2 e os resultados obtidos mostraram que o índice de
divisão celular (IDN) foi entre 1,39 ± 0,06 e 1,74 ± 0,02 e o número de MN foi entre 1,60 ±
1,07 e 17,31 ± 7,74 por mil células binucleadas. Os tratamentos não apresentaram diferença
significativa em relação ao controle sem tratamento, demonstrando que as amostras não foram
genotóxicas. Já a avaliação da permeabilidade intestinal foi realizada por meio do modelo
Caco-2, sendo que os resultados dessa análise, foram obtidos pela quantificação de MOX por
Cromatografia Líquida de Alta Eficiência (HPLC), e mostraram que não ocorreu
permeabilidade do fármaco na forma lipossomal (LipMox) e nem livre, enquanto as imagens
da microscopia de fluorescência mostram uma monocamada de células íntegras e lipossomas
sobre as células. Por fim, as imagens de MEV demonstraram a interação dos macrófagos com
os lipossomas, indicando que a fagocitose dos lipossomas pelos macrófagos, que são células
muito importantes na patogênese de TB. Portanto, conclui-se que os métodos empregados e os
resultados obtidos neste trabalho permitiram analisar a interação entre células eucarióticas e
lipossomas e foram de fundamental importância para determinar o potencial dessa
nanoestrutura para futura aplicação.
Tuberculosis (TB) is primarily caused by Mycobacterium tuberculosis and remains a public health concern worldwide, mainly due to the development of resistan strains to available antibiotics and the particular pathogenesis of the disease. In this context, using the strategy of nanoencapsulating drugs to improve their biological performance, liposomes containing moxifloxacin (MOX) were developed to propose a new drug delivery approach against TB. Based on this, this study aimed to evaluate the cytotoxicity of these liposomes with and without moxifloxacin, LipMox and LipV, respectively, against four mammalian cell lines, namely, J774.A1, MRC-5, HepG2 and Caco-2, mutagenicity by the micronucleus (MN) method, permeability in intestinal cells in vitro and the interaction of liposomes with macrophages by means of scanning electron microscopy (SEM). LipV presented IC50 greater than 19% (v/v) for the four cell lines analyzed. LipMox presented IC50 between 12 and 23% (v/v). These results show that both LipV and LipMox are safe against the cell lines analyzed. The MN test was performed on HepG2 cells and the results obtained showed that the cell division index (NDI) was between 1.39 ± 0.06 and 1.74 ± 0.02 and the number of MN was between 1.60 ± 1.07 and 17.31 ± 7.74 per thousand binucleated cells. The treatments showed no significant difference in relation to the untreated control, demonstrating that the samples were not genotoxic. The evaluation of intestinal permeability was performed using the Caco-2 model, and the results of this analysis, obtained by the quantification of MOX by High Performance Liquid Chromatography (HPLC), showed that there was no permeability of MOX in the liposomal (LipMox) or free form, while the Fluorescence microscopy images show a monolayer of intact cells and liposomes on top of the cells. Finally, SEM images showed the interaction of macrophages with liposomes, indicating that phagocytosis of liposomes by macrophages, which are cells very important in the pathogenesis of TB. Therefore, it is concluded that the methods used and the results obtained in this work allowed analyzing the interaction between eukaryotic cells and liposomes and were of fundamental importance to determine the potential of this nanoparticle for future administration.
Tuberculosis (TB) is primarily caused by Mycobacterium tuberculosis and remains a public health concern worldwide, mainly due to the development of resistan strains to available antibiotics and the particular pathogenesis of the disease. In this context, using the strategy of nanoencapsulating drugs to improve their biological performance, liposomes containing moxifloxacin (MOX) were developed to propose a new drug delivery approach against TB. Based on this, this study aimed to evaluate the cytotoxicity of these liposomes with and without moxifloxacin, LipMox and LipV, respectively, against four mammalian cell lines, namely, J774.A1, MRC-5, HepG2 and Caco-2, mutagenicity by the micronucleus (MN) method, permeability in intestinal cells in vitro and the interaction of liposomes with macrophages by means of scanning electron microscopy (SEM). LipV presented IC50 greater than 19% (v/v) for the four cell lines analyzed. LipMox presented IC50 between 12 and 23% (v/v). These results show that both LipV and LipMox are safe against the cell lines analyzed. The MN test was performed on HepG2 cells and the results obtained showed that the cell division index (NDI) was between 1.39 ± 0.06 and 1.74 ± 0.02 and the number of MN was between 1.60 ± 1.07 and 17.31 ± 7.74 per thousand binucleated cells. The treatments showed no significant difference in relation to the untreated control, demonstrating that the samples were not genotoxic. The evaluation of intestinal permeability was performed using the Caco-2 model, and the results of this analysis, obtained by the quantification of MOX by High Performance Liquid Chromatography (HPLC), showed that there was no permeability of MOX in the liposomal (LipMox) or free form, while the Fluorescence microscopy images show a monolayer of intact cells and liposomes on top of the cells. Finally, SEM images showed the interaction of macrophages with liposomes, indicating that phagocytosis of liposomes by macrophages, which are cells very important in the pathogenesis of TB. Therefore, it is concluded that the methods used and the results obtained in this work allowed analyzing the interaction between eukaryotic cells and liposomes and were of fundamental importance to determine the potential of this nanoparticle for future administration.
Descrição
Palavras-chave
tuberculose, citotoxicidade, avaliação toxicológica, micronúcleo, nanotecnologia farmacêutica, lipossomas, permeabilidade intestinal, microscopia eletrônica, “drug delivery”