RESUMEN
PURPOSE: The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. MATERIALS AND METHODS: Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. RESULTS: Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. CONCLUSIONS: Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.
Asunto(s)
Antineoplásicos/farmacología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/metabolismo , Exosomas/metabolismo , Paclitaxel/farmacología , Animales , Animales Modificados Genéticamente , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Permeabilidad Capilar , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Modelos Animales de Enfermedad , Doxorrubicina/química , Doxorrubicina/metabolismo , Endocitosis , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Xenoinjertos , Humanos , Trasplante de Neoplasias , Paclitaxel/química , Paclitaxel/metabolismo , Tamaño de la Partícula , Tecnología Farmacéutica/métodos , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Pez CebraRESUMEN
Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.