뇌종양 치료를 위한 항암제와 siRNA 복합전달

Abstract

Glioblastoma is one of the most common and fatal malignant tumors in human, which cannot be effectively treated with the conventional chemotherapy. Thus, effective strategies are urgently needed for its treatment. Co-delivery of bis-chloroethylnitrosourea (BCNU) and VEGF small interfering RNA (VEGF-siRNA) into the glioblastoma cells by using non-toxic peptide based carrier might be effective therapeutic method. The R3V6 peptides, which are composed of 3-arginine and 6-valine, can form self-assembled micelles in aqueous solution. In addition, these peptides were able to deliver siRNA and hydrophobic anti-cancer drugs into cells. The aim of this study is to investigate the tumor suppressive effect of the complex of bis-chloroethylnitrosourea (BCNU) loaded R3V6 (R3V6-BCNU) and VEGF small interfering RNA (VEGF-siRNA). The cytotoxicity of VEGF-siRNA/R3V6-BCNU complex against C6 rat glioblastoma cells in vitro suggested that VEGF-siRNA/R3V6-BCNU complexes had higher anti-cancer effect than BCNU alone. In vitro transfection assay in rat glioblastoma cells, VEGF-siRNA formed with R3V6-BCNU had the highest transfection efficiency at a 1:20 weight ratio (VEGF-siRNA:R3V6-BCNU). In a gel retardation assay, the BCNU/R3V6 completely retarded siRNA at a 1:20 weight ratio (VEGF-siRNA:R3V6-BCNU). The heparin competition assay showed that siRNA-VEGF with BCNU/R3V6 peptide at 1:5 ratio (VEGF-siRNA:R3V6-BCNU). In an in vitro silencing assay in rat glioblasto macells, VEGF-siRNA/R3V6-BCNU complexes had higher transfection efficiency than lipofectamine or naked siRNA. The fluorescent microscopy and fluorescence activated cell sorter (FACS) were used to measure the siRNA cellular uptake efficiency in rat glioblastoma cells. The fluoresce in isothiocyanate labeled siRNA (FITC-siRNA)/R3V6-BCNU complexes had the highest uptake efficiency. These results showed the promising effect of VEGF-siRNA/R3V6-BCNU complexes.