The gene-silencing efficiency achieved 30% when the weight ratio of NP/siRNA was 4. cell viabilities were inhibited and the migration capacities were repressed remarkably, analyzed by cell counting kit-8 and transwell assay separately. In this study, we shown the PEI-coated Fe3O4 nanoparticle as a vehicle for restorative siRNA delivery, at an appropriate NP/siRNA weight percentage for REST silencing in GBM cells, inhibiting cell proliferation and migration efficiently. These might represent a novel potential treatment strategy for GBM. < 0.05 compared with the 0 group; (C) the cellular uptake of the NP/siRNA complexes in U-87 cells was verified by prussian blue staining, fluorescence labeling and circulation cytometry, P2 offered positive proportions. Pub shows 100 m. 2.3. Cellular Uptake of the NP/siRNA Complexes To verify the siRNA delivery into GBM cells from the PEI-coated Fe3O4 NPs, two human being GBM Lidocaine (Alphacaine) cell lines U-87 and U251 cells were used. The cells were incubated with NP/siRNA complexes in the percentage of 0, 2, 4, 6 and 8 Lidocaine (Alphacaine) for 6 h, respectively. The cellular uptakes of the NPs stained with prussian blue are demonstrated in Number 2C and Number 3. It was observed that almost all the cells were stained blue and darker when the NP/siRNA percentage was 4 or 6. Furthermore, 5-Carboxyfluorescein (FAM)-conjunct siRNA was used to analyze the cellular uptake of siRNA delivered from the NPs. Under the fluorescence microscope, the fluorescence labeling of siRNA was observed, indicating the cellular uptake of the siRNA. The labeling efficiencies were recognized using circulation cytometry and it showed a NP/siRNA ratio-dependent behavior. Nevertheless, the efficiencies recognized by circulation cytometry were lower than the results of prussian blue staining and fluorescence labeling. This could be due to the detection sensitivity and the quenching of fluorescein by Lidocaine (Alphacaine) NP. These results indicated efficient delivery of siRNA in to GBM cells from the PEI-coated Fe3O4 NPs. Open in a separate window Number 3 The cellular uptake of the NP/siRNA complexes in U-251 cells. The cellular uptake of the NP/siRNA complexes in U-251 cells was verified by prussian blue staining, fluorescence labeling and circulation cytometry, P2 offered positive proportions. Pub shows 100 m. 2.4. REST (Repressor Element 1-Silencing Transcription Element) Silencing Mediated by NP/siRNA Complexes To verify the gene silencing mediated by NP/siRNA complexes in GBM cells, the cells were incubated with NP/siRNA complexes in the percentage of 4 for 24 h, real-time polymerase chain reaction (PCR) and Ptgfr Western blotting were carried out. Lidocaine (Alphacaine) As demonstrated in Number 4A, the mRNA levels of REST in U-87 and U-251 cells treated with NP/siRNA focusing on REST was significantly reduced as compared to control experiments. Consistent with the tendency of realtime PCR results, Western blotting showed stronger REST knockdown in U-87 and U-251 cells (Number 4B,C), indicating that Lidocaine (Alphacaine) REST was silenced by NP/siRNA complexes primarily in transcription and translation levels. Open in a separate window Number 4 Repressor element 1-silencing transcription element (REST) silencing mediated by NP/siRNA complexes in GBM cells. (A) The mRNA levels of REST in U-87 and U-251 cells incubated with NP/siRNA complexes (in the percentage of 4 h for 24 h) focusing on REST, assayed by real-time polymerase chain reaction (PCR); (B) The protein levels in U-87 cells treated with NP/siRNA complexes focusing on REST were detected by Western blotting; (C) The protein levels in U-251 cells treated with NP/siRNA complexes focusing on REST were detected by western blotting. * < 0.05 compared with the control group. 2.5. Anti-Tumor Activity of NP/siRNA Complexes Cell viability and migration are crucial to GBM development and metastasis. The anti-tumor activity of REST-silencing mediated from the PEI-coated Fe3O4 NPs was identified using CCK-8 assay and transwell assay in U-87 and U-251 GBM cells. In the cell viability assay, the concentration of the NP/siRNA was 200 ng/50 ng. In Number 5A,B, the results of the CCK-8 assay offered significant reduction of the cell viabilities upon siRNA against REST delivery from the PEI-coated Fe3O4 NPs, both in U-87 and U-251 cells. Moreover, the cell migration capacities of U-87 and U-251 cells were significantly inhibited from the NP/siRNA complexes focusing on REST (Number 5C,D). These data have proved the PEI-coated Fe3O4 NPs like a novel delivery.
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