Magneto-Fluorescent Carbon Nanotube-Mediated siRNA for Gastrin-Releasing Peptide Receptor Silencing in Neuroblastoma

Abstract

We demonstrate a newly-developed magneto-fluorescent carbon nanotube (CNT) mediated siRNA (CNT-siRNA) delivery system, which significantly silences our target of interest, gastrin-releasing peptide receptor (GRP-R), in neuroblastoma. CNT-siGRP-R resulted in a 50% silencing efficiency and a sustained efficacy of 9 days for one-time siRNA treatment in vitro, whereas siRNA delivered by the commercial transfection reagent couldn't knockdown GRP-R expression. We further show that CNT-siRNA efficiently inhibits the growth of subcutaneous xenograft tumors in vivo. This system allows us to track the CNT-siRNA distribution via both near-infrared fluorescence and magnetic resonance imaging. Moreover, our delivery system can be used to knockdown GRP-R expression in other cancer cell types, such as human breast cancer cells. The high efficiency and sustained efficacy may indicate that the natural stacking interactions between CNTs and siRNAs can protect siRNAs from degradation and enhance their stability during the delivery process.

Keywords: CNT; Fluorescence; GRP-R; MRI; Neuroblastoma; siRNA.

Figure 1

Schematic diagrams of various CNT-siRNA. (A) ssDNA (green) was absorbed on the sidewall of a CNT to suspend the CNT in solution and siRNA oligonucleotides (blue) wrapped around the CNT through non-covalent aromatic interactions. (B) An amine-terminated PEG-CNT was linked with siRNA via covalent bonds. (C) A PEG-CNT without any active functional group was conjugated to siRNA through non-covalent stacking interactions. Red lines represent PEGs.

Figure 2

Dual-modality imaging of CNTs. MRI T₂ maps of (A) PEG-CNT water suspensions with different concentration (39.50, 19.75, 9.88, 4.94, and 0.00 μg/ml) and (B) cells with (left) and without (right) CNT-siRNA treatments; (C) a fluorescence image of cells treated with CNT-siGRP-R overlaid by the corresponding optical image. The scale bar is 50 μm.

Figure 3

CNT-mediated GRP-R silencing in neuroblastoma in vitro and in vivo. (A) BE(2)-C cells were treated with LIPO-siRNA, CNT-siRNA, and naked-siRNA for 2 and 9 days, respectively. Protein expression was detected by Western blotting. GRP-R expression was significantly silenced by CNT-siGRP-R, when compared to commercial transfection reagent LIPO and naked-siRNA. Relative levels of GRP-R were calculated by densitometry and listed below each band. β-actin was used as a loading control. (B, C) CNT-siRNA was injected locally into BE(2)-C subcutaneous xenografts. Bioluminescence images were determined of mice treated with CNT-siCON or CNT-siGRP-R; CNT-siGRP-R significantly reduced the tumor size and inhibited the tumor growth. (D, E) Representative immunohistochemical staining of GRP-R in tumors treated with CNT-siCON or CNT-siGRP-R. The expression of target GRP-R (brown staining) was significantly decreased in CNT-siGRP-R treated tumor sections. (F, G) Representative H&E-stained tumor sections from mice treated with CNT-siCON or CNT-siGRP-R. (H, I) Paraffin embedded sections were stained with anti-human phospho-Histone H3 (Ser10) antibody followed by Alexa Fluor 568 Dye (Red). DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride, bue) was used for staining nucleus. CNT-siGRP-R treatments resulted in the loss of cell-cell adhesion (G) and a reduced number of mitotic cells (I), leading to decreased tumor cell proliferation and inhibition of tumor growth.

Figure 4

High efficiency in other types of cancer cells. Human breast cancer MDA-MB-231 cells were transfected with CNT-siGRP-R or CNT-siCON for 72 h (A) and 96 h (B), respectively. Target GRP-R silencing was examined by Western blotting. LIPO-siGRP-R and LIPO-siCON were used as transfection control. CNT-siRNA distinctly silenced target GRP-R at both 72 h and 96 h time points. AKT2, a downstream target of GRP-R, was significantly decreased with CNT-siGRP-R. Relative levels of GRP-R were calculated by densitometry and listed below each band. β-actin was used as a loading control.