Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide

Abstract

The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

Keywords: C-X-C chemokine receptor type 4 (CXCR4); Cancer cell migration; Graphene oxide (GO); Polyethylenimine (PEI); Small interfering RNA (siRNA).

Fig. 1

Transmission electron microscopy images of pristine GO and PEI-GO. The surface morphology of pristine GO (a, b) was compared with that of PEI-GO (c, d) by a JEOL 2000FX TEM at different scales

Fig. 2

Scanning electron microscopy images of pristine GO and PEI-GO. The surface morphology of pristine GO (a, c, e) was compared with that of PEI-GO (b, d, f) by a JSM-6500 F SEM at different scales

Fig. 3

Average particle size and zeta potential of PEI-GO analyzed by dynamic light scattering. a The average particle diameter of 1 mg/ml PEI-GO was compared at 4, 25, and 37 °C. b The zeta potential of 1 mg/ml PEI-GO was compared to that off pristine GO at 25 °C and neutral pH. Error bars represent standard deviations (n ≥ 3)

Fig. 4

Binding capacity of PEI-GO toward siRNA. PEI-GO was reacted with Dharmacon siGENOME GAPD control siRNA at various mass ratios, followed by electrophoretic mobility shift assay (EMSA)

Fig. 5

Cytotoxicity of PEI-GO in MDA-MB-231 cells. Human breast carcinoma cells MDA-MB-231 were treated with 0–20 μg/ml of PEI-GO for 48 h. Cell viability was determined by WST-1 assay and quantitated spectrophotometrically by measuring the optical density at 450 nm, with a reference wavelength of 650 nm. Error bars represent standard deviations (n ≥ 3). *p < 0.05 and **p < 0.01 compared to the control

Fig. 6

Relative CXCR4 mRNA expression of MDA-MB-231 cells transfected with PEI-GO:siCXCR4 complexes. PEI-GO was incubated with siCXCR4 at mass ratios of 0.3:1, 0.5:1, and 1:1 for 20 min at room temperature before cultured with MDA-MB-231 cells to achieve a final siCXCR4 concentration of 25 nM. Two days after siRNA transfection, cells were harvested for RNA extraction and real-time PCR analysis. Control, MDA-MB-231 cells cultured in growth medium for 48 h; Lipofectamine, MDA-MB-231 cells transfected with siCXCR4 using Lipofectamine 2000 as transfection reagent; PEI-GO:siMOCK, MDA-MB-231 cells transfected with PEI-GO:siMOCK at a mass ratio of 1:1. Error bars represent standard deviations (n ≥ 3). *p < 0.05 and **p < 0.01 compared to the control; ^## p < 0.01 compared to Lipofectamine

Fig. 7

Wound healing assay of MDA-MB-231 cells transfected with PEI-GO:siCXCR4 complexes. MDA-MB-231 cells were allowed to migrate over a cell-free gap (bordered by the pair of black lines) after treated with PEI-GO:siCXCR4 complexes of mass ratios 0.3:1 (d), 0.5:1 (e), and 1:1 (f) for 48 h. The results were compared with untreated cells (a) and those treated with Lipofectamine 2000:siCXCR4 (b) or PEI-GO:siMOCK (c). Migrated cells per field from three independent experiments were quantitated as shown in the bar graph (g)