Multifunctional gold nanorods for siRNA gene silencing and photothermal therapy

Abstract

Cancer is a complex disease that usually requires several treatment modalities. A multifunctional nanotherapeutic system is designed, incorporating small interfering RNA (siRNA) and gold nanorods (Au NRs) for photothermal therapy. Surface-engineered Au NRs with polyethylenimine are synthesized using a layer-by-layer assembly and siRNA is absorbed on the surface. The siRNA is efficiently delivered into breast cancer cells, resulting in subsequent gene silencing. Cells are then irradiated with near-infrared (NIR) light, causing heat-induced anticancer activity. The combination of gene silencing and photothermal therapy results in effective inhibition of cell proliferation.

Keywords: gold nanorods; layer-by-layer assembly; photothermal therapy; siRNA.

Figure 1

Characterization of PEI-Au NRs delivery sysytem. (a) Transmission electron microscopy (TEM) images of PEI-Au NRs. (b) Size distribution of PEI-Au NRs measured by dynamic light scattering. (c) Zeta potential of particles at various stages of fabrication. Results are presented as the mean of five measurements ± standard deviation. (d) Ultraviolet visible (UV-Vis) spectra of PEI Au NRs in water solution. (e) Heat-generation kinetics of free PEI-Au NRs with serial concentrations in response to near infrared light. (f) Confocal microscope images of PEI-Au NR/siRNA complexes. Red fluorescence originates from Alexa555-conjugated siRNA.

Figure 2

Optimization and siRNA release from the PEI-Au NR/siRNA. (a) Left panel: agarose gel retardation assay of PEI-Au NRs/siRNA complexes under various weight ratios (5:1, 10:1, 15:1, 20:1, 25:1, and 30:1). Right panel: protection of siRNA against RNAse digestion and siRNA release by competitive binding sodium dodecyl sulfate (SDS) with carriers after degradation. Naked siRNA was used as a control. (b) Zeta potential changes with different weight ratios. Results are presented as the mean of five measurements ± standard deviation. (c) Profile of siRNA release from PEI-Au NR/siRNA complexes prepared at a weight ratio of 15:1.

Figure 3

Cellular uptake and transfection efficiency of PEI-Au NRs/siRNA complexes. (a) Time-dependent release of Alexa555-siRNA (red) inside MDA-MB-231 and SUM-159 breast cancer cells. The cell membrane and nuclei were stained with AF 488 Phalloidin (green) and DAPI (blue), respectively. (b) Transmission electron microscopy (TEM) images of the cellular distribution of PEI-Au NR/siRNA complexes. (c) Quantitative flow cytometry analysis of fluorescence intensity after transfection with PEI-Au NR/Alexa555-siRNA complex. The cell only was used as a control.

Figure 4

Intracellular trafficking of polyethylenimine (PEI)-gold (Au)-nanorod (NR)/small interfering RNA (siRNA) complexes. Cells were harvested at the indicated time points and stained with Lysotracker for imaging the late endosome/lysosome (in red) and DAPI for imaging the nuclei (in blue). Fluorescent images were captured with a confocal microscope.

Figure 5

Evaluation of the cytotoxicity and immunotoxicity of polyethylenimine (PEI)-gold (Au)-nanorod (NR)/small interfering RNA (siRNA) complexes. (a) Cell viability of MDA MB-231 and SUM-159 breast cancer cells was evaluated using an MTS assay. (b) Immunotoxicity was evaluated in Raw-264.7 mouse macrophage cells by measuring the levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in the supernatant by ELISA. Lipopolysaccharide (LPS) was used as a positive control. Experiments were performed in triplicates and data is presented as mean + standard deviation. **p <0.01.

Figure 6

Pyruvate kinase isoenzyme type M2 (PKM2) protein expression and cell viability in response to gene silencing and thermal therapy using polyethylenimine (PEI)-gold (Au)- nanorod (NR)/small interfering RNA (siRNA) complexes. (a) Western blot analyses of PKM2 protein expression in MDA-MB-231 and SUM-159 breast cancer cells. β-actin was used as a loading control. Oligofectamine and PEI represented positive controls. (b) Cell viability (MTS assay) in response to PEI-Au NR/siRNA and near infrared light. (c) Cell viability staining after NIR treatment of MDA-MB-231 and SUM-159cells incubated with PEI-Au NRs/siRNA and treated with NIR. Live cells were stained green with calcein AM, and dead cells were stained red with EthD-1. The boundary of NIR laser beam was marked with a white line in each well. Experiments were performed in triplicates and data is presented as mean + standard deviation. **p <0.01.

Scheme 1

Schematic illustration of the fabrication and function of PEI-Au NRs for the delivery of siRNA.