Trimethyl-Chitosan Coated Gold Nanoparticles Enhance Delivery, Cellular Uptake and Gene Silencing Effect of EGFR-siRNA in Breast Cancer Cells

Abstract

Purpose: Despite the promising therapeutic effects of gene silencing with small interfering RNAs (siRNAs), the challenges associated with delivery of siRNAs to the tumor cells in vivo, has greatly limited its clinical application. To overcome these challenges, we employed gold nanoparticles modified with trimethyl chitosan (TMC) as an effective delivery carrier to improve the stability and cellular uptake of siRNAs against epidermal growth factor receptor (EGFR) that is implicated in breast cancer. Methods: AuNPs were prepared by the simple aqueous reduction of chloroauric acid (HAuCl₄) with ascorbic acid and coated with synthesized TMC. EGFR-siRNA was then complexed with the AuNPs-TMC via electrostatic interaction to make AuNPs-TMC/EGFR-siRNA with a w/w ratio of 10:1. Nanoparticles were assessed for physicochemical characteristics and in vitro cellular behavior on MCF-7 breast cancer cell line. Results: Spherical and positively charged AuNPs-TMC (67 nm, +45 mV) were successfully complexed with EGFR-siRNA (82 nm, +11 mV) which were able to retard the gene migration completely. Confocal microscopy and flow cytometry analysis demonstrated complete cellular uptake of Cy5 labeled AuNPs-TMC in the MCF-7 cells after 4 h incubation. MTT test after 48 h incubation showed that the AuNPs-TMC were safe but when combined with EGFR-siRNA exert significant cytotoxicity while the cell viability was about 50%. These nanocomplexes also showed a high gene expression knockdown (86%) of EGFR and also a high apoptosis rate (Q2 + Q3 = 18.5%) after 24 h incubation. Conclusion: This study suggests that the simply synthesized AuNPs-TMC are novel, effective, and promising nanocarriers for siRNA delivery, and AuNPs-TMC/EGFR-siRNA appears to be a potential therapeutic agent for breast cancer treatment.

Keywords: EGFR-siRNA; breast cancer; gold nanoparticles (AuNPs); siRNA delivery; trimethyl chitosan (TMC).

FIGURE 1

Synthesis and characterization of the AuNPs-TMC: (A) FTIR spectrum of TMC. (B) NMR Spectrum of TMC. (C) UV–Vis spectrum of AuNPs and AuNPs-TMC. (D) TEM image of AuNPs. (E) TEM image of AuNPs-TMC. (F) SEM image of AuNPs-TMC.

FIGURE 2

Stability, release profile and cellular uptake of AuNPs-TMC/siRNA. (A) Agarose gel electrophoresis for naked siRNA and different w/w ratios of AuNPs-TMC/siRNA. (B) Release Profile of siRNA from AuNPs-TMC/siRNA in 10:1 w/w ratio in 10% FBS aqueous media at 37°C. The percentage of free siRNA was measured at 0, 1, 2, 4, 6, and 24 h time points, data are presented as mean ± SD of triplicates. Cellular uptake of AuNPs-TMC/Cy5-labeled siRNA (w/w ratio 10:1) in MCF-7 cells after 4 h incubation. (C) Confocal microscopy images (The cell nuclei were stained by DAPI). (D) Flow cytometry analysis; which is shown in histogram with the X-axis indicating the mean fluorescence intensity and the Y-axis indicating the cell count.

FIGURE 3

Therapeutic effects of AuNPs-TMC/siRNA. (A) Cell viability percentage of MCF-7 cells after 48 h incubation with AuNPs-TMC, Free siRNA and AuNPs-TMC/siRNA (w/w ratio 10:1) at different concentration of NPs (28, 56 and 112 μg/ml) and siRNA (50, 100 and 200 nM). Data is shown as mean ± SD (N = 3). Statistical analysis: Two-way ANOVA, post-test Sidak, (***p < 0.001 significantly difference between columns). (B) Relative gene expression percent of EGFR in MCF-7 cells after 24 h incubation with AuNPs-TMC/EGFR-siRNA in w/w ratio of 10:1 (56 μg/ml/100 nM) using RT-PCR method. (KD = knockdown). (C) Apoptosis assay in MCF-7 cancer cells following no treatment and treatment with AuNPs-TMC, Free siRNA and AuNPs-TMC/EGFR-siRNA (56 μg/ml/100 nM) after 24 h using annexin V-FITC/PI staining.