An assessment of gold nanoclusters in the delivery of therapeutic siRNA to breast cancer cells in vitro.

Abstract

Strategies that inhibit the expression of aberrant genes have potential in the treatment of triple-negative breast cancer (TNBC), a highly heterogeneous subtype of breast cancer that lacks targeted treatments. Small interfering RNA (siRNA)-mediated knockdown of the inflammatory cytokine tumour necrosis factor-alpha (TNF-α) and the oncogene c-MYC has emerged as a potential novel TNBC therapy. siRNA, however, requires a vector to ensure safe and successful delivery into the target cells. Gold nanoclusters (AuNC) are novel ultrasmall nanoparticles (NPs) that have been widely investigated as imaging agents due to their unique optical properties, and have recently gained attention as vectors for gene delivery. This study aimed to synthesize and functionalize AuNC and investigate their potential as vectors for the delivery of siRNA in selected breast cancer cell lines. AuNC were synthesized through glutathione reduction of chloroauric acid and functionalized with chitosan and polyethylene glycol in weight ratios of 1% and 2%. The synthesized AuNC and its functionalized counterpart (FAuNC) were characterized using UV-visible and Fourier transform infrared (FTIR) spectroscopy, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Interactions between the FAuNC and siRNA were investigated using the gel retardation, ethidium bromide intercalation, and nuclease protection assays. In vitro studies were conducted in three cell lines, the hormone receptor positive MCF-7, TNBC MDA-MB-231, and non-cancer HEK293. Cytotoxicity and cellular uptake of the FAuNC were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay and fluorescence microscopy, respectively. The in vitro effects of anti-TNF-α siRNA delivery were investigated using flow cytometry to determine the effects on the apoptosis and oxidative stress levels, and cell cycle distribution in treated cells. Knockdown efficiency was evaluated in vitro using anti-c-MYC siRNA, targeting the upregulated MYC oncogene, in MCF-7 cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the enzyme-linked immunosorbent assay (ELISA) were conducted to assess gene knockdown at the mRNA and protein levels, respectively. UV spectroscopy and TEM confirmed the synthesis of nanoclusters less than 2 nm in diameter. NTA analysis showed all FAuNC to display appropriate hydrodynamic diameters for cellular uptake and good colloidal stability. All FAuNC were capable of effectively binding and condensing siRNA, and providing protection from complete degradation by RNase A. The MTT cytotoxicity assay showed all FAuNC to be well-tolerated in all cell lines. Cellular uptake studies indicated successful interactions between FAuNC and cells, suggesting successful uptake. Flow cytometry assays showed delivery of anti-TNF-α siRNA to have little effect on the growth of HEK293 and MCF-7 cells; however, delivery in MDA-MB-231 cells was observed to lead to slight increases in apoptosis and oxidative stress levels, and slight shifts in cell cycle distribution. Based on these results, the AuCS-2% PEG were chosen as vectors for gene silencing studies. From qPCR and ELISA, the AuCS-2% PEG did not induce significant knockdown of the c-MYC gene. Overall, the FAuNC synthesized in this study showed favourable physicochemical characteristics and low cytotoxicity in vitro, but the results obtained warrant further optimization of the PEGylated FAuNC should they be considered for delivery of siRNA.