Functionalised gold nanoparticle delivery for c-MYC siRNA cancer gene therapy.

RNA interference (RNAi), which can be induced by chemically synthesized small interfering RNA (siRNA), has emerged as a powerful tool in molecular biology for the treatment of genetic disorders.However, due to the challenges that exist in delivery of these siRNA molecules, which include poor cellular uptake and instability, an efficient, stable and non-toxic delivery vehicle needs to be designed and developed for the introduction of siRNA to target cells.Due to the rapid progress achieved in the field of nanotechnology for drug and gene delivery, gold nanoparticles (AuNPs) have presented themselves as potential candidates for the therapeutic delivery of siRNA.In this study, five cationic AuNP-based delivery systems were formulated, their cellular uptake efficiency assessed in four different mammalian cell lines in vitro and their ability to introduce the c-MYC oncogene targeted siRNA for gene silencing in a cancer cell culture model was investigated.AuNPs were synthesized using the modified Turckevich-Frens citrate reduction method and were functionalised with the natural cationic polymer, chitosan.The AuNP formulations were further modified with 0 -5 % ( w /w) of either polyethylene-glycol 400 (PEG400) or polyethylene-glycol 2000 (PEG2000), which afforded sterically stabilized AuNP formulations.The shape, size and zeta potential of the functionalised AuNPs (FAuNPs) and their corresponding nanocomplexes with siRNA were evaluated by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), with synthesis and functionalisation being confirmed by UV spectroscopy and Fourier Transformed Infra-red (FTIR) spectroscopy.The ability of these FAuNPs to bind, condense and protect siRNA were assessed using the band shift, SYBR Green II dye displacement and RNase A protection assays, respectively.In vitro cytotoxicity of the nanocomplexes was determined using the MTT, AlamarBlue® and acridine orange/ethidium bromide dual staining in the human embryonic kidney (HEK293), epithelial colorectal adenocarcinoma (Caco 2), breast adenocarcinoma (MCF-7) and colon adenocarcinoma (HT-29) cell lines.Cellular uptake studies were conducted using FITC-labelled siRNA in all 4 cell lines and the fluorescence quantitatively evaluated.The gene silencing efficiency of the FAuNP formulations were further assessed at the mRNA and protein levels by qRT-PCR and ELISA analysis following the introduction of c-MYC siRNA to the MCF-7 cell line, in vitro.All FAuNP formulations were capable of successfully binding, condensing and protecting the siRNA against degradation.The FAuNPs were well tolerated by all the cell lines being tested iii in vitro with observed cell death found to be apoptotic in nature.Enhanced cellular uptake of the siRNA nanocomplexes was observed which corresponded to successful silencing of the c-MYC gene at the mRNA level, exceeding that of commercially available Lipofectamine 3000 ® .PEG400 Chito-AuNPs were the most successful with up to a 7 -fold decrease in c-MYC expression at the mRNA level, and up to 94.6% reduction in gene expression at the protein level.Overall, these FAuNP nanocomplexes have shown great potential as vehicles for enhancing cellular uptake and c-MYC siRNA gene silencing in the tested cancer cell culture model.