Significance of the balance between intracellular glutathione and polyethylene glycol for successful release of small interfering RNA from gold nanoparticles

Abstract

The therapeutic promise of small interfering RNAs (siRNAs) for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery platform, such as gold nanoparticles, offers tremendous potential for treating diseases and advancing our understanding of cellular processes. Their success or failure is dependent on both the uptake of the nanoparticles into the cells and subsequent intracellular release of the functional siRNA. In this study, utilizing gold nanoparticle siRNA-mediated delivery against C-MYC, we aimed to determine if we could achieve knockdown in a cancer cell line with low levels of intracellular glutathione, and determine the influence, if any, of polyethylene glycol (PEG) ligand density on knockdown, with a view to determining the optimal nanoparticle design to achieve C-MYC knockdown. We demonstrate that, regardless of the PEG density, knockdown in cells with relatively low glutathione levels can be achieved, as well as the possible effect of steric hindrance of PEG on the availability of the siRNA for cleavage in the intracellular environment. Gold nanoparticle uptake was demonstrated via transmission electron microscopy and mass spectroscopy, while knockdown was determined at the protein and physiological levels (cells in S-phase) by in-cell westerns and BrdU incorporation, respectively.

Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells

“…This was most notable with the osteo-committed MG63s, where a 5A-mediated reduction in C-Myc expression alongside an increase in runx2 and SMAD3 indicated that the cells were perhaps reducing proliferation to focus on enhancing commitment along the osteoblast lineage ( Fig 5A ). Also with the more primitive MSCs, BMPR-2 and BMP-R1A, which are co-involved in the very early cell signalling pathways for osteogenesis [ 22 ], transcript changes may suggest early stages of osteo-commitment ( Fig 5B ).…”

“…RNAhybrid predicts the minimum free energy hybridization of two RNA sequences, allowing for miR target prediction. Hybridization is assessed by domain analysis with one sequence hybridized to the best fitting part of another [ 22 ].…”

“…Other groups have used lipofectamine to deliver miRs, however these liposomes tend to be cytotoxic and can leak their miR cargo. We believe gold nanoparticles (GNPs) are excellent candidates, as they have exceptionally low toxicity, many copies of the miR can be attached to the nanoparticle and GNPs have successfully delivered siRNA in vitro and in vivo [ 21 , 22 ]. We therefore designed thiolated antagomiRs against miR-31, with both 5’ and 3’ reading direction (5A and 3A respectively), which were conjugated onto GNPs for delivery into cells.…”

RNA interference for glioblastoma therapy: Innovation ladder from the bench to clinical trials

“…Gold nanoparticles (AuNP) are biologically inert, easily synthesized, commercially available and highly stable composites [173,174]. Over the last decade, they have gained great popularity in the biomedical field.…”

<p>Brain Targeted Gold Liposomes Improve RNAi Delivery for Glioblastoma</p>

“…29,[32][33][34] AuNPs are biologically inert, easily synthesized, commercially available, and highly stable composites. 35,36 They possess feasible characteristics that enable their application in diagnostics, imaging, and therapy. 16,32,36,37 The surface of AuNPs can enable multiple coupling with drugs, CNS-specific ligands, and oligonucleotides.…”