Lipid nanoparticles as nano-Trojan-horses for siRNA delivery and gene-knockdown

Abstract

The therapeutic messenger RNA strategies, such as those using small interfering RNAs, take several advantages (versatility, efficiency and selectivity) over plasmid DNA-based strategies. However, the challenge remains to f ind nanovectors capable of properly loading the genetic material, transporting it through troublesome environments, like a tumoral site, and delivering it into the cytoplasm of target cells. Here, lipid nanoparticles, consisting of a gemini cationic/neutral helper lipid mixture, are proposed as siRNA nanovector. Cells from cervical and brain cancer overexpressing the green fluorescent protein (GFP) were chosen to analyse the biological response as well as the efficiency and safety of the siRNA-loaded nanovector according to the cell phenotype. Flow cytometry and epifluorescence or confocal microscopy were used to follow the gene knockdown in these overexpressed cells. The effect of the nanovector on cellular proliferation was evaluated with cytotoxicity assays while their potential oxidative stress generation was determined by quantifying the generation of reactive oxygen species. To explore the mechanism of cellular uptake, different inhibitors of endocytic pathways were used during incubation with cells. Finally, nanovectors were incubated in 3D-grown cells (spheroids) to see