Evaluation of Various Polyethylenimine Formulations for Light-Controlled Gene Silencing using Small Interfering RNA Molecules

Abstract

Photochemical internalization (PCI) is a technology based on a photosensitizer that photochemically destabilizes endosomal membranes after illumination, resulting in the release of endocytosed material into the cytosol. In this study, we investigated the potential of using polyethylenimine (PEI) for light-controlled delivery of small interfering RNA (siRNA) molecules via the endocytic pathway. PEI formulations with different molecular weights (MW) and chemical forms (linear [L]/branched [B]) were investigated for their capacity to deliver siRNA molecules with or without PCI at variable nitrogen/phosphorus (N/P) ratios and illumination doses. By targeting the S100A4 gene in an osteosarcoma cell model system, potent gene silencing was observed in samples treated with PCI compared with samples not treated with PCI. The effect of light-controlled gene silencing was dependent on several factors, including light-doses and MW, chemical form, as well as on the N/P ratio of the PEI formulations. This study demonstrates the first success in using PEI formulations as siRNA carriers for light-controlled gene silencing with the objective of future use in in vivo applications.

Cytotoxicity of polycations: Relationship of molecular weight and the hydrolytic theory of the mechanism of toxicity

“…no polycations between 2.5 and 25 kDa) and lack of LDH or similar assays make further interpretation difficult. (Boe S, 2008) In none of these cases the degree of cell membrane disruption has been assessed (i.e. by the lactose dehydrogenase assay), and since the toxicity is clearly due to the disruption and poration of cellular membranes this is clearly a major gap in our knowledge, which will be addressed in the current work.…”

A Novel Photosensitizer for Light-Controlled Gene Silencing

“…When delivering siRNA molecules, care was taken to obtain high endosomal entrapment prior to illumination of the PS. The endosmolytic capacity of B-PEI in combination with S100A4 5¢UTR siRNA molecules under various N/P ratios have been extensively validated in our previous study (Boe et al, 2008).…”

“…NM_002961) were ordered from Eurogentec. The S100A4 5¢UTR siRNA molecules (481-499; 5-UGAGCAA GUUCAAUAAAGA-3, 3-ACUCGUUCAAGUUAUUUCU-5) were chosen based on effective gene silencing in previous studies (Boe et al, 2007(Boe et al, , 2008(Boe et al, , 2010. For the S100A4 5¢UTR siRNA binding site, see Supplementary Fig.…”

Enhanced Transfection Rates of Small-Interfering RNA Using Dioleylglutamide-Based Magnetic Lipoplexes

“…Polyethylenimine (PEI), a cationic polymer that has been widely used as a nonviral vector (Boe et al, 2008), has previously been used to enhance the transfection efficiencies of magnetic nanoparticles (McBain et al, 2007), such as for the intracellular delivery of plasmid DNA (Huth et al, 2004). However, a recent study reported that PEI shows cytotoxicity and genotoxicity when used as a gene carrier (Choi et al, 2010).…”