Photochemical internalization enhances silencing of epidermal growth factor receptor through improved endosomal escape of siRNA

Abstract

Photochemical internalization (PCI) has been employed as a tool for site-specific intracellular delivery of a variety of molecules. In this study, for the first time, PCI has been employed to facilitate the endosomal escape of small interfering RNA (siRNA) molecules, which are the functional mediators of RNA interference (RNAi). In order to interact with the machinery that will induce post-transcriptional gene silencing, siRNA molecules need to enter the cytoplasm of the cells. This study shows that one of the important rate-limiting steps of siRNA silencing efficiency is the ability of siRNA molecules and/or complexes to escape from the endosomes into the cytosol of the cells. The target of this study, the epidermal growth factor receptor (EGFR), is known as an attractive target for cancer therapy. In this study, a 10-fold increased efficiency in knockdown of the EGFR protein was obtained when anti-EGFR siRNA treatment was combined with PCI as compared to siRNA treatment alone. The fact that this combined treatment resulted in a stronger silencing efficiency indicates that lower doses of siRNA can be used when PCI is employed to augment siRNA delivery. Lowering doses of siRNA would prevent saturation of the RNAi machinery and reduce off-target effects. In addition, local illumination of target tissue would only induce PCI in the desired cells, which can further increase the specificity of the treatment, supporting PCI as an attractive strategy to improve siRNA silencing efficiency.

Photochemically stimulated drug delivery increases the cytotoxicity and specificity of EGF–saporin

“…PCI in vitro has also been reported as an intracellular delivery system for peptides (Berg et al 1996, Berg et al 1999, PNAs (Shiraishi & Nielsen 2006, siRNA (Oliveira et al 2007) and some chemotherapeutics, such as bleomycin , doxorubicin , Lai et al 2007) and mitoxantrone (Adigbli et al 2007). PCI has been demonstrated in vivo with the protein toxin gelonin (Selbo et al 2001b, a nonviral p53 gene (Ndoye et al 2006), and has also been shown to increase the therapeutic effect of bleomycin .…”

Breaking down the barriers: siRNA delivery and endosome escape

“…Efficient light-directed delivery of siRNA to the cytosol has been shown by Oliveira and colleagues. The authors used PCI to facilitate endosomal escape of siRNA targeting EGFR (Oliveira et al, 2007a). Addition of a photosensitizer [meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings (TPPS 2a )] to Lipofectamine TM increased the silencing efficiency tenfold compared with this transfection reagent alone.…”

Photodynamic therapy with an endocytically located photosensitizer cause a rapid activation of the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun NH2 terminal kinase with opposing effects on cell survival

“…1). PCI has been shown to stimulate intracellular delivery of a large variety of drugs that do not readily penetrate the plasma membrane, including type I ribosome inactivation proteins, ribosome inactivation protein -based immunotoxins, and DNA delivered as gene-encoding plasmids, or by means of adenoviruses or adenoassociated viruses, peptide nucleic acids, small interfering RNA, and chemotherapeutic agents such as bleomycin (3,4). The PCI principle has also been documented in several animal models in vivo (5 -8).…”