Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency

Victoria Capel,Driton Vllasaliu,Peter Watts,Snow Stolnik

doi:10.1016/j.bbrc.2016.06.054

Victoria Capel, Driton Vllasaliu + Show 2 more

Open Access

https://doi.org/10.1016/j.bbrc.2016.06.054

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Abstract

Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a ‘classical’ condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system’s silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the ‘stratified siRNA silencing vector’ requires.

Highlights

Small interfering RNA-based therapeutics hold promise in the management of a wide variety of diseases, but their full potential rests on development of clinically suitable delivery carriers [1]
Cells were pre-incubated with inhibitors for 1 h at a concentration at which high viability was preserved in H1299 cells (Supporting Information, Fig. S2) followed by incubation with Small interfering RNA (siRNA)-polyplexes in serum-free medium containing the corresponding inhibitor
We investigated the mechanisms by which siRNA-polyplexes enter different types of lung-derived epithelial cells, as means of probing the potential relationship between cell entry pathways and silencing efficiency

Summary

Introduction

Small interfering RNA (siRNA)-based therapeutics hold promise in the management of a wide variety of diseases, but their full potential rests on development of clinically suitable delivery carriers [1]. Understanding the cell entry processes of siRNA delivery systems is essential in enabling the prediction, design and optimization of transfection efficiency. The cell entry pathway of a delivery system is likely to affect intracellular processing and the silencing efficiency. Despite extensive research in developing siRNA delivery systems, understanding of the cell uptake mechanism of siRNA carriers is often inadequate, leading to an inability to predict or explain transfection efficiencies. This is important as different materials have been proposed as siRNA-. By employing well-established inhibitors of endocytosis processes in combination with cell imaging, we aimed to uncover differences in the internalization pathways and relate these to the systems’ silencing efficiency

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