Non-viral RNAi Delivery

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Although viral RNAi delivery has proven effective in vitro, its clinical application has largely been deterred by problems associated with its oncogenic potential, lack of tumour-specific targeting, high immunogenicity and difficulty of large scale production. As a result, non-viral RNAi delivery systems, which can potentially circumvent or minimise these problems, have attracted considerable attention in recent years [77].

Several important parameters need to be considered in the design of a non-viral RNAi delivery system, including:

·      Size: Particles should be 10-100 nm in diameter to avoid rapid renal clearance [78], and to accumulate preferentially at disease sites characterised by increased vascular permeability, such as sites of infection or tumour [79, 80]. This effect is known as enhanced permeability and retention (EPR).

·      Zeta potential: Zeta potential is a measure of the magnitude of the electrostatic attraction or repulsion between particles, and is related to their surface charge. To minimize nonspecific electrostatic interactions with cells, which typically display a zeta potential within the range of -5 to -20 mV [81, 82], and to reduce macrophage uptake, a small negative zeta potential is generally desirable for nanoparticles [83].

·      Immunomasking: A highly immunogenic delivery system can trigger potentially lethal inflammatory responses in patients, as seen with adenoviruses, and also prohibits repeated dosing. This can be minimised in non-viral delivery systems via surface coverage with PEG molecules, which mask epitopes from the immune system [84].

·      Targeting: One common problem with many conventional cancer drugs in use today is a lack of specificity. They are readily internalised by healthy as well as cancer cells, thus producing side effects. To this end, ligands that bind cancer cell surface markers can be incorporated into non-viral RNAi delivery systems. These markers are typically receptors which are overexpressed on cancer cells, such as EGFR, and receptors for transferrin (Tf) and folate [77]. Targeting agents can be natural receptor ligands like Tf or synthetic ones like peptide or nucleic acid aptamers [85]. Aside from preferential binding to cancer cells, targeting ligands also offer a mechanism of cell entry by receptor-mediated endocytosis [86].

·      Endosomal escape: Following endocytosis, the delivery system and sxRNA must escape the endosome into the cytosol, where the sxRNA mediates its effect. This may be achieved by incorporating endosomal buffering ligands, such as histidine-rich peptides and polyethyleneimine. These ligands can absorb protons as they are pumped into the endosome, acting as ‘proton sponges’, and promoting further proton influx. This in turn is coupled to an increased influx of chloride ions and water, resulting in the osmotic swelling and eventual rupture of the endosome, which can then release its contents into the cytosol [87].  

A diverse range of non-viral RNAi delivery systems have been tested and some are already in advanced stages of clinical trials. A selection of some of the most promising formulations is highlighted below.