Abstract
Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.
Highlights
Small interfering RNA can be used to knock down a wide range of protein targets in viral and cancer cells, raising the possibility that siRNA can be used as a therapy for the treatment of cancers and viral infections
Both nanoparticles carrying siRNA and plasmid DNA must both escape from the endosome, plasmid DNA has to reach the nucleus in order for gene expression to occur, whereas siRNA has the advantage of only having to be released into the cytoplasm where it will be loaded onto the multi-protein RNAinducing silencing complex (RISC) thereby initiating the process of sequence-specific degradation of the mRNA targets.[4]
We have carried out a comprehensive comparative study of the siRNA delivery and gene knockdown properties of nanoparticles formulated from different cationic peptide sequences, a 1 : 1 mixture of the lipids DOTMA and DOPE, and siRNA
Summary
Small interfering RNA (siRNA) can be used to knock down a wide range of protein targets in viral and cancer cells, raising the possibility that siRNA can be used as a therapy for the treatment of cancers and viral infections. Whereas plasmid DNA can be highly condensed by complexation with cationic lipids or polymers to form lipoplexes or polyplexes, siRNA (21 base pairs) is unlikely to be significantly condensed, preferring instead to behave as a rigid rod. Both nanoparticles carrying siRNA and plasmid DNA must both escape from the endosome, plasmid DNA has to reach the nucleus in order for gene expression to occur, whereas siRNA has the advantage of only having to be released into the cytoplasm where it will be loaded onto the multi-protein RNAinducing silencing complex (RISC) thereby initiating the process of sequence-specific degradation of the mRNA targets.[4]
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