Abstract
Small interfering RNA (siRNA) based RNA interference (RNAi) is an important therapeutic strategy in treatment of cancer and hereditary diseases. RNAi is composed of several important stages, the first being siRNA delivery into cells. Polymeric carriers are used in delivery to aggregate siRNA molecules into nano-scale polyplexes. Upon delivery of polyplexes into cytosol, siRNA has to be released to trigger RNAi. However, mechanism of siRNA release from polyplexes is not clear; how endogenous molecules may affect the stability of polyplexes in cytosol is yet to be explored. In this study, we performed a series of molecular dynamics (MD) simulations to study the behavior of siRNA polyplexes in the presence of microRNA (miRNA), the most abundant nucleic acid inside cells. Polyethylenimine (PEI), known as a ‘gold-standard’ among polymeric delivery vehicles, was used as the carrier. miRNAs were introduced into pre-formed siRNA-PEI polyplexes. Quantitative analysis of the MD trajectories was carried out to characterize the change in the structure, compactness and charge distribution of the polyplex upon miRNA addition. PEI affinity toward siRNA and miRNA was determined and compared through individual simulations on siRNA-PEI and miRNA-PEI complexation. Data obtained from the MD simulations was compared with gel electrophoresis assays of siRNA-PEI-miRNA mixtures as well as siRNA-PEI-heparin mixtures; the latter served as a control due to the known capability of heparin to cause siRNA release. Our results revealed the ability of miRNAs to bind to pre-formed siRNA polyplexes through electrostatic interactions with PEI nitrogens. Therefore, upon the addition of miRNA, the size of the polyplex increased and its charge became negative. In agreement with the simulations, in gel electrophoresis, at the same concentrations, heparin was able to release siRNA while polyplexes maintained their integrity in the presence of miRNA.
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