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Polyanionic Drugs Reduce Intracellular Bioavailability and Transfection Efficiency of Cationic siRNA Lipoplex: Quantitative Pharmacology Studies

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes, and several such agents are undergoing clinical evaluation. The present study evaluated the effects of polyanionic drugs (suramin, heparin) on the in vitro stability, cellular processing, and biological activity of Lipoplex, a complex between small interfering RNA (siRNA) and cationic liposome (50% DOTAP, 30% Cholesterol, 19% DOPE, 1% DSPE‐PEG). The resulting Lipoplex had a diameter of 195 nm and surface charge of +33.9 mV. Two siRNA, against survivin and metadherin, were used. The destabilization products of Lipoplex were separated, identified, and quantified using ultrafiltration, gel electrophoresis, and RT‐qPCR (quantitative reverse transcription polymerase chain reaction). Binding and endocytosis of Lipoplex in cells, and the amount of siRNA located in its target site RISC (RNA‐induced silencing complex) were evaluated using endocytosis markers, confocal microscopy, quantitative image analysis, immunoprecipitation, and RT‐qPCR. The results show suramin and heparin exerted several effects that were dependent on their concentrations as well as Lipoplex concentration. These agents altered the Lipoplex physical properties (i.e., reduced particle size, changed surface charge from positive to negative, removed protein‐biocorona or modified its composition). These changes are associated with Lipoplex destabilization and release of lipids and siRNA (ds/ss siRNA) and formation smaller Lipoplex with reduced siRNA content. The Lipoplex destabilization products due to suramin and heparin were different from each other and from those caused by serum proteins, both qualitatively and quantitatively. In addition, suramin and heparin prevented the cell surface binding and internalization of Lipoplex, diminished the intracellular bioavailability of siRNA in RISC, and retarded the mRNA knockdown by Lipoplex. Quantitative pharmacology analysis using experimental results and computational simulations indicated that polyanion:siRNA molar concentration ratio (Ratio) determined the relative contribution of two mechanisms to the reduction of Lipoplex‐conferred gene silencing; the Lipoplex destabilization and loss of siRNA cargo had a greater contribution compared to the inhibition of Lipoplex cellular binding and endocytosis at lower Ratio (e.g., 17:1), whereas the latter had a greater contribution at higher Ratio (e.g., 128:1). In summary, interactions with polyanionic drugs destabilize Lipoplex result in different products for different drugs, and the three‐way interactions between polyanionic drugs, Lipoplex and cell membrane binding sites diminish the Lipoplex biological activity.