Quantitative contributions of processes by which polyanion drugs reduce intracellular bioavailability and transfection efficiency of cationic siRNA lipoplex

Abstract

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes; several RNAi are undergoing clinical evaluation in various diseases. The present study identified the relative contributions of three mechanisms by which polyanion drugs reduced the gene silencing activity of Lipoplex, a complex of small interfering RNA (siRNA) and cationic liposomes. The study used a siRNA against the chemoresistance gene survivin and two model polyanion drugs (suramin, heparin). Products of Lipoplex destabilization were separated, identified, and/or quantified using ultrafiltration, gel electrophoresis, and RT-qPCR (quantitative reverse transcription polymerase chain reaction). Cell binding and endocytosis of fluorescence-labeled Lipoplex and the amount of siRNA at its site of action 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 multiple concentration-dependent effects. First, these agents altered several Lipoplex properties (i.e., reduced particle size, changed surface charge, modified composition of protein biocorona). Second, both caused Lipoplex destabilization to release double- and single-strand siRNA and/or smaller siRNA-lipid complexes with reduced siRNA cargo. Third, both prevented the cell surface binding and internalization of Lipoplex, diminished the siRNA concentration in RISC, and retarded the mRNA knockdown. Suramin and heparin yielded qualitatively and quantitatively different results. Analysis of the experimental results of suramin using quantitative pharmacology (QP) modeling indicated the major cause of gene silencing activity loss depended on drug concentration, changing from inhibition of endocytosis at lower concentration (accounting for 60% loss at ~9μM) to inhibition of cell surface binding and loss of siRNA cargo at higher concentrations (accounting for 64% and 27%, respectively, at 70μM). In summary, the present study demonstrates the complex and dynamic interactions between polyanions and Lipoplex, and the use of QP modeling to delineate the contributions of three mechanisms to the eventual loss of gene silencing activity.

Poly(lactide-co-glycolide) Nanoparticles Mediate Sustained Gene Silencing and Improved Biocompatibility of siRNA Delivery Systems in Mouse Lungs after Pulmonary Administration

“…For LPNs, the suppressions of EGFP were 0, 17, and 45% at a siRNA concentration of 20 nM; 0, 35, and 68% at a siRNA concentration of 40 nM; 6, 55, and 81% at a siRNA concentration of 80 nM; and 10, 70, and 90% at a siRNA concentration of 120 nM, when the N/P ratios of the formulations were 5, 10, and 62, respectively (Figure A). The results exhibited that the higher the N/P ratio, the higher the efficiency of gene silencing when the concentration of siRNA of LPNs and LPXs was kept the same, which can be attributed to the high concentration of cationic lipid leading to enhanced cell surface binding and subsequent cellular internalization of siRNA . Compared to LPNs at the same N/P ratio and siRNA concentration, the enhanced gene silencing efficiency of LPXs may partly be because of the smaller size and higher zeta potential of LPXs, which could facilitate the interaction with the cell membrane and endosomal escape. , …”

“…To set the siRNA free from the bounded form, HD solution composed of 1 mg/mL of heparin and 100 mM OG was used to dissociate the nanocomplexes. Heparin is a commonly used reagent for the decomplexation of siRNA from LPXs or polyplexes. , OG is a surfactant, which contributes to the decomplexation of LPXs or polyplexes. In the spiking experiment (Supporting Information Table S1), it was proved that the addition of the HD solution in the LPX suspension could make all siRNA (including free and bounded siRNA) free to bind to RiboGreen RNA reagent to exert fluorescence.…”

“…The results exhibited that the higher the N/P ratio, the higher the efficiency of gene silencing when the concentration of siRNA of LPNs and LPXs was kept the same, which can be attributed to the high concentration of cationic lipid leading to enhanced cell surface binding and subsequent cellular internalization of siRNA. 33 Compared to LPNs at the same N/P ratio and siRNA concentration, the enhanced gene silencing efficiency of LPXs may partly be because of the smaller size and higher zeta potential of LPXs, which could facilitate the interaction with the cell membrane and endosomal escape. 34,35 Despite the higher gene silencing efficiency being achieved, the higher N/P ratio also led to lower cell viability when the concentration of siRNA of LPNs and LPXs was kept the same (Figure 2B).…”

Mechanistic Insights into the Superior DNA Delivery Efficiency of Multicomponent Lipid Nanoparticles: An In Vitro and In Vivo Study

“…This is an unusual behavior for lipid systems (e.g., Lipofectamine 3000) as lipoplexes are usually observed as single spots in the cytosol. 47 On the other hand, the behavior of LNP15 resembles that of enveloped viruses that fuse with cellular membranes to infect cells. 48 With respect to viral particles, LNP15 exploits electrostatic attractions with the plasma membrane to lower the barrier due to the repulsive "hydration force" between hydrated lipid bilayers.…”

“…Fluorescence confocal microscopy revealed that a portion of administered LNP15 underwent lysosomal degradation, while another one produced a diffuse intracellular fluorescence from labeled lipids (Figure ). This is an unusual behavior for lipid systems (e.g., Lipofectamine 3000) as lipoplexes are usually observed as single spots in the cytosol . On the other hand, the behavior of LNP15 resembles that of enveloped viruses that fuse with cellular membranes to infect cells .…”