Enhancing microRNA activity through increased endosomal release mediated by nigericin

Abstract

The therapeutic promise of small RNA therapeutics (siRNAs, miRNAs) is not only limited by the lack of delivery vehicles, but also by the inability of the small RNAs to reach intracellular compartments where they can be biologically active. We previously reported successful delivery of functionally active miRNAs via receptor-mediated endocytosis1. This type of targeted therapy still faces one of the major challenges in the delivery field, endosomal sequestration. Here, a new method has been developed to promote endosomal escape of delivered miRNA. The strategy relies on the difference in solute contents between nascent endosomes and the cytoplasm: early endosomes are rich in sodium ions (Na+) while the intracellular fluid is rich is potassium ions (K+). Exploiting this difference through favoring the influx of K+ into the endosomes without the exchange for a osmotically active ion (Na+), results in an osmotic differential leading to endosome swelling and bursting. One molecule that is able to exchange K+ for an osmotically inactive hydrogen ion is the ionophore nigericin. Via generating an intramolecular miRNA delivery vehicle, containing a ligand, in this case folate, and nigericin we achieve escape of folate-RNA conjugates (e.g. FolamiRs) from their entrapping endosomes into the cytoplasm where they bind the RNA Induced Silencing complex (RISC) and activate the RNAi response.