New Mechanism for Release of Endosomal Contents: Osmotic Lysis via Nigericin-Mediated K⁺/H⁺ Exchange

Abstract

Although peptides, antibodies/antibody fragments, siRNAs, antisense DNAs, enzymes, and aptamers are all under development as possible therapeutic agents, the breadth of their applications has been severely compromised by their inability to reach intracellular targets. Thus, while macromolecules can often enter cells by receptor-mediated endocytosis, their missions frequently fail due to an inability to escape their entrapping endosomes. In this paper, we describe a general method for promoting release of any biologic material from any entrapping endosome. The strategy relies on the fact that all nascent endosomes contain extracellular (Na+-enriched) medium, but are surrounded by intracellular (K+-enriched) fluid in the cytoplasm. Osmotic swelling and rupture of endosomes will therefore be facilitated if the flow of K+ down its concentration gradient from the cytosol into the endosome can be facilitated without allowing downhill flow of Na+ from the endosome into the cytosol. While any K+ selective ionophore can promote the K+ specific influx, the ideal K+ ionophore will also exchange influxed K+ for an osmotically inactive proton (H+) in order to prevent buildup of an electrical potential that would rapidly halt K+ influx. The only ionophore that catalyzes this exchange of K+ for H+ efficiently is nigericin. We demonstrate here that ligand-targeted delivery of nigericin into endosomes that contain an otherwise impermeable fluorescent dye can augment release of the dye into the cell cytosol via swelling/bursting of the entrapping endosomes. We further show that nigericin-facilitated escape of a folate-targeted luciferase siRNA conjugate from its entrapping endosomes promotes rapid suppression of the intended luciferase reporter gene. Taken together, we propose that ionophore-catalyzed entry of K+ into endosomal compartments can promote the release of otherwise impermeable contents from their encapsulating endosomes.

Enhancing microRNA activity through increased endosomal release mediated by nigericin

“…We reasoned that the inclusion of an inter-molecular endosomal escape mechanism could ensure robust cytosolic delivery of the RNA cargo and lead to enhanced miRNA activity. This approach relies on the use of a small molecule, nigericin, that upon cellular internalization gets cleaved from the folate carrier and localizes to endosomal membrane where it exchanges K + and water for osmotically inactive proton (H + ) causing endosomal swelling 15 . To be effective, nigericin release has to occur in a controlled manner and only within the endosomes.…”

“…We recently described a novel method to facilitate the escape of biologics from entrapping endosomes and in that study provided preliminary evidence for the potential use of this method to improve the escape of small RNAs from endosomes 15 . The strategy relies on the difference in solute contents between nascent endosomes and the cytoplasm: endosomes are rich in sodium ions (Na + ) while the intracellular fluid is rich is potassium ions (K + ).…”

“…The strategy relies on the difference in solute contents between nascent endosomes and the cytoplasm: endosomes are rich in sodium ions (Na + ) while the intracellular fluid is rich is potassium ions (K + ). The method exploits this difference by using a small molecule, nigericin 16,17 , that exchanges K + for osmotically inactive proton (H + ) to achieve balance in charge without release of a compensatory solute (Na + ) thus causing a osmotic differential that leads to endosome swelling and bursting 15 .…”

“…Authentication of MB-23, KB, and A549 cells was performed using short tandem repeat profiling [American Type Culture Collection (ATCC)].Preparation of pyridyldisulfide amide derivative of nigericinFolate-ethylediamine (EDA), folate-cys and folate-dybenzocyclooctyne (DBCO) synthesis was performed as previously described 1 . Folate-nigericin synthesis was performed as described in15 , nigericin free acid (0.035 mmol), Py-SS-(CH 2 ) 2 NH 2 (0.052 mmol), hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU; 0.052 mmol), and N,N-Diisopropylethylamine (DIPEA; 0.069 mmol) were dissolved in anhydrous CH 2 Cl 2 (2.0 mL) and stirred under argon at room temperature overnight. Progress of the reaction was monitored by liquid chromatographymass spectrometry (LC-MS).…”

“…Progress of the reaction was monitored by LC-MS. After complete conversion of folate-nigericin, the crude reaction mixture was purified by RP-HPLC, (mobile phase A = 10 mM ammonium acetate, pH = 7; organic phase B = acetonitrile; method: 0% B to 40% B in 35 min at 13 ml/min) and furnished folate-nigericin 65% yield. LC-MS (A = 10 mM ammonium bicarbonate, pH = 7; organic phase B = acetonitrile; method: 0% B to 100% B in 7 min) RT = 4.0 min (M +H + = 1440.0).Preparation of folate-nigericin-DBCOFolate-nigericin-DBCO synthesis was performed as previously described15 : N,N-Diisopropylethylamine (DIPEA; 0.0013 g, 0.010 mmol, 1.5 eq.) was added dropwise to a stirred solution of folate-nigericin (0.010 g, 0.0069 mmol, 1 eq.)…”