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Tangential flow filtration for isolating exomeres and other nanoscale extracellular particles
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DOI：10.1039/D6NR01765G.png)
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Abstract
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Extracellular particles; including extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs); enable intercellular communication by transferring regulatory miRNAs and other biomolecules. While EVs have been studied for drug delivery; NVEPs remain relatively unexplored. Exomeres; a recently discovered class of NVEPs enriched in RNAi proteins; preferentially carry miRNAs and deliver them to cells more effectively than EVs; underscoring their potential as vehicles for therapeutic RNAs. One current limitation to studying and applying exomeres for therapeutic RNA delivery is the lack of scalable; cost-effective; and rapid isolation methods. Here; we investigated whether tangential flow filtration (TFF); a common bioseparation approach that separates species by size; would effectively isolate exomeres from conditioned media with comparable purity and identity to exomeres isolated by differential ultracentrifugation. TFF successfully isolated exomeres that were enriched in RNAi components including Argonaute-2 (AGO2); heat shock protein (HSP)90AB1; and a unique set of miRNAs not abundant in EVs. Remarkably; exomere-encapsulated miRNAs were resistant to nuclease degradation even after treatment with protease and surfactant; suggesting that exomeres are highly stable; non-vesicular complexes with potentially extended circulating half-lives. Together; our results establish TFF as an efficient bench-scale method for isolating exomeres; and further demonstrate that TFF could potentially be applied in a bioprocess for exomere-based RNA therapeutic production. This study is also the first to demonstrate that exomere miRNAs are highly resistant to nuclease degradation; suggesting that exomeres could complement and potentially outperform current clinical standards for RNA delivery.
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