Combinatorial microRNA Loading into Extracellular Vesicles for Increased Anti-Inflammatory Efficacy

Abstract

Extracellular vesicles (EVs) have emerged as promising therapeutic entities in part due to their potential to regulate multiple signaling pathways in target cells. This potential is derived from the broad array of constituent and/or cargo molecules associated with EVs. Among these, microRNAs (miRNAs) are commonly implicated as important and have been associated with a wide variety of EV-induced biological phenomena. While controlled loading of single miRNAs is a well-documented approach for enhancing EV bioactivity, loading of multiple miRNAs has not been fully leveraged to maximize the potential of EV-based therapies. Here, an established approach to extrinsic nucleic acid loading of EVs, sonication, was utilized to load multiple miRNAs in HEK293T EVs. Combinations of miRNAs were compared to single miRNAs with respect to anti-inflammatory outcomes in assays of increasing stringency, with the combination of miR-146a, miR-155, and miR-223 found to have the most potential amongst the tested groups.

Harnessing Extracellular microRNAs for Diagnostics and Therapeutics in Acute Systemic Inflammation

“…Since the discovery of miRNA in 1993 and extracellular miRNA in the 2000s, novel discoveries continue to emerge and expand our understanding of the impact miRNA can have not only on intracellular activity but also on cell-cell communication and immune regulation [ 1 , 11 , 52 ]. With a greater understanding of miRNA’s function outside of the cell, researchers have evaluated several different species of miRNA as both diagnostic biomarkers of inflammation and targets for the treatment of diseases [ 10 , 40 , 77 , 79 , 80 , 81 , 82 , 98 ].…”

“…Extracellular miRNAs can be combined (e.g., loaded into the same vesicle) to have different effects from each individual miRNA on its own [ 77 ]. Pottash et al combined miR-146a,-155,-223 to reduce inflammation in LPS-treated macrophages and mice [ 77 ].…”

“…Extracellular miRNAs can be combined (e.g., loaded into the same vesicle) to have different effects from each individual miRNA on its own [ 77 ]. Pottash et al combined miR-146a,-155,-223 to reduce inflammation in LPS-treated macrophages and mice [ 77 ]. While these miRNAs can have pro-inflammatory effects individually, there is an observed synergistic anti-inflammatory effect when all three are loaded into the same vesicle, evidenced by a reduction in IL-6 and other inflammatory markers [ 33 , 77 , 78 , 79 ].…”

“…Pottash et al combined miR-146a,-155,-223 to reduce inflammation in LPS-treated macrophages and mice [ 77 ]. While these miRNAs can have pro-inflammatory effects individually, there is an observed synergistic anti-inflammatory effect when all three are loaded into the same vesicle, evidenced by a reduction in IL-6 and other inflammatory markers [ 33 , 77 , 78 , 79 ]. However, other proinflammatory cytokines had variable expression.…”

MicroRNA Nano-Shuttles: Engineering Extracellular Vesicles as a Cutting-Edge Biotechnology Platform for Clinical Use in Therapeutics

“…Other direct mechanistic approaches to destabilize the bilipid membrane of EVs are predominantly performed through sonication and electroporation. For instance, recent reports from Pottash et al used sonication-mediated EV loading techniques to incorporate anti-inflammatory miRNAs (miR-146a, miR-155, and miR-223) into HEK293T EVs for downstream use in inflammation-related diseases [ 96 ]. The focus of sonication is on disrupting the membrane rigidity and microviscosity through ultrasound waves, while the electroporation of EVs is highly dependent upon their subjection to a high-voltage pulsing that dismembers its pores.…”