Exogenous loading of miRNAs into small extracellular vesicles

Abstract

Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently‐labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent‐based compound (saponin) or cholesterol‐modification of the miRNA, Exo‐Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT‐PCR data showed that, compared with native sEVs, Exo‐Fect modulation led to a > 1000‐fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR‐155‐5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo‐Fect‐loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo‐Fect‐loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.

Neurotoxic Astrocytes Directly Converted from Sporadic and Familial ALS Patient Fibroblasts Reveal Signature Diversities and miR-146a Theragnostic Potential in Specific Subtypes

“…We were particularly interested in the content of miRNAs in sEVs, since they are promising biomarkers and potential delivery carriers of miRNA mimics and inhibitors [ 37 , 67 , 68 , 69 ]. In fact, miRNA inclusion in sEVs is more stable than its free species [ 68 ], and sEVs are known to travel longer distances and to cross the blood–brain barrier [ 70 ].…”

Endogenous Controls for the Evaluation of Osteoarthritis-Related miRNAs in Extracellular Vesicles from Bone-Marrow-Derived Mesenchymal Stromal Cells and the Impact of Osteoarthritis Synovial Fluid

“…Similarly, ASC inflammatory priming leaded to a more pronounced cartilage regenerative and anti-inflammatory EV phenotype [ 25 ]. Moreover, customized drug-loaded EVs are gradually applied in the field of OA therapy by either loading miRNA into the secreting cells or directly mixing EV with purified particles [ 61 , 62 , 63 ], although this technology has to be improved in terms of efficiency [ 64 ]. Eventually, differential tissue regenerative and inflammatory modulating properties related to EV-miRNAs emerged comparing ASCs and hAMSCs [ 41 ].…”

Challenges in the Development of Drug Delivery Systems Based on Small Extracellular Vesicles for Therapy of Brain Diseases

“…Lately, efficient delivery of siRNAs and miRNAs into sEVs was shown to be achieved with commercial transfection kits, as the Exo-Fect TM from System Biosciences (SBI; System Biosciences, Palo Alto, CA) ( Chivero et al, 2020 ). It was demonstrated to be more efficient than electroporation, heat shock, saponin, or cholesterol-mediated, at least for miRNAs, where more than 1000-fold upregulation was achieved, as compared to native sEVs ( de Abreu et al, 2021 ).…”