McCann, Jenna2020-03-062020-03-062020-03-06http://hdl.handle.net/10393/40234http://dx.doi.org/10.20381/ruor-24467Small extracellular vesicles are endogenous delivery vehicles produced by most cells. They have been shown to carry miRNA to recipient cells to modulate them. As a result, they have attracted much attention for their potential use as delivery vehicles for therapeutics. Silencing RNAs are a potent drug class but toxicity from the delivery vehicles used and the elevated doses they require prevented many clinical trials from moving forward. Additionally, their therapeutic effect is short-lived but can be extended by the addition of chemical modifications. Therefore, it is beneficial to determine a method that enables chemically stabilized siRNAs to be packaged in small extracellular vesicles. Few miRNA are packaged in these vesicles and their selective packaging is not well understood, making it difficult to promote the loading of specific siRNAs into small extracellular vesicles. Since chemically modified RNAs cannot be expressed, transfection of extracellular vesicle-producing cells was attempted to load siRNA in small extracellular vesicles. Here, we provide evidence that small extracellular vesicle preparations isolated from transfected cells contain larger particles than ones isolated from untransfected cells, suggesting that the vesicles may fuse with transfection complexes. Transfected siRNA co-localized with the late endosomes where some vesicle subtypes, called exosomes, are synthesized. This suggests that transfection complexes may accumulate in the late endosome where they may fuse with or be released alongside small extracellular vesicles. Importantly, extracellular vesicles released by transfected cells also appear to be less efficient at delivering siRNA cargoes than those produced by untransfected cells. Therefore, transfection complexes or fusions of transfection complexes and the vesicles may be a major contaminant of the preparations of small extracellular vesicles from transfected cells. This is likely a major concern as a significant amount of published literature has used transfection of cells to load RNAs in small extracellular vesicles.enExtracellular VesiclesTransfection ComplexesThesis