In vitro evaluation of endothelial exosomes as carriers for small interfering ribonucleic acid delivery

Abstract

Exosomes, one subpopulation of nanosize extracellular vesicles derived from multivesicular bodies, ranging from 30 to 150 nm in size, emerged as promising carriers for small interfering ribonucleic acid (siRNA) delivery, as they are capable of transmitting molecular messages between cells through carried small noncoding RNAs, messenger RNAs, deoxyribonucleic acids, and proteins. Endothelial cells are involved in a number of important biological processes, and are a major source of circulating exosomes. In this study, we prepared exosomes from endothelial cells and evaluated their capacity to deliver siRNA into primary endothelial cells. Exosomes were isolated and purified by sequential centrifugation and ultracentrifugation from cultured mouse aortic endothelial cells. Similar to exosome particles from other cell sources, endothelial exosomes are nanometer-size vesicles, examined by both the NanoSight instrument and transmission electron microscopy. Enzyme-linked immunosorbent assay analysis confirmed the expression of two exosome markers: CD9 and CD63. Flow cytometry and fluorescence microscopy studies demonstrated that endothelial exosomes were heterogeneously distributed within cells. In a gene-silencing study with luciferase-expressing endothelial cells, exosomes loaded with siRNA inhibited luciferase expression by more than 40%. In contrast, siRNA alone and control siRNA only suppressed luciferase expression by less than 15%. In conclusion, we demonstrated that endothelial exosomes have the capability to accommodate and deliver short foreign nucleic acids into endothelial cells.

Extracellular Vesicles Function as Bioactive Molecular Transmitters in the Mammalian Oviduct: An Inspiration for Optimizing in Vitro Culture Systems and Improving Delivery of Exogenous Nucleic Acids during Preimplantation Embryonic Development

“…Briefly, to load EVs with heterologous siRNAs, isolated EVs are mixed with the siRNAs, then incubated on ice for 10 min and exposed to an electric field pulse [15]. Importantly, other studies have also shown that heterologous small RNAs, delivered by EVs, are functional in recipient cells [102,107,108].…”

Extracellular vesicles of the blood-brain barrier

“…For example, exosomes from primary aortic endothelial cells were reported to successfully deliver siRNA against luciferase into endothelial cells in a gene-silencing study. 34 However, additional studies are needed to explore if endothelial ECV can successfully deliver different cargo molecules into the brain as well.…”

CNS-Derived Blood Exosomes as a Promising Source of Biomarkers: Opportunities and Challenges

“…Transmission electron microscopy (TEM) images of negatively stained exosomes initially indicated a cup-shaped morphology, yet later cryo-electron microscopy images of unfixed exosomes, including in a study by Banizs et al, comparing negative-stain EM and unstained cryo-EM of the same exosome preparation, showed a spherical shape, suggesting that the cup-shaped morphology might have resulted from the fixation process in conventional TEM (Figure 2; Théry et al, 2006;Raposo and Stoorvogel, 2013;Banizs et al, 2014).…”