Authors: Logan Whitney, Jaren Wilson, Elley Colledge, Jamie Thygerson
Mentors: Jessica Pullan
Insitution: Southern Utah University
Exosomes are nanosized (40-150 nm) extracellular vesicles (EVs) secreted from cells that play a vital role in the human body's intracellular communication.1 The use of bovine-derived exosomes as a drug delivery vehicle provides an innovative approach to targeted drug delivery4 due to their stability, nontoxicity, non-immunogenic properties, and cost efficiency.5,6,7,8,9 The purpose of our research is to establish and refine a raw bovine milk exosome isolation protocol to determine the quality of exosomes. Current standards for small EV identification are currently being established and regulated by the International Society of Extracellular Vesicles (ISEV) but center around three main categories: proteins, nucleic acids, and lipids.2 To refer to EVs as exosomes, they must be of endosome origin, otherwise they are referred to as ectosomes, and must be appropriately referred to by their size.13 Upon isolation, we verified the morphology of the small EV’s using Scanning Electron Microscopy, allowing visualization of the size and shape of the small EVs. Our research methods for the quantification of surface proteins include Rose Bengal Assay12 and Bradford Assay. Examination using exosome-specific cell surface markers, such as CD63, demonstrates protein functionality of the small EVs. Quantification of lipids allows for comparison of lipid concentration to surface protein concentration for further evidence of exosome identification. Lastly, RNA quantification ensured the small EVs met the concentration threshold to be defined as exosomes. The combination of these methods will further the work in bovine milk extracellular vesicles by yielding a low-cost, high-yield protocol for the isolation and characterization of EVs that can be further used as biomarkers, drug carriers, and a variety of platforms of delivery to physiological targets.
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