Purification of extracellular vesicles from dairy whey using membrane-based techniques

Extracellular vesicles (EVs) are a diverse group of nanoparticles of various shapes and sizes that are naturally released from both prokaryotic and eukaryotic cells. They are enclosed in a phospholipid bilayer membrane with integrated proteins, and encapsulate and protect a variety of bioactive compounds including proteins, lipids and ribonucleic acids, from degradation. To date, EVs have primarily been isolated from various types of human cell fluids. However, their cultivation is limited by several factors, such as senescence and the requirement for a large surface area for growth. Due to these issues, researchers have begun exploring alternative sources of EVs, such as bovine milk, due to its accessibility, affordability and scalability potential. Bovine milk-derived extracellular vesicles are gaining interest as they are characterized by low toxicity, increase the oral bioavailability of drugs, and are suitable for delivering therapeutic and nutritional bioactive compounds. It is also possible to use dairy whey, a by-product of cheese production, as a source of EVs. This alternative source is economically and environmentally sustainable and has a lower content of impurities due to the removal of caseins and fats during milk processing. The purpose of this experimental work is to develop a scalable membrane-based process for purifying extracellular vesicles from dairy whey derived from the production of Parmigiano Reggiano cheese. The results show that the best process among those proposed is tangential flow microfiltration followed by tangential flow constant volume diafiltration, as it effectively removes almost all impurities originally present in the feed. Additionally, it was demonstrated that differential ultracentrifugation is unable to reach the same level of impurity removal.