Abstract
Bovine milk-derived extracellular vesicles (BM-EVs) are recognized as promising nanoscale delivery vectors owing to their large availability. However, few isolation methods can achieve high purity and yield simultaneously. Therefore, we developed a novel and cost-effective procedure to separate BM-EVs via “salting-out.” First, BM-EVs were isolated from skimmed milk using ammonium sulfate. The majority of BM-EVs were precipitated between 30 and 40% saturation and 34% had a relatively augmented purity. The separated BM-EVs showed a spherical shape with a diameter of 60–150 nm and expressed the marker proteins CD63, TSG101, and Hsp70. The purity and yield were comparable to the BM-EVs isolated via ultracentrifugation while ExoQuick failed to separate a relatively pure fraction of BM-EVs. The uptake of BM-EVs into endothelial cells was dose- and time-dependent without significant cytotoxicity. The levels of endothelial nitric oxide syntheses were regulated by BM-EVs loaded with icariside II and miRNA-155-5p, suggesting their functions as delivery vehicles. These findings have demonstrated that it is an efficient procedure to isolate BM-EVs via “salting-out,” holding great promise toward therapeutic applications.
Highlights
Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer membraneenclosed nanoscale particles released by various cells [1, 2]
We have described the development of a salting-out method to isolate Bovine milk-derived extracellular vesicles (BM-EVs) from whey and provided preliminary evidence of their function as therapeutic carriers
The BM-EVs precipitated by ammonium sulfate have several characterizations that were consistent with previous knowledge, including nanoscale spherical shape, proteins, and nucleic acid cargo
Summary
Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer membraneenclosed nanoscale particles released by various cells [1, 2]. They exist in biological fluids and carry important information originating from their parent cells, which play a key and unique role in cell-cell communications. EVs have distinct advantages as effective vectors for delivery of drugs and functional RNAs: vast and natural sources, nano-sized dimension, protection of the cargo within a lipid bilayer, a membrane platform with ligands. Bovine milk-derived extracellular vesicles (BM-EVs) have demonstrated biocompatibility and may serve as novel drug delivery carriers with less cytotoxic effects and immunogenic profiles [11,12,13]. EVs can protect the cargo from acids and enzymes in the gastrointestinal tract, meaning that oral administration is reasonably practicable [14, 15]
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