Biocompatibility of highly purified bovine milk‐derived extracellular vesicles

Abstract

Extracellular vesicles (EVs) deliver biologically active cargos from donor cells to recipient cells for intercellular communication. Since the existence of RNA cargo was discovered, EVs have been considered to be useful drug-delivery systems. Specifically, EVs from bovine milk (mEV) are one of the most promising platforms, since bovine milk is a scalable source of EVs for mass production. However, it is still difficult to isolate pure EVs from bovine milk owing to the complexity of raw materials. Furthermore, the biocompatibility and immunotoxicity of mEVs are still unclear. In this study, we developed a new method for isolating bovine milk-derived EVs by employing acid treatment and ultracentrifugation. Isolated mEVs are spherical in shape, measure 120 nm in diameter and contain typical EV marker proteins, such as tetraspanins. Compared with the previously reported method, our method can isolate purer mEVs. When mEVs are contacted with the mouse macrophage cell line Raw264.7, mEVs are readily taken up by the cells without a cytotoxic effect, suggesting that mEVs can deliver the cargo molecules into cells. While systemic administration of mEVs into mice resulted in the absence of systemic toxicity, certain types of cytokines were slightly induced. No anaphylaxis effect was observed after serial administration of mEVs in mice. Thus, mEVs isolated using our method are well tolerated in vivo and may be useful for the drug-delivery application.

Milk MicroRNAs in Health and Disease

“…In this case, milk EVs are referred to as “exosomes” and harbor several exosome‐associated proteins like TSG‐101, Alix, HSP70, CD9, CD63, and CD81 among others (Benmoussa et al., , ; Kowal et al., ; Reinhardt et al., ). Some groups define exosomes as a special subset of EVs defined by the presence of proteins derived from MVB trafficking such as TSG101, ALIX, and others (Kahn et al., ; Manca et al., ; Martin, Patel, Williams, Arora, & Sims, ; Somiya, Yoshioka, & Ochiya, ; Zempleni et al., ).…”

“…According to more than 30 studies, from different teams around the world, milk EVs resisted in vitro digestion (Benmoussa et al., ; Kahn et al., ; Liao et al., ; Rani et al., ; Shandilya et al., ; Vashisht et al., ) were internalized by recipient cells upon co‐incubation (Admyre et al., , 2008; Aqil et al., , ; Badawy et al., ; Golan‐Gerstl et al., ; Hock et al., ; Kahn et al., ; Liao et al., ; Maburutse, Park, Oh, & Kim, ; Martin et al., ; Matsuda & Patel, ; Mobley et al., ; Munagala et al., ; Naslund et al., ; Qin et al., ; Vashisht, Rani, Onteru, & Singh, ; Yu et al., ), transduced through the intestinal wall in vitro (Rani et al., ; Shandilya et al., ; Wolf et al., ), and were detected in animal models after ingestion (Manca et al., ; Munagala et al., ) or intravenous injection (Somiya, Yoshioka, & Ochiya, ).…”

“…Twenty‐one studies involving milk EVs from humans, rat, camels, and most often cow milks reported milk EVs bioactivity in vitro on cells (Admyre et al., ; Aqil et al., , ; Badawy et al., ; Golan‐Gerstl et al., ; Hock et al., ; Martin et al., ; Matsuda & Patel, ; Mobley et al., ; Munagala et al., ; Naslund et al., ; Qin et al., ; Vashisht et al., ; Yu et al., ) and in vivo in animal models (Agrawal et al., ; Aqil et al., , ; Badawy et al., ; Leiferman et al., ; Munagala et al., ; Munagala et al., ; Somiya et al., ).…”

“…Functionality of milk EVs has been explored following their intrinsic biological activity or their potential as drug carriers for cancer treatment (Agrawal et al., ; Aqil et al., , ; Badawy et al., ; Munagala et al., , ; Qin et al., ; Somiya et al., ), for the modulation of inflammation (Admyre et al., ; Arntz et al., ; Badawy et al., ; Martin et al., ; Vashisht et al., ), for gut, bone, and muscle development (Chen et al., ; Hock et al., ; Leiferman et al., ; Martin et al., ; Mobley et al., ; Oliveira et al., , ), bacterial proliferation (Yu et al., ), and in the prevention of HIV infection (Naslund et al., ).…”

“…Therefore, the bulk of evidence supports milk EV stability, bioaccessibility, bioavailability, and biological activity and recent evidence supports their biocompatibility (that is, the lack of side effects associated with their consumption or their direct injection into the circulatory system in vivo ; Mentkowski, Snitzer, Rusnak, & Lang, ; Munagala et al., ; Somiya et al., ). Nevertheless, concerns about the biological content of EVs, and environmental context of their use, were raised and must be taken into consideration when using milk EVs as therapeutic tools (Melnik, ; Melnik & Schmitz, ; Melnik et al., ; Nordgren et al., ).…”