Novel Protocols for Scalable Production of High Quality Purified Small Extracellular Vesicles from Bovine Milk

Abstract

Extracellular Vesicles (EVs) are cell-secreted nanovesicles that have unique potential for encapsulating and targeting "difficult-to-drug" therapeutic cargos. Milk provides an enriched source of EVs, and of particular interest to the drug delivery field, small EVs. Small EVs are distinguished from large EVs by membrane components, biogenesis mechanism and downstream functionality - in particular, small EVs are primarily composed of exosomes, which show high stability in vivo and naturally function in the targeted delivery of biological materials to cells. Moreover, bovine milk is abundantly produced by the dairy industry, widely consumed, and generally well tolerated by humans. Importantly, there is evidence that milk exosomes and small EVs are efficiently taken up into the circulation from the gut, providing the opportunity for their use in administration of therapeutics such as microRNAs or peptides not typically available via an oral route. Unfortunately, present methods for isolation do not efficiently separate EVs from milk proteins, resulting in contamination that is not desirable in a clinical-grade therapeutic. Herein, we present novel EV purification methods focused on optimized timing and levels of temperature and divalent cation chelation. Incorporation of these solubilization steps into centrifugation- and tangential flow filtration-based methods provide large amounts of purified small EVs at ultra-dense concentrations, which are substantially free from contaminating milk proteins. Remarkably, these ultra-dense isolates equal 10 to 15% of the starting volume of milk indicating a prodigious rate of small EV production by mammary glands. Our approach enables gentle, scalable production of ultrastructurally and functionally intact small EVs from milk, providing a path to their industrial scale purification for oral delivery of therapeutic biologics and small drugs.

Keywords: Bovine Milk Extracellular Vesicles; Drug Delivery vehicle; Exosomes; Small Extracellular Vesicle Isolation; Small Extracellular Vesicles; Tangential Flow Filtration.

Figure 1

Overview of steps in the optimized Ultracentrifugation (UC)-based method of small EV isolation from milk. Chemical chelation with EDTA at 37°C was found to be optimally placed prior to SEC separation.

Figure 2

Overview of steps in the optimized Tangential Flow Filtration (TFF)-based protocol for isolation of small EVs from milk. Chemical chelation with EDTA 37°C was found to be optimally placed prior to TFF.

Figure 3

Characterization of the Ultracentrifuge-based method for small EV (sEV) isolation. A) Sequential fractions collected during the SEC filtration step, with protein concentrations in mg/ml. B) Western blot of sEV markers CD-81, CD-9 and Syntenin, along with non-small EV markers: Casein, Arf6 (microvesicle marker) and calnexin (endoplasmic reticulum and apoptotic body marker). Peak sEV SEC fractions occur between fractions 8 and 9. Contaminating proteins, including Casein, predominate after fraction 12. Lysates from HeLa cells are included as comparative controls. C) Nanoparticle Tracking Analysis (NTA) data for sEV isolates. Concentration is shown under NTA graph. D) Negative stain electron microscopy of final isolates, showing ultra-dense accumulation of sEVs in peak SEC fractions, and E) high levels of Casein macrostructures in a later SEC isolate (fraction 17).

Figure 4

Characterization of TFF-based protocol for small EV isolation. A) Sequential fractions collected during the SEC filtration step, with protein concentrations in mg/ml. B) Western blot of sEV markers CD-81, CD-9 and Syntenin, along with non-small EV markers Casein, Arf6 and calnexin. Peak sEV SEC fractions occur between fractions 8 and 9. Contaminating proteins, including Casein, predominate after fraction 12. Lysates from HeLa cells are included as comparative controls. C) Nanoparticle Tracking Analysis data for sEV isolates. Concentration is shown under NTA analysis graph. D) Negative stain electron microscopy of final isolates, showing ultra-dense accumulation of sEVs in peak SEC fractions 8.5 and 9, and E) high levels of Casein macrostructures in a later SEC isolate (fraction 17).

Figure 5

Representative TEM image of post-SEC, EV-containing fraction number 8.5.

Figure 6

Time-dependent uptake of esterified Calcein AM dye into TFF-isolated milk EVs. Peak small EV containing SEC fractions were diluted 1:10 in Hepes buffer. The images show uptake resulting from 1-, 2-, 3- and 4-hour incubations in Calcein-AM. Dye uptake indicate that the sEVs contain esterase activity and are capable of retaining increasing amounts of de-esterified calcein molecules over at least a 4-hour time course. Scale bars in bottom right of each image represent 1 µm.