Cells of the multicellular organisms communicate with each other in many different ways, among which extracellular vesicles play a unique role. Almost all cell types secrete vesicles into the extracellular space and deliver their contents to recipient cells. Today, one of the groups of extracellular vesicles that is of particular interest for studying is exosomes-membrane vesicles with a diameter of 40-100 nm. Exosomes are secreted by cells and found in various biological fluids-blood, tears, saliva, urine, cerebrospinal fluid, and milk. Exosomes provide not only targeted delivery of molecular signals to recipient cells but also carry unique markers, which makes them a promising substrate in diagnostic studies, primarily due to their small RNA and protein contents. The milk of cows, horses, humans, and other mammals is a unique source of exosomes since these organisms can produce liters of milk per day, which is much higher than the volume of exosomes produced in cell culture fluid or blood plasma. Unfortunately, milk exosomes are currently much less studied than exosomes of blood or culture fluid. This review examines the methods of the isolation, biochemical analysis (composition of proteins, lipids, and nucleic acids), morphology, and prospects for the use of milk exosomes.

Exosomes are nanovesicles with a 40–150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

Exosomes are natural nanocontainers actively secreted by the body’s cells and transmitting molecular signals of various types to recipient cells. Cellular mechanisms of exosomes’ biogenesis involve specific sorting of RNA for incorporation into them. As a result, the molecular composition of exosomes is closely related to the donor cell’s functional state, and this makes exosomes an important diagnostic and prognostic marker in a number of diseases (primarily oncological). The ability of exosomes to transport biologically active molecules and to protect the cargo from degradation makes them nearly ideal candidates as delivery carriers of RNA in therapeutic or prophylactic regimes. Potential of exosomal surface functionalization enables improved targeting to specific organs, tissues and cells. However, the development of an effective technology for RNA’s loading into exosomes cannot be considered resolved. This review is focused on experimental data on the use of exosomes as vehicles for the delivery of therapeutic and prophylactic RNAs. We briefly consider the biogenesis and functions of exosomes, focusing on those biological properties that make them formidable candidates in the race to develop effective delivery carriers. Furthermore, we describe various techniques of cargo loading into exosomes. Prospects of exosomes application as therapeutic delivery system for siRNAs, miRNAs, and long RNAs are considered.