Introduction
Extracellular vesicles (EVs) including exosomes, microvesicles, and apoptotic vesicles, are membrane-bound small structures released from cells into the surrounding environment (1). They play essential roles in intercellular communication, particularly exosomes, which contain several constituents from the cells that secrete them, and appear to be involved in the pathogenesis of various disorders, including cancer, neurodegeneration, and inflammatory diseases (2). Interestingly, the biocompatibility of exosome, their circulating stability, and bioavailability in vivo, allowed them to gain increasing attention as an emerging drug delivery methodology over the last decade (3). They represent an important pathway to transfer information between cells, and thus, might be developed to package and deliver therapeutic molecules. Although exosomes are not nanoparticles derived from the nanotechnology due to its non-mankind nature, they may act as a Nano-carrier owing to their particle diameter (4), which is estimate to be between 30-100nm, and thus, can be used to load a variety of small bioactive molecules to improve bioavailability. Therefore, exosome’s particle size allows them penetrate deep into the tissues and overcome barriers such as the blood-brain barrier and the deformable cytoskeleton. Another important character is that they have a slightly negative zeta potential, which guarantees their long circulation (5). In addition, some exosomes are capable of escaping from the immune system and have shown to have a low immunogenicity, and high stability in the blood, which prolongs drug circulation within the body (6).
Generally, exosomes are excreted from different types of cells and found abundantly in animal milk, which confirmed to contain relatively a stable structure and were shown to contain different miRNAs, including miRNA-148a-3p (7). Since bovine milk exosomes are protected from degradation by stomach acids (2, 8), milk exosomes are, therefore, considered to be useful for drug-delivery systems (DDSs), hence, milk exosome-encapsulated formulations can be used for therapeutic purposes (9). Therefore, milk-derived exosomes are seeing as promising new drug carriers for reaching distant tissues (10, 11).
Mare’s milk is the national drink of the indigenous population in Central Asia, including Kazakhstan. Recent scientific data on the characteristics of the composition of horse milk and their potential properties that contribute to improving health have increased interest in this dietary source (12). Mare’s milk shares some similarities to human breast milk and therefore, may have some valuable therapeutic properties (13). Mare’s milk has a very good hygienic and sanitary status, differs from the milk of other farm animals in that it has the lowest somatic cell content and a very low total number of microorganisms (14). There are no studies examine the use of mare’s milk-derived exosomes as a form of therapeutic carriers. Therefore, the current study aims to extract mare’s milk-derived EVs, and isolate high content exosomes to test their suitability as a reliable form of therapeutic drug carrier. However, milk-derived EVs significantly different in size ranging from 30nm-10µm, and possibly contain various constituents, thus we do not exactly know which EVs extraction methods would allow us to have the optimum exosomal size. Thus, there is a need to compare and characterize different methods that allows isolation of the highest concentration of exosomes with the most homogenous shapes and sizes.