**3.2 Lipids of exosomes**

*Extracellular Vesicles and Their Importance in Human Health*

**Number of proteins described**

**Method of protein analysis**

2299 Trypsinolysis, LC-MS/MS

2698 SDS-PAGE, trypsinolysis,

8 SDS-PAGE,

571 SDS-PAGE, trypsinolysis,

with iTRAQ

LC-MS/MS

2D-electrophoresis, trypsinolysis, MALDI-TOF-MS/MS

LC-MS/MS

2107 Trypsinolysis, LC-MS/MS Exosomes and

115 Trypsinolysis, LC-MS/MS During 12 months of

**Protein content compared in**

milk fat globule membrane

lactation

Norm and mastitis

Extracellular vesicles and high-density complexes

Before and after gel filtration

**Most represented proteins**

Xanthine oxidase Adipophilin Lactadherin

Lactoferrin Tenascin Serum albumin β-Casein Xanthine dehydrogenase Polymeric Ig receptor

Xanthine dehydrogenase Lactadherin Fatty acid synthase

CD9, CD63, CD81 Flotilin Lactadherin Annexins G-protein subunits Ras-related proteins Rab syntenin

> Lactadherin Actin Butyrophilin Lactoferrin

Thrombospondin Albumin Lactotransferrin Ceruloplasmin Complement C4 α-Glucosidase

Cow [26] Butyrophilin

Cow [13] Butyrophilin

Horse [8] β-Lactoglobulin

Swine [33] Fibronectin

**Source of milk exosomes**

Human [36]

Human [35]

**Table 1.**

The data on the protein composition of milk exosomes gives new information on the structure and biological significance of these vesicles and reveals the potential role of exosomes in the physiology of the mammary gland. Investigation of the proteome of highly purified milk exosomes compared to milk proteome can shed light on the real protein composition of exosomes; these data may be translated to the exosomes obtained from other biological liquids. Results of milk exosome proteome analysis will possibly lead to their use in medicine as biocompatible carriers of

However, as shown above, not all proteins found in crude vesicle preparations are proper exosome proteins. At the same time, it is possible that some proteins associated with the surface of the vesicles may also play some unique role in the exosomes' functioning. The identification of hundreds and thousands of proteins

**24**

drugs or personal therapy tools.

*Most represented proteins of milk exosomes.*

The exosome membrane is enriched with specific lipids (phosphatidylcholine, cholesterol, sphingomyelin, ceramides) and has a unique protein composition that characterizes them as independent compartments [53, 54]. The minimum size of exosomes depends on the structure of the lipid bilayer, which is about 5 nm thick and has sufficient rigidity to form vesicles of 40 nm in size [55]. The lipid composition of exosomes greatly varies, due to differences in cell types, conditions of cell growth and development, as well as the use of different methods for isolating exosomes and analyzing them.

The first works on the lipid composition of exosomes were carried out using a thin layer and gas-liquid chromatography. The results obtained using these methods cannot be considered fully quantitative, since the phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidic acids migrate together and are presented in the single band after separation. Similarly, sphingomyelin and ganglioside GM3 are not separated and, therefore, are taken into account together in the quantitative analysis. Today, these methods are considered obsolete for the analysis of lipids of exosomes, and most studies use modern mass spectrometry technology [56].

It was found that different classes of lipids are asymmetrically distributed in the membrane of exosomes, so sphingomyelin and other sphingolipids, as well as phosphatidylcholines, are mainly located in the outer layer of the membrane, while the different classes of lipids are located in the inner layer [57]. However, the established asymmetry of the membrane bilayer can be altered by the action of particular enzymes, such as flippases, floppases, and scramblases [58, 59].

Phosphatidylserine in exosomes, as in the plasma membrane, is located in the inner lipid layer [57], but several studies have shown its presence also in the outer layer together with annexin 5. It is known that the presence of phosphatidylserine in the outer lipid layer activates blood cells and acts as a signal to macrophages to capture [60].

The composition of certain classes of lipids in the exosomal membranes secreted by different cell types may be similar or not to the parental cells. Exosomes contain up to 2–3 times more cholesterol, sphingomyelin, glycosphingolipids, serine, and saturated fatty acids. In addition, GM3 ganglioside [56, 61], ceramides, and their derivatives [57, 62, 63] are also present in exosomes in significant amounts. In most cases, exosomes contain fewer phosphatidylcholines than their parent cells, and no noticeable differences in the content of phosphatidylserine in exosomes and parent cells were found [64].

The bis(monoacylglycero)phosphates (BMP) are present in the membranes of the intraluminal vesicles of multivesicular bodies and, as stated in [57], can also be contained in the exosomes. It was shown that BMP are not transferred to exosomes but, with high probability, are included in the intraluminal vesicles of those multivesicular bodies that are associated with lysosomes. In this regard, it is believed that the primary purpose of the BMP is to promote the stability and integrity of the lysosomes. Also, it is a necessary cofactor in the process of catabolism of sphingolipids in lysosomes [65].

The stiffness of exosomal membranes increases with the transition from acidic to neutral pH values, suggesting that during the secretion of the exosomes from the multivesicular bodies, some reorganization of the membrane occurs. At neutral pH, the packaging of lipids on the surface of exosomes is dense, but the transmembrane movement of lipids increases. Such a flip-flop effect (the transition of an individual molecule from one layer to another) disrupts the asymmetric distribution of lipids between the membrane layers. A uniform distribution of phosphatidylethanolamine between the two layers of the exosomal membrane [66] was shown; on the contrary, in the plasma membrane, it is located mainly in the inner lipid layer [67].

The lipids of the exosomal membranes are not inert molecules but participate in the biogenesis of vesicles and affect their biological activity. Besides, exosomes carry carbohydrate groups on the outer surface; the presence of mannose, polylactosamine, α-2,6 sialic acid, and complex N-linked glycans was shown [68].

Lipids form the basis of the exosome membrane; the composition of exosomal lipids significantly differs from the composition of lipids of non-vesicular structures.

The above data reflect the content of lipids and oligosaccharides in the composition of mainly crude preparations of exosomes. However, as noted above, the second peak after gel filtration of exosome preparations also contains various lipids and oligosaccharides. Therefore, it cannot be excluded that part of the lipids and polysaccharides found in the vesicles will be attributed in the future to the highmolecular complexes co-isolating with exosomes during different centrifugations.

#### **3.3 Nucleic acids of milk exosomes**

Several works show that milk exosomes contain different types of nucleic acids, including functional mRNA with a poly(A) tract at the 3′-end [33, 34, 41] and microRNA [34, 69, 70]. Currently the composition of nucleic acids in milk exosomes is described in the case of human [71, 72], cow [73, 74], porcine [33, 75], and rat [10] milk.

As we have noted above, the number of individual RNA molecules in some papers may be overestimated due to various reasons. For example, it has been shown that exosomes of porcine milk contain up to 16,304 mRNA. Most of these molecules may be involved in the development of the immune system, cell proliferation, and intercellular signal transduction. Protein products of identified mRNAs might be involved in the regulation of metabolism and the growth of the piglet's intestines [33].

Among the 19,230 mRNAs found in cow milk exosomes, the most common mRNA molecules are various isoforms of the mRNA of milk and ribosomal proteins: inositol 1,4,5-triphosphate receptor type 1; α-lactalbumin; β-lactoglobulin; caseins β, κ, and α; ribosomal proteins S28, S3, P0, L32, L21, and H1 histone; and many others. Since the expression level of most of the 50 most represented transcripts in exosomes exceeds the standard in supernatants obtained by ultracentrifugation, the authors of the [34] conclude that the mRNAs are concentrated in the exosomes of milk. Unfortunately, it is difficult to agree with this statement, since the process of enrichment of exosomes with mRNA transcript molecules during their biogenesis is entirely unclear, as well as it is difficult to suggest a mechanism by which this unimaginable number of mRNA molecules can fit into an exosome with a diameter of 40–100 nm. At the same time, human and porcine milk exosomes contain little or no 18S and 28S rRNA [41, 70], which correlate with the mechanism of exosome biogenesis.

**27**

*Milk Exosomes: Isolation, Biochemistry, Morphology, and Perspectives of Use*

It is shown that miRNA is ubiquitous in tissues and biological fluids and was previously isolated from and associated with exosomes formed from various biological fluids (serum, saliva, urine) and homogenates of body tissues, including the mammary gland [76]. Some studies have shown that the exclusion of milk exosomes and their contents, including exosomal miRNAs, from nutrition in newborns leads to impaired purine metabolism, as well as impaired spatial learning and memory in humans and mice [72, 77]. These data indicate the undesirability of feeding newborns with infant formulas since their content of microRNAs is absent or much

Analysis of nucleic acids isolated from human milk exosomes revealed about 452 pre-miRNAs, which is approximately 32% of 1424 miRNAs described for humans. These 452 pre-miRNAs lead to the generation of 639 mature miRNAs, many of which are involved in the regulation of immune responses. At the same time, the distribution of different miRNAs in human milk exosomes is irregular; some miRNAs are represented in a million copies and others in single molecules. Ten miRNAs compose up to 62% of the total number of miRNA; the most represented miRNAs are miR-30b-5p, miR-141-3p, miR-148a-3p, miR-182-5p, miRs let-7a-5p and let-7f-5p, miR-29a-3p, miR-146b-5p, miR-182-5p, miR-200a-3p, and miR-378a-3p [79]. Interestingly miR-148a-3p may comprise up to 35% of the total number of miRNAs of human milk exosomes. miR-148a specifically controls the expression of several genes, including the TGIF2, which encodes a transcription factor, inducing the expression of various transporters and drug-metabolizing enzymes [80], and the DNMT3B gene, which encodes DNA

Transcriptome of cow milk exosomes contain various miRNAs, the most common of which are bta-miR-320a-1, bta-miR-193a, bta-miR-2284x, bta-mir-181b-1, bta -miR-19b-2, bta-miR-135a-1, bta-miR-200c, bta-miR-142, bta-miR-2887-1, bta-miR-30b, bta-miR-let7i, and bta-miR-6522 [72]. It was shown that cow milk exosomes penetrate intestinal and choroidal epithelial cells [82, 83] and macrophages [34], accumulate in peripheral tissues [84, 85], and transfer miRNA to the recipient cells [86]. Analysis of exosome bioavailability between species showed that microRNA of cow milk exosomes, after oral delivery to other organisms, is protected under the low pH, RNase, and other factors of the gastrointestinal tract

Analysis of miRNA content in porcine milk exosomes revealed 366 premiRNAs, which can give rise to 315 mature miRNAs, and 176 of them were described in other sources. Functional analysis of porcine milk miRNA indicates their role in immune responses, and 14 of 20 of most represented miRNAs may be involved in the regulation of milk IgA production [69]. Also, it was shown that miR-148a, widely represented in the exosomes of human [71] and cow [89] milk, is also highly expressed during lactation in exosomes of porcine milk [70]. Other highly expressed miRNAs of porcine milk are miR-181 family (181a/181b/181c/181d), miR-30 family (b/c/d/e), let-7 family (a/b/d/f), and miR-98 family. Thus, miRNAs included in these families can participate in the develop-

Nucleic acids play an essential role in biological functions of milk exosomes. The further investigations of milk exosome miRNA and mRNA variety will significantly expand the prospects of their practical use. However, when analyzing different RNA in exosomes, it should not be forgotten that exosomal preparations used in most of the papers described above were crude. Therefore, some of the detected RNA may be in the fraction with co-isolating proteins and their complexes, which

*DOI: http://dx.doi.org/10.5772/intechopen.85416*

lower than breast milk [78].

methyltransferase [81].

ment of the digestive tract in piglets [69].

may be separated from exosomes with gel filtration.

[78, 87, 88].

#### *Milk Exosomes: Isolation, Biochemistry, Morphology, and Perspectives of Use DOI: http://dx.doi.org/10.5772/intechopen.85416*

*Extracellular Vesicles and Their Importance in Human Health*

inner lipid layer [67].

**3.3 Nucleic acids of milk exosomes**

mechanism of exosome biogenesis.

structures.

rat [10] milk.

intestines [33].

The stiffness of exosomal membranes increases with the transition from acidic to neutral pH values, suggesting that during the secretion of the exosomes from the multivesicular bodies, some reorganization of the membrane occurs. At neutral pH, the packaging of lipids on the surface of exosomes is dense, but the transmembrane movement of lipids increases. Such a flip-flop effect (the transition of an individual molecule from one layer to another) disrupts the asymmetric distribution of lipids between the membrane layers. A uniform distribution of phosphatidylethanolamine between the two layers of the exosomal membrane [66] was shown; on the contrary, in the plasma membrane, it is located mainly in the

The lipids of the exosomal membranes are not inert molecules but participate in the biogenesis of vesicles and affect their biological activity. Besides, exosomes carry carbohydrate groups on the outer surface; the presence of mannose, polylac-

The above data reflect the content of lipids and oligosaccharides in the composition of mainly crude preparations of exosomes. However, as noted above, the second peak after gel filtration of exosome preparations also contains various lipids and oligosaccharides. Therefore, it cannot be excluded that part of the lipids and polysaccharides found in the vesicles will be attributed in the future to the highmolecular complexes co-isolating with exosomes during different centrifugations.

Several works show that milk exosomes contain different types of nucleic acids,

including functional mRNA with a poly(A) tract at the 3′-end [33, 34, 41] and microRNA [34, 69, 70]. Currently the composition of nucleic acids in milk exosomes is described in the case of human [71, 72], cow [73, 74], porcine [33, 75], and

As we have noted above, the number of individual RNA molecules in some papers may be overestimated due to various reasons. For example, it has been shown that exosomes of porcine milk contain up to 16,304 mRNA. Most of these molecules may be involved in the development of the immune system, cell proliferation, and intercellular signal transduction. Protein products of identified mRNAs might be involved in the regulation of metabolism and the growth of the piglet's

Among the 19,230 mRNAs found in cow milk exosomes, the most common mRNA molecules are various isoforms of the mRNA of milk and ribosomal proteins: inositol 1,4,5-triphosphate receptor type 1; α-lactalbumin; β-lactoglobulin; caseins β, κ, and α; ribosomal proteins S28, S3, P0, L32, L21, and H1 histone; and many others. Since the expression level of most of the 50 most represented transcripts in exosomes exceeds the standard in supernatants obtained by ultracentrifugation, the authors of the [34] conclude that the mRNAs are concentrated in the exosomes of milk. Unfortunately, it is difficult to agree with this statement, since the process of enrichment of exosomes with mRNA transcript molecules during their biogenesis is entirely unclear, as well as it is difficult to suggest a mechanism by which this unimaginable number of mRNA molecules can fit into an exosome with a diameter of 40–100 nm. At the same time, human and porcine milk exosomes contain little or no 18S and 28S rRNA [41, 70], which correlate with the

tosamine, α-2,6 sialic acid, and complex N-linked glycans was shown [68]. Lipids form the basis of the exosome membrane; the composition of exosomal lipids significantly differs from the composition of lipids of non-vesicular

**26**

It is shown that miRNA is ubiquitous in tissues and biological fluids and was previously isolated from and associated with exosomes formed from various biological fluids (serum, saliva, urine) and homogenates of body tissues, including the mammary gland [76]. Some studies have shown that the exclusion of milk exosomes and their contents, including exosomal miRNAs, from nutrition in newborns leads to impaired purine metabolism, as well as impaired spatial learning and memory in humans and mice [72, 77]. These data indicate the undesirability of feeding newborns with infant formulas since their content of microRNAs is absent or much lower than breast milk [78].

Analysis of nucleic acids isolated from human milk exosomes revealed about 452 pre-miRNAs, which is approximately 32% of 1424 miRNAs described for humans. These 452 pre-miRNAs lead to the generation of 639 mature miRNAs, many of which are involved in the regulation of immune responses. At the same time, the distribution of different miRNAs in human milk exosomes is irregular; some miRNAs are represented in a million copies and others in single molecules. Ten miRNAs compose up to 62% of the total number of miRNA; the most represented miRNAs are miR-30b-5p, miR-141-3p, miR-148a-3p, miR-182-5p, miRs let-7a-5p and let-7f-5p, miR-29a-3p, miR-146b-5p, miR-182-5p, miR-200a-3p, and miR-378a-3p [79]. Interestingly miR-148a-3p may comprise up to 35% of the total number of miRNAs of human milk exosomes. miR-148a specifically controls the expression of several genes, including the TGIF2, which encodes a transcription factor, inducing the expression of various transporters and drug-metabolizing enzymes [80], and the DNMT3B gene, which encodes DNA methyltransferase [81].

Transcriptome of cow milk exosomes contain various miRNAs, the most common of which are bta-miR-320a-1, bta-miR-193a, bta-miR-2284x, bta-mir-181b-1, bta -miR-19b-2, bta-miR-135a-1, bta-miR-200c, bta-miR-142, bta-miR-2887-1, bta-miR-30b, bta-miR-let7i, and bta-miR-6522 [72]. It was shown that cow milk exosomes penetrate intestinal and choroidal epithelial cells [82, 83] and macrophages [34], accumulate in peripheral tissues [84, 85], and transfer miRNA to the recipient cells [86]. Analysis of exosome bioavailability between species showed that microRNA of cow milk exosomes, after oral delivery to other organisms, is protected under the low pH, RNase, and other factors of the gastrointestinal tract [78, 87, 88].

Analysis of miRNA content in porcine milk exosomes revealed 366 premiRNAs, which can give rise to 315 mature miRNAs, and 176 of them were described in other sources. Functional analysis of porcine milk miRNA indicates their role in immune responses, and 14 of 20 of most represented miRNAs may be involved in the regulation of milk IgA production [69]. Also, it was shown that miR-148a, widely represented in the exosomes of human [71] and cow [89] milk, is also highly expressed during lactation in exosomes of porcine milk [70]. Other highly expressed miRNAs of porcine milk are miR-181 family (181a/181b/181c/181d), miR-30 family (b/c/d/e), let-7 family (a/b/d/f), and miR-98 family. Thus, miRNAs included in these families can participate in the development of the digestive tract in piglets [69].

Nucleic acids play an essential role in biological functions of milk exosomes. The further investigations of milk exosome miRNA and mRNA variety will significantly expand the prospects of their practical use. However, when analyzing different RNA in exosomes, it should not be forgotten that exosomal preparations used in most of the papers described above were crude. Therefore, some of the detected RNA may be in the fraction with co-isolating proteins and their complexes, which may be separated from exosomes with gel filtration.
