Milk Exosomes Research Articles

The absorption of bovine milk small extracellular vesicles largely depends on galectin 3 and galactose ligands in human intestinal cells and C57BL/6J mice.

Small extracellular vesicles in milk (sMEVs) have attracted attention in drug delivery and as bioactive food compounds. Previous studies implicate galactose residues on the sMEV surface in sMEV transport across intestinal and endothelial barriers in humans, but details of glycoprotein-dependent transport are unknown. We used a combination of cell biology and genetics protocols to identify glycoproteins on the sMEV surface that facilitate sMEV absorption. We identified 256 proteins on the bovine sMEVs surface by using LC-MS/MS, and bioinformatics analysis suggested that 42, 13, and 13 surface proteins were N-, O-, and 13 C-glycosylated, respectively. Lectin blots confirmed the presence of mannose, galactose, N-acetyl galactose, fucose, and neuraminate. When surface proteins were removed by various treatment with various proteases, sMEV uptake decreased by up to 58% and 67% in FHs-74 Int and Caco-2 cells, respectively, compared with controls (P < 0.05). When glycans were removed by treatment with various glycosidases, sMEV uptake decreased by up to 54% and 74% in FHs-74 Int and Caco-2 cells, respectively (P < 0.05). When galactose and N-acetyl galactosamine residues were blocked with agglutinins, sMEV uptake decreased by more than 50% in FHs-74 Int cells (P < 0.05). When bovine sMEVs were administered to Galectin-3 knockout mice by oral gavage, hepatic sMEV accumulation decreased by 56% compared with wild-type mice (P < 0.05), consistent with a role of β-galactoside glycan structures in the absorption of sMEVs. We conclude that sMEVs are decorated with glycoproteins, and Galectin-3 and its galactose ligands are particularly important for sMEV absorption.NEW & NOTEWORTHY This is the first paper to assess the role of unique glycans and their Galectin-3 receptor in the transport and distribution of small extracellular vesicles ("exosomes") from milk in mammals. The research assessed milk exosome transport and distribution by using multiple approaches and platforms including cell cultures, various exosome labels, knockout and mutant mice, enzymatic removal of surface proteins and glycans, and lectin blocking of glycans.

Application of Milk Exosomes for Musculoskeletal Health: Talking Points in Recent Outcomes.

Milk is a nutrient-rich food source, and among the various milks, breast milk is a nutrient source provided by mothers to newborns in many mammals. Exosomes are nano-sized membranous extracellular vesicles that play important roles in cell-to-cell communication. Exosomes originate from endogenous synthesis and dietary sources such as milk. Discovered through electron microscopy as floating vesicles, the existence of exosomes in human milk was confirmed owing to a density between 1.10 and 1.18 g/mL in a sucrose gradient corresponding to the known density of exosomes and detection of MHC classes I and II, CD63, CD81, and CD86 on the vesicles. To date, milk exosomes have been used for treating many diseases, including cancers, and are widely proposed as promising carriers for the delivery of chemotherapeutic agents. However, few studies on milk exosomes focus on geriatric health, especially sarcopenia and osteoporosis related to bone and muscle. Therefore, the present study focused on milk exosomes and their cargoes, which are potential candidates for dietary supplements, and when combined with drugs, they can be effective in treating musculoskeletal diseases. In this review, we introduce the basic concepts, including the definition, various sources, and cargoes of milk exosomes, and exosome isolation and characterization methods. Additionally, we review recent literature on the musculoskeletal system and milk exosomes. Since inflammation and oxidative stress underly musculoskeletal disorders, studies reporting the antioxidant and anti-inflammatory properties of milk exosomes are also summarized. Finally, the therapeutic potential of milk exosomes in targeting muscle and bone health is proposed.

Instead of Calories, Should We Be Counting our Consumption of Exosomes and MicroRNAs?

The specific foods to eat for optimal nutrition remain ill-defined. Studies using plantbased diets or milk suggest that vesicles, termed exosomes, and small RNAs termed microRNAs (miRNAs) are health promoting components in foods. However, numerous studies refute the potential of dietary cross-kingdom communication of exosomes and miRNAs. While research reinforces that plant-based diets and milk are healthy components of a well-rounded diet, the bioavailability and bioactivity of the exosomes and miRNAs present in plant-based diets and milk remain unclear. Further investigations of plant-based diet and milk exosome like particles may open a new era in application of food for overall health enhancement. In addition, the potential biotechnological plantbased diet and milk exosome like particles can aid in cancer treatment.

Sequencing and analysis of micro RNAs incamelmilk exosomes.

In order to study the species and functions of micro (mi)RNAs in the exosomes of camel milk, non-coding small (s)RNAs were sequenced and identified by Illumina sequencing technology, and the miRNA fraction was analysed by bioinformatics. After quality control, the average length of sRNA of camel milk exosomes was 18-24 nucleotides. A total of 2,659 miRNAs were identified, including 2,458 known, and 201 new miRNAs. Among the known miRNAs, miR-148a and let-7i had the highest expression levels. The results of gene ontology enrichment analysis indicated that the target genes of camel milk exosome miRNAs were involved in multicellular organismal, catabolic and other biological processes. They play role in the extracellular region, in the cytoskeleton and other cell components, in protein binding, but also have structural molecule activity and other molecular functions. According to the results of the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, the target genes of camel milk exosome miRNAs are involved in Alzheimer's disease, non-alcoholic fatty liver disease, Staphylococcus aureus infection and other pathological pathways. We speculate that the reportedbeneficial effect of camel milk in various pathologic conditions may be closely related to the regulatory function of the exosomal miRNAs exerted on target genes of the diseases.

Bovine Milk Proteome: Milk Fat Globule Membrane Protein Is the Most Sensitive Fraction in Response to High Somatic Cell Count.

The impacts of high milk somatic cell count (SCC) on different milk fractions are not well understood. In this study, proteins in milk exosomes, milk fat globule membrane (MFGM), and whey from cows with low (<105 cells/mL, CG) and high SCC (>5 × 105 cells/mL, HSG) were identified using a tandem mass tag proteomic approach. In total, 1568, 2160, and 1002 proteins were identified, with 65, 552, and 98 proteins being altered by high SCC in exosomes, MFGM, and whey, respectively. With high SCC, the exosome marker (ACTB) was increased in the exosomes of HSG. The main MFGM proteins (BTN1A1, PLIN3, FABP3, and MFGE8) and functional proteins (MUC1, IGSF5, TLR5, and CD36/14) were decreased, while the lipid/energy metabolism-related proteins were increased in the MFGM of HSG. The glycolysis-related proteins were increased in the whey of HSG. Also, the host defense/inflammation-related proteins were changed in three fractions under high SCCs. MFGM was the most sensitive fraction to a high SCC, followed by whey. These findings provide guidance for the early detection of unhealthy mammary glands.

Isolation of Bovine and Human Milk Extracellular Vesicles.

Extracellular vesicles such as exosomes are small-sized, bilayered extracellular biovesicles generated by almost every cell and released into the surrounding body fluids upon the fusion of multivesicular bodies and the plasma membrane. Based on their origin, they are enriched with a variety of biologically active components including proteins, lipids, nucleic acids, cellular metabolites, and many other constituents. They can either attach or fuse with the membrane of a target cell, or alternatively be taking up via endocytosis by a recipient cell. In particular, milk exosomes have been recently shown to be a fundamental factor supporting infant growth, health, and development. In addition, exosomes derived from different cell types have been shown to possess regenerative, immunomodulatory, and anti-inflammatory properties, suggesting that they are a potential therapeutic tool in modulating the pathogenesis of diverse diseases. Therefore, efficient protocols for the isolation of milk exosomes in a high quantity and purity are the basis for establishing clinical applications. Here, we present an easy-to-follow protocol for exosome isolation from bovine and human milk. Electron microscopic analysis and nanoparticle tracking analysis reveal that the protocols allow the isolation of highly enriched fractions of exosomes. The purified exosomes express the typical exosomal protein markers, CD81 and ALIX.

Immunomodulatory and regenerative potentials of breast milk, prematurity and children’s heart

Recent expansion of fundamental knowledge on the physiology of lactation, and breast milk exosomes, stem cell biology, mother-child interactions from prenatal period to postnatal development requires a progressive, dynamic view from the scientific community and practicing physicians when analyzing known, generally accepted clinical phenomena and patterns (development of the immune system of infants and young children, natural and artificial feeding, features of postnatal development and growth of organs and tissues in children born prematurely). The components of the mother-breast-milk-infant triad are closely related to each other and influence developmental trajectory of the infant. According to modern concepts, breast milk of a nursing woman is a “living, metabolic / endocrine signaling system”, which may be considered an “immune organ” significant for postnatal growth and body programming of a premature baby. A valuable phenomenon of early postnatal development is actively discussed in the special literature, i,e., “microchimerism” caused by breastfeeding which, according to modern concepts, may play a key role in development of immune system and the whole body. Absence of protective (immunomodulatory and regenerative) effects of breast milk from a nursing woman on the spontaneous, uncorrectable impact of adverse factors of prematurity is likely predispose for remodeling and dysfunction of heart in prematurely born children, and, at longer range, in adults. The young children born prematurely show a unique cardiac phenotype characterized by reduced biventricular volume, relatively lower systolic and diastolic function, disproportionate muscle mass gain, clinically manifesting by increased risk of cardiovascular disease, hypertension, and decreased exercise tolerance. Hence, the premature birth may be considered a chronic disease state. Therefore, the natural feeding which provides a natural evolutionarily protective mechanism for the child’s heart should be attributed to the fundamental factors that play a vital role in prevention of cardiovascular diseases in prematurely born children and at later life periods.

CircABPD1 alleviates oxidative lung injury of bronchopulmonary dysplasia through regulating miR-330-3p/HIF1α axis

In the NICU, bronchopulmonary dysplasia (BPD) is a concerning common respiratory complication in preterm and low birth-weight infants. Clinical studies have confirmed that human milk has an important nutritional role for children with BPD, therefore, dentification of beneficial components in human milk that prevent BPD is urgently needed. Our previous work showed that human milk exosomes (HM-Exos) could inhibit apoptosis of alveolar type II epithelial cells (AT II), and the circular RNA (circRNA)-circABPD1 were highly expressed in preterm colostrum milk exosomes. Exosomes transport circRNAs that are stable and may exert anti-inflammatory and immune effects attracted the attention of researchers, but the role and mechanism of human milk exosome-derived circABPD1 in BPD remains unclear. Here, we constructed BPD in vivo and in vitro models through exposure to hyperoxia, verified the effect of circABPD1 and revealed its mechanism through rescue experiments. We found that circABPD1 had circRNA properties, and overexpression of circABPD1 could improve reduced alveolar number, enlarged the alveolar linear intercept in vivo models of BPD, promote cell proliferation, reduce oxidative stress levels and alleviate lung epithelial cell damage in vivo and in vitro models. Mechanistically, circABPD1 targets miR-330–3p and regulates the expression of HIF1α. These results suggest that circABPD1 can improve the pathologoical changes of bronchopulmonary dysplasia, promote cell proliferation, inhibit oxidative stress level, and alleviate lung injury by targeting the miR-330–3p/HIF1α axis, which provides a new idea for the prevention and treatment of bronchopulmonary dysplasia.

Potential Epigenetic Effects of Human Milk on Infants' Neurodevelopment.

The advantages of human milk feeding, especially in preterm babies, are well recognized. Infants' feeding with breast milk lowers the likelihood of developing a diverse range of non-communicable diseases later in life and it is also associated with improved neurodevelopmental outcomes. Although the precise mechanisms through which human milk feeding is linked with infants' neurodevelopment are still unknown, potential epigenetic effects of breast milk through its bioactive components, including non-coding RNAs, stem cells and microbiome, could at least partly explain this association. Micro- and long-non-coding RNAs, enclosed in milk exosomes, as well as breast milk stem cells, survive digestion, reach the circulation and can cross the blood-brain barrier. Certain non-coding RNAs potentially regulate genes implicated in brain development and function, whereas nestin-positive stem cells can possibly differentiate into neural cells or/and act as epigenetic regulators in the brain. Furthermore, breast milk microbiota contributes to the establishment of infant's gut microbiome, which is implicated in brain development via epigenetic modifications and key molecules' regulation. This narrative review provides an updated analysis of the relationship between breast milk feeding and infants' neurodevelopment via epigenetics, pointing out how breast milk's bioactive components could have an impact on the neurodevelopment of both full-term and preterm babies.

Identification and analysis of miRNAs expression profiles in human, bovine, and donkey milk exosomes

The purpose of this study is to identify and characterize mirnas in mammalian exosomes. Using Illumina sequencing technology, we sequenced miRNAs in the exosomes of mammalian human milk, bovine milk, and donkey milk. 36 known mature miRNAs and 256 novel miRNAs were identified in human milk. 61 known mature miRNAs and 346 novel miRNAs were identified in milk. 16 known mature miRNAs and 196 novel miRNAs were identified in donkey milk, and miRNAs target genes were predicted. Gene Ontology analysis showed that the miRNAs of human, bovine and donkey milk exosomes all labeled the functions related to body metabolism. Kyoto Encyclopedia pathway analysis showed that human, bovine and donkey milk miRNAs enriched AGE-RAGE signaling pathway in Complications of diabetes. Diabetes is a Metabolic disorder. Based on this pathway, we screened out hsa-miR-8485, bta-miR-342, miR-29c and other genes related to diabetes. This study has a new understanding of the physiological function of mammalian milk miRNAs, and also provides a new way to explore diabetes related miRNAs.

A d-peptide-based oral nanotherapeutic modulates the PD-1/PD-L1 interaction for tumor immunotherapy.

PD-1/PD-L1 immune checkpoint inhibitors are currently the most commonly utilized agents in clinical practice, which elicit an immunostimulatory response to combat malignancies. However, all these inhibitors are currently administered via injection using antibody-based therapies, while there is a growing need for oral alternatives. This study has developed and synthesized exosome-wrapped gold-peptide nanocomplexes with low immunogenicity, which can target PD-L1 and activate antitumor immunity in vivo through oral absorption. The SuperPDL1exo was characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and gel silver staining. The transmembrane ability of SuperPDL1exo was evaluated by flow cytometry and immunofluorescence. Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay. ELISA experiments were conducted to detect serum and tissue inflammatory factors, as well as serum biochemical indicators. Tissue sections were stained with H&E for the evaluation of the safety of SuperPDL1exo. An MC38 colon cancer model was established in immunocompetent C56BL/6 mice to evaluate the effects of SuperPDL1exo on tumor growth in vivo. Immunohistochemistry (IHC) staining was performed to detect cytotoxicity factors such as perforin and granzymes. First, SuperPDL1 was successfully synthesized, and milk exosome membranes were encapsulated through ultrasound, repeated freeze-thaw cycles, and extrusion, resulting in the synthesis of SuperPDL1exo. Multiple characterization results confirmed the successful synthesis of SuperPDL1exo nanoparticles. Furthermore, our data demonstrated that SuperPDL1exo exhibited excellent colloidal stability and superior cell transmembrane ability. In vitro and in vivo experiments revealed that SuperPDL1exo did not cause damage to multiple systemic organs, demonstrating its good biocompatibility. Finally, in the MC38 colon cancer mouse model, it was discovered that SuperPDL1exo could inhibit the progression of colon cancer, and this tumor-suppressive effect was mediated through the activation of tumor-specific cytotoxic T lymphocyte (CTL)-related immune responses. This study has successfully designed and synthesized an oral nanotherapeutic, SuperPDL1exo, which demonstrates small particle size, excellent colloidal stability, transmembrane ability in tumor cells, and biocompatibility. In vivo experiments have shown that it effectively activates T-cell immunity and exerts antitumor effects.

Human Milk Exosomes Induce ZO-1 Expression via Inhibition of REDD1 Expression in Human Intestinal Epithelial Cells.

Human milk exosomes (HMEs) enhance intestinal barrier function and contribute to an improvement in inflammation and mucosal injury, such as necrotizing enteritis (NEC), in infants. Here, we aimed to elucidate the intracellular factors involved in HME-induced expression of zonula occludens-1 (ZO-1), a tight junction protein, in Caco-2 human intestinal epithelial cells. HME treatment for 72 h significantly increased transepithelial electrical resistance in these cells. The mean ZO-1 protein levels in cells treated with HME for 72 h were significantly higher than those in the control cells. The mRNA and protein levels of regulated in development and DNA damage response 1 (REDD1) in HME-treated cells were significantly lower than those in the control cells. Although HME treatment did not increase the mechanistic target of rapamycin (mTOR) level in Caco-2 cells, it significantly increased the phosphorylated mTOR (p-mTOR) level and p-mTOR/mTOR ratio. The ZO-1 protein levels in cells treated with an inducer of REDD1, cobalt chloride (CoCl2) alone were significantly lower than those in the control cells. However, ZO-1 protein levels in cells co-treated with HME and CoCl2 were significantly higher than those in cells treated with CoCl2 alone. Additionally, REDD1 protein levels in cells treated with CoCl2 alone were significantly higher than those in the control cells. However, REDD1 protein levels in cells co-treated with HME and CoCl2 were significantly lower than those in cells treated with CoCl2 alone. This HME-mediated effect may contribute to the development of barrier function in the infant intestine and protect infants from diseases.

Exosomes as an Emerging Plasmid Delivery Vehicle for Gene Therapy.

Despite its introduction more than three decades ago, gene therapy has fallen short of its expected potential for the treatment of a broad spectrum of diseases and continues to lack widespread clinical use. The fundamental limitation in clinical translatability of this therapeutic modality has always been an effective delivery system that circumvents degradation of the therapeutic nucleic acids, ensuring they reach the intended disease target. Plasmid DNA (pDNA) for the purpose of introducing exogenous genes presents an additional challenge due to its size and potential immunogenicity. Current pDNA methods include naked pDNA accompanied by electroporation or ultrasound, liposomes, other nanoparticles, and cell-penetrating peptides, to name a few. While the topic of numerous reviews, each of these methods has its own unique set of limitations, side effects, and efficacy concerns. In this review, we highlight emerging uses of exosomes for the delivery of pDNA for gene therapy. We specifically focus on bovine milk and colostrum-derived exosomes as a nano-delivery "platform". Milk/colostrum represents an abundant, scalable, and cost-effective natural source of exosomes that can be loaded with nucleic acids for targeted delivery to a variety of tissue types in the body. These nanoparticles can be functionalized and loaded with pDNA for the exogenous expression of genes to target a wide variety of disease phenotypes, overcoming many of the limitations of current gene therapy delivery techniques.

Milk Exosomes from Gestational Diabetes Mellitus (GDM) and Healthy Parturient Exhibit Differential miRNAs Profiles and Distinct Regulatory Bioactivity on Hepatocyte Proliferation.

Exosomes, a novel type of bioactive component in human milk (HM), affect infant development, growth, and health. Recent studies indicate that HM exosomes and miRNAs relate to gestational diabetes mellitus (GDM). However, the miRNAs profiles and functionalities of HM exosomes from GDM parturient remain unclear. This study aims to compare the differential miRNAs in HM exosomes from GDM and healthy parturient, and investigate the HM exosomes bioactivities in regulating hepatocyte proliferation and insulin sensitivity. This study extracted HM exosomes from GDM (GDM-EXO) and healthy (NOR-EXO) parturient by ultracentrifugation, high-throughput sequenced and compared the exosomal miRNAs profiles, and explored the regulatory bioactivities on hepatocyte proliferation in HepG2 cells and Balb/c mice. As compared to NOR-EXO, GDM-EXO has similar morphology, size, concentration, and exosome-specific markers (CD9 and TSG101) expression. GDM-EXO and NOR-EXO specifically harbor 1299 and 8 miRNAs, respectively. Moreover, GDM-EXO had 176 upregulated and 47 downregulated miRNAs compared with NOR-EXO. Both GDM-EXO and NOR-EXO were absorbed in cultured HepG2 hepatocytes and mice liver. GDM-EXO inhibited hepatocytes proliferation by downregulating mammalian target of rapamycin (mTOR) possibly via exosomal miR-101-3p delivery. HM exosomes from GDM and healthy parturient exhibit differential miRNAs profiles and distinct regulatory bioactivity on hepatocyte proliferation.

Milk Exosomes: Next-Generation Agents for Delivery of Anticancer Drugs and Therapeutic Nucleic Acids.

Exosomes are nanovesicles 40-120 nm in diameter secreted by almost all cell types and providing humoral intercellular interactions. Given the natural origin and high biocompatibility, the potential for loading various anticancer molecules and therapeutic nucleic acids inside, and the surface modification possibility for targeted delivery, exosomes are considered to be a promising means of delivery to cell cultures and experimental animal organisms. Milk is a unique natural source of exosomes available in semi-preparative and preparative quantities. Milk exosomes are highly resistant to the harsh conditions of the gastrointestinal tract. In vitro studies have demonstrated that milk exosomes have an affinity to epithelial cells, are digested by cells by endocytosis mechanism, and can be used for oral delivery. With milk exosome membranes containing hydrophilic and hydrophobic components, exosomes can be loaded with hydrophilic and lipophilic drugs. This review covers a number of scalable protocols for isolating and purifying exosomes from human, cow, and horse milk. Additionally, it considers passive and active methods for drug loading into exosomes, as well as methods for modifying and functionalizing the surface of milk exosomes with specific molecules for more efficient and specific delivery to target cells. In addition, the review considers various approaches to visualize exosomes and determine cellular localization and bio-distribution of loaded drug molecules in tissues. In conclusion, we outline new challenges for studying milk exosomes, a new generation of targeted delivery agents.

Bovine milk-derived exosomes attenuate NLRP3 inflammasome and NF-κB signaling in the lung during neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC), an inflammatory intestinal disease common in premature infants, has been associated with the development of lung damage. Toll-like receptor 4 has been shown to regulate inflammation in the NEC lungs, however, other important inflammatory mechanisms have not been thoroughly investigated. In addition, we reported that milk-derived exosomes were able to attenuate intestinal injury and inflammation in experimental NEC. This study aims to (i) investigate the role of the NLRP3 inflammasome and NF-κB pathway in regulating lung damage during experimental NEC; and (ii) evaluate the therapeutic potential of bovine milk exosomes in reducing lung inflammation and injury during NEC. NEC was induced by gavage feeding of hyperosmolar formula, hypoxia, and lipopolysaccharide administration in neonatal mice from postnatal days 5-9. Exosomes were obtained by ultracentrifugation of bovine milk and administered during each formula feed. The lung of NEC pups showed increased inflammation, tissue damage, NLRP3 inflammasome expression, and NF-κB pathway activation, which were attenuated upon exosome administration. Our findings suggest that the lung undergoes significant inflammation and injury following experimental NEC which are attenuated by bovine milk-derived exosomes. This emphasizes the therapeutic potential of exosomes not just on the intestine but also on the lung.

Influence of Estrus on Dairy Cow Milk Exosomal miRNAs and Their Role in Hormone Secretion by Granulosa Cells.

Estrus is crucial for cow fertility in modern dairy farms, but almost 50% of cows do not show the behavioral signs of estrus due to silent estrus and lack of suitable and high-accuracy methods to detect estrus. MiRNA and exosomes play essential roles in reproductive function and may be developed as novel biomarkers in estrus detection. Thus, we analyzed the miRNA expression patterns in milk exosomes during estrus and the effect of milk exosomes on hormone secretion in cultured bovine granulosa cells in vitro. We found that the number of exosomes and the exosome protein concentration in estrous cow milk were significantly lower than in non-estrous cow milk. Moreover, 133 differentially expressed exosomal miRNAs were identified in estrous cow milk vs. non-estrous cow milk. Functional enrichment analyses indicated that exosomal miRNAs were involved in reproduction and hormone-synthesis-related pathways, such as cholesterol metabolism, FoxO signaling pathway, Hippo signaling pathway, mTOR signaling pathway, steroid hormone biosynthesis, Wnt signaling pathway and GnRH signaling pathway. Consistent with the enrichment signaling pathways, exosomes derived from estrous and non-estrous cow milk both could promote the secretion of estradiol and progesterone in cultured bovine granulosa cells. Furthermore, genes related to hormonal synthesis (CYP19A1, CYP11A1, HSD3B1 and RUNX2) were up-regulated after exosome treatment, while exosomes inhibited the expression of StAR. Moreover, estrous and non-estrous cow-milk-derived exosomes both could increase the expression of bcl2 and decrease the expression of p53, and did not influence the expression of caspase-3. To our knowledge, this is the first study to investigate exosomal miRNA expression patterns during dairy cow estrus and the role of exosomes in hormone secretion by bovine granulosa cells. Our findings provide a theoretical basis for further investigating milk-derived exosomes and exosomal miRNA effects on ovary function and reproduction. Moreover, bovine milk exosomes may have effects on the ovaries of human consumers of pasteurized cow milk. These differential miRNAs might provide candidate biomarkers for the diagnosis of dairy cow estrus and will assist in developing new therapeutic targets for cow infertility.

Isolation of Bovine Milk Exosome Using Electrophoretic Oscillation Assisted Tangential Flow Filtration with Antifouling of Micro-ultrafiltration Membrane Filters

Tangent flow-driven ultrafiltration (TF-UF) is an efficient isolation process of milk exosomes without morphological deformation. However, the TF-UF approach with micro-ultrafiltration SiNx membrane filters suffers from the clogging and fouling of micro-ultrafiltration membrane filter pores with large bioparticles. Thus, it is limited in the long term, continuous isolation of large quantities of exosomes. In this work, we introduced electrophoretic oscillation (EPO) in the TF-UF approach to remove pore clogging and fouling of with micro-ultrafiltration SiNx membrane filters by large bioparticles. As a result, the combined EPO-assisted TF (EPOTF) filtration can isolate large quantities of bovine milk exosomes without deformation. Furthermore, several morphological and biological analyses confirmed that the EPOTF filtration approach could isolate the milk exosomes in high concentrations with high purity and intact morphology. In addition, the uptake test of fluorescent-labeled exosomes by the keratinocyte cells visualized the biological function of purified exosomes. Hence, compared to the TF-UF process, the EPOTF filtration produced a higher yield of bovine milk exosomes without stopping the filtering process for over 200 h. Therefore, this isolation process enables scalable and continuous production of morphologically intact exosomes from bovine milk, suggesting that high-quality exosome purification is possible for future applications such as drug nanocarriers, diagnosis, and treatments.

The potential therapeutic role of camel milk exosomes – A review

Abstract Exosomes (EXOs) are naturally occurring nano-sized vesicles released into the extracellular environment by exocytosis. EXOs contribute to intercellular communications by regulating DNA, mRNA, and protein levels. EXOs are considered good vehicles for delivering therapeutic agents in multiple diseases. Camel milk (CM) is a valuable food with a rich source of EXOs. Camel milk exosomes (CMEXOs) have unique ingredients compared to other animal milks. It is reported that CMEXOs reduce the growth of cancer cells by inducing apoptosis, and inhibiting oxidative stress and inflammation. CMEXOs can block stress signaling pathways including inflammation and apoptosis which could be resulted in adverse effects if medication levels exceed the therapeutic range. Moreover, CMEXOs improve the antioxidant capability in both in vitro and in vivo experiments. On the other hand, the anti-angiogenesis property of CMEXOs has been reported via decrease in expression of the angiogenesis-related gene of vascular endothelial growth factor (VEGF). It is predicted that exosomal lactoferrin (LF) and kappa casein (KC) mRNAs are crucial parts of CMEXOs mediating their anticancer effects. The immunomodulatory effect of CMEXOs may be attributed to their high contents of LF and KC. According to previous works, CMEXOs are promising alternatives in developing new therapeutic models for multipile diseases. This review aims to provide an overview of the isolation, characterization and biological activities of the exosomes derived from camel milk for addressing their possible use in therapeutics.

Cow milk-derived exosomes enhance the activity of the Na+/K+ ATPase in Caco-2 cells via EP3 and EP4 receptors

The increase in bovine dairy consumption has been correlated with the development of many inflammatory diseases. Emerging evidence points to milk exosomes as potential causative agents. The cargo of exosome consists of cytokines that are elevated in inflammatory bowel disease and modulate the activity of the colonic Na+/K+ ATPase. Whether a cause effect relationship exists between the exosomes and the Na+/K+ ATPase is a question that has not been addressed before and constitutes the aim of this work, using Caco-2 cells as a model. Exosomes were extracted from skim milk by a series of ultra-centrifugations and filtrations. The activity of the pump was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. The exosomes increased dose and time-dependently the activity of the ATPase with a maximal effect observed at 1 h. This increase was mediated via PGE2 and still appeared in presence of a blocker of EP1 and EP2, was enhanced further when EP4 was blocked, and changed to an inhibition in presence of an EP3 antagonist, suggesting an involvement of both EP3 and EP4. The activation of the Na+/K + ATPase was significantly enhanced or reduced when cells were treated respectively with an inhibitor or an activator of PKA. Since EP4 and EP3 act via Gs and Gi respectively, they exert opposite effects on PKA and consequently on the ATPase. The stimulatory effect of EP3 on the ATPase predominates over that of EP4 resulting in a net activation of the pump.