Current Research Status of Milk Exosomes

Exosomes are membrane-associated vesicles excreted by cells, and they have been identified in various physiological fluids - blood, urine, saliva, milk, ascites. Exosomes enable inter-cell communication by transporting their cargo from the source cell to the destination cell. Due to their high biocompatibility, exosomes present a promising potential as drug delivery vehicles. Milk exosomes can be extracted from colostrum and milk from cows, porcine milk, rat milk, ovine milk, kangaroo milk, and human milk. These exosomes can be isolated in both semi-preparative and preparative scales. Milk exosomes are highly resistant to the digestive tract, a promising target for oral drug delivery, which has gained popular attention.

Cargo of Milk Exosomes

MicroRNA (miRNA): Exosomes of milk contain many microRNAs that are involved in the regulation of the mammary gland and the immune system. Colostrum has been found to contain more immune- and development-related miRNAs than mature milk. Some miRNAs are shared between species, but others depend on species and the health of the mother.
Proteins: The proteins present in milk exosomes are divided into membrane-bound and intraluminal proteins. These membrane-bound proteins include conserved proteins like CD6, CD81, and CD9, or even special proteins like CD47, and those that prevent macrophages from binding to and expelling exosomes. In general, colostrum exosomes are high in proteins related to immune activity, inflammation, antimicrobial peptides, cell growth and complement signalling. The exosomes of adult milk, on the other hand, contain proteins involved in transport and death.
Nucleic acids: Milk exosomes contain nucleic acids such as DNA, mRNA, rRNA and lncRNA, which are bound in the exosome's lipid bilayer. Research on DNA within milk exosomes is still limited, and its specific functions remain unclear. Most milk-derived RNAs are encapsulated within exosomes, with over 19,000 mRNAs detected in milk exosomes, involved in protein synthesis and cell signaling pathways.
Lipids: Only a few reports describe the amount of lipids in milk exosomes, and further studies are needed to explain their function. But we know that lipids are essential for maintaining the structural integrity and stability of exosomes.

Schematic diagram of cargo transport of milk exosomes. Fig. 1. Graphical representation of milk exosomes showing general exosomal cargos (Feng, X., Chen, X., et al., 2021).

Isolation of Milk Exosomes

Isolation of milk exosomes is performed in the same steps as for other exosomes, such as differential centrifugation, tangential flow filtration, density gradient centrifugation, ultracentrifugation, immune affinity capture, gel filtration and other chromatographic methods. The point is that because milk is extremely saturated in lipids and proteins, they must be removed before the exosome isolated. Relying on a single isolation technique may result in low-quality exosomes; therefore, combining multiple methods often yields higher quality exosomes.

The following table outlines common methods for isolating milk exosomes, along with their respective advantages and disadvantages.

Table. 1. Extraction and purification of exosomes from milk and other biological fluids: advantages and disadvantages (Timofeeva AM, Paramonik AP., et al., 2023).

Methods of Isolation	Principle of Isolation	Advantages	Disadvantages
Centrifugation	Differential centrifugation, density gradient centrifugation, isoelectric deposition	Method simplicity, high yield	Time-consuming, low purity, exosomes can be destroyed, high cost of ultracentrifuges
Ultrafiltration	By vesicle size	Low cost, no expensive equipment required	Low purity
Immunological	Binding of antibodies to specific exosome markers	High purity, high specificity, low equipment cost, easy to operate	High reagent cost, low yield
Microfluidic devices	Size, density, surface antigens	High purity, portability, easy to operate, time-saving, automation, speed of extraction	Small sample volume, no method validation
Chromatography	Gel filtration, affinity chromatography	High purity, scalability	Requires special equipment, sorbents
Co-precipitation	Precipitation with commercial reagents	Easy to use, time-saving	Expensive, low purity, small sample volume, difficult to scale

Identification of Milk Exosomes

Physical characteristics: Common methods for identification include transmission electron microscopy (TEM), dynamic light scattering (DLS), tunable resistive pulse sensing (tRPS), and nanoparticle tracking analysis (NTA). These techniques are used to determine the size, morphology, and other fundamental structural characteristics of milk exosomes (MDEs). Typically, milk exosomes have a cup-shaped structure and range in size from 30 to 150 nanometers.
Biochemical characteristics: Biochemical identification usually involves techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry to identify specific surface proteins and internal contents of exosomes.

Applications of Milk Exosomes

Mastitis biomarkers: Milk exosomes can vary with disease progression, and can thus serve as biomarkers for a range of disorders. In bovine mastitis, for instance, miRNAs are differentially expressed in exosomes: miR-223 and miR-142-5p are significantly altered, suggesting that they might serve as markers for mastitis.
Immune regulation: Exosomes of milk are rich in immune-regulating miRNAs that regulate the immune system. The milk exosomes can also be used to suppress the expression of DNMT1 and DNMT3, potentially preventing colon cancer.
Gut genesis: Milk exosomes are non-competitive against protease and ribonuclease digestion, so they easily slip through the digestive tract and get absorbed. Evidence suggests that these exosomes can control gut epithelial permeability, barrier function, and proliferation.
Drug delivery: Exosomes deliver siRNA, miRNA, proteins, small molecules, and nanoparticles. Milk exosomes have the additional benefit of being resistant to bad gut health, making them candidates for oral delivery.

Advantages of Milk Exosomes

Rich source: Milk is a readily accessible and cost-effective source of exosomes.
High safety: As milk is generally consumed, the milk exosomes are highly safe when used as transporters.
Resistance: The surface glycoproteins (XDH, BTN, MUC1) and surface proteins (FLOT1, ICAM1, ALIX, EpCAM) in milk exosomes have gastric protease resistance, enhancing stability and cross-biological integration for oral and minimally invasive delivery.
Biocompatibility: Milk exosomes are highly biocompatible, low toxic and non-immunogenic properties..
Anti-cytotoxic: Milk exosomes can inhibit cisplatin-induced cytotoxicity in macrophages and have been shown to reduce the toxicity of anticancer drugs.
Stability: Milk exosomes exhibit stability in the bloodstream, facilitating the efficient systemic distribution of therapeutic agents.

Products & Services	Description
Exosome Analysis	Creative Bioarray provides diverse exosomal species analysis to help you understand your exosome compositions.
Exosome Identification	Creative Bioarray provides comprehensive support for your exosome identification by including the morphology assay, purity, and quantity assay, particle size distribution analysis, and exosome-specific markers expression.
Exosome Isolation and Purification	Creative Bioarray provides reliable techniques for exosome isolation from different sample matrices. We also provide exosome isolation using specified techniques if required by customers.

References

Feng, X., et al. Latest trend of milk derived exosomes: cargos, functions, and applications. Frontiers in nutrition, 2021. 8, 747294.
Timofeeva AM., et al. Milk exosomes: next-generation agents for delivery of anticancer drugs and therapeutic nucleic acids. Int J Mol Sci. 2023. 15;24(12):10194.

For research use only. Not for any other purpose.