Milk-derived Extracellular Vesicles Research Articles

Phospholipid-Anchored Ligand Conjugation on Extracellular Vesicles for Enhanced Cancer Targeting.

Extracellular vesicles (EVs) are recognized as potential candidates for next-generation drug delivery systems. However, the inherent cancer-targeting efficiency is unsatisfactory, necessitating surface modification to attach cell-binding ligands. By utilizing phospholipase D from Streptomyces in combination with maleimide-containing primary alcohol, the authors successfully anchored ligands onto milk-derived EVs (mEVs), overcoming the issues of ligand leakage or functional alteration seen in traditional methods. Quantitative nano-flow cytometry demonstrated that over 90% of mEVs are effectively modified with hundreds to thousands of ligands. The resulting mEV formulations exhibited remarkable long-term stability in conjugation proportion, ligand number, size distribution, and particle concentration, even after months of storage. It is further shown that conjugating transferrin onto mEVs significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel. Overall, this study presents a highly efficient, stable, cost-effective, and scalable ligand conjugation approach, offering a promising strategy for targeted drug delivery of EVs.

Harnessing Nature's Defence: The Antimicrobial Efficacy of Pasteurised Cattle Milk-Derived Extracellular Vesicles on Staphylococcus aureus ATCC 25923.

Increasing antimicrobial resistance (AMR) challenges conventional antibiotics, prompting the search for alternatives. Extracellular vesicles (EVs) from pasteurised cattle milk offer promise, due to their unique properties. This study investigates their efficacy against five pathogenic bacteria, including Staphylococcus aureus ATCC 25923, aiming to combat AMR and to develop new therapies. EVs were characterised and tested using various methods. Co-culture experiments with S. aureus showed significant growth inhibition, with colony-forming units decreasing from 2.4 × 105 CFU/mL (single dose) to 7.4 × 104 CFU/mL (triple doses) after 12 h. Milk EVs extended lag time (6 to 9 h) and increased generation time (2.8 to 4.8 h) dose-dependently, compared to controls. In conclusion, milk EVs exhibit dose-dependent inhibition against S. aureus, prolonging lag and generation times. Despite limitations, this suggests their potential in addressing AMR.

Milk-Derived Extracellular Vesicles: Biomedical Applications, Current Challenges, and Future Perspectives.

Extracellular vesicles (EVs) are nano to-micrometer-sized sacs that are released by almost all animal and plant cells and act as intercellular communicators by transferring their cargos between the source and target cells. As a safe and scalable alternative to conditioned medium-derived EVs, milk-derived EVs (miEVs) have recently gained a great deal of popularity. Numerous studies have shown that miEVs have intrinsic therapeutic actions that can treat diseases and enhance human health. Additionally, they can be used as natural drug carriers and novel classes of biomarkers. However, due to the complexity of the milk, the successful translation of miEVs from benchtop to bedside still faces several unfilled gaps, especially a lack of standardized protocols for the isolation of high-purity miEVs. In this work, by comprehensively reviewing the bovine miEVs studies, we provide an overview of current knowledge and research on miEVs while highlighting their challenges and enormous promise as a novel class of theranostics. It is hoped that this study will pave the way for clinical applications of miEVs by addressing their challenges and opportunities.

The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1β). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Effects of milk extracellular vesicles from bovine, human, and caprine origin on rotavirus infectivity in cultured human intestinal cells

Rotavirus is the leading cause of severe gastroenteritis in neonates representing a global health burden. Milk-derived extracellular vesicles [EVs] have emerged as potential antivirals, offering a promising avenue for intervention. The present study evaluated the antiviral activity of EVs sourced from bovine, human, and caprine milk against the infection of the human Wa and bovine WC3 rotavirus strains. EVs induced an endogenous effect in Caco-2 cells, resulting in a significant reduction of rotaviral infection. The antiviral properties of the milk EVs, were found to be associated with synergistic interactions at pre- and post-attachment stages of the infection. Finally, the effect of milk processing on bovine milk EVs efficacy was assessed. It was found that after high-temperature short-time pasteurization EVs retained their activity, while homogenization resulted in no antiviral effect at all. Overall, our findings demonstrate a significant reduction in rotavirus infection in cultured Caco-2 cells when treated with milk EVs.

Extracellular vesicles separated from goat milk by differential centrifugation coupled with sodium citrate pretreatments

Satisfactory separation of milk-derived extracellular vesicles (MEVs) is important for the downstream analysis of the functions and properties of MEVs. However, the presence of abundant proteins in milk hindered the separation of MEVs. In this study, three pretreatment methods, including sodium citrate (SC), acetic acid (AA), and high-speed centrifugation, were adopted to separate MEVs from goat milk while minimizing the impact of protein. The MEVs were then characterized by nanoparticle tracking, transmission electron microscopy and western blotting experiments. The results indicated that pretreatments with AA and SC greatly decreased the impact of casein, but AA pretreatment damaged the surface structure of MEVs. Additionally, the differential centrifugation process resulted in a slight loss of MEVs. Overall, MEVs with small size and high purity can be obtained under 125 k × g centrifugation combined with SC pretreatment, which suggests a promising method for separation of MEVs from goat milk.

Maternal fiber-rich diet promotes early-life intestinal development in offspring through milk-derived extracellular vesicles carrying miR-146a-5p

BackgroundsThe intestinal development in early life is profoundly influenced by multiple biological components of breast milk, in which milk-derived extracellular vesicles (mEVs) contain a large amount of vertically transmitted signal from the mother. However, little is known about how maternal fiber-rich diet regulates offspring intestinal development by influencing the mEVs.ResultsIn this study, we found that maternal resistant starch (RS) consumption during late gestation and lactation improved the growth and intestinal health of offspring. The mEVs in breast milk are the primary factor driving these beneficial effects, especially enhancing intestinal cell proliferation and migration. To be specific, administration of mEVs after maternal RS intake enhanced intestinal cell proliferation and migration in vivo (performed in mice model and indicated by intestinal histological observation, EdU assay, and the quantification of cyclin proteins) and in vitro (indicated by CCK8, MTT, EdU, and wound healing experiments). Noteworthily, miR-146a-5p was found to be highly expressed in the mEVs from maternal RS group, which also promotes intestinal cell proliferation in cells and mice models. Mechanically, miR-146a-5p target to silence the expression of ubiquitin ligase 3 gene NEDD4L, thereby inhibiting DVL2 ubiquitination, activating the Wnt pathway, and promoting intestinal development.ConclusionThese findings demonstrated the beneficial role of mEVs in the connection between maternal fiber rich diet and offspring intestinal growth. In addition, we identified a novel miRNA-146a-5p-NEDD4L-β-catenin/Wnt signaling axis in regulating early intestinal development. This work provided a new perspective for studying the influence of maternal diet on offspring development.Graphical

Potentials of Milk-Derived Extracellular Vesicles as Carriers for Oral Delivery of Active Phytoconstituents.

Extracellular vesicles (EVs) play a crucial role in intercellular communication and have the potential to serve as in vivo carriers for delivering active molecules. The biocompatibility advantages of EVs over artificial nanocarriers create new frontiers for delivering modern active molecules. Milk is a favorable source of EVs because of its high bioavailability, low immunogenicity, and commercial producibility. In this study, we analyzed the advantages of milk-derived EVs in the oral delivery of active molecules, discussed their research progress in delivering active phytoconstituents, and summarized the necessary technologies and critical unit operations required for the development of an oral delivery system based on EVs. The review aims to provide innovative ideas and fundamental quality control guidelines for developing the next-generation oral drug delivery system based on milk-derived EVs. Expected final online publication date for the Annual Review of Food Science and Technology, Volume 15 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Particles in Raw Sheep Milk Can Modulate the Inflammatory Response in THP-1, a Human Monocyte Cell Line, In Vitro

Background: The UK dairy sheep industry is relatively small but growing, particularly for cheese and yogurt products. Anecdotally, sheep milk (SM) may be better tolerated by humans than cows’ milk and could have environmental as well as health benefits. All milk contains sub-micron particles called extracellular vesicles (EVs) which are mainly derived from the mammary epithelium. Physiologically, milk-derived EVs are thought to aid in the development of infant immunity and the microbiome, but may also have health benefits to adult humans. The purpose of this study was to determine whether EVs could be isolated from raw sheep milk and whether they have any effect on inflammatory responses in THP-1, a human monocyte cell line, in vitro. Methods: Using sequential ultracentrifugation, vesicles of <1 µm (LEV) followed by <200 nm (sEVs) were isolated from six individual sheep during mid-lactation. RNA was extracted and microRNA analyzed by RTqPCR for sequences previously identified in cows’ milk. Human THP-1 monocytes were differentiated into macrophages and incubated with SM-derived LEVs and sEVs in the presence of pro-inflammatory LPS to measure the effects on the secretion of the chemokine CCL-2 or in the presence of DMNQ and fluorescent dihydrorhodamine-1,2,3 to measure reactive oxygen species. Results: LEVs induced an increase in ROS in both monocytes and macrophages, whilst sEVs decreased DMNQ-mediated ROS in macrophages but not monocytes. Interestingly, the LEVs did not induce CCL2 release; however, they increased LPS-induced CCL2 secretion in monocytes but not macrophages. miR26a, miR92a, miR125b, miR155 and miR223 were identified in both sEVs and LEVs by RT-qPCR and could be responsible for the modulation of ROS and CCL2 expression. Conclusions: These findings suggest that like cows’ milk, sheep milk contains EVs, and they can influence human monocyte/macrophage responses, and so is worthy of further investigation for its potential human- and non-human-animal health benefits.

Oral Milk-Derived Extracellular Vesicles Inhibit Osteoclastogenesis and Ameliorate Bone Loss in Ovariectomized Mice by Improving Gut Microbiota.

Milk-derived extracellular vesicles can improve intestinal health and have antiosteoporosis potential. In this paper, we explored the effects of bovine raw milk-derived extracellular vesicles (mEVs) on ovariectomized (OVX) osteoporotic mice from the perspective of the gut-bone axis. mEVs could inhibit osteoclast differentiation and improve microarchitecture. The level of osteoporotic biomarkers in OVX mice was restored after the mEVs intervened. Compared with OVX mice, mEVs could enhance intestinal permeability, reduce endotoxin levels, and improve the expression of TNF-α, IL-17, and IL-10. 16S rDNA sequencing indicated that mEVs altered the composition of gut microbiota, specifically for Bacteroides associated with short-chain fatty acids (SCFAs). In-depth analysis of SCFAs demonstrated that mEVs could restore acetic acid, propionic acid, valeric acid, and isovaleric acid levels in OVX mice. Correlation analysis revealed that changed gut microbiota and SCFAs were significantly associated with gut inflammation and osteoporotic biomarkers. This study demonstrated that mEVs could inhibit osteoclast differentiation and improve osteoporosis by reshaping the gut microbiota, increasing SCFAs, and decreasing the level of pro-inflammatory cytokines and osteoclast differentiation-related factors in OVX mice. These findings provide evidence for the use of mEVs as a food supplement for osteoporosis.

Applications of emerging extracellular vesicles technologies in the treatment of inflammatory diseases.

The emerging extracellular vesicles technologies is an advanced therapeutic approach showing promising potential for addressing inflammatory diseases. These techniques have been proven to have positive effects on immune modulation and anti-inflammatory responses. With these advancements, a comprehensive review and update on the role of extracellular vesicles in inflammatory diseases have become timely. This review aims to summarize the research progress of extracellular vesicle technologies such as plant-derived extracellular vesicles, milk-derived extracellular vesicles, mesenchymal stem cell-derived extracellular vesicles, macrophage-derived extracellular vesicles, etc., in the treatment of inflammatory diseases. It elucidates their potential significance in regulating inflammation, promoting tissue repair, and treating diseases. The goal is to provide insights for future research in this field, fostering the application and development of extracellular vesicle technology in the treatment of inflammatory diseases.

Toward the nanoscale chemical and physical probing of milk-derived extracellular vesicles using Raman and tip-enhanced Raman spectroscopy.

Tip-enhanced Raman spectroscopy (TERS) is an advanced technique to perform local chemical analysis of the surface of a sample through the improvement of the sensitivity and the spatial resolution of Raman spectroscopy by plasmonic enhancement of the electromagnetic signal in correspondence with the nanometer-sized tip of an atomic force microscope (AFM). In this work, TERS is demonstrated to represent an innovative and powerful approach for studying extracellular vesicles, in particular bovine milk-derived extracellular vesicles (mEVs), which are nanostructures with considerable potential in drug delivery and therapeutic applications. Raman spectroscopy has been used to analyze mEVs at the micrometric and sub-micrometric scales to obtain a detailed Raman spectrum in order to identify the 'signature' of mEVs in terms of their characteristic molecular vibrations and, therefore, their chemical compositions. With the ability to improve lateral resolution, TERS has been used to study individual mEVs, demonstrating the possibility of investigating a single mEV selected on the surface of the sample and, moreover, analyzing specific locations on the selected mEV with nanometer lateral resolution. TERS potentially allows one to reveal local differences in the composition of mEVs providing new insights into their structure. Also, thanks to the intrinsic properties of TERS to acquire the signal from only the first few nanometers of the surface, chemical investigation of the lipid membrane in correspondence with the various locations of the selected mEV could be performed by analyzing the peaks of the Raman shift in the relevant range of the spectrum (2800-3000 cm-1). Despite being limited to mEVs, this work demonstrates the potential of TERS in the analysis of extracellular vesicles.

Treatment of milk with various chemicals differentially affects the physicochemical and functional characteristics of extracellular vesicles

Isolation of high-purity milk-derived extracellular vesicles (miEV) is complicated by protein impurities, such as casein. The focus of this study was to investigate whether the treatment of milk with acetic acid (AA), ethylenediaminetetraacetic acid (EDTA), and sodium citrate (SC), which are known chemicals to deplete caseins from miEV, alters their physicochemical and functional characteristics differently. For this purpose, following the treatment of milk with AA, EDTA, and SC, various subpopulations (12 K, 35 K, 70 K, and 100 K) of miEV were isolated by differential ultracentrifugation and characterized by protein quantification assay, gel electrophoresis, western blotting, dynamic light scattering, and scanning electron microscopy. In addition, the cellular uptake and immunomodulatory potential of miEV were also assessed. The results indicated that protein impurities, including caseins, were differentially depleted from miEV subpopulations by AA, EDTA, and SC. However, the acidification of milk induced partial protein aggregation. Additionally, miEV from both AA- and EDTA-treated milk exhibited a less smooth surface and higher uptake compared to those from SC-treated milk. Although all miEV significantly reduced T cell proliferation, a higher immunomodulatory potential was found in miEV from AA-treated milk. In conclusion, AA, SC, and EDTA have different efficiencies in improving the purity of miEV and can affect their physicochemical and functional characteristics differently. This issue may be critical for the efficient isolation of pure subpopulations of miEV for biomedical research.

Site- and Structure-Specific Glycosylation Signatures of Bovine, Caprine, Porcine, and Human Milk-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane-bound vesicles released by living cells. As vesicles for macromolecule transmission and intercellular communication, EVs are broadly applied in clinical diagnosis and biomimetic drug delivery. Milk-derived EVs (MEVs) are an ideal choice for scale-up applications because they exhibit biocompatibility and are easily obtained. Herein, intact glycopeptides in MEVs from bovines, caprines, porcines, and humans were comprehensively analyzed by high-resolution mass spectrometry using the sceHCD, followed by the EThcD fragment method, revealing that protein glycosylation is abundant and heterogeneous in MEVs. The dominant glycans in all MEVs were sialic acid-modified N-linked glycans (over 50%). A couple of species-specific glycans were also characterized, which are potentially markers of different original EVs. Interestingly, the Neu5Gc-modified glycans were enriched in caprine milk-derived EVs (58 ± 2%). Heterogeneity of MEV protein glycosylation was observed for glycosites and glycan compositions, and the structural heterogeneity of protein glycosylation was also identified and validated. The glycosignatures of EV biogenesis- and endocytosis-related proteins (CD63 and MFGE8) were significantly different in these four species. Overall, we comprehensively characterized the glycosylation signature of MEVs from four different species and provided insight into protein glycosylation related to drug target delivery.

Identification of omega-3 oxylipins in human milk-derived extracellular vesicles with pro-resolutive actions in gastrointestinal inflammation.

Premature infants (PIs) are at risk of suffering necrotizing enterocolitis (NEC), and infants consuming human milk (HM) show a lower incidence than infants receiving formula. The composition of HM has been studied in depth, but the lipid content of HM-derived small extracellular vesicles (HM sEVs) remains unexplored. Identifying these molecules and their biological effects has potential for the treatment of intestinal disorders in PIs and could contribute to the development of HM-based fortified formulas. We isolated HM sEVs from HM samples and analyzed their oxylipin content using liquid chromatography coupled to mass spectrometry, which revealed the presence of anti-inflammatory oxylipins. We then examined the efficacy of a mixture of these oxylipins in combating inflammation and fibrosis, in vitro and in a murine model of inflammatory bowel disease (IBD). HM-related sEVs contained higher concentrations of oxylipins derived from docosahexaenoic acid, an omega-3 fatty acid. Three anti-inflammatory oxylipins, 14-HDHA, 17-HDHA, and 19,20-DiHDPA (ω3 OXLP), demonstrated similar efficacy to HM sEVs in preventing cell injury, inducing re-epithelialization, mitigating fibrosis, and modulating immune responses. Both ω3 OXLP and HM sEVs effectively reduced inflammation in IBD-model mice, preventing colon shortening, infiltration of inflammatory cells and tissue fibrosis. Incorporating this unique cocktail of oxylipins into fortified milk formulas might reduce the risk of NEC in PIs and also provide immunological and neurodevelopmental support.

Antioxidant-Engineered Milk-Derived Extracellular Vesicles for Accelerating Wound Healing via Regulation of the PI3K-AKT Signaling Pathway.

Inspired by the experience of relieving inflammation in infants with milk, antioxidant-engineered milk-derived extracellular vesicles (MEVs) are developed to evaluate their potential for accelerating wound healing. In this work, MEVs with polydopamines (PDA) are engineered using the co-extrusion method. Subsequently, the authors incorporated them into a Schiff-based crosslink hydrogel, forming a skin dosage form that could facilitate the wound healing process. The antioxidant properties of PDA assist in the anti-inflammatory function of engineered MEVs, while the gel provides better skin residency. The PDA@MEVs+GEL formulation exhibits excellent biocompatibility, pro-angiogenic capacity, and antioxidant ability in vitro. Furthermore, in vivo experiments demonstrate its efficacy in wound repair and inflammation inhibition. Mechanistically, PDA@MEVs+GEL simultaneously promotes the growth, migration, and anti-inflammation of 3T3 cells by activating PI3K-AKT pathway. Moreover, PDA@MEVs+GEL exhibits enhanced functionality in promoting wound healing in vivo, attributed to its ability to inhibit inflammation, stimulate angiogenesis, and promote collagen synthesis. In conclusion, this study delves into the mechanism of MEVs and underscores the improved efficacy of engineered extracellular vesicles. Additionally, the feasibility and prospect of engineered MEVs in treating skin wounds are verified, suggesting that antioxidant-engineered MEVs could be a promising therapeutic agent for wound healing applications.

Human milk extracellular vesicles enhance muscle growth and physical performance of immature mice associating with Akt/mTOR/p70s6k signaling pathway

Extracellular vesicles (EVs) play an important role in human and bovine milk composition. According to excellent published studies, it also exerts various functions in the gut, bone, or immune system. However, the effects of milk-derived EVs on skeletal muscle growth and performance have yet to be fully explored. Firstly, the current study examined the amino acids profile in human milk EVs (HME) and bovine milk EVs (BME) using targeted metabolomics. Secondly, HME and BME were injected in the quadriceps of mice for four weeks (1 time/3 days). Then, related muscle performance, muscle growth markers/pathways, and amino acids profile were detected or measured by grip strength analysis, rotarod performance testing, Jenner-Giemsa/H&E staining, Western blotting, and targeted metabolomics, respectively. Finally, HME and BME were co-cultured with C2C12 cells to detect the above-related indexes and further testify relative phenomena. Our findings mainly demonstrated that HME and BME significantly increase the diameter of C2C12 myotubes. HME treatment demonstrates higher exercise performance and muscle fiber densities than BME treatment. Besides, after KEGG and correlation analyses with biological function after HME and BME treatment, results showed L-Ornithine acts as a “notable marker” after HME treatment to affect mouse skeletal muscle growth or functions. Otherwise, L-Ornithine also significantly positively correlates with the activation of the AKT/mTOR pathway and myogenic regulatory factors (MRFs) and can also be observed in muscle and C2C12 cells after HME treatment. Overall, our study not only provides a novel result for the amino acid composition of HME and BME, but the current study also indicates the advantage of human milk on skeletal muscle growth and performance.

Comparison of goat and cow milk-derived extracellular vesicle miRNomes

miRNAs present in milk are mainly found in extracellular vesicles (EVs), which are nanosized membrane vesicles released by most of the cell types to ensure intercellular communication. The majority of the studies performed so far on these vesicles have been conducted on human and cow’s milk and focused on their miRNA content. The objectives of this study were to profile the miRNA content of purified EVs from five healthy goats and to compare their miRNome to those obtained from five healthy cows, at an early stage of lactation. EV populations were morphologically characterized using Transmission Electron Microscopy and Nanoparticle Tracking Analysis. The presence of EV protein markers checked by Western blotting and the absence of contamination of preparations by milk proteins. The size distribution and concentration of bovine and goat milk-derived EVs were similar. RNA-sequencing were performed, and all sequences were mapped to the cow genome identifying a total of 295 miRNAs. This study reports for the first-time a goat miRNome from milk EVs and its validation using cow miRNomes.

Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30–1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120–210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1–13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Bovine milk-derived extracellular vesicles enhance doxorubicin sensitivity in triple negative breast cancer cells by targeting metabolism and STAT signalling.

Metastatic triple-negative breast cancer (TNBC) has a low 5-year survival rate of below 30% with systemic chemotherapy being the most widely used treatment. Bovine milk-derived extracellular vesicles (MEVs) have been previously demonstrated to have anti-cancer attributes. In this study, we isolated bovine MEVs from commercial milk and characterised them according to MISEV guidelines. Bovine MEVs sensitised TNBC cells to doxorubicin, resulting in reduced metabolic potential and cell-viability. Label-free quantitative proteomics of cells treated with MEVs and/or doxorubicin suggested that combinatorial treatment depleted various pro-tumorigenic interferon-inducible gene products and proteins with metabolic function, previously identified as therapeutic targets in TNBC. Combinatorial treatment also led to reduced abundance of various STAT proteins and their downstream oncogenic targets with roles in cell-cycle and apoptosis. Taken together, this study highlights the ability of bovine MEVs to sensitise TNBC cells to standard-of-care therapeutic drug doxorubicin, paving the way for novel treatment regimens.