Discussion
The current study investigated the feasibility of isolating milk-derived exosomes from mare’s milk derived EVs and tested their suitability as therapeutic carriers. There are other milk and dietary derived EVs available, but this study focuses on testing the concept of mare’s milk, a widely consumed milk in central Asia. Different exosome isolation methods were used and compared based on the presence of exosomal markers confirming particle isolation using immunoblotting, particle size as confirmed by proteomic profile (Zetasizer), exosome morphology using TEM, and protein concentration (bicinchoninic acid assay, BCA). There are a number of methods available to extract milk-derived exosomes including centrifugation, and ultracentrifugation- density gradient (20), but this study aims to find a cheap, easy to use and reliable extraction method. Thus, out of the three tested methods IP, SEC, TEI, the latter proved to be the most suitable methodology capable of isolating high quantity and good quality exosomes.
The suitability of mare’s milk-derived exosomes as therapeutic carriers was tested in terms of an increase in the therapeutic effects compared to free drug (quercetin only). For example, quercetin loaded exosomes showed significantly higher antioxidant activity compared to quercetin alone in doxorubicin treated cells, demonstrating the potential of exosomes in improving drug bioavailability in vitro. This is due to their ability to easily pass through biological barriers, such as blood-brain barrier, intestinal and placental barriers (21), which allows them to play an important role in cellular communication and enable them to deliver their mRNA, miRNAs, proteins, and small molecules into other cells (22).
This theory was further supported in the animal work, where exosomes isolated from mare’s milk were loaded with quercetin and administered to ageing rats. The analysis showed that exosomes loaded with quercetin has more bioavailability than quercetin alone and that they were efficiently absorbed in different organs. This finding is in an agreement with previous reports that showed the use of milk-derived exosomes as Nano-carriers increased drug bioavailability. For example, Munagala et al. 2016, reported that milk-derived exosomes loaded with withaferin A significantly enhanced the tumor reduction of the drug (23), as well as Aqil et al., 2016, which showed the use of exosomes increased the therapeutic response of Celastrol against lung cancer (24).
Interestingly, a range of histopathological changes identified in the heart, liver, and kidney sections of old rats across all groups compared to the group of young rats. These changes included cellular damage (tinctorial and morphological), tissue structure alterations, and cellular infiltrate associated with organismal aging. For example, in the heart, the most common age-related lesion observed was fibrosis with loss and hypertrophy of cardiomyocytes. The kidneys exhibited focal chronic progressive glomerulonephropathies, including mild sclerosis of the capsule and mesangium of glomeruli. The liver cells displayed widespread variations in the size of hepatocyte nuclei, chromatin distribution within the nucleus, vesicular nuclei, basophilic, fine-grained or vacuolated cytoplasm. The observed damages in the old animals were of acute, subacute and chronic damages. While the latter is consistent with age-related damage (25), the acute and subacute are the results of doxirubucin treatment, confirming the success of the establishing an ageing model using this drug.
However, animals treated with exosomes loaded with quercetin showed significantly less frequent patterns of acute and subacute damage in the myocardium, kidneys, and liver compared to the control group of old males without treatment (p<0.05). These findings observed in the exosomes loaded with quercetin treatment group, but not with quercetin only or exosomes only groups. While, this might be used as a supporting evidence for the suitability of mare’s milk-derived exosomes as potential drug carriers, it must be interpreted with caution, as exosomes alone were reportedly involved in regulating oxidative stress (OS) (26). A number of studies indicated that exosomes might not only transport proteins, RNA, and other molecules, but also participate in OS-related conditions, such as ischemia–reperfusion, atherosclerosis, and cardiac remodeling by reducing reactive oxygen species (ROS) through inhibiting protein synthesis and mRNA degradation (27, 28). For instance, Wang et al., 2019, reported that mir-126 derived from exosomes reduced apoptosis and lessened OS in ischemia and reperfusion injuries (29).
However, the amounts of proteins, mRNA, and RNA found in the exosomes as well as their therapeutic potential depend on the source of the exosomes (e.g. milk) and the isolation methodology used. There were approximately 200 different proteins found in dendritic cell derived exosomes extracted using crude preparation (30), and more than 2000 different proteins, including major protein markers, identified from bovine milk-derived exosomes isolated by centrifugation (31). While, the protein components of the isolated exosomes were not analyzed as this is beyond the scope of the current study, we can assume that no proteins or any other component was involved in OS. Thus, it is safe to assume that due to their ability in crossing biological barriers, mare’s milk-derived exosomes increased the bioavailability of quercetin, and hence, they might be used as a reliable form of a therapeutic carrier to improve drug delivery.
Nevertheless, the findings confirm the absorption of exosomes by different organs and show an increase of quercetin bioavailability at the target site. However, was the observed effect due to the increased bioavailability of quercetin? Alternatively, did the drug and the exosome achieve it synergistically? Further research is needed to determine whether synergistic effects between exosome and quercetin exist. It would also be interesting to investigate the roles of exosomes isolated from mare’s milk alone, or their components in different pathologies with or without drugs. This will likely shed more lights on their biological roles.
In conclusion, the current study supports the concept of using exosomes as a potentially reliable form of a drug carrier. The results demonstrated that mare’s milk-derived exosomes, can easily be absorbed by different tissues, and that their use as a drug carrier, presumably, increased the bioavailability of quercetin. However, follow up studies are needed to identify the components of mare’s milk-derived exosomes such as proteins, mRNA and miRNA, as well as to determine their biological roles.
Data Availability Statement. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Competing Interests’ Statement. None
Funding. This research was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP13067844) and Nazarbayev University Collaborative Research Program 2021–2023 (Award no.OPCRP2021006).
Conflict of Interest. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.