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.