Page 13 - EXOSOMES

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nanoparticles, principally liposomes. [6,7] Current barriers to the nanovesicles per million cells could be isolated, [5,21,22] thus
clinical use of biofunctionalized nanoparticles include lack of highlighting the potential of milk as an economic and scalable
batch to batch reproducibility and toxicity. Naturally derived source of exosomes.
nanoparticles such as exosomes are increasingly being pro- Transmission electron microscopy (TEM) images confirmed
posed as an alternative to synthetic nanoparticles to overcome a non-aggregated population of exosomes with typical “cup-
some of those limitations, thanks to their natural origin, shape” morphology. Large extracellular vesicles (distinguishable
(phospho)lipid bilayer structure, and nanometric size. [8] by their irregular shape and electron-dense appearance, [8,23]
Among the possible sources, milk exosomes are notable for cellular fragments, or protein clusters are often found in sus-
their easy production and high sample volumes in comparison pensions of isolated milk exosomes, [24] but these contaminants
with culture fluid or blood plasma. With this approximation, were not identified in the micrographs (Figure 1a). These
a high yield, harmless, and cost-effective production of non- results evidence the successful combination of ultracentrifuga-
[9]
tumor exosomes can be obtained for use as a scalable source. tion, precipitation, and size exclusion chromatography for the
In addition, the non-toxic and non-immunogenic behavior of isolation of goat milk exosomes. The protocol also removes
milk exosomes in healthy models has been demonstrated, [10,11] other contaminants, such as milk fat.
as has as their application as carriers for chemotherapeutic/ Quantitative measurement of the hydrodynamic size by
[12]
chemopreventive agents. Although these characteristics have dynamic light scattering (DLS) established a mean size of
been evaluated in exosomes from different milk sources such 125.70 ± 3.25 nm (Figure 1b), with a polydispersity index of 0.14,
as human, pig, rat, camel, and horse, to the authors’ knowl- confirming a monodisperse suspension of exosomes. Nanopar-
[8]
edge goat milk exosomes have not yet been investigated. ticle tracking analysis (NTA) showed modal average values of
Aside from the great potential of EVs in cancer therapy, [5,6] 124.44 ± 8.54 nm, similar to the results achieved by DLS. This
milk exosomes have been implicated in the regulation of size distribution matches the profile described for exosomes
[13]
inflammatory processes through miRNA trafficking. More- isolated from cow milk, one of the most employed sources for
over, milk exosomes are rapidly trapped by macrophages [14,15] as the collection of milk extracellular vesicles. [12,25] NTA also dem-
part of the clearance activity that these cells, along with neu- onstrated the high content of nanovesicles in the exosome sus-
11
11
trophils, carry out when foreign agents are detected. [16–18] This pension, reporting a concentration of 3.32 × 10 ± 3.25 × 10
−1
natural targeting and their nanometric size support the use particles mL (Figure 1c).
of milk exosomes in the diagnosis and therapy of inflamma-
tion underlying the pathogenesis of diseases such as athero-
sclerosis, chronic obstructive pulmonary disease, asthma, and 2.2. Proteomic Evaluation of Milk Exosomes
cancer. [19,20]
The main aims of this work were a full physicochemical To determine the composition of milk exosomes, extensive
characterization of exosomes isolated from goat milk and their LC-MS/MS proteomic analyses were performed. A total of ≈900
further evaluation for use as natural nanoprobes in the detec- proteins (derived from ≈4500 peptides) were identified in all
tion of inflammatory processes. This evaluation was performed three independent isolations of goat milk exosomal samples.
through their fluorescent labeling and further in vitro and in GO analyses using the top 100 most abundant proteins
vivo assessment by means of optical imaging. The internaliza- revealed enrichment in terms such as extracellular exosome,
tion of these fluorescent exosomes in inflammatory M0, M1, extracellular region part, vesicle, and membrane-bounded ves-
and M2 macrophages in vitro was evaluated by means of con- icle, confirming the identity of the samples as EVs (Figure S1,
focal imaging and flow cytometry. Once the capacity of mac- Supporting Information).
rophages to internalize the exosomes was demonstrated in In addition, PANTHER overrepresentation analyses
vitro, it was evaluated in vivo in a mouse peritonitis model in showed that the identified proteins were involved in pro-
comparison with healthy mice by assessing the biodistribution cesses such as neutrophil degranulation, the innate immune
of the probe through optical imaging. Finally, exudates from system, the immune system, hemostasis, and platelet activa-
the peritonitis model were analyzed using flow cytometry and tion. This kind of activity related to the immune system has
confocal imaging to confirm the in vivo uptake of the nanopar- been described for other milk-derived exosomes. [25] A 15-fold
ticles by specific inflammatory cell populations (macrophages or greater enrichment was observed for the ECRT (endosomal
and neutrophils). sorting complex required for transport), endosomal/vacuole
pathway, and regulation of the complement cascade, among
others.
2. Results and Discussion Similarly, proteins were involved in several molecular
functions including membrane transport and binding activity,
2.1. Isolation and Characterization of Milk Exosomes protein transport, vesicle-mediated transport, adhesion, or met-
abolic processes. These kinds of processes are related to general
Our isolation protocol enabled us to collect pure goat’s milk cell function and growth or to the endocytic pathway of the vesi-
−1
exosomes in suspension at 2.29 ± 0.25 mg mL , as quanti- cles themselves. Other processes such as endosomal transport
fied by Coomassie-Bradford assay, from an initial goat’s milk via the multivesicular body sorting pathway, that pathway itself,
volume of 60 mL. Previous publications detailing exosome iso- late endosome-to-vacuole transport, and response to reactive
lation techniques from culture media of dendritic and murine oxygen species were enriched more than tenfold. Cellular com-
tumor cell lines have reported that only 0.2–2.0 µg of these partment results showed an over-representation of “endosome”;

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