Results
Comparison of exosome isolation methods
A comparison of the three different methods of exosome isolation
performed to evaluate the morphology, properties of the obtained
exosomes and their applicability as drug carriers. The morphology
assessment of the isolated exosomes performed using TEM, shows that
exosomes isolated using the different methods have morphologically dense
vesicular structure indicated with arrows (Figure 1). It can be clearly
observed that some debris can be observed in EVs obtained by IP and SEC,
but not the in the EVs isolated using TEI method, which might be due to
the PBS dilution during the isolation process. Nanoparticle tracking
illustrating the size distribution of the particles shows the
distribution of particles close to 100 nm (Figure 2). Particles isolated
by SEC showed less variation in particle distribution than IP and TEI.
No significant difference in the mean size of exosomes across the
different isolation methods SEC, IP and TEIK (110nm, 118nm and 131.5nm,
respectively).
Western blot using the membrane markers, MEFG-8 monoclonal, MEFG-8
polyclonal and CD63 antibodies performed to measure confirm the identity
of exosomes present in each of the isolates (Figure 3). The protein
content in IP isolate is shown to be the highest as measured by MEFE-8
and CD63 antibodies. The TEI showed a significant band against MEGD-8
monoclonal antibody, but no significant differences in other antibodies
tested in comparison with SEC. However, for protein content, isolates
from TEI showed the highest, followed by IP and then SEC (Figure 4).
Protein contents from TEI method (mean protein concentration of 0.1914)
are significantly higher (p= 0.012) than the negative control PBS (mean
protein concentration is 0.0085 mg/ml).