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).