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Grant, Ryan Conrad (2016) Development of novel isolation methodologies for microvesicles and exosomes, as potential biomarkers for health (Therapeutic Lifestyle Changes), and use of microvesicle-delivered β-Gly in erythroleukaemia. Doctoral thesis, London Metropolitan University.
Both MVs and exosomes are isolated by similar methodologies such as differential centrifugation of cell culture supernatant (S/N), filtration, floating on chemical gradients or using antibody labelled magnetic beads. Hence, the isolation techniques, commonly in use, provide low levels of purification and therefore require improvements and an in-depth analysis of observed results. This research has established a protocol for isolation of these vesicles and a means to identify them using known markers and their respective properties. The isolation techniques used and developed in this research include differential centrifugation, sucrose gradient centrifugation, and filtration. MVs and exosomes were later identified and distinguished according to their relative ability to bind FITC and PE conjugated annexin V, anti-LAMP1, anti-CD63 and anti-CD11b antibodies using flow cytometry. Protein gels were run with MVs and exosome samples isolated by different techniques to identify some distinct proteins in MVs/exosomes that may be uniquely present on one or either and hence that could be used as a marker to distinguish MVs from exosomes.
To prevent exosomal contamination of MV preparations, in a further improvement of the newly developed reverse filtration technique, gentle disruption of exosome aggregates by gentle water sonication was used and measurements of plasma MV levels made from a population of healthy donors. This found plasma MV levels to range between 0.51 and 2.82 x 105 MVs/ml. The MVs were characterised as phosphatidylserine-positive and ≥0.2 μm in diameter. Most of the variables looked at in this study including freezing at -20ºC, gender and subject age did not alter MV absolute counts significantly. For the first time the effect of fasting on MV levels has been studied. Fasting individuals had a wider spread and appeared to have higher MV levels (2.8x105-5.8x105 MVs/ml). This could be defined as the normal baseline fasting reference range, which is almost 3-fold higher compared to the non-fasting group reference range (0.9-1.5x105) MVs/ml. These results suggest that when analysing total MVs, further investigations should use fasting subjects, as for quantification of other fasting analytes.
Prospective markers of (monocyte-derived) MVs, IL-1α, RANTES, G-CSF, CCL-1 and IL-17E are proposed for further investigation. MVs were shown to inhibit phagocytosis of apoptotic bodies more effectively than exosomes. With a view to understanding likely pathways being stimulated during MV release, and to also shortlist possible pharmacological reagents capable of being used to therapeutically inhibit Y27632, calpeptin and methyl-β cyclodextrin were shown to inhibit MV release. MV release from K562 cells was inhibited by Y27632 and calpeptin but less effectively by chlorpromazine or methyl-β-cyclodextrin, MV release from THP-1 acute monocytic leukaemia cells was greater than from peripheral blood monocytes which were in turn shown to express more phosphotidylserine than on exosomes.
With a view to using MVs as drug delivery vehicles, THP-1 MVs were found to fuse/hemifuse to K562 erythroleukaemia cells, but not at 4°C or if the MV surface was blocked with annexin V. β-Gly (10 μM) (as well as MVs carrying β-Gly [β-Gly MVs]) was found to inhibit proliferation at least 3-fold compared to untreated control. β-Gly and β-Gly MVs increased the doubling time but neither induced apoptosis of K562 cells (unlike MVs from apoptotic cells). Finally, β-Gly increased the percentage of cells in G0/G1 whilst decreasing the cells in G2/M and appears to induce erythroid differentiation as seen by the increase in the percentage of benzidine-stained cells.
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