| Zusammenfassung: | <p>Extracellular vesicles (EVs) are membranous nanoparticles released by cells that are important mediators of intercellular communication via transfer of their cargoes to recipient cells. For this reason, EVs have been harnessed as a novel targeted therapeutic delivery platform. However, EV research faces numerous challenges that need to be addressed if their potential as therapeutic devices is to be fully realised.</p>
<p>EV heterogeneity is arguably the most crucial challenge to be addressed. EVs differ in size, biogenesis, and composition, and these characteristic differences have been linked to functional differences which may impact EV therapeutic activity. Here, functionally heterogeneous EV populations were identified among size-based and subclonal EV populations. EVs were characterised revealing differences in marker and functional protein expression, including integrins, cell adhesion proteins and EV marker proteins. These differences may explain functional differences between EV subpopulations and could aid selective purification of therapeutically advantageous EVs. Furthermore, the subclonal nature of the analysed EV populations also indicated that EV heterogeneity might be linked to cellular heterogeneity. Separately, changes in EV protein expression were documented among EVs harvested at different time points, highlighting another critical consideration for EV therapeutic development.</p>
<p>Advancement of in vivo EV biodistribution and purification techniques was also sought to improve EV therapeutic development and characterisation. While requiring further experimentation, EV DNA barcoding, a technique that labels EVs with synthetic DNA identifiers, showed potential as a novel EV biodistribution and therapeutic siRNA delivery tool. Additionally, Capto Core binding chromatography showed significant improvement over size exclusion chromatography for purification of high concentrations of EVs from high cell density cultures.</p>
<p>Overall, this research offers insights into EV heterogeneity and novel techniques to aid therapeutics EV production. It also highlights the importance of understanding EV biology and overcoming the challenges associated with EV research to improve EV therapeutic development.</p>
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