Characterization of extracellular vesicles and their behavior: Method development and applications

Abstract

Extracellular vesicles (EVs) can transfer RNA molecules from one cell to another, thereby changing the phenotype of recipient cells. EVs play a role in several (patho)physiological conditions, which makes them attractive candidates for biomarker research. Moreover, the ability of EVs to functionally transfer biomolecules from one cell to another has caught the attention of the drug delivery field. Biomolecules such as RNA and (recombinant) proteins are very promising therapeutics, but their clinical application can be hindered because of inefficient delivery of these molecules to their site of action. For RNA, this is especially challenging as their site of activity is in the cytosol of target cells. Therefore, many researchers are inspired by EVs in drug delivery. In this thesis, two approaches for exploiting EVs in drug delivery were described and discussed. One is focused on the use of endogenous EVs as drug delivery vehicles, by decorating them with targeting ligands and loading them with therapeutic (bio)molecules. The second approach is incorporating EV components, that are important for efficient cargo delivery, into existing drug delivery systems. In order to achieve this, thorough understanding of EV behavior, in particular of their interaction with cells, is required. Fluorescence microscopy is often used for this purpose. To compare different labeling strategies for investigating the interaction of EVs with cells, we labeled EVs with lipid, surface protein and luminal labels. Our results indicate that no release of luminal content in the cytosol had taken place. It may be hypothesized that, within a population of EVs, only a small fraction of EVs is able to functionally transfer luminal content and more sensitive methods might be required to observe this. In most studies, EV interaction with cells is investigated under static conditions. Bodily fluids, in contrast, are constantly moving, which is likely to influence EV-cell interaction. Therefore, we developed a method to investigate binding and uptake of EVs by cells under dynamic flow conditions. One proposed mechanism for EV cargo transfer is membrane fusion at the plasma membrane of cells. In this thesis, we described the challenges and technical hurdles of using the R18 lipid mixing assay to investigate fusion of EVs with cells. In addition to EV composition, EV mechanics might have an influence on EV-cell interaction. We used atomic force microscopy to determine the mechanic properties of EVs derived from red blood cells (RBCs). We found a difference in bending when comparing RBC EVs from a patient with RBC EVs derived from healthy donors, which might have implications for RBC EV behavior. Apart from their attractiveness as drug delivery systems, EVs are also interesting from a diagnostic viewpoint. We treated cancer cells with cetuximab and observed that the EVs released by the cancer cells had an altered composition after treatment. This implicates that EVs could be used to monitor cetuximab treatment. In conclusion, this thesis provides insides in the caveats that are encountered in unraveling the interaction of EVs with target cells and provides tools to further analyze their activity. Thereby, this thesis may contribute to the clinical development of EV-based therapeutics.