Extracellular vesicle engineering for drug delivery

Abstract

Inhibition of expression of disease-causing genes with small interfering RNA (siRNA) is a promising therapeutic approach for the treatment of various diseases, including cancer. However, protective formulation into a suitable carrier system is necessary in order to fully exploit the therapeutic potential of siRNA, since its physicochemical properties limit stability in the circulation and target cell entry. Over the past decades, numerous synthetic nanoparticulate systems have been investigated as carriers of siRNA and other (biological) therapeutic cargoes, but often fail to meet the requirements for clinical application. To overcome the limitations of these systems, it may be useful to borrow a leaf from nature’s book. Extracellular vesicles (EVs) are secreted from virtually all cells in our body, typically have sizes of 50-200 nm, and contain proteins and nucleic acids which are encapsulated by a phospholipid bilayer membrane. EVs play a role in intercellular communication by functionally transferring their cargo from one cell to another. These unique properties have raised interest into whether EVs could also be exploited for the biocompatible, efficient, and safe delivery of therapeutics, including siRNA. However, in order to apply EVs for drug delivery purposes, their properties may need to be engineered. In this thesis, we investigated how natural EVs could be loaded with siRNA and targeted to tumor cells, without compromising EV integrity. We employed a previously described electroporation method for evaluating siRNA loading into EVs. Surprisingly, electroporation was found to not result in siRNA loading into EVs, but in siRNA aggregation and precipitation instead. The obtained aggregates obscured the amount of siRNA actually loaded into EVs, which was found to be negligible. This illustrates that electroporation is far less efficient than previously anticipated, and highlights the necessity for alternative methods to prepare siRNA-loaded EVs, for example by engineering of EV-secreting cells. To promote the specific interactions of EVs with tumor cells, we devised three strategies to decorate the surface of EVs with targeting ligands. We employed anti-epidermal growth factor receptor (EGFR) nanobodies as targeting ligands for tumor cells, and grafted these on EVs by (I) fusion to glycosylphosphatidylinositol (GPI) anchors, (II) coupling to PEGylated lipids, which were inserted into the EV membrane, or (III) fusion to C1C2 domains of the lactadherin protein, which self-associate with phosphatidylserine on the EV surface. All three strategies were found to be effective for the introduction of nanobodies on EVs and resulted in increased association of EVs with EGFR-overexpressing tumor cells. Each technique displayed advantages and disadvantages, and further research should show which strategy is most suitable for the scalable and reproducible generation of tumor cell-targeted EVs. In addition to engineering EVs for drug delivery purposes, we also discussed how EV components could possibly be used to improve synthetic drug delivery systems. Taken together, this thesis provides valuable clues for the design and development of biocompatible, efficient and targeted EV-inspired carrier systems for the delivery of (biological) therapeutics in cancer.