Final Report Summary - MINDS (Microvesicle-inspired drug delivery systems) Biologicals, like proteins peptides and nucleic acids represent promising therapeutic molecules for the treatment of various diseases, including cancer. However, protective formulation into a suitable carrier system is necessary in order to fully exploit their therapeutic potential. Particularly for nucleic acids, like siRNA, their 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. Therefore, we investigated how natural EVs could be harvested, labeled, loaded with siRNA and targeted to tumor cells, without compromising EV integrity. We demonstrated that harvesting method was important for functional characteristics of EVs, the golden standard (i.e. centrifugation) was shown to compromise EV activity. Instead chromatography preserved activity. For labeling, to be able to track EVs', we were successful in labeling proteins lipids or interior of the EVs poviding a comprehensive overview of all the components. One lableing method was described in literature to be useful to determine fusion of EVs with other cells. However we determined that this assay is not useful. Also we showed that the golden standard loading method of RNA in to EVs (i.e. electroporation) was based on an artefact.We developed three methods to equip EVs with targeting ligands, which were all successful in vitro and to a certain degree in vivo. Subsequent research will need to show if one technique outperforms the others. Together with partners we were the first to show mRNA transfer by EVs between tissues, underlining their drug delivery utility. Finally we also studied the targeting under flow conditions and studied the mechanics of EVs.
