Abstract
Extracellular Vesicles (EVs) are nanoparticles produced by cells and are key mediators of intercellular communication. Their ability to vectorize biomolecules from one cell to another makes them promising vectors for therapeutic protein delivery. However, loading of EVs with exogenous molecules is complex and the currently developed methods expose limitations, such as reproducibility issues and low loading rates—features that are poorly compatible with therapeutic applications. In this context, the aim of this PhD was to develop effective EV-loading approaches for protein cargos in order to enable their functional cell delivery. We initially used Physical Protocols (PP) such as freeze/thaw or ultrasonication to permeabilize EV membrane and enable the diffusion of a fluorescent Single-Chain Variable Fragment of antibody (scFv) inside EVs. We obtained results similar to the literature suggesting successful EV loading and cell vectorization (in murine mesenchymal stem cells or pancreatic carcinoma PANC-1 cells) using PP. However, after additional controls and development of adequate separation methods, we showed that these observations were actually artifacts due to protein aggregation. We therefore changed our approach and used a strategy based on a lipid anchor (LA) to insert the protein of interest into EV membrane. Horse Radish Peroxidase (HRP) was used as model enzyme for its very sensitive readout, enabling protein activity evaluation and characterization of loading metrics. HRP was conjugated with different LA based on two lipids (cholesterol (CLS) and DSPE) and different polyethylene glycol (PEG) molecular weights (1k, 2k and 5k g/mol). Using a range of complementary methods, based on both bulk and single particle analysis approaches, we were able to demonstrate the effectiveness of this approach. Up to 1000 HRP per EV and 35-50% of initial HRP was associated to EVs using CLS lipid, which is among best EV loading capacities and efficiencies obtained until now. Interestingly, effective cell delivery was obtained with low molecular weight PEG while PEG 5k limited cell internalization by steric hindrance. To our knowledge, this is the first study characterizing the intracellular vectorization of a protein using EVs based on this approach. Finally, we demonstrated the versatility of this approach by associating HRP with Lipid-Raft derived nanoparticles from human placenta-derived mesenchymal stromal cells. This work should pave the way for the efficient loading of a therapeutic anti-RAS scFv to EVs, before functional delivery in tumoral cells.