Résumé
Background: Despite the proof of concept of their efficiency as drug delivery systems (DDS) compared to synthetic nanoparticles, the rationale of using extracellular vesicles (EVs) still requires numerous improvements (yield of production, drug loading, pharmacokinetics). In this context, our team aims at overcoming these hurdles by using its pharmaceutical/physico-chemical skills to perform post-production modifications of EVs, in order to create a potent DDS. Methods: We proved ablility to produce, isolate (differential ultracentrifugation) and characterize (dynamic light scattering, nanoparticle tracking analysis (NTA), protein dosage, western blot, proteomics, cryoTEM) EVs from murine MSC with yields coherent with their use in this project. Importantly, we developed a freeze-drying protocol for their long-term storage, with no impact on vesicle numbers, structure (cryoTEM) and content (proteomic). After labelling with a lypophilic dye, EVs were incubated with the parent cells or foreign cells (NIH3T3), in the presence of endocytosis inhibitors, and tracked by flow cytometry. All experiments were also performed on liposomal commercial standards (PC/Chol) as a comparison. Results: EVs were 94 ± 11 nm (NTA, n = 9) with a production yield of 3.41 pg protein and 9.48.108 particles/106 cells (n = 9). The western blot and proteomics analysis evidenced the presence of EV-specific markers such as TSG101, CD81 and ADAM10. The EVs were internalized to a greater extent than their liposomal counterparts in both target cells (n = 3). Our preliminary data suggest that they could follow different endocytic routes. Among the processes evaluated for drug loading, EVs were extruded through 50 nm membranes without damage. We are currently investigating whether the performed modifications impact their internalization rate and pathway. Summary/conclusion: Our team has been able to reproducibly isolate, characterize and label mMSC-derived EVs. The EVs show increased internalization in vitro compared to liposomes currently used as DDS, whatever the target cell type, and EVs may follow a different endocytic route than liposomes. We propose here to present our latest results regarding the rationale of using EVs as vectors for drug delivery.