Evaluating and optimizing loading methods of single-chain variable fragment protein into extracellular vesicles

• Κύριος συγγραφέας: Mohamed, Khaled
• Άλλοι συγγραφείς: Κλεπετσάνης, Παύλος
• Μορφή: Thesis
• Γλώσσα: English
• Έκδοση: 2020
• Θέματα: Nanosized extracellular vesicles; Protein transfer; FDA; Νανοσωματιδιακά εξωκυτταρικά κυστίδια; Μεταφορά πρωτεϊνών

by the FDA. While commercially available proteins have an extracellular activity, intracellular targets could be a powerful alternative to treat many diseases (cancer, inflammatory disorders,...). Nevertheless, proteins face many barriers to reach their intracellular targets (internalisation, endosomal escape, cytoplasm trafficking) so efficient cytoplasmic delivery of exogenous protein remains a long-standing challenge. In this context, based on their natural properties to vectorize proteins, extracellular vesicles (EVs) are a promising candidate. Biological techniques that aim to encapsulate proteins into EVs (such as transfection of a plasmid to overexpress the protein in EVs producing cells) encounter issues regarding the reproducibility and the control of the drug loading, so we tried to change our perspective of these biological vesicles to a more physico-chemical one, and to view and manipulate EVs as synthetic vectors. Therefore, we have chosen to evaluate the use of nanosized EVs produced from murine mesenchymal stem cells (mMSC) (sEV-mMSC) for antibody fragments (single-chain variable fragment, scFv) transfer into the cell cytoplasm. To establish a proof of concept of scFv encapsulation into sEV-mMSC, as well as the effective intracellular release of functional scFv, we have selected a GFP-tagged scFv that binds to intracellular tubulin (GFP-scFv/αtub) as a reporter protein. Indeed, this GFP-scFv/αtub could allow dual tracking of loaded protein and evaluation of its activity by the tubulin staining ability. To associate GFP-scFv/αtub into sEV-mMSC we evaluated several physical methods inspired by the liposome research field (extrusion, bath sonication cycles, freeze drying, freeze-thaw cycles and several combinations of these methods). Impact of these processes was evaluated on sEV-mMSC (concentration, size, structure) and GFP-scFv/αtub (stability) individually before evaluating scFv loading into sEV-mMSC. Next, we evaluated gel permeation chromatography aiming for separation of EV from free scFv and calculation of encapsulation yield. Finally, GFP-scFv/αtub internalisation and its ability to attach to intracellular tubulin was evaluated in human MSC by fluorescence microscopy. Since antibody fragments are highly liable for aggregation, we particularly focused on protein stabilization. Polysorbate 80 (PS 80) at 0.01% was found to be the most effective stabilizing agent compared to arginine 1M and trehalose 25mM. Interestingly, such stabilizer has also been shown to be useful for avoiding sEV-mMSC aggregation. We managed to identify protocols leading to less than 20% loss in terms of sEV-mMSC and GFP-scFv/αtub concentrations. sEV-mMSC and GFP-scFv/αtub were well-separated thanks to gel permeation chromatography. Remarkably, scFv alone was shown as unable to enter target cells, while for one of our combined protocols, tubulin staining was observed. These preliminary results confirm the interest of associating scFv with sEV-mMSC for intracellular delivery of a functional, biologically active protein.