Extracellular Vesicles as an Innovative Tool for siRNA Delivery to Block the Pathological Communication between Tumor and Microenvironment.

Multiple myeloma (MM) is a plasma cell malignancy in which monoclonal plasma cells proliferate in bone marrow (BM). The role of Notch aberrant activation in tumor development and progression has been established by many studies. The interplay between MM cells and the BM niche does not only rely on direct cell-cell interaction, but a critical role is played by MM-derived extracellular vesicles (MM-EV). In fact, recently we proved that MM-EVs mediate the communication between MM and BM microenvironment in a NOTCH-dependent way. Due to the relevant role of Notch signaling in MM progression, we aim to provide the proof-of-concept for a siRNA-mediated therapeutic approach to inhibit Notch signaling in MM cells using EVs as a drug delivery tool. Here, we present a part of a methodological set-up. For the EV-mediated delivery set-up, EVs were isolated from the MM cell line by ultracentrifugation. MM-EVs specific delivery to the tumor was assessed using a xenograft mice model. We got advantage of the MM-luc cells which were injected subcutaneously in C57BL/6 mice and the health status of the mice was monitored daily. When the tumor size reached the appropriate size, the mice were intravenously injected with MM-EVs. In vivo analysis of the xenograft mice was indicative of the tropism of the MM-EVs selectively recognizing the neoplastic tissue. For siRNA loading into MM-EVs, we took advantage of sonication. The uploading setup was divided into 3 different steps 1) identification of the conditions of sonication which induce fewer EV alterations using nanoparticle tracking analysis and flow cytometry; 2) evaluation of the conditions of EVs sonication with siRNA which guarantee the higher siRNA uploading, and 3) in vitro evaluation of siRNA-loaded EVs up take in MM cells. The first results of set-up indicate that sonication of the EVs does not significantly change their dimensional profile and quantity, while the uptake experiment showed that sonicated EVs can be uptaken less (~30%) in MM cells compared to the not sonicated ones. In conclusion, the experiment of the xenograft model suggests that EVs can be used as a specific siRNA delivery tool. In addition, the preliminary results of the EVs sonication setup revealed that sonication does not significantly affect the EVs quality and quantity, but it can affect EV uptake by the MM-cells. In the future, we will sonicate EVs in the presence of siRNA and evaluate its efficacy and biological impacts in MM in vitro and in vivo.