Targeting the tumor microenvironment: a click chemistry-based surface-functionalization method and a therapeutic-loading strategy for plasma- and erythrocyte-derived extracellular vesicles

Thanks to their role in intercellular communication and their natural ability to transport functional cargoes, extracellular vesicles (EVs) have been recently considered valuable therapeutic nanocarriers. Low immunological response, intrinsic targeting potential, especially for autologous EVs, and the capability to cross various biological barriers represent some of the advantages that make EVs an optimal alternative to synthetic nanoparticles for targeted drug delivery. In this project, we developed two methods for EV-engineering based on their loading with a medicinal cargo and their surface functionalization with a fluorescent peptide targeting the extracellular matrix antigen Extra domain B fibronectin (ED-B FN). The latter represents one of the most investigated FN variants for tumor-targeting strategies since it has been demonstrated to play a key role in tumorigenesis, angiogenesis, metastasis formation, and epithelial-to-mesenchymal transition (EMT). Two different EV populations have been considered in this study as an ideal proposal for both an autologous and heterologous context: plasma-derived EVs and artificial red blood cell-derived EVs (nanoerythrosomes, NanoEs). The EV membrane functionalization strategy has been based on the “traditional” click chemistry reaction (Huisgen cycloaddition) of an alkyne, in this case tied to EV surface, and an azide, owned by the anti-ED-B FN fluorescent peptide. The achievement of functionalization, evaluated by flow cytometry analysis, revealed that about 50% of EVs are peptide-clicked. In addition, after confirming that our membrane-engineering approach didn’t affect EV identity, we observed that peptide-clicked EVs were efficiently internalized by responder cells (MDA-MB 231). We developed a strategy to load Paclitaxel into nanoparticles by sonication with an efficiency of about 0.1% for plasma-EVs and 1.5% for NanoEs (HPLC-MS analysis). In addition, either loaded plasma-EVs or NanoEs showed a significant cytotoxic effect on MDA-MB 231 cells when compared with their empty counterpart (MTT assay). Copper-free click chemistry and sonication turned out to be effective approaches for EV surface functionalization and therapeutic encapsulation, respectively. The development and standardization of these protocols lay the foundation for potential applications as new innovative targeted therapies in either an allogenic or an autologous scenario. In addition, targeting a tumor microenvironment antigen such as ED-B, which is expressed in a wide range of tumor types, would allow the development of therapies potentially relevant for different oncological applications.