DEVELOPMENT OF A NANOPARTICLE-BASED PLATFORM FOR THE DELIVERY OF THERANOSTIC AGENTS TO TUMOR TISSUE: TOWARD CLINICAL TRANSLATION

The selective delivery of anticancer agents to tumor tissue remains a major challenge in oncology, as many clinically effective drugs are limited by poor tumor specificity and dose-limiting systemic toxicity. Previous studies from the laboratory have explored the use of tumor-derived and patient-derived EVs as efficient, natural, and biocompatible vehicles displaying inherent tropism toward transformed cells, for the selective delivery of diagnostic agents to neoplastic tissue. These studies led to the evaluation of autologous, patient-derived EVs (PDEVs) for the intraoperative detection of tumor margins in a Phase I/II clinical trial. This thesis focuses on the development of a technological platform for personalized anticancer therapy based on the use of EVs as delivery tools for anticancer agents. To this aim, conventional anticancer treatments, together with innovative radiotherapeutic and precision-oncology approaches, were selected as proofs of concept for the encapsulation protocol and for efficacy studies in preclinical models, and innovative strategies for EV preservation were also developed. In particular, the EV formulation was applied to the following antineoplastic agents: i) doxorubicin, a classical broad-spectrum chemotherapeutic agent whose clinical application is constrained by cardiotoxicity; ii) sodium borocaptate (BSH), used in boron neutron capture therapy (BNCT), which can be applied only when sufficiently high concentrations of boron are reached in neoplastic tissue; and iii) small interfering RNA, as an example of agents relevant to emerging gene-silencing strategies in the treatment of cancer. In these experiments, tumor-derived EVs were employed as a model system to develop protocols that will, in the future, be applied to PDEVs for clinical translation. The results demonstrate efficient and highly significant encapsulation of all agents into EVs which, considering the efficiency of targeted cargo delivery to neoplastic tissue, may greatly enhance their anticancer activity. This was clearly demonstrated with EV-loaded doxorubicin, which increased antitumor efficacy compared with the current formulation of the drug in both in vitro and in vivo murine models of colon adenocarcinoma. Moreover, the experiments clearly demonstrated the feasibility of lyophilization, supporting long-term storage, as well as the feasibility of repeated treatment regimens for the clinical application of autologous EV protocols in anticancer therapies. Altogether, the results indicate that the EV formulations developed in this thesis may contribute to the development of innovative, personalized therapeutic platforms for oncological applications. Despite their high therapeutic potential, however, the clinical translation of EV-based systems still faces significant challenges related to standardization, large-scale production, and regulatory compliance. To address these limitations, the laboratory is working to reproduce targeted delivery of antineoplastic agents using synthetic lipid nanoparticles; preliminary experiments in this direction were carried out in the final part of this thesis.