IMMUNE-DERIVED EXOSOMES MIMETICS (IDEM) AS AN INNOVATIVE, VERSATILE AND BIOMIMETIC NANOPARTICLE SYSTEM FOR CANCER TREATMENT

Exosomes are extracellular nanovesicles naturally produced by inward of the cell membrane and are involved in cell-to-cell communication and content exchange. They have specific characteristics in terms of dimension, structure and surface proteins expression and they can be loaded with drugs for specific tumor cell targeting. These features allow them to avoid immune response, to enhance the immune system against cancer cells, to take advantage of EPR effect (for accumulation of drug in a specific area, for diagnosis) and to efficiently target the therapy to tumor cells, with consequentially less drug side effects and more protection of the drug or biological compound. Natural exosomes are currently exploited as delivery systems for drugs and small compounds to efficiently induce cell death or phenotype changes in target cells. However, their applicability in clinical settings is limited by the low yield, scarce scalability, poor batch-to-batch reproducibility and time-consuming synthesis. The purpose of this thesis is to develop a strategy to obtain versatile and smart nanoparticles bioinspired by exosomes with the potential to be used as carriers of chemotherapeutics for cancer treatment. These nanoparticles were defined as Immune Derived Exosomes Mimetics (IDEM) as they were synthetized by sequentially forcing 8.5x106 THP-1 immune cells through 8um and 10um filters and cycles of centrifugation. We synthetized and characterized IDEM in comparison with exosomes extracted from cell culture media. All nanoparticles were analyzed by western blot and flow cytometry for protein expression, whereas Scanning Electron Microscope (SEM) and Nanoparticle Tracking analysis (NTA) were used for morphological characterization and particle concentration, respectively. The IDEM approach provided a greater number of nanoparticles compared to natural exosomes (9.4x109 exosomes than 2.3x109 IDEM). Furthermore, IDEM presented a higher diameter than exosomes (177nm versus 112nm, respectively) but still fell within the exosomal dimensional range. We also confirmed the similarity of the two formulations in terms of protein content (i.e. tetraspanins CD63, CD81, ALIX and TSG101, typical of exosomal formulations). Afterwards, Doxorubicin was loaded inside both the nanoparticle formulations and the encapsulation efficiency (EE%) and drug release profile studies were performed. IDEM showed an increase in EE% and a similar drug release profile with exosomes, presenting a burst release of 32% of encapsulated drug in the first 2 hours. We then tested the cytotoxic effect of different doses of doxorubicin-loaded IDEM on SKOV3 human ovarian cancer cell line as compared to doxorubicin-loaded exosomes over a 96-hour period. Alamar Blue assay and the measurement of impedance of adherent cells by the xCelligence Sight were used for cell viability assessment. We also verified the drug uptake by cells by flow cytometry and confocal microscopy. Data obtained revealed that a lower concentration of IDEM and exosomes (1ug/mL) compared to free Doxorubicin (10ug/mL) was sufficient to kill cancer cells. With this work we can conclude that IDEM hold potential for innovative cancer treatments as they couple the advantages of natural exosomes with the higher reproducibility and production yields.