Evaluating multimodal therapeutic effects exploiting inorganic nanoparticles on cancer cells.

The present thesis dissertation presents my doctoral work carried out during the last three years at the Italian Institute of Technology (IIT) and the University of Genoa. The work was focused on the evaluation of novel inorganic nanoparticles able to fulfill multiple therapeutic and imaging actions for a multi-modal approach to tackle and diagnosed cancer cells. This relies on the fact that single therapies often turned out to be not enough for a complete tumor eradication, due to complex tumor resistance based on multiple factors, including the tumor microenvironment (TME) stiffness, acidity hypoxia and heterogeneity of cancer cells. Furthermore, cancer cells are able to undergo genetic mutations when exposed continuously to a therapy, ultimately bringing them to develop a resistance, which also contributes to the failure in cancer cells killing. In this context, thermal therapies, such as magnetic hyperthermia treatment (MHT) and photothermal therapies (PTT), are able to induce cancer cells death through apoptotic or necrotic processes as well as to reshape TME, making cancer cells more sensitive to the effect of drugs, particle radiations, and immune cells response. As such, in the first chapter I investigated the use of ferrite-based NPs for MHT in combination with either chemotherapy or with immunotherapy. For combining MHT with chemotherapy, I explored three delivering systems, which differ in terms of complexity. In a first approach, cubic-shaped Zinc Ferrite NPs were intratumorally injected and used as heat mediators in an in vivo adenocarcinoma mouse model, where the chemotherapeutic drug Doxorubicin (DOXO) was intravenously injected. The evaluation of tumoral growth showed that the combination with MHT and DOXO was the most effective treatment. This study confirmed that the damages induced by the heat together with the cytotoxicity of DOXO, even when systemically administered, provided a synergic effect with respect to the single therapies. In a second approach, seeking to exploit MHT as a mechanism for drug release, I employed a system based on cubic shaped iron oxide nanoparticles (IONCs) coated with a thermo-responsive polymer loaded with DOXO. This nanoplatform was studied against glioblastoma patient’s derived cells, known to be enriched with quiescent tumor subpopulation, i.e. Cancer stem cells (CSCs), which are responsible for tumor regression and metastasis. The local drug release at the tumor during MHT enabled to affect the whole cancer cells population, but more importantly to hit CSCs, which turned to be particularly sensitive to the synergistic effect of the heat (T:45°C) combined to the toxicity of the drug. In a third approach, as-prepared IONCs and free DOXO were co-encapsulated within an electrospun polycaprolactone scaffold, which serves as reservoir of NPs and drug for repeated treatments. Upon exposure to MHT, this system was boosting the release of the drug, which resulted in the highest cancer cells mortality percentage, alongside a continuous drug release over time due to the biodegradability of the scaffold. MHT was also investigated in combination with immunotherapy. In particular, we examined the interaction of IONCs with a specific subset of immune cells, the Natural killer (NK) cells. Being part of the first line of defense against tumor, NK cells have been widely investigated in adoptive cell transfer therapy, in which cells, usually obtained from peripheral blood, are expanded ex vivo, and then reinfused into patients. We here thought to exploit an immune-delivery system based on NK cells loaded with IONCs that could be potentially used to accumulate both NPs and NK cells at the tumor target, owing to the possibility to magnetically activate and imaging guide the magnetic loaded NK cells. To this aim, an internalization study of IONCs was conducted using either a cell line commercially available or NK cells collected from healthy donors. Herein, the parameters to promote cellular uptake without compromising NK cells functions were determined. In the second chapter, the thermal therapy PTT was investigated in combination with drug release and internal radiotherapy. PTT owes its toxicity effect by the increase of temperature upon light-to-heat conversion of some inorganic nanoparticles when exposed to laser irradiation. However, the limited penetration depth of light often leads to incomplete treatment of tumors outside the PTT range. In this regard, here I investigated a novel nanomaterial for PTT, i.e. chalcopyrite (CuFeS2) NPs, which were explored for merging PTT with either drug delivery or internal radiotherapy. In a first part, CuFeS2 NPs were functionalized with a thermo-responsive polymer for the controlled release of DOXO upon laser exposure. This smart system proved to have an effective anti-cancer action, in in vitro assay, against both A431 and U87. In a second part, CuFeS2 NPs were radiolabeled using a cation exchange protocol with the radioisotope 64Cu to provide a nanoplatform for PTT as well as internal 64Cu -radiotherapy. The in vivo efficacy results obtained in an adenocarcinoma mouse model showed that this therapeutic combination was extremely efficient in killing cancer cells, bringing to a total tumor eradication in 15 days. Finally, this system was also proved to be useful in PET imaging, revealing that radiolabeled NPs injected intravenously were able to target the tumor site even in the absence of targeting moieties, due to the EPR effect.