Multifunctional Nanomaterials for Tumor Diagnosis and Treatment

This thesis presents the research carried out over the last three years of my PhD studies at the Italian Institute of Technology (IIT) and the University of Genoa. The work focuses on the development of functional inorganic nanoparticles for multimodal therapeutic and diagnostic applications targeting cancer cells. Chapter 1 describes the development of a promising multifunctional nanoplatform based on iron oxide nanocubes (IONCs), designed as magnetic carriers for a multimodal therapeutic strategy that combines magnetic hyperthermia treatment (MHT) with Pt-based chemotherapy to enhance efficacy against αvβ3 integrin expressing cancer, specifically glioblastoma multiforme (GBM). The main objective was to engineer IONCs as smart nanocarriers capable of inducing localized heating for MHT, while concurrently allowing the loading and pH-responsive release of chemotherapeutic agents with high specificity at targeted tumor cells. IONCs with edge lengths ranging from 17 to 19 nm, were selected as heat-mediating magnetic nanoplatforms due to their demonstrated heating efficiency under clinically relevant MHT conditions. The drug delivery strategy developed in this work centers on a PEGylated oxaliplatin derivative, Amino-PEG-Pt-DACH, specifically designed for pH-responsive release of active oxaliplatin chemotherapeutic drug. In this system, the platinum-based prodrug is covalently attached to a PEG backbone, which is subsequently conjugated to the IONCs. The targeting strategy relies on integrin recognition and was achieved by functionalizing the IONCs with a cyclic arginine-glycine-aspartic acid (cRGD) peptide-derived, selected for its strong affinity and selectivity toward the αvβ3 integrin, which is significantly overexpressed in GBM cells. To evaluate the features of the functionalized IONCs in synergistic multiple therapeutic approach, combining targeted drug delivery, pH-dependent release of Pt drug and MHT, a comprehensive material characterization and in vitro cell study was performed. The effectiveness of the targeting agent in enhancing the nanoparticles and Pt uptake and chemotherapeutic effect through intracellular drug release was demonstrated by using positive and negative cells expressing different level of αvβ3 integrins. Additionally, the heating properties of the particles upon exposure to AMF were assessed, confirming their ability to function as heat mediators even when functionalized with Pt and cRDG and, also, when exposed to the cellular environment. Finally, the role of MHT in sensitizing tumor cells thus further enhancing the overall therapeutic efficacy of Pt and RGD-mediated uptake, was also observed in vitro on U87 glioblastoma cells. Chapter 2 focuses on the development of copper-based and Copper-64-based nanoclusters (CuNCs and ⁶⁴CuNCs) for diagnostic imaging and cancer therapy. A straightforward, rapid, and scalable protocol was developed to produce orange-emitting fluorescent CuNCs using a synthesized multidentate thiol-based ligand (Cys-PIMA-PEG) and ascorbic acid (Vitamin C) as the reducing agent. The resulting CuNCs exhibit an average diameter of 1.6 ± 0.2 nm, orange fluorescence emission, good quantum yield (QY), and good photostability. Owing to their biocompatibility, and favorable optical properties, the synthesized CuNCs were employed for in vitro bioimaging studies on Glioblastoma cells. Using the same protocol with a ⁶⁴Cu radioisotope precursor, radioactive ⁶⁴CuNCs were also prepared. In vitro studies demonstrated their enhanced anticancer activity against glioblastoma cells compared to free ⁶⁴Cu. A preliminary in vivo study in a xenograft murine tumor model further confirmed the ability of intravenously administered ⁶⁴CuNCs to accumulate at the tumor site, even in the absence of active targeting ligands. However, stability assessments revealed that ⁶⁴CuNCs retain structural integrity in physiological conditions for only few hours which, although limited, is sufficient to support their use in short-term diagnostic applications such as PET imaging.