Application of Nanostructures for Treatment and Diagnosis of Solid Tumors

In the modern era, technology has a crucial impact on everyday life. It helps us to adapt to the natural environment, perform specific and complex tasks, solve problems, and improve the quality of our life. The arising of nanotechnology, that is the understanding and control of matter at dimensions on nanometre, is further revolutionising technological applications, since such reduced dimensions confer new properties that are different from those of the bulk material. Among several fields of application for nanotechnologies, nanomedicine is of great interest because of its implications in healthcare and especially in cancer management. Indeed, an antitumor drug has to face several hurdles in order to reach its site of action, mainly due to organism immune surveillance, abnormal vascular supply at cancerous tissues, hostile tumour microenvironment and intracellular pathways for its uptake. Some of these mentioned problems limit also the accumulation of tracers applied for diagnostic imaging procedures. The employment of nanostructures to deliver drugs and/or tracers at the tumor lesions offers several advantages as they improve stability, pharmacokinetics and biodistribution of the molecule of interest, and protect the drug from the biological environment. Moreover, it is possible to produce different types of nanostructures with different properties, offering the choice of what is better to use according to the needs of the specific medical purpose. Nanotechnology can also improve the creation of devices or methodologies to develop new ex vivo analysis with diagnostic purposes. A new tool in cancer management can be derived from the so-called circulating tumor cells (CTCs), which are cells detached from the primary tumor and circulating into the bloodstream. They are critical in tumor progression since their extravasation in distant organs can give rise to establishment of secondary tumors (i.e., metastatization process). The number of CTCs detected in the blood of patients has a prognostic value in metastatic breast, prostate and colorectal cancer, but they can be found also in other tumor types. Furthermore, changes in CTC counts during treatment can predict survival outcomes, highlighting the potential use of CTCs as a cancer biomarker in the new concept of “liquid biopsy”. The main challenge of CTC analysis relays in their extremely low number also in patients with metastatic disease, and their heterogeneous phenotype as they can have a more epithelial-like or mesenchymal-like phenotype according to the stage of metastatic development. Several platforms are emerging for CTCs isolation and detection, first of all for their enumeration, some exploiting their biological properties (i.e., tumor specific markers) and others their physical properties (i.e., size and dielectric properties), and nanotechnologies can offer a significant help with this challenge. The aim of this work is to create, evaluate and implement new diagnostic strategies for cancer management with the application of innovative nanotechnologies, which could offer also the possibility to translate the developed diagnostic nanostructures into therapeutic approaches for the disease treatment. Multiple types of nanosystems have been developed during this PhD project in order to approach some limitations of the current in vivo and ex vivo diagnostic methodologies applied into cancer management. In particular, we have synthesized and assessed a new class of nanostructures, the multifunctional Au-Fe alloy nanoparticles. The goal of these nanoparticles is that they offer a good performance in magnetic resonance imaging (MRI), computed X-ray tomography (CT) and surface enhanced Raman spectroscopy (SERS) at the same time, to obtain both anatomical and physiological information (MRI and CT) and high spatial resolution for intrasurgical imaging (SERS). Their features of biological compatibility and exploitability have been confirmed both in vitro and in vivo, where our nanoparticles show also the capability to accumulate at cancerous lesions by enhanced permeability and retention (EPR) effect. We have also investigated a new intriguing strategy to target tumors by means of macrophage cells as carriers, since macrophages migrate at inflammatory sites as tumor microenvironments. We have developed nanosystems that own both superparamagnetic properties and Raman activity by an appropriate mixing of gold and iron oxide nanoparticles in one single tool. By using these nanostructures, we are able to load macrophage cells and then separate this population of interest from other cell populations, in order to exploit it for in vivo administration in syngeneic mouse tumor models. Moreover, we have further implemented this typology of nanostructures in a new core-shell-satellite architecture to be able to link antibodies as driving moieties for an active tumor targeting. These new nanoparticles are able to recognize and separate the antigen expressing population from surrounding environment thanks to their magnetic properties. The last part of this work is dedicated to the isolation, enumeration and phenotypic analysis of the CTCs present in the blood of oncologic patients. We think that a molecular investigation of antigens expressed on these cells can be crucial for clinicians to define a comprehensive diagnosis and even to guide treatment decision, beyond their simple enumeration. In order to overcome the limitations due to the rarity of CTCs in patient blood, we have exploited the ultra-sensitivity of Raman spectroscopy associated to SERS effect; this is able to increase the signal of Raman dyes loaded on plasmonic nanoparticles, in order to detect even very low signal intensities at the single cell level. The Raman-SERS methodology allow us to study also the possibility of multiplexing analysis, where different cellular markers can be studied on the same cells at the same time and/or different cell populations can be investigated for different phenotype markers at the same time. Our optimized protocol allow to trap CTCs with an efficiency >80% and the phenotype analysis by Raman-SERS spectroscopy in an automatized way is possible thanks to the development of an appropriate computer tool. Our approach has been preliminary validated by applying the new chip for CTC capture with spiked samples (blood mononuclear cells plus tumor cells) that mimic the real CTC samples.