Multi-materials nano-heterostructures for combined therapy and diagnosis

The present dissertation is resulting from the work performed during the Ph.D. research activity carried out at the Italian Institute of Technology (IIT) under the supervision of Dr. Teresa Pellegrino (Nanomaterials for biomedical research line) of the Italian Institute of Technology and Fabio Canepa of the University of Genova. The thesis has been conducted in the framework of the ERC-founded project ICARO (ERC starting grant n° 678109, Principal Investigator: Dr. Teresa Pellegrino), whose main purpose is the development of novel inorganic nanostructures for radiotherapy and chemotherapy of cancer. Thus, this thesis aims to progress the field of nanomedicine. In particular, the first goal of this work is to synthesize innovative water stable chalcogenide nanoparticles, with the purpose of achieving nano-sized platforms capable of incorporating radioactive 64Cu ions, which would make such systems suitable for the use in radiotherapy and for positron emission tomography. The second goal is to explore the coupling of such chalcogenide nanocrystals with magnetic nanoparticles, which are already part of the nanoparticles’ portfolio available in the research group where this thesis was carried out. These nanoparticles have shown great potential for magnetic hyperthermia treatment of cancer and, in combination with radiotherapy, could result in a synergic and more effective cancer treatment. Thus, this thesis provides new ground in the rational design of multifunctional nano-heterostructures for cancer diagnosis and therapy. The first chapter of this thesis deals with the synthesis, water transfer and radiolabeling of ZnS nanoparticles. A non-hydrolitycal thermal decomposition synthesis route was exploited in order to obtain quasi-spherical nanoparticles. Such nanoparticles have hydrophobic ligands on their surface and thus are stable in organic solvents. In order to successfully transfer them to water phase, the ligands were exchanged through a procedure that employs a multi-dentate amphiphilic polymer (cysteamine-poly(isobutylene-alt-maleic anhydride)-polyethylene glycol, CYS-PIMA-PEG), which resulted in inorganic colloids with perfect stability in aqueous phase. On this system, cation exchange reactions with both radioactive and non-radioactive copper were carried out. The optimized protocol for the radiolabeling of ZnS nanocrystals with 64Cu permitted to obtain high values of radiochemical yield (93%), defined as the percentage of the total activity used that is incorporated in the crystals, without losing colloidal stability during radiolabeling reaction or subsequent concentration and purification process. The results obtained indicate ZnS nanoparticles as an efficient nano-platform for the use in radiotherapy and positron emission tomography, given the fast, reproducible and easily clinical-translatable radiolabeling procedure resulting in quantitative incorporation of 64Cu ions. In the second chapter we report the research activity carried out to couple in a single nano-heterostructure, the ZnS nanoparticles or copper-deficient copper sulfide nanoparticles (previously reported to be exploitable as radioisotopes carriers) with highly performing magnetic nanoparticles (iron oxide nanocubes, IONCs) or nano-heterostructures (gold-iron oxide dimers, Au@FeOy). Although the direct growth of ZnS domains on IONCs, through colloidal two-pot seeded-growth synthesis procedures, was not possible, this thesis succeeds on merging the different nanoparticles in a single nano-platform by exploiting the use of gold NPs as “linkers” between magnetic iron oxide domains and copper deficient Cu2-xS domains, using Au@FeOy dimers as seeds for the growth of copper sulfide domains. Thus, this procedure resulted in the production of FeOy@Au@Cu2-xS2 trimers, with the additional possibility to tune the size of the Cu2-xS domain by changing precursors’ concentration. These trimers were thoroughly characterized through diverse structural and magnetic analysis techniques (transition electron microscopy, X-ray diffraction, SQUID magnetometry and UV-VIS-NIR spectroscopy). In particular, magnetic properties measurements, allowed to conclude that the magnetic properties of the FeOy@Au@Cu2-xS trimers are comparable to the ones of the Au@FeOy dimers (used as seeds for the subsequent reaction of growth of Cu2-xS domain). In addition, the trimers display two localized surface plasmon resonance absorption bands, one assigned to the gold domain and the other assignable to the copper deficient copper sulfide domain, respectively localized in the first and second NIR biological windows and, consequently, exploitable in photothermal therapy. In the third chapter, the newly synthesized trimers were transferred to water phase and tested for the application as carriers for 64Cu and as heating probes in magnetic hyperthermia and photothermal therapy. Different strategies were explored in order to develop a reproducible and high-yield water transfer protocol. Among them, a two-step ligand exchange procedure employing methoxy-poly(ethylene glycol)-thiol and poly(catechol)-poly(ethylene glycol) as amphiphilic ligands resulted in aqueous phase stable trimers with high water transfer procedure yield (> 80 %). FeOy@Au@Cu2-xS trimers were also successfully transferred to water, although with lower yields if compared to previous procedure, using a polymer coating procedure and employing commercially available and cost-effective poly-(maleic anhydride-alt-1-octadecene). Lastly, the developed nano-platform showed great relevance in the field of nanomedicine. Indeed, when employed in magnetic hyperthermia, trimers resulted in high SAR values, preserving the excellent hyperthermia performances of the Au@FeOy used as seeds for their synthesis and thus in line with the best magnetic nano-heterostructures reported so far. Radiolabeling reactions were performed on trimers, resulting in a radiochemical yield of 97 %, higher than any value reported so far for 64Cu incorporation in water stable nanocrystals. Furthermore, the stability of the trimers during the radiolabeling and subsequent purification procedures was likewise ensured by the use of CYS-PIMA-PEG as stabilizing agent, permitting to recover quantitatively the nanocrystals and the associated radioactivity. The performances of FeOy@Au@Cu2-xS trimers when used in photothermal heating were also tested under the exposure to 808 nm laser irradiation. Although when using high power density (4.67 W/cm2), a temperature increase of 33°C in five minutes was registered, the performances obtained with lower laser’s power density were limited. However, the possibility to tune the absorption wavelengths by means of changing gold and copper sulfide domain’s properties, gives space to further improvements for these multifunctional nano-heterostructures. To the best of our knowledge, the here described FeOy@Au@Cu2-xS trimers are the first ever reported nano-heterostructures able to combine in one single nano-object the possibility to perform magnetic hyperthermia, photothermal therapy and radiotherapy/positron emission tomography, thus allowing the possible development of more efficient cancer treatments.