During the three years of my Ph.D, Ph.D cycle XXXII (2016-2019), at the Fondazione Istituto Italiano di Tecnologia, under the supervision of Dr.Teresa Pellegrino and the co-supervision of Prof.Orietta Monticelli (University of Genova), my focus was mainly on developing colloidally stable nanoclusters assembled at well-defined geometries produced from benchmark iron oxide nanocubes. These nanoclusters were designed, exploited and characterized for their potential use in theranostic applications comprising their exploitation in Magnetic Hyperthermia, Magnetic Resonance Imaging (MRI) and Magnetic Nanoparticles Imaging.
As the first aim, my focus was on building a two-dimensional nanoplatform based on highly efficient iron oxide nanocubes enwrapped with a bacteria extracted, biodegradable and biocompatible polyhydroxyalkanoate copolymer. Moreover, these magnetic polymeric clusters exhibit the unique feature to disassemble upon exposure to an intracellular rich lytic enzyme solution thus providing a gradual change in the cluster configuration accompanied by a gradual increase of magnetic heat performances in comparison to the initial 2D-clusters and to the individual iron oxide nanocubes used as building blocks for the cluster preparation. Indeed, comparing magnetic heat properties of the 2D assemblies with three dimensional centro-symmetrical assemblies (3D-MNBs) or single iron oxide nanocubes from same batch of cubes, emphasize how the initial 2D-assembly of iron oxide nanocubes s (2D-MNBs) dispersed in water are more advanced than the 3D-assemblies, but worse with respect to individual nanocubes. In addition, the heat abilities of these 2D clusters progressively increased when incubated in presence of esterase enzyme under physiological temperature, after 3 hours of incubation the specific absorption rate values, a measure of the heat-ability of the nanoparticles under a radio frequency were almost double than that of single cubes. Such an increase corresponds to disassembling of 2D-MNBs into short chain-like clusters of few nanocubes. Remarkably, our 2D-MNBs did not exhibit any variations in heat performance even after inducing an intentional aggregation. This is not the case for individual nanocubes. Magnetophoresis measurements suggest a faster response of 3D and 2D clusters to external magnets (0.3T) than that of individual nanocubes. This feature is desirable for the physical accumulation of magnetic materials under external magnetic field gradients. To the best of our knowledge, this is the first example of a nanoplatform, which combined enzymatic cleavable properties to a clear enhancement of the magnetic heat losses. In addition to this cluster study, I have also contributed to characterize other chain-like assemblies, named Dimer/Trimer obtained by assembling low interacting core-shell of wustite/magnetite iron oxide nanocubes into an amphiphilic copolymer, poly(styrene-co-maleic anhydride) cumene-terminated. Interestingly, by modulating the amount of polymer to nanoparticle surface ratio, the geometry of the same clusters could be modulated from a single structure to Dimer/Trimer to centrosymmetric structures. The short chains of nanocubes exhibit even in this case an enhanced specific absorption rate value with respect to single cubes and centrosymmetric clusters. Overall these studies reveal the significance of particle arrangement as a means to improve magnetic heating performances of the same building blocks, the nanocubes in our case.
According to our second aim, the above nanoclusters (developed for magnetic hyperthermia mentioned) were investigated as magnetic tracers, to unveil their diagnostic features for, recently emerging magnetic nanoparticles imaging (MPI) and for Magnetic resonance imaging (MRI). The multimodal imaging models with combined MPI and MRI properties could assist in real-time mapping of tissues that expected to improve the diagnostic accuracy. We found that the 2D-MNBs based on high interacting Iron oxide nanocubes exhibit poor MPI signal than that of standard Resovist. However, this signal of 2D-MNBs underwent a progressive increase upon incubation with esterase enzyme under physiological temperature (almost doubled) starting from their initial state, which attributes to the splitting of 2D beads into a small chain-like configuration. These results show a similar trend to the enzymatic triggered increase in heat performance, as mentioned above. Moreover, the 2D-MNBs possess a remarkable transverse relaxation rate (r2), indicating an efficient negative contrast of 2D-MNBs as agents for MRI. This value reduced by half upon exposure to lytic enzyme providing a significant T2-signal change upon to a stimulus triggered change (the enzymatic degradation). On the other side, among the nanoclusters based on core-shell iron oxide nanocubes; single structure, Dimer/Trimer to centrosymmetric structures, Dimer/Trimer exhibit a very remarkable MPI signal in comparison to the nanocube assemblies and to the individual nanoparticles and with respect to Resovist the most accepted FDA approved standard. Complementing the signal dominance in short chains of 2D-MNBs, the increase of MPI signal in Dimer/Trimer can also corresponds to their short uniaxial configuration. In addition, they have given a very significant transverse relaxation rate (r2) than many other superparamagnetic iron oxide nanoparticles. This kind of nanovectors with multifunctional theranostic features of MRI, MPI and magnetic hyperthermia are beneficial to improve thermo-therapy treatment of cancerous tissues while offering at the same time a potential readable and changing signal for image mapping.
Finally, as reported in chapter 3, we aim to develop an assembled nanoplatform made of magnetic iron oxide nanocube-based clusters and gadolinium-based nanoparticles that make the assembly responsive to the tumor microenvironment. This will enable to track tumor accumulation and disassembly of the nanoplatform for efficient thermotherapy based on T1 gadolinium-changing signal. For this purpose, we synthesize multicomponent nanostructures starting from iron oxide nanocubes embedded in a polymeric bead (MNBs) with a surface negative charge and decorated with Sodium gadolinium fluoride nanoparticles (NaGdF4 NPs), placed in between enzyme-degradable polymer spacers. Our hybrid structure achieved desired heating abilities under an alternative magnetic field of biological relevance. In addition to prominent T2 properties coming from MNBs, we demonstrated disassembling and detaching of polymer and NaGdF4 NPs from the surface of the MNBs upon exposure to enzymes that in turn improved water accessibility to NaGdF4 NP surface with a corresponding increase of T1 signal. In this way, we tracked the morphological changes of the systems at different time points of incubation in the presence of an enzyme, by MRI changing signal. This data was also confirmed by observing structural changes using TEM imaging. The integration of diagnostic tools to benchmark therapeutic probes could be a smart approach that enables to track the nanoparticle accumulation through artifact-free diagnosis and improve the heat efficiency of the magnetic hyperthermia treatment at the tumor
