MESOSCALE ASSEMBLIES OF INORGANIC NANOPARTICLES FOR THERANOSTIC APPLICATIONS

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

Magnetic nanoparticles and clusters for magnetic hyperthermia : Optimizing their heat performance and developing combinatorial therapies to tackle cancer

Magnetic hyperthermia (MHT) is a therapeutic modality for the treatment of solid tumors that has now accumulated more than 30 years of experience. In the ongoing MHT clinical trials for the treatment of brain and prostate tumors, iron oxide nanoparticles are employed as intra-Tumoral MHT agents under a patient-safe 100 kHz alternating magnetic field (AMF) applicator. Although iron oxide nanoparticles are currently approved by FDA for imaging purposes and for the treatment of anemia, magnetic nanoparticles (MNPs) designed for the efficient treatment of MHT must respond to specific physical-chemical properties in terms of magneto-energy conversion, heat dose production, surface chemistry and aggregation state. Accordingly, in the past few decades, these requirements have boosted the development of a new generation of MNPs specifically aimed for MHT. In this review, we present an overview on MNPs and their assemblies produced via different synthetic routes, focusing on which MNP features have allowed unprecedented heating efficiency levels to be achieved in MHT and highlighting nanoplatforms that prevent magnetic heat loss in the intracellular environment. Moreover, we review the advances on MNP-based nanoplatforms that embrace the concept of multimodal therapy, which aims to combine MHT with chemotherapy, radiotherapy, immunotherapy, photodynamic or phototherapy. Next, for a better control of the therapeutic temperature at the tumor, we focus on the studies that have optimized MNPs to maintain gold-standard MHT performance and are also tackling MNP imaging with the aim to quantitatively assess the amount of nanoparticles accumulated at the tumor site and regulate the MHT field conditions. To conclude, future perspectives with guidance on how to advance MHT therapy will be provided. This journal i

Uncovering the Magnetic Particle Imaging and Magnetic Resonance Imaging Features of Iron Oxide Nanocube Clusters

Multifunctional imaging nanoprobes continue to garner strong interest for their great potential in the detection and monitoring of cancer. In this study, we investigate a series of spatially arranged iron oxide nanocube-based clusters (i.e., chain-like dimer/trimer, centrosymmetric clusters, and enzymatically cleavable two-dimensional clusters) as magnetic particle imaging and magnetic resonance imaging probes. Our findings demonstrate that the short nanocube chain assemblies exhibit remarkable magnetic particle imaging signal enhancement with respect to the individually dispersed or the centrosymmetric cluster analogues. This result can be attributed to the beneficial uniaxial magnetic dipolar coupling occurring in the chain-like nanocube assembly. Moreover, we could effectively synthesize enzymatically cleavable two-dimensional nanocube clusters, which upon exposure to a lytic enzyme, exhibit a progressive increase in magnetic particle imaging signal at well-defined incubation time points. The increase in magnetic particle imaging signal can be used to trace the disassembly of the large planar clusters into smaller nanocube chains by enzymatic polymer degradation. These studies demonstrate that chain-like assemblies of iron oxide nanocubes offer the best spatial arrangement to improve magnetic particle imaging signals. In addition, the nanocube clusters synthesized in this study also show remarkable transverse magnetic resonance imaging relaxation signals. These nanoprobes, previously showcased for their outstanding heat performance in magnetic hyperthermia applications, have great potential as dual imaging probes and could be employed to improve the tumor thermo-therapeutic efficacy, while offering a readable magnetic signal for image mapping of material disassemblies at tumor sites

Co-loading of doxorubicin and iron oxide nanocubes in polycaprolactone fibers for combining Magneto-Thermal and chemotherapeutic effects on cancer cells

none11Among the strategies to fight cancer, multi-therapeutic approaches are considered as a wise choice to put in place multiple weapons to suppress tumors. In this work, to combine chemotherapeutic effects to magnetic hyperthermia when using biocompatible scaffolds, we have established an electrospinning method to produce nanofibers of polycaprolactone loaded with magnetic nanoparticles as heat mediators to be selectively activated under alternating magnetic field and doxorubicin as a chemotherapeutic drug. Production of the fibers was investigated with iron oxide nanoparticles of peculiar cubic shape (at 15 and 23 nm in cube edges) as they provide benchmark heat performance under clinical magnetic hyperthermia conditions. With 23 nm nanocubes when included into the fibers, an arrangement in chains was obtained. This linear configuration of magnetic nanoparticles resemble that of the magnetosomes, produced by magnetotactic bacteria, and our magnetic fibers exhibited remarkable heating effects as the magnetosomes. Magnetic fiber scaffolds showed excellent biocompatibility on fibroblast cells when missing the chemotherapeutic agent and when not exposed to magnetic hyperthermia as shown by viability assays. On the contrary, the fibers containing both magnetic nanocubes and doxorubicin showed significant cytotoxic effects on cervical cancer cells following the exposure to magnetic hyperthermia. Notably, these tests were conducted at magnetic hyperthermia field conditions of clinical use. As here shown, on the doxorubicin sensitive cervical cancer cells, the combination of heat damage by magnetic hyperthermia with enhanced diffusion of doxorubicin at therapeutic temperature are responsible for a more effective oncotherapy.10 pages, 7 figuresSerio, Francesca; Silvestri, Niccolò; Kumar Avugadda, Sahitya; Nucci, Giulia E P; Nitti, Simone; Onesto, Valentina; Catalano, Federico; D'Amone, Eliana; Gigli, Giuseppe; Del Mercato, Loretta L; Pellegrino, TeresaSerio, Francesca; Silvestri, Niccolò; Kumar Avugadda, Sahitya; Nucci, Giulia E P; Nitti, Simone; Onesto, Valentina; Catalano, Federico; D'Amone, Eliana; Gigli, Giuseppe; Del Mercato, Loretta L; Pellegrino, Teres

Multifunctional Magnetic and Upconverting Nanobeads as Dual Modal Imaging Tools.

We report the fabrication of aqueous multimodal imaging nanocomposites based on superparamagnetic nanoparticles (MNPs) and two different sizes of photoluminescent upconverting nanoparticles (UCNPs). The controlled and simultaneous incorporation of both types of nanoparticles (NPs) was obtained by controlling the solvent composition and the addition rate of the destabilizing solvent. The magnetic properties of the MNPs remained unaltered after their encapsulation into the polymeric beads as shown by the T2 relaxivity measurements. The UCNPs maintain photoluminescent properties even when embedded with the MNPs into the polymer bead. Moreover, the light emitted by the magnetic and upconverting nanobeads (MUCNBs) under NIR excitation (λexc = 980 nm) was clearly observed through different thicknesses of agarose gel or through a mouse skin layer. The comparison with magnetic and luminescent nanobeads based on red-emitting quantum dots (QDs) demonstrated that while the QD-based beads show significant autofluorescence background from the skin, the signal obtained by the MUCNBs allows a decrease in this background. In summary, these results indicate that MUCNBs are good magnetic and optical probes for in vivo multimodal imaging sensors

Multifunctional Magnetic and Upconverting Nanobeads as Dual Modal Imaging Tools

We report the fabrication of aqueous multimodal imaging nanocomposites based on superparamagnetic nanoparticles (MNPs) and two different sizes of photoluminescent upconverting nanoparticles (UCNPs). The controlled and simultaneous incorporation of both types of nanoparticles (NPs) was obtained by controlling the solvent composition and the addition rate of the destabilizing solvent. The magnetic properties of the MNPs remained unaltered after their encapsulation into the polymeric beads as shown by the T2 relaxivity measurements. The UCNPs maintain photoluminescent properties even when embedded with the MNPs into the polymer bead. Moreover, the light emitted by the magnetic and upconverting nanobeads (MUCNBs) under NIR excitation (λ_exc = 980 nm) was clearly observed through different thicknesses of agarose gel or through a mouse skin layer. The comparison with magnetic and luminescent nanobeads based on red-emitting quantum dots (QDs) demonstrated that while the QD-based beads show significant autofluorescence background from the skin, the signal obtained by the MUCNBs allows a decrease in this background. In summary, these results indicate that MUCNBs are good magnetic and optical probes for in vivo multimodal imaging sensors