Iron oxide nanocubes in nanomedicine: theranostic approach for cancer treatment and diagnosis

The present dissertation discloses the doctoral work carried out during the past three years at the Italian Institute of Technology (IIT) and the University of Genoa. The work was focused on the functionalization of iron oxide nanocubes (NCs) for different biological applications. Tuning the surface features of these magnetic nanocubes, as well as their assembly and intrinsic chemical and physical properties, resulted in the development of suitable nanotools for cancer theranostic, i.e. the combination of diagnosis and cancer therapy. The first chapter deals with the synthesis of magnetic nanoclusters, referred as magnetic nanobeads (MNBs). Here, maghemite (\u3b3-Fe2O3) nanocubes were tightly enwrapped into an amphiphilic polymer able to solubilize and stabilize them in water-based solutions. The synthetic route, reported in literature, was improved in order to obtain more stable polymeric shells that can be further functionalized with PEG-derivatized molecules. Due to the higher magnetic moment found for the MNBs, compared to that of single nanocubes, they were investigated for magnetic cell sorting. Therefore, a targeting feature was added to their surface by attaching a PEG molecule derivatized with folic acid (PEG-FA). This approach provides: 1) the binding of the MNBs to folate receptors overexpressed on the cell membrane of some cancer cells; 2) stability in complex biological media; 3) distance of the FA from the polymeric surface; 4) degree of freedom to the bioactive folic acid. A cancer cell line having high folate receptor expression profile (KB cell line) was chosen as a model for testing the sorting ability of the MNBs. The results obtained showed a significantly higher sorting efficiency for the MNBs functionalized with PEG-FA in comparison to the one observed for the MNBs functionalized with a non-biologically active PEG. This outcome reveals the potential of PEG-FA functionalized MNBs for the isolation of relevant folate receptor positive cancer cells, e.g. ovarian cancer cells, from biological tissues. In the second chapter, the structural transformation of core-shell wuestite/maghemite (FeO/\u3b3-Fe2O3) nanocubes into maghemite is reported. Here, an approach that is not often applied by material scientists was followed for the transformation under aqueous conditions. The non-interacting nature of core-shell nanocubes, due to their low magnetization, allowed for an easy and quantitative transfer in water, using a standard protocol for the coating with an amphiphilic polymer. Then, it was found that a mild oxidation process, carried out in water at 80 \ub0C, promoted the conversion of the core-shell structure into fully maghemite nanocubes, enhancing their magnetic features, especially the specific absorption rate (SAR). Thus, the nanocubes were functionalized with PEG-FA and the annealing treatment was repeated. Noteworthy, the oxidation strategy developed did not compromise the bio-functionality of the PEG-FA molecule. Unfortunately, the SAR values of the obtained one-phase nanocubes were found to be viscosity dependent, discouraging their use for magnetic hyperthermia in cellular environment. Instead, because annealing increased the magnetic moment of the nanocubes, they were efficiently used for the magnetic sorting of KB cells. The sorting efficiency found for these nanocubes was comparable to that of the MNBs reported in chapter 1, suggesting that a high amount of single nanocubes bound to the cell membrane increases the magnetic moment of the whole nanocubes-cell system. Thus, the methodology adopted for tuning the magnetic properties of core-shell iron oxide-based materials into one single phase NPs was proven appropriate for the preparation of nanocubes for magnetic driven cell sorting. The third chapter discusses the use of maghemite (Fe2O3) nanocubes for developing multimodal nanotherapeutics to treat ovarian cancer. The intriguing high heat performance of the nanocubes was exploited to perform magnetic hyperthermia. At the same time, the high surface area available on the nanocubes has been used for drug delivery and specific antibody-mediated tumor targeting towards ovarian cancer cells. The NCs were functionalized with both an oxaliplatin-derivatized PEG (PEG-Pt) and a PEG-Bis(carboxymethyl)-lysine (a nitrilotriacetic derived molecule) complex for the binding of an his-tag antibody fragment (scFv) specific for folate receptor \u3b1 (\u3b1FR). The functionalized nanocubes were able to recognize their target and to be efficiently internalized by the desired cells via endocytosis pathway. Once inside the cells, the nanocubes delivered the Pt compound, which induced toxicity by intercalating the DNA. Thanks to their crystal structure and size, these nanocubes exhibited a viscosityindependent behavior, keeping high SAR values even in highly viscous media. Indeed, once incubated with the cells, the nanocubes were able to heat the tumor mass up to 42 \ub0C, promoting cell death. The contribution of the cytotoxicity from both Pt delivery and hyperthermia highlighted the use of these nanocubes for cancer multitherapy. Thus, combining targeting, drug delivery and magnetic hyperthermia, a suitable platform for a synergistic treatment of cancer has been developed

A Fast Alternating Minimization Algorithm for Total Variation Deblurring Without Boundary Artifacts

Recently, a fast alternating minimization algorithm for total variation image deblurring (FTVd) has been presented by Wang, Yang, Yin, and Zhang [{\em SIAM J. Imaging Sci.}, 1 (2008), pp. 248--272]. The method in a nutshell consists of a discrete Fourier transform-based alternating minimization algorithm with periodic boundary conditions and in which two fast Fourier transforms (FFTs) are required per iteration. In this paper, we propose an alternating minimization algorithm for the continuous version of the total variation image deblurring problem. We establish convergence of the proposed continuous alternating minimization algorithm. The continuous setting is very useful to have a unifying representation of the algorithm, independently of the discrete approximation of the deconvolution problem, in particular concerning the strategies for dealing with boundary artifacts. Indeed, an accurate restoration of blurred and noisy images requires a proper treatment of the boundary. A discrete version of our continuous alternating minimization algorithm is obtained following two different strategies: the imposition of appropriate boundary conditions and the enlargement of the domain. The first one is computationally useful in the case of a symmetric blur, while the second one can be efficiently applied for a nonsymmetric blur. Numerical tests show that our algorithm generates higher quality images in comparable running times with respect to the Fast Total Variation deconvolution algorithm

Belousov-Zhabotinsky type reactions: the non-linear behavior of chemical systems

AbstractChemical oscillators are open systems characterized by periodic variations of some reaction species concentration due to complex physico-chemical phenomena that may cause bistability, rise of limit cycle attractors, birth of spiral waves and Turing patterns and finally deterministic chaos. Specifically, the Belousov-Zhabotinsky reaction is a noteworthy example of non-linear behavior of chemical systems occurring in homogenous media. This reaction can take place in several variants and may offer an overview on chemical oscillators, owing to its simplicity of mathematical handling and several more complex deriving phenomena. This work provides an overview of Belousov-Zhabotinsky-type reactions, focusing on modeling under different operating conditions, from the most simple to the most widely applicable models presented during the years. In particular, the stability of simplified models as a function of bifurcation parameters is studied as causes of several complex behaviors. Rise of waves and fronts is mathematically explained as well as birth and evolution issues of the chaotic ODEs system describing the Györgyi-Field model of the Belousov-Zhabotinsky reaction. This review provides not only the general information about oscillatory reactions, but also provides the mathematical solutions in order to be used in future biochemical reactions and reactor designs

Inversion dynamics of class manifolds in deep learning reveals tradeoffs underlying generalisation

To achieve near-zero training error in a classification problem, the layers of a feed-forward network have to disentangle the manifolds of data points with different labels, to facilitate the discrimination. However, excessive class separation can bring to overfitting since good generalisation requires learning invariant features, which involve some level of entanglement. We report on numerical experiments showing how the optimisation dynamics finds representations that balance these opposing tendencies with a non-monotonic trend. After a fast segregation phase, a slower rearrangement (conserved across data sets and architectures) increases the class entanglement.The training error at the inversion is stable under subsampling, and across network initialisations and optimisers, which characterises it as a property solely of the data structure and (very weakly) of the architecture. The inversion is the manifestation of tradeoffs elicited by well-defined and maximally stable elements of the training set, coined ``stragglers'', particularly influential for generalisation

Neuroanatomical and neuropsychological correlates of resting-state EEG diagnostic features in patients with Alzheimer's disease

Background: In the search for accurate, low cost biomarkers for Alzheimer’s disease (AD) and other dementias, quantitative electroencephalography (EEG) may offer a solution. In a recent multisite study by Cognision, patients with AD were assessed using the Alzheimer Disease Neuroimaging Initiative protocol, plus EEG assessment. The primary objective of the current analysis, was to examine the relationships between a resting-state (rs)EEG feature set (that best discriminated AD patients from controls) and neuroanatomical measures. The second objective was to identify the rsEEG measures that reflected disease staging. Method: Eighty-nine patients with mild AD (MMSE 21-26) were evaluated using a comprehensive neuropsychological assessment battery, 5 minute eyes-open rsEEG, and structural MRI. Correlations (Spearman’s) were assessed between the 35 rsEEG features (that most accurately discriminated the AD patients), neuroanatomical measures (derived using Freesurfer), and neuropsychological test results. Result: Cortical Thickness (CT) measures within the left posterior cingulate and right precuneus were related to alpha features. Beta features were associated with regions including the right entorhinal cortex, middle temporal, supramarginal, lingual, and paracentral cortex, in addition to the anterior cingulate cortex (ACC) and precuneus, bilaterally. Gamma features correlated with regions that included the right ACC and fronto-parietal cortex. Delta features were linked to the left fronto-parietal and right entorhinal cortex. Theta features were associated with the left ACC and visual cortex. In relation to disease staging – Clinical Dementia Rating scores were correlated with gamma features at frontal electrode sites, and with power over frequency bands, delta to beta, at Fz. Alpha features were associated with hippocampal volume (bilaterally), whereas some delta and theta features were linked to left hippocampal volume. Conclusion: These preliminary correlation analyses highlight multiple brain regions that appear to underpin the rsEEG abnormalities that occur due to AD. Given the rich data offered by both rsEEG and by structural MRI, future studies could investigate the combined potential for these techniques to classify the dementias

Bone Marrow Clonogenic Capability, Cytokine Production, and Thymic Output in Patients with Common Variable Immunodeficiency

AbstractIn patients with primary Ab deficiencies, hematological and immunological abnormalities are frequently observed. A regenerative failure of hemopoietic stem/progenitor cells has been hypothesized. We evaluated in the bone marrow (BM) of 11 patients with common variable immunodeficiency, the phenotype of BM progenitors and their in vitro growth by colony-forming cell (CFC) and long-term culture (LTC) assays. A significant decrease in erythroid and mixed CFC and, to a greater extent, in primitive LTC-CFC progenitors was observed in patients compared with healthy controls. The frequency of BM pre-B and pro-B cells correlated directly with the absolute number of CD19+ lymphocytes. BM cells cultured in vitro produced spontaneously lower amounts of IL-2 and elevated levels of TNF-α compared with controls, indicating a skewing toward a proapoptotic cytokine pattern. In addition, stromal cells generated after BM LTC secreted less IL-7 and displayed by immunohistochemistry an altered phenotype. These findings were associated with a significant decrease in naive Th cells coexpressing CD31 in the peripheral blood. These results indicate an impaired growth and differentiation capacity of progenitor cells in patients with common variable immunodeficiency

Asymmetric Assembling of Iron Oxide Nanocubes for Improving Magnetic Hyperthermia Performance

Magnetic hyperthermia (MH) based on magnetic nanoparticles (MNPs) is a promising adjuvant therapy for cancer treatment. Particle clustering leading to complex magnetic interactions affects the heat generated by MNPs during MH. The heat efficiencies, theoretically predicted, are still poorly understood because of a lack of control of the fabrication of such clusters with defined geometries and thus their functionality. This study aims to correlate the heating efficiency under MH of individually coated iron oxide nanocubes (IONCs) versus soft colloidal nanoclusters made of small groupings of nanocubes arranged in different geometries. The controlled clustering of alkyl-stabilized IONCs is achieved here during the water transfer procedure by tuning the fraction of the amphiphilic copolymer, poly(styrene-co-maleic anhydride) cumene-terminated, to the nanoparticle surface. It is found that increasing the polymer-to-nanoparticle surface ratio leads to the formation of increasingly large nanoclusters with defined geometries. When compared to the individual nanocubes, we show here that controlled grouping of nanoparticles - so-called "dimers" and "trimers" composed of two and three nanocubes, respectively - increases specific absorption rate (SAR) values, while conversely, forming centrosymmetric clusters having more than four nanocubes leads to lower SAR values. Magnetization measurements and Monte Carlo-based simulations support the observed SAR trend and reveal the importance of the dipolar interaction effect and its dependence on the details of the particle arrangements within the different clusters

Cooperative dynamics of DNA-grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami

Magnetic nanoparticles (MNPs) provide new opportunities for enzyme-free biosensing of nucleic acid biomarkers and magnetic actuation by patterning on DNA origami, yet how the DNA grafting density affects their dynamics and accessibility remains poorly understood. Here, we performed surface functionalization of MNPs with single-stranded DNA (ssDNA) via click chemistry with a tunable grafting density, which enables the encapsulation of single MNPs inside a functional polymeric layer. We used several complementary methods to show that particle translational and rotational dynamics exhibit a sigmoidal dependence on the ssDNA grafting density. At low densities, ssDNA strands adopt a coiled conformation that results in minor alterations to particle dynamics, while at high densities, they organize into polymer brushes that collectively influence particle dynamics. Intermediate ssDNA densities, where the dynamics are most sensitive to changes, show the highest magnetic biosensing sensitivity for the detection of target nucleic acids. Finally, we demonstrate that MNPs with high ssDNA grafting densities are required to efficiently couple to DNA origami. Our results establish ssDNA grafting density as a critical parameter for the functionalization of MNPs for magnetic biosensing and functionalization of DNA nanostructures

Multimodal prediction of Alzheimer's disease severity level based on resting-state EEG and structural MRI

While several biomarkers have been developed for the detection of Alzheimer's disease (AD), not many are available for the prediction of disease severity, particularly for patients in the mild stages of AD. In this paper, we explore the multimodal prediction of Mini-Mental State Examination (MMSE) scores using resting-state electroencephalography (EEG) and structural magnetic resonance imaging (MRI) scans. Analyses were carried out on a dataset comprised of EEG and MRI data collected from 89 patients diagnosed with minimal-mild AD. Three feature selection algorithms were assessed alongside four machine learning algorithms. Results showed that while MRI features alone outperformed EEG features, when both modalities were combined, improved results were achieved. The top-selected EEG features conveyed information about amplitude modulation rate-of-change, whereas top-MRI features comprised information about cortical area and white matter volume. Overall, a root mean square error between predicted MMSE values and true MMSE scores of 1.682 was achieved with a multimodal system and a random forest regression model

Moduli Stabilization and Cosmology of Type IIB on SU(2)-Structure Orientifolds

We consider type IIB flux compactifications on six-dimensional SU(2)-structure manifolds with O5- and O7-planes. These six-dimensional spaces allow not only for F_3 and H_3 fluxes but also for F_1 and F_5 fluxes. We derive the four-dimensional N=1 scalar potential for such compactifications and present one explicit example of a fully stabilized AdS vacuum with large volume and small string coupling. We then discuss cosmological aspects of these compactifications and derive several no-go theorems that forbid dS vacua and slow-roll inflation under certain conditions. We also study concrete examples of cosets and twisted tori and find that our no-go theorems forbid dS vacua and slow-roll inflation in all but one of them. For the latter we find a dS critical point with \epsilon numerically zero. However, the point has two tachyons and eta-parameter \eta \approx -3.1.Comment: 35 pages + appendices, LaTeX2e; v2: numerical dS extremum added, typos corrected, references adde