Fe3 O4 @Au@Cu2-x S Heterostructures Designed for Tri-Modal Therapy: Photo- Magnetic Hyperthermia and 64 Cu Radio-Insertion.

Here, the synthesis and proof of exploitation of three-material inorganic heterostructures made of iron oxide-gold-copper sulfide (Fe3 O4 @Au@Cu2-x S) are reported. Starting with Fe3 O4 -Au dumbbell heterostructure as seeds, a third Cu2-x S domain is selectively grown on the Au domain. The as-synthesized trimers are transferred to water by a two-step ligand exchange procedure exploiting thiol-polyethylene glycol to coordinate Au and Cu2-x S surfaces and polycatechol-polyethylene glycol to bind the Fe3 O4 surface. The saline stable trimers possess multi-functional properties: the Fe3 O4 domain, of appropriate size and crystallinity, guarantees optimal heating losses in magnetic hyperthermia (MHT) under magnetic field conditions of clinical use. These trimers have indeed record values of specific adsorption rate among the inorganic-heterostructures so far reported. The presence of Au and Cu2-x S domains ensures a large adsorption which falls in the first near-infrared (NIR) biological window and is here exploited, under laser excitation at 808 nm, to produce photo-thermal heat alone or in combination with MHT obtained from the Fe3 O4 domain. Finally, an intercalation protocol with radioactive 64 Cu ions is developed on the Cu2-x S domain, reaching high radiochemical yield and specific activity making the Fe3 O4 @Au@Cu2-x S trimers suitable as carriers for 64 Cu in internal radiotherapy (iRT) and traceable by positron emission tomography (PET).

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.

Elongated magnetic nanoparticles with high-aspect ratio: a nuclear relaxation and specific absorption rate investigation.

Medical application of nanotechnology implies the development of nanomaterials capable of being functional in different biological environments. In this sense, elongated nanoparticles (e-MNPs) with high-aspect ratio have demonstrated more effective particle cellular internalization, which is favoured by the increased surface area. This paper makes use of an environmentally friendly hydrothermal method to produce magnetic iron oxide e-MNPs, starting from goethite precursors. At high temperatures (Td) goethite transforms into hematite, which subsequently reduces to magnetite when exposed to a hydrogen atmosphere for a certain time. It is shown that by adjusting Td it is possible to obtain Fe3O4 e-MNPs with partially controlled specific surface area and magnetic properties, attributed to different porosity of the samples. The particles' efficiencies for diagnostic and therapeutic purposes (in magnetic resonance imaging and magnetic fluid hyperthermia, respectively) are very good in terms of clinical standards, some samples showing transversal proton nuclear relaxivity r2 (B0 = 1.33 T) = 340 s-1 mM-1 and specific absorption rate SAR > 370 W g-1 at high field amplitudes (B0 = 55 mT). Direct correlations between the SAR, relaxivity, magnetic properties and porosity of the samples are found, and the physico-chemical processes underneath these correlations are investigated. Our results open the possibility of using very efficient high-aspect ratio elongated nanoparticles with optimized chemico-physical properties for biomedical applications.

Anisotropic magnetic nanoparticles for biomedicine: bridging frequency separated AC-field controlled domains of actuation.

Magnetic nanoparticles (MNPs) constitute promising nanomedicine tools based on the possibility of obtaining different actuations (for example, heating or mechanical response) triggered by safe remote stimuli. Particularly, the possibility of performing different tasks using the same entity constitutes a main research target towards optimizing the treatment. But such a goal represents, in general, a very difficult step because the requisites for achieving efficient responses for separate actuations are often disparate - if not completely incompatible. An example of this is the response of MNPs to external AC fields, which could in principle be exploited for either magneto-mechanical actuation (MMA) at low frequencies (tens of Hz); or heat release at high frequency (0.1-1 MHz range) for magnetic fluid hyperthermia (MFH). The problem is that efficient MMA involves large torque, the required material parameters for which are detrimental to high heating, thus hindering the possibility of effective alternation between both responses. To overcome such apparent incompatibility, we propose a simple approach based on the use of anisotropic MNPs. The key idea is that the AC-frequency change must be concurrent with a field-amplitude variation able to promote - or impede - the reversal over the shape-determined anisotropy energy barrier. This way it is possible to switch the particle response between an efficient (magnetically dissipationless) rotation regime at low-f, for MMA, and a "frozen" (non-rotatable) high-energy-dissipation regime at high-f, for MFH. Furthermore, we show that such an alternation can also be achieved within the same high-f MFH regime. We use combined Brownian dynamics and micromagnetic simulations, based on real experimental samples, to show how such a field threshold can be tuned to working conditions with hexagonal-disk shape anisotropy.

Scale-up approach for the preparation of magnetic ferrite nanocubes and other shapes with benchmark performance for magnetic hyperthermia applications.

Magnetic nanoparticles are increasingly used in medical applications, including cancer treatment by magnetic hyperthermia. This protocol describes a solvothermal-based process to prepare, at the gram scale, ferrite nanoparticles with well-defined shape, i.e., nanocubes, nanostars and other faceted nanoparticles, and with fine control of structural/magnetic properties to achieve point-of-reference magnetic hyperthermia performance. This straightforward method comprises simple steps: (i) making a homogeneous alcoholic solution of a surfactant and an alkyl amine; (ii) adding an organometallic metal precursor together with an aldehyde molecule, which acts as the key shape directing agent; and (iii) reacting the mixture in an autoclave for solvothermal crystallization. The shape of the ferrite nanoparticles can be controlled by the structure of the aldehyde ligand. Benzaldehyde and its aromatic derivatives favor the formation of cubic ferrite nanoparticles while aliphatic aldehydes result in spherical nanoparticles. The replacement of the primary amine, used in the nanocubes synthesis, with a secondary/tertiary amine results in nanoparticles with star-like shape. The well-defined control in terms of shape, narrow size distribution (below 5%), compositional tuning and crystallinity guarantees the preparation, at the gram scale, of nanocubes/star-like nanoparticles that possess, under magnetic field conditions of clinical use, specific adsorption rates comparable to or even superior to those obtained through thermal decomposition methods, which are typically prepared at the milligram scale. Here, gram-scale nanoparticle products with benchmark features for magnetic hyperthermia applications can be prepared in ~10 h with an average level of expertise in chemistry.

Clickable Polymer Ligand-Functionalized Iron Oxide Nanocubes: A Promising Nanoplatform for 'Local Hot Spots' Magnetically Triggered Drug Release.

Exploiting the local heat on the surface of magnetic nanoparticles (MNPs) upon exposure to an alternating magnetic field (AMF) to cleave thermal labile bonds represents an interesting approach in the context of remotely triggered drug delivery. Here, taking advantages of a simple and scalable two-step ligand exchange reaction, we have prepared iron oxide nanocubes (IONCs) functionalized with a novel multifunctional polymer ligand having multiple catechol moieties, furfuryl pendants, and polyethylene glycol (PEG) side chains. Catechol groups ensure a strong binding of the polymer ligands to the IONCs surface, while the PEG chains provide good colloidal stability to the polymer-coated IONCs. More importantly, furfuryl pendants on the polymer enable to click the molecules of interest (either maleimide-fluorescein or maleimide-doxorubicin) via a thermal labile Diels-Alder adduct. The resulting IONCs functionalized with a fluorescein/doxorubicin-conjugated polymer ligand exhibit good colloidal stability in buffer saline and serum solution along with outstanding heating performance in aqueous solution or even in viscous media (81% glycerol/water) when exposed to the AMF of clinical use. The release of conjugated bioactive molecules such as fluorescein and doxorubicin could be boosted by applying AMF conditions of clinical use (16 kAm-1 and 110 kHz). It is remarkable that the magnetic hyperthermia-mediated release of the dye/drug falls in the concentration range 1.0-5.0 µM at an IONCs dose as low as 0.5 gFe/L and at no macroscopical temperature change. This local release effect makes this magnetic nanoplatform a potential tool for drug delivery with remote magnetic hyperthermia actuation and with a dose-independent action of MNPs.

A deep learning approach using synthetic images for segmenting and estimating 3D orientation of nanoparticles in EM images.

BACKGROUND AND OBJECTIVE: Nanoparticles present properties that can be applied to a wide range of fields such as biomedicine, electronics or optics. The type of properties depends on several characteristics, being some of them related with the particle structure. A proper characterization of nanoparticles is crucial since it could affect their applications. To characterize a particle shape and size, the nanotechnologists employ Electron Microscopy (EM) to obtain images of nanoparticles and perform measures over them. This task could be tedious, repetitive and slow, we present a Deep Learning method based on Convolutional Neural Networks (CNNs) to detect, segment, infer orientations and reconstruct microscope images of nanoparticles. Since machine learning algorithms depend on annotated data and there is a lack of annotated datasets of nanoparticles, our work makes use of artificial datasets of images resembling real nanoparticles photographs. METHODS: Our work is divided into three tasks. Firstly, a method to create annotated datasets of artificial images resembling Scanning Electron Microscope (SEM). Secondly, two models of convolutional neural networks are trained using the artificial datasets previously generated, the first one is in charge of the detection and segmentation of the nanoparticles while the second one will infer the nanoparticle orientation. Finally, the 3D reconstruction module will recreate in a 3D scene the set of detected particles. RESULTS: We have tested our method with five different shapes of basic nanoparticles: spheres, cubes, ellipsoids, hexagonal discs and octahedrons. An analysis of the reconstructions was conducted by manually comparing each of them with the real images. The results obtained have been promising, the particles are segmented and reconstructed accordingly to their shapes and orientations. CONCLUSIONS: We have developed a method for nanoparticle detection and segmentation in microscope images. Moreover, we can also infer an approximation of the 3D orientation of the particles and, in conjunction with the detections, create a 3D reconstruction of the photographs. The novelty of our approximation lies in the dataset used. Instead of using annotated images, we have created the datasets simulating the microscope images by using basic geometrical objects that imitate real nanoparticles.

Elucidating the Innate Immunological Effects of Mild Magnetic Hyperthermia on U87 Human Glioblastoma Cells: An In Vitro Study.

Cancer immunotherapies have been approved as standard second-line or in some cases even as first-line treatment for a wide range of cancers. However, immunotherapy has not shown clinically relevant success in glioblastoma (GBM). This is principally due to the brain's "immune-privileged" status and the peculiar tumor microenvironment (TME) of GBM characterized by a lack of tumor-infiltrating lymphocytes and the establishment of immunosuppressive mechanisms. Herein, we explore a local mild thermal treatment, generated via cubic-shaped iron oxide magnetic nanoparticles (size ~17 nm) when exposed to an external alternating magnetic field (AMF), to induce immunogenic cell death (ICD) in U87 glioblastoma cells. In accordance with what has been observed with other tumor types, we found that mild magnetic hyperthermia (MHT) modulates the immunological profile of U87 glioblastoma cells by inducing stress-associated signals leading to enhanced phagocytosis and killing of U87 cells by macrophages. At the same time, we demonstrated that mild magnetic hyperthermia on U87 cells has a modulatory effect on the expression of inhibitory and activating NK cell ligands. Interestingly, this alteration in the expression of NK ligands in U87 cells upon MHT treatment increased their susceptibility to NK cell killing and enhanced NK cell functionality. The overall findings demonstrate that mild MHT stimulates ICD and sensitizes GBM cells to NK-mediated killing by inducing the upregulation of specific stress ligands, providing a novel immunotherapeutic approach for GBM treatment, with potential to synergize with existing NK cell-based therapies thus improving their therapeutic outcomes.

Di- and tri-component spinel ferrite nanocubes: synthesis and their comparative characterization for theranostic applications.

Spinel ferrite nanocubes (NCs), consisting of pure iron oxide or mixed ferrites, are largely acknowledged for their outstanding performance in magnetic hyperthermia treatment (MHT) or magnetic resonance imaging (MRI) applications while their magnetic particle imaging (MPI) properties, particularly for this peculiar shape different from the conventional spherical nanoparticles (NPs), are relatively less investigated. In this work, we report on a non-hydrolytic synthesis approach to prepare mixed transition metal ferrite NCs. A series of NCs of mixed zinc-cobalt-ferrite were prepared and their magnetic theranostic properties were compared to those of cobalt ferrite or zinc ferrite NCs of similar sizes. For each of the nanomaterials, the synthesis parameters were adjusted to obtain NCs in the size range from 8 up to 15 nm. The chemical and structural nature of the different NCs was correlated to their magnetic properties. In particular, to evaluate magnetic losses, we compared the data obtained from calorimetric measurements to the data measured by dynamic magnetic hysteresis obtained under alternating magnetic field (AMF) excitation. Cobalt-ferrite and zinc-cobalt ferrite NCs showed high specific adsorption rate (SAR) values in aqueous solutions but their heating ability was drastically suppressed once in viscous media even for NCs as small as 12 nm. On the other hand, non-stoichiometric zinc-ferrite NCs showed significant but lower SAR values than the other ferrites, but these zinc-ferrite NCs preserved almost unaltered their heating trend in viscous environments. Also, the presence of zinc in the crystal lattice of zinc-cobalt ferrite NCs showed increased contrast enhancement for MRI with the highest T2 relaxation time and in the MPI signal with the best point spread function and signal-to-noise ratio in comparison to the analogue cobalt-ferrite NC. Among the different compositions investigated, non-stoichiometric zinc-ferrite NCs can be considered the most promising material as a multifunctional theranostic platform for MHT, MPI and MRI regardless of the media viscosity in which they will be applied, while ensuring the best biocompatibility with respect to the cobalt ferrite NCs.

Revealing a masked Verwey transition in nanoparticles of coexisting Fe-oxide phases.

The attractive electronic and magnetic properties together with their biocompatibility make iron-oxide nanoparticles appear as functional materials. In Fe-oxide nanoparticle (IONP) ensembles, it is crucial to enhance their performance thanks to controlled size, shape, and stoichiometry ensembles. In light of this, we conduct a comprehensive investigation in an ensemble of ca. 28 nm cuboid-shaped IONPs in which all the analyses concur with the coexistence of magnetite/maghemite phases in their cores. Here, we are disclosing the Verwey transition by temperature dependent (4-210 K) Raman spectroscopy.

Design strategies for shape-controlled magnetic iron oxide nanoparticles.

Ferrimagnetic iron oxide nanoparticles (magnetite or maghemite) have been the subject of an intense research, not only for fundamental research but also for their potentiality in a widespread number of practical applications. Most of these studies were focused on nanoparticles with spherical morphology but recently there is an emerging interest on anisometric nanoparticles. This review is focused on the synthesis routes for the production of uniform anisometric magnetite/maghemite nanoparticles with different morphologies like cubes, rods, disks, flowers and many others, such as hollow spheres, worms, stars or tetrapods. We critically analyzed those procedures, detected the key parameters governing the production of these nanoparticles with particular emphasis in the role of the ligands in the final nanoparticle morphology. The main structural and magnetic features as well as the nanotoxicity as a function of the nanoparticle morphology are also described. Finally, the impact of each morphology on the different biomedical applications (hyperthermia, magnetic resonance imaging and drug delivery) are analysed in detail. We would like to dedicate this work to Professor Carlos J. Serna, Instituto de Ciencia de Materiales de Madrid, ICMM/CSIC, for his outstanding contribution in the field of monodispersed colloids and iron oxide nanoparticles. We would like to express our gratitude for all these years of support and inspiration on the occasion of his retirement.

Understanding the Influence of a Bifunctional Polyethylene Glycol Derivative in Protein Corona Formation around Iron Oxide Nanoparticles.

Superparamagnetic iron oxide nanoparticles are one of the most prominent agents used in theranostic applications, with MRI imaging the main application assessed. The biomolecular interface formed on the surface of a nanoparticle in a biological medium determines its behaviour in vitro and in vivo. In this study, we have compared the formation of the protein corona on highly monodisperse iron oxide nanoparticles with two different coatings, dimercaptosuccinic acid (DMSA), and after conjugation, with a bifunctional polyethylene glycol (PEG)-derived molecule (2000 Da) in the presence of Wistar rat plasma. The protein fingerprints around the nanoparticles were analysed in an extensive proteomic study. The results presented in this work indicate that the composition of the protein corona is very difficult to predict. Proteins from different functional categories-cell components, lipoproteins, complement, coagulation, immunoglobulins, enzymes and transport proteins-were identified in all samples with very small variability. Although both types of nanoparticles have similar amounts of bonded proteins, very slight differences in the composition of the corona might explain the variation observed in the uptake and biotransformation of these nanoparticles in Caco-2 and RAW 264.7 cells. Cytotoxicity was also studied using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Controlling nanoparticles' reactivity to the biological environment by deciding on its surface functionalization may suggest new routes in the control of the biodistribution, biodegradation and clearance of multifunctional nanomedicines.

Confining Iron Oxide Nanocubes inside Submicrometric Cavities as a Key Strategy To Preserve Magnetic Heat Losses in an Intracellular Environment.

The design of magnetic nanostructures whose magnetic heating efficiency remains unaffected at the tumor site is a fundamental requirement to further advance magnetic hyperthermia in the clinic. This work demonstrates that the confinement of magnetic nanoparticles (NPs) into a sub-micrometer cavity is a key strategy to enable a certain degree of nanoparticle motion and minimize aggregation effects, consequently preserving the magnetic heat loss of iron oxide nanocubes (IONCs) under different conditions, including intracellular environments. We fabricated magnetic layer-by-layer (LbL) self-assembled polyelectrolyte sub-micrometer capsules using three different approaches, and we studied their heating efficiency as obtained in aqueous dispersions and after internalization by tumor cells. First, IONCs were added to the hollow cavities of LbL submicrocapsules, allowing the IONCs to move to a certain extent in the capsule cavities. Second, IONCs were coencapsulated into solid calcium carbonate cores coated with LbL polymer shells. Third, IONCs were incorporated within the polymer layers of the LbL capsule walls. In aqueous solution, higher specific absorption rate (SAR) values were related to those of free IONCs, while lower SAR values were recorded for capsule/core assemblies. However, after uptake by cancer cell lines (SKOV-3 cells), the SAR values of the free IONCs were significantly lower than those observed for capsule/core assemblies, especially after prolonged incubation periods (24 and 48 h). These results show that IONCs packed into submicrocavities preserve the magnetic losses, as the SAR values remained almost invariable. Conversely, free IONCs without the protective capsule shell agglomerated and their magnetic losses were strongly reduced. Indeed, IONC-loaded capsules and free IONCs reside inside endosomal and lysosomal compartments after cellular uptake and show strongly reduced magnetic losses due to the immobilization and aggregation in centrosymmetrical structures in the intracellular vesicles. The confinement of IONCs into sub-micrometer cavities is a key strategy to provide a sustained and predictable heating dose inside biological matrices.

Formation Mechanism of Maghemite Nanoflowers Synthesized by a Polyol-Mediated Process.

Magnetic nanoparticles are being developed as structural and functional materials for use in diverse areas, including biomedical applications. Here, we report the synthesis of maghemite (γ-Fe2O3) nanoparticles with distinct morphologies: single-core and multicore, including hollow spheres and nanoflowers, prepared by the polyol process. We have used sodium acetate to control the nucleation and assembly process to obtain the different particle morphologies. Moreover, from samples obtained at different time steps during the synthesis, we have elucidated the formation mechanism of the nanoflowers: the initial phases of the reaction present a lepidocrocite (γ-FeOOH) structure, which suffers a fast dehydroxylation, transforming to an intermediate "undescribed" phase, possibly a partly dehydroxylated lepidocrocite, which after some incubation time evolves to maghemite nanoflowers. Once the nanoflowers have been formed, a crystallization process takes place, where the γ-Fe2O3 crystallites within the nanoflowers grow in size (from ∼11 to 23 nm), but the particle size of the flower remains essentially unchanged (∼60 nm). Samples with different morphologies were coated with citric acid and their heating capacity in an alternating magnetic field was evaluated. We observe that nanoflowers with large cores (23 nm, controlled by annealing) densely packed (tuned by low NaAc concentration) offer 5 times enhanced heating capacity compared to that of the nanoflowers with smaller core sizes (15 nm), 4 times enhanced heating effect compared to that of the hollow spheres, and 1.5 times enhanced heating effect compared to that of single-core nanoparticles (36 nm) used in this work.

Time-course assessment of the aggregation and metabolization of magnetic nanoparticles.

To successfully develop biomedical applications for magnetic nanoparticles, it is imperative that these nanoreagents maintain their magnetic properties in vivo and that their by-products are safely metabolized. When placed in biological milieu or internalized into cells, nanoparticle aggregation degree can increase which could affect magnetic properties and metabolization. To evaluate these aggregation effects, we synthesized citric acid-coated iron oxide nanoparticles whose magnetic susceptibility can be modified by aggregation in agar dilutions and dextran-layered counterparts that maintain their magnetic properties unchanged. Macrophage models were used for in vitro uptake and metabolization studies, as these cells control iron homeostasis in the organism. Electron microscopy and magnetic susceptibility studies revealed a cellular mechanism of nanoparticle degradation, in which a small fraction of the particles is rapidly degraded while the remaining ones maintain their size. Both nanoparticle types produced similar iron metabolic profiles but these profiles differed in each macrophage model. Thus, nanoparticles induced iron responses that depended on macrophage programming. In vivo studies showed that nanoparticles susceptible to changes in magnetic properties through aggregation effects had different behavior in lungs, liver and spleen. Liver ferritin levels increased in these animals showing that nanoparticles are degraded and their by-products incorporated into normal metabolic routes. These data show that nanoparticle iron metabolization depends on cell type and highlight the necessity to assess nanoparticle aggregation in complex biological systems to develop effective in vivo biomedical applications. STATEMENT OF SIGNIFICANCE: Magnetic iron oxide nanoparticles have great potential for biomedical applications. It is however imperative that these nanoreagents preserve their magnetic properties once inoculated, and that their degradation products can be eliminated. When placed in a biological milieu nanoparticles can aggregate and this can affect their magnetic properties and their degradation. In this work, we showed that iron oxide nanoparticles trigger the iron metabolism in macrophages, the main cell type involved in iron homeostasis in the organism. We also show that aggregation can affect nanoparticle magnetic properties when inoculated in animal models. This work confirms iron oxide nanoparticle biocompatibility and highlights the necessity to assess in vivo nanoparticle aggregation to successfully develop biomedical applications.