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Ferroptosis, a form of iron-dependent, lipid peroxidation-driven cell death, has been extensively investigated in recent years, and several studies have suggested that the ferroptosis-inducing properties of iron-containing nanomaterials could be harnessed for cancer treatment. Here we evaluated the potential cytotoxicity of iron oxide nanoparticles, with and without cobalt functionalization (Fe2O3 and Fe2O3@Co-PEG), using an established, ferroptosis-sensitive fibrosarcoma cell line (HT1080) and a normal fibroblast cell line (BJ). In addition, we evaluated poly (ethylene glycol) (PEG)-poly(lactic-co-glycolic acid) (PLGA)-coated iron oxide nanoparticles (Fe3O4-PEG-PLGA). Our results showed that all the nanoparticles tested were essentially non-cytotoxic at concentrations up to 100 μg/mL. However, when the cells were exposed to higher concentrations (200–400 μg/mL), cell death with features of ferroptosis was observed, and this was more pronounced for the Co-functionalized nanoparticles. Furthermore, evidence was provided that the cell death triggered by the nanoparticles was autophagy-dependent. Taken together, the exposure to high concentrations of polymer-coated iron oxide nanoparticles triggers ferroptosis in susceptible human cancer cells.

Enzymes are selective, specific, efficient, and more sustainable catalysts than their chemical counterparts. From an industrial perspective, multienzymatic systems to produce chemical compounds are particularly attractive since they comply with most green production principles including the minimization of wastes and their ecological footprints. However, the number of industrial processes based on the use of enzymes as catalysts remains limited. Indeed, incompatibility issues of enzymes with respect to reaction conditions or cross-reactivity are still challenging aspects that make one-pot enzyme cascade reactions hard to perform. The urgent need for more sustainable industrial processes has made different research fields, not accustomed to working together, join efforts to develop innovative technologies that could be key enabling to overcome these challenges. Among these technologies are those that seek to synergistically integrate the specific recognition and unique catalytic properties of enzymes with the size-dependent unique properties of nanoparticles. However, rather than giving a complete overview of the use of nanomaterials in biocatalysts, here it will be discussed the feasibility of triggering magnetic nanoparticles (MNPs) into hotspots by remote nanoactuation for the thermal modulation of discrete enzyme molecules activity with minimal collateral heating., The possibility of heating discrete and defined local regions within a sample offers the potential to introduce a game-changing breakthrough in industrial biocatalysis by achieving coordinated enzyme function and thus concurrent multienzyme reactions. Therefore, not only the principles behind the activation of MNPs as hotspots, but also those examples that report the selective control of enzyme activity by magnetic heating, together with the foreseen challenges for the transfer of this technology to the industry, will be discussed.

Herein, we have developed nanohybrids (nHs) to remotely activate a therapeutic enzyme for its use in Directed Enzyme Prodrug Therapy (DEPT). The coencapsulation of magnetic nanoparticles (MNPs) with horseradish peroxidase (HRP) using biomimetic silica as an entrapment matrix was optimized to obtain nanosized hybrids (∼150 nm) for remote activation of the therapeutic enzyme. HRP converts indole-3-acetic acid (3IAA) into peroxylated radicals, whereas MNPs respond to alternating magnetic fields (AMFs) becoming local hotspots. The AMF application triggered an increase in the bioconversion rate of HRP matching the activity displayed at the optimal temperature of the nHs (Topt = 50 °C) without altering the temperature of the reaction media. This showed that enzyme nanoactuation is possible with MNPs even if they are not covalently bound. After an extensive physicochemical/magnetic characterization, the spatial location of each component of the nH was deciphered, and an insulating role of the silica matrix was suggested as critical for introducing remote control over HRP. In vitro assays, using a human pancreatic cancer cell line (MIA PaCa-2), showed that only upon exposure to AMF and in the presence of the prodrug, the enzyme-loaded nHs triggered cell death. Moreover, in vivo experiments showed higher reductions in the tumor volume growth in those animals treated with nHs in the presence of 3IAA when exposed to AMF. Thus, this work demonstrates the feasibility of developing a spatiotemporally controlled DEPT strategy to overcome unwanted off-target effects.

Despite the progress in the field of nanotoxicology, much about the cellular mechanisms that mediate the adverse effects of nanoparticles (NPs) and, in particular, the possible role of epigenetics in nanotoxicity, remains to be clarified. Therefore, we studied the changes occurring in the genome-wide distribution of H3K27ac, H3K4me1, H3K9me2, and H3K27me3 histone modifications and compared them with the transcriptome after exposing NIH3T3 cells to iron-based magnetic NPs (i.e., Fe2O3 and Fe2O3@Co NPs). We found that the transcription response is mainly due to changes in the genomic distribution of H3K27ac that can modulate the activity of enhancers. We propose that alteration of the epigenetic landscape is a key mechanism in defining the gene expression program changes resulting in nanotoxicity. With this approach, it is possible to construct a data set of genomic regions that could be useful for defining toxicity in a manner that is more comprehensive than what is possible with the present toxicology assays.

[Background]: Trauma-associated peripheral nerve injury is a widespread clinical problem causing sensory and motor disabilities. Schwann cells (SCs) contribute to nerve regeneration, mainly by secreting nerve growth factor (NGF) and brain-derived neurotrophic factor. In the last years, adipose-derived stem cells (ASCs) differentiated into SCs (SC-ASCs) were considered as promising cell therapy. However, the cell trans-differentiation process has not been effectively showed and presents several drawbacks, thus an alternative approach for increasing ASCs neurotrophic properties is highly demanded. In the context of human cell-based therapies, Good Manufacturing Practice directions indicate that FBS should be substituted with a xenogeneic-free supplement, such as Human Platelet Lysate (HPL). Previously, we demonstrated that neurotrophic properties of HPL-cultured ASCs were superior compared to undifferentiated FBS-cultured ASCs. Therefore, as following step, here we compared the neurotrophic properties of differentiated SC-like ASCs and HPL-cultured ASCs., [Methods]: Both cell groups were investigated for gene expression level of neurotrophic factors, their receptors and neuronal markers. Moreover, the expression of nestin was quantitatively evaluated by flow cytometry. The commitment toward the SC phenotype was assessed with immunofluorescence pictures. Proteomics analysis was performed on both cells and their conditioned media to compare the differential protein profile. Finally, neurotrophic abilities of both groups were evaluated with a functional co-culture assay, assessing dorsal root ganglia survival and neurite outgrowth., [Results]: HPL-cultured ASCs demonstrated higher gene expression of NGF and lower expression of S100B. Moreover, nestin was present in almost all HPL-cultured ASCs and only in one quarter of SC-ASCs. Immunofluorescence confirmed that S100B was not present in HPL-cultured ASCs. Proteomics analysis validated the higher expression of nestin and the increase in cytoskeletal and ECM proteins involved in neural regeneration processes. The co-culture assay highlighted that neurite outgrowth was higher in the presence of HPL-ASCs or their conditioned medium compared to SC-ASCs., [Conclusions]: All together, our results show that HPL-ASCs were more neurotrophic than SC-ASCs. We highlighted that the HPL triggers an immature neuro-induction state of ASCs, while keeping their stem properties, paving the way for innovative therapies for nerve regeneration.

Magnetic nanoparticles (MNPs) are widely used for immobilizing complex biomolecules such as proteins due to their high loading capacity, biocompatibility, and easy magnetic harvesting. However, there is not a universal solution to achieve the oriented immobilization of the protein of interest. One common strategy involves the selective binding of poly-histidine-tagged (His-tag) proteins to divalent transition metal centers (TM2+) chelated by organic molecules like nitrilotriacetic acid (NTA). This article describes an innovative procedure to functionalize MNPs by simply growing of a thin layer of TM2+ hydroxides on the MNP surface, which increases the number of coordination centers and enhances the surface interaction with the protein surface. The immobilization yields of two sets of MNPs coated with Co2+, Ni2+ and Cu2+ hydroxides were compared to the conventional coating using the same inorganic cores but coated and functionalized with NTA-TM2+ moieties. It is shown that the one-step Co coated MNPs showed the highest selectivity and specificity towards His-tags. Thus, this coating not only reduce the functionalization steps to drive an oriented protein binding but also achieve an increment of immobilization yields over 1.5-fold respect to NTA-Co2+ analogous for his-tagged proteins with different 3D complexity while preserving similar or even higher enzymatic activity.

The present invention relates to a process to carry out two or more enzymatic reactions in a reaction medium, wherein the process comprises providing a system comprising at least two substantially homogeneous and colloidal populations of magnetic nanoparticles (MNPs) and applying one or more external alternating magnetic field to produce the simultaneous or sequential activation of the enzymes functionalized on the surface of each population of MNPs so that the enzymatic reactions can be thermally activated.

[EN] The present invention relates to a process to carry out two or more enzymatic reactions in a reaction medium, wherein the process comprises providing a system comprising at least two substantially homogeneous and colloidal populations of magnetic nanoparticles (MNPs) and applying one or more external alternating magnetic field to produce the simultaneous or sequential activation of the enzymes functionalized on the surface of each population of MNPs so that the enzymatic reactions can be thermally activated., [FR] La présente invention concerne un procédé pour effectuer deux ou plusieurs réactions enzymatiques dans un milieu réactionnel, le procédé comprenant la fourniture d'un système comprenant au moins deux populations sensiblement homogènes et colloïdales de nanoparticules magnétiques (MNPs) et l'application d'un ou plusieurs champs magnétiques alternatifs externes pour produire l'activation simultanée ou séquentielle des enzymes fonctionnalisées sur la surface de chaque population de MNPs de telle sorte que les réactions enzymatiques peuvent être activées thermiquement.

Nanomedicine has been considered a promising tool for biomedical research and clinical practice in the 21st century because of the great impact nanomaterials could have on human health. The generation of new smart nanomaterials, which enable time- and space-controlled drug delivery, improve the limitations of conventional treatments, such as non-specific targeting, poor biodistribution and permeability. These smart nanomaterials can respond to internal biological stimuli (pH, enzyme expression and redox potential) and/or external stimuli (such as temperature, ultrasound, magnetic field and light) to further the precision of therapies. To this end, photonic and magnetic nanoparticles, such as gold, silver and iron oxide, have been used to increase sensitivity and responsiveness to external stimuli. In this review, we aim to report the main and most recent systems that involve photonic or magnetic nanomaterials for external stimulus-responsive drug release. The uniqueness of this review lies in highlighting the versatility of integrating these materials within different carriers. This leads to enhanced performance in terms of in vitro and in vivo efficacy, stability and toxicity. We also point out the current regulatory challenges for the translation of these systems from the bench to the bedside, as well as the yet unresolved matter regarding the standardization of these materials.

It is well known that magnetic nanoparticles (MNPs) absorb magnetic energy when exposed to an alternating magnetic field (AMF) and therefore dissipate heat in their local environment. So far, the heat dissipated by the MNPs has been widely used in biomedicine for cancer treatment and drug delivery. The FET-OPEN project HOTZYMES proposes instead to exploit this property for the biotechnological production of pharmaceuticals and bio-commodities, by conjugating thermophilic enzymes on the MNPs surface in order to selectively tune their activity by triggering this highly localized magnetic heating while maintaining the reaction media at low temperature., To this aim superparamagnetic iron oxide nanoparticles coated with different organic shells (dimercaptusuccinic acid, DMSA; polyacrylic acid, PAA) have been selected due to their good colloidal stability, high density of functionalizable chemical groups (-COOH) and a good combination of different heating efficiencies. They have been further functionalized with NTA-Cu2+, a chelating agent commonly used in protein purification by metal affinity interaction. Herein, we demonstrate that we can selectively bind a His-tag variant of the superfolded GFP protein, thanks to the high affinity of the immobilized copper to the 6xhistidine tag genetically fused to the protein. Furthermore, using the same strategy, we were able to bind the tetrameric recombinant His-tag variant of the B. stearothermophilus alcohol dehydrogenase (ADH) with also high specificity. Additionally, we demonstrate that the application of an AMF to the selected protein@MNPs derivatives can lead to the generation of different temperature gradients on the MNPs surface by studying both the GFP fluorescence (that strongly depends on temperature), and the effect of the triggered local heating on ADH activity. These first results suggest the feasibility of tuning remotely protein properties by combining their coupling to MNPs with the application of alternating magnetic fields.

The histidine tag (His-tag) is one of the most used affinity-tag for protein purification due to its small size and versatility. Agarose and sepharose beads containing nitriloacetic acid (NTA) transition metal derivatives are widely used for the purification of His-tagged proteins, thanks to their high affinity to the His-tag genetically fused to the protein of interest [1]. The same chemistry can be used to conjugate enzymes to magnetic nanoparticles (MNPs) with the aim of tuning their activity by magnetic heating [2, 3]. Within the frame of the FET-OPEN project HOTZYMES (https://www.hotzymes.eu), different MNPs have been synthetized and coated with polyacrylic acid and dimercaptosuccinic acid, and then were further functionalized with NTA-Cu2+ as His-tag chelating agent. Different proteins were expressed as His-tag variants and immobilized on the MNPs, including monomeric (superfolded GFP), dimeric (C. violaceum transaminase, CvTA; C. uda cellobiose phosphorylase, CuCbP), and tetrameric (B. stearothermophilus alcohol dehydrogenase, ADH) variants. While for the monomeric protein selected as model no difficulties in the bioconjugation processes were observed, when using dimeric or tetrameric enzymes the aggregation of the MNPs occurs very easily due to crosslinking between the nanoparticles. This colloidal destabilization of the MNPs is favored due to its high surface area and the presence of several tags per enzyme molecule. To avoid this situation, different strategies have been developed: saturation of the binding site of the nanoparticles, presence of a small percentage of imidazole in reaction, changes in the incubation conditions (pH, ionic strength, …). First positive results confirm that by playing with different factors it is possible to conjugate different His-tagged enzymes to very different MNPs in terms of size, shape, surface area, and colloidal stability. Actually, it is possible to avoid protein and MNPs aggregation while obtaining good activity yiel