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The directed migration of cellular clusters enables morphogenesis, wound healing and collective cancer invasion. Gradients of substrate stiffness direct the migration of cellular clusters in a process called collective durotaxis, but the underlying mechanisms remain unclear. Here we unveil a connection between collective durotaxis and the wetting properties of cellular clusters. We show that clusters of cancer cells dewet soft substrates and wet stiff ones. At intermediate stiffness—at the crossover from low to high wettability—clusters on uniform-stiffness substrates become maximally motile, and clusters on stiffness gradients exhibit optimal durotaxis. Durotactic velocity increases with cluster size, stiffness gradient and actomyosin activity. We demonstrate this behaviour on substrates coated with the cell–cell adhesion protein E-cadherin and then establish its generality on substrates coated with extracellular matrix. We develop an active wetting model that explains collective durotaxis in terms of a balance between in-plane active traction and tissue contractility and out-of-plane surface tension. Finally, we show that the distribution of cluster displacements has a heavy tail, with infrequent but large cellular hops that contribute to durotactic migration. Our study demonstrates a physical mechanism of collective durotaxis, through both cell–cell and cell–substrate adhesion ligands, based on the wetting properties of active droplets.

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.

Nanometric magnetic iron oxide nanoparticles are very useful for biotechnological applications. We are interested in their use for the detection and control of the pathogenic fungus Fusarium oxysporum. Many F. oxysporum formae especiales are a major concern for agriculture and food industry, as they can infect a number of major crops (cereals, potato, etc.) and produce mycotoxins that may give rise to allergic symptoms or be carcinogenic in long-term consumption. These soil borne pathogens can survive as thick-walled chlamydospores, which remain viable in the soil for many years and hence control is problematic. In addition, several ff. spp. are non-pathogenic and even beneficial for agriculture exerting a bio-control effect on other pathogenic strains. Therefore it is crucial to provide control methods effective and directed to the pathogenic ff. spp. We present the synthesis and characterization of superparamagnetic magnetite nanoparticles as well as a functionalization strategy, necessary for the study of the nanoparticles internalization by the Fusarium spp. Superparamagnetic Fe3O4 nanoparticles coated with an ultra-thin (~1 nm) MgO layer (total size ¿ 25 nm and hydrodynamic diameter ¿ 30 nm) have been synthesized combining co-precipitation and sol-gel methods. A thorough chemical and structural characterization has been carried out by HRTEM, XRD, EDS, DLS and TGA. Aberration corrected HRTEM experiments with sub-angstrom spatial resolution have allowed us to distinguish the ultra-thin MgO shell that grows epitaxially on the magnetic cores. The MgO shell protects the magnetic nuclei from oxidation, preserving the superparamagnetism even after calcination at high temperatures (¿ 600ºC)1. To improve their water stability and to obtain a suitable surface for biochemical functionalization, our synthesized particles have been encapsulated in an aminated silica coating (also through a sol-gel process). After that the particles were bio-functionalized with G protein/ant

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.

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.

Oriented and covalent immobilization of proteins on magnetic nanoparticles (MNPs) is particularly challenging as it requires both the functionality of the protein and the colloidal stability of the MNPs to be preserved. Here, we describe a simple, straightforward, and efficient strategy for MNP functionalization with proteins using metal affinity binding. Our method involves a single-step process where MNPs are functionalized using a preformed, ready-to-use nitrilotriacetic acid-divalent metal cation (NTA-M2+) complex and polyethylene glycol (PEG) molecules. As a proof-of-concept, we demonstrate the oriented immobilization of a recombinant cadherin fragment engineered with a hexahistidine tag (6His-tag) onto the MNPs. Our developed methodology is simple and direct, enabling the oriented bioconjugation of His-tagged cadherins to MNPs while preserving protein functionality and the colloidal stability of the MNPs, and could be extended to other proteins expressing a polyhistidine tag. When compared to the traditional method where NTA is first conjugated to the MNPs and afterward free metal ions are added to form the complex, this novel strategy results in a higher functionalization efficiency while avoiding MNP aggregation. Additionally, our method allows for covalent bonding of the cadherin fragments to the MNP surface while preserving functionality, making it highly versatile. Finally, our strategy not only ensures the correct orientation of the protein fragments on the MNPs but also allows for the precise control of their density. This feature enables the selective targeting of E-cadherin-expressing cells only when MNPs are decorated with a high density of cadherin fragments.

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 an in vitro method for detecting at least one target poly- nucleotide in a test sample, wherein the method comprises the steps of contacting the test sample with a first oligonucleotide and a second oligonucleotide and detecting the hybridization between the first and the second oligonucleotides to the at least one target polynucleotide. The present invention also relates to devices or systems to implement the method, and kits of parts comprising said oligonucleotides., [FR] La présente invention concerne un procédé in vitro pour détecter au moins un poly-nucléotide cible dans un échantillon de test, le procédé comprenant les étapes consistant à mettre en contact l'échantillon de test avec un premier oligonucléotide et un second oligonucléotide et à détecter l'hybridation entre le premier et le second oligonucléotides avec le au moins un polynucléotide cible. La présente invention concerne également des dispositifs ou systèmes permettant de mettre en œuvre le procédé, et des kits d'éléments comprenant lesdits oligonucléotides.

[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.

Figure S1, AC magnetic susceptibility characterization of different organs (tumor, liver, skin, and blood); Figure S2, scaled out-of-phase magnetic susceptibility of several tissues and nanoparticle standards scaled to their maximum to compare the location in temperature of the maxima.

Instrumentation, functionalization and characterization of MNPs, MNP stability assays, characterization of the click reactivity of the MNPs in suspension and on surfaces, metabolic glycoengineering, and MNP–cell interactions.

In this work, we report the use of bioorthogonal chemistry, specifically the strain-promoted click azide–alkyne cycloaddition (SPAAC) for the covalent attachment of magnetic nanoparticles (MNPs) on living cell membranes. Four types of MNPs were prepared, functionalized with two different stabilizing/passivation agents (a polyethylene glycol derivative and a glucopyranoside derivative, respectively) and two types of strained alkynes with different reactivities: a cyclooctyne (CO) derivative and a dibenzocyclooctyne (DBCO) derivative. The MNPs were extensively characterized in terms of physicochemical characteristics, colloidal stability, and click reactivity in suspension. Then, the reactivity of the MNPs toward azide-modified surfaces was evaluated as a closer approach to their final application in a living cell scenario. Finally, the DBCO-modified MNPs, showing superior reactivity in suspension and on surfaces, were selected for cell membrane immobilization via the SPAAC reaction on the membranes of cells engineered to express azide artificial reporters. Overall, our work provides useful insights into the appropriate surface engineering of nanoparticles to ensure a high performance in terms of bioorthogonal reactivity for biological applications.

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

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.

The simultaneous detection and quantification of several iron-containing species in biological matrices is a challenging issue. Especially in the frame of studies using magnetic nanoparticles for biomedical applications, no gold-standard technique has been described yet and combinations of different techniques are generally used. In this work, AC magnetic susceptibility measurements are used to analyze different organs from an animal model that received a single intratumor administration of magnetic nanoparticles. The protocol used for the quantification of iron associated with the magnetic nanoparticles is carefully described, including the description of the preparation of several calibration standard samples of nanoparticle suspensions with different degrees of dipolar interactions. The details for the quantitative analysis of other endogenous iron-containing species such as ferritin or hemoglobin are also described. Among the advantages of this technique are that tissue sample preparation is minimal and that large amounts of tissue can be characterized each time (up to hundreds of milligrams). In addition, the very high specificity of the magnetic measurements allows for tracking of the nanoparticle transformations. Furthermore, the high sensitivity of the instrumentation results in very low limits of detection for some of the iron-containing species. Therefore, the presented technique is an extremely valuable tool to track iron oxide magnetic nanoparticles in samples of biological origin.