From Bench to Cell: A Roadmap for Assessing the Bioorthogonal "Click" Reactivity of Magnetic Nanoparticles for Cell Surface Engineering.

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.

Selective Magnetic Nanoheating: Combining Iron Oxide Nanoparticles for Multi-Hot-Spot Induction and Sequential Regulation.

The contactless heating capacity of magnetic nanoparticles (MNPs) has been exploited in fields such as hyperthermia cancer therapy, catalysis, and enzymatic thermal regulation. Herein, we propose an advanced technology to generate multiple local temperatures in a single-pot reactor by exploiting the unique nanoheating features of iron oxide MNPs exposed to alternating magnetic fields (AMFs). The heating power of the MNPs depends on their magnetic features but also on the intensity and frequency conditions of the AMF. Using a mixture of diluted colloids of MNPs we were able to generate a multi-hot-spot reactor in which each population of MNPs can be selectively activated by adjusting the AMF conditions. The maximum temperature reached at the surface of each MNP was registered using independent fluorescent thermometers that mimic the molecular link between enzymes and MNPs. This technology paves the path for the implementation of a selective regulation of multienzymatic reactions.

Dysprosium and Holmium Vanadate Nanoprobes as High-Performance Contrast Agents for High-Field Magnetic Resonance and Computed Tomography Imaging.

We describe a wet chemical method for the synthesis of uniform and well-dispersed dysprosium vanadate (DyVO4) and holmium vanadate (HoVO4) nanoparticles with an almost spherical shape and a mean size of ∼60 nm and their functionalization with poly(acrylic acid). The transverse magnetic relaxivity of both systems at 9.4 T is analyzed on the basis of magnetic susceptibility and magnetization measurements in order to evaluate their potential for application as high-field MRI contrast agents. In addition, the X-ray attenuation properties of these systems are also studied to determine their capabilities as computed tomography contrast agent. Finally, the colloidal stability under physiological pH conditions and the cytotoxicity of the functionalized NPs are also addressed to assess their suitability for bioimaging applications.

Au-siRNA@ aptamer nanocages as a high-efficiency drug and gene delivery system for targeted lung cancer therapy.

BACKGROUND: Gene and chemical therapy has become one of the rising stars in the field of molecular medicine during the last two decades. However, there are still numerous challenges in the development of efficient, targeted, and safe delivery systems that can avoid siRNA degradation and reduce the toxicity and adverse effects of chemotherapy medicine. RESULTS: In this paper, a highly efficient AS1411 aptamer modified, dsDNA and MMP-2 cleavable peptide-fabricated gold nanocage vehicle, which could load doxorubicin hydrochloride (DOX) and siRNAs to achieve a combination of tumor responsive genetic therapy, chemotherapy, and photothermal treatment is presented. Our results show that this combined treatment achieved targeted gene silencing and tumor inhibition. After nearly one month of treatment with DOX-loaded Au-siRNA-PAA-AS1411 nanoparticles with one dose every three days in mice, a synergistic effect promoting the eradication of long-lived tumors was observed along with an increased survival rate of mice. The combined genetic, chemotherapeutic, and photothermal treatment group exhibited more than 90% tumor inhibition ratio (tumor signal) and a ~ 67% survival rate compared with a 30% tumor inhibition ratio and a 0% survival rate in the passive genetic treatment group. CONCLUSIONS: The development of nanocarriers with double-stranded DNA and MMP-2 cleavable peptides provides a new strategy for the combined delivery of gene and chemotherapy medicine. Au-siRNA-PAA-AS1411 exerts high anticancer activities on lung cancer, indicating immense potentials for clinical application.

On-POM Ring-Opening Polymerisation of N-Carboxyanhydrides.

The ring-opening polymerisation of α-amino acid N-carboxyanhydrides (NCAs) offers a simple and scalable route to polypeptides with predicted and narrow molecular weight distributions. Here we show how polyoxometalates (POMs)-redox-active molecular metal-oxide anions-can serve as inorganic scaffold initiators for such NCA polymerisations. This "On-POM polymerisation" strategy serves as an innovative platform to design hybrid materials with additive or synergistic properties stemming from the inorganic and polypeptide component parts. We have used this synthetic approach to synthesise a library of bactericidal poly(lysine)-POM hybrid derivatives that can be used to prevent biofilm formation. This versatile "On-POM polymerisation" method provides a flexible synthetic approach for combining inorganic scaffolds with amino acids, and the potential to tailor and improve the specificity and performance of hybrid antimicrobial materials.

Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally.

Aggregation effects on the magnetic properties of iron oxide colloids.

Magnetic nanoparticles (MNPs), and in particular iron oxide nanoparticles (mainly magnetite and maghemite), are being widely used in the form of aqueous colloids for biomedical applications. In such colloids, nanoparticles tend to form assemblies, either aggregates, if the union is permanent, or agglomerates, if it is reversible. These clustering processes have a strong impact on the MNPs' properties that are often not well understood. In this review, the causes and consequences of MNPs aggregation/agglomeration are reviewed and discussed. Special attention has been paid to the impact of the MNPs aggregation/agglomeration on their magnetic properties and heating properties, when exposed to an alternating magnetic field in the frame of magnetic hyperthermia. In addition, a model system with MNPs of two different sizes coated with three different molecules oleic acid, meso-2, 3-dimercaptosuccinic acid and poly(maleic anhydride-alt-1-octadecene) has been characterized and the results used to support the ideas reviewed.

A simple and universal enzyme-free approach for the detection of multiple microRNAs using a single nanostructured enhancer of surface plasmon resonance imaging.

Here we describe a simple approach for the simultaneous detection of multiple microRNAs (miRNAs) using a single nanostructured reagent as surface plasmon resonance imaging (SPRi) enhancer and without using enzymatic reactions, sequence specific enhancers or multiple enhancing steps as normally reported in similar studies. The strategy involves the preparation and optimisation of neutravidin-coated gold nanospheres (nGNSs) functionalised with a previously biotinylated antibody (Ab) against DNA/RNA hybrids. The Ab guarantees the recognition of any miRNA sequence adsorbed on a surface properly functionalised with different DNA probes; at the same time, gold nanoparticles permit to detect this interaction, thus producing enough SPRi signal even at a low ligand concentration. After a careful optimisation of the nanoenhancer and after its characterisation, the final assay allowed the simultaneous detection of four miRNAs with a limit of detection (LOD) of up to 0.5 pM (equal to 275 attomoles in 500 µL) by performing a single enhancing injection. The proposed strategy shows good signal specificity and permits to discriminate wild-type, single- and triple-mutated sequences much better than non-enhanced SPRi. Finally, the method works properly in complex samples (total RNA extracted from blood) as demonstrated by the detection of four miRNAs potentially related to multiple sclerosis used as case study. This proof-of-concept study confirms that the approach provides the possibility to detect a theoretically unlimited number of miRNAs using a simple protocol and an easily prepared enhancing reagent, and may further facilitate the development of affordable multiplexing miRNA screening for clinical purposes.

Tri-mannose grafting of chitosan nanocarriers remodels the macrophage response to bacterial infection.

BACKGROUND: Infectious diseases are still a leading cause of death and, with the emergence of drug resistance, pose a great threat to human health. New drugs and strategies are thus urgently needed to improve treatment efficacy and limit drug-associated side effects. Nanotechnology-based drug delivery systems are promising approaches, offering hope in the fight against drug resistant bacteria. However, how nanocarriers influence the response of innate immune cells to bacterial infection is mostly unknown. RESULTS: Here, we used Mycobacterium tuberculosis as a model of bacterial infection to examine the impact of mannose functionalization of chitosan nanocarriers (CS-NCs) on the human macrophage response. Both ungrafted and grafted CS-NCs were similarly internalized by macrophages, via an actin cytoskeleton-dependent process. Although tri-mannose ligands did not modify the capacity of CS-NCs to escape lysosomal degradation, they profoundly remodeled the response of M. tuberculosis-infected macrophages. mRNA sequencing showed nearly 900 genes to be differentially expressed due to tri-mannose grafting. Unexpectedly, the set of modulated genes was enriched for pathways involved in cell metabolism, particularly oxidative phosphorylation and sugar metabolism. CONCLUSIONS: The ability to modulate cell metabolism by grafting ligands at the surface of nanoparticles may thus be a promising strategy to reprogram immune cells and improve the efficacy of encapsulated drugs.

Natural Polysaccharides for siRNA Delivery: Nanocarriers Based on Chitosan, Hyaluronic Acid, and Their Derivatives.

Natural polysaccharides are frequently used in the design of drug delivery systems due to their biocompatibility, biodegradability, and low toxicity. Moreover, they are diverse in structure, size, and charge, and their chemical functional groups can be easily modified to match the needs of the final application and mode of administration. This review focuses on polysaccharidic nanocarriers based on chitosan and hyaluronic acid for small interfering RNA (siRNA) delivery, which are highly positively and negatively charged, respectively. The key properties, strengths, and drawbacks of each polysaccharide are discussed. In addition, their use as efficient nanodelivery systems for gene silencing applications is put into context using the most recent examples from the literature. The latest advances in this field illustrate effectively how chitosan and hyaluronic acid can be modified or associated with other molecules in order to overcome their limitations to produce optimized siRNA delivery systems with promising in vitro and in vivo results.

Nanoparticles engineered to bind cellular motors for efficient delivery.

BACKGROUND: Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. RESULTS: Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. CONCLUSIONS: The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Polyoxometalate-Ionic Liquids (POM-ILs) as Anticorrosion and Antibacterial Coatings for Natural Stones.

Corrosion of stone by acid rain and deterioration from biofilms are global problems for industrial and residential buildings as well as cultural heritage, such as statues or historic buildings. Herein we show how typical building stones can be protected from corrosion ("weathering") and biofilm formation ("biodeterioration") by application of thin films of polyoxometalate-based ionic liquids (POM-ILs). Stone samples are coated with hydrophobic, acid resistant POM-ILs featuring biocidal properties. Exposure of the samples to simulated acid rain showed negligible corrosion compared to the significant deterioration of unprotected samples; in addition the biocidal properties of the POM-ILs suppress the formation of biofilms on coated stone slabs. A new class of modular molecular materials for protecting stones can now be developed for use in construction, environmental protection, and cultural heritage preservation.

Reversible Monolayer-Bilayer Transition in Supported Phospholipid LB Films under the Presence of Water: Morphological and Nanomechanical Behavior.

Mixed monolayer Langmuir-Blodgett (LB) films of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) in the 1:1 ratio have been prepared onto solid mica substrates. Upon immersion in water or in an aqueous HEPES solution (pH 7.4) the monolayer LB films were spontaneously converted into well-organized bilayers leaving free mica areas. The process has been demonstrated to be reversible upon removal of the aqueous solution, resulting in remarkably free of defects monolayers that are homogeneously distributed onto the mica. In addition, the nanomechanical properties exhibited by the as-formed bilayers have been determined by means of AFM breakthrough force studies. The bilayers formed by immersion of the monolayer in an aqueous media exhibit nanomechanical properties and stability under compression analogous to those of DPPC:Chol supported bilayers obtained by other methods previously described in the literature. Consequently, the hydration of a monolayer LB film has been revealed as an easy method to produce well-ordered bilayers that mimic the cell membrane and that could be used as model cell membranes.

Multiparametric analysis of anti-proliferative and apoptotic effects of gold nanoprisms on mouse and human primary and transformed cells, biodistribution and toxicity in vivo.

BACKGROUND: The special physicochemical properties of gold nanoprisms make them very useful for biomedical applications including biosensing and cancer therapy. However, it is not clear how gold nanoprisms may affect cellular physiology including viability and other critical functions. We report a multiparametric investigation on the impact of gold-nanoprisms on mice and human, transformed and primary cells as well as tissue distribution and toxicity in vivo after parental injection. METHODS: Cellular uptake of the gold-nanoprisms (NPRs) and the most crucial parameters of cell fitness such as generation of reactive oxygen species (ROS), mitochondria membrane potential, cell morphology and apoptosis were systematically assayed in cells. Organ distribution and toxicity including inflammatory response were analysed in vivo in mice at 3 days or 4 months after parental administration. RESULTS: Internalized gold-nanoprisms have a significant impact in cell morphology, mitochondrial function and ROS production, which however do not affect the potential of cells to proliferate and form colonies. In vivo NPRs were only detected in spleen and liver at 3 days and 4 months after administration, which correlated with some changes in tissue architecture. However, the main serum biochemical markers of organ damage and inflammation (TNFα and IFNγ) remained unaltered even after 4 months. In addition, animals did not show any macroscopic sign of toxicity and remained healthy during all the study period. CONCLUSION: Our data indicate that these gold-nanoprisms are neither cytotoxic nor cytostatic in transformed and primary cells, and suggest that extensive parameters should be analysed in different cell types to draw useful conclusions on nanomaterials safety. Moreover, although there is a tendency for the NPRs to accumulate in liver and spleen, there is no observable negative impact on animal health.

Removal of Multiple Contaminants from Water by Polyoxometalate Supported Ionic Liquid Phases (POM-SILPs).

The simultaneous removal of organic, inorganic, and microbial contaminants from water by one material offers significant advantages when fast, facile, and robust water purification is required. Herein, we present a supported ionic liquid phase (SILP) composite where each component targets a specific type of water contaminant: a polyoxometalate-ionic liquid (POM-IL) is immobilized on porous silica, giving the heterogeneous SILP. The water-insoluble POM-IL is composed of antimicrobial alkylammonium cations and lacunary polyoxometalate anions with heavy-metal binding sites. The lipophilicity of the POM-IL enables adsorption of organic contaminants. The silica support can bind radionuclides. Using the POM-SILP in filtration columns enables one-step multi-contaminant water purification. The results show how multi-functional POM-SILPs can be designed for advanced purification applications.

Ligand-Free Synthesis of Tunable Size Ln:BaGdF5 (Ln = Eu³âº and Nd³âº) Nanoparticles: Luminescence, Magnetic Properties, and Biocompatibility.

Bifunctional and highly uniform Ln:BaGdF5 (Ln = Eu(3+) and Nd(3+)) nanoparticles have been successfully synthesized using a solvothermal method consisting of the aging at 120 °C of a glycerol solution containing the corresponding Lanthanide acetylacetonates and butylmethylimidazolium tetrafluoroborate. The absence of any surfactant in the synthesis process rendered hydrophilic nanospheres (with tunable diameter from 45 nm 85 nm, depending on the cations concentration of the starting solution) which are suitable for bioapplications. The particles are bifunctional because they showed both optical and magnetic properties due to the presence of the optically active lanthanides (Eu(3+) in the visible and Nd(3+) in the NIR regions of the electromagnetic spectrum) and the paramagnetic gadolinium ion, respectively. The luminescence decay curves of the nanospheres doped with different amounts of Eu(3+) and Nd(3+) have been recorded in order to determine the optimum dopant concentration in each case, which turned out to be 5% Eu(3+) and 0.5% Nd(3+). Likewise, proton relaxation times were measured at 1.5 T in water suspensions of the optimum particles found in the luminescence study. The values obtained suggested that both kinds of particles could be used as positive contrast agents for MRI. Finally, it was demonstrated that both the 5% Eu(3+) and 0.5% Nd(3+)-doped BaGdF5 nanospheres showed negligible cytotoxicity for VERO cells for concentrations up to 0.25 mg mL(-1).

Recent advances in biosensing using magnetic glyconanoparticles.

In this critical review we discuss the most recent advances in the field of biosensing applications of magnetic glyconanoparticles. We first give an overview of the main synthetic routes to obtain magnetic-nanoparticle-carbohydrate conjugates and then we highlight their most promising applications for magnetic relaxation switching sensing, cell and pathogen detection, cell targeting and magnetic resonance imaging. We end with a critical perspective of the field, identifying the main challenges to be overcome, but also the areas where the most promising developments are likely to happen in the coming decades.

Controlling Properties and Cytotoxicity of Chitosan Nanocapsules by Chemical Grafting.

The tunability of the properties of chitosan-based carriers opens new ways for the application of drugs with low water-stability or high adverse effects. In this work, the combination of a nanoemulsion with a chitosan hydrogel coating and the following poly (ethylene glycol) (PEG) grafting is proven to be a promising strategy to obtain a flexible and versatile nanocarrier with an improved stability. Thanks to chitosan amino groups, a new easy and reproducible method to obtain nanocapsule grafting with PEG has been developed in this work, allowing a very good control and tunability of the properties of nanocapsule surface. Two different PEG densities of coverage are studied and the nanocapsule systems obtained are characterized at all steps of the optimization in terms of diameter, Z potential and surface charge (amino group analysis). Results obtained are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after covalent linking through their amino terminal moiety. An improvement in nanocapsule stability in physiological medium is observed with the highest PEG coverage density obtained. Cytotoxicity tests also demonstrate that grafting with PEG is an effective strategy to modulate the cytotoxicity of developed nanocapsules. Such results indicate the suitability of chitosan as protective coating for future studies oriented toward drug delivery.

Magnetic-Responsive Release Controlled by Hot Spot Effect.

Magnetically triggered drug delivery nanodevices have attracted great attention in nanomedicine, as they can feature as smart carriers releasing their payload at clinician's will. The key principle of these devices is based on the properties of magnetic cores to generate thermal energy in the presence of an alternating magnetic field. Then, the temperature increase triggers the drug release. Despite this potential, the rapid heat dissipation in living tissues is a serious hindrance for their clinical application. It is hypothesized that magnetic cores could act as hot spots, this is, produce enough heat to trigger the release without the necessity to increase the global temperature. Herein, a nanocarrier has been designed to respond when the temperature reaches 43 °C. This material has been able to release its payload under an alternating magnetic field without the need of increasing the global temperature of the environment, proving the efficacy of the hot spot mechanism in magnetic-responsive drug delivery devices.

Protein-templated biomimetic silica nanoparticles.

Biomimetic silica particles can be synthesized as a nanosized material within minutes in a process mimicked from living organisms such as diatoms and sponges. In this work, we have studied the effect of bovine serum albumin (BSA) as a template to direct the synthesis of silica nanoparticles (NPs) with the potential to associate proteins on its surface. Our approach enables the formation of spheres with different physicochemical properties. Particles using BSA as a protein template were smaller (∼250-380 nm) and were more monodisperse than those lacking the proteic core (∼700-1000 nm) as seen by dynamic light scattering (DLS), scanning electron microscopy (SEM), and environmental scanning electron microscopy (ESEM) analysis. The absence of BSA during synthesis produced silica nanoparticles without any porosity that was detectable by nitrogen adsorption, whereas particles containing BSA developed porosity in the range of 4 to 5 nm which collapsed on the removal of BSA, thus producing smaller pores. These results were in accordance with the pore size calculated by high-resolution transmission electron microscopy (HTEM). The reproducibility of the BSA-templated nanoparticle properties was determined by analyzing four batches of independent synthesizing experiments that maintained their properties. The high positive superficial charge of the nanoparticles facilitated adsorption under mild conditions of a range of proteins from an E. coli extract and a commercial preparation of laccase from Trametes versicolor. All of the proteins were quantitatively desorbed. Experiments conducted showed the reusability of the particles as supports for the ionic adsorption of the biomolecules. The protein loading capacity of the BSA-based biomimetic particles was determined using laccase as 98.7 ± 6.6 mg·g(-1) of particles.