Disruption of the mitochondrial network in a mouse model of Huntington's disease visualized by in-tissue multiscale 3D electron microscopy.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the coding sequence of huntingtin protein. Initially, it predominantly affects medium-sized spiny neurons (MSSNs) of the corpus striatum. No effective treatment is still available, thus urging the identification of potential therapeutic targets. While evidence of mitochondrial structural alterations in HD exists, previous studies mainly employed 2D approaches and were performed outside the strictly native brain context. In this study, we adopted a novel multiscale approach to conduct a comprehensive 3D in situ structural analysis of mitochondrial disturbances in a mouse model of HD. We investigated MSSNs within brain tissue under optimal structural conditions utilizing state-of-the-art 3D imaging technologies, specifically FIB/SEM for the complete imaging of neuronal somas and Electron Tomography for detailed morphological examination, and image processing-based quantitative analysis. Our findings suggest a disruption of the mitochondrial network towards fragmentation in HD. The network of interlaced, slim and long mitochondria observed in healthy conditions transforms into isolated, swollen and short entities, with internal cristae disorganization, cavities and abnormally large matrix granules.

Genotoxicity and heating Performance of VxFe3-xO4 nanoparticles in Health applications.

The applications of magnetic nanoparticles (MNPs) as biocatalysts in different biomedical areas have been evolved very recently. One of the main challenges in this field is to design affective MNPs surfaces with catalytically active atomic centres, while producing minimal toxicological side effects on the hosting cell or tissues. MNPs of vanadium spinel ferrite (VFe2O4) are a promising material for mimicking the action of natural enzymes in degrading harmful substrates due to the presence of active V5+ centres. However, the toxicity of this material has not been yet studied in detail enough to grant biomedical safety. In this work, we have extensively measured the structural, compositional, and magnetic properties of a series of VxFe3-xO4 spinel ferrite MNPs to assess the surface composition and oxidation state of V atoms, and also performed systematic and extensive in vitro cytotoxicity and genotoxicity testing required to assess their safety in potential clinical applications. We could establish the presence of V5+ at the particle surface even in water-based colloidal samples at pH 7, as well as different amounts of V2+ and V3+ substitution at the A and B sites of the spinel structure. All samples showed large heating efficiency with Specific Loss Power values up to 400 W/g (H0 = 30 kA/m; f = 700 kHz). Samples analysed for safety in human hepatocellular carcinoma (HepG2) cell line with up to 24h of exposure showed that these MNPs did not induce major genomic abnormalities such as micronuclei, nuclear buds, or nucleoplasmic bridges (MNIs, NBUDs, and NPBs), nor did they cause DNA double-strand breaks (DSBs) or aneugenic effects-types of damage considered most harmful to cellular genetic material. The present study is an essential step towards the use of these type of nanomaterials in any biomedical or clinical application.

Enhanced Cellular Transduction of Nanoparticles Resistant to Rapidly Forming Plasma Protein Coronas.

Nanoparticles (NPs) are increasingly being developed as biomedical platforms for drug/nucleic acid delivery and imaging. However, in biological fluids, NPs interact with a wide range of proteins that form a coating known as protein corona. Coronae can critically influence self-interaction and binding of other molecules, which can affect toxicity, promote cell activation, and inhibit general or specific cellular uptake. Glycosaminoglycan (GAG)-binding enhanced transduction (GET) is developed to efficiently deliver a variety of cargoes intracellularly; employing GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs) which enhance endocytotic cell internalization. Herein, it is demonstrated that GET peptide coatings can mediate sustained intracellular transduction of magnetic NPs (MNPs), even in the presence of serum or plasma. NP colloidal stability, physicochemical properties, toxicity and cellular uptake are investigated. Using label-free snapshot proteomics, time-resolved profiles of human plasma coronas formed on functionalized GET-MNPs demonstrate that coronae quickly form (<1 min), with their composition relatively stable but evolving. Importantly GET-MNPs present a subtly different corona composition to MNPs alone, consistent with GAG-binding activities. Understanding how NPs interact with biological systems and can retain enhanced intracellular transduction will facilitate novel drug delivery approaches for cell-type specific targeting of new nanomaterials.

PolishEM: image enhancement in FIB-SEM.

SUMMARY: We have developed a software tool to improve the image quality in focused ion beam-scanning electron microscopy (FIB-SEM) stacks: PolishEM. Based on a Gaussian blur model, it automatically estimates and compensates for the blur affecting each individual image. It also includes correction for artifacts commonly arising in FIB-SEM (e.g. curtaining). PolishEM has been optimized for an efficient processing of huge FIB-SEM stacks on standard computers. AVAILABILITY AND IMPLEMENTATION: PolishEM has been developed in C. GPL source code and binaries for Linux, OSX and Windows are available at http://www.cnb.csic.es/%7ejjfernandez/polishem. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effects of biological buffer solutions on the peroxidase-like catalytic activity of Fe3O4 nanoparticles.

Iron oxide nanoparticles (IONPs) are frequently used in biomedical applications due to their magnetic properties and putative chemical stability. Nevertheless, their well-known ability to mimic some features of the peroxidase enzyme activity under specific conditions of pH and temperature could lead to the formation of potentially harmful free radical species. In addition to the intrinsic enzyme-like activity of IONPs, the buffer solution is an important external factor that can alter dramatically the IONP activity because the buffer species can interact with the surface of the particles. In our study, IONP activity was evaluated in different buffering solutions under different experimental conditions and predominant free radical species were measured by electron paramagnetic resonance using the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The formation kinetics of the reactive oxygen species were studied by UV-visible spectroscopy with TMB and DAB peroxidase substrates. We found that the highest catalytic oxidation of peroxidase substrates and free radical generation were achieved in acetate buffer, while phosphate buffer inhibited the peroxidase-like activity of IONPs in a concentration dependent manner. When emulating the physiological conditions, a lower catalytic activity was observed at pH 7.4 when compared to that at pH 5.0. Also, in phosphate buffered saline (PBS), we observed an enhancement in the peroxidase substrate oxidation rate that was not accompanied by an increase in DMPO/adduct formation which could be related to a non-specific oxidation catalyzed by the chloride ion. Similar observations were found after the addition of a bicarbonate to HEPES buffer. TMB oxidation did not occur when the reaction was conducted with free iron ions from metal salts with the same concentration of the IONPs (0.33 Fe2+ and 0.66 Fe3+). However, we observed even higher catalytic activities than those when doubling the IONP concentration when they are combined with the free iron salts. These results indicate that biological buffering solutions need to be carefully considered when evaluating IONP catalytic activity and their potential toxicological effects since under physiological conditions of pH, salinity and buffering species, the peroxidase-like activity of IONPs is dramatically reduced.

Ultra-fast direct growth of metallic micro- and nano-structures by focused ion beam irradiation.

An ultra-fast method to directly grow metallic micro- and nano-structures is introduced. It relies on a Focused Ion Beam (FIB) and a condensed layer of suitable precursor material formed on the substrate under cryogenic conditions. The technique implies cooling the substrate below the condensation temperature of the gaseous precursor material, subsequently irradiating with ions according to the wanted pattern, and posteriorly heating the substrate above the condensation temperature. Here, using W(CO)6 as the precursor material, a Ga+ FIB, and a substrate temperature of -100 °C, W-C metallic layers and nanowires with resolution down to 38 nm have been grown by Cryogenic Focused Ion Beam Induced Deposition (Cryo-FIBID). The most important advantages of Cryo-FIBID are the fast growth rate (about 600 times higher than conventional FIBID with the precursor material in gas phase) and the low ion irradiation dose required (∼50 µC/cm2), which gives rise to very low Ga concentrations in the grown material and in the substrate (≤0.2%). Electrical measurements indicate that W-C layers and nanowires grown by Cryo-FIBID exhibit metallic resistivity. These features pave the way for the use of Cryo-FIBID in various applications in micro- and nano-lithography such as circuit editing, photomask repair, hard masks, and the growth of nanowires and contacts. As a proof of concept, we show the use of Cryo-FIBID to grow metallic contacts on a Pt-C nanowire and investigate its transport properties. The contacts have been grown in less than one minute, which is considerably faster than the time needed to grow the same contacts with conventional FIBID, around 10 hours.

The relevance of Brownian relaxation as power absorption mechanism in Magnetic Hyperthermia.

The Linear Response Theory (LRT) is a widely accepted framework to analyze the power absorption of magnetic nanoparticles for magnetic fluid hyperthermia. Its validity is restricted to low applied fields and/or to highly anisotropic magnetic nanoparticles. Here, we present a systematic experimental analysis and numerical calculations of the specific power absorption for highly anisotropic cobalt ferrite (CoFe2O4) magnetic nanoparticles with different average sizes and in different viscous media. The predominance of Brownian relaxation as the origin of the magnetic losses in these particles is established, and the changes of the Specific Power Absorption (SPA) with the viscosity of the carrier liquid are consistent with the LRT approximation. The impact of viscosity on SPA is relevant for the design of MNPs to heat the intracellular medium during in vitro and in vivo experiments. The combined numerical and experimental analyses presented here shed light on the underlying mechanisms that make highly anisotropic MNPs unsuitable for magnetic hyperthermia.

Controlling the dominant magnetic relaxation mechanisms for magnetic hyperthermia in bimagnetic core-shell nanoparticles.

We report a simple and effective way to control the heat generation of a magnetic colloid under alternate magnetic fields by changing the shell composition of bimagnetic core-shell Fe3O4/ZnxCo1-xFe2O4 nanoparticles. The core-shell structure constitutes a magnetically-coupled biphase system, with an effective anisotropy that can be tuned by the substitution of Co2+ by Zn2+ ions in the shell. Magnetic hyperthermia experiments of nanoparticles dispersed in hexane and butter oil showed that the magnetic relaxation is dominated by Brown relaxation mechanism in samples with higher anisotropy (i.e., larger concentration of Co within the shell) yielding high specific power absorption values in low viscosity media as hexane. Increasing the Zn concentration of the shell, diminishes the magnetic anisotropy, which results in a change to a Néel relaxation that dominates the process when the nanoparticles are dispersed in a high-viscosity medium. We demonstrate that tuning the Zn contents at the shell of these exchange-coupled core/shell nanoparticles provides a way to control the magnetic anisotropy without loss of saturation magnetization. This ability is an essential prerequisite for most biomedical applications, where high viscosities and capturing mechanisms are present.

Cell damage produced by magnetic fluid hyperthermia on microglial BV2 cells.

We present evidence on the effects of exogenous heating by water bath (WB) and magnetic hyperthermia (MHT) on a glial micro-tumor phantom. To this, magnetic nanoparticles (MNPs) of 30-40 nm were designed to obtain particle sizes for maximum heating efficiency. The specific power absorption (SPA) values (f = 560 kHz, H = 23.9 kA/m) for as prepared colloids (533-605 W/g) dropped to 98-279 W/g in culture medium. The analysis of the intracellular MNPs distribution showed vesicle-trapped MNPs agglomerates spread along the cytoplasm, as well as large (~0.5-0.9 µm) clusters attached to the cell membrane. Immediately after WB and MHT (T = 46 °C for 30 min) the cell viability was ≈70% and, after 4.5 h, decreased to 20-25%, demonstrating that metabolic processes are involved in cell killing. The analysis of the cell structures after MHT revealed a significant damage of the cell membrane that is correlated to the location of MNPs clusters, while local cell damage were less noticeable after WB without MNPs. In spite of the similar thermal effects of WB and MHT on the cell viability, our results suggest that there is an additional mechanism of cell damage related to the presence of MNPs at the intracellular space.

Magnetic hyperthermia enhances cell toxicity with respect to exogenous heating.

Magnetic hyperthermia is a new type of cancer treatment designed for overcoming resistance to chemotherapy during the treatment of solid, inaccessible human tumors. The main challenge of this technology is increasing the local tumoral temperature with minimal side effects on the surrounding healthy tissue. This work consists of an in vitro study that compared the effect of hyperthermia in response to the application of exogenous heating (EHT) sources with the corresponding effect produced by magnetic hyperthermia (MHT) at the same target temperatures. Human neuroblastoma SH-SY5Y cells were loaded with magnetic nanoparticles (MNPs) and packed into dense pellets to generate an environment that is crudely similar to that expected in solid micro-tumors, and the above-mentioned protocols were applied to these cells. These experiments showed that for the same target temperatures, MHT induces a decrease in cell viability that is larger than the corresponding EHT, up to a maximum difference of approximately 45% at T = 46 °C. An analysis of the data in terms of temperature efficiency demonstrated that MHT requires an average temperature that is 6 °C lower than that required with EHT to produce a similar cytotoxic effect. An analysis of electron microscopy images of the cells after the EHT and MHT treatments indicated that the enhanced effectiveness observed with MHT is associated with local cell destruction triggered by the magnetic nano-heaters. The present study is an essential step toward the development of innovative adjuvant anti-cancer therapies based on local hyperthermia treatments using magnetic particles as nano-heaters.

The orientation of the neuronal growth process can be directed via magnetic nanoparticles under an applied magnetic field.

There is a growing body of evidence indicating the importance of physical stimuli for neuronal growth and development. Specifically, results from published experimental studies indicate that forces, when carefully controlled, can modulate neuronal regeneration. Here, we validate a non-invasive approach for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles (MNPs) and magnetic fields (Ms). The concept is that the application of a tensile force to a neuronal cell can stimulate neurite initiation or axon elongation in the desired direction, the MNPs being used to generate this tensile force under the effect of a static external magnetic field providing the required directional orientation. In a neuron-like cell line, we have confirmed that MNPs direct the neurite outgrowth preferentially along the direction imposed by an external magnetic field, by inducing a net angle displacement (about 30°) of neurite direction. From the clinical editor: This study validates that non-invasive approaches for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles and magnetic fields are possible. The hypothesis was confirmed by observing preferential neurite outgrowth in a cell culture system along the direction imposed by an external magnetic field.

Neuronal cells loaded with PEI-coated Fe3O4 nanoparticles for magnetically guided nerve regeneration.

We report a one-step synthesis protocol for obtaining polymer-coated magnetic nanoparticles (MNPs) engineered for uploading neural cells. Polyethyleneimine-coated Fe3O4 nanoparticles (PEI-MNPs) with sizes of 25 ± 5 nm were prepared by oxidation of Fe(OH)2 by nitrate in basic aqueous media and adding PEI in situ during synthesis. The obtained PEI-MNP cores displayed a neat octahedral morphology and high crystallinity. The resulting nanoparticles were coated with a thin polymer layer of about 0.7-0.9 nm, and displayed a saturation magnetization value MS = 58 A m2 kg-1 at 250 K (64 A m2 kg-1 for T = 10 K). Cell uptake experiments on a neuroblastoma-derived SH-SY5Y cell line were undertaken over a wide time and MNP concentration range. The results showed a small decrease in cell viability for 24 h incubation (down to 70% viability for 100 µg ml-1), increasing the toxic effects with incubation time (30% cell survival at 100 µg ml-1 for 7 days of incubation). On the other hand, primary neuronal cells displayed higher sensitivity to PEI-MNPs, with a cell viability reduction of 44% of the control cells after 3 days of incubation with 50 µg ml-1. The amount of PEI-MNPs uploaded by SH-SY5Y cells was found to have a linear dependence on concentration. The intracellular distribution of the PEI-MNPs analyzed at the single-cell level by the dual-beam (FIB/SEM) technique revealed the coexistence of both fully incorporated PEI-MNPs and partially internalized PEI-MNP-clusters crossing the cell membrane. The resulting MNP-cluster distributions open the possibility of using these PEI-MNPs for magnetically driven axonal re-growth in neural cells.