Magnetic nanoparticles (MNPs) are being developed for a number of biomedical technologies that involve their direct contact with blood. This includes magnetic hyperthermia, targeted thrombolysis, and magnetic separation platforms. However, the potential thrombogenicity of MNPs remains an important safety concern, as nanoparticle physicochemical properties can often trigger activation of platelets and coagulation factors. Experimental evaluation of nanoparticle-induced coagulation is commonly performed in freshly donated human blood, although access to such biological material may present logistical and regulatory challenges, particularly during early stages of nanoparticle development.In this work, we explored the feasibility of using commercially available horse blood as an alternative to human blood for preliminary assessment of MNP thrombogenicity. Glycine-coated iron oxide nanoparticles (IONPs) were synthesised via co-precipitation of iron salts and physicochemically characterised by transmission electron microscopy (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and electrophoretic light scattering (ELS). Magnetic behaviour was further evaluated using AC magnetometry and AC magnetic susceptibility measurements. Coagulation assays performed in recalcified horse blood revealed that all synthesised nanoparticles accelerated clot formation compared to untreated control samples. Importantly, this procoagulant behaviour was largely attenuated when nanoparticles were pre-incubated with bovine serum albumin (BSA), indicating the influence of the nanoparticle surface in triggering blood coagulation. These coagulation trends observed in horse blood closely align with known effects seen in freshly donated human blood using analogous nanoparticle formulations.These findings suggest that horse blood may provide a practical and accessible experimental platform to perform early-stage screening of nanoparticle thrombogenicity prior to pre-clinical human testing.

The ability to control the growth and orientation of neurites over long distances has significant implications for regenerative therapies and the development of physiologically relevant brain tissue models. In this study, the forces generated on magnetic nanoparticles internalised within intracellular endosomes are used to direct the orientation of neuronal outgrowth in cell cultures. Following differentiation, neurite orientation was observed after 3 days application of magnetic forces to human neuroblastoma (SH-SY5Y) cells, and after 4 days application to rat cortical primary neurons. The direction of neurite outgrowth was quantified using a 2D Fourier transform analysis, showing agreement with the derived magnetic force vectors. Orientation control was found to be effective over areas >1cm2 using modest forces of ∼10 fN per endosome, apparently limited only by the local confluence of the cells. A bioinformatics analysis of protein expression in cells exposed to magnetic forces revealed changes to cell signaling and metabolic pathways resulting in enhanced carbohydrate metabolism, as well as the perturbation of processes related to cellular organisation and proliferation. Additionally, in cell culture regions where the measured force vectors converged, large (∼100 µm) SH-SY5Y neuroclusters loaded with nanoparticles were found, connected by unusually thick linear neurite fibres. This could suggest a magnetically driven enhancement of neurocluster growth, with the clusters themselves contributing to the local forces that direct outgrowth. Such structures, which have not been previously observed, could provide new insights into the development and possible enhancement of neural circuitry. Statement of Significance A magnetic force approach for directing outgrowth in neuronal cells over macroscopic areas is successfully demonstrated. Cells were incubated with magnetic nanoparticles which were sequestered into intracellular compartments. Permanent magnet arrays created local intracellular magnetic force vectors mediated via the internalized nanoparticles, which were found to precisely guide neurite orientation. Analysis of cellular protein expression suggested the mechanism for directed growth involved specific cell signaling and metabolic pathways. In addition, highly unusual straight and thick neural fibers were observed that connected large ‘magnetic’ spherical cell clusters. The results reported will advance nanotechnology and cell therapy for neuro-regeneration where magnetic forces could help to reconnect damaged neurons, or even build artificial neuronal architectures.

Normal functioning of the human brain is dependent on adequate regulation of essential metal nutrients. However, it is also highly sensitive to metal-mediated toxicity, linked to various neurodegenerative disorders. Exposure to environmental metal sources (especially to particulate air pollution) can stimulate toxicity and neuropathologic effects, which is particularly evident in populations chronically exposed to high levels of air pollution. Identifying the sources of metal-rich deposits in the human brain is important in not only distinguishing the effects of environmentally acquired metals from endogenous metal dysregulation, but also for tracing pollutant sources which may be subject to exposure control. This perspective reviews evidence for key physicochemical properties (size/morphology, chemical composition, oxidation state, magnetic properties, and isotopic composition) concerning their capacity to distinguish sources of metals in the brain. The scope for combining analytical techniques to study properties in tandem is also discussed.

The accumulation of amyloid plaques and increased brain redox burdens are neuropathological hallmarks of Alzheimer's disease. Altered metabolism of essential biometals is another feature of Alzheimer's, with amyloid plaques representing sites of disturbed metal homeostasis. Despite these observations, metal-targeting disease treatments have not been therapeutically effective to date. A better understanding of amyloid plaque composition and the role of the metals associated with them is critical. To establish this knowledge, the ability to resolve chemical variations at nanometer length scales relevant to biology is essential. Here, we present a methodology for the label-free, nanoscale chemical characterization of amyloid plaques within human Alzheimer's disease tissue using synchrotron X-ray spectromicroscopy. Our approach exploits a C-H carbon absorption feature, consistent with the presence of lipids, to visualize amyloid plaques selectively against the tissue background, allowing chemical analysis to be performed without the addition of amyloid dyes that alter the native sample chemistry. Using this approach, we show that amyloid plaques contain elevated levels of calcium, carbonates, and iron compared to the surrounding brain tissue. Chemical analysis of iron within plaques revealed the presence of chemically reduced, low-oxidation-state phases, including ferromagnetic metallic iron. The zero-oxidation state of ferromagnetic iron determines its high chemical reactivity and so may contribute to the redox burden in the Alzheimer's brain and thus drive neurodegeneration. Ferromagnetic metallic iron has no established physiological function in the brain and may represent a target for therapies designed to lower redox burdens in Alzheimer's disease. Additionally, ferromagnetic metallic iron has magnetic properties that are distinct from the iron oxide forms predominant in tissue, which might be exploitable for the in vivo detection of amyloid pathologies using magnetically sensitive imaging. We anticipate that this label-free X-ray imaging approach will provide further insights into the chemical composition of amyloid plaques, facilitating better understanding of how plaques influence the course of Alzheimer's disease.

Neuromelanin-pigmented neurons of the substantia nigra are selectively lost during the progression of Parkinson's disease. These neurons accumulate iron in the disease state, and iron-mediated neuron damage is implicated in cell death. Animal models of Parkinson's have evidenced iron loading inside the nucleoli of nigral neurons, however the nature of intranuclear iron deposition in the melanised neurons of the human substantia nigra is not understood. Here, scanning transmission x-ray microscopy (STXM) is used to probe iron foci in relation to the surrounding ultrastructure in melanised neurons of human substantia nigra from a confirmed Parkinson's case. In addition to the expected neuromelanin-bound iron, iron deposits are also associated with the edge of the cell nucleolus. Speciation analysis confirms these deposits to be ferric (Fe3+) iron. The function of intranuclear iron in these cells remains unresolved, although both damaging and protective mechanisms are considered. This finding shows that STXM is a powerful label-free tool for the in situ, nanoscale chemical characterisation of both organic and inorganic intracellular components. Future applications are likely to shed new light on incompletely understood biochemical mechanisms, such as metal dysregulation and morphological changes to cell nucleoli, that are important in understanding the pathogenesis of Parkinson's. Showcasing the application of scanning transmission x-ray microscopy to study the oxidation state of intranuclear iron in nigral neurons. Implications and potential future applications are discussed.

The study of exogenous and endogenous nanoscale magnetic material in biology is important for developing biomedical nanotechnology as well as for understanding fundamental biological processes such as iron metabolism and biomineralization. Here, we exploit the magneto-optical Faraday effect to probe intracellular magnetic properties and perform magnetic imaging, revealing the location-specific magnetization dynamics of exogenous magnetic nanoparticles within cells. The opportunities enabled by this method are shown in the context of magnetic hyperthermia; an effect where local heating is generated in magnetic nanoparticles exposed to high-frequency AC magnetic fields. Magnetic hyperthermia has the potential to be used as a cellular-level thermotherapy for cancer, as well as for other biomedical applications that target heat-sensitive cellular function. However, previous experiments have suggested that the cellular environment modifies the magnetization dynamics of nanoparticles, thus dramatically altering their heating efficiency. By combining magneto-optical and fluorescence measurements, we demonstrate a form of biological microscopy that we used here to study the magnetization dynamics of nanoparticles in situ, in both histological samples and living cancer cells. Correlative magnetic and fluorescence imaging identified aggregated magnetic nanoparticles colocalized with cellular lysosomes. Nanoparticles aggregated within these lysosomes displayed reduced AC magnetic coercivity compared to the same particles measured in an aqueous suspension or aggregated in other areas of the cells. Such measurements reveal the power of this approach, enabling investigations of how cellular location, nanoparticle aggregation, and interparticle magnetic interactions affect the magnetization dynamics and consequently the heating response of nanoparticles in the biological milieu.

The synchrotron x-ray spectromicroscopy technique Scanning Transmission X-ray Microscopy (STXM) offers a powerful means to examine the underlying biochemistry of biological systems, owing to its combined chemical sensitivity and nanoscale spatial resolution. Here we introduce and demonstrate methodology for the use of STXM to examine the biochemistry of the human brain. We then discuss how this approach can help us better understand the biochemical changes that occur during the development of degenerative brain disorders, potentially facilitating the development of new therapies for disease diagnosis and treatment.

Thrombotic events are a major cause of death and disability worldwide. Thrombolysis with the clot busting drug tissue plasminogen activator (tPA) or thromboprophylaxis using anticoagulants such as heparin are frontline treatments for thrombotic diseases. However, they account for important limitations in terms of effectivity and safety, producing in some cases unwanted bleeding episodes in patients. Magnetic hyperthermia (MH) mediated by magnetic nanoparticles, targeted to localized tumors, has been used to treat cancer. Here we demonstrate that MH mediated by functionalized magnetic nanoparticles can act to improve the efficacy of antithrombotic and thrombolytic treatments. Iron oxide nanoparticles (IONPs) functionalised with the PAC-1 antibody (f-IONPs) were prepared for clot targeting, and the potential of MH to enhance diffusion of antithrombotic drugs into the clot was demonstrated. We found that clot-targeted MH increased the efficacy of clot-busting drugs in ex vivo-derived human blood clots, leading to a significant reduction in clot weight. Our results demonstrate that surface-localized MH could be applied to enhance the action of antithrombotic and clot-busting drugs.

Magnetic nanoparticles (MNPs) are promising as local heat generators for magnetic hyperthermia under AC magnetic fields. The heating efficacy of MNPs is determined by the AC hysteresis loop area, which in turn is affected by the dynamic magnetic properties of the nanoparticles. Whilst inductive-based AC magnetometers can measure the average magnetic behavior of samples, the use of the magneto-optical Faraday effect with a focused laser spot allows point-probe measurements to be made, and without some of the magnetic field limitations imposed by inductive methods. In this work, the AC magnetic properties of different sized iron oxide MNPs in suspension were measured by AC magnetometry and AC susceptibility techniques. AC hysteresis loops measured by magneto-optical magnetometry were validated using a commercial inductive AC magnetometer, and compared to the magnetization relaxation behavior revealed by fitting the AC susceptibility data. The spatial sensitivity of the point-probe magneto-optical method is also demonstrated by measuring the AC hysteresis loop from large (>1 μm) MNP aggregates dried onto glass slides. These aggregated particles are found to be magnetically softer than in their suspension form, suggesting interparticle coupling mechanisms could occur when the nanoparticles form dense aggregates.

Blood clotting disorders are among the main causes of death worldwide. Magnetic coagulometry provides a new method to test coagulation in human whole blood ex vivo, offering grounds for the improvement of current methods in research and clinic.

TWIK-related K+ 1 (TREK1) is a potassium channel expressed in the nervous system with multiple functions including neurotransmission and is a prime pharmacological target for neurological disorders. TREK1 gating is controlled by a wide range of external stimuli including mechanical forces. Previous work has demonstrated that TREK1 can be mechano-activated using magnetic nanoparticles (MNP) functionalised with antibodies targeted to TREK1 channels. Once the MNP are bound, external dynamic magnetic fields are used to generate forces on the TREK channel. This approach has been shown to drive cell differentiation in cells from multiple tissues. In this work we investigated the effect of MNP-mediated TREK1 mechano-activation on early stress response pathways along with the differentiation and connectivity of neuronal cells using the model neuronal cell line SH-SY5Y. Results showed that TREK1 is well expressed in SH-SY5Y and that TREK1-MNP initiate c-Myc/NF-κB stress response pathways as well as Nitrite production after magnetic stimulation, indicative of the cellular response to mechanical cues. Results also showed that TREK1 mechano-activation had no overall effect on neuronal morphology or expression of the neuronal marker βIII-Tubulin in Retinoic Acid (RA)/Brain-derived Neurotrophic factor (BDNF) differentiated SH-SY5Y but did increase neurite number. These results suggest that TREK1 is involved in cellular stress response signalling in neuronal cells, which leads to increased neurite production, but is not involved in regulating RA/BDNF mediated neuronal differentiation.

BACKGROUND:Thrombolysis is a frontline treatment for stroke, which involves the application of tissue plasminogen activator (tPA) to trigger endogenous clot-degradation pathways. However, it is only effective within 4.5 h of symptom onset because of clot contraction preventing tPA permeation into the clot. Magnetic hyperthermia (MH) mediated by tumor-targeted magnetic nanoparticles is used to treat cancer by using local heat generation to trigger apoptosis of cancer cells. OBJECTIVES:To develop clot-targeting magnetic nanoparticles to deliver MH to the surface of human blood clots, and to assess whether this can improve the efficacy of thrombolysis of contracted blood clots. METHODS:Clot-targeting magnetic nanoparticles were developed by functionalizing iron oxide nanoparticles with an antibody recognizing activated integrin αIIbβ3 (PAC-1). The magnetic properties of the PAC-1-tagged magnetic nanoparticles were characterized and optimized to deliver clot-targeted MH. RESULTS:Clot-targeted MH increases the efficacy of tPA-mediated thrombolysis in contracted human blood clots, leading to a reduction in clot weight. MH increases the permeability of the clots to tPA, facilitating their breakdown. Scanning electron microscopy reveals that this effect is elicited through enhanced fibrin breakdown and triggering the disruption of red blood cells on the surface of the clot. Importantly, endothelial cells viability in a three-dimensional blood vessel model is unaffected by exposure to MH. CONCLUSIONS:This study demonstrates that clot-targeted MH can enhance the thrombolysis of contracted human blood clots and can be safely applied to enhance the timeframe in which thrombolysis is effective.

A challenge in current stem cell therapies for Parkinson's disease (PD) is controlling neuronal outgrowth from the substantia nigra towards the targeted area where connectivity is required in the striatum. Here we present progress towards controlling directional neurite extensions through the application of iron-oxide magnetic nanoparticles (MNPs) labelled neuronal cells combined with a magnetic array generating large spatially variant field gradients (greater than 20 T m −1 ). We investigated the viability of this approach in both two-dimensional and organotypic brain slice models and validated the observed changes in neurite directionality using mathematical models. Results showed that MNP-labelled cells exhibited a shift in directional neurite outgrowth when cultured in a magnetic field gradient, which broadly agreed with mathematical modelling of the magnetic force gradients and predicted MNP force direction. We translated our approach to an ex vivo rat brain slice where we observed directional neurite outgrowth of transplanted MNP-labelled cells from the substantia nigra towards the striatum. The improved directionality highlights the viability of this approach as a remote-control methodology for the control and manipulation of cellular growth for regenerative medicine applications. This study presents a new tool to overcome challenges faced in the development of new therapies for PD.

Magnetic nanoparticles (MNP) are extremely versatile tools in bioengineering and medicine with diverse uses ranging from magnetic resonance contrast agents to drug delivery vehicles. Recently, MNP have been adapted to target and regulate cell signalling pathways for control of cell behaviour. This approach has been applied to stem and progenitor cells to orchestrate tissue development in tissue engineering. This review introduces the bio-functionalisation mechanisms for MNP and highlights the recent advances in MNP-mediated cell signalling activation. We also explore how the application of this technology has novel uses for stem cell control in the context of tissue engineering and regenerative medicine.

The chemistry of copper and iron plays a critical role in normal brain function. A variety of enzymes and proteins containing positively charged Cu+, Cu2+, Fe2+, and Fe3+ control key processes, catalyzing oxidative metabolism and neurotransmitter and neuropeptide production. Here, we report the discovery of elemental (zero-oxidation state) metallic Cu-0 accompanying ferromagnetic elemental Fe-0 in the human brain. These nanoscale biometal deposits were identified within amyloid plaque cores isolated from Alzheimer's disease subjects, using synchrotron x-ray spectromicroscopy. The surfaces of nanodeposits of metallic copper and iron are highly reactive, with distinctly different chemical and magnetic properties from their predominant oxide counterparts. The discovery of metals in their elemental form in the brain raises new questions regarding their generation and their role in neurochemistry, neurobiology, and the etiology of neurodegenerative disease.

Nanoscale resolution X-ray spectromicroscopy shows the co-incubation of β-amyloid (Aβ) and iron(iii) to result in aggregate structures displaying nanoscale heterogeneity in Aβ and iron chemistry, including the formation of potentially cytotoxic Fe0.

Recommendations are given concerning the terminology of concepts and methods used in spectroscopy in analytical chemistry, covering nuclear magnetic resonance spectroscopy, atomic spectroscopy, and vibrational spectroscopy.

Characterization of magnetic nanoparticles in solution is challenging due to the interplay between magnetic relaxation and agglomeration. The AC magnetic susceptibility of magnetite nanoparticles in water has been studied using magneto-optical methods in the frequency range of 10 Hz–250 kHz. The Faraday effect is detected simultaneously with changes in the fluid configuration. It is shown that the relative sensitivity to the magnetic and structural response can be adjusted by varying the wavelength, paving the way toward spatially resolved studies at the micro-scale.

The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising, since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following the passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.