Label-Free In Situ Chemical Characterization of Amyloid Plaques in Human Brain Tissues.

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.

Optical Microscopy Using the Faraday Effect Reveals in Situ Magnetization Dynamics of Magnetic Nanoparticles in Biological Samples.

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.

Magnetic coagulometry: towards a new nanotechnological tool for ex vivo monitoring coagulation in human whole blood.

Blood clotting disorders consisting of unwanted blood clot formation or excessive bleeding are some of the main causes of death worldwide. However, there are significant limitations in the current methods used to clinically monitor the dynamics of clot formation in human whole blood ex vivo. Here a new magnetic coagulometry platform for testing ex vivo coagulation is described. This platform exploits the sensitivity of the out-of-phase component of alternating current (AC) magnetic susceptibility (χ'') to variations in mobility and agglomeration of magnetic nanoparticles when trapped during blood clot formation. By labelling human whole blood with magnetic nanoparticles, the out-of-phase component of AC magnetic susceptibility shows that the dynamics of blood clot formation correlates with a decrease in the out-of-phase component χ'' over time activation of coagulation. This is caused by a rapid immobilisation of nanoparticles upon blood coagulation and compaction. In contrast, this rapid fall in the out-of-phase component χ'' is significantly slowed down when blood is pre-treated with three different anticoagulant drugs. Remarkably, the system showed sensitivity towards the effect of clinically used direct oral anticoagulation (DOAC) drugs in whole blood coagulation, in contrast to the inability of clinical routine tests prothrombin time (PT) and partial thromboplastin time (PTT) to efficiently monitor this effect. Translation of this nanomagnetic approach into clinic can provide a superior method for monitoring blood coagulation and improve the efficiency of the current diagnostic techniques.

Magnetic activation of TREK1 triggers stress signalling and regulates neuronal branching in SH-SY5Y cells.

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 ßIII-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.

Clot-targeted magnetic hyperthermia permeabilizes blood clots to make them more susceptible to thrombolysis.

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 αIIbß3 (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.

Biogenic metallic elements in the human brain?

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 Cu0 accompanying ferromagnetic elemental Fe0 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.

Parallelized Manipulation of Adherent Living Cells by Magnetic Nanoparticles-Mediated Forces.

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.

Iron stored in ferritin is chemically reduced in the presence of aggregating Aß(1-42).

Atypical low-oxidation-state iron phases in Alzheimer's disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide ß-amyloid (Aß) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aß and ferritin resulted in the conversion of ferritin's inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aß in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.

Analysis of neuronal iron deposits in Parkinson's disease brain tissue by synchrotron x-ray spectromicroscopy.

BACKGROUND: Neuromelanin-pigmented neurons, which are highly susceptible to neurodegeneration in the Parkinson's disease substantia nigra, harbour elevated iron levels in the diseased state. Whilst it is widely believed that neuronal iron is stored in an inert, ferric form, perturbations to normal metal homeostasis could potentially generate more reactive forms of iron capable of stimulating toxicity and cell death. However, non-disruptive analysis of brain metals is inherently challenging, since use of stains or chemical fixatives, for example, can significantly influence metal ion distributions and/or concentrations in tissues. AIMS: The aim of this study was to apply synchrotron soft x-ray spectromicroscopy to the characterisation of iron deposits and their local environment within neuromelanin-containing neurons of Parkinson's disease substantia nigra. METHODS: Soft x-ray spectromicroscopy was applied in the form of Scanning Transmission X-ray Microscopy (STXM) to analyse resin-embedded tissue, without requirement for chemically disruptive processing or staining. Measurements were performed at the oxygen and iron K-edges in order to characterise both organic and inorganic components of anatomical tissue using a single label-free method. RESULTS: STXM revealed evidence for mixed oxidation states of neuronal iron deposits associated with neuromelanin clusters in Parkinson's disease substantia nigra. The excellent sensitivity, specificity and spatial resolution of these STXM measurements showed that the iron oxidation state varies across sub-micron length scales. CONCLUSIONS: The label-free STXM approach is highly suited to characterising the distributions of both inorganic and organic components of anatomical tissue, and provides a proof-of-concept for investigating trace metal speciation within Parkinson's disease neuromelanin-containing neurons.

Label-Free Nanoimaging of Neuromelanin in the Brain by Soft X-ray Spectromicroscopy.

A hallmark of Parkinson's disease is the death of neuromelanin-pigmented neurons, but the role of neuromelanin is unclear. The in situ characterization of neuromelanin remains dependent on detectable pigmentation, rather than direct quantification of neuromelanin. We show that direct, label-free nanoscale visualization of neuromelanin and associated metal ions in human brain tissue can be achieved using synchrotron scanning transmission x-ray microscopy (STXM), through a characteristic feature in the neuromelanin x-ray absorption spectrum at 287.4 eV that is also present in iron-free and iron-laden synthetic neuromelanin. This is confirmed in consecutive brain sections by correlating STXM neuromelanin imaging with silver nitrate-stained neuromelanin. Analysis suggests that the 1s-σ* (C-S) transition in benzothiazine groups accounts for this feature. This method illustrates the wider potential of STXM as a label-free spectromicroscopy technique applicable to both organic and inorganic materials.

Controlling human platelet activation with calcium-binding nanoparticles.

Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration. Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation and prevent the unwanted blood clots that causes heart attack and strokes. Previously we have predicted that calcium accumulation within the lumen of an infolded portion of the platelet plasma membrane called the open canalicular system (OCS) is essential for maintaining this cytosolic calcium rise. Due to its nanometer dimensions of the OCS, it has been difficult to measure or interfere with the predicted luminal calcium accumulation. Here we utilise iron oxide magnetic nanoparticles coated with the known calcium chelator, citrate, to create calcium-binding nanoparticles. These were used to assess whether an OCS calcium store plays a role in controlling the dynamics of human platelet activation and aggregation. We demonstrate that citrate-coated nanoparticles are rapidly and selectively uptaken into the OCS of activated human platelets, where they act to buffer the accumulation of calcium there. Treatment with these calcium-binding nanoparticles reduced thrombin-evoked cytosolic calcium rises, and slowed platelet aggregation and clot retraction in human platelets. In contrast, nanoparticles that cannot bind calcium have no effect. This study demonstrates that the OCS acts as a key source of calcium for maintaining cytosolic calcium rises and accelerating platelet aggregation, and that calcium-binding nanoparticles targeted to the OCS could provide an anti-platelet therapy to treat patients at risk of suffering heart attacks or strokes.

Emerging Approaches to Investigate the Influence of Transition Metals in the Proteinopathies.

Transition metals have essential roles in brain structure and function, and are associated with pathological processes in neurodegenerative disorders classed as proteinopathies. Synchrotron X-ray techniques, coupled with ultrahigh-resolution mass spectrometry, have been applied to study iron and copper interactions with amyloid ß (1-42) or α-synuclein. Ex vivo tissue and in vitro systems were investigated, showing the capability to identify metal oxidation states, probe local chemical environments, and localize metal-peptide binding sites. Synchrotron experiments showed that the chemical reduction of ferric (Fe3+) iron and cupric (Cu2+) copper can occur in vitro after incubating each metal in the presence of Aß for one week, and to a lesser extent for ferric iron incubated with α-syn. Nanoscale chemical speciation mapping of Aß-Fe complexes revealed a spatial heterogeneity in chemical reduction of iron within individual aggregates. Mass spectrometry allowed the determination of the highest-affinity binding region in all four metal-biomolecule complexes. Iron and copper were coordinated by the same N-terminal region of Aß, likely through histidine residues. Fe3+ bound to a C-terminal region of α-syn, rich in aspartic and glutamic acid residues, and Cu2+ to the N-terminal region of α-syn. Elucidating the biochemistry of these metal-biomolecule complexes and identifying drivers of chemical reduction processes for which there is evidence ex-vivo, are critical to the advanced understanding of disease aetiology.

Metal Ion Binding to the Amyloid ß Monomer Studied by Native Top-Down FTICR Mass Spectrometry.

Native top-down mass spectrometry is a fast, robust biophysical technique that can provide molecular-scale information on the interaction between proteins or peptides and ligands, including metal cations. Here we have analyzed complexes of the full-length amyloid ß (1-42) monomer with a range of (patho)physiologically relevant metal cations using native Fourier transform ion cyclotron resonance mass spectrometry and three different fragmentation methods-collision-induced dissociation, electron capture dissociation, and infrared multiphoton dissociation-all yielding consistent results. Amyloid ß is of particular interest as its oligomerization and aggregation are major events in the etiology of Alzheimer's disease, and it is known that interactions between the peptide and bioavailable metal cations have the potential to significantly damage neurons. Those metals which exhibited the strongest binding to the peptide (Cu2+, Co2+, Ni2+) all shared a very similar binding region containing two of the histidine residues near the N-terminus (His6, His13). Notably, Fe3+ bound to the peptide only when stabilized toward hydrolysis, aggregation, and precipitation by a chelating ligand, binding in the region between Ser8 and Gly25. We also identified two additional binding regions near the flexible, hydrophobic C-terminus, where other metals (Mg2+, Ca2+, Mn2+, Na+, and K+) bound more weakly-one centered on Leu34, and one on Gly38. Unexpectedly, collisional activation of the complex formed between the peptide and [CoIII(NH3)6]3+ induced gas-phase reduction of the metal to CoII, allowing the peptide to fragment via radical-based dissociation pathways. This work demonstrates how native mass spectrometry can provide new insights into the interactions between amyloid ß and metal cations.

Nanoparticle-mediated magnetic hyperthermia is an effective method for killing the human-infective protozoan parasite Leishmania mexicana in vitro.

Cutaneous leishmaniasis is a neglected tropical disease characterized by disfiguring skin lesions. Current chemotherapeutic options depend on toxic, expensive drugs that are both difficult to administer and becoming less effective due to increasing levels of resistance. In comparison, thermotherapy displays greater patient compliance and less adverse systemic effects, but there are still significant issues associated with this. The procedure is painful, requiring local anaesthetic, and is less effective against large lesions. Using nanoparticles to controllably generate heat in a localized manner may provide an alternative solution. Here we evaluate magnetic hyperthermia, using iron oxide magnetic nanoparticles, as a localized, heat-based method to kill the human-infective parasite in vitro. We assessed the effectiveness of this method against the differentiated, amastigote form of the parasite using three distinct viability assays: PrestoBlue, Live/Dead stain and a novel luciferase-based assay. Changes in amastigote morphology and ultrastructure were assessed by immunofluorescence, scanning and transmission electron microscopy. Our findings show that magnetic hyperthermia is an effective method to kill host-infective amastigotes, with morphological changes consistent with heat treatment. This method has the potential to be a step-change for research into new therapeutic options that moves away from the expensive chemotherapeutics currently dominating the research climate.

Magnetic Mechanoactivation of Wnt Signaling Augments Dopaminergic Differentiation of Neuronal Cells.

Wnt signaling is a key developmental pathway that regulates dopaminergic progenitor cell proliferation and differentiation during neuronal development. This makes Wnt signaling an important therapeutic target for neurodegenerative conditions such as Parkinson's disease. Wnt signaling can be modulated using peptides such as UM206, which bind to the Wnt receptor Frizzled. Previous work has demonstrated remote activation of the Wnt pathway through Frizzled using peptide-functionalized magnetic nanoparticles (MNPs) with magnetic field stimulation. Using this technology, Wnt signaling is remotely activated in the neuronal cell line SH-SY5Y, and the phenotypic response to stimulation is assessed. Results indicate ß-catenin translocalization and activation of TCF/LEF responsive transcription in response to MNP and magnetic fields, which result in dopaminergic marker expression when synergistically combined with differentiation factors retinoic acid and the phorbol ester phorbol 12-myristate 13-acetate. This approach is translated into ex vivo postnatal rat brain slices modeling the developing nigrostriatal pathway. Dopaminergic marker expression is maintained in MNP-labeled SH-SY5Y cells after injection and magnetic stimulation. These results demonstrate the translational value of remote control of signal transduction for controlling neuronal precursor cell behavior and highlight the potential applications for controlled cell differentiation as part of cell therapies for neurodegenerative disease.

The potential of magnetic hyperthermia for triggering the differentiation of cancer cells.

Magnetic hyperthermia is a potential technique for cancer therapy that exploits heat generated by magnetic nanoparticles to kill cancerous cells. Many studies have shown that magnetic hyperthermia is effective at killing cancer cells both in vitro and in vivo, however little attention has been paid to the cellular functioning of the surviving cells. We report here new evidence demonstrating the onset of thermally triggered differentiation in osteosarcoma cancer cells that survive magnetic hyperthermia treatment. This raises the possibility that in addition to causing cell death, magnetic hyperthermia could induce surviving cancer cells to form more mature cell types and thereby inhibit their capacity to self-renew. Such processes could prove to be as important as cell death when considering magnetic hyperthermia for treating cancer.

Alternating current (AC) susceptibility as a particle-focused probe of coating and clustering behaviour in magnetic nanoparticle suspensions.

HYPOTHESIS: The functionality of magnetic nanoparticles (MNPs) relies heavily on their surface coating, which in turn affects the interactions between MNPs, and the formation of single-core particles or multi-core clusters. In this study we assessed the use of AC susceptibility (ACS) as a magnetic probe of the kinetics of coating and agglomeration of functionalised nanoparticles. We demonstrate the precision and sensitivity of ACS measurements to small changes in MNP coating using arginine-glycine-aspartic acid (RGD) tripeptide binding, and subsequently discuss how ACS can be used to optimise the preparation of polyethyleneimine (PEI) functionalised MNPs aimed at nanomagnetic transfection applications. EXPERIMENTS: We varied the PEI loading of suspensions of MNPs exhibiting a combination of Brownian and Néel relaxation, and used dialysis to study the movement of excess PEI during the coating process. Numerical ACS simulations were employed to determine particle cluster sizes and polydispersity and the results compared with conventional dynamic light scattering (DLS) size measurements. FINDINGS: ACS provided information on the MNP coating and agglomeration process that was not accessible through DLS due to the additional presence of non-magnetic polymer particulates in the suspensions. We consequently derived a simple method to obtain dense, uniform PEI coatings affording high-stability suspensions without excessive quantities of unbound PEI.

Nanoscale synchrotron X-ray speciation of iron and calcium compounds in amyloid plaque cores from Alzheimer's disease subjects.

Altered metabolism of biometals in the brain is a key feature of Alzheimer's disease, and biometal interactions with amyloid-ß are linked to amyloid plaque formation. Iron-rich aggregates, including evidence for the mixed-valence iron oxide magnetite, are associated with amyloid plaques. To test the hypothesis that increased chemical reduction of iron, as observed in vitro in the presence of aggregating amyloid-ß, may occur at sites of amyloid plaque formation in the human brain, the nanoscale distribution and physicochemical states of biometals, particularly iron, were characterised in isolated amyloid plaque cores from human Alzheimer's disease cases using synchrotron X-ray spectromicroscopy. In situ X-ray magnetic circular dichroism revealed the presence of magnetite: a finding supported by ptychographic observation of an iron oxide crystal with the morphology of biogenic magnetite. The exceptional sensitivity and specificity of X-ray spectromicroscopy, combining chemical and magnetic probes, allowed enhanced differentiation of the iron oxides phases present. This facilitated the discovery and speciation of ferrous-rich phases and lower oxidation state phases resembling zero-valent iron as well as magnetite. Sequestered calcium was discovered in two distinct mineral forms suggesting a dynamic process of amyloid plaque calcification in vivo. The range of iron oxidation states present and the direct observation of biogenic magnetite provide unparalleled support for the hypothesis that chemical reduction of iron arises in conjunction with the formation of amyloid plaques. These new findings raise challenging questions about the relative impacts of amyloid-ß aggregation, plaque formation, and disrupted metal homeostasis on the oxidative burden observed in Alzheimer's disease.

Translation of remote control regenerative technologies for bone repair.

The role of biomechanical stimuli, or mechanotransduction, in normal bone homeostasis and repair is understood to facilitate effective osteogenesis of mesenchymal stem cells (MSCs) in vitro. Mechanotransduction has been integrated into a multitude of in vitro bone tissue engineering strategies and provides an effective means of controlling cell behaviour towards therapeutic outcomes. However, the delivery of mechanical stimuli to exogenous MSC populations, post implantation, poses a significant translational hurdle. Here, we describe an innovative bio-magnetic strategy, MICA, where magnetic nanoparticles (MNPs) are used to remotely deliver mechanical stimuli to the mechano-receptor, TREK-1, resulting in activation and downstream signalling via an external magnetic array. In these studies, we have translated MICA to a pre-clinical ovine model of bone injury to evaluate functional bone repair. We describe the development of a magnetic array capable of in vivo MNP manipulation and subsequent osteogenesis at equivalent field strengths in vitro. We further demonstrate that the viability of MICA-activated MSCs in vivo is unaffected 48 h post implantation. We present evidence to support early accelerated repair and preliminary enhanced bone growth in MICA-activated defects within individuals compared to internal controls. The variability in donor responses to MICA-activation was evaluated in vitro revealing that donors with poor osteogenic potential were most improved by MICA-activation. Our results demonstrate a clear relationship between responders to MICA in vitro and in vivo. These unique experiments offer exciting clinical applications for cell-based therapies as a practical in vivo source of dynamic loading, in real-time, in the absence of pharmacological agents.

Dynamical Magnetic Response of Iron Oxide Nanoparticles Inside Live Cells.

Magnetic nanoparticles exposed to alternating magnetic fields have shown a great potential acting as magnetic hyperthermia mediators for cancer treatment. However, a dramatic and unexplained reduction of the nanoparticle magnetic heating efficiency has been evidenced when nanoparticles are located inside cells or tissues. Recent studies suggest the enhancement of nanoparticle clustering and/or immobilization after interaction with cells as possible causes, although a quantitative description of the influence of biological matrices on the magnetic response of magnetic nanoparticles under AC magnetic fields is still lacking. Here, we studied the effect of cell internalization on the dynamical magnetic response of iron oxide nanoparticles (IONPs). AC magnetometry and magnetic susceptibility measurements of two magnetic core sizes (11 and 21 nm) underscored differences in the dynamical magnetic response following cell uptake with effects more pronounced for larger sizes. Two methodologies have been employed for experimentally determining the magnetic heat losses of magnetic nanoparticles inside live cells without risking their viability as well as the suitability of magnetic nanostructures for in vitro hyperthermia studies. Our experimental results-supported by theoretical calculations-reveal that the enhancement of intracellular IONP clustering mainly drives the cell internalization effects rather than intracellular IONP immobilization. Understanding the effects related to the nanoparticle transit into live cells on their magnetic response will allow the design of nanostructures containing magnetic nanoparticles whose dynamical magnetic response will remain invariable in any biological environments, allowing sustained and predictable in vivo heating efficiency.