Engineered Ferritin for Magnetogenetic Manipulation of Proteins and Organelles Inside Living Cells

Magnetogenetics is emerging as a novel approach for remote‐controlled manipulation of cellular functions in tissues and organisms with high spatial and temporal resolution. A critical, still challenging issue for these techniques is to conjugate target proteins with magnetic probes that can satisfy multiple colloidal and biofunctional constraints. Here, semisynthetic magnetic nanoparticles are tailored based on human ferritin coupled to monomeric enhanced green fluorescent protein (mEGFP) for magnetic manipulation of proteins inside living cells. This study demonstrates efficient delivery, intracellular stealth properties, and rapid subcellular targeting of those magnetic nanoparticles via GFP–nanobody interactions. By means of magnetic field gradients, rapid spatial reorganization in the cytosol of proteins captured to the nanoparticle surface is achieved. Moreover, exploiting efficient nanoparticle targeting to intracellular membranes, remote‐controlled arrest of mitochondrial dynamics using magnetic fields is demonstrated. The studies establish subcellular control of proteins and organelles with unprecedented spatial and temporal resolution, thus opening new prospects for magnetogenetic applications in fundamental cell biology and nanomedicine.     

Biodegradation Mechanisms of Iron Oxide Monocrystalline Nanoflowers and Tunable Shield Effect of Gold Coating

Understanding the relation between the structure and the reactivity of nanomaterials in the organism is a crucial step towards efficient and safe biomedical applications. The multi‐scale approach reported here, allows following the magnetic and structural transformations of multicore maghemite nanoflowers in a medium mimicking intracellular lysosomal environment. By confronting atomic‐scale and macroscopic information on the biodegradation of these complex nanostuctures, we can unravel the mechanisms involved in the critical alterations of their hyperthermic power and their Magnetic Resonance imaging T₁ and T₂ contrast effect. This transformation of multicore nanoparticles with outstanding magnetic properties into poorly magnetic single core clusters highlights the harmful influence of cellular medium on the therapeutic and diagnosis effectiveness of iron oxide‐based nanomaterials. As biodegradation occurs through surface reactivity mechanism, we demonstrate that the inert activity of gold nanoshells can be exploited to protect iron oxide nanostructures. Such inorganic nanoshields could be a relevant strategy to modulate the degradability and ultimately the long term fate of nanomaterials in the organism.     

Nanopatterning of ferritin molecules and the controlled size reduction of their magnetic cores

A nanopatterning method to deposit ferritin proteins with nanoscale accuracy over large areas is reported. Selective deposition is driven by the electrostatic interactions existing between the proteins and nanoscale features. Upon deposition, the protein shell can be removed by heating the patterns in an oxygen atmosphere. This leaves exposed the iron oxide core, which can be further reduced in size by plasma-etching methods. In this way, the initial ferritin molecules, which have a nominal size of 12 nm, are reduced to 2 nm nanoparticles. Magnetic force measurements confirm the magnetic activity of the as-deposited and etched nanoparticles.     

Designing Non‐Native Iron‐Binding Site on a Protein Cage for Biological Synthesis of Nanoparticles

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self‐assembly of 60 subunits, has been functionalized with non‐native iron‐mineralization capability by incorporating two types of iron‐binding peptides. The non‐native peptides introduced at the interior surface do not affect the self‐assembly of E2 protein subunits. In contrast to the wild‐type, the engineered E2 protein cages can serve as size‐ and shape‐constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host‐guest encapsulation properties of protein cages.     

Ferritin surplus in mouse spleen 14 months after intravenous injection of iron oxide nanoparticles at clinical dose

In this study,we followed the biodegradation of ultra-small superparamagnetic iron oxide nanoparticles injected intravenously at clinical doses in mice.An advanced fitting procedure for magnetic susceptibility curves and lowtemperature hysteresis loops was used to fully characterize the magnetic size distribution as well as the magnetic anisotropy energy of the injected P904 nanoparticles (Guerbet Laboratory).Additional magnetometry measurements and transmission electronic microscopy observations were systematically performed to examine dehydrated samples from the spleen and liver of healthy C57B16 mice after nanoparticle injection,with sacrifice of the mice for up to 14 months.At 3 months after injection,the magnetic properties of the spleen and liver were dramatically different.While the liver showed no magnetic signals other than those also present in the reference species,the spleen showed an increased magnetic signal attributed to ferritin.This surplus of ferritin remained constant up to 14 months after injection.     

Formation of water-soluble iron oxide nanoparticles derived from iron storage protein

This paper reports novel findings of an investigation of the formation of water-soluble iron oxide nanoparticles from iron-storage protein ferritin. The strategy couples thermal removal of the protein shell on a planar substrate and subsequent sonication in aqueous solution under controlled temperature. Advantages of using ferritin as a precursor include well-defined core size, core composition, water-solubility and processibility. The formation of the nanoparticles was characterized using TEM, UV-Vis and FTIR techniques. Iron oxide nanoparticles in the size range of 5-20 nm diameters were produced. In addition to thermal treatment conditions, the sonication temperature of the nanoparticles in water was found to play an important role in determining the resulting particle size. This simple and effective route has important implications to the design of composite nanoparticles for potential magnetic, catalytic, biomedical sensing and other nanotechnological applications.     

Cellular uptake of protein-bound magnetic nanoparticles in pulsed magnetic field

A method for fast delivery of proteins conjugated to superparamagnetic iron oxide nanoparticles (SPION) into mammalian cells by applying a strong magnetic field in pulses was proposed. Firstly, SPION were prepared from an alkaline solution of divalent and trivalent iron ions and covalently bound with protein through the activation of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC). After fluorescently labelling, the protein-nanoparticle conjugate was mixed with mammalian cell line and exposed to a pulsed magnetic field for short durations of few milliseconds. Results suggested that superparamagnetic nanoparticles were able to carry proteins into living cells immediately. Cellular internalization of the fluorescently labelled protein-nanoparticle conjugate was proved by the observation of cell fluorescence in a fluorescent microscopy, as well as cell analysis by a flow cytometer. We found that the cellular uptake was accomplished dominantly by the process of bombardment of magnetic nanoparticles.     

Synthesis,self-assembly,and characterization of PEG-coated iron oxide nanoparticles as potential MRI contrast agent

Aim: Investigated the self-assembly and characterization of novel antifouling polyethylene glycol (PEG)-coated iron oxide nanoparticles as nanoprobes for magnetic resonance imaging (MRI) contrast agent. Method: Monodisperse oleic acid-coated superparamagnetic iron oxide cores are synthesized by thermal decomposition of iron oleate. The self-assembly behavior between iron oxide cores and PEG-lipid conjugates in water and their characteristics are confirmed by transmission electron microscope, X-ray diffraction, thermogravimetric analysis, Fourier transform infrared spectroscopy, and vibrating sample magnetometer. Result: Dynamic light scattering shows superparamagnetic iron oxide nanoparticles coated with PEG are stable in water for pH of 3–10 and ionic strengths up to 0.3 M NaCl, and are protein resistant in physiological conditions. Additionally, in vitro MRI study demonstrates the efficient magnetic resonance imaging contrast characteristics of the iron oxide nanoparticles. Conclusion: The result indicates that the novel antifouling PEG-coated superparamagnetic iron oxide nanoparticles could potentially be used in a wide range of applications such as biotechnology, MRI, and magnetic fluid hyperthermia.     

Synthesis of ordered iron oxide nanoparticle arrays in planar DNA complexes

The formation of iron oxide nanoparticles in planar DNA complexes immobilized on substrates has been studied in reactions involving only biogenic reagents (ferritin and ascorbic acid) in aqueous solutions under normal conditions. Using transmission electron microscopy, we revealed ordered quasi-linear arrays of iron oxide nanoparticles 2–4 nm in diameter, which probably resulted from nanoparticle binding to linear DNA molecules. The electron diffraction patterns of the synthesized nanoparticles are characteristic of polycrystalline magnetic iron oxide (magnetite of maghemite) nanoparticles and point to good crystallinity of the nanoparticles. Our results demonstrate the feasibility of the synthesis of ordered arrays of iron oxide nanoparticles using DNA complexes and may have direct implications for the understanding of biomineralization processes and iron metabolism in living systems.     

纳米铁颗粒及其复合材料的界面设计及环境修复应用

近年来, 纳米铁颗粒(纳米零价铁)因其优异的催化/还原性能, 并且价廉、环境友好, 已成为主要的环境修复材料之一。目前, 纳米铁颗粒主要用于水体修复, 如: 重金属离子去除、有机物污染物降解和无机阴离子催化还原等。纳米铁颗粒易团聚和结构单一等问题会导致其活性低、稳定性差和去除种类单一。为了克服上述问题, 迫切需要研究纳米铁颗粒的界面设计。本文重点阐述纳米铁颗粒及其复合材料的可控制备、界面设计、在重金属去除和硝酸根去除转化中的应用以及在环境修复中的未来发展方向。     

Facile synthesis of magnetic iron oxide nanoparticles and their characterization

Magnetic iron oxide nanoparticles are synthesized by suitable modification of the standard synthetic procedure without use of inert atmosphere and at room temperature. The facile synthesis procedure can be easily scaled up and is of important from industrial point of view for the commercial large scale production of magnetic iron oxide nanoparticles. The synthesized nanoparticles were characterized by thermal, dynamic light scattering, scanning electron microscopy and transmission electron microscopy analyses.     

Iron oxide shell as the oxidation-resistant layer in SmCo5 @ Fe2O3 core-shell magnetic nanoparticles

This paper presents a synthesis of magnetic nanoparticles of samarium cobalt alloys and the use of iron oxide as a coating layer to prevent the rapid oxidation of as-made Sm-Co nanoparticles. The colloidal nanoparticles of Sm-Co alloys were made in octyl ether using samarium acetylacetonate and dicobalt octacarbonyl as precursors in a mixture of 1,2-hexadecanediol, oleic acid, and trioctylphosphine oxide (TOPO). Such Sm-Co nanoparticle could be readily oxidized by air and formed a CoO antiferromagnetic layer. Exchange biasing was observed for the surface oxidized nanoparticles. In situ thermal decomposition of iron pentacarbonyl was used to create iron oxide shells on the Sm-Co nanoparticles. The iron oxide shell could prevent Sm-Co nanoparticles from rapid oxidation upon the exposure to air at ambient conditions.     

Intracellular Nanoparticle Coating Stability Determines Nanoparticle Diagnostics Efficacy and Cell Functionality

Iron oxide nanoparticles (NPs) are frequently employed in biomedical research as magnetic resonance (MR) contrast agents where high intracellular levels are required to clearly depict signal alterations. To date, the toxicity and applicability of these particles have not been completely unraveled. Here, we show that endosomal localization of different iron oxide particles results in their degradation and in reduced MR contrast, the rate of which is governed mainly by the stability of the coating. The release of ferric iron generates reactive species, which greatly affect cell functionality. Lipid‐coated NPs display the highest stability and furthermore exhibit intracellular clustering, which significantly enhances their MR properties and intracellular persistence. These findings are of considerable importance because, depending on the nature of the coating, particles can be rapidly degraded, thus completely annihilating their MR contrast to levels not detectable when compared to controls and greatly impeding cell functionality, thereby hindering their application in functional in vivo studies.     

Design of iron oxide/silica/alginate hybrid magnetic carriers (HYMAC)

A large number of natural and synthetic polymers have already been evaluated for the design of nanomaterials incorporating magnetic nanoparticles for biomedical applications. The possibility to use hybrid (bio)-organic/inorganic nano-carriers have been much less studied. Here we describe the design of Hybrid MAgnetic Carriers (HYMAC) consisting of alginate/silica nanocomposites incorporating magnetite nanoparticles, based on a spray-drying approach. Transmission electron microscopy and X-ray energy dispersive spectrometry confirm the successful incorporation of magnetic colloids within homogeneous hybrid capsules. X-ray diffraction data suggest that surface iron ions are partially desorbed by the spray-drying process, leading to the formation of lepidocrocite and of an iron silicate phase. Magnetic measurements show that the resulting nanocomposites exhibit a superparamagnetic behaviour with a blocking temperature close to 225 K. Comparison with un-silicified capsules indicate that the mineral phase enhances the thermal stability of the polymer network and do not modify of the amount of incorporated iron oxide nanoparticles. Moreover, evaluation of nanocomposite up-take by fibroblasts indicates their possible internalization. A selective intracellular alginate degradation is observed, suggesting that these HYMAC nanomaterials may exhibit interesting properties for the design of drug delivery devices.     

Epirubicin‐Loaded Superparamagnetic Iron‐Oxide Nanoparticles for Transdermal Delivery: Cancer Therapy by Circumventing the Skin Barrier

The transdermal administration of chemotherapeutic agents is a persistent challenge for tumor treatments. A model anticancer agent, epirubicin (EPI), is attached to functionalized superparamagnetic iron‐oxide nanoparticles (SPION). The covalent modification of the SPION results in EPI–SPION, a potential drug delivery vector that uses magnetism for the targeted transdermal chemotherapy of skin tumors. The spherical EPI–SPION composite exhibits excellent magnetic responsiveness with a saturation magnetization intensity of 77.8 emu g^?1. They feature specific pH‐sensitive drug release, targeting the acidic microenvironment typical in common tumor tissues or endosomes/lysosomes. Cellular uptake studies using human keratinocyte HaCaT cells and melanoma WM266 cells demonstrate that SPION have good biocompatibility. After conjugation with EPI, the nanoparticles can inhibit WM266 cell proliferation; its inhibitory effect on tumor proliferation is determined to be dose‐dependent. In vitro transdermal studies demonstrate that the EPI–SPION composites can penetrate deep inside the skin driven by an external magnetic field. The magnetic‐field‐assisted SPION transdermal vector can circumvent the stratum corneum via follicular pathways. The study indicates the potential of a SPION‐based vector for feasible transdermal therapy of skin cancer.     

Self-assembled virus-like particles with magnetic cores

Efficient encapsulation of functionalized spherical nanoparticles by viral protein cages was found to occur even if the nanoparticle is larger than the inner cavity of the native capsid. This result raises the intriguing possibility of reprogramming the self-assembly of viral structural proteins. The iron oxide nanotemplates used in this work are superparamagnetic, with a blocking temperature of about 250 K, making these virus-like particles interesting for applications such as magnetic resonance imaging and biomagnetic materials. Another novel feature of the virus-like particle assembly described in this work is the use of an anionic lipid micelle coat instead of a molecular layer covalently bound to the inorganic nanotemplate. Differences between the two functionalization strategies are discussed.     

Core-shell iron-iron oxide nanoparticles synthesized by laser-induced pyrolysis

Passivated iron nanoparticles (10-30 nm) have been synthesized by laser pyrolysis of a mixture of iron pentacarbonyl and ethylene vapors followed by controlled oxidation. The nanoparticles show a well-constructed iron-iron oxide core-shell structure, in which the thickness and nature (structure similar to maghemite, gamma-Fe2O3) of the shell is found to be independent of the initial conditions. On the other hand, the composition of the core is found to change with the particle size from the alpha-Fe structure to a highly disordered Fe phase (probably containing C atoms in its structure). The dependence of the magnetic properties on the particle size, iron oxide fraction, and temperature was also investigated. In the case of smaller particles, the magnetic data indicate the existence at low temperature of a large exchange anisotropy field, the magnitude of which increases with decreasing temperature in correspondence with the freezing of magnetic moments in the oxide shell.     

Alignment under Magnetic Field of Mixed Fe2O3/SiO2 Colloidal Mesoporous Particles Induced by Shape Anisotropy

When using the bottom‐up approach with anisotropic building‐blocks, an important goal is to find simple methods to elaborate nanocomposite materials with a truly macroscopic anisotropy. Here, micrometer size colloidal mesoporous particles with a highly anisotropic rod‐like shape (aspect ratio ≈ 10) have been fabricated from silica (SiO₂) and iron oxide (Fe₂O₃). When dispersed in a solvent, these particles can be easily oriented using a magnetic field (≈200 mT). A macroscopic orientation of the particles is achieved, with their long axis parallel to the field, due to the shape anisotropy of the magnetic component of the particles. The iron oxide nanocrystals are confined inside the porosity and they form columns in the nanochannels. Two different polymorphs of Fe₂O₃ iron oxide have been stabilized, the superparamagnetic γ‐phase and the rarest multiferroic ε‐phase. The phase transformation between these two polymorphs occurs around 900 °C. Because growth occurs under confinement, a preferred crystallographic orientation of iron oxide is obtained, and structural relationships between the two polymorphs are revealed. These findings open completely new possibilities for the design of macroscopically oriented mesoporous nanocomposites, using such strongly anisotropic Fe₂O₃/silica particles. Moreover, in the case of the ε‐phase, nanocomposites with original anisotropic magnetic properties are in view.     

Structure control synthesis of iron oxide polymorph nanoparticles through an epoxide precipitation route

Iron oxide polymorph nanoparticles (magnetite, goethite and lepidocrocite) were synthesised through a novel epoxide precipitation route. Epoxide, as a precipitating agent, induced the precipitation of iron(II) ions in the form of hydroxide through its reaction with aquocomplexes of Fe²⁺ ions. The organic intermediate, which was produced from the ring-opening reaction of epoxide, functionalised the surface of the formed precipitate in situ, resulting in the minimisation of the nanoparticles’ aggregation. Another advantage of this route is its simple procedure for the preparation of iron oxide nanoparticles with different structures and their well-defined morphology. Magnetite, goethite and lepidocrocite nanoparticles were obtained just by the pH regulation and air oxidation at room temperature.     

Metal–Organic Framework Nanoparticles Induce Pyroptosis in Cells Controlled by the Extracellular pH

Ion homeostasis is essential for cellular survival, and elevated concentrations of specific ions are used to start distinct forms of programmed cell death. However, investigating the influence of certain ions on cells in a controlled way has been hampered due to the tight regulation of ion import by cells. Here, it is shown that lipid-coated iron-based metal–organic framework nanoparticles are able to deliver and release high amounts of iron ions into cells. While high concentrations of iron often trigger ferroptosis, here, the released iron induces pyroptosis, a form of cell death involving the immune system. The iron release occurs only in slightly acidic extracellular environments restricting cell death to cells in acidic microenvironments and allowing for external control. The release mechanism is based on endocytosis facilitated by the lipid-coating followed by degradation of the nanoparticle in the lysosome via cysteine-mediated reduction, which is enhanced in slightly acidic extracellular environment. Thus, a new functionality of hybrid nanoparticles is demonstrated, which uses their nanoarchitecture to facilitate controlled ion delivery into cells. Based on the selectivity for acidic microenvironments, the described nanoparticles may also be used for immunotherapy: the nanoparticles may directly affect the primary tumor and the induced pyroptosis activates the immune system.