Constructing Spatiotemporally Controllable Biocatalytic Cascade in RBC Nanovesicles for Precise Tumor Therapy Based on Reversibly Induced Glucose Oxidase-Magnetoferritin Dimers.

Chemodynamic therapy is a promising tumor treatment strategy. However, it remains a great challenge to overcome the unavoidable off-target damage to normal tissues. In this work, it is discovered that magnetoferritin (M-HFn, biomimic peroxidase) can form nanocomplexes with glucose oxidase (GOD) in the presence of glucose, thus inhibiting the enzyme activity of GOD. Interestingly, GOD&M-HFn (G-M) nanocomplexes can dissociate under near-infrared (NIR) laser, reactivating the enzyme cascade. Based on this new finding, a spatiotemporally controllable biocatalytic cascade in red blood cell (RBC) nanovesicles (G-M@RBC-A) is fabricated for precise tumor therapy, which in situ inhibits enzyme cascade between GOD and M-HFn during blood circulation and reactivates the cascade activity in tumor site by NIR laser irradiation. In RBC nanovesicles, GOD is grabbed by M-HFn to form G-M nanocomplexes in the presence of glucose, thus inhibiting the Fenton reaction and reducing side effects. However, after NIR laser irradiation, G-M nanocomplexes are spatiotemporally dissociated and the cascade activity is reactivated in the tumor site, initiating reactive oxygen species damage to cancer cells in vivo. Therefore, this work provides new insight into the fabrication of spatiotemporally controllable biocatalytic cascade for precise cancer therapy in the future.

Destruction of Lysozyme Amyloid Fibrils Induced by Magnetoferritin and Reconstructed Ferritin.

Neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), or systemic amyloidosis, are characterized by the specific protein transformation from the native state to stable insoluble deposits, e.g., amyloid plaques. The design of potential therapeutic agents and drugs focuses on the destabilization of the bonds in their beta-rich structures. Surprisingly, ferritin derivatives have recently been proposed to destabilize fibril structures. Using atomic force microscopy (AFM) and fluorescence spectrophotometry, we confirmed the destructive effect of reconstructed ferritin (RF) and magnetoferritin (MF) on lysosome amyloid fibrils (LAF). The presence of iron was shown to be the main factor responsible for the destruction of LAF. Moreover, we found that the interaction of RF and MF with LAF caused a significant increase in the release of potentially harmful ferrous ions. Zeta potential and UV spectroscopic measurements of LAF and ferritin derivative mixtures revealed a considerable difference in RF compared to MF. Our results contribute to a better understanding of the mechanism of fibril destabilization by ferritin-like proteins. From this point of view, ferritin derivatives seem to have a dual effect: therapeutic (fibril destruction) and adverse (oxidative stress initiated by increased Fe2+ release). Thus, ferritins may play a significant role in various future biomedical applications.

Enhanced Magnetic Hyperthermia of Magnetoferritin through Synthesis at Elevated Temperature.

Iron oxide nanoparticles have attracted a great deal of research interest in recent years for magnetic hyperthermia therapy owing to their biocompatibility and superior thermal conversion efficiency. Magnetoferritin is a type of biomimetic superparamagnetic iron oxide nanoparticle in a ferritin cage with good monodispersity, biocompatibility, and natural hydrophilicity. However, the magnetic hyperthermic efficiency of this kind of nanoparticle is limited by the small size of the mineral core as well as its low synthesis temperature. Here, we synthesized a novel magnetoferritin particle by using a recombinant ferritin from the hyperthermophilic archaeon Pyrococcus furiosus as a template with high iron atom loading of 9517 under a designated temperature of 90 °C. Compared with the magnetoferritins synthesized at 45 and 65 °C, the one synthesized at 90 °C displays a larger average magnetite and/or maghemite core size of 10.3 nm. This yields an increased saturation magnetization of up to 49.6 emu g-1 and an enhanced specific absorption rate (SAR) of 805.3 W g-1 in an alternating magnetic field of 485.7 kHz and 49 kA m-1. The maximum intrinsic loss power (ILP) value is 1.36 nHm2 kg-1. These results provide new insights into the biomimetic synthesis of magnetoferritins with enhanced hyperthermic efficiency and demonstrate the potential application of magnetoferritin in the magnetic hyperthermia of tumors.

Relaxivity properties of magnetoferritin: The iron loading effect.

Self-assembling ferritin protein cages have been used as a template for magnetic iron oxide nanoparticle synthesis within its 8 nm cavity to be explored as a potential magnetic resonance imaging contrast agent. Here in, magnetic nanocores with various iron content were successfully synthesized using recombinant human H-chain ferritin (HFn) by a controlled mineralization reaction. r1 and r2 relaxivities of the synthesized magnetoferritin nanoparticles were measured and the effect of iron loading factor on the r1 and r2 relaxivity was investigated by using a quite large range of 10 different iron loadings per protein cage (500-5000) at 90 MHz and 300 MHz. The sample with the highest iron loading of 5329 Fe/cage has r2 value of 165.2 mM-1 s-1 and r1 value of 1.98 mM-1 s-1 at 300 MHz. This high r2 value together with a very low protein and iron concentrations (0.03-0.2 mg/mL and 0.15 mM, respectively) renders magnetoferritin very effective T2 contrast agents. However, r1 values were found to be smaller than literature values suggesting that magnetoferritin may not serve as T1 contrast agent in MRI. Moreover, magnetoferritin showed an increase in r2 relaxivity with the iron loadings while r1 values have not been affected by the number of Fe atoms loaded as much as r2 values. This result also sheds light on understanding the formation mechanism of iron oxide core and its contribution on relaxation in MRI.

The Impact of Redox, Hydrolysis and Dehydration Chemistry on the Structural and Magnetic Properties of Magnetoferritin Prepared in Variable Thermal Conditions.

Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plants, animals, and humans. Various ferritin iron core compositions in organisms are associated with specific living requirements, health state, and different biochemical roles of ferritin isomers. Magnetoferritin, a synthetic ferritin derivative, serves as an artificial model system of unusual iron phase structures found in humans. We present the results of a complex structural study of magnetoferritins prepared by controlled in vitro synthesis. Using various complementary methods, it was observed that manipulation of the synthesis technology can improve the physicochemical parameters of the system, which is useful in applications. Thus, a higher synthesis temperature leads to an increase in magnetization due to the formation of the magnetite phase. An increase in the iron loading factor has a more pronounced impact on the protein shell structure in comparison with the pH of the aqueous medium. On the other hand, a higher loading factor at physiological temperature enhances the formation of an amorphous phase instead of magnetite crystallization. It was confirmed that the iron-overloading effect alone (observed during pathological events) cannot contribute to the formation of magnetite.

Magnetic Nanoprobes for Spatio-Mechanical Manipulation in Single Cells.

Magnetic nanoparticles (MNPs) are widely known as valuable agents for biomedical applications. Recently, MNPs were further suggested to be used for a remote and non-invasive manipulation, where their spatial redistribution or force response in a magnetic field provides a fine-tunable stimulus to a cell. Here, we investigated the properties of two different MNPs and assessed their suitability for spatio-mechanical manipulations: semisynthetic magnetoferritin nanoparticles and fully synthetic 'nanoflower'-shaped iron oxide nanoparticles. As well as confirming their monodispersity in terms of structure, surface potential, and magnetic response, we monitored the MNP performance in a living cell environment using fluorescence microscopy and asserted their biocompatibility. We then demonstrated facilitated spatial redistribution of magnetoferritin compared to 'nanoflower'-NPs after microinjection, and a higher magnetic force response of these NPs compared to magnetoferritin inside a cell. Our remote manipulation assays present these tailored magnetic materials as suitable agents for applications in magnetogenetics, biomedicine, or nanomaterial research.

Magnetoferritin enhances T2 contrast in magnetic resonance imaging of macrophages.

Imaging of immune cells has wide implications in understanding disease progression and staging. While optical imaging is limited in penetration depth due to light properties, magnetic resonance (MR) imaging provides a more powerful tool for the imaging of deep tissues where immune cells reside. Due to poor MR signal to noise ratio, tracking of such cells typically requires contrast agents. This report presents an in-depth physical characterization and application of archaeal magnetoferritin for MR imaging of macrophages - an important component of the innate immune system that is the first line of defense and first responder in acute inflammation. Magnetoferritin is synthesized by loading iron in apoferritin in anaerobic condition at 65 °C. The loading method results in one order of magnitude enhancement of r1 and r2 relaxivities compared to standard ferritin synthesized by aerobic loading of iron at room temperature. Detailed characterizations of the magnetoferritin revealed a crystalline core structure that is distinct from previously reported ones indicating magnetite form. The magnetite core is more stable in the presence of reducing agents and has higher peroxidase-like activities compared to the core in standard loading. Co-incubation of macrophage cells with magnetoferritin in-vitro shows significantly higher enhancement in T2-MRI contrast of the immune cells compared to standard ferritin.

Longitudinal and Transverse Relaxivity Analysis of Native Ferritin and Magnetoferritin at 7 T MRI.

Magnetite mineralization in human tissue is associated with various pathological processes, especially neurodegenerative disorders. Ferritin's mineral core is believed to be a precursor of magnetite mineralization. Magnetoferritin (MF) was prepared with different iron loading factors (LFs) as a model system for pathological ferritin to analyze its MRI relaxivity properties compared to those of native ferritin (NF). The results revealed that MF differs statistically significantly from NF, with the same LF, for all studied relaxation parameters at 7 T: r1, r2, r2*, r2/r1, r2*/r1. Distinguishability of MF from NF may be useful in non-invasive MRI diagnosis of pathological processes associated with iron accumulation and magnetite mineralization (e.g., neurodegenerative disorders, cancer, and diseases of the heart, lung and liver). In addition, it was found that MF samples possess very strong correlation and MF's relaxivity is linearly dependent on the LF, and the transverse and longitudinal ratios r2/r1 and r2*/r1 possess complementary information. This is useful in eliminating false-positive hypointensive artefacts and diagnosis of the different stages of pathology. These findings could contribute to the exploitation of MRI techniques in the non-invasive diagnosis of iron-related pathological processes in human tissue.

Quantification of Iron Release from Native Ferritin and Magnetoferritin Induced by Vitamins B2 and C.

Various pathological processes in humans are associated with biogenic iron accumulation and the mineralization of iron oxide nanoparticles, especially magnetite. Ferritin has been proposed as a precursor to pathological magnetite mineralization. This study quantifies spectroscopically the release of ferrous ions from native ferritin and magnetoferritin as a model system for pathological ferritin in the presence of potent natural reducing agents (vitamins C and B2) over time. Ferrous cations are required for the transformation of ferrihydrite (physiological) into a magnetite (pathological) mineral core and are considered toxic at elevated levels. The study shows a significant difference in the reduction and iron release from native ferritin compared to magnetoferritin for both vitamins. The amount of reduced iron formed from a magnetoferritin mineral core is two to five times higher than from native ferritin. Surprisingly, increasing the concentration of the reducing agent affects only iron release from native ferritin. Magnetoferritin cores with different loading factors seem to be insensitive to different concentrations of vitamins. An alternative hypothesis of human tissue magnetite mineralization and the process of iron-induced pathology is proposed. The results could contribute to evidence of the molecular mechanisms of various iron-related pathologies, including neurodegeneration.

Dechlorination of 2,4,4'-trichlorobiphenyl by magnetoferritin with different loading factors.

Polychlorinated biphenyls are synthetic industrial organic substances. These persistent pollutants occur in nature causing high ecological risks and damage to human health. Magnetoferritin nanoparticles composed of apoferritin protein shell surrounding synthetically prepared iron-based nanoparticles seem to be a promising candidate for polychlorinated biphenyls elimination. Properties of magnetoferritin, as a redox activity, a biocompatible character, high application possibilities and a close relationship with the human body promoted ours in vitro investigation of the magnetoferritin catalytic activity in the presence of representative 2,4,4'-trichlorobiphenyl. Basic physico-chemical properties of magnetoferritin were determined by ultraviolet and visible spectrophotometry, dynamic light scattering, zeta potential measurements, superconducting quantum interference device magnetometry and atomic force microscopy. The remediation effect of magnetoferritin on 2,4,4'-trichlorobiphenyl was demonstrated by the use of gas chromatography in combination with infrared spectroscopy.

Peptide-Mediated Immobilization on Magnetoferritin for Enzyme Recycling.

Ferritin possess favorable properties because its exterior and interior surface can be applied to generate functional nanomaterials, which make them possible for enzyme immobilization and recycling. Here, we report the noncovalent immobilization of a genetically modified ß-glucosidase onto the outer surface of synthetic magnetoferritin through the electrostatic interaction of a heterodimeric coiled-coil protein formed by coils containing lysine residues (K-coils) and coils containing glutamic acid (E-coils). The immobilized enzyme was characterized, and its enzymatic properties were evaluated. Furthermore, reusability of immobilized enzyme was demonstrated in aqueous solution under an applied magnetic field. The results showed that magnetoferritin was successfully prepared and it was an excellent support for enzyme immobilization. After three times usages, the retention rates were 93.75%, 82.5%, and 56.25%, respectively, demonstrating that immobilized enzyme possessed good retention efficiency and could be used as potential carrier for other biomolecules. The strategy of enzyme immobilization developed in this work can be applied, in general, to many other target molecules.

Magnetoferritin: Process, Prospects, and Their Biomedical Applications.

Ferritin is a spherical iron storage protein composed of 24 subunits and an iron core. Using biomimetic mineralization, magnetic iron oxide can be synthesized in the cavity of ferritin to form magnetoferritin (MFt). MFt, also known as a superparamagnetic protein, is a novel magnetic nanomaterial with good biocompatibility and flexibility for biomedical applications. Recently, it has been demonstrated that MFt had tumor targetability and a peroxidase-like catalytic activity. Thus, MFt, with its many unique properties, provides a powerful platform for tumor diagnosis and therapy. In this review, we discuss the biomimetic synthesis and biomedical applications of MFt.

Proposal to use superparamagnetic nanoparticles to test the role of cryptochrome in magnetoreception.

Evidence is accumulating to support the hypothesis that some animals use light-induced radical pairs to detect the direction of the Earth's magnetic field. Cryptochrome proteins seem to be involved in the sensory pathway but it is not yet clear if they are the magnetic sensors: they could, instead, play a non-magnetic role as signal transducers downstream of the primary sensor. Here we propose an experiment with the potential to distinguish these functions. The principle is to use superparamagnetic nanoparticles to disable any magnetic sensing role by enhancing the electron spin relaxation of the radicals so as to destroy their spin correlation. We use spin dynamics simulations to show that magnetoferritin, a synthetic, protein-based nanoparticle, has the required properties. If cryptochrome is the primary sensor, then it should be inactivated by a magnetoferritin particle placed 12-16 nm away. This would prevent a bird from using its magnetic compass in behavioural tests and abolish magnetically sensitive neuronal firing in the retina. The key advantage of such an experiment is that any signal transduction role should be completely unaffected by the tiny magnetic interactions (≪kBT) required to enhance the spin relaxation of the radical pair.

Cellular uptake and apoptotic potential of rhenium labeled magnetic protein cages in MDA-MB-231 cells.

188Re-magnetoferritin nanoparticles (NPs) provide an attractive platform for localized radiation therapy due to their magnetic targeting capability while enhancing contrast in magnetic resonans imaging (MRI) signals. In this study, cellular uptake, in vitro cytotoxicity, apoptotic potential of a non-radioactive isotope of rhenium in the form of 187Re-magnetoferritin NPs were evaluated in both human normal mammary epithelial and breast metastatic adenocarcinoma cell lines. The results showed that, NP administration into the cells is through receptor mediated endocytosis and cancer cells displayed significantly higher uptake and cytotoxicity compared to normal cells. IC50 values of nanoparticles were calculated as 0.96 mg/mL for cancer and 1.73 mg/mL for normal cells. Annexin V/ Propidium Iodide (PI) staining also showed that, NPs induced higher apoptotic rates in cancer cells compared to normal cells. Gene expression analyses confirming the results showed that, pro-apoptotic PUMA and BAX genes were significantly up-regulated while anti-apoptotic BCL-2 and SURVIVIN genes were down-regulated in cancer cells compared to normal cells. Overall, these in vitro results suggest that, 187Re-magnetoferritin NPs have a promising potential for cancer therapy and can be used for imaging and diagnostic purposes for breast cancer at concentrations lower than 0.96 mg/mL. At concentrations above 1 mg/mL, NPs induce apoptosis which can also be used for cancer treatments.

Application of cationized magnetoferritin for magnetic field-assisted delivery of short interfering RNA in vitro.

Cationized magnetoferritin is used for development of a simple, efficient, and fast delivery of short interference RNA into cells using combination of magnetophoresis for pre-concentration of siRNA-magnetoferritin complex on the surface of plated cells with subsequent application of nanosecond laser pulses producing stress waves in transfection chamber, which permeabilize cell membrane for the facilitated delivery of siRNA into the cell interior. As has been quantified using siRNA inducing cell death assay, by combination of these two physical factors we have obtained high efficiency for tested three different human carcinoma cells. Proposed method of gene silencing based on cationized magnetoferritin is a versatile and easily accessible platform with many possible applications in gene therapy.

Physiological origin of biogenic magnetic nanoparticles in health and disease: from bacteria to humans.

The discovery of biogenic magnetic nanoparticles (BMNPs) in the human brain gives a strong impulse to study and understand their origin. Although knowledge of the subject is increasing continuously, much remains to be done for further development to help our society fight a number of pathologies related to BMNPs. This review provides an insight into the puzzle of the physiological origin of BMNPs in organisms of all three domains of life: prokaryotes, archaea, and eukaryotes, including humans. Predictions based on comparative genomic studies are presented along with experimental data obtained by physical methods. State-of-the-art understanding of the genetic control of biomineralization of BMNPs and their properties are discussed in detail. We present data on the differences in BMNP levels in health and disease (cancer, neurodegenerative disorders, and atherosclerosis), and discuss the existing hypotheses on the biological functions of BMNPs, with special attention paid to the role of the ferritin core and apoferritin.

The effect of solution pH on the structural stability of magnetoferritin.

The structural stability of magnetoferritin, a synthetic analogue of ferritin, at various pH levels is assessed here. The structural and electrical properties of the complexes were determined by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS) and zeta potential measurements. At pH 3-6 a reduction of electrostatic repulsion on the suspended colloids resulted in aggregation and sedimentation of magnetoferritin. At neutral to slightly alkaline conditions (pH 7-9) the magnetoferritin structure was stable for lower iron loadings. Higher solution pH 10-12 induced destabilization of the protein structure and dissociation of subunits. Increasing the loading factor in the MFer complex leads to decrease of the stability versus pH changes.

Bioengineered Magnetoferritin Nanoprobes for Single-Dose Nuclear-Magnetic Resonance Tumor Imaging.

Despite all the advances in multimodal imaging, it remains a significant challenge to acquire both magnetic resonance and nuclear imaging in a single dose because of the enormous difference in sensitivity. Indeed, nuclear imaging is almost 10(6)-fold more sensitive than magnetic resonance imaging (MRI); thus, repeated injections are generally required to obtain sufficient MR signals after nuclear imaging. Here, we show that strategically engineered magnetoferritin nanoprobes can image tumors with high sensitivity and specificity using SPECT and MRI in living mice after a single intravenous injection. The magnetoferritin nanoprobes composed of (125)I radionuclide-conjugated human H-ferritin iron nanocages ((125)I-M-HFn) internalize robustly into cancer cells via a novel tumor-specific HFn-TfR1 pathway. In particular, the endocytic recycling characteristic of TfR1 transporters solves the nuclear signal blocking issue caused by the high dose nanoprobes injected for MRI, thus enabling simultaneous functional and morphological tumor imaging without reliance on multi-injections.

Enhanced magnetic resonance imaging and staining of cancer cells using ferrimagnetic H-ferritin nanoparticles with increasing core size.

PURPOSE: This study is to demonstrate the nanoscale size effect of ferrimagnetic H-ferritin (M-HFn) nanoparticles on magnetic properties, relaxivity, enzyme mimetic activities, and application in magnetic resonance imaging (MRI) and immunohistochemical staining of cancer cells. MATERIALS AND METHODS: M-HFn nanoparticles with different sizes of magnetite cores in the range of 2.7-5.3 nm were synthesized through loading different amounts of iron into recombinant human H chain ferritin (HFn) shells. Core size, crystallinity, and magnetic properties of those M-HFn nanoparticles were analyzed by transmission electron microscope and low-temperature magnetic measurements. The MDA-MB-231 cancer cells were incubated with synthesized M-HFn nanoparticles for 24 hours in Dulbecco's Modified Eagle's Medium. In vitro MRI of cell pellets after M-HFn labeling was performed at 7 T. Iron uptake of cells was analyzed by Prussian blue staining and inductively coupled plasma mass spectrometry. Immunohistochemical staining by using the peroxidase-like activity of M-HFn nanoparticles was carried out on MDA-MB-231 tumor tissue paraffin sections. RESULTS: The saturation magnetization (M(s)), relaxivity, and peroxidase-like activity of synthesized M-HFn nanoparticles were monotonously increased with the size of ferrimagnetic cores. The M-HFn nanoparticles with the largest core size of 5.3 nm exhibit the strongest saturation magnetization, the highest peroxidase activity in immunohistochemical staining, and the highest r2 of 321 mM(-1) s(-1), allowing to detect MDA-MB-231 breast cancer cells as low as 10(4) cells mL(-1). CONCLUSION: The magnetic properties, relaxivity, and peroxidase-like activity of M-HFn nanoparticles are size dependent, which indicates that M-HFn nanoparticles with larger magnetite core can significantly enhance performance in MRI and staining of cancer cells.