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.

Synthesis, characterization and application of magnetoferritin nanoparticle by using human H chain ferritin expressed by Pichia pastoris.

Protein-based nanoparticles have developed rapidly in areas such as drug delivery, biomedical imaging and biocatalysis. Ferritin possesses unique properties that make it attractive as a potential platform for a variety of nanobiotechnological applications. Here we synthesized magnetoferritin (P-MHFn) nanoparticles for the first time by using the human H chain of ferritin that was expressed by Pichia pastoris (P-HFn). Western blot results showed that recombinant P-HFn was successfully expressed after methanol induction. Transmission electron microscopy (TEM) showed the spherical cage-like shape and monodispersion of P-HFn. The synthesized magnetoferritin (P-MHFn) retained the properties of magnetoferritin nanoparticles synthesized using HFn expressed by E. coli (E-MHFn): superparamagnetism under ambient conditions and peroxidase-like activity. It is stable under a wider range of pH values (from 5.0 to 11.0), likely due to post-translational modifications such as N-glycosylation on P-HFn. In vivo near-infrared fluorescence imaging experiments revealed that P-MHFn nanoparticles can accumulate in tumors, which suggests that P-MHFn could be used in tumor imaging and therapy. An acute toxicity study of P-MHFn in Sprague Dawley rats showed no abnormalities at a dose up to 20 mg Fe Kg-1 body weight. Therefore, this study shed light on the development of magnetoferritin nanoparticles using therapeutic HFn expressed by Pichia pastoris for biomedical applications.

Enhanced peroxidase activity and tumour tissue visualization by cobalt-doped magnetoferritin nanoparticles.

Magnetoferritin (M-HFn) is a biomimetic magnetic nanoparticle with a human heavy-chain ferritin (HFn) shell, trapping a magnetite (Fe3O4) core that has inherited peroxidase-like activity. In this study, cobalt-doped M-HFn nanoparticles (M-HFn-Co x Fe3-x O4) with different amounts of cobalt were successfully synthesized. Experimental results indicate that the controlled doping of a certain amount of cobalt into the magnetite cores of M-HFn nanoparticles enhances its peroxidase-like catalytic activity and efficacy for visualizing tumour tissues. For example, compared with sample Co0 (without cobalt doping), the peroxidase-like activity of the cobalt-doped nanoparticle sample Co60 (with a cobalt doping molar percentage of ∼34.2%) increases 1.7 times, and has the maximal reaction velocity (V max) values. Moreover, after a one-step incubation with Co60 nanoparticles, and using the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB) for colour development, the tumour tissues of breast, colorectal, stomach and pancreas tumours showed a deeper brown colour with clear boundaries between the healthy and tumourous cells. Therefore, this suggests that the cobalt-doped magnetoferritin nanoparticles enhance peroxidase activity and tumour tissue visualization.

Identification of ferrous-ferric Fe3O4 nanoparticles in recombinant human ferritin cages.

Recombinant ferritin is an excellent template for the synthesis of magnetic nanoparticles. This paper describes carefully performed experiments both to identify ironoxides within nanoparticles and to measure the number of iron atoms in the cores of recombinant human H-chain ferritin (HFn), based on spectroscopy techniques. Using electron energy-loss spectroscopy (EELS) analysis, magnetite (Fe3O4) has been unequivocally identified as the ironoxide formed within HFn cores under special preparation conditions. Atom counting analysis by EELS and high-angle annular dark-field imaging further allowed the correlation of the particle sizes to the real Fe atom numbers in a quantitative manner. These results help clarify some structural confusion between magnetite and maghemite (γ-Fe2O3), and also provide standard data for the number of Fe atoms within Fe3O4 particles of a given size, whose use is not limited to cases of magnetite synthesized in the cores of recombinant human ferritin.

A solar-driven degradation-evaporation strategy for membrane self-cleaning in the efficient separation of viscous crude oil/water emulsions.

Membrane separation techniques are promising methods for effectively treating hazardous emulsified oily wastewater, but membrane fouling remains a serious challenge because the high viscosity and complex composition of crude oil make it easy to adhere to membranes and difficult to be removed by conventional physical or chemical cleaning means. Herein, a two-stage solar-driven (photo-Fenton degradation/evaporation) strategy was proposed to realize the self-cleaning of membranes fouled by viscous crude oil (>60,000 mPa s), wherein the photo-Fenton process helped to degrade the heavy components into light components, and all light components removed during the solar-driven evaporation process. A 1D/2D heterostructure membrane with photo-Fenton activity and anti-crude-oil-fouling performance was prepared via a facile self-assembly vacuum-assist method. The addition of rod-like g-C3N4 (RCN) increased the interlayer distance of α-FeOOH/porous g-C3N4 (FPCN) nanosheets, resulting in a high permeation flux. The FPCN-RCN membrane exhibited both high permeation flux of 779 ± 19 L m-2h-1bar-1 and a separation efficiency of 99.4% for highly viscous crude oil-in-water emulsion. Importantly, the viscous crude oil fouled on the membrane was completely removed by the photo-Fenton degradation/solar-driven evaporation strategy, and the flux recovery rate of the membrane was ∼100%. Therefore, the FPCN-RCN membrane combined with the novel self-cleaning strategy exhibits great potential for practical emulsified oily wastewater treatment.

The influence of reaction temperature on biomineralization of ferrihydrite cores in human H-ferritin.

Ferritin is not only important for iron storage and detoxification in living organisms, but a multifunctional size-constrained nanoplatform for biomimetic nanoparticles. In order to tailor the biomimetic nanoparticles for future applications, it is essential to investigate the effects of external factors such as temperature on the particle size and structure of reconstituted cores in ferritin. In this study, we systematically investigated the mineral composition, crystallinity, and particle size of human H-ferritin (HuHF) reconstituted at four different temperatures (25, 30, 37, and 42°C) by integrated magnetic and transmission electron microscopy analyses. Our results showed that the particle size of reconstituted ferrihydrite cores (~5 nm) in HuHF was temperature-independent. However, the significant changes of the induced magnetization at 5 T field (M(5T)) and remanent magnetization (M(r)) at 5 K clearly showed that the crystallinity of reconstituted cores increased with increasing temperature, indicating that the reaction temperature deeply affects the structural order of reconstituted ferrihydrite cores rather than the particle size, and the reconstituted cores become more ordered at higher reaction temperatures. Our findings provide useful insights into biomineralization of ferritin under in vivo fever condition as well as in biomimetic synthesis of nanomaterials using ferritin. Furthermore, the rock magnetic methods should be very useful approaches for characterizing finite ferritin nanoparticles.

Thermostable iron oxide nanoparticle synthesis within recombinant ferritins from the hyperthermophile Pyrococcus yayanosii CH1.

Thermostable nanoparticles have numerous applications in catalysis and in the oil/gas industry. However, synthesizing these nanoparticles requires expensive polymers. Here, a novel thermostable ferritin named PcFn, originally from the hyperthermophilic archaeon Pyrococcus yayanosii CH1, was overexpressed in Escherichia coli, purified and characterized, which could successfully direct the synthesis of thermostable magnetoferritins (M-PcFn) with monodispersed iron oxide nanoparticles in one step. Transmission electron microscopy and magnetic measurements show that the cores of the M-PcFn have an average diameter of 4.7 nm, are well-crystalline and superparamagnetic. Both the PcFn and M-PcFn can resist temperatures up to 110 °C, which is significantly higher than for human H-chain ferritin (HFn) and M-HFn, and comparable to temperatures previously reported for Pyrococcus furiosus ferritin (PfFn) and M-PfFn. After heating at 110 °C for 30 minutes, PcFn and M-PcFn maintained their secondary structures and PcFn retained 87.4% of its iron uptake activity. This remarkable thermostability of PcFn and M-PcFn suggests potential applications in elevated temperature environments.

Positive magnetic resonance angiography using ultrafine ferritin-based iron oxide nanoparticles.

Iron oxide nanoparticles with good biocompatibility can serve as safe magnetic resonance imaging contrast agents. Herein, we report that ultrafine ferritin-based iron oxide (hematite/maghemite) nanoparticles synthesized by controlled biomimetic mineralization using genetically recombinant human H chain ferritin can be used as a positive contrast agent in magnetic resonance angiography. The synthesized magnetoferritin with an averaged core size of 2.2 ± 0.7 nm (hereafter named M-HFn-2.2) shows a r1 value of 0.86 mM-1 s-1 and a r2/r1 ratio of 25.1 at a 7 T magnetic field. Blood pool imaging on mice using the M-HFn-2.2 nanoparticles that were injected through a tail vein by single injection at a dose of 0.54 mM Fe per kg mouse body weight enabled detecting detailed vascular nets at 3 minutes post-injection; the MR signal intensity continuously enhanced up to 2 hours post-injection, which is much longer than that of the commercial magnevist (Gd-DTPA) contrast. Moreover, biodistribution examination indicates that organs such as liver, spleen and kidney safely cleared the injected nanoparticles within one day after the injection, demonstrating no risk of iron overload in test mice. Therefore, this study sheds light on developing high-performance gadolinium free positive magnetic resonance contrast agents for biomedical applications.

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.

Targeted in vivo imaging of microscopic tumors with ferritin-based nanoprobes across biological barriers.

The avascular microscopic breast and brain tumors (<1-2 mm diameter) can be noninvasively detected by designing human heavy-chain ferritin (HFn)-based nanoparticles as molecular probes for near-infrared fluorescence and magnetic resonance imaging. The intravenously injected HFn-based nano-particles (Cy5.5-HFn and M-HFn) can cross the endothelium, epithelium, and blood-brain barriers and be internalized into tumor cells.

Magnetoferritin nanoparticles for targeting and visualizing tumour tissues.

Engineered nanoparticles have been used to provide diagnostic, therapeutic and prognostic information about the status of disease. Nanoparticles developed for these purposes are typically modified with targeting ligands (such as antibodies, peptides or small molecules) or contrast agents using complicated processes and expensive reagents. Moreover, this approach can lead to an excess of ligands on the nanoparticle surface, and this causes non-specific binding and aggregation of nanoparticles, which decreases detection sensitivity. Here, we show that magnetoferritin nanoparticles (M-HFn) can be used to target and visualize tumour tissues without the use of any targeting ligands or contrast agents. Iron oxide nanoparticles are encapsulated inside a recombinant human heavy-chain ferritin (HFn) protein shell, which binds to tumour cells that overexpress transferrin receptor 1 (TfR1). The iron oxide core catalyses the oxidation of peroxidase substrates in the presence of hydrogen peroxide to produce a colour reaction that is used to visualize tumour tissues. We examined 474 clinical specimens from patients with nine types of cancer and verified that these nanoparticles can distinguish cancerous cells from normal cells with a sensitivity of 98% and specificity of 95%.