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Human cytoplasmic ferritins are heteropolymers of H and L subunits containing a catalytic ferroxidase center and a nucleation site for iron biomineralization, respectively. Here, ESI-MS successfully detected labile metal-protein interactions revealing the formation of tetra- and octa-iron clusters bound to L subunits, as previously underscored by X-ray crystallography.

Due to its unique structure, recent years have witnessed the use of apo-ferritin to accumulate various non-natural metal ions as a scaffold for nanomaterial synthesis. However, the transport mechanism of metal ions into the cavity of apo-ferritin is still unclear, limiting the rational design and controllable preparation of nanomaterials. Here, we conducted all-atom classical molecular dynamics (MD) simulations combined with Markov state models (MSMs) to explore the transportation behavior of Au(iii) ions. We exhibited the complete transportation paths of Au(iii) from solution into the apo-ferritin cage at the atomic level. We also revealed that the transportation of Au(iii) ions is accompanied by coupled protein structural changes. It is shown that the 3-fold axis channel serves as the only entrance with the longest residence time of Au(iii) ions. Besides, there are eight binding clusters and five 3-fold structural metastable states, which are important during Au(iii) transportation. The conformational changes of His118, Asp127, and Glu130, acting as doors, were observed to highly correlate with the Au(iii) ion's position. The MSM analysis and Potential Mean Force (PMF) calculation suggest a remarkable energy barrier near Glu130, making it the rate-limiting step of the whole process. The dominant transportation pathway is from cluster 3 in the 3-fold channel to the inner cavity to cluster 5 on the inner surface, and then to cluster 6. These findings provide inspiration and theoretical guidance for the further rational design and preparation of new nanomaterials using apo-ferritin.

The surface engineering of the apoferritin shell by means of traditional chemical modifications usually suffers from site inaccuracy and insufficient conjugation. This report describes a non-covalent method for precise modulation of the apoferritin surface without alteration of amino acid residues. A bifunctional macromolecule, structured as azide-poly(ethylene glycol)-porphyrin (termed TPA), was synthesized. TPA was observed to be able to recognize and bind apoferritin in a 12 : 1 stoichiometry with a higher binding affinity than arachidonate, thanks to the specific host-guest interaction between the pocket of each two-fold channel and the porphyrin moiety. This method allows for site-specific engineering of the apoferritin surface with on demand functionalities and optimization of drug encapsulation.

The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.

Currently, bioengineered apoferritin nanocages with flexible protein shells and functionalized modifications have become an attractive approach for efficient anti-tumor therapy. Here, we modified the N-terminus of H-chain subunits in apoferritin with different amounts of lysine via genetic recombination to obtain a poly(l-lysine) modified H-chain apoferritin (nL-HFn) nanocage for siRNA delivery and gene therapy. To achieve excellent cellular affinity and uptake, the nanocarriers were internalized through transferrin receptor-mediated endocytosis, then escaped from the endosome for cytoplasmic transport. Compared with natural apoferritin, the siRNA-loaded genetic recombination NPs modified with lysine exhibit stronger RNA-interference and antitumor efficiency both in vitro and in 4T1 tumor model mice. Therefore, bioengineered apoferritin nanocages modified with lysine might be a promising platform for nucleic acid drug delivery.

Ferritin, a naturally occuring iron-storage protein, plays an important role in nanoengineering and biomedical applications. Upon iron removal, apoferritin was shown to allow the encapsulation of an artificial transfer hydrogenase (ATHase) based on the streptavidin-biotin technology. The third coordination sphere, provided by ferritin, significantly influences the catalytic activity of an ATHase for the reduction of cyclic imines.

The effect of chitosan decoration on the transport of epigallocatechin (EGC)-encapsulated ferritin cage across the Caco-2 cells was investigated. After the encapsulation of EGC in apo-red bean (adzuki) ferritin (apoRBF), the EGC-loaded apoRBF nanoparticle (ER) was fabricated with an encapsulation ratio of 11.6% (w/w). The results indicated that different chitosan molecules (with molecular weights of 980, 4600, 46 000, and 210 000 Da) could attach onto the apoRBF via electrostatic interactions to form ER-chitosan complexes (ERCs) (ERCs980, ERCs4600, ERCs46000, and ERCs210000). ERCs980 and ERCs4600 retained the typical shell-like morphology of ferritin with a size distribution of 12 nm and showed weak negative zeta-potentials at pH 6.7, while ERCs46000 and ERCs210000 significantly aggregated. Furthermore, the transport of EGC in ERCs980 and ERCs4600 across the Caco-2 cells was enhanced by the transferrin receptor 1 (TfR-1)-mediated absorption pathway, demonstrating that chitosan molecules with low molecular weights of 980 and 4600 Da were beneficial to the absorption of EGC based on the ferritin cage. This study will facilitate the application of ferritin-chitosan materials for fabricating the core-shell platform for encapsulation and bioavailability enhancement of bioactive molecules.

Apoferritin caged gold nanoparticles (AuNPs) were assembled into flower-like structures by precise base pairing of the attached DNA molecules. The key step was to use the eight hydrophilic channels through the apoferritin to control the exact number and locations of the DNA molecules that grafted onto the caged AuNP.

Natural protein-based microcompartments containing multiple enzymes promote cascade reactions within cells. We use the apo-ferritin protein cage to mimic such biocompartments by immobilizing two organometallic Ir and Pd complexes into the single protein cage. Precise locations of the metals and their accumulation mechanism were studied by X-ray crystallography.

In this paper, we proposed a naturally derived protein cage based pH-responsive delivery system for intracellular prodrug controlled release. The drug delivery system is based on apoferritin as delivery vehicles to encapsulate the anticancer drug daunomycin (DN) and alleviate the side effect. The hydrophobic drug DN was encapsulated into the interior of apoferritin by the hydrophobic channels of the cage with swelling at slight acidic pH and electrostatic adsorption. The negatively charged poly-l-aspartic acid (PLAA) was further introduced into the apoferritin to absorb the positively charged DN. The mixture of PLAA and DN easily flew into the apoferritin cage and was stably stored in the physiological fluids. PLAA protected the leakage of DN and encapsulated a sufficient amount of drug molecules in the cage. To specifically target the tumor cells, the surface of apoferritin was modified with hyaluronic acid (HA) which can easily bind to the HA-receptor CD44. Here, human embryonic lung MRC-5 cells and lung cancer A549 cells were used to observe the specific binding of HA and morphological changes in vitro and examine the antitumor activity. This unique protein based drug delivery platform using the apoferritin cage shows great potential in the therapeutic administration of the anti-cancer agents.

The first specific functionality of the light-chain (L-chain) subunit of the universal iron storage protein ferritin was identified. The electrons released during iron-oxidation were transported across the ferritin cage specifically through the L-chains and the inverted electron transport through the L-chains also accelerated the demineralization of ferritin.

The use of (57)Fe-isotopically enriched ferritin for the accurate measurement of Fe : ferritin ratios is proposed for metabolic studies. Thus, the synthesis of (57)Fe-isotopically enriched ferritin from horse apo-ferritin and isotopically enriched (NH4)2(57)Fe(II)(SO4)2 (Mohr's salt) is conducted. Size exclusion chromatography on-line with UV-VIS absorption (at 380 nm) is used in order to monitor the loading process of apo-ferritin. These studies revealed that the Fe-incorporation process involves also the formation of protein aggregates (oligomers) showing higher molecular mass than ferritin. A final optimized protocol involving incubation of the synthesized standard with guanidine hydrochloride (pH 3.5) has provided the best conditions for maintaining a stable protein structure without aggregates. Such (57)Fe-isotopically enriched ferritin was characterized and contained an average of 2200 atoms of Fe per mole of ferritin. The evaluation of the Fe-core after saturation with (57)Fe by Transmission Electron Microscopy (TEM) has revealed the formation of (57)Fe nanoparticles with a similar diameter to that of the commercial Fe-containing ferritin, confirming the process of Fe uptake, oxidation and mineralization within the protein cavity. The synthesized (57)Fe-ferritin shows great potential as a nanometabolic tracer to study the kinetics of Fe release in the cases of iron metabolic disorders.

Luminescent europium (Eu) and dysprosium (Dy) doped yttrium-vanadate (Y-V) nanoparticles (NPs) were synthesized in the cavity of the protein, apoferritin. Y-V NPs were synthesized by incubating a solution of apoferritin with Y(3+) and VO3(-) ions in the presence of ethylene diamine-N-N'-diacetic acid (EDDA). EDDA plays an important role in preventing Y-vanadate precipitation in bulk solution by chelating the Y(3+) ions. Using high resolution electron microscopy, the obtained NPs in the apoferritin cavities were confirmed to be amorphous, and to consist of Y and V. Eu-doped Y-V (Y-V:Eu) NPs were synthesized by the same procedure as Y-V NPs, except that Eu(NO3)3 was added. Y-V:Eu NPs exhibited a strong absorption peak due to the O-V charge transfer transition and remarkable luminescence at 618 nm due to the (5)D0 → (7)F2 transition. The luminescence lifetime of Y:Eu and Y-V:Eu NPs measured in H2O and D2O solution showed reduction of non-radiative transition to the O-H vibration in Y-V:Eu NPs. Accordingly, Y-V NPs showed strong luminescence compared to Y:Eu NPs. Dy-doped Y-V NPs were also synthesized in apoferritin cavities and showed luminescence peaks at 482 nm and 572 nm, corresponding to (4)F9/2 → (6)H15/2 and (4)F9/2 → (6)H13/2 transitions. These NPs stably dispersed in water solution since their aggregation was prevented by the protein shell. NPs encapsulated in the protein are likely to be biocompatible and would have significant potential for biological imaging applications.

Light-chain apoferritin lacks ferroxidase activity, which can be supplemented with Pt-nanoparticles. The hybrid bioinorganic nanoparticle outperforms its heavy-chain pendant in terms of ferroxidase activity, mineralization ability and inhibition resistance. Being active in a cellular environment it regulates the iron homeostasis.

A selective and specific inhibition of the catalase mimetic activity but not SOD-like activity of Pt-apoferritin can be achieved through the choice of the inhibitor. The recovery of activity using a reducing agent was explored and proven to successfully restore the surface-chemistry of NPs.

A novel sandwich-type electrochemiluminescent immunoassay utilizing apoferritin-templated poly(ethylenimine) (PEI) nanoparticles as labels based on the in situ release of the co-reactant of PEI was developed for sensitive detection of HCG with a low detection limit of 0.17 μIU mL(-1).

4.5 nm nanoceria particles are successfully encapsulated into the apoferritin cavity via a dissociation-reconstruction route. The apoferritin coating not only improves the biocompatibility and changes the cellular uptake route of nanoceria, but also manipulates the electron localization at the surface of the nanoparticle thereby ameliorating the ROS-scavenging activity., (This journal is © The Royal Society of Chemistry 2012)

Mn(III)-loaded apoferritin is promptly reduced to Mn(II)-apoferritin by the oxidation of L-DOPA to melanin. The process is nicely witnessed by a marked relaxation enhancement of water proton relaxation rate that has been detected both in cultured melanoma cells and in tumor animal models., (This journal is © The Royal Society of Chemistry 2012)

This perspective paper is intended to give some insights into the recently proposed technique of NMR of solutes sedimented by ultracentrifugation in a rotor used for solid state NMR experiments. Sedimented "states" correspond to molecules with very little reorientational capability in extremely concentrated solutions. They provide solid state NMR spectra comparable in quality with those of the best microcrystalline samples. Here we report some experiments to look for chemicals which affect the properties of the sediment, and we show that it is possible to fill the rotor in a true ultracentrifuge and then record the spectra. The latter possibility opens new horizons for NMR of sedimented systems.

An artificial luminescent protein, apoferritin-encapsulated luminescent europium complex, has been designed/fabricated and displays good biocompatibility and long-lived luminescence, which means it can be used as a bioprobe to image living cells with a time-gated mode.

Spherical protein cages such as an iron storage protein, ferritin, have great potential as nanometer-scale capsules to assemble and store metal ions and complexes. We report herein the synthesis of a composite of an apo-ferritin cage and Ru(p-cymene) complexes. Ru complexes were efficiently incorporated into the ferritin cavity without degradation of its cage structure. X-Ray crystallography revealed that the Ru complexes were immobilized on the interior surface of the cage mainly by the coordination of histidine residues.

Apo-ferritin (apo-Fr) mutants are used as scaffolds to accommodate palladium (allyl) complexes. Various coordination arrangements of the Pd complexes are achieved by adjusting the positions of cysteine and histidine residues on the interior surface of the apo-Fr cage.

We have succeeded in preparing Au/Pd core-shell nanoparticles in apo-ferritin and improving the catalytic reactivity of olefin hydrogenation relative to Pd0 nanoparticles in the cage.

Apoferritin-coated photoluminescent CdSe nanoparticles generated by an EDTA-mediated in situ method are photostable, water soluble and biocompatible.

We have prepared water-soluble gadolinium oxide nanoparticles that show potential as MRI contrast agents. The particles were built into the apoferritin cavity and have an average size of 5 nm. After seven days a loss of 5% of Gd was detected compared with the as-prepared samples; after that the Gd remained constant and stabilized inside the apoferritin, indicating that the apoferritin capsid acts as a Gd store, avoiding metal delivery and consequent toxicity. The NMR longitudinal and transverse relaxivities resulted about 10 and 70 times higher than the ones of clinically approved paramagnetic Gd-chelates, thus indicating the possible route for synthesizing a novel class of MRI contrast agents.

Apo-ferritin was used for the incorporation of functionalized ferrocene derivatives and the resulting composites were studied by means of single-crystal X-ray diffraction and electrochemical analysis, revealing a different incorporation behaviour of the ferrocenes governed by the functional group present.

A methylene blue-encapsulated apoferritin complex shows cytotoxic effects on MCF-7 human breast adenocarcinoma cells when irradiated at the appropriate wavelength.

Nanocrystalline metals display interesting basic and technological properties related to their chemical and structural properties. Among other properties, they display a richer phase diagram due to the additional degree of freedom introduced by the nanoparticles surface. Metal nanoparticles encapsulated within biological macromolecules have the additional advantage of biocompatibility. In this paper we investigate the thermal evolution of the structure and dynamics of apoferritin encapsulated nanocrystalline copper. We determined the occurrence of a yet unexpected phase transition from a low temperature FCC to a complex high temperature phase including a (putative) amorphous precursor. The occurrence of a FCC-icosahedral transition is also discussed as a possible explanation to our results. The lattice dynamics of the FCC phase (monitored by its Debye temperature) differs from the behaviour expected for nanosized structures.

Apoferritin derived from the native protein ferritin was employed to encapsulate anticancer drugs cisplatin and carboplatin.

Horse spleen apoferritin, the hollow protein shell derived from ferritin, a special biological nanoparticle, can be chemoselectively modified at the lysine residues, which affords a robust scaffold for further chemical reactions including Cu(i)-catalyzed azide-alkyne cycloaddition reaction and atom transfer radical polymerization reaction.

Copper and CuFe Prussian Blue nanoparticles have been prepared by using a Cu(II)-loaded apoferritin as a chemically and spatially confined environment for their construction.

One of key aims of modern chemistry is to explore and exploit the phenomenal complexity of matter that seems to arise spontaneously within ambient environments like that of our planet. Biology, of course, is a ultimate manifestation of a universal chemical canon encompassing interactions both at the molecular and supramolecular level. Inorganic building blocks play a significant role in the self-organised assembly of many biological structures at various scale-lengths but the details of their chemical behaviour and interaction with organic compounds are still not well-understood. Stephen Mann of the University of Bristol is a pioneer in the area; his team is probing the ground rules for biomineralisation in the hope of applying them to the self-assembly of practical materials with complex hierarchical structures.