Nose-to-brain selective drug delivery to glioma via ferritin-based nanovectors reduces tumor growth and improves survival rate.

Gliomas are among the most fatal tumors, and the available therapeutic options are very limited. Additionally, the blood-brain barrier (BBB) prevents most drugs from entering the brain. We designed and produced a ferritin-based stimuli-sensitive nanocarrier with high biocompatibility and water solubility. It can incorporate high amounts of the potent topoisomerase 1 inhibitor Genz-644282. Here, we show that this nanocarrier, named The-0504, can cross the BBB and specifically deliver the payload to gliomas that express high amounts of the ferritin/transferrin receptor TfR1 (CD71). Intranasal or intravenous administration of The-0504 both reduce tumor growth and improve the survival rate of glioma-bearing mice. However, nose-to-brain administration is a simpler and less invasive route that may spare most of the healthy tissues compared to intravenous injections. For this reason, the data reported here could pave the way towards a new, safe, and direct ferritin-based drug delivery method for brain diseases, especially brain tumors.

Widespread in vivo efficacy of The-0504: A conditionally-activatable nanoferritin for tumor-agnostic targeting of CD71-expressing cancers.

Background: Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The-0504) that selectively delivers the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors. The-0504 has so far been evaluated on four different human tumor xenotransplant models (breast, colorectal, pancreatic and liver cancers). Methods: Herein, we extend our studies, by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations. Results: Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker γH2AX. Expression increases up to 4-fold and is more persistent as compared to free Genz-644282. In vivo studies confirmed a remarkable anti-tumor activity of The-0504, resulting in tumor eradication in most murine xenograft models, regardless of embryological origin (e.g. epithelial, mesenchymal or neuroendocrine), and molecular subtypes. PK studies demonstrated a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug), with a 400-fold increase in peak concentrations as compared to the free drug. On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice. Conclusion: The results presented here confirm that The-0504 is highly active against several human tumor xenotransplants, even when administered less frequently than previously reported. The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting.

Mitoxantrone-Loaded Nanoferritin Slows Tumor Growth and Improves the Overall Survival Rate in a Subcutaneous Pancreatic Cancer Mouse Model.

Pancreatic cancer (PC) represents an intriguing topic for researchers. To date, the prognosis of metastasized PC is poor with just 7% of patients exceeding a five-year survival period. Thus, molecular modifications of existing drugs should be developed to change the course of the disease. Our previously generated nanocages of Mitoxantrone (MIT) encapsulated in human H-chain Ferritin (HFt), designated as HFt-MP-PASE-MIT, has shown excellent tumor distribution and extended serum half-life meriting further investigation for PC treatment. Thus, in this study, we used the same nano-formulation to test its cytotoxicity using both in vitro and in vivo assays. Interestingly, both encapsulated and free-MIT drugs demonstrated similar killing capabilities on PaCa44 cell line. Conversely, in vivo assessment in a subcutaneous PaCa44 tumor model of PC demonstrated a remarkable capability for encapsulated MIT to control tumor growth and improve mouse survival with a median survival rate of 65 vs. 33 days for loaded and free-MIT, respectively. Interestingly, throughout the course of mice treatment, MIT encapsulation did not present any adverse side effects as confirmed by histological analysis of various murine tissue organs and body mass weights. Our results are promising and pave the way to effective PC targeted chemotherapy using our HFt nanodelivery platforms.

High activity and low toxicity of a novel CD71-targeting nanotherapeutic named The-0504 on preclinical models of several human aggressive tumors.

BACKGROUND: Ferritin receptor (CD71) is an example of a very attractive cancer target, since it is highly expressed in virtually all tumor types, including metastatic loci. However, this target can be considered to be inaccessible to conventional target therapies, due to its presence in many healthy tissues. Here, we describe the preclinical evaluation of a tumor proteases-activatable human ferritin (HFt)-based drug carrier (The-0504) that is able to selectively deliver the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors, preventing the limiting toxic effects associated with CD71-targeting therapies. METHODS: CD71 expression was evaluated using flow cytometry and immunohistochemistry techniques. The-0504 antiproliferative activity towards several cancer cell lines was assessed in vitro. The-0504 antitumor efficacy and survival benefit were evaluated in different human tumors, which had been grown either as xenografts or patient-derived xenografts in mice. The-0504 toxicology profile was investigated in multiple-cycle repeat-dose study in rodents. RESULTS: In vitro studies indicate that The-0504 is highly specific for CD71 expressing cells, and that there is a relationship between CD71 levels and The-0504 anticancer activity. In vivo treatments with The-0504 showed a remarkable efficacy, eradicating several human tumors of very diverse and aggressive histotypes, such as pancreas, liver and colorectal carcinomas, and triple-negative breast cancer. CONCLUSIONS: Durable disease-free survival, persistent antitumor responses after discontinuation of treatment and favorable toxicology profile make The-0504 an ideal candidate for clinical development as a novel, CD71-targeted, low-toxicity alternative to chemotherapy.

Engineered Human Nanoferritin Bearing the Drug Genz-644282 for Cancer Therapy.

Gastrointestinal tumors, including pancreatic and colorectal cancers, represent one of the greatest public health issues worldwide, leading to a million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different types of drugs in its cavity and can bind to its receptor, CD71, in several solid and hematological tumors, thus highlighting the potential use of ferritin for tumor-targeting therapies. Here, we describe the development and characterization of a novel nanomedicine based on the HFt that is named The-0504. In particular, this novel system is a nano-assembly comprising an engineered version of HFt that entraps about 80 molecules of a potent, wide-spectrum, non-camptothecin topoisomerase I inhibitor (Genz-644282). The-0504 can be produced by a standardized pre-industrial process as a pure and homogeneously formulated product with favourable lyophilization properties. The preliminary anticancer activity was evaluated in cultured cancer cells and in a mouse model of pancreatic cancer. Overall results reported here make The-0504 a candidate for further preclinical development against CD-71 expressing deadly tumors.

Cryo-EM structure of the human ferritin-transferrin receptor 1 complex.

Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.

Increasing the Magnetic Anisotropy of a Natural System: Co-Doped Magnetite Mineralized in Ferritin Shells.

Iron oxide nanoparticles mineralized within the internal cavity of Ferritin protein cage are extremely appealing for the realization of multifunctional therapeutic and diagnostic agents for cancer treatment by drug delivery, magnetic fluid hyperthermia (MFH) and magnetic resonance imaging. Being the maximum mean size imposed by the internal diameter of the protein shell (ca. 8 nm) too small for the use of these systems in MFH, a valuable strategy for the improvement of the hyperthermic efficiency is increasing the magnetic anisotropy by doping the iron oxide with divalent Co ions. This strategy has been demonstrated to be highly efficient in the case of iron oxide nanoparticles mineralized in Human Ferritin (HFt). However, a deterioration of nanoparticles crystallinity and consequently a reduction of the hyperthermic efficiency were observed with increasing Co-doping. In this contribution, we compare two series of Co-doped iron oxide nanoparticles (Co-doping level up to 15%) mineralized into HFt and into Ferritin from the archaea Pirococcus Furiosus (PfFt), the protein structure of which differs for the nucleation sites, with the aim of increasing the crystalline quality of the inorganic cores for larger Co doping. Highly monodisperse nanoparticles of 6-7 nm were obtained in both series. The structural and magnetic characterization indicate that the PfFt series is less subjected to crystallinity deterioration with increasing Co content with respect to the HFt one. Such difference is reflected in the hyperthermic efficiency, which reaches the maximum value for different intermediate Co-doping (10% and 5% for PfFt and HFt, respectively), and goes to zero for further Co-doping increments.

Very low intensity ultrasounds as a new strategy to improve selective delivery of nanoparticles-complexes in cancer cells.

BACKGROUND: The possibility to combine Low Intensity UltraSound (LIUS) and Nanoparticles (NP) could represent a promising strategy for drugs delivery in tumors difficult to treat overcoming resistance to therapies. On one side the NP can carry drugs that specifically target the tumors on the other the LIUS can facilitate and direct the delivery to the tumor cells. In this study, we investigated whether Very Low Intensity UltraSound (VLIUS), at intensities lower than 120 mW/cm2, might constitute a novel strategy to improve delivery to tumor cells. Thus, in order to verify the efficacy of this novel modality in terms of increase selective uptake in tumoral cells and translate speedily in clinical practice, we investigated VLIUS in three different in vitro experimental tumor models and normal cells adopting three different therapeutic strategies. METHODS: VLIUS at different intensities and exposure time were applied to tumor and normal cells to evaluate the efficiency in uptake of labeled human ferritin (HFt)-based NP, the delivery of NP complexed Firefly luciferase reported gene (lipoplex-LUC), and the tumor-killing of chemotherapeutic agent. RESULTS: Specifically, we found that specific VLIUS intensity (120 mW/cm2) increases tumor cell uptake of HFt-based NPs at specific concentration (0.5 mg/ml). Similarly, VLIUS treatments increase significantly tumor cells delivery of lipoplex-LUC cargos. Furthermore, of interest, VLIUS increases tumor killing of chemotherapy drug trabectedin in a time dependent fashion. Noteworthy, VLIUS treatments are well tolerated in normal cells with not significant effects on cell survival, NPs delivery and drug-induced toxicity, suggesting a tumor specific fashion. CONCLUSIONS: Our data shed novel lights on the potential application of VLIUS for the design and development of novel therapeutic strategies aiming to efficiently deliver NP loaded cargos or anticancer drugs into more aggressive and unresponsive tumors niche.

The presence of glutamate residues on the PAS sequence of the stimuli-sensitive nano-ferritin improves in vivo biodistribution and mitoxantrone encapsulation homogeneity.

A genetically engineered human ferritin heavy chain (HFt)-based construct has been recently shown by our group to efficiently entrap and deliver doxorubicin to cancer cells. This construct, named HFt-MP-PAS, contained a tumor-selective sequence (MP) responsive to proteolytic cleavage by tumor proteases (MMPs), located between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). HFt-MP-PAS displayed excellent therapeutic efficacy in xenogenic pancreatic and head and neck cancer models in vivo, leading to a significant increase in overall animal survivals. Here we report a new construct obtained by the genetic insertion of two glutamate residues in the PAS sequence of HFt-MP-PAS. Such new construct, named HFt-MP-PASE, is characterized by improved performances as drug biodistribution in a xenogenic pancreatic cancer model in vivo. Moreover, HFt-MP-PASE efficiently encapsulates the anti-cancer drug mitoxantrone (MIT), and the resulting MIT-loaded nanoparticles proved to be more soluble and monodispersed than the HFt-MP-PAS counterparts. Importantly, in vitro MIT-loaded HFt-MP-PASE kills several cancer cell lines of different origin (colon, breast, sarcoma and pancreas) at least as efficiently as the free drug. Finally, our MIT loaded protein nanocages allowed in vivo an impressive incrementing of the drug accumulation in the tumor with respect to the free drug.

Therapeutic Efficacy of the Novel Stimuli-Sensitive Nano-Ferritins Containing Doxorubicin in a Head and Neck Cancer Model.

Doxorubicin is employed alone or in combination for the treatment of several hematological and solid malignancies; despite its efficacy, there are associated cardiotoxicity limits both in its application in patients with heart disease risk factors and also in its long-term use. HFt-MP-PAS40 is a genetically engineered human ferritin heavy chain (HFt)-based construct able to efficiently entrap and deliver doxorubicin to cancer cells. HF-MP-PAS contains a short motif sequence (defined as MP) responsive to proteolytic cleavage by tumor matrix metalloproteases (MMPs), located between each HFt subunit and a masking polypeptide sequence rich in proline (P), alanine (A), and serine (S) residues (PAS). This carrier displayed excellent therapeutic efficacy in a xenogenic pancreatic cancer model in vivo, leading to a significant increase in overall animal survival in treated mice. Herein, we describe the HFt-MP-PAS40-Dox efficacy against squamous cell carcinomas of the head and neck (HNSCC) with the goal of validating the application of our nano-drug for the treatment of different solid tumors. In addition, a tolerability study in healthy mice was also performed. The results indicate that HFt-MP-PAS40-Dox produced increased anti-tumor effects both in vitro and in vivo in comparison to the free drug in several HNSCC cell lines. In the acute toxicity studies, the maximum tolerated dose (MTD) of HFt-MP-PAS40-Dox was about 3.5 higher than the free drug: 25 mg/kg versus 7 mg/kg doxorubicin equivalents. Importantly, evaluation of heart tissues provided evidence that doxorubicin is less cardio-toxic when encapsulated inside the ferritin carrier. In conclusion, HFt-MP-PAS40-Dox may be administered safely at higher doses compared with the free drug, resulting in superior efficacy to control HNSCC malignancies.

Selective delivery of doxorubicin by novel stimuli-sensitive nano-ferritins overcomes tumor refractoriness.

Human ferritin heavy chain (HFt) has been demonstrated to possess considerable potential for targeted delivery of drugs and diagnostic agents to cancer cells. Here, we report the development of a novel HFt-based genetic construct (HFt-MP-PAS) containing a short peptide linker (MP) between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). The peptide linker contains a matrix-metalloproteinases (MMPs) cleavage site that permits the protective PAS shield to be removed by tumor-driven proteolytic cleavage within the tumor microenvironment. For the first time HFt-MP-PAS ability to deliver doxorubicin to cancer cells, subcellular localization, and therapeutic efficacy on a xenogeneic mouse model of a highly refractory to conventional chemotherapeutics type of cancer were evaluated. HFt-MP-PAS-DOXO performance was compared with the novel albumin-based drug delivery system INNO-206, currently in phase III clinical trials. The results of this work provide solid evidence indicating that the stimuli-sensitive, long-circulating HFt-MP-PAS nanocarriers described herein have the potential to be exploited in cancer therapy.

Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin-Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements.

A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.

In Vivo Targeting of Cutaneous Melanoma Using an Melanoma Stimulating Hormone-Engineered Human Protein Cage with Fluorophore and Magnetic Resonance Imaging Tracers.

Nanoparticle (NP)-based materials are promising agents for enhancing cancer diagnosis and treatment. Once functionalized for selective targeting of tumor-expressed molecules, they can specifically deliver drugs and diagnostic molecules inside tumor cells. In the present work, we evaluated the in vivo melanoma-targeting ability of a nanovector (HFt-MSH-PEG) based on human protein ferritin (HFt), functionalized with both melanoma-targeting melanoma stimulating hormone (α-MSH) and stabilizing poly(ethylene glycol) (PEG) molecules. Independent and complementary techniques, such as whole-specimen confocal microscopy and magnetic resonance imaging, were used to detect in vivo localization of NP constructs with suitable tracers (i.e., fluorophores or magnetic metals). Targeted HFt-MSH-PEG NPs accumulated persistently at the level of primary melanoma and with high selectivity with respect to other organs. Melanoma localization of untargeted HFt-PEG NPs, which lack the α-MSH moiety, was less pronounced. Furthermore, HFt-MSH-PEG NPs accumulated to a significantly lower extent and with a different distribution in a diverse type of tumor (TS/A adenocarcinoma), which does not express α-MSH receptors. Finally, in a spontaneous lung metastasis model, HFt-MSH-PEG NPs localized at the metastasis level as well. These results suggest that HFt-MSH-PEG NPs are suitable carriers for selective in vivo delivery of diagnostic or therapeutic agents to cutaneous melanoma.

A smart platform for hyperthermia application in cancer treatment: cobalt-doped ferrite nanoparticles mineralized in human ferritin cages.

Magnetic nanoparticles, MNPs, mineralized within a human ferritin protein cage, HFt, can represent an appealing platform to realize smart therapeutic agents for cancer treatment by drug delivery and magnetic fluid hyperthermia, MFH. However, the constraint imposed by the inner diameter of the protein shell (ca. 8 nm) prevents its use as heat mediator in MFH when the MNPs comprise pure iron oxide. In this contribution, we demonstrate how this limitation can be overcome through the controlled doping of the core with small amount of Co(II). Highly monodisperse doped iron oxide NPs with average size of 7 nm are mineralized inside a genetically modified variant of HFt, carrying several copies of α-melanocyte-stimulating hormone peptide, which has already been demonstrated to have excellent targeting properties toward melanoma cells. HFt is also conjugated to poly(ethylene glycol) molecules to increase its in vivo stability. The investigation of hyperthermic properties of HFt-NPs shows that a Co doping of 5% is enough to strongly enhance the magnetic anisotropy and thus the hyperthermic efficiency with respect to the undoped sample. In vitro tests performed on B16 melanoma cell line demonstrate a strong reduction of the cell viability after treatment with Co doped HFt-NPs and exposure to the alternating magnetic field. Clear indications of an advanced stage of apoptotic process is also observed from immunocytochemistry analysis. The obtained data suggest this system represents a promising candidate for the development of a protein-based theranostic nanoplatform.

Antibody-drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin.

A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4(+) melanoma cell line, but not to a CSPG4(-) breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.

Electrostatic assembly of binary nanoparticle superlattices using protein cages.

Binary nanoparticle superlattices are periodic nanostructures with lattice constants much shorter than the wavelength of light and could be used to prepare multifunctional metamaterials. Such superlattices are typically made from synthetic nanoparticles, and although biohybrid structures have been developed, incorporating biological building blocks into binary nanoparticle superlattices remains challenging. Protein-based nanocages provide a complex yet monodisperse and geometrically well-defined hollow cage that can be used to encapsulate different materials. Such protein cages have been used to program the self-assembly of encapsulated materials to form free-standing crystals and superlattices at interfaces or in solution. Here, we show that electrostatically patchy protein cages--cowpea chlorotic mottle virus and ferritin cages--can be used to direct the self-assembly of three-dimensional binary superlattices. The negatively charged cages can encapsulate RNA or superparamagnetic iron oxide nanoparticles, and the superlattices are formed through tunable electrostatic interactions with positively charged gold nanoparticles. Gold nanoparticles and viruses form an AB(8)(fcc) crystal structure that is not isostructural with any known atomic or molecular crystal structure and has previously been observed only with large colloidal polymer particles. Gold nanoparticles and empty or nanoparticle-loaded ferritin cages form an interpenetrating simple cubic AB structure (isostructural with CsCl). We also show that these magnetic assemblies provide contrast enhancement in magnetic resonance imaging.

Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles.

BACKGROUND: Nanoparticle-based systems are promising for the development of imaging and therapeutic agents. The main advantage of nanoparticles over traditional systems lies in the possibility of loading multiple functionalities onto a single molecule, which are useful for therapeutic and/or diagnostic purposes. These functionalities include targeting moieties which are able to recognize receptors overexpressed by specific cells and tissues. However, targeted delivery of nanoparticles requires an accurate system design. We present here a rationally designed, genetically engineered, and chemically modified protein-based nanoplatform for cell/tissue-specific targeting. METHODS: Our nanoparticle constructs were based on the heavy chain of the human protein ferritin (HFt), a highly symmetrical assembly of 24 subunits enclosing a hollow cavity. HFt-based nanoparticles were produced using both genetic engineering and chemical functionalization methods to impart several functionalities, ie, the α-melanocyte-stimulating hormone peptide as a melanoma-targeting moiety, stabilizing and HFt-masking polyethylene glycol molecules, rhodamine fluorophores, and magnetic resonance imaging agents. The constructs produced were extensively characterized by a number of physicochemical techniques, and assayed for selective melanoma-targeting in vitro and in vivo. RESULTS: Our HFt-based nanoparticle constructs functionalized with the α-melanocyte-stimulating hormone peptide moiety and polyethylene glycol molecules were specifically taken up by melanoma cells but not by other cancer cell types in vitro. Moreover, experiments in melanoma-bearing mice indicate that these constructs have an excellent tumor-targeting profile and a long circulation time in vivo. CONCLUSION: By masking human HFt with polyethylene glycol and targeting it with an α-melanocyte-stimulating hormone peptide, we developed an HFt-based melanoma-targeting nanoplatform for application in melanoma diagnosis and treatment. These results could be of general interest, because the same strategy can be exploited to develop ad hoc nanoplatforms for specific delivery towards any cell/tissue type for which a suitable targeting moiety is available.

Hierarchical self-assembly and optical disassembly for controlled switching of magnetoferritin nanoparticle magnetism.

Protein cages such as ferritin and viral capsids are interesting building blocks for nanotechnology due to their monodisperse structure and ability to encapsulate various functional moieties. Here we show that recombinant ferritin protein cages encapsulating Fe(3)O(4)-γ-Fe(2)O(3) iron oxide (magnetoferritin) nanoparticles and photodegradable Newkome-type dendrons self-assemble into micrometer-sized complexes with a face-centered-cubic (fcc) superstructure and a lattice constant of 13.1 nm. The magnetic properties of the magnetoferritin particles are affected directly by the hierarchical organization. Magnetoferritin nanoparticles dispersed in water exhibit typical magnetism of single domain noninteracting nanoparticles; however, the same nanoparticles organized into fcc superstructures show clearly the effects of the altered magnetostatic (e.g., dipole-dipole) interactions by exhibiting, for example, different hysteresis of the field-dependent magnetization. The magnetoferritin-dendron assemblies can be efficiently disassembled by a short optical stimulus resulting in release of free magnetoferritin particles. After the triggered release the nanomagnetic properties of the pristine magnetoferritin nanoparticles are regained.

Effect of the charge distribution along the "ferritin-like" pores of the proteins from the Dps family on the iron incorporation process.

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the "ferritin-like" pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.

The characterization of Thermotoga maritima ferritin reveals an unusual subunit dissociation behavior and efficient DNA protection from iron-mediated oxidative stress.

Ferritin from the hyperthermophilic anaerobe Thermotoga maritima, a bacterium of ancient phylogenetic origin, is structurally similar to known bacterial and eukaryotic ferritins: 24 identical subunits assemble into a shell having octahedral symmetry and a Mr of about 460 kDa. T. maritima ferritin (TmFtn), purified to homogeneity as a recombinant protein, contains approximately 2-3 iron atoms and can incorporate efficiently up to 3,500 atoms in the form of a ferric oxy-hydroxide mineral at 80°C, the optimal growth temperature of the bacterium. The 24-mer unexpectedly dissociates reversibly into dimers at low ionic strengths. In turn, dimers re-associate into the native 24-mer assembly at high protein concentrations and upon incorporation of iron micelles containing at least 500 Fe(III). TmFtn uses O(2) as efficient iron oxidant. The reaction stoichiometry is 3-4 O(2):Fe(II) as in all bacterial ferritins. Accordingly no H(2)O(2) is released into solution, a feature reflected in the in vitro ability of TmFtn to reduce significantly iron-mediated oxidative damage to DNA at 80°C. A similar TmFtn-mediated ROS detoxifying role likely occurs in the bacterium which lacks the SOD/catalase defense systems of the aerobic world.