Effect of Spin Clustering on Basic and Relaxometric Properties of Magnetic Nanoparticles.

An increasing awareness about novel medical applications of smaller, inorganic-based nanoparticles, possessing unique properties at the nanoscale, has led to a burst of research activities in the development of "nanoprobes" for diagnostic medicine and agents for novel, externally activated therapies. In this research field magnetic nanoparticles are prominent due to fundamental peculiar properties particularly appealing for their use in materials and biomedical applications. Aiming to study the relationship between the topology of the magnetic nanoparticles and their efficacy as MRI contrast agents (relaxometric properties), we prepared three different stable colloidal suspension (ferrofluid) of magnetic nanobeads (MNBs) constituted by a discrete number of maghemite nanoparticles, arranged in disordered clusters or ordered in a polymeric matrix. An accurate morpho-dimensional and magnetic characterization displays the close correlation between the magnetic fundamental properties and the topology of our spin systems. The NMR relaxometry profiles confirmed the nature of the physical mechanisms inducing the increase of nuclear relaxation rates at low (magnetic anisotropy) and high (Curie relaxation) magnetic fields. Moreover the transverse relaxivity (r2) values for all the MNBs are higher than those of common contrast agents and the differences between the three MNBs are suggested to be due to the spin topology effect.

Local spin dynamics of iron oxide magnetic nanoparticles dispersed in different solvents with variable size and shape: A 1H NMR study.

Colloidal magnetic nanoparticles (MNPs) based on a nearly monodisperse iron oxide core and capped by oleic acid have been used as model systems for investigating the superparamagnetic spin dynamics by means of magnetometry measurements and nuclear magnetic resonance (1H NMR) relaxometry. The key magnetic properties (saturation magnetization, coercive field, and frequency dependent "blocking" temperature) of MNPs with different core size (3.5 nm, 8.5 nm, and 17.5 nm), shape (spherical and cubic), and dispersant (hexane and water-based formulation) have been determined. 1H NMR dispersion profiles obtained by measuring the r1 (longitudinal) and r2 (transverse) nuclear relaxivities in the frequency range 0.01-60 MHz confirmed that in all samples the physical mechanisms that drive the nuclear relaxation are the Néel reversal at low temperature and the Curie relaxation at high frequency. The magnetization reversal time at room temperature extracted from the fitting of NMR data falls in the typical range of superparamagnetic systems (10-9-10-10 s). Furthermore, from the distance of minimum approach we could conclude that water molecules do not arrive in close vicinity of the magnetic core. Our findings contribute to elucidate the local spin dynamics mechanisms in colloidal superparamagnetic nanoparticles which are useful in biomedical application as, e.g., contrast agents for magnetic resonance imaging.

Characterization of magnetic nanoparticles from Magnetospirillum Gryphiswaldense as potential theranostics tools.

We investigated the theranostic properties of magnetosomes (MNs) extracted from magnetotactic bacteria, promising for nanomedicine applications. Besides a physico-chemical characterization, their potentiality as mediators for magnetic fluid hyperthermia and contrast agents for magnetic resonance imaging, both in vitro and in vivo, are here singled out. The MNs, constituted by magnetite nanocrystals arranged in chains, show a superparamagnetic behaviour and a clear evidence of Verwey transition, as signature of magnetite presence. The phospholipid membrane provides a good protection against oxidation and the MNs oxidation state is stable over months. Using an alternate magnetic field, the specific absorption rate was measured, resulting among the highest reported in literature. The MRI contrast efficiency was evaluated by means of the acquisition of complete NMRD profiles. The transverse relaxivity resulted as high as the one of a former commercial contrast agent. The MNs were inoculated into an animal model of tumour and their presence was detected by magnetic resonance images two weeks after the injection in the tumour mass.

A possible novel objective intraoperative measurement of maxillary bone density.

AIM: Implant survival and success rates are strictly related to the density of the bone they are placed in. Bone density, in fact, affects both implant primary stability and implant micromovements after implant positioning. Current bone density classifications rely on subjective, scarcely reproducible evaluations. A novel implant micro motor featuring a bone density measurement probe has been recently introduced. The objective of this study was to test such bone density measurement system for its capability of distinguishing different bone density areas in the upper and in the lower jaw. METHODS: 1254 implant placement sites had their bone density measured during standard implant placement at a single clinical facility. After data collection bone density distribution was statistically analyzed in order to test the hypothesis of a non-homogeneous distribution in four different predefined anatomical maxillary zones, namely pre-antral (between teeth from 14 to 24) and sub-antral (more distally) in the upper maxilla and interforaminal (between and including teeth from 34 to 44) and retroforaminal (more distally) zone. RESULTS: Measured bone density values, organized according the named four anatomical zones, produced a statistically significant inhomogeneous pattern (P<0.001). Density distribution was consistent with data from literature, but not always corresponding with the one achieved by applying the well known Misch classification. CONCLUSION: The measuring system we tested allowed to distinguish different and clinically significant anatomical zones according to their different bone density, and can represent a fundamental diagnostic tool to plan the proper implant placement steps.

NMR-D study of the local spin dynamics and magnetic anisotropy in different nearly monodispersed ferrite nanoparticles.

We present a systematic experimental comparison of the superparamagnetic relaxation time constants obtained by means of dynamic magnetic measurements and (1)H-NMR relaxometry, on ferrite-based nanosystems with different composition, various core sizes and dispersed in different solvents. The application of a heuristic model for the relaxivity allowed a comparison between the reversal time of magnetization as seen by NMR and the results from the AC susceptibility experiments, and an estimation of fundamental microscopic properties. A good agreement between the NMR and AC results was found when fitting the AC data to a Vogel-Fulcher law. Key parameters obtained from the model have been exploited to evaluate the impact of the contribution from magnetic anisotropy to the relaxivity curves and estimate the minimum approach distance of the bulk solvent.

Atomic force microscopy imaging of lipid rafts of human breast cancer cells.

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.

Multifunctional nanoparticles for dual imaging.

For imaging with different modalities, labels, which provide contrast for all modalities, are required. Colloidal nanoparticles composed out of an inorganic core and a polymer shell offer progress in this direction. Both, the core and the polymer shell, can be synthesized to be fluorescent, magnetic, or radioactive. When different cores are combined with different polymer shells, different types of particles for dual imaging can be obtained, as for example, fluorescent cores with radioactive polymer shells. Properties and perspectives of such nanoparticles for multimodal imaging are discussed.

Water-soluble rhamnose-coated Fe3O4 nanoparticles.

Water-soluble biocompatible rhamnose-coated Fe(3)O(4) nanoparticles of 4.0 nm are obtained by covalent anchorage of rhamnose on the nanoparticles surface via a phosphate linker. These nanoparticles present superparamagnetic behavior and nuclear relaxivities in the same order of magnitude as Endorem that make them potential magnetic resonance imaging (MRI) contrast agents of a second generation, where the saccharides represent also specific ligands able to target lectins on skin cells.

Intermittent contact mode AFM investigation of native plasma membrane of Xenopus laevis oocyte.

Intermittent contact mode atomic force microscopy (AFM) was used to visualize the native plasma membrane of Xenopus laevis oocytes. Oocyte membranes were purified via ultracentrifugation on a sucrose gradient and adsorbed on mica leaves. AFM topographs and the corresponding phase images allowed for visualization and identification of both oocyte plasma membrane patches and pure lipid bilayer regions with a height of about 5 nm within membrane patches. The quantitative analysis showed a normal distribution for the lateral dimension and height of the protein complexes centered on 16.7 +/- 0.2 nm (mean +/- SE, n = 263) and 5.4 +/- 0.1 nm (n = 262), respectively. The phase signal, providing material-dependent information, allowed for the recognition of structural features observed in AFM topographs.

A new model of resorbable device degradation and drug release: transient 1-dimension diffusional model.

In the framework of resorbable devices, an increased demand for a better reliability in drug delivery systems emerged, based on resorbable polymers degradation control. These points influenced researchers to replace the trial-and-error approaches with model-based ones. In this context, we developed a transient 1-dimension model for degradation and drug release of resorbable polymeric drug loaded systems. Such model is thought to be applied to design specific devices based on the expected degradation time and drug delivery rates. It is based on the detailed conservation equations taking into account main physical-chemical parameters involved in polymer degradation and in drug release, all independently estimated. The model leads to a system of partial differential equations solved numerically, which predictions were verified through both literature and our data. Data of different authors, various systems and directly generated data were matched by model predictions. Reliability of parameter estimation procedures was also confirmed by accelerated life tests.

Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome.

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P < 0.001) and CD105 (P < 0.001), and lower expression of CD71 (P < 0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r = 0.89, P < 0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify > 95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.

Denatured H-ferritin subunit is a major constituent of haemosiderin in the liver of patients with iron overload.

BACKGROUND AND AIMS: Iron is stored in hepatocytes in the form of ferritin and haemosiderin. There is a marked increase in iron rich haemosiderin in iron overloaded livers, and ferric iron in amounts exceeding the ferritin and haemosiderin binding capacity may promote free radical generation, causing cellular damage. The aim of this study was to characterise hepatic haemosiderin using four antibodies specific for either native or denatured H/L-ferritin subunits. METHODS: Ferritin and haemosiderin were prepared from the livers of three patients with post-transfusional iron overload. The assembled ferritin molecules were analysed by non-denaturing polyacrylamide gel electrophoresis (PAGE)-immunoblotting. Ferritin subunits in the haemosiderin fraction were assessed by denaturing sodium dodecyl sulphate (SDS)-PAGE-immunoblotting. Distribution of native and denatured ferritin subunits in hepatocytes was examined by immunogold electron microscopy. RESULTS: Non-denaturing PAGE-immunoblot analyses showed that the assembled liver ferritins were recognised by the antibodies for native ferritins and not by those for the denatured subunits. Both SDS-PAGE-immunoblot and immunogold electron microscopic analyses disclosed that haemosiderin of iron overloaded liver reacted predominantly to the monoclonal antibody for the denatured H-ferritin subunit, to a lesser degree to that for denatured L-ferritin, and very weakly, if any, with antibodies for native H-ferritin or L-ferritin. CONCLUSIONS: These results suggest that in iron overloaded liver, haemosiderin consists predominantly of denatured H-ferritin subunits.

Structure of mouse L-chain ferritin at 1.6 A resolution.

Cubic F432 crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique with ammonium sulfate and cadmium sulfate as precipitants. The structure was refined to 2.1 and 1.6 A resolution from data obtained at room temperature and under cryogenic conditions, respectively. The structure of an eight-amino-acid loop insertion in the mouse sequence is found to be highly disordered both at room temperature and at low temperature.

Is hydrogen peroxide produced during iron(II) oxidation in mammalian apoferritins?

The ferritins are a class of iron storage and detoxification proteins that play a central role in the biological management of iron. These proteins have a catalytic site, "the ferroxidase site", located on the H-type subunit that facilitates the oxidation of Fe(II) to Fe(III) by O(2). Measurements during the past 10 years on a number of vertebrate ferritins have provided evidence that H(2)O(2) is produced at this diiron ferroxidase site. Recently reported experiments using three different analytical methods with horse spleen ferritin (HoSF) have failed to detect H(2)O(2) production in this protein [Lindsay, S., Brosnahan, D., and Watt, G. D. (2001) Biochemistry 40, 3340-3347]. These findings contrast with earlier results reporting H(2)O(2) production in HoSF [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965-19970]. Here a sensitive fluorescence assay and an assay based on O(2) evolution in the presence of catalase were used to demonstrate that H(2)O(2) is produced in HoSF as previously reported. However, because of the relatively few H-chain ferroxidase sites in HoSF and the reaction of H(2)O(2) with the protein, H(2)O(2) is more difficult to detect in this ferritin than in recombinant human H-chain ferritin (HuHF). The proper sequence of addition of reagents is important for measurement of the total amount of H(2)O(2) produced during the ferroxidation reaction.

Immunosuppressive effects of melanoma-derived heavy-chain ferritin are dependent on stimulation of IL-10 production.

Cultured melanoma cells release soluble factors that influence immune responses. Screening of a cDNA library with anti-sera from a melanoma patient identified an immunoreactive plaque, which encoded heavy-chain ferritin (H-ferritin). Previous studies have drawn attention to the immunosuppressive effects of this molecule and prompted further studies on its biochemical and functional properties in human melanoma. These studies demonstrated, firstly, that H-ferritin appeared to be secreted by melanoma cells, as shown by immunoprecipitation of a 21.5 kDa band from supernatants. It was also detected in extracts of melanoma cells by Western blotting as 43 and 64 kDa dimers and trimers of the 21.5 kDa fraction. Secondly, flow-cytometric analysis of H- and light-chain ferritin (L-ferritin) expression on melanoma showed a wide variation in L-ferritin expression and consequently of the ratio of H- to L-ferritin expression. Suppression of mitogenic responses of lymphocytes to anti-CD3 showed a correlation with the ratio of H- to L-ferritin in the supernatants and was specific for H-ferritin, as shown by inhibition studies with a monoclonal antibody (MAb) against H-ferritin. Similar results were obtained with H- and L-ferritin from other sources. Suppression of mitogenic responses of lymphocytes to anti-CD3 by H-ferritin was inhibited using a MAb against IL-10, which suggested that the immunosuppressive effect of H-ferritin was mediated by IL-10. Assays of cytokine production from anti-CD3-stimulated lymphocytes showed that H-ferritin markedly increased production of IL-10 and IFN-gamma and had only slight effects on IL-2 and IL-4 production. Our results suggest that melanoma cells may be a major source of H-ferritin and that production of the latter may account for some of the immunosuppressive effects of melanoma.

A human mitochondrial ferritin encoded by an intronless gene.

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.

Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: identification of the new mutation C14G.

BACKGROUND: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. METHODS: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5' untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. RESULTS: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 microg/L. The new mutation G14C was identified. CONCLUSIONS: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11.

Expression of the VL-domain of mouse monoclonal antibody F11 to human spleen ferritin in Escherichia coli cells is associated with the formation of insoluble protein aggregates (inclusion bodies). The aggregates were solubilized in the presence of guanidine hydrochloride and the recombinant VL-domain was purified by immobilized metal affinity chromatography (IMAC). Subsequent renaturation results in approximately 99% pure preparation with high yield. The VL-domain forms dimers at concentrations from 1 to 10 mg/ml. Monomeric form is detected only at protein concentrations below 0.5 mg/ml. Functional activity of the VL-domain was verified by two variants of ELISA. The affinity of the VL-domain ((0.2-1.2). 108 M(-1)) is similar to the affinity of the full-length parental antibody F11 because when the immobilized VL-domain was used, the binding constant of ferritin to the VL-domain was only 4-6-fold lower than that in the case of F11 antibody. In another ELISA system with immobilized ferritin, affinity was decreased 30-fold. The VL-domain of antibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in the absence of the partner VH-domain. The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other antigen-binding proteins in immunotherapy and in studies of correlations between folding, stability, and activity of immunoglobulins.

Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells.

The human monocytic cell line THP-1 differentiates along the macrophage line after phorbol-12-myristate-13-acetate (PMA) supplementation and can be stimulated to secrete tumour necrosis factor alpha (TNF-alpha) by interferon gamma (IFN-gamma) addition. We found that, in the early stage of differentiation (1-48 h), PMA induction elicited an upregulation of intracellular H ferritin and H ferritin binding sites and a downregulation of transferrin receptor. In addition, we found that iron administration to PMA-differentiating cells induced the expression of TNF-alpha mRNA and TNF-alpha secretion to levels even higher than those induced by IFN-gamma alone. The iron chelator desferrioxamine showed the opposite effect and reduced TNF-alpha release. In contrast, preincubation of the cells with iron before PMA induction resulted in a decrease of the TNF-alpha secretion induced by IFN-gamma, whereas the opposite was true after preincubation with desferrioxamine. The data support a co-ordinate interaction between iron and TNF-alpha in monocyte macrophages, with an iron-mediated upregulation of TNF-alpha in the early phase of differentiation and an iron-mediated inhibition at later stages. This complex relationship has to be considered in evaluating the effects of iron on inflammation.