WO2011107290A1

WO2011107290A1 - Magnetic nanoparticles for cancer diagnostic and treatment purposes

Info

Publication number: WO2011107290A1
Application number: PCT/EP2011/001089
Authority: WO
Inventors: Daniele Barbaro; Fabio Monzani; Lorenzo Di Bari; Claudio Evangelisti; Piero Salvadori; Giovanni Vitulli; Silvia Ursino
Original assignee: Advanced Catalysts S.R.L.; Dipartimento Di Medicina Interna; Società Medico Chirurgica Livornese
Priority date: 2010-03-04
Filing date: 2011-03-04
Publication date: 2011-09-09
Also published as: EP2542264A1; ITLI20100001A1; IT1398263B1

Abstract

Magnetic nanoparticles for radiologic or RMI investigations or as a therapeutic means in magnetic induction hyperthermia. For all these biomedical applications the metal particles are magnetically active, of size much less than 100 nm. The magnetic particles are functionalized with organic biocompatible ligands that permit biologic transport of the nanoparticles in a specific area. The magnetic particles are prepared substituting the ligand from metal atoms solvated in a solvent. A method for preparing metal nanoparticles by Metal Vapour Synthesis and their stabilization with biocompatible ligands of different nature, in particular iron/glucose nanoparticles. Internalization of the metal nanoparticles, in particular glucose-stabilized iron particles into neoplastic cells.

Description

TITLE

MAGNETIC NANOPARTICLES FOR CANCER DIAGNOSTIC AND TREATMENT PURPOSES

DESCRIPTION

Field of the invention

The present invention relates to magnetic metal nanoparticles for cancer diagnostic and treatment purposes, to an intermediate compound for obtaining said metal nanoparticles and to a method for preparing said intermediate compound and said nanoparticles.

It also relates to a diagnostic or therapeutic use of such particles for cancer diagnostic and treatment purposes.

Background of the invention

In the last years, magnetic nanoparticles (MN) have received a high attention in biomedicine due to their use as a paramagnetic contrast means for radiologic and RMI investigations in oncology. Magnetic nanoparticles are also used as a therapeutic means in such techniques as magnetic induction hyperthermia, which comprises electromagnetically induced heating of nanoparticles within the tissues (M.M.J. Modo, J.W.M. Bulte, Molecular and Cellular MR Imaging, CRC Press, Boca Raton, FL 2007 and references).

For all these biomedical applications, the metal particles must show high magnetization values, a size much less than 100 nm as well as a narrow size distribution (A.K. Gupta, M. Gupta, Biomaterial, 26(18) 2005 3995). Size control is important for their use, since the nanocrystals properties are strongly affected by particle size. Moreover, magnetic particles need surface functionalization to receive organic ligands. The ligands must be atoxic, biocompatible and adapted to carry the particles themselves to a specific body area. To this purpose, magnetic particles can be bound to active principles, proteins, enzymes, antibodies or nucleotides and can be placed into an organ, a tissue, or a tumour, by means of an external magnetic field (M. Chastellain, A. Petri, A. Gupta, K.V. Rao, H. Hofmann, Adv. Eng. Mater. 6(4) 2004 235).

For this use, glucose is particularly important due to its capability to stabilize iron particles, since cancerous cells consume much more glucose than normal cells, changing most of it to lactic acid. Enhanced uptake of glucose by tumour cells is also known as the Warburg Effect (WE), which is defined as an increase of aerobic glycolisis and a decrease in oxidative phosphorylation as the energy source, and it's the most common metabolic alteration of most tumour cells (R.J DeBerardinis, Genet Med. 10(11 ) 2008 767-777).

The use of magnetic induction hyperthermia as a cancer therapy method, is strongly limited by MN size control, intrinsic magnetism and, more importantly, by means to selective delivery to the tumour tissue. Most current therapeutic applications of MN are based on their locoregional administration, which is followed by an alternating (AC) magnetic field application, which raises the local temperature and causes tissue death.

Many methods are known for preparing magnetic nanoparticles for RMI biomedical use: iron salt chemical coprecipitation, microemulsion, sol-gel synthesis, sonochemical reactions, hydrothermal reactions, precursor hydrolysis and thermolysis, flow injection synthesis, and electronspray (S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst, R.N. Muller Chem Rev. 108(6) 2008 2064 and cited reference). The synthesis of magnetic particles is a complex process due to their colloidal nature.

Organic ligands-stabilized Iron oxide magnetic particles are known by many types of both monomeric ligands like carboxylated phosphates and sulphates, and polymeric ligands like dextran, polyethylene glycol, polyvinylalcohol, alginates, chitosane, polyvinylpyrrolidone, ethylcellulose, polylactic acid. EP0525199 discloses the preparation of 2-30 nm diameter iron oxide magnetic particles, by chemical coprecipitation of polysaccharides complexed iron salts, to be used as T2 contrast media, mainly in MRI-angiography. EP0543020 discloses the preparation of similar 20-to-30-nm core diameter particles for use as a MRI contrast media which are bound with carboxydextrane to improve their pharmacological properties.

Due to the high hydrodynamic diameter of these polymeric particles, however, the above described systems are not suitable to be received by the interstitial liquid, nor to be internalized by cells via endocytosis. By now, only few examples are described of metal nanoparticles that are functionalized by an organic ligand whose size is small enough to allow the internalization into cancerous cells. In oncology, as already stated, the particular nature of the used functionalized particles has made possible only their locoregional use, since they are not selective enough towards cancerous cells, and their size is not small enough.

A novel possibility to exploit the Warburg effect for diagnostic and therapeutic purposes provides binding glucose to metal nanoparticles. Metal nanoparticles that are coated and functionalized by organic molecules can be used as a paramagnetic radiologic contrast means and as an oncology therapeutic means, by an electromagnetically induced heating according to the endogenous hyperthermia technique. In oncology, the particular nature of the known functionalized particles has allowed only their locoregional use, since the organic ligands are not selective enough towards cancerous cells the nanoparticles size is not small enough (VS. Kalambur, E.K. Longmire, J.C: Bischof, Langmuir. 2007; 23:12329; A. Jordan, P. Wust, R. Scholz, B. Tesche, H. Fahling, T. Mitrovics, T. Vogl, J. Cervos-Navarro, R. Felix, Int J Hyperthermia, 12(6) 1996 705; K. Maier-Hauff, R. Rothe, R. Scholz, U. Gneveckow, P. Wust, B. Thiesen, A. Feussner, A. Von Deimling, N. Waldoefner, R. Felix, A. lordan, I Neurooncol. 81(19) 2007 53; P. Wust, U. Gnevechow, M. lohannsen, D: Boluner, T.. Henkel, F. Kalunann, I. Sehouli, R. Felix, I. Ricke, A. lordan; Int I Hyperthermia. 22(8) 2006; lordan, I Neurooncol. 81(19) 2007 53; P. Wust, U. Gnevechow, M. lohannsen, D: Boluner, T.. Henkel, F. Kalunann, I. Sehouli, R. Felix, I. Ricke, A. lordan; Int I Hyperthermia. 22(8) 2006 673. M. Johannsen, U. Gnevechow, B. Thiesen, K. Taymoorian, CH. Cho, N. Waldofner, R. Scholz, A. Jordan, SA. Loening, P. Wust, Eur Urol. 52(6) 20071653).

In "Size-Controlled Synthesis of Magnetite (Fe304) Nanoparticles Coated with Glucose and Gluconic Acid from a Single Fe(lll) Precursor by a Sucrose Bifunctional Hydrothermal Method" (Xiaohong Sun, et al Lab of Functional Polymer Materials, Department of Materials Chemistry, College of Chemistry, Nankai University, Tianjin 300071 , People's Republic of China - J. Phys. Chem. C, 2009, 113 (36), pp 16002-16008 DOI: 10.1021/jp9038682) published on August 18, 2009, a simple and facile hydrothermal reduction route is disclosed using a single iron precursor, FeCI3, and a combination of the inherent chemical reduction capability of sucrose decomposition products and their inorganic coordinating ability. The particle size is controlled in the range of 4-16 nm. Summary of the invention

It is therefore an object of the present invention to provide magnetic nanoparticles whose size is small enough to allow internalization of said nanoparticles into cells, in particular into tumoral cells.

It is also an object of the present invention to provide magnetic nanoparticles that are functionalized or stabilized by a ligand that is proper for internalization into cancerous cells, i.e. a ligand that can be can be assimilated by a cell.

It is a further object of the present invention to provide an intermediate compound to produce such nanoparticles in an industrially convenient way.

It is still an object of the present invention to provide a method for producing said intermediate and for changing said intermediate into said functionalized metal nanoparticle in an industrially convenient way.

It is a particular object of the invention to provide such a method which does not require such downstream purification operations as ultracentrifugation, magnetic filtration, etc, which would complicate an industrial scale process.

These and other objects are achieved by the preparation of metal atoms solvated in a solvent by Metal Vapour Synthesis, wherein the metal is selected from the group comprised of

— iron (Fe),

— cobalt (Co),

— a rare earth, in particular a lantanide (Ln),

— a combination thereof;

— a core of iron, or cobalt, or rare earth (Ln) in a shell of platinum (Pt) or gold (Au),

and wherein the solvent is a solvent selected from the group comprised of:

polar solvents:

— a carbonyl containing solvent;

— tetrahydrofuran;

— 1 ,4 dioxane;

— acetonitrile;

— a combination thereof

apolar solvents:

— an aromatic solvent, — an olefinic solvent

— a long chain ether.

— a combination thereof

In case of combination of two metals monoatomic alloys can be obtained where the first metal can be iron and the second metal can be selected among Co, Pt, Ln (rare earth metal), in order to obtain Fe/Co, Fe/Pt, Fe/Ln monoatomic alloys.

In an embodiment, the carbonyl groups-containing solvent is selected from the group comprised of: acetone, N-methylpirrolydone, an alcohol, in particular ethylic alcohol or isopropylic alcohol.

The apolar solvents may be selected from the group comprised of: mesitylene, toluene, cumene or xylene; 1-esene, 2-esene.

In an exemplary embodiment, the solvent is selected from the group consisting of acetone and wherein the metal is Iron (Fe).

In another exemplary embodiment, the solvent is mesitylene and wherein the metal is Iron (Fe).

The above objects are also attained by metal nanoparticles comprising organic ligands, the nanoparticles obtained replacing the solvent of the above solvated of claim 1 with a ligand selected from the group comprised of:

— a monosaccharide;

— dextrane;

— polyvinylpyrrolidone;

— polyvinylalcohol;

— oleic acid.

— a combination thereof

In an exemplary embodiment, the monosaccharide is glucose.

Glucose may be selected between L-glucose, D-Glucose and a combination thereof, in particular it may be a racemic mixture thereof.

Advantageously Metal_xO_y/Ligand or Metal/Ligand nanoparticles are obtained by adding to said solvated an acqueous solution of said ligand at room temperature from which said solvent is eventually removed. In particular, said solvent is removed by vacuum application. Preferably, after removal of said solvent dry water soluble solid nanoparticles are obtained by drying said solution. Alternatively, said solvent is removed by separation after precipitation of said MetalxOy/Ligand or Metal/Ligand nanoparticles.

The "in vivo" use is also disclosed for a diagnostic and therapeutic purpose of the above magnetic nanoparticles.

The "in vivo" use by intralesional, loco-regional application, or by intravenous injection is also disclosed of the above magnetic nanoparticles.

The "in vivo" use for a therapeutic purpose of the above magnetic nanoparticles is also disclosed for treating tumours, by hyperthermia.

The above objects are also attained by a method for forming a solvated of a magnetic metal with a solvent, comprising the steps of:

— prearranging a container;

— introducing in the container vapours of a solvent

— covering an internal surface of the container with a layer of the solidified solvent by cooling the container;

— creating a prefixed vacuum degree within the container;

— vaporizing an amount of the metal within the container, such that the magnetic metal by contacting the layer forms a solvate with the solid solvent;

— thawing the solidified layer such that atoms of the metal form the solvated, wherein the metal is selected from the group comprised of

— iron (Fe),

— cobalt (Co),

— a rare earth, in particular a lantanide (Ln),

— a combination thereof;

and wherein the solvent is a solvent selected from the group comprised of: polar solvents:

— a carbonyl containing solvent;

— tetrahydrofuran;

— 1 ,4 dioxane;

— acetonitrile;

— a combination thereof

apolar solvents: — an aromatic solvent,

— an olefinic solvent

— a long chain ether.

— a combination thereof.

In particular, the magnetic metal comprises at least two metal elements and wherein the step of vaporizing comprises a first step of vaporizing a first metal and a subsequent second step of vaporizing a second metal such that the second metal solidifies on the previously solidified first metal, a core formed by the first metal is covered by a formed by the second metal, in particular a monoatomic shell layer.

In particular, the magnetic metal comprises at least two metal elements and wherein the step of vaporizing comprises a step of simultaneously vaporizing the first and the second metal, such that the first and the second metal solidifies as an alloy.

Advantageously, a step is provided of controlling the temperature of a source of the first and of the second metal in a condensed state, such that the amount of the first metal and the amount of the second metal vaporize at a prefixed vaporization ratio.

At the basis of the present invention there are the following innovations: a method for preparing iron oxide nanoparticles by Metal Vapour Synthesis and stabilization of the nanoparticles by various ligands, among which glucose. The process comprises the following steps:

— condensation of iron vapours together with an organic compound, in particular an aromatic, a carbonyl-containing or an olefinic compound, within a suitable reactor, thus forming a solid matrix, i.e. a solid solution of the metal and of the organic compound;

— melting the solid matrix to obtain an iron-containing liquid solution (solvated iron clusters);

— stabilizing the solvated iron clusters with an organic ligand, which can be a polymeric ligand such as polyvinylpyrrolidone or a monomeric ligand such as glucose, by direct interaction in a solution of similar or miscible solvents, and treating with molecular or atmospheric oxygen at room temperature and at atmospheric pressure;

— isolating the stabilized iron particles by vacuum-drying; — internalizing the glucose-stabilized iron oxide particles into neoplastic cells: two thyroid carcinoma cell lines, (papillary and nondifferentiated carcinoma) as well as human normal cells (fibroblasts).

Therefore, glucose-coated metal nanoparticles having particular paramagnetic features and a very small size, are suitable for novel diagnostic and therapeutic uses.

A novel method is also provided for preparing such nanoparticles and for binding them to glucose, which makes them particularly well-suited for exploiting the Warburg effect, as shown by our in vitro essays.

For a diagnostic and therapeutic use of metal nanoparticles, it is necessary to verify that the nanoparticles enters into the cancerous cells and that a subsequent het treatment allows to destroy the cancerous cells. To this purpose, previous investigations used the electrons transmission microscope and showed that the nanoparticles are internalized at the endosomal level. On the contrary, functionalized particles provided with specific membrane receptors were observed on the cellular membrane without being internalized. Further methods have been set up for quantifying nanoparticles inlet into the cell (V.S. Kalambur, E.K. Longmire, I.C: Bischof, Langmuir. 2007; 23:12329).

Our reasoning is very simple: by means of MVS we can prepare particularly small (2nm or below) nanostructured MN; if we decorate them with glucose, we can take advantage of WE and promote a selective MN uptake by tumour cells. This accumulation will provide enhanced contrast in MRI and at the same time can be used for magnetic induction hyperthermia.

In principle, the advantage of this technique should consists in the double localization of the particles and of the focus of the magnetic field, for achieving high specificity.

We observed that a macromolecular coating used for commercial MN is dextran (or its derivatives), a polymer of glucose, which ensures that the colloidal suspension is stable and well tolerated. The high molecular weight of dextran is responsible for good stability of large MN, while glucose or other simpler saccharides may be better suited for self-assembled monolayers on smaller systems and at the same time be viable for WE, at variance with dextran. Concerning metal vapour synthesis (MVS), which is an established, method for obtaining organometallic compounds, according to the invention it is exploited to obtain very small metal solvated compounds, between 1 and 10 nm, preferably between 1 and 3 nm. From the very small metal solvated compounds, nanoparticles adapted for the use according to the invention are then obtained. It comprises evaporating a metal into a container where a high vacuum has been made, and whose walls are kept at a very low temperature, for instance, by external liquid nitrogen contacting, a solidified layer of an organic substance being provided on the inner wall of the container. This way, single solvated metal atoms deposit on the solidified layer creating a solid matrix, i.e. a solid solution of the metal and of the organic compound. Upon thawing, the solvated metal atoms give rise to nanoparticles, a process which needs careful control to achieve monodisperse systems. The nanoparticles can be coated (i.e. decorated) with suitable small-size or macromolecular ligands and/or may be exposed to oxidising agents. This method allows also for simultaneous co-evaporation of two metals and formation of nanostructured alloys.

Brief description of the drawings

Further exemplary features of the present invention will be made clearer with the following examples, exemplifying but not limitative, with reference to the attached drawings, in which:

— Fig. 1 is a diagram showing the nanoparticles absorbance after cell lysis at various nanoparticles concentrations and after various incubation times;

— Fig. 2 is a diagram showing the nanoparticles absorbance after cell lysis at various nanoparticles concentrations and after various incubation times for FB3 cell line;

— Fig. 3 is a diagram showing the per cent increase trend of NHDF tumoral cells at a prefixed nanoparticles concentration, and for two different ligands;

— Fig. 4 is a diagram showing the effect of nanoparticles concentration on NHDF tumoral cells per cent increase after a given treatment time;

— Fig. 5 is a diagram showing the per cent increase trend of FB3 tumoral cells at a prefixed nanoparticles concentration, and for two different ligands.

Description of preferred application of the invention MVS has been used to prepare a set of Magnetic Nanoparticles MN, which can be coated by glucose or other simple saccharides, polyvinylpyrrolidone; polyvinylalcohol; oleic acid, and then screened in vitro and in vivo. Preparation of monometallic systems

Very small metal particles (< 5 nm) have been prepared by means of MVS, by co-condensation at low temperature (-196°C) of metal vapours with a vapour of weakly stabilizing ligand. Metal particles of tailored size have been obtained controlling the main factors affecting metal atoms aggregation in solution i.e. organic ligand vs. metal; their molar ratio and the temperature of the solvated metal atoms.

In particular, Fe MN particles have been prepared and various oxidation protocols (O₂, peroxides, N-oxides) have been tested to obtain stable magnetic FeOx cores. Different approaches have been attained to coat or decorate them with D-glucose (D-glc) and to obtain Glucose coated magnetic nanoparticles GCMN.

Various ligands were used to replace glucose, such as other

MN characterization.

MN has been characterized with respect to relaxivity at 4.7 and 7T, hydrodynamic radius with dynamic light scattering (DLS), electron microscopy. Other measurements (magnetic susceptivity) have been evaluated.

Chemical characterization has also been performed: IR and NMR spectroscopy, AA (for determining Fe or other metal content), MS.

This protocol has been repeated on ageing material to determine its shelf life under different storage conditions

Preparation of bimetallic systems.

Different protocols to prepare bimetallic MN have been used. Firstly coevaporation, where two metals are put into separate vessels in the same reactor and they are sublimated simultaneously. The contact between the two elements occurs at the moment of thawing, i.e. at the stadium of solvated metal atoms (SMA), which is likely to result in the formation of mixed lattices (alloys). One the contrary, mixing the solutions of pre-formed SMA has led to the formation of separated sub-nanoscopic domains or, according to the procedure, to specific geometries like e.g. core-shell. These variables lead to different materials, such as Fe/Pt, Fe/Co and possibly also Fe/rare earths (RE) indicated also as Ln. In these cases the oxidation step may be not necessary or even undesirable.

To prevent it, coating of the metal core with an Au monoatomic layer has also been effected.

Cell lines and tests in vitro

Both D-Glucose and L-Glucose coated MNs have been prepared (respectively D-GCM and L-GCMN).

For tests in vitro the line of a poorly differentiated papillary thyroid cancer FB3 has been used, although no specific tests for the analysis of glucose transporters in this line had been done, as known, thyroid cancer cells express glucose transporter 1 and 3 (GLUT 1 , GLUT 3). As control we have used a normal line of fibroblast, Normal Diploid Human Fibroblasts (NHDF), Clonetics (Ogden, Utha); normal fibroblast are known to express glucose transporter 1 (GLUT1 ).

Ferro-fluid was added at different concentrations as stated after and after cell lysis analysis of iron was performed by adding ferrozyme, samples have been red by spectrophotometer at 560 nm and the absorbance has been expressed for million of cell.

The experimental steps were as follow:

-First preliminary step was to evaluate if d-glucose was internalized by FB3 and evaluate the kinetic of internalization of d-GCMN, according to the literature we performed the test at the same time already described for silane-coated MN that is 3, 12, 24, 48, 72 hour with concentration of magnetic ferro-fluid of 0.025, 0.5, 1 mg /dl

-In the second step, we decided to explore the kinetic at minor time 1 ,2,3 hours and at the same ferro-fluid concentration again in FB3.

-In the third step we investigated internalization of d-GCMN at the same time and concentration both in FB3 and NHDF. Since the internalization appeared to be almost steady at longest time (12, 24, 48 and 72 hours) we decided to investigate the internalization only at 1 , 2 and 3 hours.

-In the fourth step we investigated, at the same time and concentration as stated above (1 , 2 and 3 hour), the internalization both of d-glucose and I- glucose in FB3 and fibroblasts. The results were reported as the mean +/- SD of two points for each concentration and time

Results

Tests with d-GCNP.

For FB3 at the first hour the absorbance of the supernatant after cell lysis was double than the control (supernatant of cells not treated by nanoparticles) for the concentration of 0.025 mg /dl. At the concentration of 0.05 the absorbance increased to three times than control with no further increase at 0.1 mg/dl. For NHDF the absorbance of the supernatant at 1 hour was double than the control, at concentration of 0.05 mg/dl the absorbance increased to four times than the control with no further increase for the concentration of 0.1 mg/dl.

For both FB3 and NHDF no further increase of absorbance was noticed at longer time.

Test with l-GCNP

For FB3 the first hour the absorbance of the supernatant after cell lysis was double than the control for the concentration of 0.025. At the concentration of 0.05 the absorbance increased to eight time than the control with no further increase at the concentration of 0.1 mg/dl. For NHDF the absorbance of the supernatant at the first hour was the same of the control for all the concentrations although, without a statistical meaning, a mild trend to increase with a linear relationship could be observed.

ForFB3 the absorbance of the supernatant after the cell lysis at the second and third hour tended to be higher for each point of concentration (however with no statistical difference)

For NHDF the absorbance of the supernatant after the cell lysis at longer time permitted to show an increase of the value with a clear linear relationship both dose dependent and time dependent without kinetic of saturation.

Results of the tests are shown in Figs. 3-5.

Studies in vivo on animal models.

Tests in vivo regarding pharmacokinetic and possible toxicity of the GCMNs have been performed. Rats of the brown norway (BN) strain have been used. BN7005-H1 D2 is a single -cell clone of a rat colon carcinoma induced by 1 ,2 - dimethyl-hidralazine in a BN rat. Cells have been be cultured in the medium of growth, then washed and trypsinized. Rats have be inoculated subperitoneally and experiments initiated after 12-14 days after inoculation when tumour size was around 1 .5 cm.

Example 1

Preparing the system Fe/Acetone

Iron Vapour, obtained by heating a tungsten melting pot covered with alumina containing 200 mg of Fe were co-condensed with 100 ml of acetone at the liquid nitrogen temperature within a glass reactor during about 50 minutes, forming a solid matrix. Thereafter, the reactor was brought to the melting temperature of the solid matrix (about -90°C) and the resulting brown solution was recovered by siphoning it at a low temperature (-30X) into a Schlenk tube, under inert gas. The solution iron content, which was evaluated by ICP-OES analysis, was 1.1 mg/ml. The solution was stable under inert gas (Ar) at 30°C for some days, and no precipitation took place.

Preparing the system Fe_xO_y-Glucose

The above Fe/acetone solution wad added with 22 ml of a 50 % wt. aqueous glucose solution under inert gas and up to a temperature of 25°C; the obtained liquid contained therefore about 1 1 g of glucose, which means a Fe/GLU weight ratio of 1 %. The solution was stirred for about 1 hour under a molecular oxygen atmosphere at the pressure of 1 atm and at room temperature (25°C); afterwards, the solvent was removed by vacuum application and the solid was dried overnight by a mechanical pump. A greatly water soluble solid was obtained. Fe_xO_y-Glucose systems at different Fe/Glucose weight ratios (1 to 20 %) had been prepared by suitably changing the glucose amount added to the Fe/acetone solution. The diameter of the metal particles of such systems was comprised between 1 and 3 nm.

Example 2

Preparing the system Fe_xO_y-polyvinylpyrrolidone (PVP)

100 ml of example 1 Fe/acetone solution were added with 1 1 g of PVP K30 (average molecular weight 30000 Da) dissolved in 40 ml of absolute ethanol under inert gas, up to a temperature of 25°C. The solution was stirred for about 1 hour under a molecular oxygen atmosphere at the pressure of 1 atm and at room temperature (25°C). The solution, which was stable at room temperature, was added with 150 ml of diethyl ether, which caused the Fe_xO_y - PVP adduct to precipitate. The solid was isolated and dried by a mechanical pump overnight. A greatly water soluble Fe_xO_y-PVP 1 % solid was obtained, systems Fe_xO_y -PVP to different ratios Fe/PVP (1-20 % by weight) had been prepared by suitably changing the PVP amount added to the Fe/acetone solution.

Example 3

Preparing the system Fe/mesitilene

Iron vapours, obtained by heating a tungsten melting pot covered with alumina and containing 230 mg of Fe were co-condensed with 60 ml of mesitilene at the liquid nitrogen temperature within a glass reactor during about 50 minutes, forming a solid matrix. Thereafter, the reactor was brought to the melting temperature of the solid matrix (about -40°C) and the resulting brown solution recovered by siphoning at a low temperature (-30°C) into a Schlenk tube under inert gas. The solution iron content, evaluated by ICP-OES analysis, was 1.6 mg/ml. The solution was stable under inert gas (Ar) at 30°C for some days and no precipitation took place.

Example 4

Preparing the system Fe_xOyOleic acid (AO)

100 ml of the example 1 Fe/acetone solution were added with 2.73 ml of oleic acid (AO; molecular weight: 282.47, d = 0.89 g/ml; molar ratio AO/Fe = 3) under inert gas up to a temperature of 25°C. The solution was stirred for about 1 hour under a molecular oxygen atmosphere at the pressure of 1 atm and at room temperature (25°C). The solution, which was stable at room temperature, was added with 100 ml of methane which caused a the Fe_xO_y -AO adduct to precipitate. The solid was isolated and dried by a mechanical pump overnight. A greatly toluene-soluble Fe_xO_y-AO solid was obtained.

Example 5

Internalizing the glucose-stabilized iron oxide particles into neoplastic cells.

Papillary carcinoma FB3 cell line is responsive both to nanoparticle concentration and to exposure time. Cells have been incubated with 0.05, 0.1 , 0.5, 1 mg/ml of nanoparticles during 3, 24, 48 and 72 hours. Cells of the same kind, not incubated with nanoparticles, were used as a control. Cells were cultivated in DMEM (Dulbecco's modified Eagle medium) with addition of 10% bovine fetal serum (FBS) and of 1 % penicilline-streptomicine;

_? 6

25 cm flasks were used containing 0.6x10 cells/flask in 2 ml of ground at 37°C with a 5% CO2 flow. After the incubation, the cells have been "washed" with phosphate buffer (PBS), and then subjected to enzymatic lysis (trypsin 0.05%,

EDTA 0.02% in PBS). The nanoparticles intracellular incorporation was evaluated by a colorimetric method. The pellet was suspended again in 1 ml of distilled water and incubated at 65-70°C during two hours after addition of

0.25ml of 1.2M HCI and 0.1 ml of ascorbic acid. Thereafter, 0,1 ml of a solution containing 6.5 mM ferrozine, 13.1 mM neucoproine, 2M ascorbic acid and 5M ammonium acetate was added. The solution was left at room temperature (25°C) for 30 minutes and centrifuged (1000 rpm, 5 minutes). This process causes a colorimetric reaction due to the interaction between the iron oxide nanoparticles and a ferrozine compound. The supernatant absorbance at 560 nm was determined by a UV-vis spectrophotometer; the iron concentration was calculated by means of a standard curve. A solution of distilled water and of the above reactants was used as a blank. The absorbance was corrected taking

6 into account the final cell count, after incubation, and was normalized to 10 cells. Referring to Fig. 1 , the nanoparticles absorption kinetic shows an early saturation of the system (after 3 hours) at a very low concentration (0.05 mg/ml), and also shows a subsequent progressive dismission (after 72 hours absorbance is not much greater than the control).

Example 6

Internalizing the glucose-stabilized iron oxide particles into neoplastic cells: papillary carcinoma FB3 cell line

On the basis of the preliminary results (example 5), the test was repeated at lower nanoparticles concentrations (0.025, 0.05, 0,1 mg/ml) for shorter incubation times (1 , 2 and 3 hours). Fig. 2 shows a remarkable nanoparticles absorption already after an exposure of an hour to a concentration of 0.025 mg/ml. The absorption trend seems to be different after 1 , 2 or 3 hours of incubation. In fact, after an hour, a peak value is obtained for a concentration of 0.05 mg/ml followed by a plateau; instead, after 2 and 3 hours the peak value was obtained already at a concentration of 0.025 mg/ml and the like trend to plateau. Example 7

Use of the glucidic hypermetabolism (Warburg effect) in human diagnostics

FDG (2-(18F) fluoride-2-desoxiglucose) PET (positron emission tomography) has recently come into use. FDG is similar to glucose, therefore it is efficiently carried into the cells by the various glucose transporters (GLUT) and is phosphorilated by the hexokinase enzyme. The FDG-6-phosphate is not a glycolisis substrate and therefore tends to build up to give scintigraphic images. The above described Warburg effect is therefore a requirement for this important diagnostic method. FDG-PET/TAC, an improved version of FDG-PET technique, allows detecting neoplastic tissues and, in particular, very aggressive neoplastic tissues. Even if false positives may occur for inflammatory tissues and/or for quickly proliferating benign tumours, the method is a true diagnostic aid especially when semiquantitative extrapolations are made (SUV: standardized uptake value). By now, FDG PET and FDG PET TAC allow very useful and specific investigations, showing therefore that the glucidic hypermetabolism, the so-called Warburg effect, is a requirement for in vivo human diagnostic activities.

The foregoing description of specific embodiments and examples will so fully reveal the invention according to the conceptual point of view, so that others, by applying current knowledge, will be able to modify and/or adapt for various applications such embodiments without further research and without parting from the invention, and it is therefore to be understood that such adaptations and modifications will have to be considered as equivalent to the embodiments. The means and the materials to carry out the different functions described herein could have a different nature without, for this reason, departing from the field of the invention. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

Claims

A preparation of metal atoms solvated in a solvent by Metal Vapour Synthesis, wherein said metal is selected from the group comprised of

— iron (Fe),

— cobalt (Co),

— a rare earth, in particular a lantanide (Ln),

— a combination thereof;

— a core of iron, or cobalt, or rare earth (Ln) in a monoatomic shell layer of platinum (Pt) or gold (Au),

and wherein said solvent is a solvent selected from the group comprised of:

polar solvents

— a carbonyl containing solvent;

— tetrahydrofuran;

— 1 ,4 dioxane;

— acetonitrile;

— a combination thereof

apolar solvents

— an aromatic solvent,

— an olefinic solvent

— a long chain ether.

— a combination thereof

The preparation according to claim 1 , wherein said first metal is iron and said second metal is selected among Co, Pt, Ln (rare earth metal), in order to obtain Fe/Co, Fe/Pt, Fe/Ln monoatomic alloys.

The preparation according to claim 1 , wherein said carbonyl groups- containing solvent is selected from the group comprised of: acetone, N- methylpirrolydone, an alcohol, in particular ethylic alcohol or isopropylic alcohol.

The preparation according to claim 1 , wherein said apolar solvents are selected from the group comprised of: mesitylene, toluene, cumene or xylene; 1-esene, 2-esene.

5. The preparation of claim 1 wherein said solvent is acetone and wherein said metal is Iron (Fe).

6. The preparation of claim 1 wherein said solvent is mesitylene and wherein said metal is Iron (Fe).

7. Metal nanoparticles comprising organic ligands obtained replacing the solvent of the solvated of claim 1 with a ligand selected from the group comprised of:

— a monosaccharide;

— dextrane;

— polyvinylpyrrolidone;

— polyvinylalcohol;

— oleic acid.

— a combination thereof

8. Metal nanoparticles according to claim 7, wherein said ligand is glucose or dextrane.

9. Metal nanoparticles according to claim 7, wherein said glucose is selected between L-glucose, D-Glucose and a combination thereof, in particular a racemic mixture thereof.

10. Metal nanoparticles according to claim 7, wherein Metal_xO_y/Ligand or Metal/Ligand nanoparticles are obtained by adding to said solvated an acqueous solution of said ligand at room temperature from which said solvent is eventually removed.

11. Metal nanoparticles according to claim 10, said solvent is removed by vacuum application.

12. Metal nanoparticles according to claim 10, said solvent is removed by separation after precipitation of said Metal_xO_y/Ligand or Metal/Ligand nanoparticles.

13. Metal nanoparticles according to claim 10, wherein after removal of said solvent dry water soluble solid nanoparticles are obtained by drying said solution.

14. The "in vivo" use for a diagnostic and therapeutic purpose of magnetic nanoparticles according to claims 8 to 13.

15. The "in vivo" use by intralesional, loco-regional application or by intravenous injection of magnetic nanoparticles according to claims 8 to 13.

16. The "in vivo" use for a therapeutic purpose of magnetic nanoparticles according to claims 8 to 13, for hyperthermia of tumours.

17. A method for forming a solvated of a magnetic metal with a solvent, comprising the steps of:

— prearranging a container;

— introducing in said container vapours of a solvent

— covering an internal surface of said container with a layer of said solidified solvent by cooling said container;

— creating a prefixed vacuum degree within said container;

— vaporizing an amount of said metal within said container, such that said magnetic metal by contacting said layer forms a solvate with said solid solvent;

— thawing said solidified layer such that atoms of said metal form said solvated,

wherein said metal is selected from the group comprised of

— iron (Fe),

— cobalt (Co),

— a rare earth, in particular a lantanide (Ln),

— a combination thereof;

and wherein said solvent is a solvent selected from the group comprised of:

polar solvents

— a carbonyl containing solvent;

— tetrahydrofuran;

— 1 ,4 dioxane;

— acetonitrile; — a combination thereof

apolar solvents

— an aromatic solvent,

— an olefinic solvent

— a long chain ether.

— a combination thereof.

18. A method according to claim 17, wherein said magnetic metal comprises at least two metal elements and wherein said step of vaporizing comprises a first step of vaporizing a first metal and a subsequent second step of vaporizing a second metal such that said second metal solidifies on said previously solidified first metal, a core formed by said first metal is covered by a formed by said second metal, in particular a monoatomic shell layer.

19. A method according to claim 17, wherein said magnetic metal comprises at least two metal elements and wherein said step of vaporizing comprises a step of simultaneously vaporizing said first and said second metal, such that said first and said second metal solidifies as an alloy.

20. A method according to claim 19, wherein a step is provided of controlling the temperature of a source of said first and of said second metal in a condensed state, such that said amount of said first metal and said amount of said second metal vaporize at a prefixed vaporization ratio.