WO2016016505A1

WO2016016505A1 - Hybrid material as contrast agent in magnetic resonance images

Info

Publication number: WO2016016505A1
Application number: PCT/ES2015/070595
Authority: WO
Inventors: Pablo BOTELLA ASUNCIÓN; Alejandro CABRERA GARCÍA
Original assignee: Consejo Superior De Investigaciones Científicas (Csic); Universitat Politècnica De València (Upv)
Priority date: 2014-07-31
Filing date: 2015-07-30
Publication date: 2016-02-04
Also published as: ES2561934A1; ES2561934B1

Abstract

The invention relates to a contrast agent of magnetic resonance based on a hybrid material formed by an organo-metallic core derived from Prussian blue and a silica cover, and optionally, molecules of a poly(ethylene glycol), a fluorescent agent, a radio nucleus and/or a substance that directs to specific receptors, cells or tissues, joined by covalent bonding to the surface of the inorganic cover.

Description

HYBRID MATERIAL AS A CONTRACTING AGENT IN IMAGES OF

MAGNETIC RESONANCE

DESCRIPTION

Technical Field

The present invention is framed within the applications of Prussian blue derivatives (AP) for imaging and diagnosis. The high solubility of materials in biological fluids and the highly toxic nature of their components limits their use in clinical practice. The incorporation of an insoluble and biocompatible silica-based shell stabilizes the nanoparticles of the AP derivative and reduces its toxicity in vitro and in vivo, while promoting the accumulation of the magnetically active compound in pathogenic tissues due to the increased permeability and retention effect. and improves the contrast in the magnetic resonance imaging.

Background Magnetic resonance imaging (MRI) is a widely used non-invasive and non-radioactive clinical diagnostic technique. The intensity of the images obtained by MRI depends on the proton density of the water in the examined tissues, as well as the longitudinal (Ti) and transverse (T ₂ ) relaxation times. Usually, the difference in the density of water protons in the different tissues is small, so to obtain high resolution in the MRI images, the administration of a contrast agent (CA) is necessary. It is a compound capable of modifying the values of Ti and T ₂ in tissues. Two types of these agents are usually differentiated: those capable of reducing the value of Ti and those capable of lowering T ₂ . In the first case, these are substances that increase the brightness (positive contrast) of the MRI images, while in the second case they are compounds that obscure those regions where they accumulate (negative contrast). In this regard, from a point of view of its application in clinical diagnosis, the information provided by the Ti agents is far superior to that offered by substances that decrease T ₂ , although the combination of both may be complementary. The contrast agent for MRI can be injected intravenously (/ V) to improve the visualization of tumors, blood vessels and / or inflamed tissues, among others. Likewise, a contrast agent can be injected directly into a certain organ or tissues, or into a joint, when it is desired to obtain a specific image thereof. As mentioned, there are two types of these magnetically active materials that are capable of modifying the contrast in MRI images: paramagnetic, capable of primarily reducing Ti (Ti agents) and ferromagnetic, capable of primarily lowering T ₂ (T agents ₂ ). All Ti agents used in clinical MRI are based on paramagnetic complexes of Gd ³⁺ and polyamine polycarboxylate ligands soluble in physiological medium. The high electronic spin of this cation, with 7 missing electrons (4f ⁷ , S = _7/2 ), together with its baseline electronic state ( ⁸ S _7/2 ) and the slow electronic relaxation (10 ^"9 s), provides the Gd ³⁺ unique magnetic-nuclear properties for the reduction of the longitudinal relaxation time of water protons, however, the use of these soluble compounds of Gd ³⁺ entails a series of problems and risks that limit clinical practice. First, the low relaxivity of the Ti of these agents before the action of a high intensity magnetic field forces to administer high doses to obtain contrast, having to reach a plasma concentration higher than 0.1 mM. In addition, these small molecules have a very high plasma rinse rate, which considerably reduces the measurement time.This all complicates the clinical use of Ti agents, since Gd ³⁺ is a potent toxic system to renal, can lead to systemic nephrogenic fibrosis and fibrosing nephrogenic dermopathy, as a result of the release of the cation by the chelate. This toxicity is a consequence of the atomic radius of Gd ³⁺ (1, 02 A), very similar to that of Ca ²⁺ (1, 00 A). In this way, the presence of free Gd ³⁺ in the body causes the alteration of the functions of the calcium channels depending on the voltage of the cell membranes.

All T ₂ contrast agents approved for hospital use in MRI are based on superparamagnetic ferric oxide nanoparticles (SPIONs, 5-500 nm). Although these are materials with high sensitivity, non-toxic and with the ability to enter the cells, from the point of view of clinical diagnosis and cell imaging, the resolution of the images obtained with T ₂ agents is lower than those generated by Ti compounds and it is often difficult to distinguish them from hemorrhages, calcifications, metallic deposits and other possible formations in a damaged tissue.

On the other hand, both ΤΊ and T ₂ contrast agents are unstable in the stomach acid medium, which prevents their oral administration. In this regard, the development of a new contrast agent requires a stable system in physiological medium, with high plasma residence time and capable of accumulating in the tissues and target cells, allowing to improve the contrast in MRI images at low concentration, at The time during which the study is possible is increased. Likewise, it must be an easy elimination system, mainly by the renal or digestive route, once its mission has been fulfilled, without the intracellular processes to which it can be subjected give rise to toxic metabolites. Finally, with a view to improving the resolution of the MRI images, it would be of great interest that such a contrast agent allows the Ti and T ₂ values to be significantly reduced together.

AP derivatives doped with a Gd ³⁺ or other paramagnetic cation have utility as contrast agents ΤΊ and T ₂ . In US patent 2010/0254912 2010; Huang, SD; Li, Y .; Shokouhimehr, M., presents a material based on ultrafine AP nanoparticles doped with Gd ³⁺ , of generic formula A _4x Fe _4- _x '"[Fe" (CN) 6] 3 ₊ x nl-l20, where A it is a cation selected from the group consisting of Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , Cs ⁺ , NH ₄ ⁺ or Τ, Fe '"is substituted by Gd ³⁺ in a proportion ranging from 1 to 100%, x varies from 0 to 1 and n varies from 1 to 24, and its application as a contrast agent T However, the partial solubility of the derivatives of the AP in biological medium considerably limits the clinical use of these materials, due to the possible release of ions Gd ³⁺ and Fe (CN) ₆ ^{4 "} , both highly toxic.

In WO2012 / 108856 2012; Huang, S .; Khitrin, AK; Perea, VS; Kandanapitive, MS, describes an MRI contrast agent based on nanoparticles (4-500 nm) of compound A _x Mn _and [M (CN) ₆ ] _z nH ₂ 0, wherein A = Li, Na, K, NH ₄ o TI, M = Cr, Mn, Fe, Co or Ru, x = 0-2, y = 1 -4, z = 1-4, and n = 0 or 1-20, protected with a hydrophilic carboxylic acid shell or biocompatible polymer. Likewise, it is indicated that the in vitro release of CN ions ^" in water is less than 0.1 mM for a period of 24 h, which indicates a certain stabilizing effect of the organic shell on the nanoparticulate AP derivative. WO2012 / 1 10835 2012; Máthé, D .; Szigeti, K., claims a nanoparticle-based material with an AP metal core doped with one or more metal isotopes and a biocompatible organic shell, as well as its application as a contrast agent in MRI. The composition of said metal core responds to the formula A _x M _m [M (CN) ₆ ] n, where A can correspond to a metal selected from the group consisting of Li, Na, K, Rb, Cs, Fr and TI, M corresponds to a metal selected from the group consisting of V, Cr, Mn, Fe, Co, Ni, Cu, Ga, Zr, Nb, Mo, Ru, Cd, In, Hf, Ta, W, Os or Hg, M 'corresponds to a metal selected from the group consisting of Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Lu, Ba, Hf, Ta, W, Os, Pt, Hg, La, Eu, Gd, Tb, Dy and Ho, m varies from 0 to 5, x varies from 0 to 5 and n varies from 0.5 to 10. Regarding The biocompatible organic shell comprises one or more biocompatible materials selected from the group consisting of antibodies, dextran, polyethylene glycol, polyvinyl pyrrolidone and citrate. Again, the hospital application of this type of materials, which requires iv administration, is limited by the possible release of ions such as Gd ³⁺ and Fe (CN) ₆ ^{4 "} in plasma (no data on stability is offered of the material in biological medium), and its possible toxic effects.

All of the above leads to the need to develop new systems to improve the resolution of MRI images by increasing the contrast, which meet the following properties: i) biocompatibility, that is, not to cause a specific or nonspecific immune response; ii) high values of longitudinal (r-ι) and transverse (r ₂ ) relaxivity, allowing to increase, respectively, the positive and negative contrast in the MRI images with low doses of the product; iii) stability in human plasma, that is, that it does not release any component in physiological medium or, in case of release of any of its components, its concentration in blood must be kept below toxicity levels; iv) ability to incorporate on its surface molecules of diverse functionality (therapeutic, diagnostic, stabilizer, etc.) by stable covalent bonding.

In our case, we have found that the nanoparticles of a hybrid material composed of an organ-metallic core derived from the AP and a silica cover, have very high π and r ₂ values, while being biocompatible and very stable in physiological medium , being able to incorporate a large number of molecules that give them multifunctional character. In this regard, the encapsulation of nanoparticles of derivatives of the AP with a silica layer is strongly impeded, because these compounds dissolve virtually instantaneously in the alkaline medium in which silicate polymerization is carried out. To overcome this drawback, a method of deposition of a silica layer onto nanoparticles of AP derivatives at neutral pH has been developed by controlled hydrolysis of a silica precursor, using low molecular weight amines as silicate activating agents. As a result, an AP @ Si0 ₂ nanocomposite has been obtained in which the stability in physiological medium of the nanocrystals of the AP derivative is significantly improved, while controlling the textural parameters of the shell, which offers numerous functionalization possibilities . Description of the Invention

The present invention relates to a magnetic resonance contrast (MRI) agent based on a hybrid material that can comprise at least: - An organometric metal core of nanometer size derived from Prussian blue (AP), of generic formula

A _x M _m [M ^' (CN) ₆ ] _n

where

A is Li, Na, K, Rb, Cs, Fr, TI, NR ₄ where R = H or any alkyl radical of stoichiometry C _to H ₂ to + 2, or combinations thereof;

M is Cr, Mn, Fe, Co, Ru, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, or combinations thereof;

M ^' is Cr, Mn, Fe, Co, Ru or combinations thereof;

x = 0-2,

m = 1-4,

n = 1 -3, and

- A silica cover that homogeneously covers the core nanoparticles. According to a particular embodiment, the silica shell may be a silica shell preferably selected from unstructured and non-porous, which homogeneously coats the core nanoparticles, between unstructured and porous, with pore diameter between 1 and 100 nm, which homogeneously coats the core nanoparticles, between structured and porous, preferably mesoporous, with a pore diameter between 1 and 30 nm, which homogeneously coats the core nanoparticles.

The joint presence of an agent ΤΊ (eg, Gd ³⁺ ) and a T ₂ agent (eg, Fe ³⁺ ) in the contrast system of the present invention is beneficial to improve the overall contrast (positive and negative) of the images. of MRI and, therefore, the resolution, especially when the values of relajativity r-ι and r ₂ are very high. Thus, the contrast agent of the present invention may have values of longitudinal relaxivity (r-ι) between 5.0 and 50.0 mM ^"1 s ^{" 1} , preferably between 5.0 and 30.0 mM ^"1 s ^{" 1} , and transverse relaxivity values (r ₂ ) between 10 and 300 mM ^"1 s ^{" 1} , preferably between 10 and 200 mM ^"1 s ^{" 1} , in a magnetic force field preferably between 0.5 and 1 1.0 T, and more preferably between 1, 2 and 9.4 T.

The purpose of incorporating the silica shell onto the nanoparticles of the AP derivative is multiple; On the one hand, the inorganic layer stabilizes the nanocrystals of the AP derivative, minimizing or suppressing the dissolution in physiological medium and release of toxic ions, such as Gd ³⁺ and Fe (CN) ₆ ^{3 "} . This avoids unwanted effects caused due to the toxicity of these ions, while the accumulation of the contrast agent in the tissues and target cells is favored, which allows reducing the dose administered without losing resolution in the MRI images.Also, the silica cover prevents phenomena of uncontrolled aggregation of the nanoparticles of the organometallic compound, while preventing phenomena of extravasation, immune reactions and rapid renal elimination.On the other hand, the silica cover has a large number of surface silanole groups on which it can be attached by covalent bond many molecules that provide multifunctionality to the hybrid material, in any case, the encapsulation of the nanopar AP derivative with a silica cover does not imply any structural and / or functional alteration of the contrast agent, as shown in Figure 1. The presence of the silica surface layer is evidenced by a wide band in the infrared spectrum at 1080 cm ^"1 , which is not seen in the uncoated material, (see Figure 2).

In the contrast agent of the present invention the particles of said contrast agent may have different shapes. Preferably, its particles may have the shape selected between spherical, rod, blade or star and combinations thereof. Preferably, the particles are in the form of a blade or star.

According to the present invention the particles of the contrast agent may have an average diameter between 10 and 3000 nm, preferably between 20 and 1000 nm.

According to a particular embodiment, the silica cover of the contrast agent may have a thickness between 1 and 100 nm, preferably between 5 and 50 nm. In a further embodiment of the present invention the silica cover of the contrast agent can be functionalized with a molecule, that is, the silica cover of the contrast agent can further comprise a molecule selected from a poly (ethylene glycol) (PEG) , a fluorescent agent, a radionuclide, a directing molecule and combinations thereof.

According to the particular embodiment in which the contrast agent is functionalized with a PEG, this molecule increases the solubility and stability of the material in aqueous medium, allowing the preparation of stable colloids while reducing the interactions of the nanoparticles with plasma components. blood, increasing plasma residence time, reducing or eliminating the immune response, reducing toxicity and increasing efficacy, and promoting accumulation in specific tissues and / or cells. In the material of the present invention, the molecular weight of the PEG molecule can range from 200 to 20,000 Da and its concentration can range from 1 to 30% by weight.

According to another particular embodiment, the contrast agent is functionalized with a covalently bound fluorescent agent. The fluorescent agent molecule allows the study of the biodistribution of the contrast agent. Preferably, the fluorescent agent may be selected from 5-amino fluorescein, fluorescein isothiocyanate, NHS-fluorescein, rhodamine B isothiocyanate, tetramethylrodamine B isothiocyanate, NHS-Cy5 and / or NHS-Cy5.5 or a derivative thereof incorporating a organic ligand that facilitates covalent bonding with the inorganic shell, as well as combinations thereof. In the material of the present invention, the concentration of the fluorescent agent may range from 0.01 to 5% by weight. According to another particular embodiment of the present invention, the silica cover of the contrast agent may incorporate a radionuclide, preferably an ¹⁸ F radionuclide as a functionalizing molecule. The radioactive isotope allows the study of the biodistribution and pharmacokinetics of the pharmaceutical preparation using the positron emission tomography (PET) technique. In the material of the present invention, the concentration of the ¹⁸ F radionucle can range from 0.0001 to 1% by weight.

In another particular embodiment of the present invention, the silica coating of the contrast agent may incorporate a directing molecule, the directing molecule being understood as any molecule that favors endocytosis internalization in the target cells. The directing molecule may be selected from a synthetic, semi-synthetic, natural molecule and combinations thereof. The targeting molecule according to the present invention may be selected from proteins, carbohydrates, lipids, nucleic acids, hormones and hormonal analogs, vitamins, enzymatic co-factors and metabolites of protein synthesis, a derivative thereof that incorporates an organic ligand that facilitates the covalent attachment to the inorganic shell and combinations thereof.

The present invention also relates to a method of preparing the contrast agent defined above. This method may comprise, at least, contacting an aqueous suspension of nanoparticles of the Prussian blue derivative (AP) with a solution of at least one silicon compound preferably selected from a silicon alkoxide, an alkylsilane, sodium silicate and colloidal silica and combinations thereof, in the presence of at least one amine preferentially selected from primary, secondary, tertiary amine and combinations thereof and its molecular weight is less than 1000 Da, preferably maintaining the pH between 7 and 8 , and shaking to allow the growth of the inorganic wall that surrounds the organ-metallic core. Said method may further comprise a step of washing and lyophilization of the hybrid material obtained. Additionally, according to a particular embodiment, the formation of the inorganic shell can be carried out in the presence of an organic surfactant, which can be added to the mixture, and which will subsequently be removed, preferably by extraction with alcohols. This organic surfactant may preferably be a cationic, anionic or neutral compound, with a surfactant ratio: Si0 ₂ preferably between 0.005 and 0.65 molar, more preferably between 0.01 and 0.5 molar. As mentioned, said surfactant can be removed after obtaining the particles of the hybrid material, by refluxing with short chain alcohols.

According to another particular embodiment, the method of preparing the contrast agent may further comprise the use of an organic co-solvent, adding this to the mixture, with a preferred co-solvent ratio: water in volume between 1: 1 and 10: 1, more preferably between 2: 1 and 4: 1. According to this embodiment, said solvent may preferably be an aliphatic chain alcohol with a carbon number between 1 and 6.

The present invention also relates to the use of the contrast agent defined above and obtained by the method described above for magnetic resonance imaging (MRI), specifically to improve the resolution of MRI images, said use comprising administration to humans and animals either orally, parenterally, respiratoryly or transdermally in sufficient concentration to achieve non-selectively pathological tissues. The high stability of the contrast agent in biological medium and the increase in plasma residence time with respect to soluble compounds of Gd ³⁺ , as well as the processes of angiogenesis and partial destruction of the vascular endothelium associated with internal lesions, favor the extravasation of the nanoparticles of the contrast agent (EPR effect), allowing their accumulation in the extracellular matrix. Alternatively, the contrast agent defined above can be administered to the host by direct injection into the injured area, accumulating in the interstitial tissue.

The contrast agent described in the present invention can be used with a magnetic field of varying force between 0.5 and 1.0 T, preferably between 1.2 and 9.4 T.

The dose of the contrast agent of the present invention will depend on the host, tissue and / or cells under study and the route of administration. In general, the dose varies between 0.001 and 0.1 mmol / Kg.

The blood half-life of the contrast agent of the present invention is sufficient to be able to obtain MRI images in the different hosts, said half-life being in the range of 0.5 to 6 h, preferably between 1 and 6 h. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the X-ray diffractograms of the Gd [Fe (CN) 6] material prepared according to Example 1 (A) and Example 3 (C), and of the contrast agent of the present invention prepared according to Example 2 (B) and Example 4 (D).

Figure 2 shows the infrared spectra of the Gd [Fe (CN) 6] material prepared according to Example 1 (A) and the y of the contrast agent of the present invention prepared according to Example 2 (B).

Figure 3 is a schematic illustration of the nanoparticle preparation process of the contrast agent of the present invention.

Figure 4 shows nanoparticle transmission electron microscopy images of the contrast agent of the present invention prepared according to Example 2, showing the general morphology (A) and the silica coating deposited on the surface of the Gd material [Fe (CN) 6 ] (B).

Figure 5 shows the evolution of the longitudinal (1 / ΤΊ) and transverse (1 / T ₂ ) relaxation velocity with respect to the concentration of the contrast agent of the present invention prepared according to Example 2, as well as the calculated values of longitudinal relaxivity (n) and transverse (r ₂ ). Figure 6 shows the evolution of the longitudinal (1 / ΤΊ) and transverse (1 / T ₂ ) relaxation velocity with respect to the concentration of the contrast agent of the present invention prepared according to Example 4, as well as the calculated values of longitudinal relaxivity (n) and transverse (r ₂ ). The present invention is illustrated by the following examples that are not intended to be limiting thereof.

EXAMPLES

EXAMPLE 1. Preparation of nanoparticles of the Gd [Fe (CN) ₆ ] material of average diameter between 100 and 400 nm (modified from J. Phys. Chem. C 2009, 113, 21531-21537; Yamada, M; Yonekura, S .) - In a 500 ml balloon connected to the N ₂ line, 3.30 g (10 mmol) of K ₃ [Fe (CN) ₆ ] and 6.30 g (30 mmol) of tetraethyl ammonium bromide are introduced (Et ₄ NBr), and dissolved with stirring in 200 ml of methanol at 30 ° C for 3 days under nitrogen atmosphere. The mixture is filtered (0.45 m), and the filtrate is concentrated to about 10 ml in a rotary evaporator. The resulting solution is stirred with 100 ml of ethyl ether and the yellow precipitate obtained is filtered. The crude product is dissolved in 100 ml of acetonitrile at reflux; The solution is then allowed to cool to crystallize the purified powder of (Et ₄ N) ₃ [Fe (CN) ₆ ]. In a 250 ml balloon, 1.75 g of CH ₃ COOH (225 mmol) and Gd (N0 ₃ ) ₃ -5H ₂ 0 (1.08 g, 2.50 mmol) are dissolved in 50 ml of a mixture H ₂ 0: ethanol (2: 5 v / v). Then a solution of (Et ₄ N) ₃ [Fe (CN) ₆ ] (1.50 g, 2.50 mmol) in 15 ml of methanol is added. The mixture is kept at 25 ° C for a full day at rest. The precipitate is filtered and washed with ethanol, and dried in vacuo to obtain an orange powder of Gd [Fe (CN) ₆ ], consisting of crystals in the form of a blade and a rod of average diameter between 100 and 400 nm. The X-ray diffractogram of the material is shown in Figure 1A.

EXAMPLE 2. Preparation of a magnetic resonance contrast agent based on nanoparticles of a hybrid material composed of a Gd core [Fe (CN) ₆ ] prepared according to Example 1 and a silica cover. 3.80 g of the material Gd [Fe (CN) ₆ ] prepared according to Example 1 is dispersed in 1900 ml of a H ₂ 0 hydroalcoholic mixture: ethanol (2: 5 v / v) and 3.6 ml (0.019 mol) of cyanopropyl trimethoxysilane with vigorous magnetic stirring. After 30 minutes, 9.5 ml (0.064 mol) of tetramethyl orthosilicate (TMOS) is introduced and then 0.38 ml (0.003 mol) of triethylamine is added. The resulting dispersion is kept under stirring for 24 hours, which allows the growth of a non-porous silica wall. amorphous on the surface of the crystals of the material Gd [Fe (CN) ₆ ]. At 24 h and at 48 h, successive additions of TMOS (9.5 ml, 0.064 mol) are carried out while stirring. Finally, the suspension is centrifuged (500 g, 2 h), washed three times with a hydroalcoholic mixture H ₂ 0: ethanol (2: 5 v / v) (2000 g, 20 min). The sediment is lyophilized at -55 ° C for 16 h. An orange powder is obtained, consisting of particles of Gd [Fe (CN) ₆ ] @ Si0 ₂ of average diameter between 100-400 nm. The X-ray diffractogram of the material is shown in Figure 1 B, and electron transmission microscopy images of the particles are shown in Figure 4. EXAMPLE 3. Preparation of nanoparticles of the Gd [Fe (CN) ₆ ] material of average diameter between 20 and 200 nm (modified from J. Phys. Chem. C 2009, 113, 21531-21537; Yamada, M; Yonekura, S .) -

In a 500 ml balloon connected to the N ₂ line, 3.30 g (10 mmol) of K ₃ [Fe (CN) ₆ ] and 6.30 g (30 mmol) of tetraethyl ammonium bromide (Et _{4) are introduced} NBr), and dissolve with stirring in 200 ml of methanol at 30 ° C for 3 days under nitrogen atmosphere. The mixture is filtered (0.45 m), and the filtrate is concentrated to about 10 ml in a rotary evaporator. The resulting solution is stirred with 100 ml of ethyl ether and the yellow precipitate obtained is filtered. The crude product is dissolved in 100 ml of acetonitrile at reflux; The solution is then allowed to cool to crystallize the purified powder of (Et ₄ N) ₃ [Fe (CN) ₆ ]. In a 250 ml balloon, Gd (N0 ₃ ) ₃ -5H ₂ 0 (1.08 g, 2.50 mmol) is dissolved in 50 ml of a mixture H ₂ 0: ethanol (2: 5 v / v). Then a solution of (EtN) ₃ [Fe (CN) ₆ ] (1.50 g, 2.50 mmol) in 15 ml of methanol is added. The mixture is kept at 25 ° C for a full day at rest. The precipitate is filtered and washed with ethanol, and dried in vacuo to obtain an orange powder of Gd [Fe (CN) ₆ ], consisting of rod-shaped crystals of average diameter between 20 and 200 nm. The X-ray diffractogram of the material is shown in Figure 1 C.

EXAMPLE 4. Preparation of a magnetic resonance contrast agent based on nanoparticles of a hybrid material composed of a Gd core [Fe (CN) ₆ ] prepared according to Example 3 and a silica cover.

3.80 g of the material Gd [Fe (CN) ₆ ] prepared according to Example 3 is dispersed in 1900 ml of a H ₂ 0 hydroalcoholic mixture: ethanol (2: 5 v / v) and 3.6 ml (0.019 mol) of cyanopropyl trimethoxysilane with vigorous magnetic stirring. After 30 minutes 9.5 ml (0.064 mol) of tetramethyl orthosilicate (TMOS) is introduced and then 0.38 ml (0.003 mol) of triethylamine is added. The resulting dispersion is kept under stirring for 24 hours, which allows the growth of a wall of amorphous non-porous silica on the surface of the crystals of the material Gd [Fe (CN) ₆ ]. At 24 h and at 48 h, successive additions of TMOS (9.5 ml, 0.064 mol) are carried out while stirring. Finally, the suspension is centrifuged (500 g, 2 h), washed three times with a hydroalcoholic mixture H ₂ 0: ethanol (2: 5 v / v) (2000 g, 20 min). The sediment is lyophilized at -55 ° C for 16 h. An orange powder is obtained, consisting of particles of Gd [Fe (CN) ₆ ] @ Si0 ₂ of average diameter between 20-200 nm. The X-ray diffractogram of the material is shown in Figure 1 D.

EXAMPLE 5. Measurement of longitudinal (n) and transverse (r ₂ ) relaxivity with respect to the concentration of the contrast agent prepared in Example 2. Stable suspensions of the contrast agent Gd [Fe (CN) ₆ ] @ Si0 ₂ are prepared obtained according to the procedure described in Example 2, varying the concentration of Gd ³⁺ from 0.1 to 2.0 mM, and measuring the relaxation of the Ti and T ₂ protons in a 400 MHz NMR device (9.4 T). The results obtained expressed as normalized relaxivity versus concentration are presented in Figure 5. The values obtained from r-ι and r ₂ are also indicated in Table 1.

EXAMPLE 6. Measurement of longitudinal (n) and transverse (r ₂ ) relaxivity with respect to the concentration of the contrast agent prepared in Example 4. Stable suspensions of the contrast agent Gd [Fe (CN) ₆ ] @ Si0 ₂ are prepared obtained according to the procedure described in Example 4, varying the concentration of Gd ³⁺ from 0.1 to 2.0 mM, and the relaxation of the Ti and T ₂ protons in a 400 MHz NMR device (9.4 T). The results obtained expressed as normalized relaxivity versus concentration are presented in Figure 6. The values obtained from r-ι and r ₂ are also indicated in Table 1.

Table 1. Longitudinal and transverse relaxivity values measured at 9.4 T for contrast agents prepared according to the procedures described in the present invention. Example Composition Particle Size nr ₂

2 Gd [Fe (CN) ₆ ] @ Si0 ₂ 100-400 1 1, 7 58.6

2 Gd [Fe (CN) ₆ ] @ Si0 ₂ 20-200 13.2 66.6

Claims

1. Magnetic resonance contrast agent characterized in that it is based on a hybrid material comprising at least:

- An organometric metallic core of nanometric size derived from Prussian blue, of generic formula

A _x M _m [M ^' (CN) ₆ ] _n

where

M ^' is Cr, Mn, Fe, Co, Ru or combinations thereof;

x = 0-2,

m = 1-4,

n = 1 -3, and

- A silica cover that homogeneously covers the core nanoparticles.

2. Contrast agent according to claim 1, characterized in that the silica cover is an unstructured and non-porous cover.

3. Contrast agent according to claim 1, characterized in that the silica shell is an unstructured and porous shell, with a pore diameter between 1 and 100 nm.

4. Contrast agent according to claim 3, characterized in that the silica cover is a structured and porous cover, with a pore diameter between 1 and 30 nm.

5. Contrast agent according to claims 1 to 4, characterized in that it has values of longitudinal relaxivity (π) between 5.0 and 50.0 mM ^"1 s ^{" 1} , and values of transverse relaxivity (r ₂ ) between 10 and 300 mM ^"1 s ^{" 1} , in a magnetic field of force between 0.5 and 1, 0 T.

6. Contrast agent according to claim 5, characterized in that it has values of longitudinal relaxivity (r-ι) between 5.0 and 30.0 mM ^"1 s ^'

7. Contrast agent according to claim 5, characterized in that it has values of transverse relaxivity (r ₂ ) between 10 and 200 mM ^"1 s ^'

8. Contrast agent according to claim 5, characterized in that the magnetic field is a magnetic field of force between 1, 2 and 9.4 T.

9. Contrast agent according to the preceding claims, characterized in that its particles have a selected shape between spherical, rod, blade or star and combinations thereof.

10. Contrast agent according to claim 9, characterized in that the particles have a blade or rod shape.

eleven . Contrast agent according to the preceding claims, characterized in that the particles have an average diameter between 10 and 3000 nm.

12. Contrast agent according to claim 1, characterized in that the particles have an average diameter between 20 and 1000 nm.

13. Contrast agent according to the preceding claims, characterized in that the silica shell has a thickness between 1 and 100 nm.

14. Contrast agent according to claim 13, characterized in that the silica shell has a thickness between 5 and 50 nm.

15. Contrast agent according to the preceding claims, characterized in that the silica shell further comprises a molecule selected from a poly (ethylene glycol), a fluorescent agent, a radionuclide, a targeting molecule and combinations thereof.

16. Contrast agent according to claim 15, characterized in that the molecule is a covalently bonded poly (ethylene glycol) of selected molecular weight between 200 and 20,000 Da and a weight concentration between 1 and 30%.

17. Contrast agent according to claim 15, characterized in that the molecule is a covalently bound fluorescent agent selected from 5-amino fluorescein, fluorescein isothiocyanate, NHS-fluorescein, rhodamine B isothiocyanate, tetramethylrodamine B isothiocyanate, NHS-Cy5 and / or NHS-Cy5.5 or a derivative thereof that incorporates an organic ligand that facilitates covalent bonding with the inorganic shell and combinations thereof.

18. Contrast agent according to claim 15, characterized in that the molecule is an ¹⁸ F radionucle.

19. Contrast agent according to claim 15, characterized in that the molecule is a directing molecule selected from synthetic, semi-synthetic, natural molecule and combinations thereof.

20. Contrast agent according to claim 19, characterized in that the targeting molecule is selected from proteins, carbohydrates, lipids, nucleic acids, hormones and hormonal analogs, vitamins, enzymatic co-factors and metabolites of protein synthesis, or a derivative of These incorporate an organic ligand that facilitates covalent binding to the inorganic shell, and combinations thereof.

twenty-one . Method of preparing the contrast agent described according to claims 1 to 20, characterized in that it comprises at least contacting an aqueous suspension of nanoparticles of the Prussian blue derivative with a solution of at least one silicon compound in the presence of the minus an amine, keeping the pH between 7 and 8 and stirring.

22. Method of preparing the contrast agent according to claim 21, characterized in that it further comprises washing and lyophilizing the obtained hybrid material.

23. Method of preparing the contrast agent according to claims 21 and 22, characterized in that it further comprises adding an organic surfactant to the mixture.

24. Method of preparing the contrast agent according to claim 21, characterized in that the silicon compound is selected from a silicon alkoxide, an alkylsilane, sodium silicate, colloidal silica and combinations thereof.

25. Method of preparing the contrast agent according to claim 21, characterized in that the amine is selected from primary, secondary, tertiary amine and combinations thereof and its molecular weight is less than 1000 Da.

26. Method of preparing the contrast agent according to claim 21, characterized in that it further comprises adding an organic co-solvent to the mixture, with a co-solvent: water volume ratio between 1: 1 and 10: 1.

27. Method of preparing the contrast agent according to claim 21, characterized in that it further comprises adding an organic surfactant to the mixture with a surfactant ratio: Si0 ₂ between 0.005 and 0.65 molar.

28. Use of the contrast agent described in claims 1-20 and obtained according to the method described in claims 21 to 27 for magnetic resonance.

29. Use of the contrast agent according to claim 28, characterized in that the variable magnetic field strength is between 0.5 and 1, 0 T.