WO2018154165A1 - Nanoparticles modified with alkoxy-silane derivatives - Google Patents

Abstract

The invention relates to a nanoparticle obtained by means of thermal decomposition, comprising at least two alkoxy-silane derivatives. The invention also relates to a composition, the uses thereof and a synthesis method.

Description

 Nanoparticles modified with alkoxy silane derivatives

FIELD OF THE INVENTION

The present invention is within the field of medicine (Nanomedicine), chemistry and biochemistry, and refers to a superparamagnetic nanoparticle, preferably iron oxide (SPION) obtained by thermal decomposition, superficially modified by an exchange of linking with two alkoxy silane derivatives simultaneously. The present invention also relates to the compositions, the method of modifying the nanoparticle and its uses.

BACKGROUND OF THE INVENTION

The phenomenon of nuclear magnetic resonance (NMR) occurs because the nuclei of different atoms absorb different energies in the radiofrequency domain, resonating at specific frequencies when the applied magnetic field is changed periodically. Hydrogen is one of the most appropriate elements for the phenomenon of nuclear magnetic resonance, and is the most common element contained in the human body. For these reasons, magnetic resonance imaging is capable of providing high resolution images of soft tissues with detailed anatomical information. The images are obtained by placing the subject in a magnetic field and observing the interactions between the magnetic spins of the subject's water protons and the radiofrequency of radiation applied. The image is solved by applying orthogonal magnetic field gradients that ultimately encode spatially the three coordinates of each pixel in the image. The magnetic spins in the sample release the energy acquired during excitation, such as an exponentially decreasing oscillating magnetic field that induces a small current in a receiving coil. Two parameters, called proton relaxation times, are of fundamental importance in the generation of the image: T1 (longitudinal relaxation time) and T2 (transverse relaxation time). T1 or spin-net relaxation time represents the transfer of energy between the spins of the observed proton and the surrounding network, and T2 or spin-spin relaxation time is the transfer of energy between different spins or protons. An additional parameter, called relaxation time T2 *, is also necessary to properly describe the total decay of the magnetic induction. This decay includes both the decay of T2 and the additional phase-out processes caused by the inevitable lack of homogeneity in the magnetic field they produce, variations in local magnetic susceptibility. For this reason, T2 * is always shorter than T2. The detected magnetic resonance (MR) signal includes, by both, a combination of relaxation times T1, T2 and T2 *, as well as the contribution of proton density.

An advantage of this technique is that it does not use ionizing radiation, providing high quality images without exposing the patient to any type of harmful radiation. However, endogenous and inherent NMR contrasts are, in many cases, insufficient to adequately resolve small anatomical lesions or properly characterize tissue physiology. For this reason, specific series of exogenous agents have been developed to enhance the T1, T2 or T2 * components of the image, respectively. Although significant advances have been made in T1 and T2 enhancing agents, much less is known about the investigation of T2 * potentiation, which could make possible the image of tissue and tumor perfusion with greatly increased resolution and sensitivity.

The contrast agents for magnetic resonance imaging (IMR) are divided into two general classes of magnetically active materials: Paramagnetic and superparamagnetic or ferromagnetic materials. Paramagnetic contrast agents include substances based on small gadolinium (III) chelates (Gd-DTPA, Gd-DTPA-BMA, Gd-DOTA, Gd-D03A) [E. Toth et al. , 2001. The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, John Wiley & Sons, 45], and superparamagnetic contrast agents are based on nanoparticles (NPs) with iron oxide core (Fe ₃ 0 ₄ , Fe ₂ 0 ₃ ) very small (<10 nm, USPIO-ultrasmall superparamagnetic ¡ron oxide particles) or reduced (<100 nm, SPIO-superparamagnetic ¡ron oxide) [Kharissova OV et al., 2013. RSC Advances 3, 22648-22682 ].

Paramagnetic agents induce an increase in MR image intensity in T1-weighted sequences (positive contrast enhancement), and superparamagnetic agents induce a decrease in the magnetic resonance signal in T2-weighted sequences (negative contrast enhancement). The sensitivity and specificity of both types of agents is very different. While gadolinium chelates have a relaxivity that requires millimolar concentrations of the compound in the target tissue, superparamagnetic NPs, due to their greater molecular weight, are effective in micromolar or nanomolar ranges.

Superparamagnetic nanostructured materials were developed as a contrast agent for MRI since their nanoscale structure profoundly modified the relaxation time of protons, thereby enhancing the sensitivity of MRI diagnosis. Furthermore, by modifications on the surface of the NPs with specific biologically active vectors, such as monoclonal or polyclonal antibodies or avidin-biotin systems, the specificity of the MRI diagnosis can also be increased. The quality of the particles used as an MRI contrast agent is determined by the magnetic properties of the core of the material, the particle size distribution, the particle's loading surface, stability in almost neutral solvents or physiological serum, as well. as the chemical and functional properties of immobilized molecules on the surface. In addition, pharmacokinetic behavior is an important determinant in magnetic resonance imaging applications, since the agent should ideally remain in the target tissue only during the MRI exam, and be quickly removed afterwards, without accumulating anywhere in the body.

Commercial products are synthesized by coprecipitation (core size of 5-10 nm) in aqueous medium. This method of simple and sustainable synthesis produces non-toxic magnetic NPs (MNP) of small size (<10nm), which can be easily maintained in colloidal suspension, but have significantly large distributions (> 20%). The hydrodynamic size and chemical nature of the coating influence the distribution of NPM and therefore the organ or tissue accumulation. SPIO nanoparticles coated with dextran (Feridex) and Carboxydextran (Resovist®) with hydrodynamic sizes greater than 100 nm have been used for liver imaging, while USPIO nanoparticles with hydrodynamic sizes less than 50 nm have been used for angiographies and applications of tumor permeability. However, dextran or carboxydextran coatings result in a significant and non-specific binding by absorption of these particles to vascular and tissue surfaces, limiting the effective removal of these particles once the imaging study has been performed being required. relatively long waiting times until complete elimination and eventual readministration. For these reasons, the production and characterization of magnetic NPs with poor tissue and vascular adhesion that favor rapid elimination and low tissue accumulation are currently of great relevance.

The procedures for making contrast agents of type T2 are described in the literature.

An appropriate protocol for producing magnetic iron oxide NPs comprises coprecipitation of ferric and ferrous salts in an alkaline medium in the absence or presence of surfactants. The NPs thus obtained have a core with a diameter between 1 and 50 nm.

The coating of the magnetic NPs with biocompatible polymers or copolymers is carried out through covalent bonding by activation of the NPs with carbodiimide. NPs with a coated core structure have a hydrodynamic diameter between 1 and 150 nm. Superparamagnetism is a physical property that only occurs in NPs of MFe ₂ 0 ₄ (M = Fe, Co, Mn) with sizes below 20-30 nm. The efficient obtaining of this type of inorganic NPs with an adequate size, shape, homogeneity and crystallinity is achieved using synthesis methods based on the thermal decomposition of their organic iron precursors, and require the use of organic solvents, surfactants and elevated temperatures. However, the superparamagnetic iron oxide NPs (SPIONs, Superparamagnetic ron oxide nanoparticles) generated by these methods are hydrophobic, being coated with surfactant ligands, and therefore stable in apolar solvents.

In order to use these hydrophobic SPIONs in any industrial and clinical application it is necessary to make them hydrophilic, especially in those cases in which their use necessarily takes place in aqueous media (such as those oriented to biology, biotechnology and biomedicine). One of the most used methods to change the solubility of these NPs are those of "Ligand Exchange", which are based on the replacement of hydrophobic ligands on their surface with other ligands that have a reactive end with the surface of the particle and another extreme with hydrophilic groups [De Palma et al., 2007. Chem. Mater. 19, 1821-1831]. Generally, this method is carried out by adding an excess of the new ligand to a very dilute solution of the NPs in a certain organic solvent, which causes a displacement of the original hydrophobic ligand by concentration gradient. Some of the most commonly used ligand exchange ligands usually contain carboxylic acid groups (eg Citrate) [Lattuada M. et al., 2007. Langmuir 23, 2158-2168], phosphonate or bisphosphonate [Sandiford L. et al., 2013. ACS Nano 7, 500-512], alcohol (eg Dextrano) [López-Cruz A. et al., 2009, J. Mater. Chem. 19, 6870-6876], or catechol (eg Dopamine) [Lak. A. et al., 2013. Nanoscale 5, 11447-1 1455], etc. at one of its ends, which allow the new ligand to be chemically adsorbed on the surface of the particle forming a protective layer. However, the aqueous stability of the resulting SPIONs with these ligands is quite limited over time, especially in biological media, mainly because this "chemical adsorption" is a non-covalent bond and the ligands are easily desorbed from the surface of the particle as a function of its dissociation constant (that is, progressively displaced by other molecules in the surrounding environment). In addition, the SPIONs obtained in this way can lose any functionalization to which they have been subjected during the synthesis process, since this type of non-covalent binding can be affected (compromised) by the conditions in which the reaction takes place and / or in the processes required for purification (especially in the case of the use of Sephadex® columns), which induce the separation of molecules associated with the particle, necessary for its correct function (clinical, biotechnological, industrial, etc.). BRIEF DESCRIPTION OF THE INVENTION

In order to improve the T2-MRI contrast properties and the circulation time of the SPIONs, and at the same time maintain excellent structural and colloidal stability of the NPs, the surface modification of the SPIONs obtained by thermal decomposition was proposed with the minus two alkoxy silane derivatives simultaneously. In particular, the present invention describes how the use of a combination of 60% Si (OCH2CH3) 4 (TEOS) and 40% NH2- (CH2) 3-Si (OCH2CH3) 3 (APTES), for surface modification of These hydrophobic SPIONs, and their subsequent PEGylation, allow to generate particles with better T2-MRI contrast properties than their predecessors obtained only with APTES (100%). PEGylated SPIONs produced by this method maintain good properties of structural and colloidal stability, low cytotoxicity and ease of subsequent functionalization. These results exemplify the previous point (the usefulness and potential of using mixtures of alkoxy silanes to provide new physical chemical properties to NPs, maintaining and even improving their colloidal and structural stability) and open the door to a new strategy for surface modification. of SPIONs, with thin layers of silica, for the development and optimization of these NPs for a high number of applications, both in cancer diagnosis and in their therapy.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic illustration of the synthesis of structural and colloidal stable SPIONs. Both molecules and NPs are not drawn to scale.

Figure 2. TEM images of the NPs (A) SPION-OA, (B) SPION-TEOS / APTES and (C) SPION-TEOS / APTES-PEG.

Figure 3. Colloidal stability of SPION-TEOS / APTES NPs in MES buffer solution at pH 6.0 (A) and SPION-TEOS / APTES-PEG in water (C). (B) Variation of the hydrodynamic size of the SPION-TEOS / APTES NPs in water or 0.25 M PBS (pH 7.4) or 0.05 M MES (pH 6.0) or 30 mg / mL BSA at 0.25 M PBS (pH 7.4) over time For 5 days. (D) Same as (B) for SPION-TEOS / APTES-PEG NPs over time for 7 days.

Figure 4. (A) FTIR and (B) TGA of the SPION-OA (black), SPION-TEOS / APTES (blue) and SPION-TEOS / APTES-PEG NPs (red). (C) XPS of NPs SPION-APTES (black) and SPION-TEOS / APTES (red). (D) Zeta-potential variation as a function of pH for NPs SPION-APTES-PEG (black) and SPION-TEOS / APTES-PEG (red). Figure 5. (A) VSM analysis of the SPION-OA (black), SPION-APTES (blue) and SPION-APTES-PEG (red) NPs. (B) MRI phantom images showing the positive (T1) and negative (T2) contrast produced by the SPION-TEOS / APTES-PEG NPs and / or the SPION-APTES-PEG at the indicated concentrations at a magnetic field of 9.4 T.

Figure 6. Cytotoxicity of HepG2 cells treated with SPION-APTES / TEOS-PEG NPs. (A) Representative images of the HepG2 cell line treated with the indicated concentrations of SPION-APTES / TEOS-PEG NPs for 48 hours and showing the total cells (blue staining) and dead cells (yellow staining). Scale = 0.05 cm. (B) Total number of cells in culture treated with the indicated concentrations of NPs for 24 and 48 hours. (C) Total number of dead cells in culture analyzed in B. (D) Percentage of dead cells in the same cells analyzed in B.

Figure 7. (AL) Representative T2W images obtained from mice before and after intravenous administration of 9-11 mg Fe Kg-1 of SPION-APTES-PEG (AF) and SPION-APTES / TEOS-PEG (GL ) at the indicated times. The two transverse images of the animal at each time show certain organs. (MN) T2 relaxation time ratios in the different tissues measured from the MRI maps after intravenous injection of NPs SPION-APTES-PEG (M) and SPION-APTES / TEOS-PEG (N). (O) Ratio of T2 relaxivity time in the renal medulla - muscle at the times indicated after the iv administration of NPs SPION-APTES-PEG and SPION-APTES / TEOS-PEG. Figure 8. Representative T2W images, with color coding, obtained before and after iv administration of 9-11 mg Fe-Kg-1 in mice of both SPION-APTES-PEG (A) and SPION-TEOS / APTES- NPs PEG (B) at the indicated times. (C) Example of T2W image highlighting the different areas measured in the kidney (ROIs). Figure 9. (A) UV-visible spectrum of NPs SPION-TEOS / APTES-PEG-Cy5 (pink), SPION-TEOS / APTES-PEG (red), SPION-TEOS / APTES (blue) and a blank as control sample (black) treated with ninhydrin. (B) Schematic illustration of the functionalization of the SPION-TEOS / APTES-PEG NPs with Cy5-NHS ester. (C) UV-visible absorption spectrum of the SPION-TEOS / APTES-PEG-Cy5 NPs (pink), SPION-TEOS / APTES-PEG (red) and free Cy5 (green). (D) Fluorescence phantom image showing the emission of SPION-TEOS / APTES-PEG NPs before and after functionalizing with Cy5 (Aex = 600 nm and Aem = 700 nm).

Figure 10. Representation of some alkoxy silane derivatives that can be used for chemical modification of different surfaces.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have developed a method for water solubilization of nano particles, preferably SPIONs obtained by thermal decomposition, which is based on an exchange of ligand with at least two alkoxy silane derivatives simultaneously in a determined proportion such as for example , but without representing a limitation, 60% TEOS and 40% APTES, and its preferable subsequent PEGylation by an amidation reaction with a polyethylene glycol derivative (a-Methoxy-w-carboxy PEG). Nano particles, preferably PEGylated, generated in this way have better T2-MRI contrast properties and longer circulation time than their predecessors obtained only with APTES (100%). The NPs, preferably PEGylated, produced by this method maintain excellent properties of structural and colloidal stability, low cytotoxicity and ease of subsequent functionalization. These results open the door to a new strategy for surface modification of nano particles, with thin layers of silica, which allows the development and optimization of these nano particles for certain applications, both in the diagnosis of cancer and in its therapy, as well as for other cosmetic, industrial, biotechnological, etc. applications, such as but not limited to, catalysis, detoxification, decontamination, purification of biomolecules, cells, etc. NANOPARTICLE OF THE INVENTION

Therefore, a first aspect of the invention relates to a nanoparticle (NP), preferably SPIONs obtained by thermal decomposition, comprising at least two different alkoxy silane derivatives. The NPs can be of any inorganic nanomaterial, preferably obtained by "thermal decomposition", such as, but not limited to, magnetic NPs (eg, MFe ₂ 0 ₄ with M = Fe, Co, Mn), fluorescent (eg, Quantum dots), phosphorescent (eg, rare earths: NaYbF ₄ ), of noble metals (Gold, Silver, Platinum, etc.), etc. Preferably, the NPs are SPIONs. In this report, "thermal decomposition" is understood as the process of synthesis of inorganic nanocrystals or NPs with a controlled size and a homogeneous distribution of NPs through the thermal decomposition of organometallic compounds, in high boiling organic solvents containing surfactants stabilizers The thermal decomposition allows a very high degree of control in the size and difference of sizes in the NPs, which gives rise to a very homogeneous behavior in terms of their physicochemical characteristics. This is important to transfer NPs to clinical practice.

In this specification, "alkoxy silane derivatives" means chemical compounds derived from silicon that are characterized by the presence of Si-O-C radicals (alkoxy). Preferably, alkoxy silanes derivatives are understood as any compound having at least one hydrolyzable silicon group, OR, which is crosslinked by "silane polycondensation" in the presence of moisture. More particularly, the present invention is illustrated by the use of the following two "alkoxy silane derivatives" simultaneously: Si (OCH2CH3) 4 (TEOS) and NH2- (CH2) 3-Si (OCH2CH3) 3 (APTES).

Non-limitingly, a possible formula of alkoxy silane derivatives useful in the present invention is illustrated below:

RmSiX (4-n) where R is a chemical group selected from the list consisting of: an alkyl, alkene, alkyne, aromatic, alkoxides, organofunctional or any combination thereof; preferably R is an organofunctional or functional group, preferably an amino group, attached to a C1 to C20 alkyl group, more preferably C1 to C10, more preferably C1 to C6, more preferably C1 to C3, more preferably C2. Preferably R is an amino group attached to a C3 group or an alkoxy of the methoxy- or ethoxy type; where m can be 0, 1, 2 or 3; where n represents a natural number between 0 and 3; and where X represents an alkoxide group, preferably a methoxy- or an ethoxk

In the present invention, "organofunctional" or "functional" group is understood as the site where most chemical reactions take place. In this regard, the double bond in the alkenes and the triple bond in the aiquino are also considered as functional groups. The main functional groups are selected from the list consisting of aliens, alkenes, alkynes, alcohols, ethers, aldehydes, ketones, carboxylic acids, esters, amines, amides and any combination thereof,

In a preferred embodiment of this aspect of the invention, the NP of the invention is superficially modified with two alkoxy silane derivatives by a ligand exchange process. In this report, "ligand exchange" is understood as the chemical change that occurs when one ligand X is displaced by another Y, a ligand being a chemically linked molecule to an NP.

In a preferred embodiment of this aspect of the invention, the NP of the invention is magnetic, and more preferably super-paramagnetic.

According to an embodiment of the invention, the magnetic NP consists of one or more of the following components: i) an inorganic core containing one or more of the elements selected from transition metals, including but not limited to iron, cobalt, manganese, copper and magnesium; or ii) an inorganic core composed of an alloy containing elements selected from transition metals, including but not limited to iron, cobalt, manganese, copper and magnesium.

In a particular embodiment of the invention the inorganic core of the magnetic NP is selected from the group consisting of iron oxide, cobalt ferrite, manganese ferrite, magnesium ferrite and combinations thereof. In a more particular embodiment of the invention, the inorganic core of the magnetic NP is iron oxide.

The NP obtained by thermal decomposition that is hydrophobic is converted into hydrophilic by a surface modification process based on an exchange of ligand with two alkoxy silane derivatives simultaneously. Therefore, in a preferred embodiment of this aspect of the invention, the NP of the invention is hydrophilic.

In another preferred embodiment of this aspect of the invention, the alkoxy silane derivatives are selected from the list consisting of: Tetraethylorthosilicate (TEOS), 3-aminopropyl-triethoxysilane (APTES), 3-aminopropyl-trimethoxysilane (APTMS), 3- mercaptopropyl triethoxysilane (MPTES), 3- mercaptopropyl trimethoxysilane (MPTMS), 3-Glycidoxypropyl triethoxysilane, 3-Glycidoxypropyl trimethoxysilane, 3-Cyanatopropyl triethoxysilane (CPTES), 3-Cianatropropyl trimethoxysilane (CPTMS), 3-Azianapropyl trimethoxypropyl and silylpropyl silylpropyl silylpropyl simethoxyethylpropyl triethoxyethylene w-methoxy polyethylene glycol, or derivatives or analogs. In another more preferred embodiment of this aspect, the NP of the invention comprises two alkoxy silane derivatives that are selected from: Tetraethylorthosilicate (TEOS) and 3- aminopropyl triethoxysilane (APTES), and more preferably in a proportion of 60% TEOS and 40% APTES or in other proportions close to said proportion 60/40 TEOS / APTES as they are any +/- 20% proportion of said proportion 60/40, such as 40/60 or 80/20 proportions and any other intermediate proportion Between these two values.

Preferably, the NPs of the invention are characterized by having an average particle size of less than 50 nm, more preferably less than 30 nm, preferably having an average size between 5 and 25 nm, and even more preferably between 10 and 20 nm.

By "average size", or "average diameter" is meant the average diameter of the population of NPs dispersed in an aqueous medium. The average diameter of these systems can be measured by standard procedures known to the person skilled in the art, and which are described, for example, in the examples below.

There are several procedures to improve the solubility of NPs. Control in solubilization is difficult to achieve so that NPs remain sufficiently stable over time. The NP in which the exchange of ligand with the alkoxy silanes has been made, the colloidal stability can still be increased (prevents the formation of aggregates). Therefore, you need to incorporate groups that improve solubility. Thus, in a preferred embodiment, the NPs of the invention are PEGylated. More preferably, with the polyethylene glycol derivative a-Methoxy-w-carboxy PEG. More preferably, PEGylation is performed by an amidation reaction.

COMPOSITION OF THE INVENTION

A second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising the NP of the invention. In a preferred embodiment of this aspect of the invention, the composition of the invention is a pharmaceutical composition, more particularly for medical diagnosis in vivo. In another preferred embodiment, the composition is a cosmetic composition. The NPs of the invention can have multiple applications, such as, but not limited to, industrial, biotechnological applications, etc.

The pharmaceutical compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art. Thus, the pharmaceutical compositions of the invention include, but are not limited to, any liquid composition (suspension of the system including NPs in water or in water with additives such as viscosizers, pH buffers, etc.) or solid (the system including Lyophilized or atomized NPs forming a powder that can be used to make granules, tablets or capsules) for administration either orally, orally or sublingually, topically, or in liquid or semi-solid form for administration transdermally, ocularly, nasal, vaginal or parenteral. In the case of non-parenteral pathways, the contact of the NPs with the skin or mucous membranes can be improved by giving the particles a significant positive charge, which will favor their interaction with the aforementioned negatively charged surfaces. In the case of parenteral routes, more specifically for intravenous administration, these systems offer the possibility of modulating the in vivo distribution of associated drugs or molecules. They may also be suspensions in biological fluids, such as serum. Aqueous suspensions may be buffered or unbuffered and may have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives, including, but not limited to, microbial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals. The compositions may be combined with various inert vehicles or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch, etc.

In a preferred embodiment of this aspect of the invention, the composition of the invention further comprises at least one biologically active compound or molecule, a therapeutic agent or drug, or a labeling agent.

The term "biologically active molecule" has a broad meaning and comprises molecules such as high, or more preferably, low molecular weight drugs, polysaccharides, proteins, peptides, lipids, oligonucleotides and nucleic acids, as well as combinations thereof. In a variant of the invention, the biologically active molecule has the function of preventing, alleviating, curing or diagnosing diseases. In another variant of the invention the biologically active molecule has a cosmetic function. In this report the term "biologically active molecule" also includes the terms "active substance", "active substance", "pharmaceutically active substance", "ingredient active, "" therapeutic agent, "" drug, "" agent or molecule for diagnostic purposes in vivo, "or" pharmaceutically active ingredient, "meaning any component that potentially provides a pharmacological activity or other different diagnostic effect, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.It also includes cosmetics, diagnostics as well as industrial ones (enzymes, chelating molecules, etc.).

The NPs of the invention may also comprise other biologically active molecules and other agents, for industrial uses, such as enzymes and chelating agents

Additionally, the NP of the invention may comprise more than one distinct biologically active molecule.

Said additional active principle or principles may be included in the system for the transport of biologically active molecules of the invention or be part of the pharmaceutical composition of the invention without being part of the transport system.

In a preferred embodiment of this aspect of the invention, the composition of the invention further comprises a pharmaceutically acceptable excipient.

USE OF THE NANOPARTICLE OF THE INVENTION AND THE COMPOSITION OF THE INVENTION

Depending on the silane layer and its composition, the properties of the NP can vary, with different levels of diagnostic capacity in vivo and therapeutic, as well as with different biotechnological, industrial, etc. applications. It is noted that both the nanoparticle of the invention and the pharmaceutical composition of the invention are useful for diagnosis and therapy, in particular for in vivo diagnosis.

Thus, a third aspect of the invention relates to the use of the NP of the invention or of the composition of the invention, in the preparation of a medicament, or alternatively, to the nanoparticle of the invention or the composition of the invention for its use as a medicine or as a useful agent in diagnosis in vivo.

Additionally, the NPs of the invention can serve as active ingredient vehicles.

Therefore, another preferred embodiment of this aspect of the invention relates to the use of the NP or the composition of the invention in the administration of active ingredients.

In addition, it can be used to perform perfusion measurements in normal tissue and in pathologies that affect the vascular system, such as tissue ischemia, or other systems, such as neurodegeneration, inflammation, edema or cancer, in animals or humans by means of magnetic resonance imaging.

Therefore, another preferred embodiment of this aspect of the invention relates to the use of the nanoparticle or the composition of the invention in the in vivo diagnosis by nuclear magnetic resonance.

METHOD OF THE INVENTION

A fourth aspect of the invention relates to a method for the synthesis of NPs of the present invention comprising: 1. Synthesis of hydrophobic NPs, preferably hydrophobic SPIONs.

By way of illustration only it is noted that said SPIONs of, for example 10 nm, spherical, monodispersed and hydrophobic, can be produced by the "thermal decomposition" method described by Park et al. [Park J. et al. , 2004. Nat. Mater. 3, 891-895]. It is noted that there are different variants of the method of synthesis of SPIONs by "Thermal Decomposition" and that they are based primarily on the use of other organic iron precursors. Although the solvent and the surfactants used can also be changed.

A list of such possible organic iron precursors would be for example the following:

- iron complexes with fatty acids (in our case, we use "Iron Oleate (III)"), - Iron (III) Acetylacetonate,

- Iron (III) carboxylate,

-Colinacitrate-iron complex (III),

-complex-iron complex (lll), and

-Pentacarbonyl iron. On the other hand, there are other methods of Synthesis that generate hydrophobic SPIONs other than the Thermal Decomposition method or any of its variants. In this sense, these methodologies are included herein.

2. Synthesis of NPs, preferably SPIONs-derived silicon alkoxysilanes from the NPs of step 1 or other identical or similar NPs with two or more alkoxy- silanes, preferably through a ligand exchange procedure executed simultaneously.

Preferably for the synthesis of these particles the combination of alkoxy silanes of interest is added to the SPIONs suspended in an oily medium. For example, 0.15% (v / v) of TEOS (300 μΙ_), 0.10% (v / v) APTES (200 μΙ_), and 0.01% (v / v) acetic acid (20 μΙ_) were added to 40 mg of SPION-OA suspended in 200 mL of n-hexane. This mixture was stirred orbitally for 72 h at room temperature to allow progressive displacement of oleic acid molecules in the SPION-OA through the 2 alkoxy silane derivatives used (TEOS and APTES). The resulting NPs (SPION-TEOS / APTES) are insoluble in n-hexane and therefore precipitate when the ligand exchange process is complete. These NPs were separated with the help of a neodynium magnet and washed three times with 50 mL of n-hexane to remove excess TEOS and APTES and any other unreacted reagents. Finally, the particles were dried at 60 ° C and under vacuum for 48 h, to allow complete condensation of the alkoxy silanes bound to the surface of the NP. 3. Optionally, a PEGylation of the NPs obtained in step 2) is carried out.

By way of illustration only, once the SPIONs of step 2) have been synthesized, PEGylation of said NPs is carried out. By way of illustration only, this step can be carried out by suspending the SPION-TEOS / APTES (30 mg) of step 2) in 10 mL of ultrapure water. On the other hand, 2 kDa CH30-PEG-COOH (180 mg), EDC HCI (20 mg) and NHS (12 mg) were mixed in 10 mL of 0.1 M MONTH MONTH (pH 6), and incubated at room temperature to allow the activation of the carboxylic acid groups of the PEG. After the activation time had elapsed, both solutions were mixed and allowed to react for 2-3 hours at room temperature and with magnetic stirring. Then, the NPs were purified by dialysis against ultrapure water using a 12-14 KDa (cut-off) membrane; and optionally 4. Functionalization of the NPs of step 3) or step 2).

By way of illustration only, once the SPIONs of step 3) have been synthesized, they are functionalized, for example, but not limited to cyanine-5. Thus, Cy5-NHS ester (2 mg) was dissolved in 200 of DMSO and slowly added to 14 mg of SPION-TEOS / APTES-PEG dissolved in 800 L of 0.25 M PBS pH 8.0. The mixture was allowed to react in the absence of light and with orbital stirring for 4 h at 4 ^o C to allow the binding of Cy5 to the amino groups on the surface of the NPs. Then, the SPION-TEOS / APTES-PEG-Cy5 NPs were purified using a Sephadex G-25 column and a solution of PBS pH 7.4 as the mobile phase.

The inventors of the present invention show in the examples included herein that PEGylated SPION generated by the method described above have better T2-MRI contrast properties and longer circulation time than their predecessors. obtained only with APTES (100%). In addition, PEGylated SPIONs produced by this method maintain good properties of structural and colloidal stability, low cytotoxicity and ease of subsequent functionalization.

KIT OR DEVICE AND USES Some nanosystems, such as the NPs of the invention, allow the nanotechnological development of new, promising and fascinating possibilities in diagnosis and medical treatment. The nanodevices used as contrast agents in medical imaging diagnosis (especially in IMR, ultrasound and tomography) have clear advantages over traditional agents in relation to better optical dispersion, better biocompatibility, a decrease in the probability of denaturation and , especially, their ability to bind ligands, which converts them into devices with multiple functions that bind to the target cells, allowing to obtain an image for diagnosis and at the same time transporting medications, allowing a more specific and efficient treatment.

Nanotechnology allows the design of multifunctional nanomaterials with a prolonged plasma half-life (thanks to the superficial incorporation of hydrophilic polymer chains), necessary to safely achieve its objective and specifically release the dose of drug vehicle in the desired place, increasing at the same time the bioavailability of the active agent in the target tissue. Regarding cancer treatment, these NPs can reach the tumor region simply by accumulation or retention (passive transport). This phenomenon is called the increased permeation and retention effect ("EPR" effect), and it is due to the differential characteristics of the tumor environment, where the vascular tissue is altered. This explains why there is a greater accumulation of NPs in the tumor mass, compared to a healthy tissue [Martínez-Soler G.l. et al., 2010. ARS Pharmaceutica 51, Supplement 3, 113-116; Núñez-Lozano R. et al., 2015. Current Opinion in Biotechnology 35, 135-140].

NPs characterized by an extensive plasma half-life can pass through the abnormal structure of blood vessels of pathological tissues (cancer, inflammation, infection). The biological fate of any drug transport colloid can be significantly improved if it has: i) a very small particle size (<100 nm) and a spherical morphology; and, ii) adequate surface electrical and thermodynamic properties (very low electrical charge, and hydrophilicity).

The composition of the invention may additionally comprise a detectable label. A "detectable label" refers to any label that can be used to locate the composition in vivo or in vitro. Examples of markers, but not limited to these, would be fluorophores (for example, Cyanine-5), chemical or protein markers that allow the visualization of a polypeptide. The visualization can be done with the naked eye or by an apparatus (such as, but not limited to a microscope) and may involve a source of energy or light. Therefore, another aspect of the invention relates to a diagnostic kit or device, comprising at least one nanoparticle of the invention, or the pharmaceutical composition of the invention.

The terms biologically active molecule, drug or therapeutic agent, or detectable label, have similar meanings and are used interchangeably in the memory of this invention. They refer to any substance that provides pharmacological activity and is used in the treatment, cure, prevention, mitigation or diagnosis of a disease or that affects the structure or function of the body of man or other animals. These biologically active molecules can include from low molecular weight drugs to molecules of the polysaccharide type, proteins, peptides, lipids, oligonucleotides and nucleic acids and combinations thereof. These molecules are well known to the person skilled in the art and include the meaning of a compound that has the characteristics that make it acceptable for use in medicine, for example and without being limited to the active ingredient in a medicine. Therefore, for example and without being limited to, these molecules can be synthesized by different organic chemistry techniques, or molecular and biochemical biology techniques. The terms used herein are understood as any compound that is administered to a patient for the treatment of a condition and that can more efficiently reach the target tissue when it is attached to the nanoparticle of the invention than when it is administered without the nanoparticle of the invention. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.

The therapeutic agent includes, but is not limited to, hydrophilic and hydrophobic compounds. Accordingly, the therapeutic agents contemplated in this invention include but are not limited to drug-like molecules, nucleic acids, proteins, peptides, antibodies, antibody fragments, aptamers and small molecules. A protein therapeutic agent includes but is not limited to peptides, enzymes, structural proteins, receptors, and other circulating or cellular proteins as well as fragments and derivatives thereof whose aberrant expression gives rise to one or more medical conditions. A therapeutic agent also includes chemotherapeutic compounds and radioactive materials. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, tolerance, etc. of the mammal. The "therapeutically effective amount" refers to the amount of active substance, or its salts, pro-drugs, derivatives or analogs or their combinations, which produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said pro-drugs, derivatives or analogs and the therapeutic effect to be achieved. . The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art.

The term "excipient" refers to a substance that aids the absorption or distribution or action of any of the active ingredients of the present invention, which stabilizes said active substance or aids in the preparation of the medicament in the sense of giving it consistency or providing flavors that make it more pleasant. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine without excluding other types of excipients not mentioned in this paragraph.

The term "pharmaceutically acceptable" excipient refers to the excipient being allowed and evaluated so as not to cause damage to the organisms to which it is administered.

In addition, the excipient must be pharmaceutically suitable, that is, a dossier that allows the activity of the active substance or of the active ingredients, that is, that is compatible with the active substance, in this case, the active substance is any of the compounds of the present invention.

A "pharmaceutically acceptable carrier" refers to those substances, or combination of substances, known in the pharmaceutical sector used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.

The vehicle, like the excipient, is a substance that is used in the medicament to dilute any of the compounds of the present invention to a certain volume or weight. The pharmaceutically acceptable carrier is an inert substance or action analogous to the active ingredients of the present invention. The function of the vehicle is to facilitate the incorporation of other compounds, allow greater dosage and administration or give consistency and form to the pharmaceutical composition. When the form of presentation is liquid, the pharmaceutically acceptable carrier is the diluent. The term "small molecule" refers to a chemical compound, for example a peptidomimetic that can be derivatized or any other organic compound of low molecular weight, natural or synthetic. Said small molecules may be therapeutically transported substances or they may be derivatized to facilitate transport. "Low molecular weight" means compounds whose molecular weight is less than 1000 Daltons, usually between 300 and 700 Daltons.

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.

The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES OF THE INVENTION To carry out the process of the present invention, a method for water solubilization of SPIONs obtained by thermal decomposition is carried out, which is based on an exchange of ligand with two alkoxy silane derivatives simultaneously in one determined proportion (60% TEOS and 40% APTES) and its subsequent PEGylation through an amidation reaction with a polyethylene glycol derivative (a-Methoxy-w-carboxy PEG).

Example 1. Synthesis and Characterization of NPs: SPION-OA, SPION-TEOS / APTES and

SPION-TEOS / APTES-PEG

Figure 1 shows a summary scheme of this procedure. The concrete manufacturing protocol of these SPION-TEOS / APTES-PEG NPs is described in the section entitled "METHOD OF THE INVENTION". First, hydrophobic, spherical and monodispersed 10 nm SPION-OA NPs were synthesized by the method of thermal decomposition of iron oleate [Park J. et al., 2004. Nat. Mater. 3, 891-895]. Figure 2A shows an image of transmission electron microscopy (TEM) obtained for a SPION-OA preparation, where it can be seen that they have the desired size, shape and homogeneity. Once purified, as described in this invention, the hydrophobic SPION-OA NPs obtained were converted to hydrophilic by means of a ligand exchange using two alkoxy silane derivatives simultaneously (60% TEOS and 40% APTES). This method displaces the oleic acid ligands of the SPION-OA and covers them with a very thin layer of silicon oxide, generating hydrophilic SPION-TEOS / APTES NPs. As can be seen in the TEM image of the NPs obtained (Figure 2B) this ligand exchange process does not produce apparent morphological changes, nor does it affect the size, or the monodispersity of the resulting particles.

The SPION-TEOS / APTES NPs obtained are water soluble. However, the progressive protonation of the amino groups on the surface of these NPs entails the establishment of hydrogen bonds between the particles, causing the progressive formation of aggregates. Although the use of a pH 6.0 buffer solution can slow this phenomenon, its aggregation is inevitable (Figure 3A and 3B). To solve this problem, partial PEGylation of the SPION-TEOS / APTES NPs was carried out. For this, the covalent binding of a-methoxy-w-carboxylate PEG (2 KDa) molecules to the surface amino groups of these particles was induced by an amidation reaction. The resulting PEGylated SPIONs were easily purified using simple dialysis. TEM images of the NPs obtained (Figure 2C) demonstrate that this PEGylation process does not produce morphological changes, nor does it affect the size, or the monodispersity of the resulting particles. On the other hand, the colloidal stability of the SPION-TEOS / APTES-PEG NPs increased dramatically compared to that of their precursor NPs (Figure 3C and 3D).

Next, a complete physical-chemical characterization of the PEGylated SPIONs and their precursors (SPION-OA and SPION-TEOS / APTES) was carried out. First, to confirm the coating with silica and subsequent PEGylation, the analytical techniques of fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were used. The FTIR spectrum of hydrophobic precursor NPs (SPION-OA) showed the characteristic Fe-0 vibration bands of the magnetite core (585 cm ^"1 ), of CH extension (2926 and 2852 cm ^"1 ) and of carboxylate vibration (1710 and 1459 cm-1) from the oleic acid molecules that cover the SPION (Figure 4A). The FTIR spectrum of the SPION-TEOS NPs / APTES resulting from the ligand exchange had a new and wide band corresponding to the Fe-O-Si bonds (800-1200 cm ^"1 ) and the NH flexural characteristic of the primary amines (1637 and 1540 cm ^{" 1} ), confirming that The ligand exchange with the aforementioned alkoxy silane mixture was successful (Figure 4A). The FTIR spectrum of the PEGylated SPIONs had a peak (-1650 cm ^"1 ) that can be assigned to the carbonyl groups of the amide bond formed with the PEG and the typical bands from the polymer (between 1718 and 837 cm ^"1 ) covalently bonded to the surface of the SPION-TEOS / APTES NPs (Figure 4A). On the other hand, the TGA curve obtained for the NPs obtained by thermal decomposition (SPION-OA) showed a weight loss of 69 .5%, corresponding to the considerable amount of oleic acid ligands that line the core of the particle (Figure 4B). The TGA curve obtained for SPION-TEOS / APTES NPs has a weight loss of 30.9%, much smaller than that obtained with SPION-OA, and usual in particles subjected to silane ligand exchange processes [Cano M. et al., 2017. Nanoscale 9, 812-822; Cano M. et al., 2016. RSC Advances 6, 70374-70382] (Figure 4B). The TGA curve obtained for PEGylated SPIONs showed a weight loss of 70.8%, which means that there is a 39.9% increase generated by PEG molecules covalently linked through the amines of the precursor SPION-TEOS / APTES NPs (Figure 4B). This value is very similar to that obtained in the case of SPION-APTES-PEG NPs [Cano M. et al. 2017, Nanoscale 9, 812-822], which indicates that the PEGylation reaction had an equivalent yield. If we consider that the theoretical mass of a SPION of Fe ₃ 0 ₄ of 10 nm diameter is -2.7-10 ^"18 g, and taking into account the weight loss generated by the stabilizing polymer molecules, it can be estimated that each SPION it is covered by about 860 PEG molecules of 2KDa. This is equivalent to a surface density of -2.73 PEG molecules per nm ² , which is according to the literature an appropriate value for the stabilization of SPIONs in physiological environments [Amstad E. et al. , 2009. Small 5, 1334; Liu D. et al., 201 1. Adv. Funct. Mater. 21, 1498-1504; Pernía M. et al., 2015. Nanoscale 7, 2050-2059; Cano M. et al., 2017. Nanoscale 9, 812-822] The results of FTIR and TGA for SPION-TEOS / APTES NPs not only confirm that the ligand exchange reaction using 2 alkoxy silane derivatives simultaneously (60% TEOS and 40 % TEOS) is produced successfully, but they also indicate that the layer of silica produced is slightly larger than for SPION-APTE NPs S obtained using a single alkoxy silane (100% APTES). To achieve a more accurate quantitative analysis of the structure of the silica layer produced in both SPION-TEOS / APTES and SPION-APTES NPs, X-ray Photoelectronic Spectroscopy (XPS) measurements were carried out. Figure 4C shows the general spectra of XPS or Survey obtained for NPs produced by ligand exchange with 2 alkoxy silane ligands (SPION-TEOS / APTES) and with a single ligand (SPION-APTES). In both cases, the characteristic atoms, such as silicon (Si2s and Si2p) and nitrogen (N1s), of the silicon layer generated on the surface of the SPIONs can be observed. In addition, since the intensity of the Si2p signal is exponentially proportional to the thickness of the silicon layer, the average thickness (f) can be estimated using the Lambert-Beer equation, described in the following publications for very thin layers [Kallury KMR et al., 1995. Anal. Chem. 67, 3362-3370; Wong AKY et al., 2005. Anal. Bioanal Chem. 383, 187-200; XPS Applications in Thin Films Research. Geng S. et al., 2002. Materials Technology 17 (4)]:

/ = I ₀ [exp (- t / l sinQ)]

Where / is the Si2p (eV) signal strength obtained for the solid support with silane layer, l ₀ is the Si2p (eV) signal strength obtained for the solid support without silane layer, / is the exhaust depth of the Si2p electrons through the Silane layer (taken as 20 Armstrong in this work) and Θ the angle of escape at which electrons are expelled from the surface. The thickness value obtained from this equation for the surfaces of NPs SPION-TEOS / APTES and SPION-APTES were 1.15 and 0.52 nm, respectively. These results confirm that using the TEOS / APTES combination proposed in this work, a thicker silane layer is obtained than when only APTES is used. On the other hand, the signal strength N1s does not decrease for the surface of SPION-TEOS / APTES, indicating that there is a similar number of amino groups than with SPION-APTES.

Because the surface amino groups contribute to the surface charge of the resulting NPs, the zeta potential of the PEGylated SPION-TEOS / APTES particles was measured at different pH values (Figure 4D). It can be seen that these SPIONs have a behavior similar to that observed for SPION-APTES-PEG (that is, their surface charge changes from positive to negative as the pH increases) and show an isoelectric point (IEP) of - 7.2, which is considered ideal for NPs used in biological applications [Cano M. et al., 2017. Nanoscale 9, 812-822].

To complete the physicochemical characterization of the PEGylated SPIONs and their precursors, their superparamagnetic properties were also analyzed using the vibrating sample magnetometry (VSM) technique. The graphic representation of the Magnetization values (M) against the applied magnetic field (H) at 300 ° K showed the absence of coercivity and remanence in all samples, confirming their superparamagnetic behavior (Figure 5A). Our analyzes revealed that the saturation values of the magnetization of the SPION-TEOS / APTES-PEG NPs were 21.31 emu-g ^'1 , a value slightly lower than those obtained for SPION-OA and SPION- TEOS / APTES (18.78 and 28.68 emu g ^"1 ). These variations were due to the different organic coating presented by each of the SPION nuclei analyzed. In any case, the magnetization value of the SPION-TEOS / APTES-PEG NPs It was similar to that obtained with SPION-APTES-PEG NPs, and therefore also suitable for use in magnetic resonance imaging and separation [Na HB et al., 2009. Adv. Mater. 21, 2133-2848] .

Example 2. Cytotoxicity studies

Cellular biocompatibility is an important factor to consider in the development of NPs for biomedical use, so we analyze the cytotoxicity of our SPION-TEOS / APTES-PEG NPs. To do this, we use the HepG2 human cell carcinoma cell line, which is a suitable model for cytotoxicity tests in pharmaceutical studies. In addition, these cells represent a good example of a target susceptible to side effects in vivo since most NPs are massively sequestered by the liver when administered intravenously [Blanco E. et al., 2015. Nature Biotech. 33, 941-951].

We quantify cytotoxicity through cell proliferation based on the integrity of the membrane (which we use as a marker of cell death) of HepG2 cells exposed to increasing concentrations of SPION-TEOS / APTES-PEG NPs for 24 and 48 hours. For this, we use two fluorescent markers with different plasma permeability, Hoechst 33342 to stain the nuclei of the total cells (blue staining in Figure 6A) and propidium iodide to stain the dead cells (yellow staining in Figure 6A), which it can only penetrate inside the cells when the integrity of its membrane is compromised) [Jan E. et al., 2008. ACS Nano 2, 928-938]. Exposure of the cells to concentrations of SPION-TEOS / APTES-PEG NPs below 100 μg Fe-mL-1 had no effect on proliferation compared to control samples, and only when the treatment of these NPs reached 200 μg Fe mL-1 showed a negative effect on cell proliferation, which was particularly evident after 48 hours (Figure 6B). In line with the above, only in this last concentration dead cells were observed above the values of the control group (Figure 6C), although these only accounted for 23.9% of the total cells. In the rest of the treatment concentrations the percentage of dead cells was very similar to that of the control cells (Figure 6D). As previously verified with the SPION-APTES-PEG NPs [Cano M. et al. 2017, Nanoscale 9, 812-822], these results demonstrated that SPION-TEOS / APTES-PEG NPs were also not cytotoxic even at iron concentrations of 100 μg. mL-1, which corroborates its potential use in biomedical applications [Singh N. et al., 2010. Nano Reviews 1, 1-15] Example 3. MRI studies (in vitro and in vivo)

We evaluate the potential of SPION-TEOS / APTES-PEG NPs as contracting agents for MRI. For this, we analyze the times of longitudinal (T1) and transverse (T2) relaxation at a low (1.5 T) and high (9.4 T) magnetic field, using a relaxometer and a preclinical MRI scanner respectively (Table 1).

Table 1. Relaxation properties of SPION-TEOS / APTES-PEG NPs at low (1.5 T) and high (9.4 T) fields.

* Longitudinal (T1) and transverse (T2) relaxation times were measured at 0.5 mM Fe (measured by ICP-AES).

The average cross-sectional relaxivity (r2) of our PEGylated SPIONs was 156.3 mM- 1 s-1 at 1.5 T and 1 10.7 mM-1 s-1 at 9.4 T. In addition, the measure of the longitudinal relaxivity (r1) of the they were 4.76 mM-1 s-1 at 1.5 T and 0.28 mM-1 s-1 at 9.4 T, giving r2 / r1 ratios of 32.8 and 395.3 to said magnetic fields respectively. These high values indicated that SPION-TEOS / APTES-PEG NPs offered a good T2 contrast for MRI both in preclinical conditions (high magnetic field) and for clinical conditions (under magnetic field) [Pernía M. et al., 2015. Nanoscale 7, 2050-2059; Cano M. et al., 2016. RSC Advances 6, 70374-70382; Weissleider R. et al, 2010. People ^'s Medical Publishing House. - USA Global Medical Publisher 1-1357]. To verify this we carried out a high-field MRI phantom using different concentrations of SPION-TEOS / APTES-PEG and SPION-APTES-PEG NPs dissolved in water (Figure 5B). This confirmed that the negative contrast (T2) was already evident in the lower NP concentrations compared to the control (water) and that this contrast was very high in increasing concentrations (T2W image in Figure 5B). It was also observed that the T2 contrast obtained for the SPION-TEOS / APTES-PEG NPs was greater than the same obtained with the SPION-APTES-PEG for the same iron concentration, which is consistent with their high field r2 / r1 values (Table 1). On the other hand, as expected, our NPs did not show any positive contrast (T1) compared to the control water (image T1W in Figure 5B).

We injected SPION-TEOS / APTES-PEG NPs intravenously into BALB / c mice. In general, any NP administered systemically in animals is retained in the liver and spleen in large proportion [Blanco E. et al., 2015. Nature Biotech. 33, 941-951; Kettiger H. et al., 2013. Int. J. Nanomedicine 8, 3255-3269; Albanese A. et al., 2012. Annu. Rev. Biomed. Eng. 14, 1-16]. Therefore, considering this observation, we determined whether our PEGylated SPIONs offered a negative T2 contract in vivo. This experiment showed that these organs became very dark after the injection of the NPs (Figure 7 GL). Comparing the decay of the T2 signal in the liver after administration of the SPION-TEOS / APTES-PEG NPs (Figure 7 GL) and SPION-APTES-PEG (Figure 7 AF), the first NPs darkened the liver much more intensely . The quantification of the changes in T2 confirmed that the liver experienced a much more exacerbated signal decay even after one hour post-administration (Figure 7N) confirming that the silanized hybrid has a much better T2-MRI contrast, so it is potentially Best contrast agent in vivo.

As for the renal distribution, in previous publications it has been shown that NPs are retained in renal corpuscles based on their size, these being located in the renal cortex [Choi C.H.C. et al., 201 1. PNAS 108, 6556-6661; Pernía M. et al., 2015. Nanoscale 7, 2050-2059] but there is no glomerular accumulation for PEGylated NPs less than 25 nm. With this in mind, the SPION-TEOS / APTES-PEG NPs, which have a hydrodynamic size around 25 nm in fetal bovine serum (medium very similar to blood), can be found both in the peritubular capillaries, which anatomically they are both in the medulla and in the renal cortex, and probably also slightly retained in the glomerulus, which is found only in the renal cortex. Therefore, only the decay of the signal observed in the renal medulla would be entirely due to the circulating NPs in blood that shorten the T2 signal when they pass through the peritubular capillaries and return to the circulatory system. In other words, the decay of the signal observed in the renal medulla could be used as an indicator of the presence of our NPs in circulation. For this reason, during the quantification of T2 that was carried out in these experiments, ROIs (Regions of interest) were selected in the kidney that differentiated the cortex from the renal medulla. In this way we excluded a probable accumulation of our NPs in the glomeruli (renal cortex). We also excluded the renal pelvis, whose T2 decay corresponded to the NPs excreted in the urine, since this is the area where the kidney concentrates the urine before being accumulated in the bladder through the ureters (Figure 8 C).

Following this proposal, the SPION-TEOS / APTES-PEG NPs were in circulation even at 4 hours after intravenous administration (Figure 7 GL, N and 8 B). The T2 contrast in the renal medulla did not recover until its normal values or at 48 hours post-injection, which was the last measurement taken, indicating that, at that time, the NPs were still in circulation. Compared to the measurements of the T2 ratio in the renal medulla after administration of the SPION-APTES-PEG NPs, they did not show a decline in T2. significant, which indicates that its circulation time was more limited (Figure 7M). This was confirmed in the corresponding T2 images (Figure 7 AF and 8 A).

In conclusion, all these comparative results suggest that the silanized hybrid improves not only the T2 contrast in vivo for MRI but also the time in circulation, which is key to allowing NPs access to the tumor once administered intravenously.

Example 4. Ease of subsequent functionalization for adaptation to specific applications.

Finally, we evaluate that the SPION PEGilagas generated by this method (SPION-TEOS / APTES-PEG), in addition to the aforementioned improvements with respect to the SPION-APTES-PEG NPs [M. Cano et al., 2017. Nanoscale 9, 812-822], continued to maintain the ability to be easily functionalized through the amino groups still available on their surface. For this, functionalization of our SPION-TEOS / APTES-PEG NPs was carried out with a commercial fluorescent marker (Cy5-NHS) activated with an N-hydroxysuccinimide group (Figure 9B). Once the SPION-TEOS / APTES-PEG-Cy5 NPs were purified, the Ninhydrin test showed a clear reduction in the amount of free amino groups compared to their precursor NP (pink line in Figure 9A). In addition, these analyzes also revealed the appearance of a new absorption peak at -650 nm corresponding to the maximum absorption wavelength of Cy5. To confirm the efficient binding of fluorophore to SPION-TEOS / APTES-PEG NPs, the absorption spectra of free Cy5 (green line) and PEGylated NPs were compared before (red line) and after functionalization (pink line in Figure 9C). Accordingly, the Fluorescence phantom image (Aex = 600 nm and Aem = 700 nm) showed that only SPION-TEOS / APTES-PEG-Cy5 NPs show emission (Figure 9D). These results demonstrate that PEGylated NPs obtained with our method (using TEOS and APTES simultaneously) can be easily functionalized, which allows the possibility of adding properties of clinical interest to them, and, in this specific example, as a multimodal imaging agent ( MRI / Optics).

Claims

1. - A nanoparticle comprising:

- a hydrophobic core, preferably of iron oxide; Y

- a functionalized surface with at least two different alkoxy silane derivatives of formula I:

RmSiX (4-n) (formula I); where R is a chemical group selected from the list consisting of an alkyl, alkenes, alkynes, aromatic, alkoxides, organofunctional or any combination thereof; where m can be 0, 1, 2 or 3; where n represents a natural number between 0 and 3; and where X represents an alkoxy group, preferably a methoxy- or an ethoxk

2. - The nanoparticle according to the preceding claim, wherein the nanoparticle is functionalized with the alkoxy silane derivatives by means of a ligand exchange procedure.

3. - The nanoparticle according to any of claims 1-2, wherein the nanoparticle is a SPION.

4. The nanoparticle according to any of claims 1-3, wherein the alkoxy silane derivatives are selected from the list consisting of: Tetraethylorthosilicate (TEOS), 3-aminopropyl-triethoxysilane (APTES), 3-aminopropyl-trimethoxysilane (APTMS ), 3-mercaptopropyl triethoxysilane (MPTES), 3-mercaptopropyl trimethoxysilane (MPTMS), 3-Glycidoxypropyl triethoxysilane, 3- Glycidoxypropyl trimethoxysilane, 3-Cyanatopropyl trieoxysilane (CPTES), 3-Cyanatopropyl triethoxyethyl (3-Cystoxyaproxyl) -Azidatopropyl trimethoxysilane, trimethoxy PEG silanes and a-silane-w-methoxy polyethylene glycol, or any of its salts, isomers, derivatives or analogs, or any combination thereof.

5. The nanoparticle according to claim 4, wherein the nanoparticle comprises two alkoxy silane derivatives that are selected from the list consisting of: a) Tetraethylorthosilicate (TEOS) and 3-AminoPropyl-TriEthoxySilane (APTES), or b) Tetraethylorthosilicate (TEOS) and 3-mercaptopropyl trimethoxysilane, oc) Tetraethyl orthosilicate (TEOS) and a-silane-w-methoxy polyethylene glycol.

6. - The nanoparticle according to claim 5, wherein the nanoparticle comprises two alkoxy silanes derivatives which are tetraethylorthosilicate (TEOS) and 3-aminopropyl-triethoxysilane

(APTES).

7. - The nanoparticle according to claim 6, wherein the concentration of TEOS is in a concentration range, on the total of the alkoxy silanes, between 40 and 80% and APTES, on the total of the alkoxy silanes, in a concentration range of between 20 and 60%.

8. - The nanoparticle according to any of claims 1-7, wherein the nanoparticles are PEGylated.

9. The nanoparticle according to claim 8, wherein the nanoparticles are PEGylated with the polyethylene glycol derivative a-Methoxy-w-carboxy PEG.

10. -. A composition comprising the nanoparticle as defined in any of claims 1-9.

1. The composition according to the preceding claim, wherein the composition is a pharmaceutical composition.

12. - The composition according to any of claims 10 to 1 1, wherein the composition further comprises at least one biologically active compound or molecule, a therapeutic agent or drug, or a labeling agent.

13. The composition according to the preceding claim, wherein the marker agent is a contrast agent.

14. - Use of the nanoparticle according to any of claims 1-9 or of the composition according to any of claims 10 to 13, in the preparation of a medicament.

15. - Use of the nanoparticle according to any of claims 1-9 or of the composition according to any of claims 10 to 13, in the preparation of a pharmaceutical composition for in vivo diagnosis by magnetic resonance.

16. - A method for the synthesis of a nanoparticles comprising

1. Synthesis of hydrophobic nanoparticles, preferably hydrophobic SPIONs synthesized by thermal decomposition;

2. Functionalization with two alkoxy silane derivatives of formula I, as this formula has been defined in claim 1, of the nanoparticles of step 1), through a simultaneously executed ligand exchange procedure;

3. Optionally, a PEGylation of the nanoparticles obtained in step 2) is carried out; and optionally

4. A functionalization of the nanoparticles of step 3) or step 2) is carried out.

17. The method according to claim 16, wherein the hydrophobic nanoparticles of step 1) are hydrophobic SPIONs synthesized by thermal decomposition and wherein the ligand exchange process is carried out with two derivatives of formula I alkoxysilanes selected from the list consisting in Tetraethylorthosilicate (TEOS), 3-aminopropyl-triethoxysilane (APTES), 3-aminopropyl-trimethoxysilane (APTMS), 3-mercaptopropyl triethoxysilane (MPTES), 3-mercaptopropyl trimethoxysilane (MPTMS), 3-mercaptopropyl trimethoxysilane, 3-triethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyethoxyaxiside 3-Cyanatopropyl trieoxysilane (CPTES), 3-Cianatropropyl trimethoxysilane (CPTMS), 3-Azidatopropyl triethoxysilane, 3-Azidatopropyl trimethoxysilane, trimethoxy PEG silanes and a-silane-w-methoxy polyethylene glycols, or any of its analogues, , or any of its combinations.

18. The method according to the preceding claim, wherein the ligand exchange process is carried out with two alkoxysilane derivatives of formula I selected from the list consisting of APTES and TEOS.

19. The method according to any of claims 16 to 18, wherein a PEGylation of the nanoparticles obtained in step 2) is carried out, preferably with the polyethylene glycol derivative a-Methoxy-w-carboxy PEG