BR102020005909B1 - Paramagnetic nanoparticles, their manufacturing process and uses. - Google Patents

Abstract

PARAMAGNETIC NANOPARTICLES, MANUFACTURING PROCESS AND USE THEREOF AS CONTRAST IN MAGNETIC RESONANCE IMAGING. The present invention describes MRI contrast agents based on amorphous diamagnetic metal oxide nanoparticles with paramagnetic metal ions bound to their surface, which form nanofluids in aqueous medium, conjugated with molecules, biomolecules, bioactive molecules, polymer derivatives and biopolymers, and which have application as T1 and T2 contrast agents, suitable for the efficient and safe diagnosis of diseases. The present invention falls within the fields of chemistry and nanotechnology applied to medicine and, more particularly, relates to contrast agents for magnetic resonance imaging diagnosis.

Description

Field of Invention:

[001] The present invention is in the fields of nanotechnology and medicine and relates to magnetic resonance imaging contrast agents characterized by being based on amorphous metal oxide nanoparticles decorated with paramagnetic metal ions/oxides coupled to their surface, whose ratio of transverse and longitudinal relaxivities is controlled by the amount of said paramagnetic ions and molecular species bound to the surface, for simultaneous application as a T1 and T2 contrast medium in the field of disease diagnosis by magnetic resonance imaging (MRI). The combination of metal oxide nanoparticles, paramagnetic metal ions, and biomolecules, polymers, and drugs was achieved in a unique way, generating a new class of contrast media with high specificity, sensitivity, and low cost. The invention can be used as a safer alternative, or alternative, to gadolinium chelate-based contrast media, widely used in medical clinics.

Fundamentals of the invention:

[002] Magnetic Resonance Imaging (MRI) is widely used in medical diagnosis. Using intense magnetic fields, this non-invasive technique allows for the evaluation of soft tissues with significant spatial and contrast resolution. Through the combination of different acquisition techniques during the examination, radiologists assess alterations and possible staging. In order to improve structural and functional evaluation, contrast agents can be used in examinations to increase differentiation between tissues or structures, as well as allowing dynamic evaluation during their passage or accumulation in specific regions of interest. Currently, the most commonly used contrast agents in clinical practice are based on gadolinium paramagnetic complexes. In specific cases, such as severe renal failure, their use is contraindicated because it increases the possibility of developing nephrogenic systemic fibrosis, harming the patient. Due to their toxicity, successive doses in a short period of time are also contraindicated, which limits recalls or repetition of measurements, especially in uncooperative populations such as children or the elderly. Therefore, the development of biocompatible contrast agents that do not present, or present reduced, adverse effects is important in this scenario.

[003] In this context, nanotechnology has great potential for the development of new materials for application in the medical field. Nanotechnology is a promising platform for the development of materials that will revolutionize current technologies. Nanoparticles are the ideal nanomaterials for such applications, since it is possible to control their size, shape and surface charge, characteristics that directly influence their properties. In addition, the surface of nanoparticles can also be modified with different classes of molecules, conferring other desirable properties.

[004] Contrast media are substances capable of improving, in the image, the differentiation of certain structures and/or tissues in relation to their surroundings. In MRI equipment there is a wide range of materials that can be used for this purpose, mainly in the field of research, such as the transition metals manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni) and copper (Cu) and the lanthanides europium (Eu), gadolinium (Gd), terbium (Tb) and dysprosium (Dy) [1]. A common property among contrast media is their efficiency in reducing the T1 and T2 relaxation times of the signals relating to tissues and fluids in Magnetic Resonance Imaging (MRI). Generally, contrast media are classified according to their effects on the types of MRI relaxation, where there are compounds that cause a significant reduction in T1, called T1 contrast media, and media that reduce the T2 relaxation time, called T2 contrast media [2]. In general, a reduction in T1 relaxation time results in increased signal intensity on T1-weighted images, and a reduction in T2 relaxation time results in decreased signal intensity on T2-weighted images, with the effects potentially overlapping.

[005] Despite its toxicity, gadolinium is the most widely used contrast agent, exhibiting a high paramagnetic characteristic, being magnetizable at high temperatures and containing 7 unpaired electrons in its atomic structure. Gadolinium is used as a contrast agent in the form of gadolinium (III) chelates, which are known as gadodiamide, gadopentetic acid, gadobenic acid, gadoxetic acid, gadoversetamide, gadoteridol, gadobutrol, and gadoteric acid. Unofficial estimates show that 90 million doses of gadolinium ion chelates were administered worldwide up to 2008 [3]. Additionally, the sensitivity of gadolinium in MRI is demonstrably higher compared to the use of other contrast agents in other imaging techniques, such as iodinated contrast agents in computed tomography, and volumes approximately 5 to 15 times smaller can be used [3]. This characteristic is cited as one of the reasons why the use of gadolinium is relatively safer than iodinated contrast, which is used in computed tomography.

[006] However, there are serious risks inherent in the use of gadolinium-based contrast media, such as causing nephrogenic systemic fibrosis according to the Committee for Medicinal Products for Human Use (CHMP) consultation, Article 31 of Directive 2001/83/EC. This problem is more serious in patients with renal insufficiency, which makes it difficult to eliminate the intravenously injected contrast medium. Another problem related to the use of gadolinium-based contrast media is the permanent accumulation of this component in the liver, kidneys, muscles and bones after its administration. In addition, recent studies have demonstrated the permanent accumulation of these contrast media in the brain as well [4, 5]. Through analysis of brain samples in autopsies using inductively coupled plasma mass spectrometry and monitoring the T1 MRI signal, McDonald et al. [6] showed the presence of gadodiamide in the brain of patients with normal renal function undergoing magnetic resonance imaging [6]. Murata et al. [7] found that the contrast media gadoteridol, gadobutrol, gadobenate and gadoxetate were present in the bones of patients with normal renal function in quantities 23 times greater than in brain regions, according to a study using a methodology similar to that described by McDonald et al [6].

[007] Despite the relative effectiveness of gadolinium-based contrast media in MRI examinations, safer and more effective alternatives have been constantly sought. In this sense, superparamagnetic iron oxide nanoparticles have attracted attention as contrast media due to their high biodegradability and non-toxicity in biological systems, when compared to gadolinium chelates [810]. There are successful works in the development of iron oxide-based materials with high spin relaxivity and high selectivity through appropriate functionalization with biomolecules. Spinel nanoparticles of MFe2O4 with high transverse relaxivity (r2) values were synthesized and applied to MRI by Lee et al. [11]. The r2 values obtained were 358, 218, 172 and 152 mM-1 s-1 for the MnFe2O4, FeFe2O4, CoFe2O4 and NiFe2O4 nanoparticles, respectively. Furthermore, MnFe2O4 nanoparticles, when associated with an antibody, showed selectivity in detecting small tumors implanted in rats, promoting a signal increase after injection of the contrast medium. Cheng and colleagues [12] developed biocompatible Fe3O4 nanoparticles with high colloidal stability and great potential for application in MRI by promoting a significant reduction in T2 relaxation times, showing r1 and r2 relaxivities of 7.2 and 82 mM-1 s-1, respectively, under a magnetic field of 1.5T.

[008] In fact, iron oxide nanoparticles are more viable alternatives in terms of biocompatibility compared to gadolinium chelate-based contrast media, in addition to exhibiting high transverse relaxivity (r2) values. However, these materials are not yet suitable for widespread clinical use in MRI because they have low longitudinal relaxivity (r1) values, i.e., they provide a significant contrast effect in T2-weighted MRI, but not in T1, limiting their application. Therefore, in this patent we claim protection for a new class of materials to act as efficient contrast media in both T1 and T2, based on metal oxide nanoparticles with paramagnetic ions adsorbed on their surface. This type of material design is highly efficient in promoting T1 and T2 spin relaxation, having great potential as an alternative to gadolinium chelate, iron oxide, and manganese chelate-based contrast media in MRI in the vast majority of clinical protocols applied in diagnosis. Focusing solely on tumor diagnosis, the socioeconomic impact of a new alternative diagnostic MRI contrast medium to Gd(III) chelates will be very significant, considering the approximately 12 million new cases and approximately 8 million deaths worldwide, with around 600,000 new cancer cases expected per year for the 2018-2019 biennium in Brazil alone (INCA).

[009] In the context of MRI imaging techniques, the Time of Flight (ToF) sequence is an alternative used in clinical examinations where the use of gadolinium chelate is contraindicated. Angiography based on the ToF sequence is widely used in brain examinations for the evaluation of vessels, such as arteries and veins. However, regions with flow in the same plane as the acquisition, turbulent flow, or very tortuous vessels are disadvantages of this technique for diagnosis. In magnetic resonance angiography, regardless of the technique used, T1 relaxation is exploited to obtain hypersignal of the vessels. In angiographies using gadolinium chelate contrast, rapid acquisition techniques allow for better evaluations of arteriovenous, cardiac, renal, and other malformations encompassed by the blood circulatory system; that is, the present invention allows for further exploration of this important class of diagnostic evaluation techniques due to its action also on T1 relaxation times.

[010] Perfusion and permeability are assessments at the level of blood microcirculation. MRI dynamically observes the passage of the gadolinium chelate contrast agent, and this dynamic behavior is related to the capillary integrity of organs and tissues. The ASL (Arterial Spin Labeling) sequence is a technique under development used as an alternative in the study of cerebral perfusions without the use of contrast agents. However, this sequence is costly to implement, the image resolutions are worse than in contrast-enhanced measurements, the coverage area is limited, it is only applicable to cerebral assessments, quantitative measurements are time-consuming, and it provides perfusion maps with less information than the maps generated by the technique using contrast agents. In this context, the present invention offers an alternative contrast agent that also presents a reduction in T2 relaxation times comparable to that of commercial gadolinium chelate-based contrasts (III), allowing, mainly due to its action on T1 relaxation times, to be an alternative that does not require adaptations or the use of additional resources in the already implemented clinical routine.

[011] In view of the above, nanotechnology presents great potential for the construction of materials for application in the field of medicine. In particular, nanobiomaterials are promising for replacing currently used contrast agents. Thanks to coating with biocompatible species and biomolecules, NBMs exhibit greater biocompatibility and reduced toxicity. Thus, the present innovation is focused on the development of MRI nanocontrast agents based on nanoparticles with suitable magnetic properties, biocompatibility and low toxicity. The combination of metal oxide nanoparticles and paramagnetic metal ions offers technological advantages because it couples the binding properties of biomolecules, polymers, and drugs on the surface of the nanoparticles with other intrinsic properties of the nanoparticle core, such as magnetic properties, thus promoting the emergence of a new class of contrast media capable of shortening relaxation times in both T1 and T2, due to their high specificity and sensitivity, low cost, and high potential for the subsequent development of theranostic agents.

[012] Ideally, nanoparticles for magnetic resonance imaging diagnostics should exhibit paramagnetic behavior and induce the greatest possible change in T1 and T2 without spontaneous aggregation or aggregation in the presence of body fluids. This behavior has been found to be exhibited by very small nanoparticles, particularly nanoparticles with diameters smaller than about 10-20 nm, preferably by ultrasmall nanoparticles with diameters less than 5 nm. In addition, they should be fully dispersible in water and biological media, be biocompatible to avoid toxic and immunological responses, and should have some chemical and/or physical pathways for the attachment/binding of molecules and biomolecules to their surface, such as antibodies or other proteins, nucleic acids or peptides, or even bioactive molecules, according to the desired application.

[013] In this context, the proposal presented here addresses an important application of diagnostic MRI, which is widely explored in the general context of magnetic resonance imaging examinations and aims to solve the problems inherent in the state of the art using nanoparticles capable of acting simultaneously as efficient T1 and T2 contrast agents for magnetic resonance imaging diagnosis.

State of the Art:

[014] The search for the history of the invention in question led to some documents related to the subject of the current exclusivity request.

[015] Patent document CN103007302A describes the application and preparation of a nanoparticle composite consisting of Gd2O3 and TiO2 comprising 20 to 80 wt% of titanium dioxide nanocrystals in the anatase phase, in sizes from 25 to 200 nm for magnetic resonance imaging. Document CN103007302A describes a composite material, where the titanium dioxide is in the anatase crystalline phase and in the size range of 25 to 200 nm, significantly different from the present invention, where the material is amorphous, contains biocompatible paramagnetic ions, and where the preferred size of the nanoparticles is less than 10 nm.

[016] Patent document CN104353075A reports the preparation and application of a water-soluble magnetic titanium dioxide. The material is formed by titanium dioxide doped with iron oxide and functionalized by hydrophilic molecules. The crystalline phase of the titanium dioxide is rutile or anatase. The magnetic TiO2 particles have a size of 1 to 1000 nm. The authors state that the material can be applied in magnetic resonance imaging. The aforementioned document reports a typically crystalline material, in the anatase or rutile phase, where iron oxide is dispersed throughout the volume of the crystalline structure, replacing TiO2 in the crystalline lattice, generating a material entirely different from the present invention, based on amorphous titanium dioxide nanoparticles having Fe(III) ions chemically adsorbed on the surface of the TiO2 nanostructures, a fundamental characteristic to confer the unique combination of properties presented by the contrast nanoagents of the present invention.

[017] Patent document KR20070058358A describes a material formed by manganese oxide nanoparticles or manganese oxide with another metal present in its structure, which acts as a contrast agent for magnetic resonance imaging. The manganese oxide-based material reported in the aforementioned prior art is entirely different from the present invention, as it is made of a distinct material and does not present the nanostructure nor the characteristics “i” to “v”, and “a” to “g”, reported in the description of the present invention.

[018] Patent document CN102125699A reports the preparation and application of a TiO2/Fe2O3 composite as a contrast agent for magnetic resonance imaging. The said TiO2/Fe2O3 composite has a size of 5 nm (p. 8, Fig. 2) and the titanium oxide and iron oxide are in the anatase and magnetite crystalline phases (p. 8, Fig. 3), respectively. That is, in CN102125699A, a material with a well-defined crystalline structure (anatase (TiO2) and magnetite (Fe2O3)) is presented, indicating the presence of anatase and magnetite nanoparticles, while in the present invention, the material is based on ultrasmall amorphous titanium dioxide nanoparticles with Fe(III) ions chemically adsorbed on the surface.

[019] Finally, patent document US6530944B2 describes a localized heat delivery diagnostic methodology and materials for in vitro and in vivo imaging. The method involves localized induction of hyperthermia in the cell or tissue through the use of nanoparticles, which absorb photons from a range of radiation, converting the corresponding energy into heat. The nanoparticles used consist of a 60 nm gold core coated with a 5-20 nm silica layer. The material described in US6530944B2 has a different constitution, structure, and purpose than those of the present invention.

Brief description of the invention:

[020] The present invention relates to amorphous nanoparticles of metal oxides such as silica and silicates, titanium dioxide and titanates, zinc oxide, and other oxides that are insoluble or poorly soluble in biological media, biocompatible and/or of low toxicity, decorated with paramagnetic ions of transition metals, lanthanides or actinides, chemically bonded/adsorbed on their surface, for application as a contrast medium for magnetic resonance imaging diagnosis. The invention can be used as an alternative to contrast media currently used in clinical medicine based on gadolinium (III) complexes, presenting high economic and technological potential. It thus differs from Fe3O4-TiO2 nanocomposites and analogous materials composed of nanocrystals of said oxides. Solutions and advantages:

[021] In fact, the combination of amorphous and ultrasmall nanoparticles of diamagnetic metal oxides and paramagnetic metal ions, also in the form of amorphous materials, enabled the synergistic coupling of the adsorption properties of biomolecules and drugs with the paramagnetic properties of metal ions, leading to an unexpected increase in T1 relaxation properties while preserving T2 relaxation properties, making them comparable to those of gadolinium(III) complexes, thus generating a new class of MRI contrast media with high specificity, sensitivity and low cost.

[022] Unexpectedly, ultrasmall amorphous nanoparticles, with diameters less than 5 nm, amorphous and with paramagnetic ions bound and chemically adsorbed on the surface, also made it possible to control the T1/T2 ratio through the relative concentrations of paramagnetic ions such as Fe and Ti, making it preferably equal to or less than about 20, more preferably less than 6 (T1/T2=5.3), in addition to making them highly efficient as MRI contrast agents.

[023] Another advantage of this class of materials is their ability to accumulate in regions of diagnostic or therapeutic interest when modified with molecules that have high specificity/selectivity for these regions, structures expressed for example by tumors, after their intravenous administration. Formulations combining nanoparticles with different conjugations, functionalizations, can add an important and novel component for personalized diagnosis and treatment, and therapeutic monitoring.

Brief description of the figures:

[024] For a better visualization of the results and the object of the present invention, the tables and figures to which reference is made are presented, as follows.

[025] FIGURE 1 shows a transmission electron microscopy (TEM) image of amorphous TiO2 nanoparticles on an “ultra-thin carbon” support.

[026] FIGURE 2 shows a histogram of the size distribution of amorphous TiO2 nanoparticles with iron(III) ions chemically adsorbed on the surface, as a function of volume percentage. The measurement was performed at a concentration of 10 mg of TiO2 per 1 mL of water.

[027] FIGURE 3 shows the X-ray diffractograms of TiO2 nanoparticles with and without iron(III) ions chemically adsorbed on the surface, compared with the diffractogram of TiO2.

[028] FIGURE 4 shows the FTIR spectra of TiO2 nanoparticles coated with paramagnetic iron(III) ions and linked to the functionalizing agents tiron, dopamine, glucose-1-phosphate, phosphoethanolamine, glycerol-3-phosphate and citrate.

[029] FIGURE 5 shows MRIs of two male Wistar rats, after intravenous injection of the commercial gadolinium chelate contrast medium (image on the left) and the TiO2NP@Fe contrast medium (image on the right), which is the subject of this patent application.

[030] FIGURE 6 shows the variation of the T1 signal in the heart as a function of time, given by the passage of the contrast medium. The measurement was performed on four animals, two using gadolinium chelate contrast and two using TiO2NP@Fe contrast medium. Baselines were corrected by subtracting the average signal in the period prior to contrast injection. The signal was calculated by averaging the signal of the region of interest, obtained manually for the heart of each animal.

[031] FIGURE 7 shows the variation of the T1 signal in the liver as a function of time, given by the passage of the contrast medium. The measurement was performed on four animals, two using gadolinium chelate contrast and two using the new TiO2NP@Fe contrast medium. The baselines were corrected by subtracting the average signal in the period prior to contrast injection. The signal was calculated by averaging the signal of the region of interest, obtained manually for the liver of each animal.

[032] FIGURE 8 shows the variation of the T1 signal in the kidneys as a function of time, given by the passage of the contrast medium. The measurement was performed on four animals, two using gadolinium chelate contrast and two using the new TiO2NP@Fe contrast medium. The baselines were corrected by subtracting the average signal in the period prior to contrast injection. The signal was calculated by averaging the signal of the region of interest, obtained manually for the kidneys of each animal.

[033] FIGURE 9 shows the variation of the T1 signal as a function of time, in minutes, before and after the injection of gadolinium chelate contrast and the new TiO2NP@Fe contrast medium. The arrows indicate the hypersignal in the kidneys, indicating this excretion pathway also by the new TiO2NP@Fe-based contrast medium, unexpectedly occurring 10 minutes after the onset of the observed for gadolinium chelate.

[034] FIGURE 10 shows the unexpected dependence of the relaxivities r1 and r2, as a function of the type of functionalizing molecule attached to the surface of the amorphous TiO2@Fe nanoparticles, at a concentration of 4 mg/mL in agarose medium.

Detailed Description of the Invention:

[035] The present invention is based on amorphous diamagnetic nanoparticles of diamagnetic metal oxides, preferably titanium dioxide (TiO2), with paramagnetic ions/oxides, preferably Fe(III) and Mn(III), chemically adsorbed/bound on the surface. This strategy can be extended to other types of nanostructured or amorphous inorganic oxide nanoparticles with high surface area, such as silicon dioxide (SiO2), silicates, titanates, and zinc oxide (ZnO), or even polymeric nanoparticles, which exhibit a high adsorption capacity, through physical or chemical bonding, or coordination, of a wide variety of paramagnetic ions such as Mn2+, Mn3+, Mn4+, Fe2+, Fe3+, Co2+, Co3+, Eu2+, Eu3+, Gd2+, Gd3+, and Tb3+, forming amorphous paramagnetic materials chemically adsorbed/bound on the surface of the ultrasmall nuclei. This allows the modulation of the magnetic properties of the resulting nanomaterials by controlling the relative concentration and nature of the paramagnetic metal ions. Furthermore, the exposed surface of metal oxide nanoparticles is fertile ground for functionalization/binding of various classes of molecules and biomolecules, allowing the efficiency and specificity of these new MRI contrast media to be adjusted so that they bind/concentrate on specific physiological targets in the body. The literature describes the use of core-shell nanoparticles and/or nanocomposites as contrast media in MRI. In contrast, in our invention, we unexpectedly found that paramagnetic metal ions adsorbed on the surface of diamagnetic and amorphous metal oxide cores exhibit a high adsorption or binding capacity, preferentially of biocompatible paramagnetic transition metal ions/oxides, allowing for greater interaction of the latter with water molecules and adequately reducing T1 and T2 relaxation times, where the r1/r2 ratio is controlled by the relative amount of paramagnetic ions. These characteristics are essential for the efficiency of an MRI contrast medium, which are claimed in the present application.

[036] Additionally, the present invention takes as a preferred example, titanium dioxide nanoparticles (TiO2NP) with diameters smaller than 20 nm, totally amorphous, dispersible and stable in aqueous medium, modified with paramagnetic ions of transition metals, preferably from the first transition series such as Fe(III) and Mn(III), and molecules on their surface, which confer, respectively, paramagnetic properties to the core and colloidal stability to the nano-contrast agent.

[037] Ultrasmall titanium dioxide nanoparticles are presented, with average sizes less than 20 nm in average diameter, preferably from 1 to 4 nm, and simultaneously functionalized with paramagnetic Fe(III) ions and with neutral or ionic molecules, biomolecules, polymers, biopolymers and bioactive molecules, such as PEG, PEG derivatives, gelatin, collagen, cellulose, gums and other natural polymers, glycerol, aminoethyl phosphate, dopamine, glucose, biotin, folic acid, thiron, citrate, among others, which are fully dispersible in aqueous medium, have no toxicity or have sufficiently low toxicity for application as an MRI contrast agent.

[038] Aiming at the development of alternative materials for the fabrication of MRI contrast media, it was also unexpectedly found that the strategy of chemical adsorption of paramagnetic ions of transition metals onto diamagnetic and amorphous metal oxide cores, forming an amorphous and paramagnetic material, can be used to successfully control the effects induced on the T1 and T2 spin-lattice relaxation times. This represents a significant advance compared to materials based on superparamagnetic iron oxide nanoparticles (magnetite, maghemite, and ferrites) in which the effect on T1 is much smaller than on T2, reducing the possibilities of their application as contrast media. Thus, efforts were concentrated on understanding and controlling the effect, in order to decrease the effect on T2 and increase it on T1 by increasing the surface area, decreasing the spin-spin coupling, and increasing the spin-lattice coupling of paramagnetic ions. Thus, an efficient strategy was developed to control the fundamental parameters for the development of MRI contrast media, as shown in TABLE 1.

[039] TABLE 1 presents T1 and T2 measurements performed with MRI nanocontrast agents based on amorphous titanium dioxide nanoparticles (TiO2NP) incorporating different proportions of Fe(III) ions chemically adsorbed on their surface, using a Gd(III) chelate currently used in the clinic as a reference.

[040] In the tests, MRI imaging procedures were performed using male rats as an animal model. The procedure consisted of performing a bolus injection of the commercial contrast agent and the present invention, separately, into Wistar rats via the penile vein during a dynamic MRI acquisition. In this way, it was proven that the invention offers image contrast similar to that of gadolinium (III) chelate-based contrast agents, as can be seen in FIGURES 5 to 9, thus demonstrating the high potential of TiO2NP@Fe, the subject of this patent application, as MRI contrast agents.

[041] It is clearly noted that the T1 and T2 values can be modulated over a wide range in such a way as to influence the former much more than the latter. Thus, it was possible to produce aqueous dispersible nanoparticles that have effects similar to those of gadolinium chelates when injected intravenously. In fact, it was possible to observe the displacement of the bolus as it traveled through the penile vein towards the heart, and then throughout the rat's body. A typical image is shown in FIGURE 5 where the results and highly favorable prospects can be clearly seen, considering the unexpected efficiency comparable to, or even superior to, that of gadolinium chelates. Furthermore, production scaling was successfully carried out and the low production cost was confirmed.

[042] All magnetic resonance images and T1 and T2 measurements presented here were obtained on a 7 Tesla scanner, with a maximum gradient power of 70 mT/m @ 200 T/m/s and a head coil with 1 transmission channel and 32 reception channels. The proper functioning of the equipment is periodically evaluated through a specific quality control program and has regular preventive maintenance.

[043] The study presented was approved by the Ethics Committee on the Use of Animals (CEUA) of the Faculty of Medicine of the University of São Paulo (FMUSP), registration numbers 1193/2018 and 966/2019.

[044] The general parameters of the images acquired in this study were:

[045] T1-Weighted Images (T1w): 2D-TSE; TR = 750 ms; TE = 9 ms; Nex = 2; ETL = 4; Spatial resolution of 0.5 x 0.5 x 1.0 mm3 and 1 x 1 x 3 mm3. With and without fat saturation.

[046] T2-Weighted (T2w) Images: 2D-TSE; TR = 5000 ms; TE = 50-70 ms; Nex = 1; ETL = 9; Spatial resolution of 0.5 x 0.5 x 1.0 mm3 and 1 x 1 x 3 mm3.

[047] TurboFlash Dynamic Images: 2D-TurboGRE; TR = 200 ms; TE = 1.3 ms; FA = 20°; Spatial resolution 0.5 x 0.5 x 3.0 mm3; 80 dynamics with 0.6Hz sampling.

[048] TWIST images: TR = 3 ms; TE = 1.4 ms; FA = 12°; Spatial resolution 0.5 x 0.5 x 0.5 mm3;

[049] Dynamic TWIST Images: TR = 3.3 ms; TE = 1.2 ms; FA = 14°; Spatial resolution 0.75 x 0.75 x 1.0 mm3; 60 dynamic images with 0.5Hz sampling.

Examples of the invention:

[050] Example 1 - TiO2 nanoparticles were produced using a titanium precursor selected from titanium chloride, titanium ethoxide, titanium isopropoxide and titanium butoxide with the addition of a saturated or unsaturated chain organic acid with 1 to 18 carbons and a solvent which can be water, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, propanetriol, polyethylene glycol, benzene, toluene, heptadecane and acetone, among other solvents, and heating the system at temperatures that can vary between 25 and 250 °C for a period of 15 to 360 minutes. After this stage, the nanoparticles were functionalized with selected organic molecules from among phosphonates with 1 to 16 carbons, polyphosphonates, phosphates or saturated or unsaturated chain organic acids with 1 to 18 carbon atoms, amines, catechols, carbohydrates, polyethylene glycol derivatives, polyvinyl alcohols, oligomers, polymers, biopolymers, biomolecules, collagen derivatives, gelatin, carboxycellulose, polylactic acid, polyglycolic acid, natural gums, xanthan gum, chitosan, carbohydrate derivatives, alginate, polysaccharides, peptides, glycoproteins, antigens, antibodies, aptamers, tyron, dopamine, glucose-1-phosphate, phosphoethanolamine, glycerol-3-phosphate and polycarboxylates, citrate, glycolate, gluconate, silicon derivatives, silanes, siloxanes, among others, and combinations thereof.

[051] Example 2 - Titanium chloride and titanium isopropoxide were added in a 10:3.3 ratio to oleic acid followed by the addition of heptadecane as described in reference [14], and the reaction mixture was heated to 110 °C for one hour. The nanoparticles were washed and separated by precipitation by the addition of ethanol. Amorphous TiO2 nanoparticles of 2 to 3 nm in diameter, functionalized with lipophilic molecules, were obtained.

[052] Example 3 - Adapting the procedure previously described in the literature [15], 1.1 mL of TiCl4 was added to 12 g of polyethylene glycol (PEG 600) at room temperature, and the system was heated in a volumetric flask at 150 °C for up to 6 hours under stirring. The nanoparticles were functionalized with organic ligands such as phosphoethanolamine, citrate, glycerol phosphate, and glucose. TiO2 nanoparticles in the anatase phase, 5 nm in size, functionalized with hydrophilic molecules, were obtained.

[053] Example 4 - The procedure described in example 3 was repeated, replacing polyethylene glycol with glycerol. 5 nm TiO2 nanoparticles in the anatase phase with hydrophilic functionalization were obtained.

[054] Example 5 - The procedures described in examples 3 and 4 were carried out by adding one or more saturated or unsaturated chain organic acids containing from 1 to 18 carbon atoms. TiO2 nanoparticles with average diameters in the range of 1 to 200 nm were obtained, with hydrophilic functionalization/coating, and titanium oxide cores in the anatase, rutile or amorphous phases, depending on the reaction temperature in the range of 50 to 250 °C.

[055] Example 6 - The adsorption of metal ions/oxides on the surface of amorphous TiO2 nanoparticles was carried out using a methodology that consists of dispersing the nanoparticles in the presence of a salt solution of the transition metal, for example Fe(III), in a solvent that can be water, methanol, ethanol, propanol, propanetriol, butanol, benzene, toluene and acetone, among others. The adsorption time can vary from 1 to 300 minutes, the temperature from 10 to 150 °C, and the hydrogen ion potential pH from 2 to 12.

[056] Example 7 - The concentration of iron and titanium was determined directly in the dry samples, or in aqueous suspension, by the X-ray fluorescence (EDX) technique, or by total reflection X-ray fluorescence (TRXF). The concentrations of phosphonate, phosphate, and sulfonate ligands in relation to the metallic elements were quantitatively determined by the aforementioned techniques. In addition, techniques such as infrared and Raman spectroscopy, and UV-Vis electronic spectroscopy, ICP-OES, and calorimetry were used to determine the Fe and ligand/molecule contents, in order to determine and monitor the composition and structure of the MRI contrast nanoagents. Techniques such as dynamic light scattering (DLS) and zeta potential measurements were used to determine the size distribution, electric charge density (zeta potential), and zero charge potential of the nanoparticles.

[057] Example 8 - The morphology and structure of ultrasmall NPs based on amorphous titanium dioxide nanoparticles coated with paramagnetic transition metal ions were characterized by various techniques. Transmission electron microscopy (TEM) images show that the contrast nanoagents of the present invention consist of spherical nanoparticles with an average size of approximately 3 nm (dark spots), monodisperse, non-agglomerated, individually dispersed on an “ultrathin carbon” support, as shown in FIGURE 1. The histogram of nanoparticle size distribution as a function of volume percentage was obtained by dynamic light scattering (DLS) and showed a unimodal distribution with an average diameter of 3.6 nm, FIGURE 2 and TABLE 2, slightly larger than the average diameter determined by TEM, as expected by the presence of functionalizing molecules and a hydration layer. Furthermore, the present invention is characterized by the high affinity of the nanoparticles for water, forming nanofluids, that is, fluids containing individually dispersed nanoparticles. The degree of crystallinity of TiO2 nanoparticles with and without iron ions adsorbed on the surface, compared to commercial reference TiO2 nanoparticles P25, was determined by X-ray diffraction and shown in FIGURE 3. The commercial material showed diffraction peaks corresponding to the crystalline planes characteristic of materials in the anatase (A(101); A(004); A(200); A(105); A(211); A(204); A(116); A(220)) and rutile (R(110); R(101); R(111)) phases. On the other hand, the 3 nm TiO2 nanoparticles with and without iron(III) semi-ions adsorbed on the surface, the subject of the present invention, did not show any X-ray diffraction peaks characteristic of crystalline material, including the peaks of materials in the anatase and rutile phases, indicating that they have a completely amorphous structure. Furthermore, the absence of peaks corresponding to crystalline iron oxides in the TiO2NP 3nm/Fe sample suggests that the iron(III) ions adsorbed onto the amorphous TiO2 cores also do not present an organized crystalline structure, generating a completely amorphous material, consistent with the adsorption strategy of individual iron(III) ions onto the amorphous titanium dioxide nanoparticles as described. The FTIR spectra of TiO2NP@Fe functionalized with thion, dopamine, glucose-1-phosphate, phosphoethanolamine, glycerol-3-phosphate, and citrate are shown in FIGURE 4, where characteristic peaks corresponding to O-H bonds can be observed on the TiO2 surface at 3340 cm-1 and 1640 cm-1; Ti-O-Ti type bonds in the region between 490 and 700 cm-1; Aromatic rings at 1630, 1590, 1470 and 1430 cm-1; O-H groups not on the surface of TiO2 at 1370 cm-1; C-O bonds at 1200 cm-1; phenolic groups at 1240 and 1290 cm-1; O-S-O bonds at 1100 cm-1 and saryl oxygens of the Ti-O bond at 1170 and 1270 cm-1.

[058] TABLE 2 presents typical size distribution data for amorphous TiO2 nanoparticles with iron(III) ions adsorbed on the surface, as a function of their respective volume percentages.

[059] Example 9 - The potential use of 3nm TiO2NPs/Fe as MRI contrast media in vitro and in vivo was evaluated, thus demonstrating the proof of concept and high efficiency of these materials for the intended application. For the in vitro evaluation, a medium composed of 3% (w/v) agarose and 0.5% (w/v) NaCl in increasing concentrations of NPs was prepared. The samples were prepared to mimic the conditions of biological tissues. The MRI image results and T1 and T2 measurements are presented in TABLE 1. For comparison purposes, the contrast agent, also known as gadoteric acid (GA), which is based on gadolinium (III) chelate, a paramagnetic species capable of interfering with the T1 magnetic relaxation time, decreasing the longitudinal relaxation time and leading to a hypersignal in T1-weighted MRI, was used. The AG was also prepared in 3% agarose and 0.5% NaCl medium for comparison purposes. Analyzing the results presented in TABLE 1, it was unexpectedly found that the T1 and T2 of the TiO2NP@Fe-based contrast medium at a concentration of 4 mg/mL in agarose medium produced greater contrast than AG at a concentration of 5 mM in agarose medium, a concentration approximately 5 times higher than the AG concentration after intravenous injection in humans.

[060] Example 10 - Comparing the relaxivities of TiO2NP@Fe at a concentration of 4 mg/mL with different functionalizations, in agarose medium, it was also unexpectedly found that r2 varies little, but that r1 is significantly dependent on the functionalizing layer, as shown in FIGURE 10.

[061] Example 11 - To demonstrate the proof of concept of the invention, an MRI imaging procedure was performed using male rats as an animal model. Briefly, the procedure consisted of performing a bolus injection of the commercial contrast agent and the present invention, separately, into Wistar rats via the penile vein during a dynamic MRI acquisition. In this way, it was proven that the invention offers image contrast similar to that of gadolinium (III) chelate-based contrast agents, as can be observed in FIGURES 5 to 9, thus demonstrating the high potential of TiO2NP@Fe, the subject of this patent application, as MRI contrast agents. REFERENCES 1. Sanchez, T.A., Preliminary characterization and application of a natural oral contrast agent for magnetic resonance imaging of the gastrointestinal tract, in Faculty of Philosophy, Sciences and Letters of Ribeirão Preto. 2005, University of São Paulo: Ribeirão Preto. 2. Frey, N.A., et al., Magnetic nanoparticles: synthesis, functionalization, and applications in bioimaging and magnetic energy storage. Chemical Society Reviews, 2009. 38(9): p. 2532-2542. 3. Elias Junior, J., et al., Complications from the use of intravenous gadolinium-based contrast agents for magnetic resonance imaging. Radiol Bras, 2008. 41: p. 263-267.4. Ramalho, J., et al., Gadolinium-Based Contrast Agent Accumulation and Toxicity: An Update. American Journal ofNeuroradiology, 2016. 37(7): p. 1192-1198.5. Rogosnitzky, M. and S. Branch, Gadolinium-based contrast agent toxicity: a review of known and proposed mechanisms.Biometals, 2016. 29: p. 365-376.6. McDonald, R.J., et al., Intracranial Gadolinium Depositionafter Contrast-enhanced MR Imaging. Radiology, 2015. 275(3): p. 772-782.7. Murata, N., et al., Macrocyclic and Other Non-Group 1Gadolinium Contrast Agents Deposit Low Levels of Gadolinium in Brain and Bone Tissue: Preliminary Results From 9Patients With Normal Renal Function. Investigative Radiology, 2016. 51(7): p. 447-453.8. Xu, C. and S. Sun, New forms of superparamagnetic nanoparticles for biomedical applications. Advanced Drug Delivery Reviews, 2013. 65(5): p. 732-743.9. Bin, N. H., S. I. Chan, and H. Taeghwan, InorganicNanoparticles for MRI Contrast Agents. Advanced Materials, 2009. 21(21): p. 2133-2148.10. Bulte, J.W.M. and D. L. Kraitchman, Iron oxide MR contrast agents for molecular and cellular imaging. NMR in Biomedicine, 2004. 17(7): p. 484-499.11. Lee, J.H., et al., Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nature Medicine, 2007. 13(1): p. 95-99. 12. Cheng, F.Y., et al., Characterization of aqueous dispersions of Fe3O4 nanoparticles and their biomedical applications. Biomaterials, 2005. 26(7): p. 729-738.13. Zeng, L., et al., Multifunctional Fe3O4-TiO2 nanocomposites for magnetic resonance imaging and potential photodynamic therapy. Nanoscale, 2013. 5(5): p. 2107-2113.14. Menzel, R., et al., Two-stage, non-hydrolytic synthesis for improved control of TiO2 nanorod formation. Journal of Materials Chemistry, 2012. 22(24): p. 12172-12178.15. Goncalves, R.H., W.H. Schreiner, and E.R. Leite, Synthesis of TiO2 Nanocrystals with a High Affinity for Amine Organic Compounds. Langmuir, 2010. 26(14): p. 11657-11662.

Claims (17)

1. Paramagnetic nanoparticles, CHARACTERIZED by comprising: (a) an amorphous metal oxide core consisting of diamagnetic metal oxides that are insoluble or poorly soluble in aqueous media and biological fluids, preferably silicon dioxide, silicates, titanates and zinc oxide, and more preferably amorphous titanium dioxide with an average diameter of 1 to 5 nm, having at least one type of paramagnetic ion/oxide selected from the transition metals manganese (Mn), iron (Fe3+), cobalt (Co), nickel (Ni) and copper (Cu), and the lanthanides europium (Eu), gadolinium (Gd), terbium (Tb), samarium (Sm) and dysprosium (Dy) attached to its surface, which is chemically adsorbed, bound to the surface generating an amorphous paramagnetic material, where the paramagnetic ions are weakly magnetically coupled to each other. (e) and (b) at least one molecule, biomolecule and/or polymer and biopolymer, having an average core diameter in the range of 1 to 200 nm, preferably less than or equal to 20 nm, and more preferably with diameters less than 5 nm, with a size distribution in water of 3.6 nm; wherein, (c) one or more molecules are covalently bonded to the surface forming a molecular layer responsible for colloidal dispersion and stabilization. 2. Nanoparticles, according to claim 1, CHARACTERIZED in that the longitudinal and transverse relaxivity and the relaxation times T1 and T2 are dependent on the relative concentration of chemically adsorbed paramagnetic ions, bound to the surface of amorphous diamagnetic metal oxide cores, and on the composition of the molecular functionalizing layer bound to the surface. 3. Nanoparticles, according to claim 2, CHARACTERIZED in that the paramagnetic Fe(III) ion/oxide is present in a concentration ranging from 0.001 to 50% by mass. 4. Nanoparticles, according to claim 2, CHARACTERIZED in that the T1/T2 ratio is less than 25, and preferably equal to or less than 6. 5. Nanoparticles, according to any one of claims 1 to 4, CHARACTERIZED by the fact that one or more dispersing/stabilizing molecules are linked/coupled to a molecular layer already previously attached, coordinated to the surface. 6. Nanoparticles, according to any one of claims 1 to 5, CHARACTERIZED in that the molecular layer is constituted by one or more types of molecular and/or polymeric species. 7. Nanoparticles, according to any one of claims 1 to 6, CHARACTERIZED in that the molecular layer on the surface of the paramagnetic amorphous cores comprises compounds having in their structure at least one cationic, neutral or anionic group. 8. Nanoparticles, according to claim 7, CHARACTERIZED in that the cationic group is amine/polyamine and/or N-heterocyclic N-alkylated or non-alkylated, and the anionic group is sulfonate, sulfate, phosphonate, phosphate and/or carboxylate, and the neutral group is alcohol or polyalcohol, amide or polyamide, diamide, ether or polyester, and/or nitrile. 9. Nanoparticles, according to claim 8, CHARACTERIZED in that the amine group is an alkylamine selected from the group consisting of alkylamines with 1 to 18 carbon atoms such as ethylamine, propylamine, butylamine, etc., in addition to aminoethyl phosphate, dopamine and glycol derivatives such as glycerol, polyethylene glycols, polyalcohols, polycarboxylic acids, polyglycolic acid, alginate, saccharides, polysaccharides, thion, among others. 10. Nanoparticles, according to any one of claims 1 to 9, CHARACTERIZED in that the molecules are bioactive and are selected from the group consisting of biotin, glucose-1-phosphate, glucose-6-phosphate, folic acid, dopamine, phosphoethanolamine, peptides, antibodies, antigens, aptamers, DNA fragments, glycoproteins, alginate, gluconic acid, glycolic acid, carbohydrate derivatives, collagen and other biopolymers. 11. Nanoparticles, according to claim 10, CHARACTERIZED in that the bioactive molecules are attached to the surface by a coupling reaction, forming a molecular layer on the surface of the amorphous metal oxide core. 12. Process for obtaining nanoparticles defined in claims 1 to 11, CHARACTERIZED by comprising the steps of: (a) preparation of the diamagnetic and amorphous metal oxide core; (b) purification of the nanoparticles obtained in a); (c) conjugation of molecules, biomolecules and/or derivatives of polymers, biopolymers and bioactive molecules to the surface; or (d) coupling reactions of molecules, biomolecules and/or derivatives of polymers, biopolymers and bioactive molecules with molecules previously conjugated to the surface preferably by means of amide, amine, ether, ester, diamide and carbon-carbon linkages; (e) modification with or coupling of at least one type of paramagnetic ion/oxide to the surface of the nanoparticles obtained in a) defined by claim 1. 13. Use of paramagnetic nanoparticles, as defined in any one of claims 1 to 11, CHARACTERIZED by being for the preparation of MRI contrast agents. 14. Use according to claim 13 CHARACTERIZED by being based on dispersions of at least one type of nanoparticle, as defined in any of claims 1 to 11. 15. Use, according to claim 14, CHARACTERIZED in that the dispersion of said nanoparticle is at a concentration of 0.01 to 20 mg/ml. 16. Use, according to claim 14, CHARACTERIZED by further comprising an accumulation/concentration step in some target region by conjugation or not of at least one vectoring molecule on the surface of the amorphous metal oxide nanoparticles. 17. Use, according to any one of claims 13 to 16, CHARACTERIZED by comprising a formulation containing combinations of amorphous metal oxide nanoparticles decorated with paramagnetic ions and conjugated to different types of molecules/biomolecules, allowing accumulation/concentration in different target regions/structures simultaneously.