METAL OXIDES NANOPARTICLES CONJUGATED WITH NAPHTHALENE DERIVATIVES AS CONTRAST AGENTS FOR THE DETECTION OF BETA AMYLOID PLAQUES BY MAGNETIC RESONANCE IMAGES

Abstract

Compounds with magnetic properties are provided herein, which belong to the category of metal oxide nanoparticles, coated and conveniently functionalized, which are conjugated with naphthalene compounds related to agglomerates and β-amyloid plaques present in neurodegenerative diseases. These new nanoparticles (NPs) are used for the non-invasive detection of agglomerates and amyloid plaques using the Magnetic Resonance Imaging (MRI) technique. The nanoparticles described here cross the blood-brain barrier (BBB), without the use of any membrane-disrupting agent. Likewise, they bind with high affinity and specificity to the agglomerates and β-amyloid plaques, and are used as contrast agents in MRI for the early detection of Alzheimer's disease (AD).

Claims

1. A magnetic nanoparticle related to the agglomerates and β-amyloid plaques for the diagnosis of Alzheimer's disease by magnetic resonance imaging, of Formula I comprising a metal oxide core coated with a multifunctional organic layer, wherein said organic layer is conjugated to a naphthalene derivative, ##STR00003## wherein: R.sub.1: is an organic coating to the metal oxide core, of polymeric type, catechol derivatives or trialkoxyalkylaminosilane; R.sub.2: is —NHCO-alkylenyl-C(O)NH-alkylenyl-R.sub.3; R.sub.3: is —COO—, —CO—, —NH, —O—, —S—, —NH-alkylenyl-NH—, or —NR.sub.4—CSS—; R.sub.4: is —H, —CH.sub.3, —CH.sub.2—CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3, and M.sub.xO.sub.y: is iron oxide (Fe.sub.3O.sub.4/γFe.sub.2O.sub.3), gadolinium oxide(III), manganese oxide(II) or copper(II) oxide; wherein the conjugated, functionalized and coated magnetic nanoparticle is capable of, when it is administered to a mammal, crossing the blood-brain barrier and specifically binding to the agglomerates and β-amyloid plaques present in brain tissue; wherein, with the nanoparticle bound to the agglomerates and β-amyloid plaques in the brain tissue, hypo- or hyper-intense signals are observed in the region of interest through magnetic resonance imaging.

2. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein the alkylenyl term in R.sub.2 and R.sub.3 is selected from the group consisting of ethylenyl (—CH.sub.2CH.sub.2—), butylenyl (—CH.sub.2CH.sub.2CH.sub.2CH.sub.2—) and hexylenyl (—CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—).

3. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein the organic coating R.sub.1 of the metal oxide core further has a terminal functional group selected from the group consisting of —SH, —OH, —NH.sub.2, —NCS, —COOH and its esters of N-hydroxysuccinimide and —Br.

4. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein R.sub.1 is a polymer selected from the group consisting of polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyethylene glycol (PEG) and its derivatives, PEG-PLA, PEG-PLGA, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropylmethylcellulose (HPMC), dextran and chitosan.

5. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein R.sub.1 is a catechol derivative selected from the group consisting of 4-(2-aminoethyl)benzene-1,2-diol, 3,4-dihydroxycinnamic acid, 3,4-dihydroxyphenethyl isothiocyanate and 4-(bromoethyl)benzene-1,2-diol.

6. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein R.sub.1 is a trialkoxyalkylaminosilane selected from the group consisting of a (2-aminoethyl)triethoxysilane, (3-aminopropyl)triethoxysilane, N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine and (4-aminobutyl)triethoxysilane.

7. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein R.sub.1 is an organic coating, comprising the mixture of a polymer selected from the group consisting of polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyethylene glycol (PEG) and its derivatives, PEG-PLA, PEG-PLGA, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropylmethylcellulose (HPMC), dextran and chitosan, and a catechol derivative of selected from the group consisting of 4-(2-aminoethyl)benzene-1,2-diol, 3,4-dihydroxycinnamic acid, 3,4-dihydroxyphenethyl isothiocyanate and 4-(bromoethyl)benzene-1,2-diol, in stoichiometric proportions from 1:0.01 to 1:1.

8. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein R.sub.1 is an organic coating, comprising a mixture of a polymer selected from the group consisting of polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyethylene glycol (PEG) and its derivatives, PEG-PLA, PEG-PLGA, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropylmethylcellulose (HPMC), dextran and chitosan, and a trialkoxyalkylaminosilane derivative selected from the group consisting of a (2-aminoethyl)triethoxysilane, (3-aminopropyl)triethoxysilane, N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine and (4-aminobutyl)triethoxysilane, in stoichiometric proportions from 1:0.01 to 1:1.

9. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein R.sub.1 is an organic coating, comprising the mixture of a catechol derivative selected from the group consisting of a 4-(2-aminoethyl)benzene-1,2-diol, 3,4-dihydroxycinnamic acid, 3,4-dihydroxyphenethyl isothiocyanate and 4-(bromoethyl)benzene-1,2-diol and a trialkoxyalkylaminosilane trialkoxyalkylaminosilane selected from the group consisting of a (2-aminoethyl)triethoxysilane, (3-aminopropyl)triethoxysilane, N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine and (4-aminobutyl)triethoxysilane, in proportions stoichiometric proportions from 1:0.01 to 1:1.

10. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein the naphthalene derivative is selected from the group consisting of N1-(2-aminoethyl)-N4-(1-naphthyl)succinamide, N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine, 6-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}hexanoic acid, N1-(2-aminobutyl)-N4-(1-naphthyl)succinamide, N-(2-hydroxyethyl)-N′-1-naphthyl succinamide, N-(3-mercaptopropyl)-N′-1-naphthysuccinamide, N-{2-[(2-aminoethyl)amino]ethyl}-N′-1-naphthysuccinamide and (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl)carbamodithioic acid sodium salt.

11. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein a hydrodynamic radius of the coated nanoparticles, functionalized and conjugated with a naphthalene derivative is less than 150 nm.

12. The magnetic nanoparticle related to the agglomerates and β-amyloid plaques of claim 1, wherein a hydrodynamic radius of the coated nanoparticles, functionalized and conjugated with a naphthalene derivative is between 100 and 5 nm.

13. A pharmaceutical composition of the magnetic nanoparticles of Formula I, ##STR00004## wherein: R.sub.1: is an organic coating to the metal oxide core, of polymeric type, catechol derivatives or trialkoxyalkylaminosilane; R.sub.2: is —NHCO-alkylenyl-C(O)NH-alkylenyl-R.sub.3; R.sub.3: is —COO—, —CO—, —NH, —O—, —S—, —NH-alkylenyl-NH—, or —NR.sub.4—CSS—; R.sub.4: is —H, —CH.sub.3, —CH.sub.2—CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3, and M.sub.xO.sub.y: is iron oxide (Fe.sub.3O.sub.4/γFe.sub.2O.sub.3), gadolinium oxide(III), manganese oxide(II) or copper(II) oxide; wherein pharmaceutically acceptable excipients are employed in the pharmaceutical composition of the nanoparticle; wherein, with the administration of the pharmaceutical composition of nanoparticle, hypo- or hyperintense images are recorded by Magnetic Resonance in a region of interest associated with the agglomerates and β-amyloid plaques present in the brain tissue of mammals.

14. The pharmaceutical composition of the magnetic nanoparticles of claim 13, wherein the formulation is administered to a mammal by a route selected from the group consisting of nasal, intracerebroventricular, intraperitoneal and intravenous.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIGS. 1A-B: show the general procedure, which includes the most significant reaction conditions, of the conjugation of NPs-1 magnetite nanoparticles functionalized with HOOC-PEG-NH.sub.2 and conjugated with N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine (A) to obtain NPs-1A.

[0050] FIG. 2: shows the FT-IR spectra of A: N-4-(1-naphthylamino)-4-oxobutanoyl-β-alanine (A), B: NPs-1 and C: NPs-1A.

[0051] FIG. 3: shows the hydrodynamic diameter of the NPs-1A, dispersed in DMSO, determined by the DLS technique. Time between measurements 2 min. The DLS profiles were obtained in a DelsaNano C spectrometer from the Beckman Coulter firm. The measurements were made at an angle of 179°.

[0052] FIGS. 4A-B: TEM micrographs of the NPs-1A, registered in a JEM-1010 electronic microscope, JEOL at 80 kV are presented.

[0053] FIGS. 5A-C: show the general procedure, which includes the most significant reaction conditions, of the conjugation of magnetite nanoparticles NPs-2, functionalized with PEG-dicarboxylated, with N.sup.1-(2-aminoethyl)-N.sup.4-(1-naphthyl)succinamide (B) to obtain NPs-2B.

[0054] FIG. 6: shows the FT-IR spectra of A: N.sup.1-(2-aminoethyl)-N.sup.4-(1-naphthyl) succinamide (B), B: NPs-2 and C: NPs-2B.

[0055] FIG. 7: shows the hydrodynamic diameter of the NPs-2B, dispersed in DMSO, determined by the DLS technique. Time between measurements 2 min. The DLS profiles were obtained in a DelsaNano C spectrometer from the Beckman Coulter firm. The measurements were made at an angle of 179°.

[0056] FIG. 8: shows the curves of thermograms (TG) and Differential Thermal Analysis (DTA) of the NPs-2B subjected to a thermal treatment from room temperature to 1000° C. under a flow of Ar. The simultaneous thermograms of ATD and TG were registered in a NETZSCH equipment, model STA 449 F3. The experimental data of the variation of the weight of the sample with the temperature were processed with the help of the program included in the equipment, “Proteus”, version 5.2.1/07.04.2001. The error of the quantitative TG analysis is 2.0%.

[0057] FIGS. 9A-C: show the general procedure, which includes the most significant reaction conditions, of the conjugation of gadolinium oxide nanoparticles functionalized with PEG-dicarboxylic (NPs-3), with N1-(2-aminoethyl)-N4-(1-naphthyl) succinamide (B) to obtain NPs-3B.

[0058] FIG. 10: shows the FT-IR spectra of A: N1-(2-aminoethyl)-N4-(1-naphthyl) succinamide (B), B: NPs-3 and C: NPs-3B.

[0059] FIG. 11: shows the hydrodynamic diameters of the NPs-3B, dispersed in DMSO, determined by the DLS technique. Time between measurements 2 min. The DLS profiles were obtained in a DelsaNano C spectrometer from the Beckman Coulter firm. The measurements were made at an angle of 179°.

[0060] FIG. 12: the curves of thermograms (TG) and Differential Thermal Analysis (DTA) of NPs-3B subjected to a thermal treatment from room temperature to 1000° C. under an Ar flow are presented. The simultaneous thermograms of ATD and TG were registered in a NETZSCH equipment, model STA 449 F3. The experimental data of the variation of the weight of the sample with the temperature were processed with the help of the program included in the equipment, “Proteus”, version 5.2.1/07.04.2001. The error of the quantitative TG analysis is 2.0%.

[0061] FIGS. 13A-B: shows the variations of the signal intensity of the NPs-1A prepared at different concentrations. These curves are generated from the measurements made in the magnetic resonance imaging (MRI) obtained with Spin Eco (SE) sequences. FIG. 13 A—shows the longitudinal relaxation curves; with a fixed Echo Time (TE=11 ms) and different values of TR, while FIG. 13 B—shows the transversal relaxation curves, with a Fixed Repetition Time (TR=10000 ms) and different TE values. C—Comparative study of relaxivities of Resovist and NPs-1A by MRI.

[0062] FIGS. 14A-B: shows the variations of the signal intensity of NPs-3B prepared at different concentrations. These curves are generated from the measurements made in the magnetic resonance images obtained with Spin Eco (SE) sequences. FIG. 14A shows the longitudinal relaxation curves; with an Echo Time (TE=11 ms) fixed and different values of TR, while FIG. 14B shows the transversal relaxation curves, with a Fixed Repetition Time (TR=10000 ms) and different TE values.

[0063] FIGS. 15A-B: intensity measurements in different areas are presented in the transgenic mouse brain images. On the left the intensities measured before contrast administration in four zones (including reference). On the right the intensities measured after the administration.

[0064] FIGS. 16A-B: representative microphotographs of the prefrontal cortex corresponding to healthy animals and transgenic APPSwe/PS1dE9 mice (scale bar=200 μm) are presented.

[0065] FIG. 17: shows an Fe calibration curve and linear adjustment used for Table 1.

DETAILED DESCRIPTION

[0066] This invention is related to Chemistry and Physics applied to the field of Medicine and refers to the use of compounds with magnetic properties, which belong to the category of metal oxide nanoparticles, coated and conveniently functionalized, which are conjugated with naphthalene compounds related to agglomerates and β-amyloid plaques present in neurodegenerative diseases. These new nanoparticles (NPs) are used for the non-invasive detection of agglomerates and amyloid plaques using the Magnetic Resonance Imaging (MRI) technique. The nanoparticles described here cross the blood-brain barrier (BBB), without the use of any membrane-disrupting agent. Likewise, they bind with high affinity and specificity to the agglomerates and β-amyloid plaques, and are used as contrast agents in MRI for the early detection of Alzheimer's disease (AD).

[0067] The present invention relates to the use of metal oxide nanoparticles with magnetic properties, coated, functionalized and conjugated to naphthalene compounds highly related to agglomerates and β-amyloid plaques. The design of the NPs presented here was based on the analysis of the structure of the senile plaques, specifically the agglomerates of the Aβ peptide, to avoid nonspecific recognition with other brain structures. To do this, different databases and computer programs were analyzed in a combined and singular way (3D structure of fibrils Aβ 1-42 of Alzheimer's, Code: 2BEG, DOI: 10.2210/pdb2beg/pdb, deposited: 2005 Oct. 24, published: 2005 Nov. 22, Wyrzykowska et al Nanotechnology 2016, 27 445702; Chen, et al., in J. Mol. Biol. 2005; 354: 760-776; Landau et al., in PLoS Biol. 2011; 9: e1001080, Hetényi et al in Biochem Biophys, Res. Commun 2002; 292: 931-936) and it was obtained that the NPs described here interact unexpectedly with the Aβ peptide, mainly with amino acid residues, essentially through interactions hydrophobic, Van der Waals forces and H-bonds. Thus, the estimated energy values ΔG (−9.8 to −6.6 kcal/mol) and the affinity constant Ki (1.33×10.sup.−7 to 2.79×10.sup.−7) of the β-amyloid peptide-organic coating complex of NPs, demonstrate the stability of these NPs with plaques. Accordingly, the NPs interact with the Aβ peptide in the region that appears to be key in the formation of the β-folding structure (Chen et al., in the Journal of Molecular Biology, 2005, 354 (4): 760-776; Hetényi et al in Biochemical and Biophysical Research Communications 2002, 292 (4): 931-936). The design of these functionalized NPs includes a carbon chain that carries different functional groups that allow the selective conjugation with the naphatalene derivatives, giving rise to a new chain that responds structurally with the synergy of both structures, and that also, surprisingly, it helps NPs claimed in this patent cross the BBB, solving the drawbacks encountered with other CA in the prior state-of-the-art and overcoming the described technique.

[0068] The present invention entails the use of new functionalized and conjugated magnetic nanoparticles to diagnose Alzheimer's disease in early stages by Magnetic Resonance Imaging. These nanoparticles of Formula I comprise a metal oxide core coated with a multifunctional organic layer, wherein said organic layer is conjugated to a naphthalene derivative related to the β-amyloid plaques,

##STR00002## [0069] wherein: [0070] R.sub.1: is an organic coating to the metal oxide core, of polymeric type, catechol derivatives and trialkoxyalkylaminosilane; [0071] R.sub.2: —NHCO-alkylenyl-C (O) NH-alkylenyl-R.sub.3; [0072] R.sub.3: —COO—, —CO—, —NH, —O—, —S—, —NH-alkylenyl-NH—, —NR.sub.4—CSS—; [0073] R.sub.4: —H, —CH.sub.3, —CH.sub.2—CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, and [0074] M.sub.xO.sub.y: iron oxide (Fe.sub.3O.sub.4/γFe.sub.2O.sub.3), gadolinium oxide (III), manganese oxide (II) and copper (II) oxide; [0075] wherein the coated, functionalized and conjugated magnetic nanoparticle is capable, when administered to a mammal, of crossing the blood-brain barrier and specifically binding to the agglomerates and amyloid plaques present in brain tissue; [0076] wherein, with the nanoparticle bound to the agglomerates and amyloid plaques in the brain tissue, hypo- or hyper-intense signals are observed in the region of interest through magnetic resonance imaging.

[0077] Through the NPs described here, the acquisition of Magnetic Resonance Imaging is carried out to detect the agglomerates and β-amyloid plaques present in the brain. These NPs cross the BBB, without the use of any membrane disrupting agent, due to the synergy of properties that arise from the combination of the use of specific coatings for each naphthalene derivative related to the β-amyloid plaques.

[0078] Unexpectedly, without being bound by theory, the singular combination of the naphthalene derivatives, related to the β-amyloid plaques, with the coatings used, allows the obtaining of CA of the type T1 and T2, by varying only the metal oxide core, which guarantees a greater precision in the diagnosis. In the state-of-the-art this property is not reported for the same compound.

[0079] The NPs of this invention can be used at low concentrations because they are highly related to β-amyloid agglomerates and their values of relaxitivities modify the contrast by more than 40%, which guarantees a high sensitivity.

[0080] In this invention, the general methods of synthesis of the new functionalized nanoparticles of metal oxides with magnetic properties, conjugated to the aforementioned naphthalene derivatives, with good yields, and their use for the diagnosis of Alzheimer's Disease are described, which should not be construed as limiting the present invention in any way. The procedures are practical, economical and can be adapted to a larger scale manufacturing.

[0081] A non-limiting example of the magnetic, and highly monodisperse synthesized NPs were NPs of iron oxide (Fe.sub.3O.sub.4/γFe.sub.2O.sub.3) coated with polyethylene glycol functionalized with carboxyl and amine groups. These coatings offer the advantage of forming an amide bond with an amino or carboxyl group, respectively, of a naphthalene derivative, related to the agglomerates and β-amyloid plaques, such as, for example, the acids: N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine (A) or 6-{([4-(1-naphthylamino)-4-oxobutanoyl] amino} hexanoic acid and the amines: N1-(2-aminoethyl)-N4-(1-naphthyl) succinamide (B) or N1-(4-aminobutyl)-N4-(1-naphthyl) succinamide, respectively. The formation of the covalent bond carried out through the method known as the Steglich reaction or the carbodiimide method (Xia et al., in Int. J. Electrochem, Sci, 2013. 8: 2459-2467). In Example 2, which is not limiting to the patent, magnetic NPs functionalized with N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine (NPs-1A) were synthesized according to the scheme shown in FIGS. 1A-B, which is not limiting. Subsequently they were isolated, washed, and dispersed in DMSO, at room temperature.

[0082] The structural characterization of NPs-1A was carried out using different analytical techniques. FIG. 2 shows the FT-IR spectra of the synthesized NPs, of N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine and NPs-1A, where the presence of N-[4-(1-naphthylamino)-4-oxobutanoyl-β-alanine (A) on the surface of the NPs synthesized is demonstrated.

[0083] In the FT-IR spectrum of the NPs-1 appears the set of typical bands of this type of system. Thus, at 3420 cm.sup.−1, a band appears corresponding to the valence vibration of the —NH.sub.2 group. At 2920 and 2850 cm.sup.−1 the valence vibrations ν.sup.as .sub.(CH) and ν.sup.s .sub.(CH) of the carbon chain of the PEG are observed. Finally, at 580 cm.sup.−1, the characteristic band of θ.sub.(Fe—O) appears confirming the presence of magnetite in the NPs.

[0084] On the other hand, in the FT-IR spectrum of the NPs-1A, the signals that corroborate the coupling of the terminal carboxyl group of N-[4-(1-naphthylamino)-4-oxobutanoyl-β-alanine (A) with the terminal amino group of NPs-1 is observed. Thus, the valence vibration bands ν.sub.(OH) and ν.sub.(CO) of N-[4-(1-naphthylamino)-4-oxobutanoyl-β-alanine (A), at 3248 cm.sup.−1 and at 1711 cm.sup.−1, respectively, disappear. This confirms that the free carboxyl group of the naphthalene derivative (A) reacts, giving rise to an amide bond, whose vibration band is observed at 1645 cm.sup.−1. There also appear, a broad band at 3370 cm.sup.−1 and another intense at 1018 cm.sup.−1, which are attributed to ν.sub.(NH) and to ν.sub.(C—O—C), respectively. The valence vibration band ν.sub.(Fe—O) is observed at 640 cm.sup.−1.

[0085] In Table 1 it is reported that the iron content in the NPs-1A, determined by Atomic Absorption, ranges between 30-45%.

TABLE-US-00001 TABLE 1 m.sub.T NPs-1A C.sub.NPs-1A C.sub.Fe m.sub.T Fe Sample (mg) (mg/L) (mg/L) (mg) % Fe 1 12.1 3 1.65 4.95 40.9% 2 15.7 3 1.57 4.71 30.0% 3 9.5 3 1.44 4.32 45.4% A = m*C.sub.Fe + n Parameter Value Error Error (%) n −0.0080 0.0004 5.0 m (L/mg) 0.0341 0.0009 2.6
The measurement of the hydrodynamic diameter (DLS profile) of the NPs-1A was made with a time interval of two minutes (FIG. 3). The value of this parameter was around 21 nm, with a polydispersity index of less than 5%. In addition, the value of the diameter remained stable at the time of measurement, which shows that no agglomerates are formed in the system. This is due to the fact that N-[4-(1-naphthylamino)-4-oxobutanoyl-β-alanine (A), which has a special amidoalkyl chain, unexpectedly gives a high stabilization to NPs due to steric hindrance, without interactions between nearby particles. According to the TEM micrographs (FIG. 4A), the diameter of the NPs-1A is 11.1±1.8 nm (FIG. 4B). In them it is not possible to appreciate the organic coating on the surface of NPs-1A.

[0086] The general procedure for the conjugation of N1-(2-aminoethyl)-N4-(1-naphthyl) succinamide (B) with the magnetite NPs coated with dicarboxylated PEG (NPs-2), is presented in FIGS. 5A-C, which is not limiting of this patent. The NPs-2B were separated magnetically and washed with DMF and subsequently with water to remove the residues from the reaction. Once the product was vacuum dried, the NPs were dispersed in DMSO and stored at room temperature until use. The chemical structure of NPs-2B could be verified through its FT-IR spectrum (FIG. 6).

[0087] In the spectrum of the NPs-2B a broad and intense band around 3432 cm.sup.−1 is observed, which corresponds to the ν.sub.(NH) in the secondary amides. Three bands are observed, at 1593 cm.sup.−1 (σ.sub.NH and ν.sub.C═O) and at 1398 cm.sup.−1 (ν.sub.CN), which show the presence of compound B in the structure, and also, at 616 cm.sup.−1 the band of ν.sub.Fe—O vibration.

[0088] In FIG. 7, the DLS profiles of the NPs-2B are observed. The measurements made by DLS reported a hydrodynamic diameter of 95-99 nm with a polydispersity index of less than 16%. The stability of the NPs-2B was evaluated through measurements by DLS, at intervals of 2 minutes (FIG. 7). According to these results, there is no appreciable variation in the values of the hydrodynamic diameters of the NPs-2B, so the system favorably presents an adequate stability during the study time in DMSO. This is due to the conjugation of B with the NPs-2, which, as in the case of the NPs-1A, does not allow other molecular interactions to take place, so there is no tendency to agglomerate the NPs-conjugates. Therefore, it can be stated that the naphthalene derivatives, related to the agglomerates of β-amyloids, unexpectedly confers stability to the magnetic NPs of iron oxides.

[0089] FIG. 8 shows the thermograms corresponding to the thermogravimetric analysis (TG), the differential thermal analysis (DTA), as well as the thermogravimetric analysis (TGD) of the NPs-2B. The TG curve is characterized by the existence of a first stage of sudden loss of mass, which corresponds to the elimination of moisture from the sample. Then, a loss of 21% of the mass occurs from 125 to 460° C., with two maximums in the curve of ATD, at 214 and at 365° C. This corresponds to an endothermic process of desorption and decomposition of the organic matter in the sample. A third loss of 4% of weight was recorded from 591° C., which is observed in the DTA curve as an endothermic process with a maximum at 632° C. Then, at 675° C., a weight gain occurs, which corresponds to a transition from the crystalline phase of magnetite to maghemite caused by oxidation. Finally, the hematite, the most stable thermodynamically crystalline phase, is obtained (Pati et al in J. Appl. Phys, 2012, 112: 210-220).

[0090] Another non-limiting example of the magnetic and highly monodisperse NPs claimed in this patent were the PEGylated Gd.sub.2O.sub.3 nanoparticles functionalized with naphthalene derivatives. These NPs can be coated with polyethylene glycol by the polyol method (Wasi Md. et al in Colloids and Surfaces A: Physicochemical and Engineering Aspects 2014, 450, 67-75.). In FIGS. 9A-C, which is not limiting, the general synthesis procedure is shown, which consists of two stages. In the first stage, the nanoparticles coated with PEG are obtained and in the second stage, the ligands are exchanged with the dicarboxylated PEG (NPs-3). The conjugation of the NPs-3 with B is carried out by the Steglich reaction.

[0091] In the FT-IR spectrum of the NPs-3B (FIG. 10), the presence of bands is observed at 1650, 1498 and 1387 cm.sup.−1, which correspond to the vibrations of the antisymmetric valence (ν.sup.as.sub.OCO) and symmetric (ν.sup.s.sub.OCO) of the carboxylate of the dicarboxylated PEG linked to the Gd.sub.2O.sub.3, overlapped with those of the amide group that takes place (σ.sub.NH, ν.sub.C═O and ν.sub.CN). At 1387, 1255 and 1095 cm.sup.−1, the bands characterizing the dicarboxylated PEG attached to the nanoparticles are observed. The broad band at 3454 cm.sup.−1 is assigned to the valence vibration ν.sub.NH of secondary amines.

[0092] The content of Gd in the NPs-3B was determined with the use of the optical emission spectrometry technique with inductively coupled plasma (ICP). The mean value obtained from two replicates was 30.29% (Table 2).

TABLE-US-00002 TABLE 2 Replica Concentration (ppm) % mass 1 6.118 30.59 2 5.997 29.99 Lineal Equation IE = 5153 × C.sub.(Gd) + 8408 Parameter Value (ppm.sup.−1) standard error (ppm.sup.−1) Slope 5153 ±68 Intercept 8408 ±314

[0093] The content of Gd was measured in an ICP-OES device, Spectro brand, Spectroflame model. The power used by the equipment was 1200 W. A nebulization flow of 1.2 L/min of Argon and a nebulization pressure of 3.8 bar was used. The auxiliary flow and the cooling flow were 1.2 L/min (Ar) and 18.8 L/min (Ar), respectively. The observation height was 15 mm with respect to the coil. Gd.sub.2O.sub.3 of 99.9% purity was used, which was dissolved in HCl (37%) to prepare the calibration curve.

[0094] The hydrodynamic diameter of NPs-3B was determined through the DLS technique (FIG. 11). The hydrodynamic diameter was 47 nm, with a polydispersity index of less than 15%. Like the NPs-2B, the NPs-3B dispersed in DMSO maintained their stability over time.

[0095] The thermal analysis provides information on the evolution of the sample against the temperature variation and allows to estimate the mass percentage of the surface coating of the Nps-3B. FIG. 12 shows the thermograms corresponding to the thermogravimetric analysis (TG), the differential thermal analysis (DTA), as well as the derivative of the thermogravimetric analysis (TGD). In the thermogram there is a small decrease in mass (0.18%) around 105° C., which is associated with the loss of hydration water in the NPs-3B. Then, and up to 900° C., a 64.34% loss of the total mass of the sample occurs, which is due to the processes of desorption and decomposition of the organic coating of the nanoparticles. As of 900° C., no significant changes in mass are observed, which corresponds to the nucleus of stable gadolinium oxide in the sample.

[0096] In order to measure the magnetic properties of the functionalized and conjugated NPs with the naphthalene derivatives of this invention, the relaxitivitie values of r.sub.1 and r.sub.2, and their relation r.sub.2/r.sub.1 (Example 7), were determined. This is a physical-chemical characteristic that reflects how the magnetic relaxation speed of a dissolution of a CA changes according to its concentration.

[0097] FIGS. 13 and 14 show the variations in the signal strength of the solutions of NPs-1A and NPs-3B prepared at different concentrations, as non-limiting examples. These curves are generated from measurements made in magnetic resonance imaging (MRI) obtained with Espin Eco sequences (Fanea L, et al. in Romanian Reports in Physics, 2011; 63 (2): 456-464).

[0098] FIG. 13A shows the longitudinal relaxation curves for a fixed Echo Time (TE=11 ms) and different TR values, while FIG. 13B shows the transverse relaxation curves, with a Repetition Time (TR=10000 ms) fixed and different TE values. The ratio of relaxitivities r.sub.2/r.sub.1 evaluated from the experimental data was r.sub.2/r.sub.1=90, for the MRI equipment of 3T (Table 2.5).

TABLE-US-00003 TABLE 2.5 Parameters NPs-1A Resovist* Hydrodynamic 21 nm 62 nm diameter r.sub.1 3.5 mg.sup.−1 s.sup.−1 L 4.6 mM.sup.−1s.sup.−1 r.sub.2 337.8 mg.sup.−1 s.sup.−1 L 143 mM.sup.−1s.sup.−1 r.sub.2/r.sub.1 90 31 *Invest Radiol 2005; 40: 715-724
In the case of the commercial negative Resovist CA, the value of r.sub.2/r.sub.1 was 31 (Rohrer, M. et al in Invest Radiol 2005, 40: 715-724 and Reimer et al., in European Radiology, 2003, 13 (6): 1266-1276). The high value of r.sub.2/r.sub.1 of the NPs-1A, compared to the Resovist, may be due to its high crystallinity, which increases the value of r.sub.2 (Levy et al., in Biomaterials, 2011, 32 (16): 3988-3999 and Salafranca et al., in Nano letters, 2012. 12 (5): 2499-2503). These results confirm that NPs-1A have excellent magnetic properties for use as a contrast agent in MRI. From the values r.sub.1 and r.sub.2 obtained from NPs-1A (Table 2.5) and from equations 1 and 2 of Example 7, the effect of the NPs-1A is calculated on the longitudinal and transverse tissue relaxation times of a brain of an APPSwe/PS1dE9 transgenic animal. The results are summarized in Table 3.

TABLE-US-00004 TABLE 3 T1.sub.t r.sub.1 C T1.sub.obs Modification of (ms) (mL/mg*s) (mg/mL) (ms) observed T1 (%) 800 3.524 0.0488 703 12.09 800 3.524 0.0244 748 6.44 T2.sub.t r.sub.2 C T2.sub.obs Modification of (ms) (mL/mg*s) (mg/mL) (ms) observed T2 (%) 80 337.8 0.0488 34.5 56.87 80 337.8 0.0244 48.2 39.74
As observed, the value of T1 in the presence of the effect of NPs-1A changes between 12% and 6%, while that of T2 changes between 56% and 40%, depending on the concentration of NPs-1A. These results confirm that NPs-1A are a negative contrast agent when the values of T2 vary significantly with respect to T1 values. If you compare these results with the one described for Resovist (a variation of 53% of the T2 of the tissue for a concentration of 0.1 mM) it confirms that NPs-1A is an excellent contrast agent.

[0099] FIG. 14A shows the longitudinal relaxation curves; with an Echo Time (TE=11 ms) fixed and different values of TR, while in FIG. 14B the transversal relaxation curves are observed, with a Fixed Repetition Time (TR=10 000 ms) and different values of TE. The relaxitivities values r.sub.1 and r.sub.2 of the NPs-3B are: r.sub.1=7.74 mg.sup.−1s.sup.−1L and r.sub.2=17.9 mg.sup.−1s.sup.−1L and its relation r.sub.2/r.sub.1 of 2.31. This value was compared with that described for Magnevist (r.sub.2/r.sub.1=1.19), a positive contrast agent for commercial use in MRI (Rohrer M, et al in Invest Radiol 2005; 40: 715-724). This shows that NPs-3B have excellent magnetic properties for use as a contrast agent in MRI.

[0100] From the values of r.sub.1 and r.sub.2 obtained from the NPs-3B, their effect on the longitudinal and transverse relaxation times of a brain tissue of a transgenic animal APPSwe/PS1dE9 was calculated. With equations 1 and 2 of Example 7, the relaxation times observed (T1.sub.obs or T2.sub.obs) in the tissue are calculated as a consequence of the accumulation of NPs-3B. The modification (expressed in percentage) of the longitudinal and transverse relaxation times observed in a brain tissue of a transgenic animal APPSwe/PS1dE9 in the presence of NPs-3B is presented in Table 4.

TABLE-US-00005 TABLE 4 T1.sub.t r.sub.1 C T1.sub.obs Modification of (ms) (mL/mg*s) (mg/mL) (ms) observed T1 (%) 800 7.743 0.09 416.5 47.96 800 7.743 0.075 444.7 44.41 T2.sub.t r.sub.2 C T2.sub.obs Modification of (ms) (mL/mg*s) (mg/mL) (ms) observed T2 (%) 80 17.9 0.09 70 12.50 80 17.9 0.075 69.4 13.25
As observed, the T1 value of the tissue under the effect of the NPs-3B changes between 44% and 47% depending on the concentration, while the T2 does it between 12% and 13%. The predominant variation in the observed T1 value demonstrates that NPs-3B is a positive contrast agent. If this result is compared with the variation of 20% caused by Magnevist on tissue T1, it is concluded that NPs-3B is an adequate positive contrast agent.

[0101] Table 5 shows changes in intensity tres Regions of Interest in a healthy animal of 18 months. The zones are 1-3 in brain and cerebellum and one of reference. In zones 1-3 the contrast change was up to 22%. In the reference area, where the contrast does not reach, there were no statistically significant changes.

TABLE-US-00006 TABLE 5 Zone Before injection After injection Variation (%) 1 802.2 613.6 23.5 2 1434.8 1487.5 3.3 Reference 43.7 45.5 0

EXAMPLES

[0102] The obtaining of the nanoparticles of this invention and their use for the diagnosis of Alzheimer's Disease is illustrated by the following examples, which should not be interpreted in any way as limiting.

Example 1: Magnetite Nanoparticles Functionalized with HOOC-PEG-NH.SUB.2 .(NPs-1)

[0103] In a 50 mL round bottom flask under an argon atmosphere, Fe(acac).sub.3 (2.5 mmol, 0.883 g), HOOC-PEG-NH.sub.2 1000 (2.5 mmol, 2.5 g) and PEG-300 (37.3 mmol, 11.2 g) are mixed with constant stirring. The reaction mixture was heated at 160° C. for 30 min. and subsequently, at 220° C. for 2 h., with vigorous stirring. The reaction mixture was cooled to room temperature and ethanol was added. The particles were separated by centrifugation at 10 000 rpm and dispersed in DMF (1 mL) to store at room temperature. FT-IR, ν (cm.sup.−1): 3420; 2920; 2850; 1603; 1070; 580 (ν.sub.Fe—O).

Example 2: Magnetite Nanoparticles Conjugated with N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine (A); (NPs-1A)

[0104] In a 50 mL round bottom flask, A (10 mg, 31.8 μmol) dissolved in DMF (1 mL) was added. Hydroxybenzotriazole (HOBTz, 4.7 mg, 35 μmol), previously dissolved in DMF (500 μL), was added to the reaction mixture. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (8.2 mg, 52.4 μmol) dissolved in DMF (500 μL) was added. The reaction mixture was stirred for 30 min, then 200 μL of a dispersion of magnetic nanoparticles NPs-1 is added. The reaction mixture is was stirred at room temperature for 4 hours. The product was magnetically separated, washed with DMF (2×250 μL) and with water (2×250 μL) and then vacuum dried over P.sub.2O.sub.5. The NPs-1A were dispersed in DMSO (1 mL) to store at room temperature. FT-IR, ν (cm.sup.−1): 3370; 1645; 1018; 640 (ν.sub.Fe—O).

Example 3: Magnetite Nanoparticles Coated with Polyethylene Glycol Dicarboxylate (NPs-2)

[0105] In a 50 mL round bottom flask under an argon atmosphere, PEG-di-COOH-600 (0.4 g, 0.7 mmol) and 10 mL of PEG-300 (33 mmol) are mixed with constant stirring. Next, a solution of Fe(acac).sub.3 (0.18 g, 0.51 mmol) in 2.5 mL PEG-300 was added. The reaction mixture was heated at 160° C. for 30 min. and subsequently, at 220° C. for 2 h., with vigorous stirring. The reaction mixture was cooled to room temperature and ethanol was added. The particles were separated by centrifugation at 10 000 rpm and dispersed in DMF (1 mL) to store at room temperature. Mass of the product obtained: 200 mg. FT-IR, ν (cm.sup.−1): 3420; 2924; 1626; 1412; 1096; 571

Example 4: Magnetite Nanoparticles Coated with Polyethylene Glycol Dicarboxylate (NPs-2) Conjugated to N1-(2-aminoethyl)-N4-(1-naphthyl)succinamide (B) (NPs-2B)

[0106] In a 50 mL round bottom flask immersed in an ice bath, 200 μL of NP-2 (dispersed in DMF (1 mL) of Example 3) and a solution of HOBTz (4.7 mg, 35 μmol) in DMF were added (500 μL). Then a solution of EDC (8.2 mg, 52.4 μmol) in DMF (500 μL) was added. The reaction mixture was stirred for 30 min to add a solution of 3 (10 mg, 35 μmol) in DMF (500 μL) and then stirring at room temperature for 48 h. Finally, the conjugated nanoparticles (NPs-2B) were magnetically separated, washed with DMF and water (2×250 μL, each one) and dried in a vacuum under P.sub.2O.sub.5 for 24 h. The particles were dispersed in DMSO (4 mL) and thus stored at room temperature. Mass of the product obtained: 40 mg. FT-IR, ν (cm.sup.−1): 3432; 2920; 1592; 1397; 616

Example 5: Gadolinium Oxide Nanoparticles Coated with Polyethylene Glycol Dicarboxylate (Gd.SUB.2.O.SUB.3.-PEGdicarboxylated) (NPs-3)

[0107] In a 50 mL round bottom flask, equipped with a reflux condenser, 3.45 g (5 mmol) of GdCl.sub.3×6H.sub.2O were dissolved in 25 mL of PEG (Mn=400), at 100° C. with stirring. To this solution was added a solution of NaOH (0.6 g, 15 mmol) in 10 mL of PEG (Mn=400). The reaction mixture was heated at 180° C. for 4 h, with constant stirring. Then, the temperature was lowered to 80° C. to add 8 mmol (4 mL) of PEGdicarboxylated (PEGD, Mn=600) and then, it was again heated at 180° C. for 4 h, with constant agitation. The reaction mixture was cooled to room temperature to add 500 mL of distilled H.sub.2O. The colloidal suspension was stirred for 10 min and then allowed to settle until the sedimentation of the particles (about one week). The supernatant liquid was decanted and the solid was dried under vacuum in a desiccator over P.sub.2O.sub.5. Mass obtained from NPs-3: 893 mg. FT-IR, ν (cm.sup.−1): 3295; 1580; 1525; 1431; 1401; 1301; 1006.

Example 6: Gd.SUB.2.O.SUB.3.-PEGdicarboxylated Nanoparticles (NPs-3) Conjugated with N1-(2-aminoethyl)-N4-(1-naphthyl) succinamide (B)) (NPs-3B)

[0108] In a 25 mL round bottom flask, 5 mg of Gd.sub.2O.sub.3-PEGD were dispersed in 4 mL of DMF and an EDC solution (40 mg, 0.26 mmol) in DMF (4 mL) was added. The reaction mixture was cooled in an ice bath and HOBT (20 mg, 0.13 mmol) and 3 (40 mg, 0.14 mmol) were added in stepped time of 30 min, between them. Then, it was stirred at room temperature for 2 days and centrifuged at 10 000 rpm for 30 min to remove the supernatant fluid. The solid was washed with ethanol (10 mL×3) and dried under vacuum in a desiccator over P.sub.2O.sub.5. Mass obtained from NPs-3B: 2.7 mg. FT-IR, ν (cm.sup.−1): 3454; 2933; 2870; 1650; 1498; 1387; 1255, 1095.

Example 7: In Vitro Characterization of the Magnetic Properties of NPs-1A and NPs-3B

[0109] The contrast agents (CA) affect both the T1 and T2 observed (T1.sub.obs, T2.sub.obs) in the tissues in which it accumulates. Equations (1) and (2) describe this phenomenon (Haacke E M, et al., in Magnetic Resonance Imaging Physical Principles and Sequence Design, 1999. United States, New York.).

[00001]$\begin{array}{cc}\begin{array}{c}{R}_{1\mathrm{obs}}1/T{1}_{obs}\equiv 1/T{1}_t+r_1*C\\ \equiv R_{1t}+r_1.Math.C\end{array}& \left(1\right)\end{array}$

Being:

[0110] C—Concentration of the CA (mM or mg/ml, depending on the availability of the substance)
R.sub.1obs—speed or relaxation rate observed (s.sup.−1). It is the relaxation rate of the tissue modified by the CA with a concentration C.
T1.sub.obs—observed T1 (ms)
T1.sub.t—T1 of the tissue (ms)
r.sub.1—longitudinal relaxitivity (mM.sup.−1s.sup.−1)
In a similar way, it is proposed for T2:

[00002]$\begin{array}{cc}\begin{array}{c}{R}_{2\mathrm{obs}}1/T{2}_{obs}\equiv 1/T{2}_t+r_2*C\\ \equiv R_{2t}+r_2.Math.C\end{array}& \left(2\right)\end{array}$

Being:

[0111] R.sub.2obs—speed or relaxation rate observed (s.sup.−1). It is the relaxation rate of the tissue modified by the CA with a concentration C
T2.sub.obs—observed T.sub.1 (ms)
T2.sub.t—T1 of the tissue (ms)
r.sub.2—Transverse relaxivity (mM.sup.−1s.sup.−1)

[0112] The improvement in the intensity of the tissue signal is not only determined by the relaxitivities r.sub.1 and r.sub.2 of the contrast agent. But also by the concentration levels of this in the tissue. In the limit case of high concentrations can lead to signal is saturation and loss of contrast (Elster A D et al in Radiology 1990; 174: 379-381). For this reason, at lower concentrations, better results are obtained.

Concentration Values of the Solutions of the NPs-1A and NPs-3B Nanoparticles Used in Relaxivity Measurements

[0113]

TABLE-US-00007 tubes NPs-1A (mg/mL) tubes NPs-3B (mg/mL) 1 0.012 1 0.09 2 0.024 2 0.075 3 0.048 3 0.060 4 0.072 4 0.0488 5 0.096 5 0.0244 5B 0.120 6 0.0124

Example 8: In Vivo Study. Modification of T1 and T2 Relaxation Times in Brain Tissue with Amyloid Plaques, in the Presence of NPs-1A

[0114] The in vivo study was performed with 5 mice (APPSwe/PS1dE9 transgenic mice, 12 months) and with 3 healthy mice of the same age. Mice were anesthetized (5 mL/kg body weight) with a mixture of 100 mg/mL ketamine and 10 mg/mL xylazine in phosphate buffered saline (PBS). The suspension of NP-1A was diluted with PBS (pH 7.4) at a dose of 5 mg/kg Fe/kg body weight immediately before injection. A total of 100 μL of diluted NP-1A was injected through the tail vein.

[0115] In-vivo measurements were made in the brain of mice to quantify the variations in intensity as a result of the administration of CA. The quantification of contrast variation was carried out according to the equation:

Contrast=100*(Area.sub.Before−Area.sub.After)/Area.sub.Before

Where Area.sub.Before is the intensity of the area before the administration of the CA and Area.sub.After is the intensity of the area after the administration.

[0116] In FIG. 15, images of the animal are observed before and after the injection of CA. These are coronal sections weighted in T2 with a spin echo sequence (TR/TE 4000/80) and a spatial resolution of 180 μm.

[0117] Table 7 shows the in-vivo effect of the application of NPs-1A. Contrast changes were achieved between 17 and 22%. This is considered a good result that corroborates NPs-1A as a negative agent because it decreases the intensity of the image.

[0118] NPs-3B is administered in a similar group and increases in signal intensity of about 25% are obtained. In this way it is corroborated that this new compound is a positive contrast agent.

Example 9: Histological Evaluation of the Detection of Plaques in the Brain Tissue of Mice

[0119] Once the imaging study was completed, the animals were deeply anesthetized and perfused with a solution of 4% paraformaldehyde in 0.01 mol/L PBS pH=7.2. After the mice died, their brains were removed, washed with saline, dried, cut in half and embedded in paraffin. Then, the brains were sectioned into 4 mm thick slices using a microtome. The sections were dewaxed, hydrated in distilled water and treated with 70% formic acid for 30 minutes. The sections were serially stained to locate the β-amyloid deposition. Cuts were treated with 3% H.sub.2O.sub.2 for 30 minutes to eliminate residual peroxidase activity, and rinsed again with 0.01 mol/L PBS (pH=7.2). Sections were incubated overnight at 4° C. with an anti-Aβ1-42 monoclonal antibody (SIGMA, USA) at a 1:1000 dilution. The slides were then rinsed with 0.01 mol/L PBS (pH=7.2) and were first incubated with a secondary antibody (SIGMA, USA) for 30 minutes and, second, with an avidin-biotin complex (SIGMA, USA) for 30 min. At room temperature. For the staining, diaminobenzidine was used for 10 min as a chromogen. The sections were contrasted with Harris's haematoxylin and mounted in aqueous medium. Brain slices from healthy mice were taken as negative controls and received the same treatment. The images were visualized with the Olympus BX51 microscope camera (Japan) (FIG. 15).

[0120] FIG. 16A shows pre-frontal cortex tissue of a healthy mouse C57. No β-amyloid plaques are observed. FIG. 16B shows pre-frontal cortex tissue of transgenic mouse 2×Tg (APP/PS1), at 18 months. β-amyloid plaques are observed.