Magnetic nanoparticles functionalized with carbohydrates

Abstract

The present invention describes metal magnetic nanoparticles, preferably iron oxides, functionalized with specific ligands, preferably belonging to carbohydrates, having high penetrability into solid tumors, in particular high-grade gliomas, in the central nervous system. The invention also relates to a synthesis method and application thereof.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the development of metallic magnetic nanoparticles (MMNPs), particularly iron oxide nanoparticles (IONPs), which have specific ligands functionalized with carbohydrates for biomedical applications such as the diagnosis and treatment of central nervous system (CNS) tumors. Background Technology

[0002] The World Health Organization (WHO) estimated in 2020 that cancer is the second to fifth leading cause of death worldwide. The most common primary tumor of the central nervous system is glioma, which is classified from benign (grade I) to highly aggressive (grade IV). Glioblastoma multiforme (GBM) is a grade IV glioma, accounting for 15% of all primary malignant CNS tumor diagnoses, and has a very poor prognosis, with a median survival of only about 15 months, making it one of the most significant challenges in neuro-oncology.

[0003] The diagnosis of GBM is based on neuroimaging examinations as well as histological and molecular information; that is, on a "comprehensive diagnosis." Magnetic resonance imaging (MRI) is the gold standard technique for evaluating CNS tumors because it provides excellent morphological and functional images in a non-invasive manner. The use of clinical contrast agents (CAs) is often necessary, but these agents have limitations due to their toxicity and low specificity.

[0004] Standard treatment options include maximal safe resection followed by radiotherapy combined with temozolomide and / or temozolomide as adjuvant therapy. Due to the aggressive nature of GBM, progression is expected in all cases, and when deciding on additional treatment, the patient's condition, tumor size and location, and the time elapsed since the first treatment must be considered.

[0005] Therefore, there is an urgent need to improve the specificity of in vivo imaging diagnosis, and more importantly, to improve the efficacy of GBM treatment.

[0006] Nanotechnology is dedicated to the monitoring, evaluation, shaping, control, and fabrication of materials in the size range of 1 to 100 nm, in which unique physicochemical phenomena emerge that enable new applications in the fields of physics, chemistry, biology, materials science, engineering, and health.

[0007] Among nanomaterials, and particularly, metallic magnetic nanoparticles (MMNPs) (iron oxide nanoparticles (IONPs)) developed to improve cancer diagnosis and treatment have attracted particular attention because they can be used for both cancer diagnosis (CA for MRI) and treatment (magnetothermal therapy or drug nanocarriers). In fact, several commercially available IONPs (Sinerem, Combidex, Farahem, Feridex and Endorem) are available on the market [1].

[0008] However, none of them are suitable for the diagnosis / treatment of GBM. This is likely due to the highly restrictive blood-brain barrier (BBB), which prevents many compounds from reaching their targets or from reaching their targets with sufficient efficacy. The blood-brain barrier (BBB) ​​has a complex structure and functional composition that makes it almost impermeable, adding additional challenges to both the diagnosis and treatment of pathological conditions of the CNS, including brain tumors [2].

[0009] In the case of IONP (or any other type of nanoparticle - NP), the enhanced permeation and retention (EPR) effect has long been considered the most efficient mechanism for their accumulation in solid tumors. This is a passive transport mechanism based on the size of the NP, which is theoretically optimal for NPs in the 20 to 100 nm range [3,4].

[0010] To date, this passive transfer mechanism has also relied heavily on NP delivery across the BBB.

[0011] However, as recently demonstrated, the EPR effect is extremely inefficient in brain tumors due to the limitations imposed by the blood-brain tumor barrier (BBTB), particularly in the case of metal NPs[5].

[0012] NanoTherm® (MAGFORCE) developed the first and only nanotechnology-based therapy approved in Europe for the treatment of brain tumors, in which IONPs are delivered directly into the wall of the tumor or resected cavity via stereotactic administration [6]. In other words, they do not use intravenous administration, although such administration is expected to result in a more homogeneous distribution of NPs in the tumor stroma (particularly in areas of high tumor vascularity). Thus, the choice of intratumoral administration is clearly related to the ineffective accumulation of IONPs passively transported via the EPR effect after intravenous administration.

[0013] Glucose transporters (GLUTs) in the BBB maintain the CNS’s persistently high demand for glucose, as it is almost entirely dependent on glucose as an energy source. Some GLUTs (mainly GLUT1) have been found to be significantly overexpressed in GBM patients [7-9], and GLUT1 is particularly significantly overexpressed in the vascular endothelium of GBM tumors

[10] .

[0014] Therefore, in principle, overexpression of GLUT1 in the tumor vasculature (blood vessels, vascular system) enables the transport and accumulation of glucose-derived NPs in the GBM (active tumor targeting).

[0015] Strategies that facilitate the passage of various compounds across the BBB have been studied in the healthy brain: the use of lipid nanoparticles functionalized with mannose derivatives (p-aminophenyl-α-d-mannopyranoside (MAN))

[11] . However, the contribution of mannose functionalization of NPs to their ability to cross the BBB is questionable, as this is likely due to the lipid nature of NPs, since lipophilicity is considered a key factor for better penetration into the brain.

[0016] It should also be considered that in the case of brain tumors, the properties of the BBB are altered, making it impossible to extrapolate the behavior of these NPs when crossing the BBB. Therefore, to date, no NPs have been reported describing any combination capable of crossing the BBB.

[0017] In addition, although GLUT1 is overexpressed in BBTB compared to BBB in healthy patients, its affinity for glucose is significantly greater than its affinity for any other glucose derivative under in vivo conditions

[12] . Therefore, NPs functionalized with glucose derivatives must compete with naturally occurring glucose, which reduces their ability to be transported across BBTB. Summary of the Invention

[0018] This invention describes IONPs functionalized with specific ligands belonging to the carbohydrate family, which have a stronger ability to penetrate solid CNS tumors (particularly high-grade gliomas, especially glioblastoma multiforme (GBM)) thanks to the specific recognition of carbohydrates by specific receptors / transporters (primarily glucose membrane transporter (GLUT)) that enable them to cross the blood-brain tumor barrier (BBTB).

[0019] IONP is functionalized using carbohydrates, preferably glucose derivatives, and more preferably glucuronic acid.

[0020] Since the facilitated transport of glucose and its derivatives through GLUT1 is saturable, a decrease in circulating glucose levels (such as that resulting from a 24-hour fast) reduces competition for glucose with GLUT1, thereby further increasing the transport of NPs functionalized with glucose derivatives (in this case, glucuronic acid) across the vascular endothelium.

[0021] Therefore, in combination with mild hypoglycemia, glucuronide-functionalized IONPs actively target high-grade gliomas across BBTB, and since GLUT1 and GLUT3 are also typically overexpressed in tumor cells (particularly in GBM stem cells (GSCs))

[13] , IONPs functionalized in this way are also suitable for active targeting of GSCs. In other words, this active targeting can reach two targets: tumor vascular endothelium (enabling active transport into the tumor interior); and tumor cells (particularly GSCs).

[0022] Therefore, they can not only cross BBTB, but also be internalized into tumor cells via GLUT1, thus forming the best therapeutic nanoplatform that can reach not only the tumor stroma, but also tumor cells (especially GSCs), which are largely the cause of GBM resistance to chemotherapy and radiotherapy

[14] .

[0023] Once accumulated in a tumor, these functionalized IONPs can be used as therapeutic nanosystems, i.e., for diagnostic purposes (where IONPs act as contrast agents for magnetic resonance imaging (MRI)) and for therapeutic purposes (where IONPs act as mediators for magnetothermal therapy or as platforms for drug transport and delivery). This treatment approach falls under the category of a "see-treat-see" strategy because the therapeutic nanosystems utilize MRI for real-time monitoring.

[0024] Therefore, they are far more effective than other types of NPs for both diagnostic and therapeutic purposes. This ability to cross the vascular endothelial barrier of gliomas and specifically accumulate in the tumor has not been previously achieved by any other known NP or compound.

[0025] Generally, the present invention provides nanoparticles or nanostructures primarily composed of iron / iron oxides or derivatives, having polymeric ligands on their surfaces to enhance their colloidal stability and facilitate subsequent functionalization. Subsequent functionalization with carbohydrates primarily derived from glucose (such as, but not limited to, glucuronic acid) facilitates the specific recognition of certain molecular targets (glucose transporters or GLUTs), enabling successful accumulation of NPs in solid tumors, particularly in high-grade gliomas, which is significantly enhanced by mild hypoglycemia (such as that resulting from a 24-hour fast).

[0026] IONPs or their derivatives have suitable sizes to avoid ferromagnetic behavior that would limit their in vivo applications.

[0027] NP / nanostructures functionalized with carbohydrates of interest in biomedicine can then be used as novel nanoplatforms because they allow for the anchoring of other types of biomolecules, thereby increasing their versatility.

[0028] The NPs covered in this invention have many advantages over existing NPs. Primarily, due to their active targeting, their accumulation in solid tumors or metastatic lesions increases rapidly over a short period, leading to a higher likelihood of achieving therapeutic doses within these tumors.

[0029] This invention also describes a process for preparing novel MRI CAs based on IONPs or their derivatives, wherein polymer-derived organic ligands functionalized with carbohydrates are firmly anchored on their surfaces. The process includes the following steps: 1. Synthesize a magnetic core from a metallic precursor (preferably an iron precursor, and especially iron oleate) by chemical or physical methods (such as thermal decomposition). For thermal decomposition, the key point is the extreme control of the ramp temperature (heating rate).

[0030] 2. Functionalization of the metallic magnetic core with polymer ligands using physical or chemical methods. Polymer ligands capable of stabilizing the metallic magnetic core in an aqueous medium are synthesized by covalent bonding between catechol and the polymer. Preferably, the anchoring molecule is gallic acid (GA), and the polymer is diamino polyethylene glycol NH2-PEG-NH2. As an example, IONP suspended in toluene is combined with GA-PEG... 2000 The -NH2 ligand was incubated in an ultrasonic bath for 1 hour, and then incubated in a temperature-controlled water bath for 4 hours. Preferably, the temperature was in the range of 20-80°C, more preferably between 40 and 60°C.

[0031] 3. Final functionalization with compounds belonging to the carbohydrate family (primarily glucuronic acid, but not limited to) using physical or chemical methods. Final functionalization with carbohydrates enables nanoparticles to specifically recognize cellular receptors / transporters, achieving active targeting.

[0032] In each step, the nanoparticles are purified by dialysis with Milli-Q water or by centrifugation using a suitable filter.

[0033] To test the IONP of this invention, in vivo experiments were conducted using mice as an animal model. MRI was used to analyze pharmacokinetics and biodistribution in vivo. Tumors were orthotopically transplanted into rodents. High-grade glioma cells were directionally transplanted into the right caudate nucleus. Once the tumor reached an appropriate size, the metallic magnetic nanoparticles of this invention were administered intravenously to animals that had been fasting for 24 hours. Dynamic and parametric MRI sequences were used for short-term and long-term monitoring of the injected NP (pharmacokinetics).

[0034] The results show that the IONPs of the present invention exhibit very high stability (over 500 hours), strong internalization ability, no cytotoxicity, and biocompatibility. They also possess excellent ability to remain in the bloodstream and selectively accumulate in tumor tissue, and this accumulation is promoted under mild hypoglycemic conditions. Furthermore, once accumulated in the tumor, the IONPs of the present invention are homogeneously distributed throughout the tumor tissue, which is also crucial for their therapeutic efficacy.

[0035] Therefore, a first aspect of the present invention relates to metallic magnetic nanoparticles, hereinafter referred to as "metallic magnetic nanoparticles of the present invention", which comprise at least one organic ligand attached to their surface, characterized in that the metallic magnetic nanoparticles are functionalized by chemical bonds of at least one molecule belonging to the carbohydrate family.

[0036] In a preferred embodiment, the metallic core is composed of iron or M-iron, wherein M is selected from the list including cobalt, manganese, zinc, nickel, gold, silver, platinum, palladium, and ruthenium, and can be in metallic form or as an oxide, more preferably an iron oxide. Preferably, the metallic core has a size between 1 and 100 nanometers.

[0037] In another preferred embodiment, the organic ligand attached to the surface of the nanoparticles is a catechol derivative, such as L-DOPA, dopamine, caffeic acid, dihydrocaffeic acid, adrenaline, norepinephrine, noradrenaline, adrenaline, protocatechuic acid, and gallic acid, preferably gallic acid, covalently bonded to a polymer selected from the list including polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, chitosan, and derivatives thereof. Preferably, the catechol derivative is gallic acid, and the polymer is polyethylene glycol or a derivative thereof, and more preferably, bis(amino) polyethylene glycol. More preferably, the polyethylene glycol has a molecular weight in the range of 200-6000 kDa, and even more preferably between 1000 and 3000 kDa.

[0038] In another preferred embodiment, the carbohydrate is selected from the list including allose, arbutin, glucose, gulose, mannose, idole, galactose, trehalose, and derivatives thereof. Preferably, the carbohydrate is a hexose, more preferably glucose or a derivative thereof, and most preferably glucuronic acid.

[0039] In a particularly preferred embodiment of the invention, the metallic magnetic nanoparticles have a metal core composed of iron oxides, the metallic magnetic nanoparticles include at least one organic complex fixed to their surface, the organic complex being derived from the covalent bonding of catechol (particularly gallic acid) and a polyethylene glycol derivative (preferably bisamino polyethylene glycol), and are characterized in that the metallic magnetic nanoparticles are functionalized with a glucose derivative (preferably glucuronic acid).

[0040] Preferably, the metallic magnetic nanoparticles of the present invention are suitable for administration via intratumoral, intranasal, intraperitoneal, intravenous, intra-arterial, anal, or oral routes.

[0041] A second aspect of the present invention relates to a method for synthesizing the metallic magnetic nanoparticles of the present invention, hereinafter referred to as "the synthesis method of the present invention," which includes the following steps: a) Synthesizing a metal nucleus from at least one metal precursor selected from the list of evaporation, condensation, laser ablation, hydrothermal method, chemical reduction, irradiation, coprecipitation, electrochemistry, microemulsion, thermal decomposition and photoreduction using a chemical or physical method selected from the list of evaporation, condensation, laser ablation, hydrothermal method, chemical reduction, irradiation, coprecipitation, electrochemistry, microemulsion, thermal decomposition and photoreduction.

[0042] b) Functionalization with polymer ligands by physical or chemical methods, wherein the polymer ligands are synthesized by covalent bonding between catechol derivatives selected from the list including gallic acid and polymers selected from the list including polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, chitosan or derivatives thereof.

[0043] c) Functionalizing with compounds belonging to a carbohydrate family selected from a list including allose, arbutin, glucose, gulose, mannose, idole, galactose, trehalose, or derivatives thereof (such as glucuronic acid).

[0044] In a preferred embodiment of the synthesis method of the present invention, the metal core is synthesized by thermal decomposition. In a more preferred embodiment, the decomposition is carried out at a high temperature (preferably in the range of 180-350°C) and in an inert atmosphere in an organic solvent. Preferably, the solvent used for the reaction is an organic compound, more preferably 1-octadecene. In an even more preferred embodiment, a stabilizer is used for the reaction, and preferably, these stabilizers are selected from oleic acid, oleyl alcohol, oleylamine, and 1,2-hexadecylthiol and mixtures thereof.

[0045] In another preferred embodiment, the metal core is synthesized from more than one metal precursor. In another preferred embodiment, the metal precursor is ferric oleate.

[0046] In a preferred embodiment, the first functionalization is performed using the polymer ligand GA-PEG-NH2.

[0047] In another preferred embodiment, the functionalization is carried out in two steps: first, by applying ultrasound, and second, by raising the reaction temperature.

[0048] In a preferred embodiment, a second functionalization is performed using glucuronic acid.

[0049] In another preferred embodiment, the linking to the carbohydrate uses carbodiimide as a coupling agent, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of N-hydroxysuccinimide.

[0050] Preferably, in each step of the synthesis method of the present invention, the nanoparticles are purified by dialysis with Milli-Q water or by centrifugation using a suitable filter.

[0051] A third aspect of the invention relates to the use of the metallic magnetic nanoparticles of the invention in methods for diagnosing, evaluating, imaging, or treating solid tumors or metastatic lesions (preferably solid tumors or metastatic lesions of the central nervous system).

[0052] In another embodiment of this aspect of the invention, the use of the metallic magnetic nanoparticles of the invention as a contrast agent for magnetic resonance imaging, preferably for evaluating solid tumors or metastatic lesions, and more preferably for tumors of the central nervous system is described.

[0053] In another embodiment of this aspect of the invention, the use of the metallic magnetic nanoparticles of the invention as a drug delivery platform, preferably for targeted therapy of drugs for solid tumors or metastatic lesions, and more preferably for tumors of the central nervous system is described.

[0054] In another embodiment of this aspect of the invention, the use of the metallic magnetic nanoparticles of the invention as a magnetothermal therapy medium is described, preferably for the treatment of solid tumors or metastatic lesions, and more preferably for the treatment of tumors of the central nervous system.

[0055] In another embodiment of this aspect of the invention, the use of the metallic magnetic nanoparticles of the invention via intratumoral, intranasal, peritoneal, intravenous, intraarterial, anal, or oral administration is described.

[0056] In another embodiment of this aspect of the invention, the use of the metallic magnetic nanoparticles of the invention in patients with mild hypoglycemia or near the lower limit of normal pre-meal blood glucose is described.

[0057] Preferably, the state arises from fasting or from prior administration of a compound selected from the list including: insulin, biguanides, sulfonylureas, glitinides, dipeptidyl peptidase IV (DPP-IV) inhibitors, sodium-glucose cotransporter-2 (SGLT2) inhibitors, glucagon-like peptide-1 (GLP-1) agonists, alpha-glucosidase inhibitors (AGIs), thiazolidinediones, or (SGLT2) inhibitors, glucagon-like peptide-1 (GLP-1) agonists, alpha-glucosidase inhibitors (AGIs), thiazolidinediones, or related compounds. Attached Figure Description

[0058] Figure 1 a) Schematic diagrams of different functionalization steps, showing the chemical structures of the various PEGylated ligands and glucuronic acid used. The size and shape of the IONPs throughout the process were evaluated by electron microscopy (representative images are shown). In all cases, the scale corresponds to 50 nm. b) Different functionalization steps were evaluated by Fourier transform infrared (FTIR). c) The hydrodynamic diameter of the IONPs with different functionalizations was measured over time.

[0059] Figure 2 For NPs@OH (a) and NPs@Gluc (b), the slope of the fitted line of R2 with respect to iron concentration is calculated as transverse relaxation (r2), which is performed under different magnetic fields: 1.44 T (blue) and 9.4 T (black). The high-field T2-weighted images corresponding to each concentration are shown at the right edge.

[0060] Figure 3 Representative optical microscopic images of HFF-1 fibroblasts obtained by combining bright-field (grey), Hoechst 33342 (blue), and TOPRO-3 iodine (red) images: (a) negative control, (b) positive control, (c) cells exposed to 100 μg / mL NPs@OH NP. Scale bar corresponds to 50 μm. (d) Total number of cells per well after exposure to increasing concentrations of NPs@OH NP. (e) Percentage of dead cells after exposure to increasing concentrations of NPs@OH NP. (f) MTT assay of cells exposed to increasing concentrations of NPs@OH NP.

[0061] Figure 4 Representative optical microscopic images of HFF-1 fibroblasts obtained by fusion of bright field (grey), Hoechst 33342 (blue), and TOPRO-3 iodine (red): (a) negative control, (b) positive control, (c) cells exposed to 100 μg / mL NPs@Gluc NP. Scale bar corresponds to 50 μm. (d) Total number of cells per well after exposure to increasing concentrations of NPs@Gluc NP. (e) Percentage of dead cells after exposure to increasing concentrations of NPs@Gluc NP. (f) MTT assay of cells exposed to increasing concentrations of NPs@Gluc NP.

[0062] Figure 5 Representative optical images of HFF1, C6, u87, and 4T1 cells exposed to NPs@OH (left) and NPs@Gluc (right) (both at an iron concentration of 100 μg / mL) for 2 and 4 hours, respectively, with Prussian blue staining of both types of NPs. Cellular uptake was observed only in NPs functionalized with glucuronide.

[0063] Figure 6 a) Western blot analysis of expression of specific glucose transporters (GLUT1 and GLUT3) in C6 cells with MW-molecular markers. Glucose competition assay: b) Representative optical images of C6 cells exposed to different glucose concentrations and a constant concentration of 100 μg / mL NPs@Gluc, stained with Prussian blue; and c) Quantitative assessment by inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS).

[0064] Figure 7 a) Short-term pharmacokinetics obtained by dynamic MRI during the first 30 minutes following intravenous administration of NPs@OH (G1), NPs@Gluc (G2), and NPs@Gluc (G3) in mice that had undergone overnight fasting, across the entire tumor (red) and at the tumor margin (blue). b) T2-weighted MR images from different experimental groups at 1 h and 2 h after NP administration, and corresponding quantitative analyses by calculating the change in relaxation rate (ΔR2) over time across the entire tumor (c) and at the tumor margin (d).

[0065] Figure 8 a) Representative electron micrographs of blood 1 hour after injection of NPs@OH (top) and NPs@Gluc (bottom); b) Changes in relaxation rate (ΔR2) of plasma samples 1 hour after intravenous injection of NPs@OH (grey) and NPs@Gluc (blue), corrected for normal plasma values.

[0066] Figure 9 Representative tissue sections of liver and tumor stained with Prussian blue 2 hours after injection of PBS (a), NPs@Oh (b), and NPs@Gluc. Scale bar corresponds to 50 μm. Detailed Implementation

[0067] The synthesis of iron oxide nanoparticles (IONP), functionalization with PEGylated ligands, and final functionalization with glucose derivatives.

[0068] Synthesis of ferric oleate (iron precursor)

[0069] A mixture of 10.8 g ferric chloride (40 mmol) and 36.5 g sodium oleate (120 mmol) was dissolved in 80 mL ethanol, 60 mL distilled water, and 140 mL hexane. The resulting solution was heated to 70°C and placed under an inert atmosphere for 4 hours, allowing the hexane to reflux. The reaction was then cooled to room temperature, and two phases were distinguished: a lower aqueous phase and an upper organic phase containing ferric oleate. The organic phase was washed three times with distilled water, and the hexane was evaporated in a rotary evaporator.

[0070] Synthesizing IONP via thermal decomposition

[0071] Weigh 1.81 g (5.3 mmol) of ferric oleate and 0.32 g (1.133 mmol) of oleic acid into a 50 mL double-necked round-bottom flask. Use 15 mL of 1-octadecene as the solvent. Preheat the reaction to 200°C under an inert atmosphere (N2) and with magnetic stirring. Increase the temperature to 320°C using a ramp temperature of 1°C / min. Maintain the temperature at 320°C for 1 h and then allow it to cool to room temperature. Wash the NP suspension three times with a mixture of acetone and ethanol (1:1), centrifuge at 8000 rpm for 20 min to remove unwanted reagents, and finally resuspend it in toluene. Store the nanoparticle suspension at 4°C.

[0072] Synthesis of PEGylated ligands

[0073] Synthesis of GA-PEG-OH with the following formula:

[0074] In summary, under a nitrogen atmosphere in a round-bottom flask, polyethylene glycol (molecular weight: 3000 g / mol, 1 mmol, 3.0 g), gallic acid (molecular weight: 170 g / mol, 1 mmol, 170 mg), and 4-(dimethylamino)pyridine (molecular weight: 122 g / mol, 200 μmol, 24 mg) were dissolved in 100 mL of tetrahydrofuran and 10 mL of dichloromethane. A tetrahydrofuran solution of dicyclohexylcarbodiimide (molecular weight: 206 g / mol, 5 mmol, 1 g) was added dropwise to the mixture. The mixture was stirred overnight at room temperature. The reaction mixture was filtered through filter paper, and the solvent was evaporated by rotation.

[0075] Synthesis of GA-PEG-NH2 with the following formula:

[0076] In summary, under a nitrogen atmosphere in a round-bottom flask, α,ω-diamino polyethylene glycol (Mw: 2000 g / mol, 1 mmol, 2.0 g), gallic acid (Mw: 170 g / mol, 1 mmol, 170 mg), and 4-(dimethylamino)pyridine (Mw: 122 g / mol, 200 μmol, 24 mg) were dissolved in 100 mL of tetrahydrofuran and 10 mL of dichloromethane. Dicyclohexylcarbodiimide (Mw: 206 g / mol, 5 mmol, 1 g) was added dropwise to the tetrahydrofuran solution. The mixture was stirred overnight at room temperature. The reaction mixture was filtered through filter paper, and the solvent was evaporated by rotation.

[0077] Ligand exchange process.

[0078] In summary, a solution containing 1.0 mL NP (10 g / L Fe), 1.0 mL GA-PEG-OH ligand (0.1 M in CHCl3), and 50 μL triethylamine was added to a glass vial. The mixture was sonicated for 1 h and maintained at 55°C for 4 h. At this point, the mixture was diluted with 5 mL toluene, 5 mL milli-Q water, and 10 mL acetone. The mixture was then shaken to transfer the functionalized NP to the aqueous phase. The aqueous phase was collected in a round-bottom flask, and the residual organic solvent was evaporated by rotary evaporation. The PEGylated NP was then purified at 450 rcf using a centrifugal filter with a molecular weight cutoff of 100 kDa. After each centrifugation, the functionalized NP was resuspended in milli-Q water. The purification steps were repeated several times until the filtered solution was clear. After purification, the PEGylated NP was resuspended in PBS buffer. Finally, to ensure the high stability of the monodisperse NPs, the solution was centrifuged at 150 rcf for 5 min and placed on a permanent magnet (0.6 T) for 5 minutes. These nanoparticles will be referred to as NPs@OH below.

[0079] Functionalization with GA-PEG-NH2 was carried out in a similar manner.

[0080] These nanoparticles will be referred to as NPs@NH2 in the following text.

[0081] Covalent reaction with carbohydrates

[0082] In a round-bottom flask, 1 mL of NPs@NH2 (0.6–0.7 mg / mL iron), 0.192 mg (1.67 μmol) NHS (N-hydroxysuccinimide), 0.192 mg (1.24 μmol) EDC (1-ethyl-3-3-dimethylaminopropylcarbodiimide), and 0.162 mg (0.834 μmol) glucuronic acid were added. The reaction was carried out on ice. After 2 hours of reaction, the mixture was purified by centrifugation at 450 rcf using a 100 kDa molecular weight cutoff filter to remove unreacted reagents. These nanoparticles will be referred to as NPs@GLUC below.

[0083] NPs were characterized by inductively coupled plasma high-resolution mass spectrometry (ICPHRMS).

[0084] The Fe concentration was determined using a NexION ICP-HRMS (Perkin-Elmer, Waltham, MA, USA). Briefly, 2.5 mL of aqua regia was added to 25 μL of the nanoparticle solution in a volumetric flask. The mixture was allowed to stand overnight. Then, Milli-Q water was added to bring the total volume to 25 mL.

[0085] NPs were characterized by transmission electron microscopy (TEM).

[0086] TEM images were obtained using a FEI Tecnai G2 Twin microscope operated at an accelerating voltage of 100 kV. TEM samples were prepared by dropping a solution of nanoparticles equivalent to approximately 1 g / L (Fe) onto a carbon-coated copper grid and allowing the solvent to evaporate. Diameters were calculated based on the average of 100 measured nanoparticles.

[0087] NPs were characterized by Fourier transform infrared spectroscopy (FTIR).

[0088] FTIR spectra were recorded using an FTIR-4100 Jasco with a single-reflection ATR accessory (MIRacle ATR, PIKE Technologies) coupled to a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector. The range was from 4000 to 800 cm⁻¹. -1 Record all spectra within the range, with a resolution of 4 cm⁻¹. -1 A total of 200 scans were performed. All samples were recorded as solid products.

[0089] NPs were characterized by dynamic light scattering (DLS).

[0090] The particle size distribution of PEGylated IONP was measured using a Zetasizer Nano ZS90 (Malvern, USA). Nanoparticles were dispersed in milli-Q water or PBS at a concentration of 100 mg / L Fe. Measurements were performed on a ZEN0118 low-capacity disposable particle size cuvette (refractive index set to 2,420, detection angle set to 90°). The measurement duration was set to automatic, and the number of measurements was set to three. A general-purpose analytical model (normal resolution) was selected.

[0091] NP is characterized by the transverse relaxation rate (r2).

[0092] Under physiological conditions at 37°C, magnetic NPs in PBS solutions with iron concentrations ranging from 0.125 to 2 mM were used for proton nuclear magnetic resonance (NMR) studies in low (1.44 T) and high (9.4 T) magnetic fields. 1Transverse relaxation time (T2) was measured by 1H NMR. T2 measurements were performed at low magnetic fields using a Carl-Purcell-Meiboom-Gill (CPMG) sequence on a Bruker Minispec system (Bruker BioSpin, Rheinstetten, Germany). At high fields, T2 values ​​were measured at 298 K on a Bruker Biospec MRI system (Bruker Biospec, Bruker BioSpin, Ettlingen, Germany), equipped with a 400 mT... m -1 Field gradient and a 40 mm orthogonal "birdcage" resonator. T2 values ​​(TE values ​​from 7.5 ms to 640 ms) were measured using a 64-echo Carl-Purcell-Meiboom-Gill (CPMG) imaging sequence. The transverse relaxation rate r2 in the two magnetic fields was calculated based on the slope of a linear fit between the relaxation rate (1 / T2) and the Fe concentration, as indicated by the following equation:

[0093] Draw a region of interest (ROI) on the first image of the image sequence, extract the intensity values, and fit them to the following equation:

[0094] Where M z and M xy M0 is the signal strength at TR or TE time, and M0 is the signal strength at equilibrium.

[0095] In vitro testing.

[0096] Various tests were performed to comprehensively analyze the cytotoxicity of IONP.

[0097] A) Cell culture.

[0098] The HFF-1 (human fibroblast) cell line was selected as a working model and used as a representative control of healthy cells. C6 (rat glioblastoma), u87 (human glioblastoma), and 4T1 (mouse breast cancer) cell lines were also selected. Standard culture conditions were used in all cases, including three different media: Duchenne Modified Eagle Medium (DMEM) for HFF-1 and C6 cells; Los Vickers Memorial Institute (RPMI) for 4T1 cells; and DMEM / F12 for u87 cells. All media were supplemented with 5% serum, penicillin / streptomycin, and L-glutamine. Cells were maintained at 37°C in an atmosphere containing 5% CO2. Cells were passaged every 2 days using trypsin.

[0099] B) Assess cellular uptake by Prussian blue staining.

[0100] At 37°C under a 5% CO2 atmosphere, 3×10 5 Cells were seeded in 24-well plates. After 24 hours, the medium was replaced with fresh medium containing PEGylated IONP. The cells were incubated again for 2 h and 4 h. After incubation with NP, the cells were washed three times with PBS to remove free NP. Subsequently, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 40 min. Then, the cells were washed three times with PBS and incubated with freshly prepared Prussian blue (4% potassium ferrocyanide [Sigma-Aldrich] / 12% HCl, 1:1, v / v) for 30 min. The cells were washed three times with PBS, stained with Sigma-Aldrich nuclear fast red, and observed using an inverted optical microscope.

[0101] C) Protein blotting.

[0102] First, protein extracts were prepared by cell lysis. Cells were cultured to confluence for this purpose. The cells were then washed three times with PBS at 4°C with lysis buffer added. The cell suspension was subsequently transferred and centrifuged in pre-cooled centrifuge tubes. Proteins remained in the supernatant. Once the extracts were obtained, the proteins were separated according to their molecular weight by gel electrophoresis. The gel was transferred to a membrane for immunoassay. Anti-GLUT1 and anti-GLUT3 antibodies were used for this work.

[0103] D) Quantify cellular uptake using inductively coupled plasma high-resolution mass spectrometry (ICP-MS).

[0104] Similarly, as mentioned above, at 37°C in an atmosphere with 5% CO2, 1×10 5 Four T1 cells were seeded in 96-well plates. After 24 h, the medium was replaced with fresh medium containing PEGylated IONP. The cells were incubated again for 2 h and 4 h. After incubation with NP, the cells were washed three times with PBS to remove any free NP. At this point, 100 μl of aqua regia was added to each well. The mixture was left to stand overnight. Milli-Q water was added to bring the total volume to 10 mL. Iron concentration was measured on a NexION ICP-HRMS (Perkin-Elmer, Waltham, MA, USA).

[0105] E) Glucose competition detection.

[0106] To demonstrate that NPs@Gluc is expected to be transported via glucose transporter (GLUT), we performed a glucose competition assay. For this assay, C6 cells were grown to confluence and incubated with a constant concentration of NPs@Gluc [100 μg / mL] while the glucose concentration was increased. After 2 hours, the cells were stained with Prussian blue as described in part B). Furthermore, quantitative analysis by ICP-MS was performed as described in part D.

[0107] F) High-content screening (HCS).

[0108] At 37°C under a 5% CO2 atmosphere, at a rate of 1×10 4 HFF-1 cells were seeded in 96-well plates at a density of 100 μL per well (replication = 5). After 24 h of culture, the medium in the wells was replaced with fresh medium containing different concentrations of NP from 0.1 to 100 μg / mL (Fe). After 24 h, Triton X-100 was added to the positive control wells. After 15 min, all wells were stained with: 1) DAPI (4',6-diamidinyl-2-phenylindole, 1:3000 dilution in PBS) for labeling cell nuclei, but with stronger labeling in live cells; 2) calcein (1:1000 in PBS) for assessing cell viability; and 3) TO-PRO-3 iodine for labeling dead cells (1:1000 dilution in PBS). Cell morphology was imaged using a Perkin Elmer Operetta high-content imaging system with a 20 × LWD 0.45NA air objective. Five replicate wells were analyzed for each case, with 10 random image fields captured in each well. For each field, in addition to bright-field images, fluorescence images of DAPI, calcein-AM, and TO-PRO-3 were also captured. The percentage of cell death was automatically calculated using Operetta Harmony software, which identifies all cell nuclei (live and dead) based on DAPI staining and determines the percentage of dead cells based on the number of nuclei showing high levels of TO-PRO-3 staining. Intracellular esterase activity was assessed by calcein-AM staining.

[0109] G) MTT testing.

[0110] In short, at 37°C in a 5% CO2 atmosphere, at a rate of 1×10 4HFF-1 cells were seeded in 96-well plates at a density of 200 μL per well (replication = 5). After 24 h of culture, the medium in each well was replaced with fresh medium containing PEGylated IONP at concentrations ranging from 0.1 μg / mL to 100 μg / mL. After 24 h, Triton X-100 was added to the positive control wells, and after 15 min, the supernatant in each well was replaced with 200 μL of fresh medium containing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (0.5 mg / mL). After incubation at 37°C and 5% CO2 for 2 h, the medium was removed, and formazan crystals were dissolved in 200 μL of DMSO. The solution was vigorously mixed to dissolve the reacted dye. The absorbance of each well was read at 550 nm using a microplate reader (Dynatech MR7000 instrument). [Abs] 孔 The relative cell viability (%) and its error were calculated using the following equation compared to the control wells containing cell culture medium without nanoparticles: RCV (%)=

[0111] error (%)= RCV 测试 ×

[0112] Where σ is the standard deviation.

[0113] In vivo testing.

[0114] In the orthotopic GBM model (mouse model), pharmacokinetics and biodistribution of intravenously PEGylated IONP were determined using dynamic and parametric MRI.

[0115] animal.

[0116] Six-week-old male Balb / c mice (20 g weight) supplied by Charles River (n = 4 per experimental group) were used for in vivo experiments. These experiments were conducted in accordance with the ethical guidelines of the local ethics committee and also in compliance with national (RD 53 / 2013) and European (2010 / 62 / EU) regulations.

[0117] Tumor transplantation.

[0118] To achieve 70-80% confluence of C6 cells, they were then subjected to trypsinization and cell counting. 10 cells were suspended in 5 μL of culture medium... 5Cells were inoculated into the right caudate nucleus of mice. Tumors were then allowed to grow until they reached 0.16 to 0.2 mm. 3 The volume. At this point, the mice were randomly divided into three groups: G1. Intravenous administration of NPs@OH.

[0119] G2. Intravenous administration of NPs@Gluc.

[0120] G3. Mice were fasted overnight and intravenously administered NPs@Gluc.

[0121] Evaluation was performed using magnetic resonance imaging (MRI).

[0122] MRI experiments were performed on a 9.4T Bruker Biospec system equipped with a 400 mT / m gradient and a mouse brain surface antenna for signal reception, as well as a volumetric coil for transmission. ParaVision 6.0 software (Bruker Medical GmbH®) was used on the Linux platform. Animals were anesthetized with 1% isoflurane, cannulated in the tail vein, and placed in a spectrometer, where respiration and body temperature were monitored throughout the MRI experiments using a small animal monitoring system (PC-SAM 32 v8.02 software). PEGylated IONP was administered intravenously via the tail vein at a concentration of 10 mg (Fe) / kg.

[0123] Pharmacokinetic and biodistribution studies were conducted using the following protocols: 1. Obtain a high-resolution T2-weighted image (0 h). 2. Obtain the magnetic susceptibility weighted image (0 h). 3. Obtain T2 parametric images (quantitative) (0 h). 4. Intravenous injection of PEGylated IONP, 5. Obtain the dynamic sequence (pharmacokinetics) of the T2-weighted image. 6. Acquire high-resolution T2-weighted images (30 min). 7. Acquire magnetic susceptibility-weighted images (30 min). 8. Acquire parametric T2 (quantitative) images (30 min). 9. Acquire high-resolution T2-weighted images (1 h). 10. Acquire magnetic susceptibility-weighted images (1 h). 11. Acquire parametric T2 (quantitative) images (1h). 12. Obtain high-resolution T2-weighted images (2 h). 13. Acquire magnetic susceptibility-weighted images (2 h). 14. Obtain parametric T2 (quantitative) images (2 h).

[0124] Magnetically susceptibility-weighted images were obtained using a FLASH sequence (TE = 16 ms, TR = 350 ms, 4 averages, 1 replicate, flip angle = 40°, FOV = 1 cm, matrix size = 512×256).

[0125] High-resolution T2-weighted images were acquired using a turbo RARE sequence with respiratory synchronization (TE = 27 ms, TR = 1000 ms, 4-fold average, 156 μm in-plane resolution, and 1 mm slice thickness).

[0126] Quantitative T2 measurements were also performed using multi-echo spin-echo CPMG sequences (TE = 7 ms to 448 ms, TR = 3500 ms, FOV = 4 cm, matrix size = 128 × 128, slice thickness = 1 mm). For short-term pharmacokinetic analysis based on T2 images, turbo-RARE sequences with the same parameters as above were used, but with only one averaging to improve temporal resolution (one image per 30 sec).

[0127] Semi-quantitative analysis of short-term pharmacokinetic data acquired during the first 30 minutes was performed using the following expression:

[0128] Where RE is the modulus of relative signal enhancement, and I t I0 is the signal intensity at a given time after nanoparticle injection, and I0 is the signal intensity before injection.

[0129] Long-term pharmacokinetics were measured at 0 h, 0.5 h, 1 h, and 2 h using parametric (quantitative) T2 imaging. Pharmacokinetics were obtained by calculating the mean values ​​within different regions of interest (ROIs) in the following tissues: the entire tumor and the tumor margin.

[0130] In vitro testing.

[0131] Analysis of blood circulation time.

[0132] One hour after IONP injection, mice were euthanized and blood was collected for further analysis. The shape and size of the PEGylated IONP were determined using transmission electron microscopy (TEM), and... 1 The transverse relaxation rate (R2) of plasma was measured by 1H NMR relaxation measurement.

[0133] Histology.

[0134] Tissues were fixed in 4% formaldehyde (Panreac, buffered to pH 7) for 48 h, with the formaldehyde being replaced every 24 h. The samples were then dehydrated using graded ethanol and embedded in paraffin (at 56°C for 2 h under stirring and vacuum). Detailed procedures are described below.

[0135] Hematoxylin and eosin (H&E): Paraffin-embedded samples were sectioned to a thickness of 7 μm, dewaxed, rehydrated, stained with H&E, dehydrated in ethanol of increasing concentration, cleared in xylene, and mounted on commercially available slides.

[0136] Prussian blue (PB): Paraffin-embedded samples are sliced ​​to a thickness of 7 μm, then dewaxed, rehydrated, immersed in 20% hydrochloric acid and 10% potassium ferrocyanide, washed with water, counterstained with "Nuclear Fast Red", dehydrated in ethanol of increasing concentration, cleared with xylene, and mounted on commercially available glass slides.

[0137] result.

[0138] In this work, IONPs were functionalized with two different PEGylated ligands (OH and NH2-terminated). In subsequent chemical reactions, NPs@NH2 were covalently bonded to glucuronic acid via an amide reaction. Neither chemical reaction affected the metal core.

[0139] Since different functionalization steps can affect the metal cores of IONPs, altering their size and / or shape, and thus their magnetic properties, detailed TEM analysis is necessary to ensure their stability. Figure 1 a). Generally, although some aggregation occurs in the case of NPs@NH2, the shape and size of IONPs are not affected by different synthetic steps. It should be noted that no aggregation is observed after covalent linkage with glucuronic acid. Furthermore, the presence of PEGylated ligands was confirmed by FTIR for both NPs@OH and NPs@NH2. Figure 1 (b) indicates a favorable ligand exchange process. The covalent bond between NPs@NH2 and glucuronic acid was also satisfactorily confirmed by FTIR. Furthermore, functionalization with PEGylated ligands should increase colloidal stability in aqueous media. However, NPs@NH2 exhibited very low colloidal stability, with visible precipitation after 48 hours. Figure 1 c). In contrast, both NPs@OH and NPs@Gluc exhibited high colloidal stability even after 500 hours.

[0140] The ability of these three IONPs as CAs for MRI was evaluated in two different magnetic fields: 1.44 T and 9.4 T. Figure 2 Both NPs@OH and NPs@Gluc exhibited excellent performance as CAs for MRI. Due to the low stability of NPS@NH2, their r2 values ​​could not be accurately determined.

[0141] Regarding cytotoxicity, NPs@OH ( Figure 3 ) or NPs@Gluc ( Figure 4 None of them showed cytotoxic effects on HFF-1 cells. For example, as observed by optical microscopy (… Figure 3 a and Figure 4 a) Total number of cells ( Figure 3 d and Figure 4 d) Percentage of dead cells ( Figure 3 e and Figure 4 e) or mitochondrial activity ( Figure 3 f and Figure 4 f) As observed, no significant changes in cell morphology were observed, confirming the absence of cytotoxicity.

[0142] The targeting and internalization capabilities of glucuronide-functionalized NPs were evaluated in healthy (HFF-1) and tumor (C6, u87, and 4T1) cells. Cells were exposed to NPs@OH or NPs@Glucose (… Figure 5 NPs@OH showed very little internalization in all cases, only observed in C6 cells after 4 h of incubation. In contrast, NPs@Gluc showed high internalization primarily in C6 and 4T1 cells after 2 h of exposure, and moderately low internalization in HFF-1 and u87 cells. Particularly for C6 cells, NPs@Gluc internalization increased after 4 h of exposure.

[0143] We investigated whether the internalization of NPs@Gluc is mediated through specific recognition and interaction with glucose transporters (GLUTs), for which we selected C6 cells. First, Western blot analysis confirmed the presence of GLUT1 and GLUT3 (which are frequently found to be overexpressed in various tumor cells), showing high expression of both GLUTs, with GLUT3 being of higher quality as expected. Figure 6 a). Once the presence of GLUTs was confirmed, glucose competition assays were performed to demonstrate their involvement in the transport of NPs@Gluc across the plasma membrane. Glucose addition inhibited NPs@Gluc entry into C6 cells in a dose-dependent manner. Figure 6 b- Figure 6 c), thus clearly demonstrating that this process is mediated by GLUT.

[0144] These data demonstrate the biocompatibility of IONP in vitro, as the results of these assays did not show statistically significant changes compared to the control group. Furthermore, glucose competition assays have demonstrated that NPs@Gluc facilitates glucose transport through specific recognition by glucose transporters in different cell lines.

[0145] However, in vivo tumor cell targeting requires prior tumor targeting, i.e., the ability of NPs to selectively accumulate in tumor tissues, which necessitates their passive or active crossing of the tumor vascular system to reach the stroma. Passive tumor targeting is particularly difficult in brain tumors because the blood-brain barrier is a major limiting factor. Therefore, an alternative strategy is active targeting. Given that GLUT1 is overexpressed in the vascular system of GBM

[15] , it is proposed to administer NPs@Gluc to GBM via GLUT-mediated active targeting. Therefore, the pharmacokinetics of NPs@Gluc and NPs@OH were monitored in vivo using MRI.

[0146] Short-term pharmacokinetics (SAP) following administration of PEGylated IONP in groups G1 (9%) and G2 (11%) Figure 7 a) A slight increase in signal was observed, which remained constant from the 10th minute until the end of the test. In contrast, in the case of G3 (24-hour fasting), the signal increased sharply (approximately 40%), indicating significant extravasation of NPs@Gluc into the tumor stroma. Therefore, mild hypoglycemia significantly increased the amount of NPs@Gluc reaching the tumor site. These data are consistent with in vitro glucose competition assays, which showed much higher internalization of NPs@Gluc at lower glucose concentrations. In other words, lowering blood glucose levels (by fasting or by using a hypoglycemic agent) promoted efficient active transport of NPs@Gluc into the tumor via GLUT. It should be noted that the increase in signal was observed both throughout the tumor and at the tumor margin. As mentioned for G1 and G2, the signal remained constant after 10 minutes.

[0147] Regarding long-term pharmacokinetics (qualitatively assessed using magnetic susceptibility-weighted imaging), no significant changes were observed over time in tumors of group G1, while group G2 showed slight darkening 1 hour after NP injection. Conversely, very pronounced darkening was detected in group G3 at this time. Tumors in both groups G2 and G3 maintained similar darkening 2 hours after injection. In summary, the 24-hour fasting period led to decreased glucose concentrations, resulting in a sharp increase in NPS@Gluc accumulation within the tumor. These results were also evaluated using quantitative MRI (T2 plot). In group G3, a statistically significant increase in R2 value was observed approximately 2 s⁻¹ 1 hour after NP injection. -1 and 5 s -1 (For the entire tumor and the tumor margin respectively) Figure 7 c- Figure 7 d). Two hours after NP injection, these values ​​were similar. It should be noted that in any case, no change in R2 was observed in the contralateral part of the brain, indicating selective accumulation in the tumor.

[0148] To enhance tumor targeting, NPs must have a moderately long residence time in the blood. Therefore, blood was analyzed using TEM and TD-NMR one hour after NP injection. Figure 8 Large amounts of NPs@OH and NPs@Gluc continued to circulate in the bloodstream, showing a relative increase in R2 in all cases (7 s). -1 up to 10 s -1 This demonstrates that these PEGylated NPs have a high degree of stealth properties in the animal immune system.

[0149] In addition, to assess the accumulation of NP in tissues, tissue sections of major organs were stained with Prussian blue. Figure 9 At the end of the experiment (2 hours after NP injection), no signs of NP accumulation were observed in the tumors or livers of control mice (injected with PBS). On the other hand, Kupffer cells from mice injected with both NPs@OH and NPs@Gluc showed significant NP accumulation. Regarding tumors, mice injected with NPs@OH showed low accumulation, while those injected with NPs@Gluc showed significantly higher levels. Furthermore, NPs@Gluc exhibited a more homogeneous distribution throughout the tumor tissue, which is crucial for the effectiveness of tumor treatment.

[0150] Based on the above, it can be concluded that NPs@Gluc possesses a surprising tumor-targeting ability in vivo. In particular, NPs@Gluc is able to overcome the limitations imposed by BBTB and effectively reach and accumulate in high-grade gliomas. Mild fasting-induced hypoglycemia significantly enhances this ability, suggesting that the observed vascular translocation of NPs@Gluc is likely mediated by GLUT1, which is known to be overexpressed in the vascular system of these tumors.

[0151] References

Claims

1. A metallic magnetic nanoparticle, wherein the metal core of the metallic magnetic nanoparticle is composed of iron oxide, and the metallic magnetic nanoparticle includes at least one organic ligand attached to its surface, said organic ligand being composed of gallic acid bonded to a polyethylene glycol derivative, characterized in that... The metallic magnetic nanoparticles are functionalized with at least one glucose derivative molecule.

2. The metallic magnetic nanoparticles according to the preceding claim, characterized in that, The glucose derivative is glucuronic acid.

3. The metallic magnetic nanoparticles according to the preceding claim, characterized in that, The polyethylene glycol derivative is bisamino polyethylene glycol.

4. A method for synthesizing metallic magnetic nanoparticles according to any one of the preceding claims, characterized in that, The method includes the following steps: a) The metal core is synthesized from iron oleate or its derivatives using a chemical or physical method selected from the following list: evaporation, condensation, laser ablation, hydrothermal method, chemical reduction, irradiation, coprecipitation, electrochemistry, microemulsion, thermal decomposition, and photoreduction. b) Functionalization with polymer ligands by physical or chemical methods, wherein the polymer ligands are synthesized via a covalent bond between gallic acid and polyethylene glycol or a diamino polyethylene glycol derivative; c) Functionalization with glucose derivatives or glucuronic acid.

5. The metallic magnetic nanoparticles according to any one of claims 1 to 3, for use as a contrast agent for magnetic resonance imaging.

6. The metallic magnetic nanoparticles according to the preceding claim, for use in methods of image acquisition, evaluation and / or diagnosis of solid tumors or metastatic lesions of the central nervous system.

7. The metallic magnetic nanoparticles according to any one of claims 1 to 3, for use as a drug delivery platform.

8. The metallic magnetic nanoparticles according to the preceding claim, for use in a method of treating solid tumors or metastatic lesions of the central nervous system.

9. The metallic magnetic nanoparticles according to any one of claims 1 to 3, for use as a magnetothermal therapy medium.

10. The metallic magnetic nanoparticles according to the preceding claim, for use in a method of treating solid tumors or metastatic lesions of the central nervous system.

11. The metallic magnetic nanoparticles according to any one of claims 1 to 3, for use via intratumoral, intranasal, intraperitoneal, intravenous, intra-arterial, anal, or oral administration.

12. The metallic magnetic nanoparticles according to any one of claims 1 to 3, or the metallic magnetic nanoparticles for use according to any one of claims 5 to 11, characterized in that, The patient is in a state of mild hypoglycemia or close to the lower limit of normal pre-meal blood glucose.

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