Magnetic nanoparticles functionalised with carbohydrates

Abstract

The present invention describes metallic magnetic nanoparticles, preferably iron oxide, functionalised with specific ligands, preferably belonging to carbohydrates, having a high capacity for penetration into solid tumours in the central nervous system, specifically in high-grade gliomas. The invention further relates to the synthesis method and uses thereof.

Description

[0001] MAGNETIC NANOPARTICLES FUNCTIONALIZED WITH CARBOHYDRATES.

[0002] DESCRIPTION

[0003] FIELD OF INVENTION

[0004] The invention falls within the field of biotechnology and relates to the development of metallic magnetic nanoparticles (MMNs), particularly iron oxide nanoparticles (IONPs), with specific carbohydrate-functionalized ligands, for biomedical applications such as the diagnosis and treatment of central nervous system (CNS) tumors.

[0005] STATE OF THE PRIOR ART

[0006] In 2020, the World Health Organization (WHO) estimated that cancer is between the second and fifth leading cause of death worldwide. The most common primary tumors of the central nervous system are gliomas, which are classified as benign (grade I) to highly aggressive (grade IV). Glioblastoma multiforme (GBM) is a grade IV glioma, responsible for 15% of all primary malignant CNS tumor diagnoses, and has a very poor prognosis, with an average survival of only about 15 months. Therefore, it represents one of the most important challenges in neuro-oncology.

[0007] The diagnosis of GBM is based on neuroimaging examinations and histological and molecular information—that is, on "integrated diagnosis." Magnetic resonance imaging (MRI) is the gold standard for the evaluation of CNS tumors, providing excellent noninvasive morphological and functional images. The use of clinical contrast agents (CAs) is often necessary, but these have limitations due to their toxicity and low specificity.

[0008] The standard therapeutic approach includes maximum safe resection, followed by radiation therapy combined with temozolomide and / or adjuvant temozolomide. Due to the aggressive nature of GBM, progression is expected in all cases, and the decision on additional treatment should take into account the patient's condition, tumor size and location, and the time elapsed since the first treatment.

[0009] Therefore, there is an urgent need to improve the diagnostic specificity of in vivo imaging and, more importantly, the therapeutic efficacy of GBM treatments.

[0010] Nanotechnology is dedicated to monitoring, evaluating, shaping, controlling, and manufacturing materials in the 1 to 100 nm size range, where unique physical and chemical phenomena emerge that promote novel applications in physics, chemistry, biology, materials science and engineering, and in the healthcare field.

[0011] Among the nanomaterials, and specifically metal magnetic nanoparticles (MMNs), developed to improve cancer diagnosis and treatment, iron oxide nanoparticles (lONPs) have attracted special interest since they can be used both for cancer diagnosis (ACs for MRI) and for its treatment (magnetic hyperthermia or drug nanocarriers). In fact, several IONPs are commercially available on the market (Sinerem, Combidex, Farahem, Fehdex and Endorem) [1].

[0012] However, none of them are suitable for the diagnosis / treatment of GBM. This is most likely due to the presence of a very restrictive blood-brain barrier (BBB), which prevents many compounds from reaching their target or doing so with sufficient efficacy. The blood-brain barrier (BBB) ​​has a complex structural and functional composition that makes it almost hermetic, which adds an additional challenge to both the diagnosis and treatment of pathological conditions of the CNS, including brain tumors [2].

[0013] In the case of lONPs (or any other type of nanoparticles - NPs), the Enhanced Permeability 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 NPs, being theoretically optimal for NPs ranging from 20 to 100 nm [3,4].

[0014] Until now, this passive transport mechanism has also been widely relied upon to deliver NPs across the BBB.

[0015] However, as recently demonstrated, the EPR effect is extremely inefficient in brain tumors, especially in the case of metallic NPs, due to the restrictions imposed by the tumor blood-brain barrier (TBBB) [5].

[0016] NanoTherm® (MAGFORCE) has developed the first and only nanotechnology-based therapy with European approval for the treatment of brain tumors, whose IONPs are introduced directly into the tumor or into the wall of the resection cavity by stereotactic administration [6]. In other words, they do not use intravenous administration, although a more homogeneous distribution of IONPs in the tumor interstitium can be expected using this route of administration, especially in the well-vascularized (more active) parts of the tumor. The choice of intratumoral administration is therefore clearly related to the inefficient accumulation of IONPs by passive transport via the EPR effect after intravenous administration. Glucose transporters (GLUTs) in the BBB maintain the continuous high glucose demands of the CNS, since it depends almost entirely on glucose as an energy source.Some GLUTs (mainly GLUT1) have been found to be significantly overexpressed in GBM patients [7-9], and GLUT1, particularly in the vascular endothelium of GBM tumors

[0010] ,.

[0017] Therefore, GLUT1 overexpression in the tumor vasculature would a priori allow NPs functionalized with glucose derivatives to be transported and accumulated in the GBM (active tumor targeting).

[0018] This strategy to facilitate the crossing of various compounds through the BBB has been studied in healthy brains, using lipid nanoparticles functionalized with the mannose derivative, p -aminophenyl-a- d -mannopyranoside (MAN)

[0011] . However, the contribution that the functionalization of NP with mannose may have on its ability to cross the BBB is questionable, since this may be largely due to the lipid nature of the NP, since lipophilicity is considered the key factor for better penetration into the brain.

[0019] It should also be noted that in brain tumors, the properties of the BBB are altered, so it is not possible to extrapolate how these NPs would behave when crossing the BHET. Therefore, to date, there are no reports describing NPs of any composition capable of crossing the BHET.

[0020] Furthermore, although GLUT1 is overexpressed in the BHET with respect to the BBB of healthy patients, under in vivo conditions, its affinity for glucose substantially exceeds its affinity for any other glucose derivative

[0012] , so that NPs functionalized with glucose derivatives must compete with the naturally present glucose, which reduces their ability to be transported through the BHET.

[0021] DESCRIPTION OF THE INVENTION

[0022] The present invention describes lONPs functionalized with specific ligands belonging to the carbohydrate family that have a greater penetration capacity in solid CNS tumors, specifically in high-grade gliomas, particularly in glioblastomas multiforme (GBM), thanks to the specific recognition of the carbohydrate by specific receptors / transporters, mainly glucose membrane transporters (GLUTs), which allow them to cross the blood-brain barrier (BBB).

[0023] The functionalization of lONPs is carried out with carbohydrates, preferably with glucose derivatives, and more preferably with glucuronic acid. Since the facilitated transport of glucose and derivatives by GLLIT1 is saturable, the decrease in circulating glucose levels, such as that produced by a 24-hour fasting period, allows for further increased transport of NPs functionalized with a glucose derivative across the vascular endothelium, in this case with glucuronic acid, by reducing the competition of glucose for GLUT1.

[0024] Thus, combined with mild hypoglycemia, glucuronic acid-functionalized lONPs are actively targeted across the BHET to high-grade gliomas, and since GLUT1 and GLUT3 are often overexpressed also in tumor cells, in particular in GBM stem cells (GSCs)

[0013] , the lONPs thus functionalized are also suitable for active targeting to GSCs, i.e., this active targeting is able to reach two targets: the tumor vascular endothelium, allowing its active transport into the tumor; and tumor cells, in particular GSCs.

[0025] Thus, they are not only able to cross the BHET, but also to be internalized in tumor cells, through GLUT1, thus constituting an optimal therapeutic nanoplatform, capable not only of reaching the tumor interstitium, but also the tumor cells, especially the GSCs, which are largely responsible for the resistance of GBM to chemotherapy and radiotherapy

[0014] ,

[0026] Once accumulated in the tumor, these functionalized lONPs can be used as theranostic nanosystems, i.e., for diagnostic purposes, where the lONPs act as contrast agents for Magnetic Resonance Imaging (MRI), and for therapeutic purposes, where the lONPs act as mediators of magnetic hyperthermia or as platforms for drug transport and delivery. This theranostic approach falls into the category of "see-treat-see" strategies, since the theranostic nanosystem is monitored in real time using MRI.

[0027] They are therefore much more effective, both for diagnostic and therapeutic use, than other types of NPs. This ability to cross the glioma vascular endothelial barrier and accumulate specifically in the tumor has not been previously achieved by any other known NP or compound.

[0028] In general terms, the present invention provides nanoparticles or nanostructures, composed mainly of iron / iron oxides or derivatives, which present polymeric ligands on their surface to improve their colloidal stability, as well as to promote subsequent functionalizations. The subsequent functionalization with carbohydrates, mainly glucose derivatives, such as glucuronic acid, but not exclusively, facilitates the specific recognition of certain molecular targets (glucose transporters or GLUTs), allowing the successful accumulation of NPs in solid tumors, specifically in high-grade gliomas, this accumulation being greatly enhanced by mild hypoglycemia, such as that produced by a 24-h fasting period.

[0029] lONPs or their derivatives have a suitable size to avoid ferromagnetic behavior, which would limit their in vivo applications.

[0030] Carbohydrate-functionalized NPs / nanostructures of biomedical interest can also be used as neo-nanoplatforms since they allow the anchoring of other types of biomolecules, thus increasing their versatility.

[0031] The NPs of the present invention offer numerous advantages over existing NPs. Mainly, their accumulation in solid tumors or metastatic lesions increases in short periods of time due to their active targeting, resulting in a greater likelihood of achieving a therapeutic dose within said tumors.

[0032] The present invention also describes a process for preparing novel MRI ACs based on lONPs, or their derivatives, on the surface of which organic ligands derived from polymers and functionalized with carbohydrates are strongly anchored. This process includes the following steps:

[0033] 1. Synthesis of the metallic magnetic core from metallic precursors, preferably an iron precursor, and in particular iron oleate, by performing a chemical or physical process, such as thermal decomposition. Regarding thermal decomposition, a critical point is extreme control of the temperature ramp.

[0034] 2. Functionalization of the metallic magnetic core with polymeric ligands by a physical or chemical method. Polymeric ligands, capable of stabilizing the metallic magnetic core in aqueous media, are synthesized by a covalent bond between a catechol and a polymer. Preferably, the anchor molecule is gallic acid (GA), and the polymer is bis-amino polyethylene glycol NH2-PEG-NH2. As an example, IONPs suspended in toluene are incubated with a GA-PEG ligand. 2Ooo-NH2 for 1 hour in an ultrasonic bath and then for 4 hours in a temperature-controlled water bath. Preferably, the temperature is in the range of 20–80°C, more preferably between 40 and 60°C.

[0035] 3. A final functionalization with a compound belonging to the carbohydrate family (primarily glucuronic acid, but not exclusively) using a physical or chemical method. The final functionalization with the carbohydrate endows the nanoparticles with the ability to specifically recognize cellular receptors / transporters, enabling active targeting. At each step, the nanoparticles are purified by dialysis against Milli-Q water or by centrifugation using appropriate filters.

[0036] In order to test the INPs of the invention, in vivo experiments were performed in mice as an animal model. Pharmacokinetics and biodistribution were analyzed in vivo by MRI. Tumors were orthotopically implanted in the rodents. High-grade glioma cells were stereotactically implanted in the right caudate nucleus. Once the tumor reached the appropriate size, the metallic magnetic nanoparticles object of the present invention were administered intravenously to animals subjected to a 24-hour fast. Dynamic and parametric MRI sequences were used for short- and long-term monitoring of the injected NPs (in vivo pharmacokinetics).

[0037] It was concluded that the lONPs of the invention exhibit very high stability (more than 500 hours), a high internalization capacity, are non-cytotoxic, and are biocompatible. They also exhibit an excellent ability to remain in the blood and accumulate selectively in tumor tissue, with this accumulation being promoted under conditions of mild hypoglycemia. Furthermore, once accumulated in the tumor, the lONPs of the invention were distributed homogeneously throughout the tumor tissue, which is also crucial for their efficacy.

[0038] Therefore, a first aspect of the invention relates to a metallic magnetic nanoparticle, hereinafter "metallic magnetic nanoparticle of the invention", which comprises at least one organic ligand fixed to its surface, characterized in that it is functionalized by chemical bonding with at least one molecule belonging to the carbohydrate family.

[0039] In a preferred embodiment, the metallic core is composed of iron or M-iron, where M is selected from the list comprising cobalt, manganese, zinc, nickel, gold, silver, platinum, palladium, and ruthenium, either in metallic form or as an oxide, and more preferably iron oxide. Preferably, its metallic core has a size between 1 and 100 nanometers.

[0040] In another preferred embodiment, the organic ligand attached to the surface of the nanoparticle is a catechol derivative, such as L-DOPA, dopamine, caffeic acid, dihydrocaffeic acid, epinephrine, norepinephrine, noradrenaline, adrenaline, protocatechuic acid and gallic acid, preferably gallic acid, covalently bonded to a polymer selected from the list comprising 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 its derivatives and more preferably it is bis-amino polyethylene glycol. More preferably, the polyethylene glycol has a molecular weight in the range 200-6000 kDa and even more preferably between 1000 and 3000 KDa.

[0041] In another preferred embodiment, the carbohydrate is selected from the list comprising allose, altrose, glucose, gulose, mannose, idose, galactose, trehalose, and derivatives thereof. Preferably, the carbohydrate is a hexose, more preferably glucose or its derivatives, and most preferably glucuronic acid.

[0042] In a particularly preferred embodiment of the invention, the metallic magnetic nanoparticle has a metallic core composed of iron oxide, comprises at least one organic ligand derived from catechol, specifically gallic acid, covalently bonded to a polyethylene glycol derivative, preferably bis-amino polyethylene glycol, fixed to the surface, and is characterized in that it is functionalized with a glucose derivative, preferably glucuronic acid.

[0043] Preferably, the metallic magnetic nanoparticle of the invention is suitable for administration via intratumoral, intranasal, intraperitoneal, intravenous, intraarterial, anal or oral routes.

[0044] A second aspect of the invention relates to the synthesis method of the metallic magnetic nanoparticle of the invention, hereinafter “synthesis method of the invention”, which comprises the following steps: a) Synthesis of the metallic core from at least one metallic precursor that is selected from the list comprising chloride, sulfate, nitrate, carbonyl, acetylacetonate, iron oleate and their derivatives, by means of a chemical or physical process that is selected from the list comprising evaporation, condensation, laser ablation, hydrothermal method, chemical reduction, irradiation, coprecipitation, electrochemistry, microemulsion, thermal decomposition and photoreduction.b) Functionalization with a polymeric ligand synthesized by means of a covalent bond between a catechol derivative selected from the list comprising gallic acid and a polymer selected from the list comprising polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, chitosan or derivatives thereof, by a physical or chemical method. c) Functionalization with a compound belonging to the carbohydrate family selected from the list comprising allose, altrose, glucose, gulose, mannose, idose, galactose, trehalose, or derivatives thereof, such as glucuronic acid.

[0045] In a preferred embodiment of the synthesis method of the invention, the synthesis of the metallic core is carried out by thermal decomposition. In a more preferred embodiment, the decomposition is carried out in an organic solvent, at high temperature, preferably in the range 180-350°C and in an inert atmosphere. Preferably, the solvent for the reaction is an organic compound, more preferably 1-octadecene. In an even more preferred embodiment, stabilizing agents are used for the reaction, and preferably these are selected from oleic acid, oleyl alcohol, oleylamine and 1,2-hexadecanethiol and mixtures thereof.

[0046] In another preferred embodiment, the synthesis of the metallic core is carried out from more than one metallic precursor. In another preferred embodiment, the metallic precursor is iron oleate.

[0047] In a preferred embodiment, the first functionalization is performed with the polymeric ligand GA-PEG-NH2.

[0048] In another preferred embodiment, said functionalization is carried out in two steps: A first step by applying ultrasound and a second step in which the reaction temperature is increased.

[0049] In a preferred embodiment, the second functionalization is carried out with glucuronic acid.

[0050] In another preferred embodiment, the union with carbohydrates is carried out using carbodiimide as the coupling agent, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of N-hydroxysuccinimide.

[0051] Preferably, in each step of the synthesis method of the invention, the nanoparticles are purified by dialysis against Milli-Q water or by centrifugation using suitable filters.

[0052] A third aspect of the invention relates to the metallic magnetic nanoparticle of the invention for use in a method for the diagnosis, evaluation, imaging or treatment of solid tumors or metastatic lesions, preferably of the central nervous system.

[0053] In another embodiment of this aspect of the invention, the metallic magnetic nanoparticle of the invention is described for use as a contrast agent for magnetic resonance imaging, preferably, directed at the evaluation of solid tumors or metastatic lesions, and more preferably, for tumors of the central nervous system.

[0054] In another embodiment of this aspect of the invention, the metallic magnetic nanoparticle of the invention is described for use as a drug delivery platform, preferably, for drugs aimed at the treatment of solid tumors or metastatic lesions, and more preferably, tumors of the central nervous system.

[0055] In another embodiment of this aspect of the invention, the metallic magnetic nanoparticle of the invention is described for use as a mediator of magnetic hyperthermia, preferably, directed at the treatment of solid tumors or metastatic lesions, and more preferably, tumors of the central nervous system.

[0056] In another embodiment of this aspect of the invention, the metallic magnetic nanoparticle of the invention is described for use via intratumoral, intranasal, intraperitoneal, intravenous, intraarterial, anal or oral routes.

[0057] In another embodiment of this aspect of the invention, the metallic magnetic nanoparticle of the invention is described for use in patients who are in a state of mild hypoglycemia or close to the lower limit of preprandial normoglycemia.

[0058] Preferably, said state is derived from fasting or is due to the previous administration of a chemical compound that is selected from the list comprising Insulin, Biguanides, Sulfonylureas, Glitinides, Dipeptidyl Peptidase IV Inhibitors (DPP-IV), Sodium-glucose cotransporter-2 Inhibitors (SGLT2), Glucagon-like peptide-1 (GLP-1) Agonists, Alpha-glucosidase Inhibitors (AGIs), Thiazolidinediones or related compounds.

[0059] DETAILED DESCRIPTION OF THE FIGURES

[0060] Figure 1. a) Diagram of the different functionalization steps, showing the chemical structure of the different PEGylated ligands used and glucuronic acid. The size and shape of the lONPs throughout the process were evaluated by electron microscopy (representative images are shown). The scale corresponds to 50 nm in all cases. b) Fourier transform infrared (FTIR) evaluation of the different functionalization steps. c) Measurement of the hydrodynamic diameter of the different functionalized lONPs over time.

[0061] Figure 2. Transverse relaxations (r2) calculated as the slope of the fitting line of R2 versus iron concentration, calculated at different magnetic fields: 1.44 T (blue) and 9.4 T (black) for NPs@OH (a) and NPs@Gluc (b). The high-field T2-weighted images corresponding to each concentration are shown in the right margin.

[0062] Figure 3. Representative optical microscopy images of HFF-1 fibroblasts resulting from the fusion of brightfield (gray), Hoechst 33342 (blue), and TOPRO-3 Iodine (red) images: (a) negative control, (b) positive control, (c) cells exposed to 100 pg / ml of NPs@OH NPs. Scale bar corresponds to 50 pm. (d) Total number of cells per well after exposure to increasing concentrations of NPs@OH NPs. (e) Percentage of dead cells after exposure to increasing concentrations of NPs@OH NPs. (f) MTT assay of cells exposed to increasing concentrations of NPs@OH NPs. Figure 4. Representative optical microscopy images of HFF-1 fibroblasts resulting from the fusion of brightfield (gray), Hoechst 33342 (blue), and TOPRO-3 Iodine (red) images: (a) negative control, (b) positive control, (c) cells exposed to 100 pg / ml of NPs@Gluc NPs. The scale bar corresponds to 50 pm.(d) Total number of cells per well after exposure to increasing concentrations of NPs@Gluc NPs. (e) Percentage of dead cells after exposure to increasing concentrations of NPs@Gluc NPs. (f) MTT assay of cells exposed to increasing concentrations of NPs@Gluc NPs.

[0063] 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 pg / ml, for 2 h and 4 h, both NPs stained with Prussian blue. Cellular uptake is only observed for NPs functionalized with glucuronic acid.

[0064] Figure 6. a) Western-blot analysis of the expression of specific glucose transporters (GLUT1 and GLUT3) in C6 vs MW - Molecular Weight cells. Glucose competition experiment; b) Representative optical images of C6 cells exposed to different glucose concentrations and to a constant concentration of 100 pg / ml of NPs@Gluc stained with Prussian blue, and c) their quantitative evaluation by inductively coupled plasma high-resolution mass spectroscopy (ICP-HRMS).

[0065] Figure 7. a) Short-term pharmacokinetics obtained by dynamic MRI in the whole tumor (red) and in the tumor periphery (blue) during the first 30 min after intravenous administration of NPs@OH (G1), NPs@Gluc (G2) and NPs@Gluc in mice subjected to overnight fasting (G3). b) T2-weighted MR images at 1 h and 2 h after administration of NPs, of the different experimental groups, and the corresponding quantitative analysis by calculating the relaxation rate variation (AR2) over time of the whole tumor (c) and the tumor periphery (d).

[0066] Figure 8. a) Representative electron microscopy images of blood 1 hour after injection of NPs@OH (top) and NPs@Gluc (bottom) b) Variation of relaxation velocity (AR2) of plasma samples 1 hour after intravenous injection of NPs@OH (gray) and NPs@Gluc (blue) corrected with normal plasma values.

[0067] Figure 9. Representative histological sections of Prussian blue staining of the liver and tumors 2 h after injection of PBS (a), NPs@Oh (b) and NPs@Gluc. The scale bar corresponds to 50 µm.

[0068] EXAMPLE OF THE INVENTION Synthesis of iron oxide nanoparticles (lONPs), their functionalization with PEGylated ligands and finally with Glucose derivatives.

[0069] Synthesis of iron oleate (iron precursor)

[0070] A mixture of 10.8 g of iron chloride (40 mmol) and 36.5 g of sodium oleate (120 mmol) was dissolved in 80 ml of ethanol, 60 ml of distilled water and 140 ml of hexane. The resulting solution was heated to 70°C and left for 4 h in an inert atmosphere 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 the iron oleate. The organic phase was washed 3 times with distilled water, and the hexane was evaporated in a rotary evaporator.

[0071] Synthesis of lONPs by thermal decomposition

[0072] 1.81 g (5.3 mmol) of iron oleate and 0.32 g (1.133 mmol) of oleic acid were weighed into a two-necked round-bottom flask with a maximum volume of 50 ml. 15 ml of 1-octadecene were used as solvent. The reaction was then preheated to 200 °C under an inert atmosphere (N2) and magnetic stirring. The temperature was increased to 320 °C using a temperature ramp of 1 °C / min. The temperature of 320 °C was maintained for 1 hour and subsequently allowed to cool to room temperature. The NP suspension was washed 3 times with a mixture of acetone:ethanol (1:1), centrifuged at 8000 rpm for 20 minutes to remove unwanted reagents and finally resuspended in toluene. The nanoparticle suspension was maintained at 4°C.

[0073] Synthesis of PEGylated ligands

[0074] Synthesis of GA-PEG-OH with formula:

[0075] Briefly, to a solution of 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 pmol, 24 mg) in 100 ml of tetrahydrofuran and 10 ml of dichloromethane, in a round-bottom flask under nitrogen atmosphere, was added dropwise a solution of dicyclohexyl carbodiimide (Molecular weight: 206 g / mol, 5 mmol, 1 g) in tetrahydrofuran. The mixture was stirred overnight at room temperature. The reaction mixture was filtered through a filter paper and the solvents were evaporated by rotation.

[0076] Synthesis of GA-PEG-NH2 with formula:

[0077] Briefly, to a solution of a.co-Bis-aminopolyethylene 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 pmol, 24 mg) in 100 ml of tetrahydrofuran and 10 ml of dichloromethane, in a round-bottom flask under nitrogen atmosphere, was added dropwise a solution of dicyclohexyl carbodiimide (Mw: 206 g / mol, 5 mmol, 1 g) in tetrahydrofuran. The mixture was stirred overnight at room temperature. The reaction mixture was filtered through a filter paper and the solvents were evaporated by rotation.

[0078] Ligand exchange process.

[0079] Briefly, a solution containing 1.0 mL of NPs (10 g / L Fe), 1.0 mL of the GA-PEG-OH ligand at a concentration of 0.1 M in CHCl3, and 50 mL of triethylamine was added to a glass vial. The mixture was sonicated for 1 h and held at 55°C for 4 h. At this point, the mixture was diluted with 5 mL of toluene, 5 mL of milli-Q water, and 10 mL of acetone. The mixture was then shaken to transfer the functionalized NPs to the aqueous phase. The aqueous phase was then collected in a round-bottom flask, and residual organic solvents were rotary evaporated. The PEGylated NPs were then purified on centrifuge filters with a molecular weight cutoff of 100 kDa at 450 rcf. During each centrifugation, the functionalized NPs were resuspended with milli-Q water. The purification step was repeated several times until the filtrate was clear. After purification, the PEGylated NPs were resuspended in PBS buffer.Finally, to ensure high stability of the monodispersed NPs, this solution was centrifuged at 150 rcf for 5 min and placed on a permanent magnet (0.6 T) for 5 min. These nanoparticles are referred to hereafter as NPs@OH.

[0080] Functionalization with GA-PEG-NH2 was performed in a similar manner.

[0081] These nanoparticles will be referred to hereafter as NPs@NH2. 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 pmol) of NHS (N-hydroxysuccinimide), 0.192 mg (1.24 pmol) of EDC (1-ethyl-3-3- dimethylaminopropylcarbodiimide), and 0.162 mg (0.834 pmol) of glucuronic acid were added. The reaction was carried out on ice. After 2 hours of reaction, the mixture was purified on centrifugal filters with a molecular weight cutoff of 100 kDa at 450 rcf to remove unreacted reactants. These nanoparticles are hereafter referred to as NPs@GLUC.

[0083] Characterization of NPs high-resolution mass with

[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 ml of a nanoparticle solution in a volumetric flask. The mixture was left overnight. Milli-Q water was then added to make the total volume of 25 ml.

[0085] Characterization of NPs electronic transmission line

[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 the nanoparticles corresponding to ~1 g / L of (Fe) onto a carbon-coated copper grid and allowing the solvent to evaporate. Diameters were calculated based on an average of 100 measured nanoparticles.

[0087] Characterization of NPs Fourier transform

[0088] FTIR spectra were recorded with a Jasco FTIR-4100 using a single-reflection ATR accessory (MIRacle ATR, PIKE Technologies) coupled to a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector. All spectra were recorded in the 4000–800 cm2 range. 1 with a resolution of 4 cm- 1 , accumulating 200 scans. All samples were recorded as solid product.

[0089] Characterization of NPs light dynamics

[0090] The size distribution measurements of PEGylated IONPs were performed on a Zetasizer Nano ZS90 (Malvern, USA). The nanoparticles were dispersed in milli-Q water or PBS at a concentration of 100 mg / L Fe. Measurements were performed in a ZEN0118 low-volume disposable size cuvette, with a refractive index of 2420 and a detection angle of 90°. The measurement duration was set to automatic, and the number of measurements was three. The general purpose (normal resolution) analysis model was chosen.

[0091] Characterization of NPs by transverse relaxation (r2).

[0092] The transverse relaxation time (T2) was measured by proton nuclear magnetic resonance. 1H NMR, at low (1.44 T) and high (9.4 T) magnetic fields, using solutions of magnetic NPs in PBS with Fe concentrations ranging from 0.125 to 2 mM under physiological conditions at 37°C. T2a measurements under magnetic field were performed on a Bruker Minispec system (Bruker BioSpin, Rheinstetten, Germany) using the Carl-Purcell-Meiboom-Gill (CPMG) sequence. In high field, T2a values ​​were measured on a Bruker Biospec MRI magnetic resonance imaging (MRI) system (Bruker Biospec, Bruker BioSpin, Ettlingen, Germany) equipped with 400 mT*m' field gradients. 1and a 40 mm “birdcage” quadrature resonator at 298 K. T2 values ​​were measured using a 64-echo Carl-Purcell-Meiboom-Gill (CPMG) imaging sequence (TE values ​​from 7.5 ms to 640 ms). The transverse relaxivity, r2, in both magnetic fields was calculated from the slope of the linear fit of the relaxation rate (1 / T2) versus Fe concentration, as given in the following equation:

[0093] Regions of interest (ROI) were drawn on the first image of the image sequence, and intensity values ​​were extracted and fitted to the following equations: where M z and Mxy are the signal intensities at time TR or TE, and M o is the signal intensity at equilibrium.

[0094] In vitro tests.

[0095] Different tests were performed for a comprehensive analysis of the cytotoxicity of IONPs.

[0096] A) Cell cultures.

[0097] The HFF-1 cell lines (human fibroblasts) were selected as working models, serving as representative controls for healthy cells, as well as C6 (rat glioblastoma), u87 (human glioblastoma), and 4T1 (mouse breast cancer). In all cases, established standard culture conditions were used, which included the use of three different culture media: Dulbecco's Modified Eagle's (DMEM) for HFF-1 and C6 cells; Roswell Park 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 a 5% CO2 atmosphere. Cells were subcultured every 2 days using trypsin.

[0098] B) Evaluation of cellular uptake by Prussian blue staining.

[0099] 3x10 were planted 5cells in a 24-well plate at 37°C in a 5% CO2 atmosphere. After 24 h, the medium was replaced with fresh medium containing the PEGylated lONPs. Cells were incubated again for 2 h and 4 h. Following incubation with NPs, cells were washed three times with PBS to remove free NPs. Cells were subsequently fixed for 40 min using 4% paraformaldehyde (Sigma-Aldrich). Cells were then 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. Cells were washed three times with PBS, stained with “nuclear fast red” (Sigma-Aldrich), and observed under an inverted light microscope.

[0100] C) Western blot.

[0101] First, the protein extract was prepared by cell lysis. To do this, the cells were grown to confluence. The cells were then washed three times with PBS at 4°C, and lysis buffer was added. The cell suspension was then transferred and centrifuged in a pre-chilled centrifuge tube. The proteins remained in the supernatant. Once the extract was obtained, the proteins were separated by gel electrophoresis based on their molecular weight. The gel was transferred to a membrane for immunodetection. Anti-GLUT1 and anti-GLUT3 antibodies were used for this work.

[0102] D) Quantification of cellular uptake by high-resolution inductively coupled plasma mass spectroscopy (ICP-MS).

[0103] Similarly, as mentioned above, 1x10 were sown 54T1 cells were grown in a 96-well well at 37°C in a 5% CO2 atmosphere. After 24 h, the medium was replaced with fresh medium containing PEGylated lONPs. Cells were incubated again for 2 h and 4 h. After incubation with NPs, cells were washed three times with PBS to remove any free NPs. At that time, 100 pl of aqua regia was added to each well. The mixture was left overnight. Milli-Q water was then added to complete the total volume of 10 ml. The Fe concentration was determined on a NexION ICP-HRMS (Perkin-Elmer, Waltham, MA, USA).

[0104] E) Glucose competition tests.

[0105] To demonstrate the predicted transport of NPs@Gluc through glucose transporters (GLUTs), 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 pg / ml] while increasing the glucose concentration. After 2 h, cells were stained with Prussian blue, as described in section B). In addition, quantitative analysis was also performed by ICP-MS, as described in section D).

[0106] F) High content screening (HCS).

[0107] HFF-1 cells were seeded at a density of 1x10 4cells / well in a 96-well plate at 37°C in a 5% CO2 atmosphere (100 µl per well, number of replicates = 5). After 24 h of culture, the medium in the wells was replaced with fresh medium containing the NPs at concentrations varying from 0.1 to 100 µg / ml (Fe). After 24 h, Triton X-100 was added to the positive control wells. All wells were stained after 15 min with: 1) DAPI (4',6-diamidino-2-phenylindole, 1:3000 dilution in PBS) to label cell nuclei, although with stronger labeling in live cells; 2) calcein (1:1000 in PBS) to assess cellular activity and 3) TO-PRO-3 iodine to label dead cells (1:1000 dilution in PBS). Cell morphology images were taken using a Perkin Elmer Operetta High Content Imaging System with a 20* LWD 0.45 NA aerial objective lens. Five replicate wells were analyzed for each condition with 10 random image fields captured per well.For each field, fluorescence images for DAPI, Calcein-AM, and TO-PRO-3, in addition to a brightfield image, were captured. Cell mortality percentages were automatically calculated using Operetta Harmony software, by which all nuclei (live and dead) were identified from DAPI staining, and the percentage of dead cells was determined from the number of nuclei showing elevated levels of TO-PRO-3 staining. Intracellular esterase activity was assessed by Calcein-AM staining.

[0108] G) MTT test.

[0109] In brief, HFF-1 cells were seeded at a density of 1x10 4cells / well in a 96-well plate at 37°C in a 5% CO2 atmosphere (200 µl per well, number of replicates = 5). After 24 h of culture, the medium in the wells was replaced with fresh medium containing PEGylated lONPs at concentrations ranging from 0.1 pg / ml to 100 pg / ml. After 24 h, Triton X-100 was added to the positive control wells and, after waiting 15 min, the supernatant of each well was replaced with 200 µl of fresh medium containing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (0.5 mg / ml). After 2 h of incubation at 37°C and 5% CO2, the medium was removed, the formazan crystals were solubilized with 200 µl of DMSO and the solution was mixed vigorously to dissolve the reacted dye. The absorbance of each well [Abs] was read in a microplate reader (Dynatech MR7000 instruments) at 550 nm.The relative cell viability (%) and its error in relation to the control wells containing cell culture medium without nanoparticles were calculated using the equations: where or is the standard deviation.

[0110] In vivo tests.

[0111] In an orthotopic model of GBM (mouse model), the pharmacokinetics and biodistribution after intravenous administration of PEGylated IONPs were determined by dynamic and parametric MRI.

[0112] Animals.

[0113] For in vivo experiments, 6-week-old male Balb / c mice (20 g weight) provided by Charles River were used (n = 4 per experimental group). These experiments were carried out in accordance with the ethical guidelines of the local ethics committee and also in line with national (RD 53 / 2013) and European (2010 / 62 / EU) regulations.

[0114] Tumor implantation.

[0115] C6 cells were brought to 70-80% confluence. At that point, they were trypsinized and the cells counted. 10 5 cells, suspended in 5 pL of culture medium, into the right caudate nucleus of mice. The tumors were then allowed to grow until they reached a volume of 0.16 to 0.2 mm 3 At this point, the mice were randomly divided into three groups:

[0116] G1. Intravenous administration of NPs@OH. G2. Intravenous administration of NPs@Gluc.

[0117] G3. Mice were fasted overnight and administered NPs@Gluc intravenously.

[0118] Magnetic resonance imaging (MRI) evaluation.

[0119] MRI experiments were performed on a 9.4 T Bruker Biospec system equipped with 400 mT / m gradients and a mouse brain surface antenna for signal reception and a volume antenna for transmission. ParaVision 6.0 software (Bruker Medical GmbH®) was used for the experiments on a Linux platform. Animals were anesthetized with 1% isoflurane, tail vein cannulated, and placed in the spectrometer, where respiration and body temperature were monitored throughout the MRI experiment using a small animal monitoring system (PC-SAM 32 v8.02 software). PEGylated lONPs were administered intravenously via the tail vein at a concentration of 10 mg (Fe) per kg.

[0120] Pharmacokinetic and biodistribution experiments were performed using the following experimental scheme:

[0121] 1. High-resolution T2-weighted imaging (0 h),

[0122] 2. Susceptibility-weighted imaging (0 h),

[0123] 3. Taking parametric T2 images (quantitative) (0 h),

[0124] 4. intravenous injection of PEGylated IONPs,

[0125] 5. Taking a dynamic sequence of T2-weighted images (pharmacokinetics),

[0126] 6. High-resolution T2-weighted imaging (30 min),

[0127] 7. Susceptibility-weighted imaging (30 min),

[0128] 8. Taking parametric T2 images (quantitative) (30 min),

[0129] 9. High-resolution T2-weighted imaging (1 h),

[0130] 10. Susceptibility-weighted imaging (1 h),

[0131] 11. Taking parametric T2 images (quantitative) (1 h),

[0132] 12. High-resolution T2-weighted imaging (2 h),

[0133] 13. Susceptibility-weighted imaging (2 h), 14. T2 parametric imaging (quantitative) (2 h).

[0134] Susceptibility-weighted images were taken using a FLASH sequence (TE = 16 ms, TR = 350 ms, 4 means, 1 repeat, flip angle = 40°, FOV = 1 cm, matrix size = 512 x 256).

[0135] High-resolution T2-weighted images were taken using a respiratory-gated turbo-RARE sequence (TE = 27 ms, TR = 1000 ms, 4 averages, 156 pm in-plane resolution, and 1 mm slice thickness).

[0136] Quantitative T2 measurements were also performed using a multi-echo spin-echo CPMG sequence (TE from 7 ms to 448 ms, TR = 3500 ms, FOV = 4 cm, matrix size = 128x128, slice thickness = 1 mm). For short-time T2-based pharmacokinetics, the turbo-RARE sequence was used with the same parameters as above, but only 1 average to improve temporal resolution (1 image every 30 seconds).

[0137] The short-term pharmacokinetics acquired during the first 30 min were analyzed semiquantitatively using the following expression: 100 where RE is the relative magnification module of the signal, l t is the signal intensity at a given time after the injection of the nanoparticle, and l0 is the signal intensity before injection.

[0138] Long-term pharmacokinetics were measured using parametric (quantitative) T2-weighted imaging at 0 h, 0.5 h, 1 h, and 2 h. Pharmacokinetics were obtained by calculating the mean values ​​within different regions of interest (ROIs) in the following tissues: whole tumor and tumor periphery.

[0139] Ex vivo tests.

[0140] Analysis of blood circulation times.

[0141] One hour after injection of lONPs, mice were sacrificed and blood was collected for further analysis. Transmission electron microscopy (TEM) was used to determine the shape and size of PEGylated IONPs, and relaxometry by 1 H NMR to measure the transverse relaxation velocity (R2) of blood plasma.

[0142] Histology. Tissues were fixed in 4% formaldehyde (Panreac, buffered to pH 7) for 48 h, with the 4% formaldehyde changed every 24 h. Samples were then dehydrated with graded ethanol and embedded in paraffin (temperature 56°C for 2 h under shaking and vacuum). Detailed procedures are described below.

[0143] Hematoxylin and eosin (H&E): Paraffin-embedded specimens were sectioned at 7 pm thickness, deparaffinized, rehydrated and stained with H&E, then dehydrated in ascending concentrations of ethanol, cleared in xylene, and mounted on commercial glass slides.

[0144] Prussian blue (PB): Paraffin-embedded specimens were sectioned at 7 µm thickness, then deparaffinized, rehydrated, immersed in 20% hydrochloric acid and 10% potassium ferrocyanide, washed with water and counterstained with “nuclear fast red”, dehydrated in ascending concentrations of ethanol, cleared with xylene and mounted on commercial glass slides.

[0145] Results.

[0146] In this work, lONPs were functionalized with two different PEGylated ligands (OH and NH2 termini). In a subsequent chemical reaction, the NPs@NH2 were covalently linked to glucuronic acid via an amide reaction. Neither chemical reaction affected the metal core.

[0147] Since the different functionalization steps could affect the metallic core of the lONPs, modifying their size and / or shape and, therefore, their magnetic properties, exhaustive TEM analysis was necessary to ensure their stability (Figure 1a). In general, neither the shape nor the size of the lONPs were affected by the different synthetic steps, despite some aggregation in the case of NPs@NH2. Notably, after covalent bonding with glucuronic acid, aggregation was no longer observed. Furthermore, the presence of the PEGylated ligand was confirmed by FTIR for NPs@OH and NPs@NH2 (Figure 1b), indicating a favorable ligand exchange procedure. The covalent bond between the 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 showed very low colloidal stability, even with precipitation observable by the naked eye at 48 h (Figure 1c). In contrast, both NPs@OH and NPs@Gluc displayed high colloidal stability, even after 500 h. The capacity of these three lONPs as MRI ACs was evaluated in two different magnetic fields: 1.44 T and 9.4 T (Figure 2). Both NPs@OH and NPs@Gluc exhibited excellent properties as MRI ACs. Due to the low stability of NPs@NH2, their r2 could not be accurately determined.

[0148] Regarding cytotoxicity, neither NPs@OH (Figure 3) nor NPs@Gluc (Figure 4) exhibited cytotoxic effects on HFF-1 cells. No significant changes were observed in cell morphology, as observed by optical microscopy (Figures 3a and 4a), total cell number (Figures 3d and 4d), percentage of dead cells (Figures 3e and 4e), or mitochondrial activity (Figures 3f and 4f), supporting the absence of cytotoxicity.

[0149] The cellular targeting and internalization capabilities of glucuronic acid-functionalized NPs were evaluated in both healthy (HFF-1) and tumor cells (C6, u87, and 4T1). Cells were exposed to either NPs@OH or NPs@Gluc (Figure 5). NPs@OH showed very little internalization in all cases, being only observable in C6 cells after 4 hours of incubation. In contrast, NPs@Gluc showed high internalization after 2 hours of exposure, mainly in C6 and 4T1 cells, and moderately lower in HFF-1 and u87 cells. NPs@Gluc internalization increases at 4 hours post-exposure, particularly for C6 cells.

[0150] The internalization of NPs@Gluc was studied through the specific recognition and interaction with glucose transporters (GLUTs) by selecting C6 cells. First, the presence of GLUT1 and GLUT3, which are frequently found overexpressed in a variety of tumor cells, was confirmed by western blot analysis, showing high expression of both GLUTs, with GLUT3 being qualitatively higher, as expected (Figure 6a). Once the presence of GLUTs was confirmed, a glucose competition assay was performed to demonstrate their involvement in the transport of NPs@Gluc across the plasma membrane. The addition of glucose inhibited the entry of NPs@Gluc into C6 cells in a dose-dependent manner (Figure 6b-c), thus clearly demonstrating that this process is mediated by GLUTs.

[0151] These data demonstrate the biocompatibility of IONPs in vitro, as the results of these assays show no statistically significant variation compared to controls. Furthermore, the specific recognition and facilitated transport of NPs@Gluc by glucose transporters in different cell lines was demonstrated using the glucose competition assays performed.

[0152] However, tumor cell targeting in vivo is necessarily preceded by tumor targeting, i.e., the ability of NPs to selectively accumulate in tumor tissue, which requires passively or actively crossing the tumor vasculature to reach its interstitium. Passive tumor targeting is particularly challenging in brain tumors, where the blood-brain barrier is a major limiting factor. Therefore, an alternative strategy is active targeting. Since GLLIT1 is overexpressed in the vasculature of GBMs

[0015] , we proposed to deliver NPs@Gluc to GBMs via GLUT-mediated active targeting. Thus, the pharmacokinetics of NPs@Gluc and NPs@OH were monitored in vivo by MRI.

[0153] Short-term pharmacokinetics (Figure 7a) showed a slight increase in signal after administration of PEGylated lONPs in groups G1 (9%) and G2 (11%), which remained constant from minute 10 to the end of the experiment. In contrast, in G3 (24-h fasting), there was a dramatic increase in signal (around 40%), indicating the large extravasation of NPs@Gluc into the tumor interstitium. Therefore, mild hypoglycemia promoted a dramatic increase in the amount of NPs@Gluc reaching the tumor. These data are consistent with in vitro glucose competition experiments, which showed a much higher internalization of NPs@Gluc at lower glucose concentrations. In other words, lowering glucose levels (fasting or by the use of hypoglycemic agents) promotes efficient GLUT-mediated active transport of NPs@Gluc into the tumor interior.It's worth noting that this increased signal was observed both throughout the tumor and at its periphery. As mentioned for G1 and G2, the signal remained constant from minute 10 onwards.

[0154] Regarding long-term pharmacokinetics (qualitatively assessed by magnetic susceptibility-weighted imaging), no obvious changes were observed over time in the tumor of group G1, while group G2 showed slight darkening 1 hour after NP injection. In contrast, group G3 showed highly significant darkening at this time point. Tumors in groups G2 and G3 maintained a similar darkening 2 hours after injection. In conclusion, the 24-h fasting period resulted in a decrease in blood glucose concentration, which led to a dramatic increase in NPS@Gluc accumulation within the tumor. These results were also assessed by quantitative MRI (T2 maps). A statistically significant increase in R2 was observed, approximately 2 and 5 s. 1(for the entire tumor and the tumor periphery, respectively) 1 h after NP injection in group G3 (Figure 7c-d). These values ​​were similar to those 2 h after NP injection. Notably, no changes in R2 were observed in any case in the contralateral part of the brain, indicating selective accumulation in the tumor. To enhance tumor targeting, NPs must have a reasonably long residence time in the blood. Therefore, blood was analyzed 1 h after NP injection by TEM and TD-NMR (Figure 8). A significant amount of both NPs@OH and NPs@Gluc remained circulating in the bloodstream, all showing a relative increase in R2 between 7 and 10 s. 1 , which demonstrates the high stealth properties of these PEGylated NPs, with respect to the animal's immune system.

[0155] Furthermore, to evaluate NP accumulation in tissues, histological sections from major organs were stained with Prussian blue (Figure 9). At the end of the experiment (2 h after NP injection), no evidence of NP accumulation was observed in either the tumor or the liver of control mice (injected with PBS). On the other hand, Kupffer cells from mice injected with both NPs@OH and NPs@Gluc denoted clear NP accumulation. Regarding the tumor, mice injected with NPs@OH showed low accumulation, while those injected with NPs@Gluc showed a significantly higher amount. Furthermore, NPs@Gluc were more homogeneously distributed throughout the tumor tissue, which is also crucial for the efficacy of tumor treatment.

[0156] Based on the above, we conclude that NPs@Gluc exhibit extraordinary tumor-targeting capacity in vivo. Specifically, NPs@Gluc were able to bypass the restrictions imposed by BHET and efficiently reach and accumulate in high-grade gliomas. Mild fasting-induced hypoglycemia greatly enhanced this ability, indicating that the observed vascular translocation of NPs@Gluc is likely mediated by GLUT1, which is known to be overexpressed in the vasculature of these tumors.

[0157] References

[0158] 1. Sivasubramanian et al. Seeing Better and Going Deeper in Cancer Nanotheranostics. International Journal of Molecular Sciences. 2019; 20(14):3490.

[0159] 2. Israel LL et al. . Magnetic iron oxide nanoparticles for imaging, targeting and treatment of primary and metastatic tumors of the brain. J Control Release. 2020 Apr 10;320:45-62.

[0160] 3. Nakamura Y. et al . Nanodrug Delivery: Is the Enhanced Permeability and Retention Effect Sufficient for Curing Cancer? Bioconjug Chem. 2016 Oct 19;27(10):2225-2238.

[0161] 4. Caro C et al. D. Polysaccharide Colloids as Smart Vehicles in Cancer Therapy. Curr Pharm Des. 2015;21(33):4822-36. doi: 10.2174 / 1381612821666150820100812. 5. C. Caro et al., Passive targeting of high-grade gliomas via the EPR effect: a closed path for metallic nanoparticles? Biomaterials Sciences 9 (2021) 7984-7995.

[0162] 6. B. Thiesen, et al., Clinical applications of magnetic nanoparticles for hyperthermia International Journal of Hyperthermia 24 (2008) 467-474.

[0163] 7. T. Nishioka, et al., Distribution of the glucose transporters in human brain tumors. Cancer Research 52 (1992) 3972-3979.

[0164] 8. F. Pistollato, et al., Intratumoral hypoxic gradient drives stem cells distribution and MGMT expression in glioblastoma. Stem Cells 28 (2010) 851-862.

[0165] 9. M. R. Guda, et al., GLLIT1 and TLIBB4 in Glioblastoma Could be Efficacious Targets. Cancers 11(9) (2019) 1308.

[0166] 10. K. Suzuki, et al., Glucose transporter 1 -mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors. Journal of Controlled Release 301 (2019) 28-41.

[0167] 11. Singh, I., Swami, R., Jeengar, M.K., Khan, W., Sistla, R., 2015. p-Aminophenyl-a-d- mannopyranoside engineered lipidie nanoparticles for effective delivery of docetaxel to brain. Chem. Phys. Lipids 188, 1-9. https: / / doi.Org / 10.1016 / j. chemphyslip.2015.03.003.

[0168] 12. Sawayama, H. & Baba, H. in Molecular Nutrition: Carbohydrates (ed Vinood B. Patel) 191- 207 (Academic Press, 2019). https: / / doi.Org / https: / / doi.org / 10.1016 / B978-0-12-849886-6.00007- 0.

[0169] 13. A. Harland, et al., Glioma Stem-Like Cells and Metabolism: Potential for Novel Therapeutic Strategies. Frontiers in Oncology 11 (2021) 743814.

[0170] 14. Blazek, et al., I nt J Radiat Oncol Biol Phys 67(1) (2007) 1-5. doi:10.1016 / j.ijrobp.2006.09.037; Liu, et al., Mol Cancer 5 (2006) 67. doi : 10.1186 / 1476-4598-5- 67.]

[0171] 15. W.-L. Yeh, et al., Enhancement of glucose transporter expression of brain endothelial cells by vascular endothelial growth factor derived from glioma exposed to hypoxia Mol Pharmacol. 73(1) (2008) 170-177.

Claims

CLAIMS 1. Metallic magnetic nanoparticle whose metallic core is composed of iron oxide comprising at least one organic ligand composed of a gallic acid bound to a polyethylene glycol derivative fixed to its surface, characterized in that it is functionalized with at least one molecule of a glucose derivative.

2. Metallic magnetic nanoparticle according to the preceding claim, characterized in that the glucose derivative is glucuronic acid.

3. Metallic magnetic nanoparticle according to the preceding claim, characterized in that the polyethylene glycol derivative is bis-amino-polyethylene glycol.

4. Method for synthesizing metallic magnetic nanoparticles according to any of the preceding claims, characterized in that it comprises the following steps: a) Synthesis of the metallic core from iron oleate or its derivatives, by means of a chemical or physical process selected from the list comprising evaporation, condensation, laser ablation, hydrothermal method, chemical reduction, irradiation, coprecipitation, electrochemistry, microemulsion, thermal decomposition and photoreduction. b) Functionalization with a polymeric ligand synthesized by means of a covalent bond between gallic acid and a polyethylene glycol or bis-amino-polyethylene glycol derivative by a physical or chemical method. c) Functionalization with a glucose derivative or glucuronic acid.

5. Metallic magnetic nanoparticle according to any of claims 1 to 3 for use as a contrast agent for magnetic resonance imaging.

6. Metallic magnetic nanoparticle according to the preceding claim for use in a method of obtaining images, evaluating and / or diagnosing solid tumors or metastatic lesions of the central nervous system.

7. Metallic magnetic nanoparticle according to any of claims 1 to 3, for use as a drug delivery platform.

8. Metallic magnetic nanoparticle according to the preceding claim for use in a method of treating solid tumors or metastatic lesions of the central nervous system.

9. Metallic magnetic nanoparticle according to any of claims 1 to 3 for use as a magnetic hyperthermia mediator.

10. Metallic magnetic nanoparticle according to the preceding claim for use in a method of treating solid tumors or metastatic lesions of the central nervous system.

11. Metallic magnetic nanoparticle according to any of claims 1 to 3 for use via intratumoral, intranasal, intraperitoneal, intravenous, intraarterial, anal or oral routes.

12. Metallic magnetic nanoparticle according to any of claims 1 to 3 for use according to any of claims 5 to 11, characterized in that the patient is in a state of mild hypoglycemia or close to the lower limit of preprandial normoglycemia.

Images