Iron nanoclusters, their production processes and their uses to combat iron deficiencies

Abstract

The present invention relates to iron nanoclusters having the following characteristics: - they are coated on their surface by a mixed layer comprising histidine (His), acetate ions (Ac) and ascorbate ions (Asc); - they are spherical in shape; - they have a hydrodynamic diameter ranging from 0.6 to 2.0 nm, and preferably less than 1.0 nm; - they have a metallic core diameter ranging from 0.5 to 1.5 nm, and preferably less than 1.0 nm; - they have a stability over time ranging from 5 to 20 weeks when the nanoclusters are in liquid form and stored at a temperature of 4 °C; - they have a stability over time of at least 12 months, and preferably from 12 to 18 months, when the nanoclusters are in dry form and stored at a temperature of 4 °C and under nitrogen;They exhibit spectrophotometric properties, with a secondary peak in the UV-visible spectrum at 300 ± 15 nm and a fluorescence spectrum with excitation wavelengths of 364 ± 15 nm and emission wavelengths of 415 ± 15 nm. These iron nanoclusters can be designated by the formula "FeNC@HisAcAsc". The invention also relates to a method for the preparation of these iron nanoclusters and their uses in combating iron deficiencies.

Description

technical field

[0001] The present invention falls within the field of chemistry, and more particularly pharmaceutical chemistry. The invention relates to iron nanoclusters, their production processes, and their uses in combating iron deficiencies, and more particularly in the prevention and / or treatment of pathologies causing iron deficiency. Previous technique

[0002] Iron deficiency, also called iron deficiency, refers to a lack of iron in the body. Iron is a metal essential for the body's functions and is obtained solely through diet. Iron deficiency is one of the most widespread mineral deficiencies in the world: more than 1.5 billion people worldwide suffer from iron deficiency.

[0003] Iron is present in all cells of the human body and is responsible for many vital functions, including oxygen transport through its presence in hemoglobin. Iron deficiency can interfere with these vital functions, the first sign of which is microcytic anemia, which in extreme cases can lead to death.

[0004] The majority of iron in the body (70%) is in heme form, meaning it is bound to hemoglobin in the blood (65%) or to myoglobin in muscles (5%). The remaining iron is in non-heme form (ferritin, transferrin, etc.).

[0005] Iron absorption is a finely regulated mechanism. When the body's iron reserves decrease, its absorption rate increases, and conversely, when iron reserves are high, absorption decreases, thus preventing excessive iron accumulation in the body (iron accumulation leads to hemochromatosis).

[0006] Diet provides an average of 10 to 15 mg of iron per day. Only 1 to 2 mg are absorbed in the upper part of the small intestine. Iron absorption depends on its type, the quality of the meal, and the individual's iron stores. 15 to 25% of heme iron is absorbed, compared to 2 to 20% of non-heme iron.

[0007] Iron deficiency can have several origins: increased needs in an individual, decreased intake, malabsorption, chronic bleeding and various pathologies.

[0008] Iron deficiencies are generally treated with oral iron preparations (tablets, capsules, powder, drops, syrup, etc.). When ingested orally, the preparation reaches the stomach, then the iron is absorbed through the intestinal lining and thus enters the bloodstream.

[0009] Oral iron supplementation can, however, have several drawbacks, such as poor digestive tolerance. Patients may experience stomach upset because some preparations release iron directly in the stomach. Since the intestine can only absorb a limited amount of iron (a maximum of 20 to 25% for heme iron), a relatively large portion of the ingested iron is excreted. Therefore, orally administered iron can cause frequent side effects, including nausea, diarrhea or constipation, and black stools; less commonly, abdominal pain, a metallic taste in the mouth, or a blackening of the teeth that disappears upon discontinuation of treatment.

[0010] Oral iron supplementation also requires consideration of potential drug and food interactions. Indeed, some medications and foods can bind oral iron in the digestive tract, forming non-absorbable complexes that significantly reduce its absorption.

[0011] Furthermore, oral iron preparations are often unsuitable for certain types of patients, such as those with iron malabsorption due to chronic inflammatory bowel disease. In some cases, oral iron preparations are simply ineffective, as iron supplementation may not improve the individual's iron deficiency, primarily because the iron cannot cross the intestinal barrier.

[0012] Thus, when it is not / no longer possible to administer oral iron preparations, for the reasons mentioned above, or because there is a clinical need to administer iron rapidly, or because the recommended iron dosage in a subject is greater than the capacity for iron absorption by the digestive tract, then it is possible to administer iron intravenously, which means that the iron is administered directly into the blood by an intravenous infusion.

[0013] The advantage of intravenous administration is that the iron enters the bloodstream directly, and from there, the entire body. However, intravenous administration carries potential side effects such as hypersensitivity reactions, hypophosphatemic osteomalacia, impaired liver or kidney function, infection, extravasation during infusion, etc., which make hospital administration essential. This necessitates hospitalization with the inherent structural needs of this type of care (available bed, medical and nursing staff). Furthermore, the iatrogenic risks of this type of infusion are not negligible.

[0014] In view of these difficulties, the need to find an alternative to intravenous or oral iron treatments remains to this day.

[0015] In the present invention, new iron nanoclusters are proposed to combat all types of iron deficiencies, said nanoclusters being advantageously administered orally, without presenting the aforementioned disadvantages.

[0016] Iron nanoclusters have already been proposed in the literature to combat iron deficiency. For example, US patent application 2016 / 0022733 describes iron oxide nanocomposites capped with folic acid, nicotinic acid, and ascorbic acid for use in the treatment of anemia, these nanocomposites being intended for oral administration. US patent application 2016 / 008292 describes iron oxide nanoparticles coated with biocompatible polymers possessing polyethylene glycol and silane groups, these groups being covalently linked via a bonding group. These nanoparticles are proposed for the parenteral treatment of anemia.

[0017] However, the inventors deserve credit for developing new iron nanoclusters with characteristics particularly suited to combating iron deficiencies. They also deserve credit for developing an original synthesis process for these iron nanoclusters. Summary

[0018] The present invention relates to iron nanoclusters which have the following characteristics: They are coated on their surface with a mixed layer comprising histidine (His), acetate ions (Ac), and ascorbate ions (Asc); they are spherical in shape; they have a hydrodynamic diameter ranging from 0.6 to 2.0 nm, and preferably less than 1.0 nm; they have a metallic core diameter ranging from 0.5 to 1.5 nm, and preferably less than 1.0 nm; they exhibit a stability over time ranging from 5 to 20 weeks when the nanoclusters are in liquid form and stored at a temperature of 4°C; they exhibit a stability over time of at least 12 months, and preferably 12 to 18 months when the nanoclusters are in dry form and stored at a temperature of 4°C under nitrogen; they exhibit spectrophotometric properties, with a shoulder on the UV-Visible spectrum at 300 ± 15 nm and a spectrum of fluorescence with excitation wavelengths of 364 ± 15 nm and emission wavelengths of 415 ± 15 nm, said iron nanoclusters being able to be designated by the formula "FeNC@HisAcAsc". The nanoclusters of the invention may, however, be referred to interchangeably in the following as "nanoclusters", "iron nanoclusters", "FeNC nanoclusters", "FeNC", "NC-Fe », (“FeNC” or “NC-Fe” meaning “iron nanoclusters”), “FeNC@HisAcAsc nanoclusters”, “FeNC@HisAcAsc”.

[0019] The formula "FeNC@HisAcAsc" is the most explicit since it describes that the iron nanocluster includes on its surface a layer comprising both histidine, acetate and ascorbate ions.

[0020] The invention also relates to a method for preparing said iron nanoclusters which comprises the following steps: reaction between iron(II) acetate and histidine to obtain a mixture of iron acetate and histidine, the molar ratio histidine / iron(II) acetate being greater than or equal to 8, preferably from 8 to 200, and more preferably from 80 to 200, reaction between the mixture of iron acetate and histidine with ascorbic acid to obtain a mixture of iron acetate, histidine and ascorbic acid, the molar ratio ascorbic acid / iron(II) acetate being greater than or equal to 12, preferably from 12 to 700, and more preferably from 130 to 700, recovery of iron nanoclusters coated on their surface with a mixed layer comprising histidine, acetate ions and ascorbate ions.

[0021] The invention also relates to iron nanoclusters for use in: in the prevention and / or treatment of pathologies generating iron deficiency (such as iron deficiency anemia), to combat iron deficiencies.

[0022] Finally, the invention also relates to a composition comprising the iron nanoclusters of the invention, said composition being a drug, a food supplement or a food composition.

[0023] The composition of the invention is further characterized in that it is in a form suitable for oral administration. Brief description of the drawings

[0024] Other features, details and advantages will become apparent upon reading the detailed description below and analyzing the attached figures. There figure 1is a schematic representation of an iron nanocluster "FeNC@HisAcAsc" of the invention, consisting of a metallic iron core surrounded by a mixed corona comprising histidine, acetate and ascorbate ions. figure 2 This is a high-performance liquid chromatography (reversed-phase partitioning) analysis of iron nanoclusters, showing the presence of acetate ions on the surface of the iron metallic core. The chromatogram was obtained on previously purified fractions (size-exclusion chromatography) of iron nanoclusters. A chromatogram of a sodium acetate reference solution was also performed. figure 3This is a high-performance liquid chromatography (reverse-phase partitioning) analysis of iron nanoclusters, showing the presence of histidine and ascorbate ions on the surface of the iron metallic core. The chromatogram was obtained on previously purified fractions (size-exclusion chromatography) of iron nanoclusters. A chromatogram of a reference solution of histidine and ascorbic acid was also performed. figure 4 illustrates the hydrodynamic diameter (in nanometers) of iron nanoclusters evaluated by dynamic light scattering. figure 5 illustrates the hydrodynamic diameter (in nanometers) of iron nanoclusters evaluated by Taylor scatter. figure 6 A UV-Visible spectrum of iron nanoclusters. figure 7 is a fluorescence spectrum of iron nanoclusters. figure 8illustrates the results of the viability test (MTT) on HepG2 cells (hepatocellular carcinoma cells) that proliferated in the presence of iron, said iron being in the form of iron(III) nitrate (control, standard) (represented by FeNO3) or in the form of the iron nanoclusters of the invention at varying concentrations (represented by NC-Fe1x; NC-Fe1 / 2, NC-Fe1 / 4, NC-Fe1 / 8, NC-Fe1 / 16 and NC-Fe1 / 32).

[0025] The "Negative" corresponds to the "IMDM" selective culture medium without iron supplementation. The histogram with horizontal hatching on the far left of each histogram group corresponds to day 1+3 of the cell viability test. The histogram immediately next to it corresponds to day 1+5, and the one after that corresponds to day 1+7. The black histogram on the far right of each histogram group corresponds to day 1+10. Detailed description Iron nanoclusters

[0026] Thus, the present invention relates to iron nanoclusters characterized in that they: are coated on their surface with a mixed layer comprising histidine (His), acetate ions (Ac) and ascorbate ions (Asc), have a spherical shape, have a hydrodynamic diameter ranging from 0.6 to 2.0 nm, and preferably less than 1.0 nm, have a metallic core diameter ranging from 0.5 to 1.5 nm, and preferably less than 1.0 nm, have a stability over time ranging from 5 to 20 weeks when the nanoclusters are in liquid form and stored at a temperature of 4°C, have a stability over time of at least 12 months, and preferably 12 to 18 months when the nanoclusters are in dry form and stored at a temperature of 4°C and under nitrogen, have spectrophotometric properties, with a shoulder on the UV-Visible spectrum at 300 ± 15 nm and a fluorescence spectrum with wavelengths excitation wavelength of 364 ± 15 nm and emission wavelengths of 415 ± 15 nm, said iron nanoclusters being able to be designated by the formula “FeNC@HisAcAsc”.

[0027] The iron nanoclusters that are the subject of the invention are metallic nanoclusters. A metallic nanocluster consists of the association of tens of atoms of a metallic element (in this case iron in the invention) with a metallic core diameter less than or equal to 2.0 nanometers (nm).

[0028] The nanoclusters of the invention consist of a metallic iron core covered / coated / surrounded by a mixed layer / crown comprising histidine, acetate ions and ascorbate ions.

[0029] The terms "crown" and "layer" may be used interchangeably in the application. The term "mixed" is used to indicate that the crown or layer surrounding the iron core comprises both histidine, acetate, and ascorbate ions.

[0030] Similarly, the verbs "cover / recover / surround" can be used interchangeably to indicate that the iron core comprises over its entire surface a layer / crown of histidine, acetate ions and ascorbate ions.

[0031] The entire iron nanocluster has a spherical shape.

[0032] The formula "FeNC@HisAcAsc" in the sense of the invention designates a nanocluster consisting of an iron metallic core coated with said mixed layer of histidine, acetate, and ascorbate ions. The iron nanoclusters of the invention thus advantageously comprise three ligands on the surface of the iron core, namely histidine, acetate, and ascorbate ions. These three ligands are linked to the iron metallic core by coordination bonds.

[0033] The mixed crown comprising histidine, acetate and ascorbate ions gives the nanoclusters of the invention in particular very high stability and low reactivity.

[0034] A "low reactivity" means low degradation, particularly related to oxidation (for example, due to oxygen in the air).

[0035] The stability of the nanoclusters of the invention means that the structure and properties of the nanoclusters are maintained over time at a storage temperature of 4°C. Maintaining the structure means, in particular, that the composition of the nanocluster (metallic core surrounded by the mixed layer / corona as defined above), its shape, and its diameter (of the metallic core and hydrodynamic core) are conserved over time.

[0036] The "liquid form" of iron nanoclusters refers to a liquid solution or mixture of iron nanoclusters. The 5- to 20-week stability mentioned above applies to iron nanoclusters in liquid form when stored at a temperature of 4°C.

[0037] The term "dry form" refers to a solid form that can be ground into powder if necessary. The 12- to 18-month stability mentioned above applies to iron nanoclusters in dry form when stored at a temperature of 4°C under nitrogen.

[0038] Depending on their form (liquid or solid), their stability over time will therefore differ. The nanoclusters of the invention exhibit spectrophotometric properties, particularly fluorescence, which are characteristic of this scale, namely a metallic core diameter of less than or equal to 2 nm, which is intermediate between the molecule and the nanoparticle.

[0039] The metallic core diameter or metallic diameter refers, as its name suggests, to the diameter formed solely by the iron metal.

[0040] The hydrodynamic diameter takes into account the diameter of the iron core plus its layer / crown of histidine, acetate, and ascorbate ions. The hydrodynamic diameter therefore refers to the diameter of the entire iron nanocluster.

[0041] According to one embodiment of the invention, the iron nanoclusters have a metallic core diameter and a hydrodynamic diameter that are almost equivalent, preferably less than 1.0 nm.

[0042] However, the diameter of the metallic core will of course always be smaller than the hydrodynamic diameter.

[0043] The metallic core diameter is evaluated by transmission electron microscopy while the hydrodynamic diameter is evaluated by dynamic light scattering and / or Taylor scattering.

[0044] According to an advantageous embodiment of the invention, the iron nanoclusters are in liquid or dry form.

[0045] The dry form of nanoclusters is advantageous in particular because it allows for easy storage, preservation and transport.

[0046] According to yet another advantageous embodiment, the iron nanoclusters of the invention are characterized in that they exhibit at least one of the following characteristics: They are able to cross the intestinal barrier, they exhibit good bioavailability, they are biocompatible, they are biodegradable, they are freeze-dried, they are not toxic to the human body, and they do not accumulate in organs such as the liver, spleen, kidneys, or lungs. According to an advantageous embodiment, the iron nanoclusters of the invention possess all the characteristics described above.

[0047] The fact that the nanoclusters of the invention are not sequestered in said organs is primarily due to their small size (hydrodynamic diameter less than or equal to 2.0 nm, and preferably less than 1.0 nm). The size of the nanoclusters of the invention allows for longer circulation in the bloodstream compared to larger compounds. More specifically, the small size of the nanoclusters allows them to cross membranes (particularly the digestive membrane) without passing through physiological absorption systems via a persorption phenomenon (spontaneous passage through the pores of a physiological system). This persorption phenomenon is the source of the toxicity risks associated with nanoclusters, but it becomes a therapeutic option if the quantity of nanocluster intake is controlled.

[0048] The nanoclusters of the invention possess surface properties enabling them to cross the intestinal barrier, which represents a significant advantage over oral iron preparations that are often unable to cross the intestinal barrier.

[0049] "Good bioavailability" means that orally administered iron nanoclusters effectively reach the general circulation and are well distributed to target organs.

[0050] "Biocompatibility" refers to the fact that iron nanoclusters are well accepted by the different organs of the body without being toxic to these organs.

[0051] The fact that nanoclusters are biodegradable means that their degradation releases substances that are metabolized or eliminated without problems by the body (iron, histidine, acetate and ascorbate).

[0052] According to an advantageous embodiment of the invention, the nanoclusters are freeze-dried. Indeed, they can be freeze-dried because they are perfectly stable. Freeze-drying thus allows for the easy storage, preservation, and transport of the nanoclusters. The stability of the nanoclusters is as defined above.

[0053] The advantageous properties of the nanoclusters of the invention are due in particular to the original combination of its constituents, namely iron, histidine, acetate and ascorbate ions.

[0054] To the Inventors' knowledge, iron nanoclusters comprising a mixed crown / layer of histidine, acetate and ascorbate ions surrounding a metallic iron core, and exhibiting the advantageous properties described above, have never been described to date. The process of preparing iron nanoclusters

[0055] The present invention also relates to a process for preparing iron nanoclusters as defined above, characterized in that it comprises the following steps: reaction between iron(II) acetate and histidine to obtain a mixture of iron acetate and histidine, the molar ratio histidine / iron(II) acetate being greater than or equal to 8, preferably from 8 to 200, and more preferably from 80 to 200. reaction between the mixture of iron acetate and histidine with ascorbic acid to obtain a mixture of iron acetate, histidine and ascorbic acid, the molar ratio ascorbic acid / iron(II) acetate being greater than or equal to 12, preferably from 12 to 700, and more preferably from 130 to 200, recovery of iron nanoclusters.

[0056] The molar ratios defined above, respectively between histidine and iron acetate, and between ascorbic acid and iron acetate, are important because they allow the histidine, acetate, and ascorbate ligands to bind to the iron core. This results in nanoclusters with three ligands on the surface of the iron core, these three ligands being linked to the iron core surface by coordination bonds.

[0057] Ascorbic acid is a reducing agent. The reaction between the mixture of iron acetate and histidine with ascorbic acid is specifically a reduction reaction of the mixture of iron acetate and histidine with ascorbic acid. Ascorbic acid notably allows the production of non-toxic iron nanoclusters.

[0058] The present invention results in particular from the Inventors' unexpected discovery that the original combination of reagents used, namely iron acetate, histidine and ascorbic acid, and in the proportions defined above, makes it possible to obtain iron nanoclusters with particularly advantageous properties.

[0059] The excellent stability of the nanoclusters of the invention is one example.

[0060] According to one embodiment of the invention, iron nanoclusters can be prepared more specifically according to the "in solution" protocol or according to the "solid phase" protocol. Each of these two synthesis routes conforms to the process described above. 1 / Protocol in solution

[0061] According to an advantageous embodiment of the invention, the preparation process as defined above is more particularly characterized in that it is carried out under an inert gas and in that: Iron acetate is in solution form and histidine is in powder form. A solution of iron acetate and histidine is prepared by adding histidine to the iron acetate solution. The iron acetate and histidine solution is adjusted to a pH value of 11 to 13, and preferably 12. Ascorbic acid is in powder form. A solution of iron acetate, histidine, and ascorbic acid is prepared by adding ascorbic acid to the iron acetate and histidine solution whose pH has been adjusted to the aforementioned values. The iron acetate, histidine, and ascorbic acid solution is stirred for 2 to 6 hours, and preferably 4 hours, at a temperature of 35°C to 45°C, and preferably 40°C. A solution containing iron nanoclusters is obtained at the end of In the previous stirring step, the solution containing the iron nanoclusters is optionally dialyzed to obtain a purified solution of iron nanoclusters.The solution containing the iron nanoclusters, possibly dialyzed, is then possibly lyophilized to obtain a dry form of iron nanoclusters.

[0062] The solution containing the iron nanoclusters, which may be dialyzed, exhibits a stability of 5 to 20 weeks at a storage temperature of 4°C. Dialysis removes any components not bound to the iron core, such as excess histidine or ascorbic acid, or any residual iron that may be present in the nanocluster solution. The layer containing histidine, acetate, and ascorbate ions is bound to the iron core by coordination bonds.

[0063] The dry form of iron nanoclusters, obtained after freeze-drying, exhibits a stability over time of at least 12 months, and preferably 12 to 18 months, at a storage temperature of 4°C and under nitrogen.

[0064] The dry form of iron nanoclusters can be reconstituted at any time by mixing it with a reconstitution solvent, such as purified water. "Reconstitution" refers to the simple process of mixing the dry form or the lyophilized powder with a solvent.

[0065] Analysis of the iron nanocluster solution, obtained after reconstitution of the dry form, shows that the iron nanoclusters exhibit all the properties defined above and are therefore exactly the same as those directly obtained after their preparation process.

[0066] The iron nanocluster solution, obtained after reconstitution of the dry form, exhibits a stability over time ranging from 5 to 12 weeks at a storage temperature of 4°C, and preferably under nitrogen.

[0067] The preparation process as defined above is further characterized in that it also comprises at least one characteristic chosen from: The inert gas is nitrogen, the iron acetate solution is prepared by adding iron acetate to ultrapure filtered water, the iron acetate solution has a concentration ranging from 0.5 to 5.0 mM, the histidine concentration is higher than the concentration of the iron acetate solution, the pH of the iron acetate and histidine solution is adjusted using sodium hydroxide, the ascorbic acid concentration is equal to the histidine concentration, the solution containing the iron nanoclusters, possibly dialyzed, has an iron concentration ranging from 14 to 112 µg / mL, the solution containing the iron nanoclusters, possibly dialyzed, is lyophilized to obtain a dry form of iron nanoclusters.

[0068] According to an advantageous embodiment, the process of the invention has all the characteristics described above. 2 / Solid-phase protocol

[0069] According to another advantageous embodiment of the invention, the process for preparing iron nanoclusters as defined above is more particularly characterized in that: Iron acetate and histidine are in powder form. A powdered mixture of iron acetate and histidine is obtained by mixing each of the powders. This powdered mixture is ground until a homogeneous, colored powder is obtained. The homogeneous powdered mixture of iron acetate and histidine is placed in a reactor. Ascorbic acid is in powder form. Ascorbic acid is added to the reactor containing the homogeneous powdered mixture of iron acetate and histidine. The resulting powdered mixture of iron acetate, histidine, and ascorbic acid is stirred, and then ultrapure, filtered water is added dropwise to the reactor. The reactor is then placed under an inert gas and protected. light, the mixture of iron acetate, histidine,Ascorbic acid and water are kept under stirring in the reactor for 16 to 36 hours, and preferably 24 hours. A liquid mixture containing iron nanoclusters is obtained at the end of the previous stirring step. The liquid mixture containing iron nanoclusters is optionally dialyzed to obtain a purified liquid mixture of iron nanoclusters. The liquid mixture containing iron nanoclusters, optionally dialyzed, is optionally lyophilized to obtain a dry form of iron nanoclusters.

[0070] The preparation process as defined above is further characterized in that it also comprises at least one characteristic chosen from: the histidine concentration is greater than the iron acetate concentration, the ascorbic acid concentration is equal to the histidine concentration, the water added to the reactor is ultrapure filtered water, the inert gas is nitrogen, the liquid mixture which includes the iron nanoclusters, possibly dialyzed, has an iron concentration ranging from 1500 to 15000 µg / mL, the liquid mixture which includes the iron nanoclusters, possibly dialyzed, is lyophilized to obtain a dry form of iron nanoclusters.

[0071] According to another embodiment of the invention, the dry form of iron nanoclusters obtained after lyophilization (according to the "solution" or "solid-phase" protocol) is stored under nitrogen, preferably in vials, and preferably at 4°C. Under such conditions, the iron nanocluster powder can be stored for a period of at least 12 months, and preferably from 12 to 18 months, without any alteration in the stability of the iron nanoclusters.

[0072] The reconstitution of the nanocluster powder at the end of this period shows that the nanoclusters are the same as those obtained directly after their preparation (according to the "solution" protocol or the "solid phase" protocol). Indeed, the iron nanoclusters exhibit all the properties defined above. The use of iron nanoclusters

[0073] The invention also relates to iron nanoclusters as defined above or obtained according to the processes as defined above, for use as a medicinal product.

[0074] More particularly, the invention relates to iron nanoclusters as defined above or obtained according to the processes as defined above, for use in the prevention and / or treatment of pathologies generating an iron deficiency.

[0075] Iron deficiency anemia is an example of a pathology that causes iron deficiency. The invention further relates to iron nanoclusters as defined above or obtained according to the processes defined above, for use in combating iron deficiencies.

[0076] In this application, iron deficiency means iron deficiency in the broadest sense, namely iron deficiency with or without iron deficiency anemia.

[0077] Another object of the invention lies in a composition characterized in that it comprises iron nanoclusters as defined above or obtained according to the processes as defined above.

[0078] The composition of the invention may be a medicinal product, a food supplement or a food composition.

[0079] The amount of iron in a given ingredient can determine whether it is considered a dietary supplement or a medication. A dietary supplement should contain less iron than a medication.

[0080] As an example of food composition, one can cite infant milks which are supplemented with iron, and more particularly with the iron nanoclusters of the invention.

[0081] According to an advantageous embodiment of the invention, the composition is in a form suitable for oral administration.

[0082] Iron nanoclusters can advantageously be administered orally because they are able to pass the intestinal barrier without difficulty, particularly due to their small size.

[0083] The use of the iron nanoclusters of the invention advantageously makes it possible to avoid the intravenous route.

[0084] According to yet another advantageous embodiment, the composition of the invention comprising the iron nanoclusters comprises a quantity of iron less than the quantity of iron usually present in a conventional oral preparation, whether it be a drug or a food supplement.

[0085] Advantageously, the composition of the invention does not present the disadvantages that can be encountered with a conventional oral preparation, whether it be a drug or a food supplement. Examples

[0086] The following examples illustrate the invention, they do not limit it in any way. Example 1 Preparation of iron nanoclusters

[0087] This example describes respectively the two possible synthesis routes for preparing the iron nanoclusters of the invention, namely the "solution protocol" and the "solid phase protocol". 1 / Protocol in solution Reagents used :

[0088] Iron (II) acetate [Fe(CH 3 COO) 2 ], M = 171.83 g / mol (Sigma Aldrich, Cas 3094-87-9); L(-)-Histidine, M=155.15 g / mol (Merck, Cas 71-00-1); Ascorbic acid, M=176.12 g / mol (Sigma Aldrich, Cas 50-81-7); 1M NaOH solution, M= 40.00 g / mol (VWR, Cas 1310-73-2). Precautions to take

[0089] The synthesis is carried out under inert gas (nitrogen). The glassware is washed with aqua regia (1 volume of 65% nitric acid to 2 volumes of 37% hydrochloric acid).

[0090] Ultra-pure water is used and filtered through a filter with a pore diameter of 0.2 µm.

[0091] Iron acetate is in powder form and is stored under nitrogen. Once the iron has been weighed, the remaining stock must be returned to nitrogen.

[0092] Since iron nanoclusters are intended to be studied in vivo, it is necessary to work under a clean hood and to clean all equipment used with 70% v / v ethanol. Preparation of a 2.5 mM iron acetate stock solution

[0093] A quantity of 42.9 mg of iron acetate is placed in a 100 mL volumetric flask. Ultrapure filtered water is added up to the flask's calibration mark. A 2.5 mM iron acetate solution is obtained.

[0094] After complete dissolution, the iron acetate solution is transferred to a suitable container. This solution can be stored for one month in a refrigerator at 4°C. Synthesis of histidine-stabilized iron nanoclusters

[0095] A quantity of 500 µL of the iron acetate stock solution, as prepared in the previous step, is added to a round-necked flask capable of holding up to 50 mL of solution. Then, 4500 µL of filtered ultrapure water is added to the flask. The resulting iron acetate solution is called 1x.

[0096] The 1x iron acetate solution is stirred at 130 rpm using a multi-plate stirrer. 39 mg of histidine is added to the iron acetate solution. The iron acetate and histidine solution is stirred for 15 minutes.

[0097] The solution takes on a slightly red tint. After 15 minutes of stirring, the pH of the iron acetate and histidine solution is adjusted to 12 with 10 drops of 1M NaOH. 139 mg of ascorbic acid is added to the reaction mixture. The ascorbic acid is allowed to dissolve completely for 2 minutes. The flask (reactor) is then placed in a water bath at 40°C with stirring (speed set at 6) for 4 hours.

[0098] At the end of the synthesis, the resulting nanocluster solution is colorless. The iron nanocluster solution thus obtained is called 1x and has an iron concentration of 14 µg / mL. It is stored in a cool place, at 4°C.

[0099] The synthesis yield is 100%: there is no residual iron (element iron) in the nanocluster solution. Iron nanocluster solutions can be lyophilized. Iron acetate solutions with concentrations ranging from 1x has 8xare prepared to obtain iron nanocluster solutions 1x has 8x which have an iron concentration ranging from 14 to 112 µg / mL.

[0100] As an example, a solution of iron acetate 2x is prepared by placing 1000 µL of iron acetate stock solution in the flask and making up to 5000 µL with filtered ultrapure water. An iron acetate solution 4x is prepared by placing a quantity of 2000 µL of iron acetate stock solution in the flask and making up to 5000 µL with ultrapure filtered water etc.

[0101] Iron nanocluster solutions 1x has 8x The products thus obtained are kept cool at 4°C. Dialysis of iron nanoclusters

[0102] The iron nanocluster solution 1x obtained in the previous step is purified by dialysis.

[0103] A dialysis cell is prepared (X12 Float-a-lyzer G2 CE MWCO 100-500 D, Reference 1511160), and a 150 mL beaker is filled with 100 mL of filtered ultrapure water. The dialysis cell is filled with the filtered ultrapure water using a Pasteur pipette. The dialysis cell is placed in the beaker and stirred (130 rpm). The cell is allowed to hydrate and wash for 1 hour.

[0104] The water is then replaced with a new 100 mL volume of ultrapure filtered water. The dialysis cell is emptied using a Pasteur pipette and then filled with the iron nanocluster solution. 1x, which is left under agitation all night (for 12 hours) at a temperature between 2 and 6°C.

[0105] The dialyzed solution of iron nanoclusters 1x The solution thus obtained is transferred into a suitable container and stored at a temperature of 4°C.

[0106] Dialysis does not affect the iron concentration of nanoclusters. Thus, the iron concentrations of dialyzed iron nanocluster solutions are identical to those of non-dialyzed solutions.

[0107] Iron nanocluster solutions, possibly dialyzed, can be lyophilized. The iron concentrations of dialyzed iron nanocluster solutions are identical to those of non-dialyzed solutions, and range from 14 to 112 µg / mL for iron acetate solutions with a concentration ranging from 1x has 8x. 2 / Solid-phase protocol Reagents used and precautions to take

[0108] Iron(II) acetate, L(-)-histidine, and ascorbic acid are the same as those used in the solution protocol. Sodium hydroxide is not required in the solid-phase protocol.

[0109] The precautions to be taken are the same as those for the solution protocol. Synthesis of histidine-stabilized iron nanoclusters

[0110] A quantity of 23 mg of iron acetate is weighed out and placed inside an agate mortar. A quantity of 1.7 g of histidine is then weighed out. One volume of histidine powder to one volume of iron acetate powder is added, taking care to thoroughly grind the powders with the pestle until a mixture of homogeneous color and appearance is obtained. This operation is repeated until all the histidine remains.

[0111] The final mixture of the two powders should be red and homogeneous. The mixture is then transferred to a 50 mL single-neck round-bottom flask (ground-neck NS 19 / 26) using a spatula. Three grams of ascorbic acid are weighed and added to the flask. An olive-shaped magnetic stirrer is placed at the bottom of the flask. Five milliliters of ultrapure water is filtered using a 5 mL plastic syringe and added dropwise to the reactor. A liquid mixture is obtained.

[0112] The reactor is sealed with a hinged, skirted stopper (19.4 mm diameter) and pressurized with nitrogen using a balloon. The reactor is then wrapped in aluminum foil and stirred at 200 rpm for the duration of the reaction. The reaction must be allowed to complete for 24 hours. At the end of the reaction, the product, a liquid, is red in color.

[0113] The resulting liquid, containing iron nanoclusters, has an iron concentration of 1500 µg / mL, and is called 100x.

[0114] The iron concentration of the nanoclusters is of course dependent on the amount of iron acetate used at the start of the process of the invention.

[0115] The operations described above are repeated to obtain iron concentrations ranging from 1500 (100x) at 15000 µg / mL (1000x)for quantities of iron acetate at the start of the process of the invention (solid phase protocol) ranging from 23 to 230 mg.

[0116] The resulting liquid containing the iron nanoclusters is then transferred into a suitable plastic container (the final volume is not 5 mL but slightly more, approximately 8.5 mL). The liquid containing the iron nanoclusters is either stored at 4°C or transferred to a freeze dryer.

[0117] Lyophilization is carried out in 1 mL fractions, without the addition of any further reagents. After lyophilization, the contents of the vial (which include the iron nanoclusters in dry form) are placed under nitrogen and stored at 4°C.

[0118] The dry form of iron nanoclusters can be reconstituted at any time in 1 mL of purified water. The reconstituted iron nanocluster solution is stored at 4°C, preferably under nitrogen. Example 2 Characterization of iron nanoclusters

[0119] Iron nanoclusters such as those obtained in Example 1, whether using the solution protocol or the solid phase protocol, are characterized with respect to their structure, sizes, spectrophotometric properties and stability. Structure of iron nanoclusters

[0120] The iron nanoclusters of the invention are particularly spherical in shape. They consist of a metallic iron core covered by a mixed corona comprising histidine, acetate, and ascorbate ions. figure 1 is a schematic representation of an iron nanocluster of the invention, which can also be designated by the formula "FeNC@HisAcAsc".

[0121] The presence of acetate ions on the surface of iron nanoclusters has been demonstrated by high-performance liquid chromatography (HPLC), more specifically by reversed-phase polarity partition chromatography.

[0122] Iron nanoclusters in the dialysis solution 1x such as obtained in example 1, see point 1 / "Protocol in solution", are destroyed (total dissolution and return to the different elements composing the structure of the nanoclusters) by a chemical process, namely dissolving in a concentrated acid (HCl) then a concentrated base (NaOH), then are analyzed by HPLC in comparison to a sodium acetate control.

[0123] The results obtained are illustrated in the chromatogram of the figure 2 . The peak at 3.6 min associated with acetate ions (control, see bottom trace) is found in iron nanoclusters (see top trace), thus showing the presence of acetate ions on the surface of the metallic core.

[0124] The presence of histidine and ascorbate ions on the surface of iron nanoclusters was also demonstrated by high-performance liquid chromatography, more specifically after purification of the nanoclusters in solution by size-exclusion chromatography, compared to a histidine and ascorbic acid control.

[0125] The results obtained are illustrated in the chromatogram of the figure 3 .

[0126] The peak at 2.1 min associated with histidine (control, see bottom trace) is found in the iron nanoclusters (see top trace), thus showing the presence of histidine on the surface of the metallic core.

[0127] The peak at 3.0 min associated with ascorbate ions (control, see bottom trace) is found in the iron nanoclusters (see top trace), thus showing the presence of ascorbate ions on the surface of the metallic core. Size of iron nanoclusters

[0128] The hydrodynamic diameter (Dh) of the iron nanoclusters was evaluated by dynamic light scattering ( figure 4 ) (173° angle, 530 nm laser, 25°C temperature on Nanosizer Malvern) and by Taylor scatter analysis ( figure 5 ).

[0129] The dynamic light scattering analysis method involves analyzing the Brownian motion of particles and modeling it using the Stokes-Einstein equation. The Taylor scattering analysis method involves injecting a strip of solute into an open capillary tube (50 µm) and mobilizing it under the influence of a hydrodynamic flow (positive pressure 1 psi, parabolic velocity profile). The principle for determining the hydrodynamic radius is based on the Taylor-Aris relationship, which establishes the link between the spread of the solute peak (modeling a Gaussian distribution) and the molecular scattering coefficient.

[0130] The metallic diameter of the iron nanoclusters was evaluated by transmission electron microscopy (deposition on nickel grids, observations under beams operating at 200 kV (LaB6 cathode) Philips CM 200).

[0131] The diameters (hydrodynamic and metallic) of the iron nanoclusters were respectively evaluated immediately after their synthesis, whether it was the protocol in solution or in solid phase.

[0132] Regarding the solution protocol, the hydrodynamic and metallic diameters were evaluated on the solution of iron nanoclusters 1x, non-dialyzed and non-lyophilized, which has an iron concentration of 14 µg / mL.

[0133] Regarding the solid-phase protocol, the hydrodynamic and metallic diameters were evaluated on lyophilized samples obtained from the iron nanocluster liquid 100x, non-dialyzed, which has an iron concentration of 1500 µg / mL.

[0134] The average hydrodynamic diameter of iron nanoclusters, like the metallic diameter, is less than 1.0 nm. More specifically, it emerges from the Figures 4 and 5 than in dynamic light diffusion ( Fig. 4 ) and in Taylor's dispersion ( Fig. 5 ) the hydrodynamic diameter of the iron nanoclusters is equal to 0.69 ± 0.06 nm. Spectrophotometric properties

[0135] The spectrophotometric properties of iron nanoclusters were evaluated by UV-Visible spectroscopy ( figure 6 ) and by fluorescence spectroscopy ( figure 7 ), immediately following their synthesis.

[0136] The UV-vis spectrum shows a shoulder at 300 ± 15 nm ( Fig. 6 ), This confirms the existence of nanoclusters.

[0137] Iron nanocluster fluorescence exists with ( Fig. 7) an excitation wavelength of 364 ± 15 nm and an emission wavelength of 415 ± 15 nm, which also confirms the existence of nanoclusters.

[0138] The iron nanoclusters of the invention exhibit optical properties, in particular fluorescence, characteristic of this intermediate scale between the molecule and the nanoparticle. Stability of iron nanoclusters

[0139] The stability of iron nanoclusters was evaluated by measuring their hydrodynamic diameter in dynamic light scattering (angle 173°, laser 530 nm, temperature 25°C on Nanosizer Malvern).

[0140] The analyses were carried out on the iron nanoclusters obtained according to the solution protocol and according to the solid phase protocol.

[0141] Regarding the solution protocol, the analyses were performed on solutions of iron nanoclusters 1x(iron concentration of 14 µg / mL). Stability was evaluated a little over 5 weeks after the synthesis of said solutions.

[0142] It was found that the hydrodynamic diameter of the iron nanoclusters was still 0.69 ± 0.06 nm more than 5 weeks after their synthesis, which demonstrates their excellent stability.

[0143] Regarding the solid-phase protocol, the analyses were performed on: lyophilized samples obtained from iron nanocluster liquid 100x, non-dialyzed (iron concentration of 1500 µg / mL), the solutions reconstituted after lyophilization of said samples, in the same volume as that of the lyophilization.

[0144] The hydrodynamic diameter of the iron nanoclusters in the lyophilized samples is 0.70 nm after more than 5 weeks of storage under nitrogen.

[0145] After reconstitution of the samples, in the same volume as that of the lyophilization, the hydrodynamic diameter of the iron nanoclusters of the reconstituted samples is 0.76 nm, which demonstrates once again their excellent stability. Example 3 Evaluation of the toxicity of iron nanoclusters

[0146] This example describes the results of the viability test (MTT test) on HepG2 cells (hepatocellular carcinoma cells) that proliferated in the presence of iron, said iron being in the form of iron (III) nitrate (control, standard) or in the form of the iron nanoclusters of the invention at varying concentrations. Iron

[0147] The iron (III) nitrate (or ferric nitrate) used as a control is the chemical compound with the semi-developed formula "Fe(NO3)3", which is more particularly used in its nonahydrate form "Fe(NO3)3.9H2O".

[0148] A solution of iron nitrate nonahydrate is prepared to a total volume of 50 mL at a concentration of 100 mg / L of water, corresponding to an iron concentration of 14 µg / mL. This solution is filtered under a fume hood.

[0149] The iron nanocluster solution 1x, which includes an iron concentration of 14 µg / mL as prepared in example 1 (point 1 / solution protocol), is most particularly used.

[0150] A cascade dilution range of the iron nanocluster solution 1x is carried out at 1 / 2, 1 / 4, 1 / 8, 1 / 16 and 1 / 32nd (see below media 4 to 8 prepared). HepG2 cells

[0151] HepG2 cells are a cell line derived from the liver tissue of a patient with hepatocellular carcinoma (HCC). MTT Test

[0152] The MTT assay is a rapid colorimetric method for quantifying live cells within a sample. The reagent used is the tetrazolium salt "MTT" ("3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide"). The tetrazolium ring it contains is reduced to formazan by mitochondrial succinate dehydrogenase in active live cells. This forms a purple precipitate in the mitochondria.

[0153] The amount of precipitate formed is proportional to the number of living cells (but also to the metabolic activity of each cell). Therefore, after incubating the cells with MTT for a certain time at 37°C (approximately three hours), simply dissolve the cells, their mitochondria, and thus the purple Formazan precipitates in 100% DMSO (dimethyl sulfoxide).

[0154] A simple measurement of the optical density at 570 nm by spectroscopy makes it possible to know the relative quantity of living and metabolically active cells.

[0155] Therefore, if the test is to be quantitative, it is necessary to create a calibration curve for each trial. The reading is taken at 570 nm using a spectrophotometer (see figure 8 ). HepG2 cell growth conditions

[0156] HepG2 cells are grown in a complete culture medium “DMEM” (Dulbecco modified Eagle medium) in 24-well plates for 24h, then the medium is changed to a selective culture medium “IMDM*” (see below) + / - iron nanoclusters of the invention versus iron nitrate nonahydrate Fe(NO3)3.9H2O.

[0157] Fe 3+< is reduced to Fe 2+< and then passes through the plasma membrane via "DMT1" ("divalent metal transporter 1"). IMDM* selective culture medium:

[0158] IMDM (Modified Dulbecco medium according to Iscove), 10% FBS (fetal calf serum) (dialyzed), 1% antibiotics (mixture of penicillin and streptomycin), 1% (1 mM) pyruvate. Eight selective media are prepared. :

[0159] Medium 1: IMDM*; Medium 2: IMDM* + 1% Fe(NO 3 ) 3.9H 2 O (stock 100 mg / L); Medium 3: IMDM* + 1% iron nanocluster solution 1x (i.e., 14 µg / mL Iron equivalent); Medium 4: Dilute 1 / 2 of Medium 3 in IMDM*; Medium 5: Dilute 1 / 2 of Medium 4 in IMDM* (i.e., 1 / 4 of Medium 3); Medium 6: Dilute 1 / 2 of Medium 5 in IMDM* (i.e., 1 / 8 of Medium 3); Medium 7: Dilute 1 / 2 of Medium 6 in IMDM* (i.e., 1 / 16 of Medium 3); Medium 8: Dilute 1 / 2 of Medium 7 in IMDM* (i.e., 1 / 32 of Medium 3). Test duration

[0160] A plate is prepared for each stage (Day 1+3, Day 1+5, Day 1+7, Day 1+10). The overall duration of the test is 11 days.

[0161] The steps are as follows: Day 0: Inoculation in p24 (triplicates), 4000 cells / cm2<, complete DMEM medium (500 µL / Well); Day 1: Change to selective IMDM medium iron nanocluster solution versus Fe(NO3)3.9H2O (0.1 mg / L); Day 1+3: 1 plate stopped for MTT, change of medium for the rest; Day 1+5: 1 plate stopped for MTT, change of medium for the rest; Day 1+7: 1 plate stopped for MTT, change of medium for the rest; Day 1+10: 1 plate stopped for MTT, end of the test.

[0162] The results obtained are described in the figure 8 , Or : “Negative” corresponds to Medium 1, “FeNO3” corresponds to Medium 2, “NC-Fe 1x” corresponds to Medium 3, “NC-Fe 1 / 2” corresponds to Medium 4, “NC-Fe 1 / 4” corresponds to Medium 5, “NC-Fe 1 / 8” corresponds to Medium 6, “NC-Fe 1 / 16” corresponds to Medium 7, “NC-Fe 1 / 32” corresponds to Medium 8.

[0163] Repeated MTT viability tests at D+3, D+5, D+7 and D+10 indicate that treatment with the iron nanoclusters of the invention is not toxic to HepG2 cells treated with an equivalent dose of iron found in standard culture media in the form of iron nitrate nonahydrate Fe(NO3)3.9H2O at a concentration of 0.1 mg / L.

[0164] The negative control was treated with a medium without added iron, but the presence of fetal calf serum may have provided enough iron to allow cell growth (so the control is not completely negative).

[0165] MTT was weighed, dissolved at 5 mg / mL in native IMDM medium, filtered to 0.2µm under a PSM hood and stored at +4°C throughout the experimental protocol.

[0166] At each stage of the MTT test, the 5 mg / mL solution was diluted to 0.5 mg / mL in a complete IMDM medium (IMDM + FBS + antibiotics + pyruvate), but without iron, then incubated for 3 hours at 37°C.

[0167] At each change of medium, the used medium was collected and frozen at -20°C for subsequent assays of transferrin and ferritin.

[0168] In conclusion, this test demonstrates that the iron nanoclusters of the invention exhibit no toxicity, regardless of their concentration. HepG2 cells cultured in an iron-free medium and then with added iron show excellent viability.

Claims

1. Iron nanoclusters, characterized in that they: - are covered on their surface with a mixed layer comprising histidine (His), acetate ions (Ac), and ascorbate ions (Asc), - have a spherical shape, - have a hydrodynamic diameter ranging from 0.6 to 2.0 nm, and preferably of less than 1.0 nm, - have a metal core diameter ranging from 0.5 to 1.5 nm, and preferably of less than 1.0 nm, - exhibit a stability duration ranging from 5 to 20 weeks when the nanoclusters are in liquid form and are stored at a temperature of 4°C, - exhibit a stability duration of at least 12 months, and preferably from 12 to 18 months, when the nanoclusters are in dry form and are stored at a temperature of 4°C and under nitrogen, - exhibit spectrophotometric properties, with a shoulder on the UV-Visible spectrum at 300 ± 15 nm and a fluorescence spectrum with excitation wavelengths of 364 ± 15 nm and emission wavelengths of 415 ± 15 nm, it being possible to refer to said iron nanoclusters by using the formula "FeNC@HisAcAsc".

2. Iron nanoclusters according to claim 1, characterized in that they are in liquid form or dry form.

3. Iron nanoclusters according to claim 1 or claim 2, characterized in that they exhibit at least one of the following characteristics: - they are able to cross the intestinal barrier, - they exhibit good bioavailability, - they are biocompatible, - they are biodegradable, - they are able to be freeze-dried, - they are non-toxic to the human body, - they do not accumulate in organs such as the liver, spleen, kidneys, or lungs.

4. A method of preparing iron nanoclusters according to any one of claims 1 to 3, characterized in that it comprises the following steps: - reaction of iron (II) acetate with histidine in order to obtain a mixture of iron acetate and histidine, the molar ratio of histidine / iron (II) acetate being greater than or equal to 8, preferably ranging from 8 to 200, and more preferably ranging from 80 to 200, - reaction of the mixture of iron acetate and histidine with ascorbic acid in order to obtain a mixture of iron acetate, histidine, and ascorbic acid, the molar ratio of ascorbic acid / iron (II) acetate being greater than or equal to 12, preferably ranging from 12 to 700, and more preferably ranging from 130 to 700, - collecting the iron nanoclusters.

5. Method of preparation according to claim 4, characterized in that it is carried out under inert gas and in that: - the iron acetate is in solution form and the histidine is in powder form, - a solution of iron acetate and histidine is prepared by adding histidine to the iron acetate solution, - the solution of iron acetate and histidine is adjusted to a pH value ranging from 11 to 13, and preferably 12, - the ascorbic acid is in powder form, - a solution of iron acetate, histidine, and ascorbic acid is prepared by adding ascorbic acid to the solution of iron acetate and histidine for which the pH has been adjusted to the aforementioned values, - the solution of iron acetate, histidine, and ascorbic acid is stirred for 2 to 6 hours, and preferably 4 hours, at a temperature ranging from 35°C to 45°C, and preferably 40°C, - a solution comprising iron nanoclusters is obtained at the end of the previous stirring step, - the solution comprising iron nanoclusters is optionally dialyzed to obtain a purified solution of iron nanoclusters, - the solution comprising iron nanoclusters, optionally dialyzed, is optionally freeze-dried to obtain a dry form of iron nanoclusters.

6. Method according to claim 5, characterized in that it further comprises at least one characteristic selected from the following: - the inert gas is nitrogen, - the iron acetate solution is prepared by adding iron acetate to filtered ultrapure water, - the iron acetate solution has a concentration ranging from 0.5 to 5.0 mM, - the histidine concentration is higher than the concentration of the iron acetate solution, - the pH of the solution of iron acetate and histidine is adjusted using sodium hydroxide, - the ascorbic acid concentration is equal to the histidine concentration, - the solution comprising iron nanoclusters, optionally dialyzed, has an iron concentration ranging from 14 to 112 µg / mL, - the solution comprising iron nanoclusters, optionally dialyzed, is freeze-dried to obtain a dry form of iron nanoclusters.

7. Method of preparation according to claim 4, characterized in that: - the iron acetate is in powder form and the histidine is in powder form, - a powder mixture of iron acetate and histidine is obtained by mixing together each of the iron acetate and histidine powders, - the powder mixture of iron acetate and histidine is ground until a powder mixture of homogeneous color is obtained, - the homogeneous powder mixture of iron acetate and histidine is placed in a reactor, - the ascorbic acid is in powder form, - the ascorbic acid is added to the reactor comprising the homogeneous powder mixture of iron acetate and histidine, - the thus obtained powder mixture of iron acetate, histidine, and ascorbic acid is stirred, then water is added dropwise into the reactor, said water being filtered ultrapure water, - the reactor is placed under inert gas and protected from light, - the mixture of iron acetate, histidine, ascorbic acid, and water is left to stir in the reactor for 16 to 36 hours, and preferably 24 hours, - a liquid mixture comprising iron nanoclusters is obtained at the end of the previous stirring step, - the liquid mixture comprising iron nanoclusters is optionally dialyzed to obtain a purified liquid mixture of iron nanoclusters, - the liquid mixture comprising iron nanoclusters, optionally dialyzed, is optionally freeze-dried to obtain a dry form of iron nanoclusters.

8. Method according to claim 7, characterized in that it further comprises at least one characteristic selected from the following: - the histidine concentration is greater than the iron acetate concentration, - the ascorbic acid concentration is equal to the histidine concentration, - the water added to the reactor is filtered ultrapure water, - the inert gas is nitrogen, - the liquid mixture comprising iron nanoclusters, optionally dialyzed, has an iron concentration ranging from 1500 to 15000 µg / mL, - the liquid mixture comprising iron nanoclusters, optionally dialyzed, is freeze-dried to obtain a dry form of iron nanoclusters.

9. Method according to any one of claims 5 to 8, characterized in that the dry form of the iron nanoclusters is stored under nitrogen, preferably in vials, and preferably at 4°C, it being possible to store said powder for a period of at least 12 months, and preferably from 12 to 18 months, with no change in the stability of the iron nanoclusters.

10. Iron nanoclusters as defined in any one of claims 1 to 3 or as obtained according to the method of any one of claims 4 to 9, for use as medication.

11. Iron nanoclusters as defined in any one of claims 1 to 3 or as obtained according to the method of any one of claims 4 to 9, for use in the prevention and / or treatment of diseases causing iron deficiency.

12. Iron nanoclusters for use according to claim 11, characterized in that the disease causing an iron deficiency is iron deficiency anemia.

13. Iron nanoclusters as defined in any one of claims 1 to 3 or as obtained according to the method of any one of claims 4 to 9, for use in combating iron deficiencies.

14. Composition characterized in that it comprises iron nanoclusters as defined in any one of claims 1 to 3 or as obtained according to the method of any one of claims 4 to 9.

15. Composition according to claim 14, characterized in that it is a medication, a dietary supplement, or a food composition.

16. Composition according to claim 14 or 15, characterized in that it is in a form suitable for oral administration.

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