Iron nanoclusters, method for obtaining same and use thereof in combating iron deficiencies

Abstract

The invention relates to an iron nanocluster having the following features:-the surface of which is covered with a mixed layer comprising histidine (His), acetate ions (Ac) and ascorbate ions (Asc),-which has a spherical shape,-the hydrodynamic diameter of which is 0.6-2.0 nm, preferably less than 1.0 nm,-the metal core diameter of which is 0.5-1.5 nm, preferably less than 1.0 nm,-the metal core diameter of which is 0.5-1.5 nm, preferably less than 1.0 nm,-the surface of which is coated with a mixed layer comprising histidine (His), acetate ions (Ac) and ascorbate ions (Asc),-which has a spherical shape. The nanocluster has a stability duration of 5 to 20 weeks when the nanocluster is in liquid form and stored at a temperature of 4 DEG C, a stability duration of at least 12 months, preferably 12 to 18 months, when the nanocluster is in dry form and stored at a temperature of 4 DEG C and under nitrogen gas, and exhibits spectrophotometric properties, and the nanocluster has a stability duration of 5 to 20 weeks when the nanocluster is in liquid form and stored at a temperature of 4 DEG C and under nitrogen gas, preferably 12 to 18 months when the nanocluster is in dry form and stored at a temperature of 4 DEG C and under nitrogen gas. The iron nanocluster is characterized in that the shoulder peak in the UV-visible spectrum is located at 300 + / -15 nm, the excitation wavelength of the fluorescence spectrum is 364 + / -15 nm, the emission wavelength is 415 + / -15 nm, and the formula FeNC-HisAcAsc can be used for representing the iron nanocluster. The invention also relates to a method for producing these iron nanoclusters, and to the use thereof for combating iron deficiencies.

Description

Technical Field

[0001] The present invention relates to the field of chemistry, more particularly to the field of medicinal chemistry. The present invention relates to iron nanoclusters, methods for obtaining the same, and their use in combating iron deficiency, more particularly to their use in preventing and / or treating diseases causing iron deficiency. Background Art

[0002] Iron deficiency, also known as hypoferremia and low iron levels, is a lack of iron in the body. Iron is an essential metal for human function and is derived entirely from food. It is one of the most common mineral deficiencies in the world: more than 15 billion people worldwide suffer from it.

[0003] Iron is present in all cells of the human body and is responsible for numerous vital functions, including oxygen transport through its presence in hemoglobin. Iron deficiency interferes with this vital function, with the first symptom being microcytic anemia and, in extreme cases, death.

[0004] Most of the iron in the human body (70%) exists in the form of heme, that is, bound to hemoglobin in the blood (65%) or myoglobin in the muscles (5%). The rest of the iron exists in non-heme forms (ferritin, transferrin, etc.).

[0005] Iron absorption is a finely regulated mechanism. When the body's iron stores are low, iron absorption increases; conversely, when iron stores are high, absorption decreases, thereby preventing excess iron accumulation in the body (which can lead to iron overload).

[0006] The average daily diet provides 10-15 mg of iron. Only 1-2 mg is absorbed in the upper small intestine. Iron absorption depends on the type of iron, the quality of the diet, and the individual's iron stores. Heme iron is absorbed 1-25%, while non-heme iron is absorbed 2-20%.

[0007] Deficiency can have several causes: increased individual needs, reduced intake, malabsorption, chronic bleeding and various diseases.

[0008] Iron deficiency is usually treated with oral iron preparations (tablets, capsules, powders, drops, syrups, etc.) When taken orally, the preparation reaches the stomach, and the iron is then absorbed by the intestinal mucosa and enters the bloodstream.

[0009] However, oral iron supplements can have some disadvantages, such as poor digestive tolerance. People receiving treatment may complain of stomach pain because some preparations release iron directly once they reach the stomach. Since the intestines can only absorb a limited amount of iron (heme iron is a maximum of 20-25%), a large part of the iron ingested is excreted. Therefore, oral iron administration often leads to side effects such as nausea, diarrhea or constipation and black stools; more rarely, abdominal pain, a metallic taste in the mouth or blackening of the teeth, which disappear when treatment is stopped.

[0010] Oral iron intake also needs to consider its potential interactions with medications and foods. Indeed, certain medications and foods can bind oral iron in the digestive tract, forming nonabsorbable complexes that significantly reduce its absorption.

[0011] Furthermore, oral iron preparations are often not used in certain types of patients, such as those with impaired iron absorption, particularly due to chronic inflammatory bowel disease. Furthermore, oral iron preparations may not be effective at all in some cases, meaning iron supplementation may not improve a patient's iron deficiency, particularly because iron cannot cross the intestinal barrier.

[0012] Therefore, when oral iron preparations cannot / are no longer possible for the reasons mentioned above, or because there is a clinical need to administer iron quickly, or because the patient's recommended iron dose is higher than the digestive tract's ability to absorb iron, iron can be administered intravenously, meaning that the iron is administered directly into the bloodstream by intravenous infusion.

[0013] The advantage of the intravenous route is that the iron enters the blood directly and thus reaches the whole body. However, intravenous administration has potential side effects, such as allergic reactions, hypophosphatemic osteomalacia, impaired liver or kidney function, infection, extravasation during infusion, etc., so iron must be administered in a hospital setting. Therefore, this requires hospitalization and the availability of appropriate medical facilities (sufficient beds, medical staff). In addition, the iatrogenic risk of such infusions is not zero.

[0014] Given these difficulties, there remains a need to find alternatives to intravenous or oral iron therapy.

[0015] The present invention proposes novel iron nanoclusters for combating all types of iron deficiency, which are advantageously administered orally without the above-mentioned disadvantages.

[0016] Iron nanoclusters have been proposed in the literature for treating iron deficiency. For example, US Patent 2016 / 0022733 describes an iron oxide nanocomposite coated with folic acid, niacin, and ascorbic acid for the treatment of anemia. These nanocomposites are intended for oral administration. US Patent 2016 / 008292 describes iron oxide nanoparticles coated with a biocompatible polymer containing polyethylene glycol and silane groups covalently bound via a linker. These nanoparticles are proposed for parenteral treatment of anemia.

[0017] However, the contribution of the present inventors is the development of new iron nanoclusters with characteristics that are particularly suitable for combating iron deficiency. The contribution of the present inventors is also the development of an original method for the synthesis of iron nanoclusters.

[0018] Summary of the Invention

[0019] The present invention relates to iron nanoclusters having the following characteristics:

[0020] - Its surface is covered with a mixed layer containing histidine (His), acetate ions (Ac) and ascorbate ions (Asc),

[0021] - it has a spherical shape,

[0022] - its hydrodynamic diameter is between 0.6 and 2.0 nm, preferably less than 1.0 nm,

[0023] - its metal core diameter is 0.5-1.5 nm, preferably less than 1.0 nm,

[0024] - when the nanoclusters are in liquid form and stored at a temperature of 4°C, they are stable for a period of 5 to 20 weeks,

[0025] - when the nanoclusters are in dry form and stored at a temperature of 4°C under nitrogen, they are stable for a period of at least 12 months, preferably 12 to 18 months,

[0026] - It exhibits spectrophotometric characteristics with a shoulder peak in the UV-visible spectrum located at 300±15nm, and a fluorescence spectrum with an excitation wavelength of 364±15nm and an emission wavelength of 415±15nm,

[0027] The iron nanoclusters may be represented by the formula “FeNC@HisAcAsc”.

[0028] However, the nanoclusters of the present invention may be referred to hereinafter as “nanoclusters,” “iron nanoclusters,” “FeNC nanoclusters,” “FeNC,” “NC-Fe” (“FeNC” or “NC-Fe” means “iron nanoclusters”), “FeNC@HisAcAsc nanoclusters,” or “FeNC@HisAcAsc.”

[0029] The formula "FeNC@HisAcAsc" is the most specific, as it describes an iron nanocluster comprising a layer containing histidine as well as acetate and ascorbate ions on its surface.

[0030] The present invention also relates to a method for preparing the iron nanoclusters, which comprises the following steps:

[0031] - reacting iron(II) acetate with histidine 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 8-200, more preferably 80-200,

[0032] - reacting a mixture of iron acetate and histidine with ascorbic acid 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 from 12 to 700, more preferably from 130 to 700,

[0033] - collecting iron nanoclusters covered on their surface with a mixed layer comprising histidine, acetate ions and ascorbate ions.

[0034] The present invention also relates to iron nanoclusters for use in:

[0035] - in the prevention and / or treatment of diseases causing iron deficiency (such as iron deficiency anemia),

[0036] - Combats iron deficiency.

[0037] Finally, the present invention also relates to a composition comprising the iron nanoclusters according to the invention, said composition being a medicament, a dietary supplement or a food composition.

[0038] The composition of the present invention is also characterized in that it is in a form suitable for oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other features, details, and advantages will be apparent from a reading of the following detailed description and an analysis of the accompanying drawings.

[0040] Figure 1 Schematic diagram of the iron nanocluster "FeNC@HisAcAsc" of the present invention, which consists of an iron metal core surrounded by a hybrid corona containing histidine, acetate ions and ascorbate ions.

[0041] Figure 2 High performance liquid chromatography analysis (reversed phase separation) of iron nanoclusters shows the presence of acetate ions on the surface of the iron metal core.

[0042] The chromatogram was obtained on a previously purified fraction of the iron nanoclusters (size exclusion chromatography).A sodium acetate reference solution was also chromatographed.

[0043] Figure 3 High performance liquid chromatography analysis (reversed phase separation) of iron nanoclusters shows the presence of histidine and ascorbate ions on the surface of the metallic iron core.

[0044] The chromatograms were obtained on previously purified fractions of the iron nanoclusters (size exclusion chromatography). Reference solutions of histidine and ascorbic acid were also chromatographed.

[0045] Figure 4 Shown are the hydrodynamic diameters (in nanometers) of the iron nanoclusters assessed by dynamic light scattering.

[0046] Figure 5 Shown are the hydrodynamic diameters (in nanometers) of the iron nanoclusters estimated by Taylor dispersion analysis.

[0047] Figure 6 is the UV-visible spectrum of iron nanoclusters.

[0048] Figure 7 is the fluorescence spectrum of iron nanoclusters.

[0049] Figure 8 Illustrated are the results of a viability assay (MTT) of HepG2 cells (human hepatoma cells) proliferating in the presence of iron in the form of iron (III) nitrate (control, standard) (represented by FeNO3) or in the form of different concentrations of iron nanoclusters according to the invention (represented by NC-Fe 1x; NC-Fe1 / 2, NC-Fe 1 / 4, NC-Fe 1 / 8, NC-Fe 1 / 16 and NC-Fe 1 / 32).

[0050] “Negative” corresponds to “IMDM” medium without iron supplementation.

[0051] The horizontally shaded histogram on the far left of each set of histograms corresponds to Day 1+3 of the cell viability assay. The histogram immediately following it corresponds to Day 1+5, and the histogram after that corresponds to Day 1+7. The black histogram on the far right of each set of histograms corresponds to Day 1+10. DETAILED DESCRIPTION

[0052] Iron nanoclusters

[0053] Therefore, the present invention relates to iron nanoclusters, characterized in that they:

[0054] - the surface is covered with a mixed layer containing histidine (His), acetate ions (Ac) and ascorbate ions (Asc),

[0055] - has a spherical shape,

[0056] - a hydrodynamic diameter of 0.6-2.0 nm, preferably less than 1.0 nm,

[0057] - the diameter of the metal core is 0.5-1.5 nm, preferably less than 1.0 nm,

[0058] - When the nanoclusters are in liquid form and stored at 4°C, they are stable for 5-20 weeks.

[0059] - when the nanoclusters are in dry form and stored at a temperature of 4°C under nitrogen, the stability duration is at least 12 months, preferably 12-18 months,

[0060] - exhibits spectrophotometric characteristics, with a shoulder peak at 300±15nm in the UV-visible spectrum, and an excitation wavelength of 364±15nm and an emission wavelength of 415±15nm in the fluorescence spectrum,

[0061] The iron nanoclusters may be represented by the formula “FeNC@HisAcAsc”.

[0062] The iron nanoclusters targeted by the present invention are metal nanoclusters, which are composed of dozens of atoms of a metal element (iron in the present invention), and have a metal core diameter of less than or equal to 2.0 nanometers (nm).

[0063] The nanoclusters of the present invention consist of an iron metal core coated / wrapped / surrounded by a mixed layer / corona comprising histidine, acetate ions and ascorbate ions.

[0064] The terms "crown" and "layer" are used interchangeably in this application.

[0065] The term "mixed" is used to indicate that the corona or layer surrounding the iron core comprises histidine, acetate ions, and ascorbate ions.

[0066] Similarly, the verbs "coat / wrap / surround" can be used interchangeably to indicate that the iron core includes a layer / crown of histidine, acetate ions, and ascorbate ions over its entire surface.

[0067] The iron nanoclusters have a spherical shape as a whole.

[0068] Within the meaning of the present invention, the formula "FeNC@HisAcAsc" represents a nanocluster composed of an iron metal core coated with a mixed layer of histidine, acetate ions, and ascorbate ions. Therefore, the iron nanoclusters of the present invention advantageously contain three ligands on the surface of the iron core: histidine, acetate ions, and ascorbate ions. These three ligands are bound to the iron metal core via coordination bonds.

[0069] In particular, the mixed corona comprising histidine, acetate ions and ascorbate ions imparts to the nanoclusters of the present invention a very high stability and low reactivity.

[0070] "Low reactivity" refers to low degradation, particularly degradation associated with oxidation (eg due to atmospheric oxygen).

[0071] The stability of the nanoclusters according to the present invention means that the structure and properties of the nanoclusters are maintained over time at a storage temperature of 4° C. Maintained structure means in particular that the composition of the nanoclusters (metal core surrounded by a hybrid layer / corona as defined above), their shape and their diameters (metal core diameter and hydrodynamic diameter) remain unchanged over time.

[0072] The "liquid form" of the iron nanoclusters refers to a solution or liquid mixture of the iron nanoclusters. The aforementioned stability of 5-20 weeks applies to the liquid form of the iron nanoclusters stored at a storage temperature of 4°C.

[0073] "Dry form" refers to a solid form which can be ground into a powder if necessary. The aforementioned stability of 12-18 months applies to the iron nanoclusters in dry form stored at a storage temperature of 4°C under nitrogen.

[0074] Therefore, depending on its form (liquid or solid), its stability duration will vary.

[0075] The nanoclusters of the present invention exhibit the characteristics of this scale, namely, spectrophotometric properties, especially fluorescence properties, that is, the diameter of the metal core is less than or equal to 2 nm, which is between molecules and nanoparticles.

[0076] As the name suggests, the metal core diameter or metal diameter refers to the diameter formed only by iron metal.

[0077] The hydrodynamic diameter includes the diameter of the iron core plus its layer / corona of histidine, acetate ions and ascorbate ions. Therefore, the hydrodynamic diameter refers to the diameter of the entire iron nanocluster.

[0078] According to one embodiment of the present invention, the metallic core diameter and the hydrodynamic diameter of the iron nanoclusters are more or less equal, preferably less than 1.0 nm.

[0079] However, of course, the metallic core diameter is always smaller than the hydrodynamic diameter.

[0080] The metal core diameter was assessed by transmission electron microscopy, and the hydrodynamic diameter was assessed by dynamic light scattering and / or Taylor dispersion analysis.

[0081] According to an advantageous embodiment of the present invention, the iron nanoclusters are in liquid form or in dry form.

[0082] Nanoclusters in dry form are advantageous, inter alia, in that they can be easily stored, preserved and transported.

[0083] 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:

[0084] -It is able to cross the intestinal barrier,

[0085] - it exhibits good bioavailability,

[0086] - it is biocompatible,

[0087] - it is biodegradable,

[0088] - it can be freeze-dried,

[0089] -It is non-toxic to the human body,

[0090] - It does not accumulate in organs such as the liver, spleen, kidneys or lungs.

[0091] According to an advantageous embodiment, the iron nanoclusters of the invention exhibit all the characteristics described above.

[0092] The nanoclusters of the present invention are not sequestered in the organs primarily due to their small size (hydrodynamic diameter less than or equal to 2.0 nm, preferably less than 1.0 nm). The size of the nanoclusters of the present invention enables them to circulate in the blood longer than larger compounds.

[0093] More specifically, the small size of nanoclusters enables them to cross cell membranes (particularly the gastrointestinal tract) through a phenomenon called supersorption (spontaneous passage through the pores of the physiological system) without having to go through the physiological absorption system. This supersorption phenomenon is where nanoclusters pose a toxicity risk, but if the amount of nanoclusters ingested is controlled, they can become a therapeutic modality.

[0094] The surface properties of the nanoclusters of the present invention enable them to cross the intestinal barrier, which is a significant advantage over oral iron preparations that generally cannot cross the intestinal barrier.

[0095] “Good bioavailability” means that orally administered iron nanoclusters can reach the systemic circulation and be well distributed to target organs.

[0096] "Biocompatibility" means that the iron nanoclusters are well accepted by various organs of the human body and do not cause toxicity to these organs.

[0097] The nanoclusters are biodegradable, meaning their degradation releases substances that can be successfully metabolized or excreted by the body (iron, histidine, acetate, and ascorbate).

[0098] According to an advantageous embodiment of the invention, the nanoclusters can be freeze-dried.

[0099] Because the nanoclusters are very stable, they can be freeze-dried. Therefore, freeze-drying allows for convenient storage, preservation, and transportation of the nanoclusters. The stability of the nanoclusters is defined as above.

[0100] The advantageous properties of the nanoclusters of the present invention are due in particular to the unique combination of their components, namely iron, histidine, acetate ions and ascorbate.

[0101] To the best of the inventors' knowledge, iron nanoclusters comprising a mixed corona / layer of histidine, acetate ions and ascorbate ions surrounding an iron metal core and exhibiting the above-mentioned advantageous properties have never been described before.

[0102] Method for preparing iron nanoclusters

[0103] The present invention also relates to a method for preparing the iron nanoclusters as defined above, characterized in that it comprises the following steps:

[0104] - reacting iron(II) acetate with histidine 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 8-200, more preferably 80-200,

[0105] - reacting a mixture of iron acetate and histidine with ascorbic acid 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 between 12 and 700, more preferably between 130 and 200,

[0106] - collecting the iron nanoclusters.

[0107] The molar ratios defined above between histidine and ferric acetate, and between ascorbic acid and ferric acetate, respectively, are important because they allow the histidine ligands, acetate ions, and ascorbate ions to bind to the iron metal core. This results in nanoclusters containing three ligands on the surface of the iron core, which are bound to the iron core surface via coordinate bonds.

[0108] Ascorbic acid is a reducing agent. The reaction of the mixture of iron acetate and histidine with ascorbic acid is more particularly a reduction reaction of the mixture of iron acetate and histidine with ascorbic acid. In particular, ascorbic acid makes it possible to obtain iron nanoclusters without any toxicity.

[0109] The present invention stems in particular from the unexpected discovery of the inventors 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 having particularly advantageous properties.

[0110] The excellent stability of the nanoclusters of the present invention is one example.

[0111] According to one embodiment of the invention, the iron nanoclusters can be prepared more particularly according to a "liquid phase" protocol or according to a "solid phase" protocol. Each of these two synthetic routes is consistent with the method described above.

[0112] 1 / Liquid phase solution

[0113] 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 inert gas and in that:

[0114] - the ferric acetate is in the form of a solution, and the histidine is in the form of a powder,

[0115] - adding histidine to the ferric acetate solution to prepare a solution of ferric acetate and histidine,

[0116] - adjusting the pH value of the solution of ferric acetate and histidine to 11-13, preferably 12,

[0117] - the ascorbic acid is in powder form,

[0118] - adding ascorbic acid to the solution of iron acetate and histidine whose pH has been adjusted to the above value to prepare a solution of iron acetate, histidine and ascorbic acid,

[0119] - stirring the solution of ferric acetate, histidine and ascorbic acid at a temperature of 35°C to 45°C, preferably 40°C, for 2 to 6 hours, preferably 4 hours,

[0120] - obtaining a solution containing iron nanoclusters at the end of the aforementioned stirring step,

[0121] - optionally, dialyzing the solution containing the iron nanoclusters to obtain a purified iron nanocluster solution,

[0122] - Optionally, freeze-drying the optionally dialyzed solution comprising iron nanoclusters to obtain iron nanoclusters in dry form.

[0123] The optionally dialyzed solution comprising iron nanoclusters is stable for a period of 5-20 weeks at a storage temperature of 4°C.

[0124] Dialysis can remove any substances not bound to the iron metal core, such as excess histidine or ascorbic acid, or any residual iron in the nanocluster solution.The layer containing histidine, acetate ions, and ascorbate ions is bound to the iron metal core through coordination bonds.

[0125] The iron nanoclusters in dry form obtained after freeze-drying are stable for a period of at least 12 months, preferably 12-18 months, at a storage temperature of 4° C. under nitrogen.

[0126] The dry form of the iron nanoclusters can be reconstituted at any time by mixing into a reconstitution solvent such as pure water. "Reconstitute" refers to a simple process of mixing the dry form or lyophilisate with a solvent.

[0127] Analysis of the iron nanocluster solution obtained after reconstitution of the dry form showed that the iron nanoclusters exhibited all the above-mentioned properties and were thus identical to the iron nanocluster solution obtained directly from their preparation method.

[0128] The iron nanocluster solution obtained after reconstitution in dry form is stable for a period of 5-12 weeks at a storage temperature of 4° C., preferably under nitrogen.

[0129] The preparation method as defined above is further characterized in that it further comprises at least one selected from the following features:

[0130] - the inert gas is nitrogen,

[0131] - adding ferric acetate to filtered ultrapure water to prepare the ferric acetate solution,

[0132] - the concentration of the ferric acetate solution is 0.5-5.0 mM,

[0133] - the concentration of the histidine is greater than the concentration of the ferric acetate solution,

[0134] - Use sodium hydroxide to adjust the pH of the solution of ferric acetate and histidine,

[0135] - the concentration of said ascorbic acid is equal to the concentration of said histidine,

[0136] - the iron concentration of the optionally dialyzed solution comprising iron nanoclusters is 14-112 μg / mL,

[0137] - freeze-drying the optionally dialyzed solution comprising iron nanoclusters to obtain the iron nanoclusters in dry form.

[0138] According to an advantageous embodiment, the method of the invention exhibits all the characteristics described above.

[0139] 2 / Solid phase solution

[0140] According to another advantageous embodiment of the invention, the method for preparing the iron nanoclusters as defined above is more particularly characterized by:

[0141] - the ferric acetate is in powder form, and the histidine is in powder form,

[0142] - mixing the ferric acetate and histidine powders together to obtain a powder mixture of ferric acetate and histidine,

[0143] - Grind the powder mixture of ferric acetate and histidine until a powder mixture of uniform color is obtained,

[0144] - placing a homogeneous powder mixture of ferric acetate and histidine in a reactor,

[0145] - the ascorbic acid is in powder form,

[0146] - adding said ascorbic acid to a reactor containing a homogeneous powder mixture of ferric acetate and histidine,

[0147] The powder mixture of iron acetate, histidine and ascorbic acid thus obtained was stirred, and then water, which was filtered ultrapure water, was added dropwise to the reactor.

[0148] - Place the reactor under inert gas and protect from light.

[0149] - Stirring the mixture of ferric acetate, histidine, ascorbic acid and water in a reactor for 16-36 hours, preferably 24 hours.

[0150] - At the end of the aforementioned stirring step, a liquid mixture containing iron nanoclusters is obtained.

[0151] - Optionally, dialyzing the liquid mixture containing the iron nanoclusters to obtain a purified liquid mixture of iron nanoclusters.

[0152] - Optionally, freeze-drying the optionally dialyzed liquid mixture comprising iron nanoclusters to obtain iron nanoclusters in dry form.

[0153] The preparation method as defined above is also characterized in that it further comprises at least one feature selected from the group consisting of:

[0154] - the concentration of the histidine is greater than the concentration of the ferric acetate,

[0155] - the concentration of said ascorbic acid is equal to the concentration of said histidine,

[0156] - The water added to the reactor is filtered ultrapure water,

[0157] - the inert gas is nitrogen,

[0158] - the iron concentration of the optionally dialyzed liquid mixture comprising iron nanoclusters is 1500-15000 μg / mL,

[0159] - freeze-drying the optionally dialyzed liquid mixture comprising iron nanoclusters to obtain iron nanoclusters in dry form.

[0160] According to another embodiment of the present invention, the iron nanoclusters obtained in dry form after freeze-drying (according to the "liquid phase" protocol or the "solid phase" protocol) are stored under nitrogen, preferably in vials, and preferably at 4° C. Under such conditions, the iron nanocluster powder can be stored for at least 12 months, preferably 12-18 months, without changes in the stability of the iron nanoclusters.

[0161] Reconstitution of the nanocluster powder at the end of this phase revealed that the nanoclusters were identical to those obtained by direct preparation (either according to the "liquid phase" or the "solid phase" protocol). In fact, the iron nanoclusters exhibited all the properties described above.

[0162] Use of iron nanoclusters

[0163] The present invention also relates to iron nanoclusters as defined above or obtained according to a process as defined above, for use as a medicament.

[0164] More particularly, the present invention relates to iron nanoclusters as defined above or iron nanoclusters obtained according to the method as defined above, for use in preventing and / or treating diseases causing iron deficiency.

[0165] An example of a disease that causes iron deficiency is iron deficiency anemia.

[0166] The present invention also relates to iron nanoclusters as defined above or obtained according to a method as defined above, for use in combating iron deficiency.

[0167] In the present application, iron deficiency refers to iron deficiency in a broad sense, that is, iron deficiency with or without iron deficiency anemia.

[0168] Another subject of the invention is a composition characterized in that it comprises iron nanoclusters as defined above or obtained according to a process as defined above.

[0169] The composition of the present invention may be a medicament, a dietary supplement or a food composition.

[0170] The amount of iron in each composition can determine whether it is a dietary supplement or a drug. Therefore, dietary supplements should contain less iron than drugs.

[0171] Examples of food compositions include, for example, infant formula supplemented with iron, more particularly with the iron nanoclusters of the present invention.

[0172] According to an advantageous embodiment of the invention, the composition is in a form suitable for oral administration.

[0173] Iron nanoclusters can advantageously be administered orally since they are able to cross the intestinal barrier without difficulty, particularly due to their small size.

[0174] The use of the iron nanoclusters of the present invention advantageously eliminates the need for intravenous administration.

[0175] According to another advantageous embodiment, the composition of the invention comprising iron nanoclusters comprises an amount of iron that is lower than the amount of iron typically present in conventional oral formulations, whether pharmaceuticals or dietary supplements.

[0176] Advantageously, the compositions of the present invention do not exhibit the disadvantages that may be encountered with conventional oral formulations, whether pharmaceuticals or dietary supplements.

[0177] Example

[0178] The following examples illustrate the present invention but do not limit it in any way.

[0179] Example 1

[0180] Preparation of iron nanoclusters

[0181] This example describes two possible synthetic routes for preparing the iron nanoclusters of the present invention, namely the "liquid phase approach" and the "solid phase approach".

[0182] 1 / Liquid phase solution

[0183] Reagents used :

[0184] - iron(II) acetate [Fe(CH3COO)2], M = 171.83 g / mol (Sigma-Aldrich, Cas3094-87-9);

[0185] -L(-)-histidine, M = 155.15 g / mol (Merck, Cas 71-00-1);

[0186] - ascorbic acid, M = 176.12 g / mol (Sigma-Aldrich, Cas 50-81-7);

[0187] - 1 M NaOH solution, M = 40.00 g / mol (VWR, Cas 1310-73-2).

[0188] Precautions

[0189] The synthesis was carried out under inert gas (nitrogen).The glassware was washed with aqua regia (1 volume of 65% nitric acid to 2 volumes of 37% hydrochloric acid).

[0190] Use ultrapure water and filter it through a 0.2 μm pore size filter.

[0191] The iron acetate is in powder form and stored under nitrogen. After the iron is weighed, the remaining raw material must be stored under nitrogen again.

[0192] Since the iron nanoclusters are used for in vivo studies, it is imperative to work under a clean fume hood and clean all used equipment with 70% v / v ethanol.

[0193] Preparation of 2.5 mM ferric acetate stock solution

[0194] Place 42.9 mg of ferric acetate in a 100 mL volumetric flask. Add filtered ultrapure water to the fill line of the flask to obtain a 2.5 mM ferric acetate solution.

[0195] Once completely dissolved, transfer the ferric acetate solution to a suitable container. This solution can be stored in a refrigerator at 4°C for one month.

[0196] Synthesis of nanoclusters stabilized with histidine

[0197] Add 500 μL of the ferric acetate stock solution prepared in the previous step to a round-bottom flask capable of holding up to 50 mL of solution. Then, add 4500 μL of filtered ultrapure water to the flask. The resulting ferric acetate solution is designated as 1x.

[0198] Stir the 1x ferric acetate solution at 130 rpm using a multiplate shaker. Add 39 mg of histidine to the ferric acetate solution. Stir the ferric acetate and histidine solution for 15 minutes.

[0199] The solution took on a reddish hue. After stirring for 15 minutes, the pH of the ferric acetate and histidine solution was adjusted to 12 with 10 drops of 1M NaOH. 139 mg of ascorbic acid was added to the reaction mixture. The ascorbic acid was allowed to completely dissolve over 2 minutes. The flask (reactor) was placed in a 40°C water bath with stirring (speed setting 6) for 4 hours.

[0200] At the end of the synthesis, the resulting nanocluster solution was colorless. The resulting iron nanocluster solution was designated 1x, with an iron concentration of 14 μg / mL. It was stored in a cool place at 4°C.

[0201] The synthesis yield was 100%: there was no residual iron (iron element) in the nanocluster solution. The iron nanocluster solution could be freeze-dried.

[0202] Ferric acetate solutions were prepared at concentrations ranging from 1x to 8x to obtain 1x to 8x solutions of iron nanoclusters with iron concentrations ranging from 14 to 112 μg / mL.

[0203] For reference, prepare a 2x ferric acetate solution by placing 1000 μL of ferric acetate stock solution in a flask and making up to 5000 μL with filtered ultrapure water. Prepare a 4x ferric acetate solution by placing 2000 μL of ferric acetate stock solution in a flask and making up to 5000 μL with filtered ultrapure water, etc., and so on.

[0204] The resulting 1x to 8x solutions of iron nanoclusters were stored in a cool place at 4 °C.

[0205] Dialysis of iron nanoclusters

[0206] A 1x solution of the iron nanoclusters obtained in the previous step was purified by dialysis.

[0207] Prepare dialysis apparatus (X12 Float-a-lyzer G2 CE MWCO 100-500D, Reference 1511160), and fill 150mL beaker with 100mL filtered ultrapure water. Use Pasteur pipette to fill dialysis apparatus with filtered ultrapure water. Dialysis apparatus is placed in beaker, stirred (130rpm) simultaneously. Make device keep moistening and wash 1 hour.

[0208] Then, the original water was replaced with 100 mL of new filtered ultrapure water.The dialysis apparatus was emptied using a Pasteur pipette and then filled with a 1x solution of iron nanoclusters, which was stirred at a temperature of 2-6°C overnight (12 hours).

[0209] The resulting 1x solution of dialyzed iron nanoclusters was transferred to a suitable container and stored at 4°C.

[0210] Dialysis did not affect the iron concentration of the nanoclusters. Therefore, the iron concentration of the dialyzed solution of the iron nanoclusters was the same as the iron concentration of the non-dialyzed solution.

[0211] If dialyzed, the solution of iron nanoclusters can be freeze-dried.

[0212] The iron concentration of the dialyzed solution of the iron nanoclusters was the same as that of the non-dialyzed solution and ranged from 14-112 μg / mL for ferric acetate solutions with concentrations ranging from 1× to 8×.

[0213] 2 / Solid phase solution

[0214] Reagents used and precautions

[0215] Iron(II) acetate, L(-)-histidine, and ascorbic acid were the same as those used in the liquid-phase protocol. Sodium hydroxide was not required in the solid-phase protocol.

[0216] The same precautions as for liquid-phase protocols apply.

[0217] Synthesis of iron nanoclusters stabilized with histidine

[0218] Weigh 23 mg of ferric acetate and place it in an agate mortar. Next, weigh 1.7 g of histidine. Add one part histidine powder to one part ferric acetate powder and carefully grind the powder thoroughly with the pestle until a mixture is uniform in color and appearance. Repeat this process until all the histidine is used up.

[0219] The final mixture of the two powders should be red, and the powder should be uniform. Then, use a spatula to transfer the mixture of the two powders to a 50mL single-necked flask (NS19 / 26 grinding port). Weigh 3g of ascorbic acid and transfer it to the flask. An olive-shaped magnetic stirrer is placed at the bottom of the flask. Use a 5mL plastic syringe to filter 100mL of ultrapure water and add it dropwise to the reactor. Obtain a liquid mixture.

[0220] The reactor was sealed with a 19.4 mm diameter flip-top cap and placed under nitrogen using a latex balloon to prevent overpressure. The reactor was wrapped in aluminum foil and stirred continuously (200 rpm) during the reaction. A 24-hour wait was required for the reaction to complete. At the end of the reaction, the product was a liquid with a reddish color.

[0221] The resulting liquid containing iron nanoclusters had an iron concentration of 1500 μg / mL and was recorded as 100×.

[0222] Of course, the iron concentration of the nanoclusters depends on the amount of ferric acetate used at the beginning of the process of the invention.

[0223] The above operation was repeated to obtain iron concentrations ranging from 1500 (100x) to 15,000 μg / mL (1000x), for an amount of ferric acetate at the start of the method of the invention (solid phase protocol) ranging from 23 to 230 mg.

[0224] The resulting liquid containing iron nanoclusters was then transferred to a suitable plastic container (the final volume was not 5 mL but slightly larger, about 8.5 mL).The liquid containing iron nanoclusters was stored at 4°C or transferred to a freeze dryer.

[0225] Freeze drying was performed in 1 mL units without adding other reagents. After freeze drying, the contents of the vials (which contained the iron nanoclusters in dry form) were placed under nitrogen and stored at 4°C.

[0226] The iron nanoclusters in dry form can be reconstituted at any time in 1 mL of pure water. The iron nanocluster solution reconstituted in this way is stored at 4°C, preferably under nitrogen.

[0227] Example 2

[0228] Characterization of iron nanoclusters

[0229] The iron nanoclusters obtained in Example 1, whether by a liquid or solid phase approach, are characterized by their structure, size, spectrophotometric properties and stability.

[0230] Structure of iron nanoclusters

[0231] The iron nanoclusters of the present invention more particularly have a spherical shape and consist of an iron metal core covered with a mixed corona comprising histidine, acetate ions and ascorbate ions. Figure 1 is a schematic diagram of the iron nanocluster of the present invention, which can also be represented by the formula "FeNC@HisAcAsc".

[0232] The presence of acetate ions on the surface of the iron nanoclusters was confirmed by high performance liquid chromatography (HPLC), more specifically, by reverse phase chromatography.

[0233] The iron nanoclusters in the 1x dialyzed solution obtained in Example 1 (see Section 1 / "Liquid Phase Protocol") were destroyed chemically (the nanocluster structure completely dissolved and returned to its individual component elements), i.e., dissolved in concentrated acid (HCl) and then in concentrated base (NaOH) and then analyzed by HPLC compared with a sodium acetate control.

[0234] The results are shown in Figure 2 The peak associated with acetate ions (control, see the lower figure) appears at 3.6 minutes and is present in the iron nanoclusters (see the upper figure), thus proving the presence of acetate ions on the surface of the metal core.

[0235] The presence of histidine and ascorbate ions on the surface of the 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 histidine and ascorbic acid controls.

[0236] The results are shown in Figure 3 chromatogram.

[0237] A peak at 2.1 minutes (see upper panel) associated with histidine (control, see lower panel) was found in the iron nanoclusters, thus confirming the presence of histidine on the metal core surface.

[0238] A peak at 3.0 min (see upper panel) associated with ascorbate ions (control, see lower panel) was found in the iron nanoclusters, thus demonstrating the presence of ascorbate ions on the surface of the metal core.

[0239] Iron nanocluster size

[0240] The hydrodynamic diameter (Dh) of the iron nanoclusters was determined by dynamic light scattering ( Figure 4 )(173° angle, 530 nm laser, 25°C temperature, Malvern Nanosizer) and Taylor dispersion analysis ( Figure 5 )Evaluate.

[0241] Methods using dynamic light scattering analysis involve analyzing the Brownian motion of particles and modeling it using the Stokes-Einstein equations.

[0242] The Taylor dispersion analysis method involves injecting a small bolus of solute into an open capillary (50 μm) and allowing it to diffuse under the influence of a hydrodynamic flow (positive pressure of 1 psi, parabolic velocity profile). The principle for determining the hydrodynamic radius is based on the Taylor-Aris relationship, which relates the peak diffusion of the solute (which simulates a Gaussian distribution) to the molecular diffusion coefficient.

[0243] The metallic core diameter of the iron nanoclusters was evaluated using transmission electron microscopy (deposited on nickel grids and observed under an electron beam of a Philips CM 200 operated at 200 kV (LaB6 cathode)).

[0244] For both liquid-phase and solid-phase protocols, the diameters (hydrodynamic and metallic core) of the iron nanoclusters were evaluated immediately after their synthesis.

[0245] For the liquid phase protocol, hydrodynamics and metal core diameter were assessed in a 1x solution of non-dialyzed and non-freeze-dried iron nanoclusters at an iron concentration of 14 μg / mL.

[0246] For the solid phase protocol, hydrodynamics and metal core diameters were evaluated in freeze-dried samples obtained from 100x liquid of non-dialyzed iron nanoclusters at an iron concentration of 1500 μg / mL.

[0247] Like the metal core diameter, the average hydrodynamic diameter of the iron nanoclusters is less than 1.0 nm. More specifically, Figure 4 and 5 It is shown that in dynamic light scattering ( Figure 4 ) and Taylor dispersion ( Figure 5 ), the hydrodynamic diameter of the iron nanoclusters is 0.69±0.06nm.

[0248] Spectrophotometric characteristics

[0249] The iron nanoclusters were immediately synthesized by UV-visible spectroscopy ( Figure 6 ) and fluorescence spectra ( Figure 7 )Evaluation of the spectrophotometric properties of iron nanoclusters.

[0250] The UV-vis spectrum shows a shoulder peak at 300±15nm ( Figure 6 ), confirming the existence of nanoclusters.

[0251] The iron nanoclusters exhibited fluorescence with an excitation wavelength of 364±15 nm and an emission wavelength of 415±15 nm ( Figure 7 ), which also confirms the existence of nanoclusters.

[0252] The iron nanoclusters of the present invention exhibit optical properties, in particular fluorescence, which are characteristic of such an intermediate scale between molecules and nanoparticles.

[0253] Stability of iron nanoclusters

[0254] The stability of the iron nanoclusters was evaluated by measuring the hydrodynamic diameter of the iron nanoclusters using dynamic light scattering (173° angle, 530 nm laser, 25°C temperature, Malvern Nanometer).

[0255] The iron nanoclusters obtained using both the liquid-phase and solid-phase approaches were analyzed.

[0256] For the solution phase protocol, the analysis was performed on a 1x solution of iron nanoclusters (iron concentration 14 μg / mL).Stability assessments were performed slightly more than 5 weeks after synthesis.

[0257] The study found that the hydrodynamic diameter of the iron nanoclusters remained at 0.69±0.06 nm more than 5 weeks after synthesis, indicating their excellent stability.

[0258] For the solid phase protocol, the analysis was performed as follows:

[0259] - Lyophilized sample obtained from 100x iron nanocluster liquid, not dialyzed (iron concentration 1500 μg / mL),

[0260] - The sample was freeze-dried and then reconstituted to the same volume as when freeze-dried.

[0261] After storage under nitrogen for more than 5 weeks, the hydrodynamic diameter of the iron nanoclusters in the freeze-dried sample was 0.70 nm.

[0262] After reconstitution of the sample to the same volume as that upon freeze-drying, the hydrodynamic diameter of the iron nanoclusters in the reconstituted sample was 0.76 nm, further demonstrating its excellent stability.

[0263] Example 3

[0264] Assessing the toxicity of iron nanoclusters

[0265] This example describes the results of a viability assay (MTT assay) of HepG2 cells (human hepatoma cells) proliferating in the presence of iron in the form of iron (III) nitrate (control, standard) or in the form of iron nanoclusters according to the invention at different concentrations.

[0266] iron

[0267] The iron (III) nitrate (or ferric nitrate) used as a reference is a compound having the semi-structural formula "Fe(NO 3 ) 3 ", which is more particularly used in its nonahydrate form "Fe(NO 3 ) 3 .9H 2 O".

[0268] A solution of ferric nitrate nonahydrate was prepared in a total volume of 50 mL at a concentration of 100 mg / L in water, corresponding to an iron concentration of 14 μg / mL. The solution was filtered under a PSM fume hood.

[0269] In particular, a 1x solution of iron nanoclusters with an iron concentration of 14 μg / mL prepared as in Example 1 (Part 1 / Liquid Phase Protocol) was used.

[0270] The 1x solution of iron nanoclusters was serially diluted to 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 (see prepared media 4-8 below).

[0271] HepG2 cells

[0272] HepG2 cells are a cell line derived from liver tissue of a patient with hepatocellular carcinoma (HCC).

[0273] MTT assay

[0274] The MTT assay is a rapid colorimetric method for quantifying viable cells in a sample. The reagent used is the tetrazolium salt "MTT" ("3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide"). The tetrazolium ring contained in MTT is reduced to formazan by mitochondrial succinate dehydrogenase in living cells. This forms a purple precipitate in the mitochondria.

[0275] The amount of precipitate formed is proportional to the number of viable cells (but also to the metabolic activity of each cell). Therefore, after incubating the cells with MTT at 37°C for a period of time (about 3 hours) sufficient to dissolve the cells, their mitochondria and the purple formazan precipitate in 100% DMSO (dimethyl sulfoxide).

[0276] By simple spectroscopic measurement of the optical density at 570 nm, the relative number of viable and metabolically active cells can be determined.

[0277] Therefore, if quantitative assays are performed, a calibration curve must be created for each assay. Readings are obtained spectrophotometrically at 570 nm (see Figure 8 ).

[0278] HepG2 cell growth conditions

[0279] HepG2 cells were grown in complete medium (DMEM-Dulcerative Colitis Modified Eagle Medium) in 24-well plates for 24 hours. The medium was then switched to a selective medium (IMDM*) (see below) + / - iron nanoclusters of the present invention vs. iron nitrate nonahydrate Fe(NO3)3.9H2O.

[0280] Fe 3+ Reduced to Fe 2+ , and then crosses the plasma membrane via DMT1 (divalent metal transporter 1).

[0281] Selective medium (IMDM*) :

[0282] IMDM (Iscove's modified Dulbecco's medium),

[0283] 10% FBS (fetal bovine serum) (dialyzed),

[0284] 1% antibiotics (a mixture of penicillin and streptomycin),

[0285] 1% (1 mM) pyruvate.

[0286] Preparation of eight selective culture media :

[0287] - Medium 1: IMDM*;

[0288] - Medium 2: IMDM* + 1% Fe(NO3)3.9H2O (100 mg / L stock solution);

[0289] - Medium 3: IMDM* + 1% 1x solution of iron nanoclusters (i.e., 14 μg / mL equivalent iron);

[0290] - Medium 4: Medium 3 diluted 1:2 in IMDM*;

[0291] - Medium 5: 1:2 dilution of Medium 4 in IMDM* (i.e., 1 / 4 Medium 3);

[0292] - Medium 6: 1:2 dilution of Medium 5 in IMDM* (i.e., 1 / 8 Medium 3);

[0293] - Medium 7: 1:2 dilution of Medium 6 in IMDM* (i.e., 1 / 16 Medium 3);

[0294] - Medium 8: Medium 7 diluted 1:2 to 1 / 2 in IMDM* (i.e., 1 / 32 Medium 3).

[0295] Measurement duration

[0296] One plate was prepared for each step (D1+3, D1+5, D1+7, D1+10). The total assay duration was 11 days.

[0297] Here are the steps:

[0298] - Day 0: Seed on p24 (repeated three times), 4000 cells / cm 2 , DMEM complete medium (500 μL / well);

[0299] - Day 1: Change to IMDM selective medium, iron nanocluster solution vs. Fe(NO3)3.9H2O (0.1 mg / L);

[0300] - Day 1+3: stop culturing one plate for MTT and replace the remaining plates with medium;

[0301] - Day 1+5: One plate was stopped for MTT and the remaining plates were replaced with medium;

[0302] - Day 1+7: One plate was stopped for MTT and the remaining plates were replaced with medium;

[0303] - Day 1+10: 1 plate is cultured and used for MTT, and the test is finished.

[0304] The results obtained in Figure 8 As described in, wherein:

[0305] - "Negative" corresponds to medium 1,

[0306] - "FeNO3" corresponds to medium 2,

[0307] - "NC-Fe 1x" corresponds to medium 3,

[0308] - "NC-Fe 1 / 2" corresponds to medium 4,

[0309] - "NC-Fe 1 / 4" corresponds to medium 5,

[0310] - "NC-Fe 1 / 8" corresponds to medium 6,

[0311] - "NC-Fe 1 / 16" corresponds to medium 7,

[0312] - "NC-Fe 1 / 32" corresponds to medium 8.

[0313] Repeated MTT viability assays on D+3, D+5, D+7 and D+10 showed that treatment with the iron nanoclusters of the present invention was not toxic to HepG2 cells treated with an equal dose of iron in the form of ferric nitrate nonahydrate Fe(NO3)3.9H2O at a concentration of 0.1 mg / L in standard culture.

[0314] Negative controls were treated with medium without added iron, but the presence of fetal bovine serum provided sufficient iron to allow some cell growth (thus, the control was not completely negative).

[0315] MTT was weighed, dissolved in native IMDM medium at 5 mg / mL, filtered at 0.2 μm under a PSM fume hood, and stored at +4°C throughout the experimental protocol.

[0316] At each step of the MTT assay, the 5 mg / mL solution was diluted to 0.5 mg / mL in IMDM complete medium (IMDM+FBS+antibiotics+pyruvate), but without iron, and then incubated at 37°C for 3 hours.

[0317] After each medium change, the spent medium was removed and frozen at -20°C for subsequent transferrin and ferritin assays.

[0318] In conclusion, this assay demonstrates that the iron nanoclusters of the present invention do not present any toxicity, regardless of their concentration.HepG2 cells cultured in medium without iron and then in medium supplemented with iron showed excellent viability.

Claims

1. Iron nanoclusters, characterized in that That: - The surface is covered with a mixed layer containing histidine (His), acetate ions (Ac) and ascorbate ions (Asc), - has a spherical shape, - a hydrodynamic diameter of 0.6-2.0 nm, preferably less than 1.0 nm, - the diameter of the metal core is 0.5-1.5 nm, preferably less than 1.0 nm, - when the nanoclusters are in liquid form and stored at a temperature of 4°C, the stability duration is 5-20 weeks, - when the nanoclusters are in dry form and stored at a temperature of 4°C under nitrogen, the stability duration is 12 months, preferably 12 to 18 months, - exhibits spectrophotometric characteristics, with a shoulder peak in the UV-visible spectrum located at 300±15nm, and an excitation wavelength of 364±15nm and an emission wavelength of 415±15nm in the fluorescence spectrum, The iron nanoclusters can be represented by the formula "FeNC@HisAcAsc".

2. The iron nanoclusters according to claim 1, characterized in that It is available in liquid or dry form.

3. The iron nanoclusters according to claim 1 or claim 2, characterized in that The iron nanoclusters exhibit at least one of the following characteristics: -It is able to cross the intestinal barrier, - it exhibits good bioavailability, - it is biocompatible, - it is biodegradable, - it can be freeze-dried, -It is non-toxic to humans, - It does not accumulate in organs such as the liver, spleen, kidneys or lungs.

4. A method for preparing the iron nanoclusters according to any one of claims 1 to 3, characterized in that: It includes the following steps: - reacting iron(II) acetate with histidine 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 8-200, more preferably 80-200, - reacting a mixture of iron acetate and histidine with ascorbic acid 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 from 12 to 700, more preferably from 130 to 700, - collecting the iron nanoclusters.

5. The preparation method according to claim 4, characterized in that The method is carried out under an inert gas and comprises: - the ferric acetate is in the form of a solution, and the histidine is in the form of a powder, - adding histidine to the ferric acetate solution to prepare a solution of ferric acetate and histidine, - adjusting the pH value of the solution of ferric acetate and histidine to 11-13, preferably 12, - the ascorbic acid is in powder form, - adding ascorbic acid to the solution of iron acetate and histidine whose pH has been adjusted to the above value to prepare a solution of iron acetate, histidine and ascorbic acid, - stirring the solution of ferric acetate, histidine and ascorbic acid at a temperature of 35°C to 45°C, preferably 40°C, for 2 to 6 hours, preferably 4 hours, - obtaining a solution comprising iron nanoclusters at the end of the previous stirring step, - optionally, dialyzing the solution containing the iron nanoclusters to obtain a purified iron nanocluster solution, - Optionally, freeze-drying the optionally dialyzed solution comprising iron nanoclusters to obtain iron nanoclusters in dry form.

6. The method according to claim 5, characterized in that It also includes at least one selected from the following features: - the inert gas is nitrogen, - adding ferric acetate to filtered ultrapure water to prepare the ferric acetate solution, - the concentration of the ferric acetate solution is 0.5-5.0 mM, - the concentration of the histidine is greater than the concentration of the ferric acetate solution, - adjusting the pH of the solution of ferric acetate and histidine using sodium hydroxide, - the concentration of said ascorbic acid is equal to the concentration of said histidine, - the iron concentration of the optionally dialyzed solution comprising iron nanoclusters is 14-112 μg / mL, - freeze-drying the optionally dialyzed solution comprising iron nanoclusters to obtain the iron nanoclusters in dry form.

7. The preparation method according to claim 4, characterized in that: - the ferric acetate is in powder form, and the histidine is in powder form, - mixing the ferric acetate and histidine powders together to obtain a powder mixture of ferric acetate and histidine, - Grind the powder mixture of ferric acetate and histidine until a powder mixture of uniform color is obtained, - placing a homogeneous powder mixture of ferric acetate and histidine in a reactor, - the ascorbic acid is in powder form, - adding the ascorbic acid to the reactor containing the homogeneous powder mixture of the ferric acetate and histidine, - stirring the powder mixture of ferric acetate, histidine and ascorbic acid thus obtained, and then adding water dropwise into the reactor, said water being filtered ultrapure water, - placing the reactor under inert gas and protected from light, - stirring the mixture of ferric acetate, histidine, ascorbic acid and water in a reactor for 16-36 hours, preferably 24 hours, - at the end of the aforementioned stirring step, a liquid mixture containing iron nanoclusters is obtained, -optionally, dialyzing the liquid mixture containing the iron nanoclusters to obtain a purified liquid mixture of iron nanoclusters, - Optionally, freeze-drying the optionally dialyzed liquid mixture comprising iron nanoclusters to obtain iron nanoclusters in dry form.

8. The method according to claim 7, characterized in that It further comprises at least one feature selected from the following: - the concentration of the histidine is greater than the concentration of the ferric acetate, - the concentration of said ascorbic acid is equal to the concentration of said histidine, - the water added to the reactor is filtered ultrapure water, - the inert gas is nitrogen, - the iron concentration of the optionally dialyzed liquid mixture comprising iron nanoclusters is 1500-15000 μg / mL, - freeze-drying the optionally dialyzed liquid mixture comprising iron nanoclusters to obtain iron nanoclusters in dry form.

9. The method according to any one of claims 5 to 8, characterized in that The iron nanoclusters are stored in dry form under nitrogen, preferably in vials, and preferably at 4°C. The powder can be stored for at least 12 months, and preferably 12-18 months, without a change in the stability of the iron nanoclusters.

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

11. The iron nanoclusters defined in any one of claims 1 to 3 or the iron nanoclusters obtained according to any one of claims 4 to 9, for use in preventing and / or treating diseases causing iron deficiency.

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

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

14. A composition characterized in that It comprises the iron nanoclusters defined in any one of claims 1 to 3, or the iron nanoclusters obtained according to the method in any one of claims 4 to 9.

15. The composition according to claim 14, characterized in that It is a medicament, a dietary supplement or a food composition.

16. The composition according to claim 14 or 15, characterized in that It is in a form suitable for oral administration.

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