Iron nanoclusters, methods for obtaining same and their use in combating iron deficiency - Patent Application 20070122999

Abstract

The present invention relates to an iron nanocluster that can be designated using the formula "FeNC@HisAcAsc", having the following properties: - its surface is covered with a mixed layer comprising histidine (His), acetate ions (Ac) and ascorbate ions (Asc); - it is spherical; - it has a hydrodynamic diameter in the range of 0.6 to 2.0 nm, preferably less than 1.0 nm; - it has a metal core diameter in the range of 0.5 to 1.5 nm, preferably less than 1.0 nm; - the nanocluster exhibits a stability period in the range of 5 to 20 weeks when stored in liquid form at a temperature of 4°C; - the nanocluster exhibits a stability period of at least 12 months, preferably 12 to 18 months when stored in dry form at a temperature of 4°C under nitrogen; - it exhibits spectrophotometric characteristics in the UV-visible spectrum with a shoulder at 300±15 nm and in the fluorescence spectrum with an excitation wavelength of 364±15 nm and an emission wavelength of 415±15 nm. The present invention also relates to a method for preparing said iron nanoclusters and to their use in treating iron deficiency.

Description

[Technical Field]

[0001] The present invention relates to the field of chemistry, more particularly to medicinal chemistry. The present invention relates to iron nanoclusters, methods for obtaining them and their use in combating iron deficiency, more particularly to their use in the prevention and / or treatment of diseases that cause iron deficiency. [Background technology]

[0002] Iron deficiency, also known as hypoferremia and iron deficiency, refers to a lack of iron in the body. Iron is a metal essential for bodily functions and can only be obtained through food. Iron deficiency is one of the most prevalent mineral deficiencies worldwide, affecting over 1.5 billion people worldwide.

[0003] Iron is present in every cell in the human body and is responsible for many vital functions, including transporting oxygen through the iron in hemoglobin. Iron deficiency can interfere with these vital functions, with the first sign being microcytic anemia, which can be fatal in severe cases.

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

[0005] Iron absorption is a tightly regulated mechanism: when body iron stores are low, iron absorption increases, and conversely, when iron stores are high, absorption decreases, preventing the accumulation of too much iron in the body (which can lead to iron overload).

[0006] The average person obtains 10-15 mg of iron per day from their diet. Only 1-2 mg of iron is absorbed in the upper small intestine. Iron absorption depends on the iron form, dietary quality, and individual iron stores. Absorption of heme iron is 15-25% while absorption of non-heme iron is 2-20%.

[0007] Iron deficiency can have a variety of causes, including increased individual needs, reduced intake, malabsorption, chronic bleeding, and various diseases.

[0008] Iron deficiency is commonly treated with oral iron preparations (tablets, capsules, powders, drops, syrups, etc.). When administered orally, the preparations reach the stomach, after which the iron is absorbed by the intestinal mucosa, thereby entering the bloodstream.

[0009] However, oral iron supplementation can have various disadvantages, including poor gastrointestinal tolerance. Some formulations release iron directly upon reaching the stomach, which can cause stomach pain in treated patients. Because the intestinal capacity for iron absorption is limited (up to 20–25% for heme iron), a relatively large proportion of ingested iron is excreted. Consequently, oral iron administration frequently causes adverse effects such as nausea, diarrhea or constipation, and black stools. More rarely, patients experience abdominal pain, a metallic taste in the mouth, or black teeth, which disappear when treatment is discontinued.

[0010] When taking oral iron, it is also important to consider potential interactions with medications and foods. In fact, certain medications and foods can significantly reduce iron absorption by binding with oral iron in the gastrointestinal tract to form non-absorbable complexes.

[0011] Furthermore, oral iron supplements are often unavailable for use in certain types of patients, such as those with impaired iron absorption, especially those with chronic inflammatory bowel disease. Oral iron supplements can also be completely ineffective in some cases, specifically because the iron does not cross the intestinal barrier, meaning that iron supplementation does not correct an individual's iron deficiency.

[0012] Therefore, when oral iron administration is not possible or is more difficult for the reasons mentioned above, or when there is a clinical need for rapid iron administration, or when the iron dose recommended for the patient exceeds the gastrointestinal capacity for iron absorption, intravenous iron administration, i.e., administration of iron directly into the bloodstream by intravenous injection, may be used.

[0013] The advantage of the intravenous route is that iron is directly absorbed into the bloodstream, from where it is distributed throughout the body. However, intravenous administration has potential side effects, including hypersensitivity reactions, hypophosphatemic osteomalacia, liver or kidney dysfunction, infections, and extravasation during injection, making it essential that iron be administered in a hospital setting. Therefore, intravenous iron administration requires hospitalization in a facility with the structural requirements (available hospital beds, medical and nursing personnel) specific to this type of care. Furthermore, this type of injection is not free from iatrogenic risks.

[0014] Given these problems, there is an ongoing need to find alternatives to intravenous or oral iron therapy.

[0015] The present invention proposes novel iron nanoclusters to combat all types of iron deficiency, said nanoclusters being advantageously administered orally and without the above mentioned disadvantages.

[0016] Iron nanoclusters have already been proposed in the literature to address iron deficiency. For example, U.S. Patent Application Publication No. 2016 / 0022733 describes iron oxide nanocomposites coated with folic acid, nicotinic acid, and ascorbic acid for use in treating anemia, and these nanocomposites are intended for oral administration. U.S. Patent Application Publication No. 2016 / 008292 describes iron oxide nanoparticles coated with a biocompatible polymer containing polyethylene glycol and silane groups, which are covalently attached via a linker. These nanoparticles are proposed for the parenteral treatment of anemia.

[0017] However, the inventors are credited with developing novel iron nanoclusters with properties that make them particularly well suited to addressing iron deficiency, and the inventors are also credited with developing the original method for synthesizing iron nanoclusters. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] U.S. Patent Application Publication No. 2016 / 0022733 [Patent Document 2] U.S. Patent Application Publication No. 2016 / 008292 Summary of the Invention [Means for solving the problem]

[0019] The present invention provides iron nanoclusters having the following properties: - its surface is covered with a mixed layer containing histidine (His), acetate ions (Ac) and ascorbate ions (Asc); - spherical, - have a hydrodynamic diameter in the range of 0.6 to 2.0 nm, preferably less than 1.0 nm; - have a metal core diameter in the range of 0.5 to 1.5 nm, preferably less than 1.0 nm; - the nanoclusters exhibit a stability period ranging from 5 to 20 weeks when stored in liquid form and at a temperature of 4°C; - the nanoclusters exhibit a stability period of at least 12 months, preferably 12 to 18 months, when stored in dry form and under nitrogen at a temperature of 4°C; - The spectrophotometric characteristics are as follows: a UV-visible spectrum with a shoulder at 300±15 nm, and a fluorescence spectrum with an excitation wavelength of 364±15 nm and an emission wavelength of 415±15 nm. and can be referred to using the formula "FeNC@HisAcAsc".

[0020] However, the nanoclusters of the present invention may hereinafter be referred to as either "nanoclusters," "iron nanoclusters," "FeNC nanoclusters," "FeNC," "NC-Fe," ("FeNC" or "NC-Fe" means "iron nanoclusters"), "FeNC@HisAcAsc nanoclusters," or "FeNC@HisAcAsc."

[0021] The formula "FeNC@HisAcAsc" is the most explicit, as it describes that the iron nanocluster contains a layer on its surface that contains histidine and acetate and ascorbate ions.

[0022] The present invention also relates to a method for producing a method for manufacturing a semiconductor device, comprising the steps of: - a step of reacting iron (II) acetate with histidine to obtain a mixture of iron acetate and histidine, in which the molar ratio of histidine / iron (II) acetate is 8 or more, preferably in the range of 8 to 200, and more preferably in the range of 80 to 200; - a step of reacting a mixture of iron acetate and histidine with ascorbic acid to obtain a mixture of iron acetate, histidine and ascorbic acid, wherein the molar ratio of ascorbic acid / iron(II) acetate is 12 or more, preferably in the range of 12 to 700, and more preferably in the range of 130 to 700; - recovering the iron nanoclusters, the surface of which is covered with a mixed layer containing histidine, acetate ions, and ascorbate ions; The present invention also relates to a method for preparing said iron nanoclusters, comprising:

[0023] The present invention also provides - Prevention and / or treatment of diseases that cause iron deficiency (such as iron deficiency anemia), and - Treating iron deficiency The present invention relates to iron nanoclusters for use in

[0024] Finally, the present invention also relates to a composition comprising the iron nanoclusters of the present invention, which is a pharmaceutical, a dietary supplement or a food composition.

[0025] Furthermore, the composition of the present invention is characterized in that it is in a form suitable for oral administration.

[0026] Other features, details and advantages will become apparent upon reading the following detailed description and examining the accompanying drawings. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram of the iron nanocluster of the present invention, “FeNC@HisAcAsc,” which consists of a metallic core of iron surrounded by a mixed corona containing histidine, acetate, and ascorbate ions. [Figure 2] This is a high-performance liquid chromatography (HPLC) analysis (reversed-phase separation) of iron nanoclusters, showing the presence of acetate ions on the surface of the iron metal core. The chromatogram was obtained using a previously purified fraction (size exclusion chromatography) of iron nanoclusters. Chromatography of a sodium acetate standard solution was also performed. [Figure 3] This is a high-performance liquid chromatography (HPLC) analysis (reversed-phase separation) of iron nanoclusters, showing the presence of histidine and ascorbate ions on the surface of the iron metal core. The chromatogram was obtained using a previously purified fraction (size exclusion chromatography) of iron nanoclusters. Chromatography of histidine and ascorbate standards was also performed. [Figure 4] FIG. 1 shows the hydrodynamic diameter (unit: nanometers) of iron nanoclusters calculated by dynamic light scattering. [Figure 5] FIG. 1 shows the hydrodynamic diameter (unit: nanometers) of iron nanoclusters calculated by Taylor variance analysis. [Figure 6] FIG. 1 shows the UV-visible spectrum of iron nanoclusters. [Figure 7] FIG. 1 shows the fluorescence spectrum of iron nanoclusters. [Figure 8]This figure shows the results of a viability assay (MTT) of HepG2 cells (human liver cancer cells) grown in the presence of iron, either in the form of iron(III) nitrate (control, standard) (denoted as FeNO3) or in the form of iron nanoclusters of the present invention at various concentrations (denoted as NC-Fe1x; NC-Fe1 / 2, NC-Fe1 / 4, NC-Fe1 / 8, NC-Fe1 / 16, and NC-Fe1 / 32). "Negative" corresponds to "IMDM" medium without added iron. In each group of histograms, the leftmost histogram with a horizontal hatch corresponds to D1+3 of the cell viability assay. The histogram immediately adjacent to it corresponds to D1+5, and the one next to it corresponds to D1+7. The rightmost black histogram in each group of histograms corresponds to D1+10. DETAILED DESCRIPTION OF THE INVENTION

[0028] Iron Nanocluster The present invention provides an iron nanocluster comprising: - its surface is covered with a mixed layer containing histidine (His), acetate ions (Ac) and ascorbate ions (Asc); - spherical shape, - have a hydrodynamic diameter in the range of 0.6 to 2.0 nm, preferably less than 1.0 nm; - have a metal core diameter in the range of 0.5 to 1.5 nm, preferably less than 1.0 nm; - the nanoclusters exhibit a stability period ranging from 5 to 20 weeks when stored in liquid form and at a temperature of 4°C; - the nanoclusters exhibit a stability period of at least 12 months, preferably 12 to 18 months, when stored in dry form and under nitrogen at a temperature of 4°C; - The spectrophotometric characteristics are as follows: a UV-visible spectrum with a shoulder at 300±15 nm, and a fluorescence spectrum with an excitation wavelength of 364±15 nm and an emission wavelength of 415±15 nm. and can be designated using the formula "FeNC@HisAcAsc".

[0029] The iron nanoclusters that are the subject of the present invention are metal nanoclusters that have a metal core diameter of 2.0 nanometers (nm) or less and consist of an aggregate of several tens of atoms of a metal element (in this case, iron).

[0030] The nanoclusters of the present invention are composed of a metallic core of iron covered / encased / surrounded by a mixed layer / mixed corona containing histidine, acetate ions, and ascorbate ions.

[0031] The terms "corona" and "layer" may be used interchangeably throughout this application.

[0032] The term "mixed" is used to indicate that the corona or layer surrounding the iron core contains histidine, acetate and ascorbate ions.

[0033] Similarly, the verbs "cover / enclose / surround" may be used interchangeably to indicate that the iron core comprises a layer / corona of histidine, acetate ions, and ascorbate ions on the entire surface of the iron core.

[0034] The iron nanoclusters are generally spherical in shape.

[0035] In the sense of the present invention, the formula "FeNC@HisAcAsc" designates a nanocluster composed of an iron metal core coated with said mixed layer of histidine, acetate and ascorbate ions. Thus, the iron nanocluster of the present invention advantageously comprises three ligands on the surface of the iron core: histidine, acetate and ascorbate ions. These three ligands are bound to the iron metal core by coordination bonds.

[0036] Specifically, the mixed corona containing histidine, acetate ions, and ascorbate ions confers very high stability and low reactivity to the nanoclusters of the present invention.

[0037] "Low reactivity" means low decomposition, especially with respect to oxidation (such as by atmospheric oxygen).

[0038] The stability of the nanoclusters of the present invention means that the structure and properties of the nanoclusters are maintained over time at a storage temperature of 4° C. Maintenance of structure means in particular that the composition (metal core surrounded by a mixed layer / mixed corona as defined above), shape and diameter (metal core diameter and hydrodynamic diameter) of the nanoclusters are maintained over time.

[0039] The "liquid form" of iron nanoclusters refers to a solution or liquid mixture of iron nanoclusters. The 5-20 week stability stated above applies to iron nanoclusters in liquid form when stored at a storage temperature of 4°C.

[0040] "Dry form" refers to a solid form that can be crushed into a powder if desired. The 12-18 month stability stated above applies to the dry form of iron nanoclusters when stored under nitrogen at a storage temperature of 4°C.

[0041] Depending on the form (liquid or solid) of the nanoclusters of the present invention, they will therefore have different periods of stability.

[0042] The nanoclusters of the present invention have a metal core diameter of 2 nm or less, which is intermediate between that of molecules and nanoparticles, and exhibit spectrophotometric properties, particularly fluorescence properties, characteristic of this scale.

[0043] As the name suggests, the metal core diameter or metal diameter refers to the diameter of the part made entirely of ferrous metal.

[0044] The hydrodynamic diameter includes the diameter of the iron core plus the diameter of the histidine, acetate, and ascorbate layers / corona, and therefore refers to the diameter of the entire iron nanocluster.

[0045] According to one embodiment of the present invention, the iron nanoclusters have a metallic core diameter and a hydrodynamic diameter that are approximately equal, preferably less than 1.0 nm.

[0046] However, the metal core diameter will of course always be smaller than the hydrodynamic diameter.

[0047] The metal core diameter is determined by transmission electron microscopy, while the hydrodynamic diameter is calculated by dynamic light scattering and / or Taylor dispersion analysis.

[0048] According to one advantageous embodiment of the invention, the iron nanoclusters are in liquid or dry form.

[0049] The dry form of the nanoclusters is particularly advantageous as it allows for easy storage, preservation and transportation.

[0050] According to yet another advantageous embodiment, the iron nanoclusters of the invention have the following properties: - Ability to cross the intestinal barrier; - exhibit good bioavailability; - be biocompatible; - be biodegradable; - freeze-drying is possible; - Non-toxic to the human body; - No accumulation in organs such as the liver, spleen, kidneys or lungs It is characterized by showing at least one of the following.

[0051] According to one advantageous embodiment, the iron nanoclusters of the invention exhibit all of the above properties.

[0052] The lack of uptake by the organs of the nanoclusters of the present invention is due in particular to their small size (hydrodynamic diameter of 2.0 nm or less, preferably less than 1.0 nm), which allows them to circulate in the blood for longer periods than larger compounds.

[0053] More specifically, the small size of nanoclusters allows them to pass through membranes (especially the gastrointestinal tract) via persorption (the natural passage through pores in physiological systems) without passing through physiological absorption systems. Although this persorption phenomenon poses a toxicity risk for nanoclusters, adjusting the quantitative aspects of oral administration of nanoclusters can provide a therapeutic approach.

[0054] The surface properties of the nanoclusters of the present invention enable them to cross the intestinal barrier, which offers a significant advantage over oral iron preparations, which often cannot cross the intestinal barrier.

[0055] "Good bioavailability" means that orally administered iron nanoclusters reach the systemic circulation and are adequately distributed to target organs.

[0056] "Biocompatible" means that the iron nanoclusters are properly accepted by the various organs of the body and are not toxic to these organs.

[0057] The nanoclusters are biodegradable, meaning that they break down, releasing substances (iron, histidine, acetate and ascorbate) that can be metabolized or eliminated from the body without hindrance.

[0058] According to one advantageous embodiment of the invention, the nanoclusters can be freeze-dried.

[0059] The nanoclusters are completely stabilized and can be freeze-dried, thus facilitating storage, preservation, and transportation of the nanoclusters. The stability of the nanoclusters is as defined above.

[0060] The advantageous properties of the nanoclusters of the present invention are due in particular to the unique combination of their components: iron, histidine, acetate, and ascorbate.

[0061] To the best of our knowledge, iron nanoclusters containing a mixed corona / mixed layer of histidine, acetate, and ascorbate ions surrounding an iron metallic core and exhibiting the above-mentioned advantageous properties have not been reported previously.

[0062] Method for preparing iron nanoclusters The present invention also relates to a method for producing a method for manufacturing a semiconductor device, comprising the steps of: - a step of reacting iron (II) acetate with histidine to obtain a mixture of iron acetate and histidine, in which the molar ratio of histidine / iron (II) acetate is 8 or more, preferably in the range of 8 to 200, and more preferably in the range of 80 to 200; - a step of reacting a mixture of iron acetate and histidine with ascorbic acid to obtain a mixture of iron acetate, histidine and ascorbic acid, wherein the molar ratio of ascorbic acid / iron(II) acetate is 12 or more, preferably in the range of 12 to 700, and more preferably in the range of 130 to 200; - recovering the iron nanoclusters; The present invention relates to a method for preparing iron nanoclusters as defined above, characterized in that it comprises:

[0063] The above-defined molar ratios between histidine and iron acetate and between ascorbic acid and iron acetate, respectively, are important in enabling the histidine ligand, acetate ions, and ascorbate ions to bind to the iron metal core, resulting in nanoclusters containing three ligands on the surface of the iron core, which are bound to the surface of the iron core by coordinate bonds.

[0064] Ascorbic acid is a reducing agent. The reaction of a mixture of iron acetate and histidine with ascorbic acid, more specifically, the reduction reaction of the mixture of iron acetate and histidine with ascorbic acid, is particularly noteworthy. Thanks to ascorbic acid, it is possible to obtain iron nanoclusters that are completely non-toxic.

[0065] The present invention results in particular from the unexpected discovery by the inventors that the unique combination of the reagents used, iron acetate, histidine and ascorbic acid, in the proportions defined above, makes it possible to obtain iron nanoclusters with particularly advantageous properties.

[0066] The excellent stability of the nanoclusters of the present invention is one example.

[0067] According to one embodiment of the present invention, iron nanoclusters can be prepared more particularly according to a "solution phase" protocol or a "solid phase" protocol, these two synthetic routes each following the methodology described above.

[0068] 1 / Solution Phase Protocol According to one advantageous embodiment of the invention, the preparation process defined above is more particularly characterized in that it is carried out under inert gas, - the iron acetate is in solution form and the histidine is in powder form; - preparing a solution of iron acetate and histidine by adding histidine to an iron acetate solution; adjusting the iron acetate and histidine solution to a pH value in the range of 11 to 13, preferably 12; - the ascorbic acid is in powder form; - preparing a solution of iron acetate, histidine and ascorbic acid by adding ascorbic acid to a solution of iron acetate and histidine whose pH has been adjusted to the aforementioned value; - stirring the solution of iron acetate, histidine and ascorbic acid at a temperature ranging from 35°C to 45°C, preferably 40°C, for 2 to 6 hours, preferably 4 hours; - at the end of the pre-stirring step, a solution containing iron nanoclusters is obtained; - by appropriately dialysis of the solution containing iron nanoclusters, a purified solution of iron nanoclusters can be obtained; - A dry form of iron nanoclusters can be obtained by appropriately freeze-drying a solution containing iron nanoclusters that has been appropriately dialyzed. It is characterized by:

[0069] Solutions containing iron nanoclusters, after appropriate dialysis, have a stability period ranging from 5 to 20 weeks at a storage temperature of 4°C.

[0070] Dialysis can remove any excess histidine or ascorbic acid, or residual iron present in the nanocluster solution, that is not bound to the iron metal core. The layer containing histidine, acetate ions, and ascorbate ions is bound to the iron metal core by coordination bonds.

[0071] The dry form of iron nanoclusters obtained after freeze-drying has a stability period of at least 12 months, preferably 12-18 months, at a storage temperature of 4°C under nitrogen.

[0072] The dried form of iron nanoclusters can be reconstituted at any time by mixing it with a reconstitution solvent such as purified water. "Reconstitution / reconstitution" refers to the simple procedure of mixing the dried or lyophilized form with a solvent.

[0073] Analysis of the solution of iron nanoclusters obtained after redissolution of the dried form showed that the iron nanoclusters exhibited all the properties defined above, indicating that the solution was exactly the same as that obtained directly by the preparation method.

[0074] The solutions of iron nanoclusters obtained after redissolution of the dried form exhibit a stability period ranging from 5 to 12 weeks at a storage temperature of 4°C, preferably under nitrogen.

[0075] The above defined preparation method further comprises: - 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 in the range of 0.5 to 5.0 mM; - the concentration of histidine is higher than that of the iron acetate solution; - the pH of the iron acetate and histidine solution is adjusted with sodium hydroxide; - The concentration of ascorbic acid is equal to the concentration of histidine, - the iron concentration of the solution containing iron nanoclusters after appropriate dialysis is in the range of 14-112 μg / mL; - A dry form of iron nanoclusters can be obtained by freeze-drying a solution containing iron nanoclusters that has been appropriately dialyzed. The present invention is characterized in that it further includes at least one characteristic selected from the following:

[0076] According to one advantageous embodiment, the method of the invention exhibits all the above mentioned characteristics.

[0077] 2 / Solid-phase protocol According to another advantageous embodiment of the invention, the method for preparing iron nanoclusters as defined above comprises more particularly: - the iron acetate is in powder form and the histidine is in powder form; - mixing the iron acetate and histidine powders together to obtain a powder mixture of iron acetate and histidine; - grinding the powder mixture of iron acetate and histidine until a powder mixture of uniform color is obtained; - placing a uniform powder mixture of iron acetate and histidine into a reactor; - the ascorbic acid is in powder form; adding ascorbic acid to a reactor containing the homogenous iron acetate and histidine powder mixture; - stirring the powder mixture of iron acetate, histidine and ascorbic acid thus obtained, and then adding water dropwise into the reactor, the water being filtered ultrapure water; - placing the reactor under inert gas and shielding it from light; - leaving the mixture of iron acetate, histidine, ascorbic acid and water in a reactor with stirring for 16 to 36 hours, preferably 24 hours; - at the end of the pre-stirring step, a liquid mixture comprising iron nanoclusters is obtained; - by appropriately subjecting the liquid mixture containing iron nanoclusters to dialysis, a purified liquid mixture of iron nanoclusters can be obtained; - A liquid mixture containing iron nanoclusters that has been appropriately dialyzed can be freeze-dried to obtain a dry form of iron nanoclusters. It is characterized by:

[0078] The above defined preparation method further comprises: - Histidine concentration is higher than that of ferric acetate, - The concentration of ascorbic acid is equal to the concentration of histidine, - The water added to the reactor is filtered ultrapure water; - the inert gas is nitrogen; - the liquid mixture containing iron nanoclusters after appropriate dialysis has an iron concentration in the range of 1500-15000 μg / mL; - freeze-drying a liquid mixture containing iron nanoclusters, which has been suitably dialyzed, to obtain a dry form of iron nanoclusters; The present invention is characterized by further including at least one feature selected from the following.

[0079] According to another embodiment of the invention, the dry form of the iron nanoclusters obtained after freeze-drying (according to the "solution phase" protocol or the "solid phase" protocol) is stored under nitrogen, preferably in glass bottles, preferably at 4° C. Under such conditions, the iron nanocluster powder can be stored for a period of at least 12 months, preferably 12-18 months, without any change in the stability of the iron nanoclusters.

[0080] At the end of this period, the redissolution of the nanocluster powders is shown to be identical to those obtained directly after their preparation (according to the "solution phase" or "solid phase" protocol). Indeed, the iron nanoclusters exhibit all the properties defined above.

[0081] Uses of iron nanoclusters The present invention also relates to iron nanoclusters as defined above or obtainable by the method defined above, for use as a medicament.

[0082] More particularly, the present invention relates to iron nanoclusters as defined above or obtainable by a method as defined above, for use in the prevention and / or treatment of diseases causing iron deficiency.

[0083] An example of a disease that causes iron deficiency is iron deficiency anemia.

[0084] The present invention also relates to iron nanoclusters as defined above or obtainable by the method defined above for use in combating iron deficiency.

[0085] In this application, iron deficiency refers to iron deficiency in the broad sense, i.e., iron deficiency with or without iron deficiency anemia.

[0086] Another subject of the present invention is a composition characterized in that it comprises iron nanoclusters as defined above or obtainable by the method defined above.

[0087] The composition of the present invention may be a pharmaceutical, a dietary supplement or a food composition.

[0088] The amount of iron per composition can determine whether the composition is a dietary supplement or a drug, and therefore dietary supplements should contain less iron than drugs.

[0089] An example of a food composition is iron-added infant formula, more specifically, one to which the iron nanocluster of the present invention has been added.

[0090] According to one advantageous embodiment of the invention, the composition is in a form suitable for oral administration.

[0091] Advantageously, iron nanoclusters can be administered orally, particularly because their small size allows them to cross the intestinal barrier without hindrance.

[0092] Advantageously, the use of the iron nanoclusters of the present invention obviates the need for intravenous administration.

[0093] According to another advantageous embodiment, the composition of the invention comprising iron nanoclusters contains an amount of iron that is less than that typically present in conventional oral formulations, whether pharmaceutical or dietary supplements.

[0094] Advantageously, the compositions of the present invention do not exhibit the disadvantages that may occur with conventional oral formulations, either pharmaceutical or dietary supplements. [Example]

[0095] The following examples illustrate the present invention but do not limit it in any way.

[0096] Example 1 Preparation of iron nanoclusters In this example, two synthetic routes for preparing the iron nanoclusters of the present invention are described: a "solution phase protocol" and a "solid phase protocol."

[0097] 1 / Solution Phase Protocol Reagents used - Iron(II) acetate [Fe(CH3COO)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=40.00g / mol (VWR, Cas 1310-73-2).

[0098] Precautions The synthesis is carried out under an inert gas (nitrogen) atmosphere. Glassware is cleaned with aqua regia (1 part 65% nitric acid and 2 parts 37% hydrochloric acid by volume).

[0099] Ultrapure water was used and filtered through a 0.2 μm pore size filter.

[0100] The ferrous acetate is in powder form and stored under nitrogen. After weighing the ferrous acetate, the remaining stock should be promptly re-stored under nitrogen.

[0101] As the iron nanoclusters are intended for in vivo testing, it is necessary to work in a clean fume hood and clean all equipment with 70% v / v ethanol.

[0102] Preparation of 2.5 mM iron acetate stock solution Place 42.9 mg of iron acetate in a 100 mL volumetric flask. Add filtered, ultrapure water up to the flask mark. This will give a 2.5 mM iron acetate solution.

[0103] Once completely dissolved, transfer the iron acetate solution to a suitable container. This solution can be stored in a refrigerator at 4°C for one month.

[0104] Synthesis of histidine-stabilized nanoclusters Add 500 µL of the iron acetate stock solution prepared in the previous step to a round-neck flask that can hold up to 50 mL of solution. Next, add 4500 µL of filtered, ultrapure water to the flask. This will make the iron acetate solution 1x.

[0105] The 1x iron acetate solution was agitated at 130 rpm using a multiplate shaker. 39 mg of histidine was added to the iron acetate solution. The iron acetate and histidine solution was agitated for 15 minutes.

[0106] The solution will have a slight reddish hue. After stirring for 15 minutes, add 10 drops of 1 M NaOH to the iron acetate and histidine solution to adjust the pH to 12. Add 139 mg of ascorbic acid to the reaction mixture. It takes 2 minutes for the ascorbic acid to completely dissolve. Place the flask (reactor) in a 40°C water bath and stir (speed setting 6) for 4 hours.

[0107] At the end of the synthesis, the resulting nanocluster solution will be colorless. The resulting iron nanocluster solution will be 1x, with an iron concentration of 14 μg / mL. Store the solution in a cool place at 4°C.

[0108] The synthesis yield is 100% and there is no residual iron (elemental iron) in the nanocluster solution, which may be freeze-dried.

[0109] Prepare iron acetate solutions with 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.

[0110] For reference, 2x iron acetate solution is prepared by adding 1000 μL of iron acetate stock solution to a flask and adding filtered ultrapure water to a volume of 5000 μL. 4x iron acetate solution is prepared by adding 2000 μL of iron acetate stock solution to a flask and adding filtered ultrapure water to a volume of 5000 μL. And so on.

[0111] The resulting 1x to 8x solutions of iron nanoclusters are stored in a cool place at 4°C.

[0112] Dialysis of iron nanoclusters The 1x solution of iron nanoclusters obtained in the previous step is purified by dialysis.

[0113] Prepare a dialysis apparatus (X12 Float-A-Riser G2 CE MWCO 100-500 D, part number 1511160) and fill a 150 mL beaker with 100 mL of filtered ultrapure water. Fill the dialysis apparatus with filtered ultrapure water using a Pasteur pipette. Place the dialysis apparatus in the beaker and stir (130 rpm). Leave the apparatus for 1 hour to allow it to soak and be washed.

[0114] Then, replace the water in the beaker with 100 mL of fresh filtered ultrapure water. Using a Pasteur pipette, empty the dialysis equipment and fill it with the 1x solution of iron nanoclusters. Leave it overnight (12 hours) at a temperature between 2 and 6 °C and stir.

[0115] The resulting 1x solution of dialyzed iron nanoclusters is transferred to a suitable container and stored at a temperature of 4°C.

[0116] Dialysis does not affect the iron concentration of the nanoclusters, so the iron concentration in the dialyzed iron nanocluster solution is the same as the iron concentration in the undialyzed solution.

[0117] Once the iron nanocluster solution has been dialyzed, it can be freeze-dried.

[0118] The iron concentration in the dialyzed solution of iron nanoclusters is identical to that in the undialyzed solution, ranging from 14 to 112 μg / mL depending on the concentration of the iron acetate solution, which ranges from 1x to 8x.

[0119] 2 / Solid-phase protocol Reagents used and precautions Iron(II) acetate, L(-)-histidine, and ascorbic acid are the same as those used in the solution-phase protocol. Sodium hydroxide is not required in the solid-phase protocol.

[0120] Similar precautions as for solution phase protocols are required.

[0121] Synthesis of histidine-stabilized iron nanoclusters Weigh out 23 mg of iron acetate and place it in an agate mortar. Then weigh out 1.7 g of histidine. Add an equal amount of histidine powder to the iron acetate powder and carefully grind the powder thoroughly with a pestle until a mixture of uniform color and appearance is obtained. Repeat this process until all the histidine is used.

[0122] The final mixture of the two powders should be red and homogeneous. Next, transfer the mixture of the two powders to a 50 mL single-neck flask (NS 19 / 26 ground neck) using a spatula. Weigh out 3 g of ascorbic acid and place it in the flask. Place an olive-shaped magnetic stirrer at the bottom of the flask. Filter 5 mL of ultrapure water using a 5 mL plastic syringe and add it dropwise to the reactor. A liquid mixture is obtained.

[0123] The reactor is sealed with a skirted flip-top cap (19.4 mm diameter) and placed under nitrogen using a latex balloon to avoid excessive pressure. The reactor is wrapped in aluminum foil and then stirred (200 rpm) for the duration of the reaction. A 24-hour wait is required for the reaction to be complete. Once the reaction is complete, the resulting product is in liquid form and red in color.

[0124] The resulting liquid containing iron nanoclusters will have an iron concentration of 1500 μg / mL, which is 100x.

[0125] Of course, the iron concentration of the nanoclusters will depend on the amount of iron acetate used at the start of the method of the present invention.

[0126] By repeating the above procedure, iron concentrations ranging from 1500 μg / mL (100x) to 15,000 μg / mL (1000x) can be obtained, depending on the amount of iron acetate, which ranges from 23 to 230 mg at the start of the method of the present invention (solid-phase protocol).

[0127] The resulting liquid containing the iron nanoclusters is then transferred to a suitable plastic container (final volume will be slightly more than 5 mL, approximately 8.5 mL). The liquid containing the iron nanoclusters can be stored at 4°C or transferred to a freeze dryer. Lyophilization is performed in 1 mL aliquots without the addition of any additional reagents. After lyophilization, the contents of the vial (containing the iron nanoclusters in dry form) are stored under nitrogen at 4°C.

[0128] The dried form of iron nanoclusters can be reconstituted in 1 mL of purified water at any time. Iron nanocluster solutions reconstituted in this manner are stored at 4° C., preferably under nitrogen.

[0129] Example 2 Characterization of iron nanoclusters The iron nanoclusters obtained in Example 1, either via solution or solid phase protocols, are characterized in terms of their structure, size, spectrophotometric properties and stability.

[0130] Structure of iron nanoclusters The iron nanoclusters of the present invention are more specifically spherical. They consist of a metallic core of iron surrounded by a mixed corona containing histidine, acetate ions, and ascorbate ions. Figure 1 shows a schematic diagram of the iron nanoclusters of the present invention, which can also be designated by the formula "FeNC@HisAcAsc."

[0131] High performance liquid chromatography (HPLC), more specifically reversed-phase chromatography, confirmed the presence of acetate ions on the surface of the iron nanoclusters.

[0132] The iron nanoclusters in the dialyzed 1x solution obtained in Example 1 (see Section 1, "Solution Phase Protocol") are disrupted (completely dissolved and the nanocluster structure is returned to its constituent parts) by a chemical process of dissolving in concentrated acid (HCl) and then in concentrated base (NaOH), and then analyzed by HPLC and compared to a sodium acetate control.

[0133] The results obtained are shown in the chromatograms in Figure 2. For the iron nanoclusters (see upper plot), a peak associated with acetate ions (control, see lower plot) is observed at 3.6 min, thus confirming the presence of acetate ions on the surface of the metal core.

[0134] The presence of histidine and ascorbate ions on the surface of iron nanoclusters was also confirmed by high-performance liquid chromatography, and more specifically, after purifying the nanoclusters in solution by size-exclusion chromatography, they were compared with histidine and ascorbate controls.

[0135] The results obtained are shown in the chromatogram in FIG.

[0136] For iron nanoclusters (see upper plot), a peak associated with histidine (control, see lower plot) is observed at 2.1 min, thus confirming the presence of histidine on the surface of the metal core.

[0137] For iron nanoclusters (see upper plot), a peak associated with ascorbate ions (control, see lower plot) is observed at 3.0 min, thus confirming the presence of ascorbate ions on the surface of the metal core.

[0138] Size of iron nanoclusters The hydrodynamic diameter (Dh) of the iron nanoclusters was calculated by dynamic light scattering (Figure 4) (angle 173°, 530 nm laser, temperature 25°C, Malvern Nanosizer) and Taylor dispersion analysis (Figure 5).

[0139] The dynamic light scattering method involves analyzing the Brownian motion of particles and modeling it using the Stokes-Einstein equation.

[0140] The Taylor dispersion analysis method involves plug-injecting a solute into an open capillary tube (50 μm) and dispersing it under the influence of hydrodynamic flow (positive pressure 1 psi, parabolic velocity distribution). The principle for measuring the hydrodynamic radius is based on the Taylor-Allis relation, which demonstrates a correlation between the broadening of the solute peak (modeling a Gaussian distribution) and the molecular diffusion coefficient.

[0141] The metallic core diameter of the iron nanoclusters was estimated using transmission electron microscopy (deposition on a nickel grid, observation under a beam operating at 200 kV (LaB6 cathode), Philips CM 200).

[0142] The diameters (hydrodynamic and metallic core) of the iron nanoclusters were calculated immediately after synthesis for both the solution-phase and solid-phase protocols.

[0143] In the solution phase protocol, the hydrodynamic diameter and metal core diameter were calculated for a 1x solution of non-dialyzed, non-lyophilized iron nanoclusters with an iron concentration of 14 μg / mL.

[0144] In the solid-phase protocol, the hydrodynamic diameter and metal core diameter were calculated on lyophilized samples obtained from a 100x solution of undialyzed iron nanoclusters with an iron concentration of 1500 μg / mL.

[0145] The average hydrodynamic diameter of the iron nanoclusters, similar to the diameter of the metal core, is less than 1.0 nm. More specifically, Figures 4 and 5 show that the hydrodynamic diameter of the iron nanoclusters is 0.69 ± 0.06 nm by dynamic light scattering (Figure 4) and Taylor scattering (Figure 5).

[0146] Spectrophotometric properties The spectrophotometric properties of the iron nanoclusters were evaluated immediately after synthesis by UV-visible spectroscopy (Figure 6) and fluorescence spectroscopy (Figure 7).

[0147] In the UV-visible spectrum, there is a shoulder at 300±15 nm (FIG. 6), confirming the presence of nanoclusters.

[0148] Furthermore, the iron nanoclusters exhibited fluorescence at an excitation wavelength of 364±15 nm and an emission wavelength of 415±15 nm (FIG. 7), confirming the presence of the nanoclusters.

[0149] The iron nanoclusters of the present invention exhibit optical properties, particularly fluorescence, characteristic of an intermediate scale between molecules and nanoparticles.

[0150] Stability of iron nanoclusters The stability of the iron nanoclusters was assessed by measuring their hydrodynamic diameter using dynamic light scattering (173° angle, 530 nm laser, 25° C. temperature, Malvern Nanosizer).

[0151] The analysis was performed on iron nanoclusters obtained using both solution and solid phase protocols.

[0152] For the solution-phase protocol, the analysis was performed on a 1x solution of iron nanoclusters (iron concentration 14 μg / mL). Stability was assessed slightly more than 5 weeks after synthesis.

[0153] The hydrodynamic diameter of the iron nanoclusters was observed to remain at 0.69 ± 0.06 nm for more than 5 weeks after synthesis, demonstrating excellent stability.

[0154] In the solid phase protocol, the analysis - Undialyzed lyophilized sample obtained from 100x solution of iron nanoclusters (iron concentration 1500 μg / mL), and - A solution obtained by reconstituting the freeze-dried sample to the same volume as before freeze-drying The survey was carried out on.

[0155] The hydrodynamic diameter of the iron nanoclusters in the freeze-dried sample was 0.70 nm, even after more than 5 weeks of storage under nitrogen.

[0156] After the sample was reconstituted to the same volume as before freeze-drying, the hydrodynamic diameter of the iron nanoclusters in the reconstituted sample was 0.76 nm, further demonstrating excellent stability.

[0157] Example 3 Toxicity evaluation of iron nanoclusters This example shows the results of a viability assay (MTT assay) of HepG2 cells (human liver cancer cells) grown in the presence of iron, either in the form of iron(III) nitrate (control, standard) or in the form of iron nanoclusters of the present invention at various concentrations.

[0158] iron The iron(III) nitrate (or ferric nitrate) used as a control is a compound with the semistructural formula "Fe(NO3)3", more specifically in the form of the nonahydrate "Fe(NO3)3.9H2O".

[0159] Prepare a solution of iron nitrate nonahydrate to a concentration of 100 mg / L in water (equivalent to an iron concentration of 14 µg / mL) in a total volume of 50 mL. Filter this solution in a PSM fume hood.

[0160] A 1x solution of iron nanoclusters containing an iron concentration of 14 μg / mL, as prepared in Example 1 (Section 1 / Solution Phase Protocol), is used in particular.

[0161] Make serial dilutions of the 1x solution of iron nanoclusters to 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 (see prepared media 4-8 below).

[0162] HepG2 cells HepG2 cells are a cell line derived from the liver tissue of a patient with hepatocellular carcinoma (HCC).

[0163] MTT assay 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 in MTT is reduced by mitochondrial succinate dehydrogenase in active, living cells to form formazan, which forms a purple precipitate within the mitochondria.

[0164] The amount of precipitate formed is proportional to the number of viable cells (but also to the metabolic activity of each cell), so after incubating the cells with MTT for a specific time (approximately 3 hours) at 37°C, it is sufficient to dissolve the cells, their mitochondria, and the resulting purple formazan precipitate with 100% DMSO (dimethyl sulfoxide).

[0165] Simple spectrophotometric measurement of optical density at 570 nm allows the determination of the relative amounts of viable and metabolically active cells.

[0166] Therefore, if the assay is to be quantitative, it is necessary to construct a standard curve for each assay, the readings of which are obtained at 570 nm using a spectrophotometer (see Figure 8).

[0167] Growth conditions for HepG2 cells HepG2 cells were grown in complete medium (DMEM-Dulbecco's Modified Eagle's Medium) in 24-well plates for 24 hours, then the medium was changed to selective medium (IMDM*) (see below) with or without the addition of our iron nanoclusters, compared with the addition of iron nitrate nonahydrate (Fe(NO3)3.9H2O).

[0168] Fe 3+ Fe 2+ After being reduced to , it crossed the cell membrane via DMT1 (divalent metal transporter 1).

[0169] Selective medium (IMDM*) IMDM (Iscove's Modified Dulbecco's Medium), 10% FBS (fetal bovine serum) (dialyzed), 1% antibiotic (a mixture of penicillin and streptomycin), ·1% (1mM) pyruvate.

[0170] Eight selective media were prepared - Medium 1: IMDM*, - Medium 2: IMDM* + 1% Fe(NO3)3.9H2O (100 mg / L stock solution), - Medium 3: IMDM* + 1% 1x solution of iron nanoclusters (i.e. equivalent to 14 μg / mL iron), - Medium 4: Medium 3 diluted 1:2 with IMDM*; - Medium 5: Medium 4 diluted 1:2 with IMDM* (i.e. 1 / 4 of Medium 3), - Medium 6: Medium 5 diluted 1:2 with IMDM* (i.e. 1 / 8 of Medium 3), - Medium 7: Medium 6 diluted 1:2 with IMDM* (i.e. 1 / 16 of Medium 3), - Medium 8: Medium 7 diluted 1:2 with IMDM* to make 1 / 2 (i.e. 1 / 32 of Medium 3).

[0171] Assay Period One plate is prepared for each step (D1+3, D1+5, D1+7, D1+10). The total assay duration is 11 days.

[0172] The process is as follows: - D0: 4000 cells / cm in DMEM complete medium (500 μL / well) 2 Inoculate p24 (triplicate) - D1: Change to IMDM selective medium and add iron nanocluster solution and Fe(NO3) 3. Compare 9H2O (0.1mg / L), - D1+3: 1 plate was stopped for MTT, the rest were changed medium, - D1+5: 1 plate was stopped for MTT, the rest were changed medium, - D1+7:1 plate was stopped for MTT, the rest were changed medium, - D1+10:1 plate was stopped for MTT and the test was completed.

[0173] The results obtained are shown in Figure 8, in which: - "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" is compatible with medium 8.

[0174] Repeated MTT viability assays on D1+3, D1+5, D1+7, and D1+10 indicate that treatment with the iron nanoclusters of the present invention is not toxic to HepG2 cells treated with doses of iron equivalent to those found in standard medium containing 0.1 mg / L concentration of iron nitrate nonahydrate form, Fe(NO3)3.9H2O.

[0175] The negative control was treated with medium without added iron, but the presence of fetal bovine serum provided enough iron to allow some cells to grow (therefore, the control was not completely negative).

[0176] MTT was weighed and dissolved at 5 mg / mL in natural IMDM medium, which was 0.2 μm filtered in a PSM fume hood and stored at +4°C throughout the experimental protocol.

[0177] For 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) without added iron and then incubated at 37°C for 3 hours.

[0178] After each medium change, the spent medium was removed and frozen at -20°C for subsequent transferrin and ferritin testing.

[0179] In conclusion, this assay demonstrates that the iron nanoclusters of the present invention are free of any toxicity, regardless of concentration. HepG2 cells cultured in iron-free medium and subsequently supplemented with iron show excellent viability.

Claims

1. An iron nanocluster, - its surface is covered with a mixed layer containing histidine (His), acetate ions (Ac) and ascorbate ions (Asc); - spherical, - have a hydrodynamic diameter in the range of 0.6 to 2.0 nm, preferably less than 1.0 nm; - have a metal core diameter in the range of 0.5 to 1.5 nm, preferably less than 1.0 nm; - the nanoclusters exhibit a stability period ranging from 5 to 20 weeks when in liquid form and stored at a temperature of 4°C; - the nanoclusters exhibit a stability period of at least 12 months, preferably 12 to 18 months, when stored in dry form and under nitrogen at a temperature of 4°C; - The spectrophotometric characteristics are as follows: a UV-visible spectrum with a shoulder at 300±15 nm, and a fluorescence spectrum with an excitation wavelength of 364±15 nm and an emission wavelength of 415±15 nm. and can be referred to using the formula "FeNC@HisAcAsc".

2. 2. The iron nanocluster according to claim 1, characterized in that it is in liquid or dry form.

3. The following characteristics: - Ability to cross the intestinal barrier; - exhibit good bioavailability; - be biocompatible; - be biodegradable; - freeze-drying is possible; - Non-toxic to the human body; - No accumulation in organs such as the liver, spleen, kidneys or lungs 3. The iron nanocluster according to claim 1, wherein the iron nanocluster exhibits at least one of the following:

4. The following process: - reacting iron(II) acetate with histidine to obtain a mixture of iron acetate and histidine, wherein the histidine / iron(II) acetate molar ratio is 8 or more, preferably in the range of 8 to 200, more preferably in the range of 80 to 200; - reacting a mixture of iron acetate and histidine with ascorbic acid to obtain a mixture of iron acetate, histidine and ascorbic acid, wherein the molar ratio of ascorbic acid / iron(II) acetate is 12 or more, preferably in the range of 12 to 700, more preferably in the range of 130 to 700; - recovering the iron nanoclusters; 4. A method for preparing iron nanoclusters according to any one of claims 1 to 3, comprising:

5. The preparation method is carried out under an inert gas atmosphere, - the iron acetate is in solution form and the histidine is in powder form; - preparing a solution of iron acetate and histidine by adding histidine to an iron acetate solution; adjusting the iron acetate and histidine solution to a pH value in the range of 11 to 13, preferably 12; - the ascorbic acid is in powder form; - preparing a solution of iron acetate, histidine and ascorbic acid by adding ascorbic acid to a solution of iron acetate and histidine whose pH has been adjusted to the aforementioned value; - stirring the solution of iron acetate, histidine and ascorbic acid at a temperature ranging from 35°C to 45°C, preferably 40°C, for 2 to 6 hours, preferably 4 hours; - at the end of the previous stirring step, a solution containing iron nanoclusters is obtained, - by appropriately dialysis of the solution containing iron nanoclusters, a purified solution of iron nanoclusters can be obtained; - A dry form of iron nanoclusters can be obtained by appropriately freeze-drying a solution containing iron nanoclusters that has been appropriately dialyzed. The preparation method according to claim 4, characterized in that

6. - 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 in the range of 0.5 to 5.0 mM; - the concentration of histidine is higher than that of the iron acetate solution; - the pH of the iron acetate and histidine solution is adjusted with sodium hydroxide; - The concentration of ascorbic acid is equal to the concentration of histidine, - the iron concentration of the iron nanocluster solution after appropriate dialysis is in the range of 14-112 μg / mL; - A dry form of iron nanoclusters can be obtained by freeze-drying a solution containing iron nanoclusters that has been appropriately dialyzed.

6. The method of claim 5, further comprising at least one feature selected from the group consisting of:

7. - the iron acetate is in powder form and the histidine is in powder form; - mixing the iron acetate and histidine powders together to obtain a powder mixture of iron acetate and histidine; - grinding the powder mixture of iron acetate and histidine until a powder mixture of uniform color is obtained; - placing a uniform powder mixture of iron acetate and histidine into a reactor; - the ascorbic acid is in powder form; adding ascorbic acid to a reactor containing the homogenous iron acetate and histidine powder mixture; - Stirring the powder mixture of iron acetate, histidine and ascorbic acid thus obtained, and then adding water dropwise into the reactor, the water being filtered ultrapure water; - placing the reactor under inert gas and shielding it from light; - leaving the mixture of iron acetate, histidine, ascorbic acid and water in a reactor under stirring for 16 to 36 hours, preferably 24 hours; - at the end of the pre-stirring step, a liquid mixture comprising iron nanoclusters is obtained; - by appropriately subjecting the liquid mixture containing iron nanoclusters to dialysis, a purified liquid mixture of iron nanoclusters can be obtained; - A liquid mixture containing iron nanoclusters that has been appropriately dialyzed can be freeze-dried to obtain a dry form of iron nanoclusters. The preparation method according to claim 4, characterized in that

8. - Histidine concentration is higher than that of ferric acetate, - The concentration of ascorbic acid is equal to the concentration of histidine, - The water added to the reactor is filtered ultrapure water; - the inert gas is nitrogen; - the liquid mixture containing iron nanoclusters, after appropriate dialysis, has an iron concentration in the range of 1500-15000 μg / mL; - freeze-drying a liquid mixture containing iron nanoclusters, which has been suitably dialyzed, to obtain a dry form of iron nanoclusters; 8. The method of claim 7, further comprising at least one feature selected from the group consisting of:

9. 9. A method according to any one of claims 5 to 8, characterized in that the dried form of the iron nanoclusters is stored under nitrogen, preferably in a glass bottle, preferably at 4°C, and that said powder can be stored for a period of at least 12 months, preferably between 12 and 18 months, without any change in the stability of the iron nanoclusters.

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

11. Iron nanoclusters according to any one of claims 1 to 3 or obtainable by the method according to 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 iron deficiency is iron deficiency anemia.

13. 10. Iron nanoclusters according to any one of claims 1 to 3 or obtainable by the method according to any one of claims 4 to 9 for use in combating iron deficiency.

14. A composition comprising iron nanoclusters according to any one of claims 1 to 3 or obtainable by the method according to any one of claims 4 to 9.

15. 15. The composition according to claim 14, characterized in that it is a pharmaceutical, nutritional supplement or food composition.

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

Images