Iron-based metallo-organic network and 5,5'-methylene diisophthalic acid

Abstract

The invention relates to a porous metal-organic network (MOF) comprising metal ions coordinated to an organic ligand, wherein at least 66% of the metal ions are iron(III) ions and the organic ligand is 5,5'-methylenediisophthalic acid, its preparation process, and its use for releasing a substance in a controlled manner. The present invention also relates to pharmaceutical compositions comprising such a metal-organic network.

Description

Title of the invention: Metal-organic network based on iron and 5,5'-methylenediisophthalic acid. TECHNICAL FIELD OF THE INVENTION

[0001] The present invention belongs to the field of porous metal-organic networks (MOFs). More specifically, the present invention relates to a porous crystalline metal-organic network based on iron and 5,5'-methylenediisophthalic acid, and its use for releasing a substance in a controlled manner. STATE OF THE ART

[0002] Metal-organic networks, also called MOFs, are ordered coordination polymers in the form of crystalline hybrid porous structures composed of inorganic nodes (isolated metal ions or assemblies into finite objects (oxoclusters), one-dimensional (chains), two- or even three-dimensional) coordinated by bridging organic ligands (spacers). Such organic ligands are generally multidentate and coordinate the metal ions through functional groups such as carboxylate, phosphate, or azole groups. The nature of the metal ions and organic compounds has a significant influence on the MOF structure, notably impacting the size and shape of the pores, as well as their surface chemistry, which in turn affect the material properties. A wide variety of MOF structures therefore exists due to the extensive range of combinable metal entities and organic ligands.

[0003] Due to their microporous and / or mesoporous architecture, MOFs are often characterized by a large specific surface area, combined with good thermal and chemical stability, enabling various applications such as the storage, separation, and controlled release of chemical elements in diverse fields like the environment (e.g., CO2 capture, fresh water production), energy (e.g., batteries, supercapacitors, membranes for fuel cells), or health (e.g., controlled drug release, contrast agents). Thus, these porous hybrid solids are attracting increasing interest thanks to their high degree of chemical and structural modularity as well as their numerous properties.

[0004] Iron-based MOFs are of great interest because they are characterized by good stability, and because iron is among the least toxic and most abundant metals that can be used to manufacture MOFs.

[0005] Iron-based MOFs can be biocompatible and biodegradable, making them excellent pharmaceutical carriers, especially since they exhibit a tunable adsorption capacity, which can be higher than that of liposomes. or polymer nanoparticles. They thus make it possible to stabilize pharmaceutical agents that have a short half-life and / or low bioavailability, thereby improving their clinical efficacy.

[0006] In this regard, MIL-100(Fe) (Lavoisier Institute material no. 100), obtained from iron(III) trimers and benzene tricarboxylate (also called trimesate), has been used to encapsulate pharmaceutical molecules, such as busulfan, azidothymidine triphosphate, doxorubicin, gemcitabine monophosphate, topotecan, theophylline, and aspirin, among others. In the case of theophylline, a molecule used in the treatment of asthma and COPD, administered via inhalation, MIL-100(Fe) exhibits a high drug loading capacity (32%) and allows for a prolonged release of the active ingredient (46%), with low toxicity, even at high concentrations. Despite these advantages, MIL-100(Fe) cannot be used to encapsulate macromolecules.Indeed, although it has mesopores in the form of cages, the access windows to these do not exceed 0.86 nm in accessible dimension, thus limiting the spectrum of active ingredients that can be adsorbed into the pores of this material.

[0007] Fe-MIL-101-NH2 is another iron-based mesoporous MOF, obtained from iron and 2-aminoterephthalic acid, which is used as an isoniazid carrier, with a drug encapsulation capacity of 12%, without exhibiting cytotoxicity. However, although it has larger pore access openings than MIL-100 (Fe), this material is unstable in air and water.

[0008] Finally, other iron-based MOFs with large pores are both unstable, require the use of polyaromatic ligands with low biocompatibility, and necessitate the use of toxic solvents (such as N,N'-dimethylformamide (DMF)) during their synthesis. The only mesoporous MOFs stable in the presence of water are primarily chromium-based, which is potentially toxic (e.g., Cr(VI)), or in some cases zirconium- or hafnium-based, which are biocompatible but non-endogenous.

[0009] Thus, there is a strong need to develop new MOFs, particularly mesoporous ones, exhibiting satisfactory stability, biocompatibility, and low toxicity so as to be compatible with pharmaceutical use, for example, for encapsulating large active ingredients, such as taxol-type pharmaceutical molecules and / or biomolecules (DNA, RNA, proteins), while allowing the controlled release of these molecules of pharmaceutical interest. Description of the invention

[0010] In the context stated above, the present invention aims to remedy, in whole or in part, the limitations of the prior art mentioned above.

[0011] The inventors have developed a new porous metal-organic material (MOF) based on iron and 5,5'-methylene diisophthalic acid exhibiting good stability in air and water, and which can be used to encapsulate molecules, such as macromolecules, for controlled release, for example in a patient.

[0012] A first object of the invention relates to a crystalline porous metal-organic network (MOF) comprising metal ions, consisting of at least 66% of Iron (III) ions, coordinated to an organic ligand, said organic ligand being 5,5'-methylene diisophthalic acid in a deprotonated form (mdip).

[0013] Advantageously, such a metal-organic network exhibits good stability in water and air, excellent biocompatibility and biodegradability, and low toxicity. Thus, a metal-organic network according to the invention is suitable for pharmaceutical use, in particular administration to a patient.

[0014] A metal-organic network according to the invention is capable of adsorbing and encapsulating at least partially in its pores a substance (organic, inorganic or hybrid), typically a pharmaceutical active ingredient, a combination of pharmaceutical active ingredients or any other molecule, in order to release it (or them) in a controlled manner.

[0015] Advantageously, and in the context of a specific application in the field of oncology, the release of the substance adsorbed in the pores of the metallo-organic network according to the invention can be accelerated, or even triggered, when said metallo-organic network is in a tumor microenvironment. Such release is induced by a high concentration of glutathione (GSH), for example, a glutathione concentration between 10 mM and 40 mM in the tumor microenvironment, particularly in the microenvironment of tumors resistant to anticancer treatments, which leads to degradation of the metallo-organic network by a reduction of Fe3+ ions to Fe2+ ions.

[0016] Furthermore, the generation of Fe2+ ions during the degradation of the metallo-organic network allows to initiate a Fenton type reaction which generates reactive oxygen species (ROS), making it possible to potentiate the effect of the active principles carried by said metallo-organic network.

[0017] According to one embodiment, the metal-organic network according to the invention comprises pores with a size between 1 nm and 5 nm, preferably with a size between 1 nm and 3 nm. Preferably, the pore size is determined on the basis of the N2 adsorption isotherm measured at 77 K.

[0018] According to one embodiment, the metallo-organic network according to the invention comprises pore access windows having a size between 1 nm and 5 nm, preferably a size between 1 nm and 3 nm.

[0019] Such a pore size, and such a pore access window size, makes it possible to adsorb and / or at least partially encapsulate large molecules, such as macromolecules or biomolecules, for example, molecules of size equal to or greater than 2 nm. A metal-organic network according to the invention comprising pores of such a size can particularly adsorb and / or encapsulate, at least partially, nucleic acids such as single- or double-stranded DNA or RNA of varying lengths; recombinant proteins, such as insulin; or monoclonal antibodies.

[0020] According to one embodiment, the pores of a metal-organic network according to the invention have at least a bimodal pore size distribution, with a first pore size equal to or less than 2 nm and a second pore size equal to or greater than 3 nm. Preferably, the distribution and pore size are determined on the basis of the N2 adsorption isotherm at 77 K.

[0021] Such bimodality in the pore size distribution advantageously allows for the adsorption and / or at least partial encapsulation within the same material of different molecules that may have different sizes. Thus, a metal-organic network according to the invention can adsorb at least one molecule in a first set of pores having a first size, and at least one molecule in a second set of pores having a second size. By way of example, a metal-organic network according to the invention can adsorb at least one nucleic acid in a first set of pores having a first size, and at least one chemotherapeutic agent in a second set of pores having a second size.

[0022] According to one embodiment, the pore volume of a metal-organic network according to the invention is between 0.8 cm³ / g and 0.9 cm³ / g. Such a pore volume can be calculated directly from the N₂ adsorption isotherm at 77K (measured capacity).

[0023] According to one embodiment, the metal ions of the metallo-organic network according to the invention are Iron(III) ions only.

[0024] Such a metal-organic network comprising only Iron(III) as metal ions advantageously exhibits improved biocompatibility, tolerance, and biodegradability, and an absence of toxicity. It is therefore compatible with pharmaceutical applications.

[0025] According to an alternative embodiment, the metal ions of the metal-organic network according to the invention further comprise Copper(II), Magnesium(II), Cobalt(II) or Indium(III) ions.

[0026] According to one embodiment, the metallo-organic network according to the invention has the formula [Fe3O(OH)(mdip)i 5]-x H2O, in which x is an integer equal to or greater than 1.

[0027] According to a preferred embodiment, the metallo-organic network according to the invention has the formula {Fe30(OH)0 25(Cl)o.75 (Ci7O8H8)i.5}. 8H2O.

[0028] A second object of the invention relates to a method for preparing a metal-organic network according to the invention, comprising the steps of:

[0029] 1) contacting 5,5'-methylenediisophthalic acid with at least a first a metallic precursor comprising iron(III), preferably comprising an iron(III) salt, for example FeCl3.6H2O, in a first reaction medium, preferably comprising an alcoholic solvent, thereby obtaining the metallo-organic network according to the invention, then

[0030] 2) recovery of the metallo-organic network obtained in step 1). The first medium reaction may preferentially include an alcoholic solvent, which has the advantage of having limited toxicity (eco-compatible synthesis).

[0031] According to one embodiment, the first reaction medium in a process according to the invention further comprises at least one second metallic precursor comprising Copper (II), Magnesium (II), Cobalt (II), Indium (III), Manganese (II), Nickel (II), Aluminium (III), Galium (III), Lanthanides (III) or Ruthenium ions.

[0032] According to one embodiment, the first reaction medium in the process according to the invention further comprises acetic acid.

[0033] The presence of acetic acid in the first reaction medium advantageously allows the kinetics of the formation of the metallo-organic network to be modulated, thus allowing optimal crystallization of the latter.

[0034] A third object of the invention relates to a pharmaceutical composition comprising at least one active ingredient and a metallo-organic network according to the invention as a pharmaceutically acceptable carrier.

[0035] At least one active ingredient is advantageously adsorbed into pores of the metallo-organic network acting as a pharmaceutically acceptable carrier, and can be released in a controlled manner by said metallo-organic network.

[0036] According to one embodiment, at least one active ingredient in a pharmaceutical composition according to the invention may comprise a nucleic acid, preferably DNA or RNA.

[0037] Advantageously, the size of the pores of the metallo-organic network, their one-dimensional shape, and the size of the access windows to the pores allow the adsorption of nucleic acids, such as single- or double-stranded DNA or RNA.

[0038] The metallo-organic network as a pharmaceutically acceptable vector in a pharmaceutical composition according to the invention makes it particularly possible to adsorb RNA and to transport it, avoiding its degradation, for example by RNases.

[0039] According to one embodiment, such a pharmaceutical composition may comprise a first active ingredient comprising a nucleic acid, and at least one second active ingredient. By way of non-limiting example of a second active ingredient in such a pharmaceutical composition, anticancer agents may be cited in particular, for example doxorubicin, gemcitabine, topotecan or methotrexate.

[0040] The metallo-organic network as a pharmaceutically acceptable carrier in such a pharmaceutical composition advantageously allows the adsorption of several active ingredients, for example of different nature or size, in particular because of the bimodality in the distribution of pore sizes that it comprises.

[0041] According to one embodiment, the metal-organic network as a pharmaceutically acceptable carrier in the pharmaceutical composition according to the invention is in the form of microparticles, preferably in the form of microparticles having a size of about 1 pm.

[0042] According to an alternative embodiment, the metal-organic network as a pharmaceutically acceptable carrier in the pharmaceutical composition according to the invention is in the form of nanoparticles, preferably in the form of nanoparticles having a size between 10 nm and 100 nm.

[0043] A fourth object of the invention relates to a method for preparing a pharmaceutical composition comprising a first active ingredient comprising a nucleic acid and at least one second active ingredient, said method comprising the steps of: a. Contacting the metallo-organic network according to the invention with a first active principle, said first active principle being at least one nucleic acid, thereby obtaining a metallo-organic network charged with the first active principle, then b. bringing into contact the metallo-organic network charged with the first active principle obtained at the end of step a) with a second active principle, said second active principle having a molecular weight less than or equal to 1 kDa, thereby obtaining a metallo-organic network charged with the first and second active principles.

[0044] Such a process in which the encapsulation of the first active principle being the nucleic acid is carried out before the encapsulation of the second active principle advantageously allows a better adsorption of the first active principle by the metallo-organic network.

[0045] Furthermore, the metallo-organic network of a pharmaceutical composition prepared by such a process will release the active ingredients more gradually.

[0046] A fifth object of the invention relates to the use of a metal-organic network according to the invention as a pharmaceutically acceptable vehicle for at least one active ingredient in a pharmaceutical composition.

[0047] Finally, a sixth object of the invention relates to the use of a metal-organic network according to the invention for releasing at least one substance in a controlled manner. A substance is typically understood to mean a pharmaceutical active ingredient, a combination of pharmaceutical active ingredients, but also any type of molecule, including those outside the pharmaceutical field. BRIEF DESCRIPTION OF THE FIGURES

[0048] [Fig. 1] are high-resolution transmission electron microscopy images of Fe-MDIP(III) synthesized according to Example 1. (Scale bar: 200 nm).

[0049] [Fig.2] is the X-ray diffraction pattern of an Fe-MDIP(III) powder synthesized according to example 1. Diagram A corresponds to Fe-MDIP(III) directly after synthesis, and diagram B corresponds to Fe-MDIP(III) after measurement of the N2 sorption isotherm at 77K.

[0050] [Fig.3] is the Fourier transform infrared (FT-IR) spectrum of a powder of Fe-MDIP(III) synthesized according to example 1. Spectrum A corresponds to Fe-MDIP(III) directly after synthesis, and spectrum B corresponds to Fe-MDIP(III) after measurement of the N2 sorption isotherm at 77K.

[0051] [Fig.4] is the thermogravimetric analysis of a synthesized Fe-MDIP(III) powder according to example 1.

[0052] [Fig.5] represents the sorption isotherms of N2 at 77K (adsorption: solid circles, and desorption; hollow circles) measured on the Fe-MDIP(III) synthesized according to example 1.

[0053] [Fig.6] represents the pore size distribution determined from the adsorption isotherm of N2 at 77K by DFT model and from the desorption isotherm of N2 at 77 K by BJT model.

[0054] [Fig.7] presents the monitoring of the colloidal and chemical stability of Fe-MFDIP(III) in aqueous solution (PBD).

[0055] [Fig.8] shows the release kinetics of doxorubicin by Fe-MDIP in (a) PBS pH 7.4; (b) PBS pH 5.5; (c) PBS pH 5.5 + 10 mM GSH.

[0056] [Fig.9] shows the release kinetics of labeled nucleic acids (siRNAcy5) by Fe-MDIP in (a) PBS pH 7.4; (b) PBS pH 5.5; (c) PBS pH 5.5 + 10 mM GSH; (d) PBS pH 5.5 + 20 mM GSH.

[0057] [Fig. 10] presents the cell viability of HEK-293 cells as a function of the concentration of Fe-MDIP in the medium.

[0058] [Fig. 11] shows the percentage of dead cells, assessed using DAPI staining, following administration of Fe-MDIP(III) loaded with doxorubicin, small interfering RNAs (siRNAs) directed against vimentin (siV), and / or small interfering RNAs directed against no eukaryotic protein (siN, negative control).

[0059] [Fig. 12] is the X-ray diffraction pattern of an Fe / Co-MDIP(III) powder synthesized according to example 2.

[0060] [Fig. 13] is the sorption isotherm of N2 at 77K (adsorption: solid circles, and desorption: hollow circles) for Fe / Co-MDIP(III) synthesized according to example 2.

[0061] [Fig. 14] is the X-ray diffraction pattern of an Fe / In-MDIP(III) powder synthesized according to Example 2.

[0062] [Fig. 15] is the sorption isotherm of N2 at 77K (adsorption: solid circles, and desorption: hollow circles) for FeZIn-MDIP(III) synthesized according to example 2.

[0063] [Fig. 16] is the X-ray diffraction pattern of an Fe / Cu-MDIP(III) powder synthesized according to example 2.

[0064] [Fig. 17] is the X-ray diffraction pattern of an Fe / Mg-MDIP(III) powder synthesized according to Example 2. DETAILED DESCRIPTION OF THE INVENTION Metal-organic network according to the invention

[0065] For the purposes of the present invention, "metal-organic network" or "MOF" means a class of materials composed of coordination polymers made up of metal ions (or metal centers) and organic ligands, thus forming three-dimensional crystalline structures exhibiting high porosity.

[0066] The metallic ions (or metallic clusters) thus form metallic nodes or sites within the material.

[0067] According to one embodiment, the metal ions of the MOF according to the invention are Iron(III) ions only.

[0068] According to an alternative embodiment, at least 66% of the metal ions in the MOF according to the invention are Iron(III) ions. In this embodiment, the MOF according to the invention may also include Iron(II), Copper(II), Magnesium(II), Cobalt(II), or Indium(III) ions. An MOF according to the invention may also include Manganese(II), Nickel(II), Aluminum(III), Galium(III), Lanthanides(III), or Ruthenium ions. The combination of the metal centers and organic ligands through coordination bonds create the cross-linked structure of MOFs, with a characteristic arrangement of meshes and pores. The shapes and sizes of these pores can be adjusted by choosing the metal and ligand, allowing the creation of MOFs with specific characteristics (pore size, surface functionality, stability, etc.) for targeted applications.

[0069] According to one embodiment, the organic ligand in a MOF according to the invention is 5,5'-methylene diisophthalic acid. 5,5'-methylene diisophthalic acid is an organic compound belonging to the family of dicarboxylic aromatic acids. It is characterized by two aromatic (isophthalic-type) rings linked by a methylene group (-CH2-). Each ring contains two carboxyl groups (-COOH) in the meta (1,3) position relative to each other. It is understood that 5,5'-methylene diisophthalic acid may be present in the MOF according to the invention in deprotonated (carboxylate) form.

[0070] According to a preferred embodiment, the metal-organic network according to the invention has the formula [Fe3O(OH)(mdip)ij5]-]x H2O, in which x is an integer equal to or greater than 1.

[0071] According to a particularly preferred embodiment, the metal-organic network according to the invention has the formula {Fe30(OH)0.25(Cl)0.75 (Ci7O8H8)i 5}-8H2O. In the present description, such a MOF is also referred to as "Fe-MDIP(III)".

[0072] According to one embodiment, the metal-organic network according to the invention can comprise pores of a size between 1 nm and 5 nm, preferably pores of a size between 1 nm and 3 nm.

[0073] The pore size of a MOF can be estimated from the N2 adsorption isotherm measured at 77K, using the theoretical calculation models NLDFT and BJH.

[0074] According to one embodiment, the pores of a MOF according to the invention may have pore access windows with a size between 1 nm and 5 nm, preferably between 1 nm and 3 nm. A "pore access window" is understood to be the opening allowing access to the internal cavities or channels of the porous material. It thus corresponds to the narrowest passage through which external molecules, for example, an active ingredient or a biological molecule, must pass to enter the pores of the MOF. Such a size of pore access windows differs from the pore size, in that the latter refers to the internal dimensions of the pores. The pore size of an MOF can be estimated from the N2 adsorption isotherm measured at 77 K, using the NLDFT and BJH theoretical calculation models, and confirmed by X-ray diffraction (XRD) analysis or by high-resolution electron microscopy (HRTEM)..

[0075] According to one embodiment, the MOF according to the invention can have at least a bimodal pore size distribution.

[0076] By "at least bimodal distribution" is meant a distribution characterized by the presence of at least two distinct populations of predominant pore sizes. The MOF exhibits at least two pore classes having significantly different dimensions, identifiable by at least two distinct peaks or maxima in a histogram or pore size distribution curve.

[0077] For example, the MOF may have pores of a first size equal to or less than 2 nm, for example about equal to 1 nm; and pores of a second size equal to or greater than 3 nm.

[0078] Advantageously, pores having a second pore size equal to or greater than 3 nm can have a one-dimensional (1-d) shape of cylindrical tunnels, having for example a hexagonal section.

[0079] According to one embodiment, the MOF according to the invention may comprise a pore volume of between 0.8 cm³ / g and 0.9 cm³ / g. Such a pore volume can be calculated from the N₂ adsorption isotherm measured at 77K, using the NLDFT and BJH theoretical calculation models.

[0080] By "about X" in this description, we mean the value X ± 10%, unless otherwise stated.

[0081] A metallo-organic network according to the invention can be obtained by a preparation process described below. Method for preparing a MOF according to the invention

[0082] The invention also relates to a method for preparing a metal-organic network, comprising the steps of:

[0083] 1) contacting 5,5'-methylenediisophthalic acid with at least a first A metallic precursor comprising Fe(III) is used in a first reaction medium, thereby obtaining the metallo-organic network according to the invention, and then

[0084] 2) recovery of the metallo-organic network obtained in step 1).

[0085] A "first metallic precursor comprising Fe(III)" usable in a process according to the invention may be a simple salt of Fe(III), a preformed ferric complex, an iron oxide, a ferric hydroxide, an organometallic precursor comprising iron, or a mixture thereof. By way of example, a metallic precursor comprising Fe(III) may be ferric chloride hexahydrate FeCl3.6H2O, or iron acetate Fe(C2H3O2)3.

[0086] The first reaction medium may preferably comprise an alcoholic solvent, which has the advantage of exhibiting limited toxicity. The synthesis of a MOF in such a solvent can then be considered eco-friendly. As such For example, an alcoholic solvent usable in a first reaction medium can be a mixture of isopropanol and benzyl alcohol.

[0087] The first reaction medium may further include acetic acid, advantageously allowing modulation of the kinetics of MOF formation, and optimal crystallization thereof.

[0088] According to an embodiment in which the prepared MOF comprises Iron (III) ions and furthermore at least one other metal ion, the first reaction medium in a process according to the invention comprises the metal precursor comprising Iron (III), and at least one second metal precursor comprising Copper (II), Magnesium (II), Cobalt (II), Indium (III), Manganese (II), Nickel (II), Aluminium (III), Galium (III), Lanthanides (III) or Ruthenium ions, for example a salt of these metal precursor cations or an organometallic precursor comprising such metals.

[0089] Such contacting steps in the MOF preparation process according to the invention can take place under ambient pressure conditions, i.e. at atmospheric pressure, without deliberate pressure control, and at a temperature between 20°C and 150°C, preferably a temperature between 100°C and 140°C.

[0090] Such contacting steps in the MOF preparation process according to the invention can be carried out under stirring, for example under mechanical stirring. The stirring speed can be chosen according to the viscosity of the reaction medium or the volume of the reaction medium.

[0091] The duration of such contacting steps in the MOF preparation process according to the invention is variable, and can be between 12h and 120h, for example between 24h and 120h, between 48h and 120h, between 72 and 96h.

[0092] Step 2) of recovery in a process according to the invention can be carried out by various techniques well known to those skilled in the art. For example, MOF recovery in a process according to the invention may include filtration. Such a filtration step may be performed after cooling the reaction medium, or while hot. Alternatively, MOF recovery in a process according to the invention may include centrifugation or decantation. The MOF recovered at the end of the recovery step can then be rinsed and dried.

[0093] Pharmaceutical composition according to the invention and preparation method

[0094] The invention also relates to a pharmaceutical composition which may comprise at least one active ingredient and a metal-organic network (MOF) according to the invention, as a pharmaceutically acceptable carrier.

[0095] For the purposes of this invention, "pharmaceutically acceptable vector" or "pharmaceutically acceptable vehicle" means a usable component to administer one or more active ingredients to an organism (for example, a patient). Such a vector ensures the stability and bioavailability of the active ingredients it carries. It can thus facilitate the transport, controlled release, or protection of the active ingredients to a defined target in the body. A pharmaceutically acceptable vector advantageously exhibits no toxicity towards the organism into which it is intended to be administered, and can therefore be described as biocompatible.

[0096] For the purposes of this invention, "controlled release" or "sustained release" refers to a method of administering one or more active ingredients into an organism gradually over a prolonged period. Such a method of administration can ensure the maintenance of stable concentrations of active ingredients in the body, for example in the blood or at a target site such as a tumor microenvironment, potentially allowing, for example, a reduction in the frequency of administration of the pharmaceutical composition, thereby improving treatment adherence, or limiting variations in therapeutic effects and side effects that could be caused by a peak concentration of active ingredients. Such controlled release can be facilitated by the 1-D tunnel shape of the MOF pores.

[0097] The MOF, as a pharmaceutically acceptable carrier, in a pharmaceutical composition according to the invention, can be in the form of microparticles, for example microparticles with a size between 0.1 pm and 100 pm.

[0098] Alternatively, the MOF, as a pharmaceutically acceptable carrier, in a pharmaceutical composition according to the invention, may be in the form of nanoparticles, for example nanoparticles with a size between 10 nm and 100 nm.

[0099] By "size" when referring to particles, we mean the d50 value of the particle size distribution (average number).

[0100] The pharmaceutical composition according to the invention may further comprise physiologically acceptable compounds for, for example, stabilizing and / or preserving the active ingredients and the pharmaceutically acceptable carrier. By way of non-limiting example, such physiologically acceptable compounds may be carbohydrates, such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The pharmaceutical composition according to the invention may also comprise other pharmaceutically acceptable adjuvants, buffer solutions, dispersing agents, and other similar substances.

[0101] Advantageously, the pharmaceutical composition may comprise at least two active ingredients, for example active ingredients of different sizes, conveyed in the metallo-organic network according to the invention as a unique pharmaceutically acceptable vector.

[0102] The pharmaceutical composition according to the invention may comprise at least one first active ingredient, which may be larger than 2 nm. Such a first active ingredient may be a biological molecule, for example, a nucleic acid with a therapeutic effect, such as single- or double-stranded DNA and RNA. Such a nucleic acid may vary in size from a few thousand to several million base pairs. By way of example, the at least one first active ingredient may be a gene (DNA) or a transcript (gene transcribed as messenger RNA).

[0103] Advantageously, such a first active ingredient is suitable for being adsorbed into the pores of the MOF according to the invention, which have a pore size equal to or greater than 3 nm. For example, the first active ingredient is suitable for being adsorbed into the one-dimensional (1-d) cylindrical tunnels of the MOF pores.

[0104] The pharmaceutical composition according to the invention may include at least one second active ingredient, which may have a molecular weight less than or equal to 1 kDa.

[0105] For example, at least one second active ingredient may be selected from among the anticancer agents. "Anticancer agent" preferably means chemical (non-biological) chemotherapeutic agents, antineoplastic agents, and cytotoxic agents. For example, an anticancer agent may be an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a microtubule inhibitor, an intercalating agent, or a platinum derivative, such as doxorubicin, gemcitabine, topotecan, or methotrexate.

[0106] Advantageously, such a second active ingredient is able to be adsorbed into the pores of the MOF which have a pore size equal to or less than 2 nm.

[0107] The MOF particles according to the invention can advantageously aggregate when in the presence of a weak acid, for example in a tumor microenvironment. Thus, a therapeutically effective quantity of active ingredients, carried by the MOF according to the invention, can be distributed in the tumor microenvironment.

[0108] Advantageously, the release of the active ingredients by the MOF particles according to the invention can be accelerated, or even triggered, in the tumor microenvironment due to the high concentration of glutathione (GSH) characteristic of this microenvironment. A high glutathione concentration is defined as a concentration greater than 10 mM, for example, a concentration between 10 mM and 40 mM. Such a glutathione concentration induces MOF degradation, which also generates Fe2+ ions capable of initiating a Fenton-type reaction which generates reactive oxygen species (ROS), potentiating the effect of the active ingredients carried by the MOF.

[0109] The pharmaceutical composition according to the invention can be prepared by a preparation process described below.

[0110] Another object according to the invention relates to a method for preparing a pharmaceutical composition according to the invention, comprising the steps of: a. bringing the metallo-organic network according to the invention into contact with a first active ingredient, said first active ingredient being at least a nucleic acid, thereby obtaining a metallo-organic network charged with the first active ingredient, b. contacting the metallo-organic network charged with the first active principle obtained in step a), with a second active principle, the second active principle having a molecular weight less than or equal to IkDa, thereby obtaining a metallo-organic network charged with the first and second active principles.

[0111] Such a step allows the first active ingredient to be adsorbed and encapsulated in the pores of the MOF having a pore size equal to or greater than 3 nm, the first active ingredient not being able to be encapsulated in the pores of the MOF having a pore size equal to or less than 2 nm. At the end of step a) of the pharmaceutical composition preparation process, the pores of the MOF having a pore size equal to or less than 2 nm thus remain free and can accommodate a second active ingredient.

[0112] The process for preparing a pharmaceutical composition according to the invention comprises, at the end of step a), a step of bringing the metallo-organic network charged with the first active ingredient obtained in step a), into contact with a second active ingredient, the second active ingredient having a molecular weight less than or equal to IkDa, thereby obtaining a metallo-organic network charged with the first and second active ingredients.

[0113] Such a step allows the second active ingredient to be adsorbed and encapsulated in the pores of the MOF having a size equal to or less than 2 nm, advantageously remaining free after step a). Uses of a MOF according to the invention:

[0114] An object of the invention also relates to the use of a metallo-organic network according to the invention as a pharmaceutically acceptable carrier, for example of one or more active ingredients, in a pharmaceutical composition.

[0115] Finally, the invention relates to the use of a metallo-organic network according to the invention to release one or more substances in a controlled manner.

[0116] The embodiments exemplified below are given only as illustrations and do not in any way constitute a limitation of the present invention. Example 1 - Synthesis of Fe-MDIP(III)

[0117] The synthesis of Fe-MDIP(III) was carried out in a solvent comprising a mixture of benzyl alcohol and isopropanol.

[0118] 0.173 g of H4MDIP were dispersed in a mixture comprising 8 ml of alcohol benzyl and 8 ml of acetic acid. The mixture was then heated to a temperature of 120°C, and kept under stirring at this temperature for 5 minutes.

[0119] 0.273 g of a FeCl3.6H2O solution (0.273 g) in 5 ml of isopropanol are added in the reactive medium. The mixture was maintained at 120 °C for 72 to 96 h. It was then cooled to room temperature. The Fe-MDIP(III), synthesized as a powder, was recovered by filtration and rinsed with water and ethanol. The Fe-MDIP(III) was then air-dried.

[0120] The synthesis of Fe-MDIP(III) can thus be described as "green" in that it does not use toxic solvents. The reaction has a yield of 78% (yield calculated on the basis of H4MDIP). Example 2: Synthesis of mixed-metal MOFs

[0121] Other MDIP-based MOFs have been synthesized according to a protocol similar to that in Example 1.

[0122] 0.173 g of H4MDIP are dispersed in a mixture comprising 8 ml of alcohol benzyl and 8 ml of acetic acid. The mixture was then heated to a temperature of 120°C, and kept under stirring at this temperature for 5 minutes.

[0123] Solutions comprising metallic salts were prepared according to proportions defined in Table 1 below, and then added to the H4MDIP mixture.

[0124] The mixtures were maintained at a temperature of 120°C for a period of between 72 h and 96 h. The mixtures were then cooled to room temperature. The MOFs, synthesized in powder form, were recovered by filtration and rinsed with water and ethanol, then air-dried.

[0125] These syntheses can be described as "green", in that they do not use toxic solvents.

[0126] [Tables 1] Synthesized MOF assay Mass of metal salts used as reagent (in 5 ml of isopropa nol) Mass of synthesized MOF Yields (calculated on the basis of H4MDIP) 2A Fe / Co- MDIP(III) 0.137 g of FeCl3.6H 204 mg 80% 2B Fe / In- MDIP(III) 0.137 g FeCl3.6H2O 0.111 g InCl3 208 mg 76% 2C Fe / Cu- MDIP(III) 0.137 g FeCl3.6H2O 0.085 g CuC % 2D Fe / Mg- MDIP(III) 0.137 g FeCl3.6H2O 0.102 g M gCl26H2O 184 mg 74%

[0127] Example 3 - Characterization of Fe-MDIP(III) Synthesized to Example 1

[0128] The synthesized Fe-MDIP(III) sample was analyzed by high-resolution transmission electron microscopy. The images obtained are shown in [Fig. 1] (scale bar corresponds to 200 nm). The synthesized Fe-MDIP(III) is in the form of rod-shaped nanoparticles with a length of approximately 1 pm and good monodispersity. The material also exhibits mesopores in the form of one-dimensional (1-d) channels with a diameter between 2 nm and 3 nm.

[0129] The synthesized Fe-MDIP(III) sample was also analyzed using an X-ray diffractometer (XCu(Ka)=1.5406 Å) (BRUKER D8 ADVANCE). The X-ray diffraction patterns are shown in [Fig. 2], and reveal peaks at 2θ = 2.1°, 3.7°, 4.2°, 5.5°, 9.3°, 10.4°, 11.2°. (Diffractogram A corresponds to Fe-MDIP(III) directly after synthesis, and diagram B corresponds to Fe-MDIP(III) after measurement of the N2 sorption isotherm at 77 K).

[0130] These analyses allow the following parameters to be calculated for Fe-MDIP(III): a=48.11 Â, c=22.56 Â (slip planes), in agreement with the following possible space groups: P3cl, P-3cl, P63cm, P-6c2 and Pôjmcm.

[0131] The synthesized Fe-MDIP(III) sample was also analyzed by Fourier transform infrared (FT-IR) spectroscopy. The FT-IR spectrum is shown in [Fig. 3] and suggests the formation of a MOF exhibiting metal-carboxylate bonds (stretching vibration at 1670–1200 cm⁻¹ in the solid product) and the formation of Fe-O bonds (peak at approximately 624 cm⁻¹ corresponding to the stretching vibration of the iron-oxygen bond).

[0132] The synthesized Fe-MDIP(III) sample was analyzed by thermogravimetric analysis (TGA). Approximately 5 mg of nanoparticles were placed in a PERKIN ELMER DIAMOND TGA / DTA STA 6000 instrument under a flow of O2 (20 ml / min). The sample was heated at a rate of 5°C / min over a temperature range of 25°C to 600°C. The observed mass loss of the sample was 57.8%, and the calculated mass loss of the sample was 58.7%.

[0133] The chemical formula for the synthesized Fe-MDIP(III) has been determined: {Fe30(OH)o.25(Cl)o.75 (C17O8H8)15}. 8H2O.

[0134] Finally, the sorption isotherms of N2 by the synthesized Fe-MDIP(III) were measured at 77 K using a Micromeritics 3Star instrument. The isotherms obtained are shown in [Fig. 5]. The pore volume measured for this sample is 0.84 cc / g, and the pore diameter is estimated to be between 2.8 nm and 3 nm. The pore size distribution, determined from the N2 adsorption isotherm at 77 K by the DFT model, and from the N2 desorption isotherm at 77 K by the B JT model, is shown in [Fig. 6]. The permanent porosity of the synthesized Fe-MDIP(III) is confirmed by the N2 sorption isotherm at 77 K, which allows the calculation of a BET surface area of ​​ca. 1255 m2 / g and a pore volume of 0.93 cc / g.The NLDFT model, applied to the adsorption branch of the isotherm, highlighted a bimodal distribution of pore size, with a first pore size of 1.3 nm, and a second pore size of 2.8 nm, consistent with the direct observation of mesopores of about 3 nm by transmission electron microscopy, with a possible one-dimensional (1-d) channel shape.

[0135] Example 4 - Colloidal and chemical stability of Fe-MDIP(III) synthesized according to example 1

[0136] The colloidal and chemical stability of Fe-MDIP(III) synthesized according to Example 1 in aqueous solution was evaluated and compared with MIL-1OO(Fe) nanoparticles. The Fe-MDIP(III) nanoparticles were dispersed in an aqueous PBS solution at pH 7.4. The polydispersity index and hydrodynamic diameter of the Fe-MDIP(III) nanoparticles were monitored for 24 h.

[0137] Fe-MDIP(III) nanoparticles disperse uniformly in aqueous solution, with a polydispersity index of 0.22 (as shown in Figure 7a), and with a hydrodynamic size of 247 nm (as shown in Figure 7b), and remain stable for at least 24 h in an aqueous PBS solution at pH 7.4 (simulating a biological fluid). In contrast, MIL-lOO(Fe) nanoparticles degrade rapidly under similar conditions.

[0138] Furthermore, the inventors have shown that Fe-MDIP(III) nanoparticles tend to aggregate in the presence of a weak acid in the environment. This property could promote the accumulation of Fe-MDIP(III) in tumor microenvironments.

[0139] Example 5 – Encapsulation of single-stranded DNA in Fe-MDIP(III)

[0140] The inventors have evaluated the ability of the Fe-MDIP(III) synthesized according to example 1 to carry biological molecules, for example single-stranded DNA.

[0141] 6 mg of single-stranded DNA (ssDNA) were dissolved in purified water (Milli- Q) (neutral pH) or in a 10 mM hydrochloric acid solution (pH = 2), to obtain a concentration of 1 mg / ml of single-stranded DNA. 30 mg of Fe-MDIP synthesized according to Example 1 were added to the solution. After stirring at room temperature for 0.5 h, 1 h, 2 h, or 3 h, the mixture was centrifuged and dried in a vacuum drying oven overnight, and the supernatants were collected and analyzed by UV-visible spectroscopy.

[0142] The results showed that a greater quantity of ssDNA could be loaded into Fe-MDIP(III) in the presence of purified water (neutral pH) than in hydrochloric acid solution (pH = 2). This can be explained by the protonation of ssDNA and the decrease in the electrostatic force between Fe-MDIP(III) and ssDNA at a pH below 4.3. It was also shown that the hydrodynamic size and polydispersity index of Fe-MDIP(III) remain unchanged after ssDNA loading, and that the surface of the nanoparticles becomes negative, suggesting the presence of ssDNA. The highest encapsulation efficiency of ssDNA by Fe-MDIP was achieved after 2 h of incubation.

[0143] Example 6 - Encapsulation of single-stranded DNA and DOX in Fe-MDIP

[0144] The inventors evaluated the ability of Fe-MDIP(III) to encapsulate both single-stranded DNA (ssDNA) and doxorubicin (DOX).

[0145] Two encapsulation protocols for these molecules in Fe-MDIP(III) were tested, changing the encapsulation order.

[0146] For the doxorubicin encapsulation step, an aqueous solution containing doxorubicin at a concentration of 5 mg / ml was prepared. 60 mg of Fe-MDIP(III) were added to 12 ml of this solution. The mixture was incubated with stirring overnight. The loaded MOF nanoparticles were then recovered by centrifugation.

[0147] For the single-strand DNA encapsulation step, the protocol shown in Example 5 was followed.

[0148] The specific surface area BET (N2 adsorption isotherm at 77K) and the pore volume were measured, the results are presented in Table 2 below.

[0149] The results confirm that ssDNA cannot be encapsulated in small pores. It is therefore more efficient to encapsulate ssDNA before doxorubicin. This may be explained by the fact that doxorubicin can occupy large pores, in which nucleic acids can exclusively reside.

[0150] [Tables2] Sample Surface area (m2 / g) Pore volume (cm3 / g) Fe-MDIP 1254- ± 6 0.83 391 ± 2 0.22 FeWIP@>0X 46 ± 1 0.022 Fs-MDIF@DQX-ssDNA 54 ± 1 0.025 Fe-MDIPO^ÜNA-DOX 37 ± 1 0.015

[0151] Example 7 - Controlled release of encapsulated DNA and DOX

[0152] The release kinetics of nucleic acids (ssDNA or siRNAcy5) and doxorubicin encapsulated in Fe-MDIP(III) were measured at 37°C under different conditions: (a) PBS pH 7.4; (b) PBS pH 5.5; (c) PBS pH 5.5 + 10 mM GSH; (d) PBS pH 5.5 + 20 mM GSH. The release kinetics of doxorubicin are shown in [Fig. 8].

[0153] A massive and rapid release of doxorubicin is observed when Fe-MDIP is prepared by first encapsulating doxorubicin, then ssDNA. The release of doxorubicin is gradual when Fe-MDIP is prepared by first encapsulating ssDNA and then doxorubicin.

[0154] The release kinetics of siRNAcy5-labeled nucleic acid are shown in [Fig. 9]. Note that nucleic acid release is slowed when doxorubicin is loaded into Fe-MDIP.

[0155] Finally, the presence of GSH in PBS significantly accelerates the release of doxorubicin and nucleic acid, demonstrating the possibility of releasing substances from Fe-MDIP(III) in a targeted manner, in a medium containing a high concentration of GSH. Example 8 - In vitro cytotoxicity of Fe-MDIP

[0156] The inventors evaluated the cytotoxicity induced by Fe-MDIP(III) prepared according to example 1 (uncharged).

[0157] HEK 293 cells were cultured in a 96-well plate at a density of 2104 cells per well for 24 h, and then treated with different concentrations of Fe-MDIP for 24 h. The culture medium was then removed, and 10 µl of a CCK-8 solution was added to each well. After 1–4 h of incubation in the dark, the absorbance at 450 nm for each well was measured.

[0158] The results are presented in [Fig. 10]. Fe-MDIP nanoparticles exhibited low cytotoxicity against HEK-293 cells, even at high concentrations. Fe-MDIP therefore demonstrates good biocompatibility.

[0159] Example 9 - In vitro co-delivery of DOX and SiV:

[0160] The inventors evaluated the cytotoxicity of Fe-MDIP co-delivering doxorubicin and small interfering RNAs directed against vimentin.

[0161] MDA-MB-231 cells were incubated in 24-well plates at a density of 8104 cells per well for 24 hours and then treated with PBS comprising Fe-MDIP encapsulating doxorubicin (Fe-MDIP@DOX), vimentin-directed small interfering RNAs (Fe-MDIP@siVIM), no-eukarya-directed small interfering RNAs and doxorubicin (negative control, Fe-MDIP@siN@DOX), and vimentin-directed small interfering RNAs and doxorubicin (Fe-MDIP@siVIM@DOX). The mass ratio of Fe-MDIP:siVIM was 30:1. After an additional 24-hour incubation, the cells were collected by adding 200 qL of EDTA containing 0.05% trypsin to each well, followed by centrifugation at 1000 rpm for 5 minutes. The cells were then resuspended in 100 pL of binding buffer and exposed to DAPI staining to measure cell death by flow cytometry.

[0162] Fig. 11 shows the percentage of dead cells, assessed using DAPI staining, following administration of Fe-MDIP loaded with doxorubicin, small interfering RNAs (siRNAs) directed against vimentin (siV), and / or small interfering RNAs directed against no eukaryotic protein (siN, negative control).

[0163] The results demonstrated that co-delivery by Fe-MDIP of doxorubicin and RNA interference directed against vimentin induced more apoptosis compared to the delivery alone of either of these two compounds.

[0164] Example 10 - Characterization of mixed-metal MOFs prepared according to Example 2,

[0165] The MOF samples prepared according to Example 2 were analyzed using an X-ray diffractometer (XCu=1.5406 Å) (BRUKER D8 ADVANCE). The X-ray diffraction patterns are shown in [Fig. 12] for Fe / Co-MDIP(III), in [Fig. 14] for Fe / In-MDIP(Ni), in [Fig. 16] for Fe / Cu-MDIP(III), and in [Fig. 17] for Fe / Mg-MDIP(III).

[0166] Finally, the adsorption of N2 by the synthesized MOFs was measured at 77K using a Micromeritics 3Star instrument. The isotherms obtained are shown in [Fig. 13] for Fe / Co-MDIP(in), and in [Fig. 15] for Fe / In-MDIP(III).

Claims

Demands

1. Crystalline porous metal-organic network (MOF) comprising metal ions coordinated to an organic ligand, wherein at least 66% of the metal ions are Iron(III) ions and the organic ligand is 5,5'-methylene diisophthalic acid in deprotonated form.

2. Metal-organic network according to claim 1, said metal-organic network comprising pores having a size between 1 nm and 5 nm.

3. Metal-organic network according to any one of the preceding claims, said metal-organic network having at least a bimodal pore size distribution, with a first pore size of about 1 nm and a second pore size between 3 nm and 5 nm, preferably about 3 nm.

4. Metal-organic network according to any one of the preceding claims, said metal-organic network comprising pore access windows having a size between 1 nm and 5 nm.

5. Metal-organic network according to any one of the preceding claims, said metal-organic network having a pore volume between 0.8 cm3 / g and 0.9 cm3 / g.

6. Metal-organic network according to any one of the preceding claims, wherein the metal ions are Iron (III) ions only.

7. Metal-organic network according to any one of claims 1 to 5, wherein the metal ions further comprise Copper(II), Magnesium(II), Cobalt(II), Indium(III), Manganese(II), Nickel(II), Aluminium(III), Galium(III), Lanthanides(III) or Ruthenium ions.

8. Metal-organic network according to any one of claims 1 to 6, of formula [Fe3O(OH)(mdip)ij5] • x H2O, in which x is an integer equal to or greater than 1.

9. A method for preparing a metal-organic network as defined in any one of claims 1 to 8, comprising the steps of: 1) contacting 5,5'-methylenediisophthalic acid with at least a first metallic precursor comprising iron(III), preferably comprising an iron(III) salt, for example FeCl3.6H2O, in a first reaction medium, preferably comprising an alcoholic solvent, by means of which the metallo-organic network as defined in any one of claims 1 to 8 is obtained, then 2) recovery of the metallo-organic network obtained in step 1).

10. A process according to claim 9, wherein the first reaction medium further comprises at least one second metallic precursor comprising Copper(II), Magnesium(II), Cobalt(II), Indium(III), Manganese(II), Nickel(II), Aluminium(III), Galium(III), Lanthanides(III) or Ruthenium ions.

11. A process according to any one of claims 9 or 10, wherein the first reaction medium further comprises acetic acid.

12. Pharmaceutical composition comprising at least one active ingredient and a metal-organic network as defined in any one of claims 1 to 8 as a pharmaceutically acceptable carrier.

13. Pharmaceutical composition according to claim 12, wherein at least one active ingredient comprises a nucleic acid, preferably DNA or RNA.

14. Pharmaceutical composition according to claim 12, comprising a first active ingredient comprising a nucleic acid and at least one second active ingredient.

15. Pharmaceutical composition according to any one of claims 12 to 14, wherein the metal-organic network is in the form of nanoparticles, preferably in the form of nanoparticles having a size of about 10 nm and 100 nm.

16. A process for preparing a pharmaceutical composition according to claim 13, comprising the steps of: a) contacting the metallo-organic network as defined in any one of claims 1 to 8 with a first active ingredient, said first active ingredient being at least one nucleic acid, thereby obtaining a metallo-organic network loaded with the first active ingredient, and then b) contacting the metallo-organic network loaded with the first active ingredient obtained at the end of step a) with a second active ingredient, said second active ingredient having a lower molecular weight or equal to 1 kDa, whereby a metallo-organic network charged with the first and second active principles is obtained.

17. Use of a metal-organic network as defined in any one of claims 1 to 8 as a pharmaceutically acceptable carrier of at least one active ingredient in a pharmaceutical composition.

18. Use of a metal-organic network as defined in any one of claims 1 to 8 to release a substance in a controlled manner.

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