Nanoassemblies based on curcumin or curcumin derivatives, and method for producing said nanoassemblies

Abstract

The present invention relates to a nanoassembly formed by the self-assembly of a compound selected from compounds of formula (Ia): [Chem. 6], R representing an alkyl group; and compounds of formula (Ib): [Chem. 7], the compound of formula (Ib) being associated with at least one superparamagnetic nanoparticle.

Description

Nano-assemblies based on curcumin or curcumin derivatives and their preparation process

[0001] The present invention relates to nano-assemblies based on curcumin or curcumin derivative, their preparation method and their use.

[0002] Magnetic liquid hyperthermia is a cancer treatment technique in which magnetic nanoparticles are administered to a patient near a tumor. The patient is then subjected to a locally applied alternating magnetic field, such that the magnetic nanoparticles heat up and emit heat in their vicinity, thereby increasing the temperature of the tumor, either to cause apoptosis of the cancer cells (in this case the local heating reaches a temperature of 42-43°C) or to sensitize them for subsequent chemotherapy treatment.

[0003] Magnetic nanoparticles can also be used in imaging methods. For example, magnetic particle imaging (MPI) is an emerging non-invasive tomographic technique offering high resolution (theoretically 200-500 μm), high sensitivity (which could approach 10 -7 -10 -8 M) and real-time imaging.

[0004] Curcumin is used in multiple therapeutic indications, as this molecule has interesting therapeutic potential, particularly as an anti-inflammatory and anti-cancer agent. However, its use for therapeutic purposes has three limitations. First, its weak hydrophilic character leads to its low solubility in water, which is a barrier to its use as a therapeutic compound. Second, its relative chemical stability, which leads to rapid decomposition of the molecule, is also a barrier to its therapeutic use. Finally, curcumin has two mesomeric forms: a diketone form, responsible for the anti-inflammatory and anti-cancer properties of the molecule, and an enol form. Unfortunately, in solution, curcumin is mainly found in its enol form.

[0005] Many publications describe the encapsulation of curcumin in a polymeric matrix. However, the size of these nano-assemblies is large, greater than 100 nm, which causes a short circulation time in the blood, very slow or even zero diffusion in extracellular viscous matrices, and very slow internalization in cells, which can lead to vesiculation phenomena. The size of nanoparticles or nano-assemblies is a critical parameter in controlling their biodistribution. Indeed, nanoparticles smaller than 10 nm are smaller than the renal filtration barrier and are therefore rapidly eliminated. Hoshyar, N.; Gray, S.; Han, H.; Bao, G. The Effect of Nanoparticle Size on in Vivo Pharmacokinetics and Cellular Interaction.Nanomed.2016,11(6), 673–692.Conversely, nanoparticles with sizes greater than 200 nm are rapidly taken up by the mononuclear phagocytic system and a size limit of 150 nm is generally accepted to avoid filtration by the spleen. It appears that a size between 10 and 150 nm allows good circulation of nanoparticles in the blood. On the other hand, for good cellular internalization by endocytosis, it is described that nanoparticles must have a size between 30 and 60 nm. Thermodynamic models even predict that the ideal size for efficient cellular internalization is 50 nm. Zhang, S.; Li, J.; Lykotrafitis, G.; Bao, G.; Suresh, S. Size-Dependent Endocytosis of Nanoparticles.Adv. Mater.2009,21(4), 419–424. This size has since been experimentally validated. Lu, F.; Wu, S.-H.; Hung, Y.; Mou, C.-Y. Size Effect on Cell Uptake in Well-Suspended, Uniform Mesoporous Silica Nanoparticles.Small2009,5(12), 1408–1413; Xu, W.; Zhou, M.; Guo, Z.; Lin, S.; Li, M.; Kang, Q.; Xu, Y.; Zhang, X.; Xie, J. Impact of Macroporous Silica Nanoparticles at Sub-50nm on Bio-Behaviors and Biosafety in Drug-Resistant Cancer Models.Colloids Surf. B ​​Biointerfaces2021,206, 111912 ; Jiang, W.; Kim, BYS; Rutka, JT; Chan, WCW Nanoparticle-Mediated Cellular Response Is Size-Dependent.Nat. Nanotechnol.2008,3(3), 145–150. However, it turns out that nano-assemblies of curcumin in this size range are not described in the literature. Moreover, curcumin is not the only species composing the nanoparticle and is generally the minority species.

[0006] Furthermore, curcumin has very poor solubility in water. By HPLC analysis, the solubility of curcumin in water or aqueous buffer at pH = 5 is 0.011 µg / mL Tønnesen, HH; Karlsen, J. Studies on Curcumin and Curcuminoids.Z. Für Lebensm.-Unters. Forsch.1985,180(2), 132–134. Studies using a UV-Visible spectrometric method of determination give a higher value of 0.6 µg / mL, Kurien, BT; Singh, A.; Matsumoto, H.; Scofield, RH Improving the Solubility and Pharmacological Efficacy of Curcumin by Heat Treatment.Assay Drug Dev. Technol.2007,5(4), 567–576. This low solubility of curcumin in water makes it difficult to use in the human body.

[0007] The present invention aims to solve these drawbacks by using a curcumin derivative improving the solubility and stability of the molecule in the aqueous phase, making it possible to lock the molecule in its biologically active diketone form. In order to increase the amount of curcumin derivative in water, the present invention does not propose to increase the hydrophilic properties of the curcumin derivative but rather to increase the hydrophobicity of the latter in order to allow its self-assembly in water. Self-assembly is a process in which chemical components such as molecules, metal nanoparticles, colloids, polymers or any other compound organize themselves into ordered structures spontaneously by physicochemical interactions such as weak non-covalent chemical bonds. Thus, the hydrophobicity of the curcumin derivative is controlled to be able to form small nano-assemblies.This formulation in the form of nano-assemblies makes it possible to increase the therapeutic index of the curcumin derivative by limiting its non-specific diffusion then observed for molecular compounds. The small size of the nano-assemblies obtained makes it possible to facilitate interactions with the body's cells and to increase their circulation speed in the blood as well as their diffusion in viscous environments, for example in extracellular matrices. It was also surprisingly discovered that the association of curcumin with a super-paramagnetic nano-particle makes it possible to form a nano-assembly with a good mass concentration in water.

[0008] For this purpose, the present invention relates to a nano-assembly formed by self-assembly of a compound chosen from the compounds of Formula (Ia):

[0009]

[0010] R representing an alkyl group; and

[0011] the compound of Formula (Ib):

[0012] (Ib),

[0013] said compound of Formula (Ib) being associated with at least one super-paramagnetic nanoparticle.

[0014] When the compound of Formula (Ia) or Formula (Ib) is associated with at least one superparamagnetic nanoparticle, the resulting nanoassembly may be referred to as a magnetic hybrid nanoassembly.

[0015] R may represent an alkyl group comprising at least 4 carbon atoms, preferably comprising between 4 and 16 carbon atoms.

[0016] In particular, R may be selected from butyl, pentyl, octyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl and octadecyl.

[0017] The nano-assembly according to the present invention may be constituted by self-assembly of the compound of Formula (Ia) only. Said nano-assembly may then be spherical. Such a nano-assembly may be called an organic nano-assembly. The nano-assembly may then have a diameter, as measured by transmission electron microscopy, of between 40 nm and 110 nm. It may also have a zeta potential, as measured by zetametry, of between -30 mV and -40 mV.

[0018] Zetametry measurement can be performed at 25°C. The value of the zeta potential can be deduced from the electrophoretic mobility using a Smoluchowski model.

[0019] The nano-assembly can be coated with a stabilizing polymer.

[0020] The stabilizing polymer may be a cationic polyelectrolyte. In particular, the cationic polyelectrolyte may be a poly-L-lysine-based polymer or a poly-L-lysine-based polymer whose side chains are functionalized with polyethylene glycol.

[0021] In addition to size, surface functionalization plays an important role in the biodistribution of nanoparticles in vivo. Indeed, nanoparticles are recognized by the mononuclear phagocytic system and rapidly internalized by macrophages. They then accumulate mainly in the liver and spleen. To circumvent recognition by the immune system, the main strategy developed is the use of stealth agents, mainly the polymer polyethylene glycol, PEG, (J.V. Jokerst, T. Lobovkina, RN Zare, S. Gambhir . Nanoparticle PEGylation for imaging and therapy ; Nanomedicine (Lond).2011;6(4),715-28.. The use of this polymer makes it possible to increase the circulation time in the blood and to reach a greater proportion of inflammatory sites, in particular the tumor microenvironment.

[0022] The stabilizing polymer can be functionalized by a function capable of being associated by bioconjugation with a complementary function, for example by click chemistry or peptide coupling, and the nano-assembly can be coupled to a molecule of interest, in particular chosen from peptides, fluorophores or biomolecules, the molecule of interest being functionalized by a function complementary to the function of the functionalized stabilizing polymer.

[0023] In particular, the stabilizing polymer can be functionalized by an alkyne function and the molecule of interest by an azide function in order to be able to conduct the bioconjugation of the molecule of interest and the stabilizing polymer by click chemistry.

[0024] In a particular embodiment, a compound of Formula (Ia) is associated with at least one super-paramagnetic nanoparticle.

[0025] The at least one superparamagnetic nanoparticle may be composed of iron oxide.

[0026] In particular, when there is more than one superparamagnetic nanoparticle, the other superparamagnetic nanoparticle(s) may be of different size and shape.

[0027] The at least one superparamagnetic nanoparticle may have:a spherical shape with a diameter of between 6 and 20 nm, as measured by transmission electron microscopy; ora cubic shape with an edge of between 10 and 70 nm, as measured by transmission electron microscopy; ora rod shape with a length of between 30 and 50 nm and a width of between 6 and 8 nm, as measured by transmission electron microscopy.

[0028] The nanoassembly can be spherical and have a diameter, as measured by transmission electron microscopy, between 40 nm and 250 nm. It can then have a zeta potential, as measured by zetametry, between +25 mV and +40 mV.

[0029] The invention also relates to a method for preparing a nano-assembly according to the invention, characterized in that a flash-nanoprecipitation is carried out by the following steps: in a first syringe, a solution of the compound of Formula (Ia) or Formula (Ib) in a water-miscible solvent, chosen from tetrahydrofuran, acetone or acetonitrile, which contains, where appropriate, at least one super-paramagnetic nanoparticle, is placed, and in a second syringe, water or a saline phosphate buffer, which contains, where appropriate, a stabilizing polymer; the contents of the two syringes are injected into a nanoprecipitation mixer at a speed of 2 to 10 mm / s, in particular at 5 mm / s; and the nano-assemblies are recovered in a receptacle bottle containing water, and where appropriate, a stabilizing polymer.

[0030] In particular, the receptacle bottle may contain water in an amount making it possible to dilute the entire reaction medium leaving the mixer by a factor of at least five, preferably at least ten.

[0031] The present invention also relates to a pharmaceutical composition comprising one or more nano-assemblies according to the present invention.

[0032] The present invention also relates to a nano-assembly according to the present invention for its use in the treatment of inflammatory diseases.

[0033] The present invention also relates to a nano-assembly according to the present invention for its use in the treatment of cancer.

[0034] The present invention also relates to a nano-assembly according to the present invention for its use in magnetic liquid hyperthermia.

[0035] The present invention also relates to a contrast agent for magnetic resonance imaging characterized in that it comprises nano-assemblies according to the present invention.

[0036] The present invention also relates to an agent for magnetic particle imaging characterized by the fact that it comprises nano-assemblies according to the present invention.

[0037] The compound of Formula (Ia) can be obtained using a two-step synthesis, the first step consisting of an amidic coupling of 4-formylbenzoic acid with an aminoalkane and the second step consisting of the Knoevenagel condensation of the compound obtained in the first step with curcumin in enol form to obtain such a compound of Formula (Ia) according to the present invention.

[0038] The general synthesis scheme of this compound of Formula (Ia) is indicated below:

[0039]

[0040] Superparamagnetic nanoparticles of spherical and cubic shape can be obtained by thermal decomposition of an iron(III) precursor, such as iron(III) acetylacetonate. The iron(III) precursor is dissolved in a solvent or a mixture of solvents with a high boiling temperature, above 280°C, in the presence of a fatty acid, such as oleic acid or decanoic acid, and optionally an amine, such as oleylamine, and the whole compound is heated to a high temperature, above 200°C.

[0041] Rod-shaped superparamagnetic nanoparticles are prepared solvothermally. For this purpose, iron pentacarbonyl is dissolved in a high-boiling solvent, such as 1-octanol, in the presence of a fatty acid, such as oleic acid, and an amine, such as hexadecylamine. All the compounds are heated in an autoclave at 200°C.

[0042] The general scheme for the synthesis of superparamagnetic nanoparticles of spherical (route A), cubic (route B) and rod-shaped (route C) shapes is presented.

[0043] The nano-assemblies according to the present invention can be prepared by flash nano-precipitation. The flash nano-precipitation process can be carried out in an apparatus shown schematically in.

[0044] In the, the device used for the synthesis of nano-assemblies according to the invention by a flash-nanoprecipitation process is schematically represented. This device comprises a mixer 1 provided with two inlet orifices 2 and an outlet orifice 3. The mixer 1 is typically a mixer with confined liquid impact jets.

[0045] One of the inlet ports 2 of the mixer 1 is in fluid communication with a first syringe 4 while the second inlet port 2 is in fluid communication with a second syringe 5. An anvil 6 is positioned above the pistons of the syringes 4 and 5 in order to be able to carry out the simultaneous injection of the contents of the syringes 4 and 5.

[0046] A receptacle bottle 7 is placed under the outlet orifice 3 of the mixer 1.

[0047] For the synthesis of organic nano-assemblies according to the invention, a solution of the compound of Formula (Ia) in a solvent such as tetrahydrofuran, acetone or acetonitrile can be placed in the first syringe 4. In the other syringe 5, water or saline phosphate buffer is placed, in the presence or absence of a stabilizing polymer to obtain at least one organic nano-assembly.

[0048] For the synthesis of the magnetic hybrid nano-assemblies according to the invention, a solution of the compound of Formula (Ia) or Formula (Ib) and at least one super-paramagnetic nanoparticle in a solvent such as tetrahydrofuran, acetone or acetonitrile can be placed in the first syringe 4. In the other syringe 5, water or saline phosphate buffer is placed, in the presence of a stabilizing polymer to obtain a magnetic hybrid nano-assembly.

[0049] By moving the anvil 6, the contents of the syringes 4 and 5 are injected simultaneously into the mixer 1. The mixing of the contents of the two syringes in the mixer 1 causes the formation of nano-assemblies which are then evacuated from said mixer through the outlet orifice 3 to be collected in the receptacle bottle 7.

[0050] Optionally, the stabilizing polymer can be functionalized to be able to graft compounds of interest to the surface of the nano-assembly by “Click” chemistry.

[0051] Typically, the grafting of compounds of interest can be done by a cycloaddition reaction. This reaction is catalyzed by Cu(I) at the interface between nano-assemblies, the latter presenting, via the stabilizing polymer, an alkyne function and a commercial molecule of interest comprising an azide function.

[0052] This is a schematic representation of an organic nanoassembly (ONA) according to the invention.

[0053] This is a schematic representation of a magnetic hybrid nano-assembly according to the invention.

[0054] On the, the magnetic hybrid nanoassembly (MHNA) contains superparamagnetic nanoparticles (SPM) and is coated by a stabilizing polymer (POLY).

[0055] The following examples illustrate the present invention without, however, limiting its scope. Example 1: Synthesis of the compound of Formula (Ia) Step 1: Amide coupling

[0056] To 2 g of 4-formylbenzoic acid (13.3 mmol) in anhydrous dichloromethane (40 mL) are added 2.9 mL of oxalyl chloride (33.3 mmol) and two or three drops of anhydrous dimethylformamide. The reaction medium is stirred at room temperature for two hours and then evaporated to dryness using a rotary evaporator. The crude reaction product is dissolved in 40 mL of anhydrous dimethylformamide and the aliphatic primary amine (20 mmol) is added. The medium is heated to 90 °C for 24 hours. After returning to room temperature, the precipitate is filtered and then washed 3 times with 1M NaOH solution to remove the 4-formylbenzoic acid reagent, then neutralized with 1M HCl solution and washed 3 times with diethyl ether to remove the excess aliphatic amine. No further purification is necessary.

[0057] The aliphatic primary amine is selected from butylamine, pentylamine, octylamine, undecylamine, dodecylamine, tridecylamine, tetradecylamine, pentadecylamine, and octadecylamine, depending on the desired alkyl chain length R. The amount of aliphatic primary amine material used is the same regardless of the amine chain length. Step 2: Knoevenagel condensation

[0058] To 0.76 g of the compound obtained in step 1 (2.2 mmol), 0.37 g of curcumin (1 mmol) are added in 40 mL of anhydride chloroform, anhydride dichloromethane with the optional addition of a small amount of dimethyl sulfoxide depending on the solubility of the compound obtained in step 1. Then, 20 µL of piperidine (0.2 mmol), 12 µL of glacial acetic acid (0.2 mmol) and 2.5 g of 4Å molecular sieve are then added. The medium is stirred at room temperature for 48 hours. After filtration to remove the molecular sieve, the medium is evaporated on a rotary evaporator to remove the chloroform and catalysts. The medium is diluted in dichloromethane and extracted several times with water. After drying over anhydrous sodium sulfate and evaporation, the reaction crude is purified on a silica chromatography column with a dichloromethane / ethyl acetate 8 / 2 mixture as eluent. Hydrophobic character of compounds of Formula (Ia)

[0059] To determine the hydrophobic character of the compounds of Formula (Ia), contact angle measurements were carried out.

[0060] A solution of curcumin or compound of Formula (Ia) was prepared at a concentration of 2.2 mM. Then, 3 successive deposits of 100 μL were made on a glass microscope slide by centrifugal coating (rotation speed of 2000 rpm, 1.5 min of drying between each deposit) in order to form a thin and homogeneous film.

[0061] For contact angle measurements, approximately 8 μL of solvent was deposited onto the thin film prepared above using an automatic injection syringe. Two solvents were used: water and diiodomethane.

[0062] Lamontre shows the evolution of the contact angle as a function of the compound studied of the curcumin derivative and more precisely, for the compounds of Formula (Ia) with R = C8, C 12 , C 14 etc 16 .

[0063] In both cases, water and diiodomethane then used as solvents, an increase in the contact angle is observed with the increase in the chain length of the R group. Curcumin has the lowest value of 77° with water. This value confirms that curcumin and its derivatives are not very hydrophilic molecules, resulting in their low solubility in water but not hydrophobic enough to form stable nano-assemblies. The film of the compound of Formula (Ia) with R = C8 has a contact angle with water of 88°. For the other compounds of Formula (Ia), the contact angles are greater than 90 with a maximum value of 103° for the compound of Formula (Ia) with R = C 16 The same trend is observed for the contact angles with the solvent diiodomethane.

[0064] Example 2: synthesis of organic nano-assemblies according to the invention Synthesis

[0065] A 0.55 mM organic solution of the compound of Formula (Ia) obtained in Example 1 is prepared in 1 mL of tetrahydrofuran.

[0066] A flash-nanoprecipitation preparation device as described above in connection with the. Mixer 1 is a confined liquid impact jet mixer.

[0067] The organic solution of the compound of Formula (Ia) is placed in syringe 4 and water is placed in the second syringe 5.

[0068] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into mixer 1 simultaneously.

[0069] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of water. Mass concentration in water

[0070] The organic nano-assemblies according to the present invention can have a molar concentration of up to 0.4 mmol / L, i.e. a mass concentration of between 200 and 290 µg / mL (depending on the curcumin derivative used). The method according to the invention therefore makes it possible to increase the mass concentration of the molecule by a factor of approximately 25,000 compared to the lowest solubility of curcumin of 0.011 µg / mL. Characterization

[0071] In order to obtain information on the morphology of the nano-assemblies, nanoscale photos are taken using a transmission electron microscope (TEM). The analysis of these photos also makes it possible to obtain the diameter of the nano-assemblies, noted d MET. For this, a size distribution of approximately 300 nano-assemblies per sample is produced. This distribution is then modeled by a log-normal function. The samples are deposited on copper grids covered with a carbon film and are then left to dry in the open air.

[0072] This is a TEM photo showing nanoassemblies according to the present invention.

[0073] The measurement of the zeta potential, noted ζ, is based on the measurement of the electrophoretic mobility of the nano-assemblies, noted U E An electric field is applied to the sample and the charged particles in suspension will migrate towards the oppositely charged electrode. The migration speed of the nano-assemblies is thus measured. It is then possible to go back to the value of the zeta potential, ζ, using Henry's equation.

[0074] Henry's equation

[0075]

[0076] With ε, dielectric constant of water, η, dynamic viscosity of water and f(κa), Henry function. Here we will make the Smoluchowskii approximation. ef(κa) = 1.5.

[0077] Table 1 below indicates the values ​​of MET diameter and zeta potential for the organic nano-assemblies according to the invention as a function of the length of the carbon chain R of the compound of Formula (Ia)

[0078] d MET (nm)ζ (mV)R = C886.9 ± 8.3-42.6 ± 9.0R = C 12 68.0 ± 13.4-33.5 ± 3.9R = C 14 48.5 ± 1.1-32.8 ± 2.8R = C 16 50.8 ± 1.0-30.9 ± 2.3

[0079] For all four systems studied, the morphology of the organic nanoassemblies is the same. Regular spheres in shape and size are observed. A correlation can be observed between the size of the nanoassemblies and the length of the side chain of the compound of Formula (Ia) that composes it. The longer the side chain of the compound of Formula (Ia), the smaller the size of the organic nanoassembly. For example, the compound with R = C8 allows nanoassemblies of 87 nm in diameter to be obtained, while compounds with R > C 14allow the production of nano-assemblies of approximately 50 nm in diameter, which is the ideal size for long circulation in the blood and good cellular internalization. The compounds of Formula (Ia) with the longest chains are the most hydrophobic. During nanoprecipitation, numerous nuclei are formed to locally limit interactions with the surrounding aqueous medium. The local concentration of free organic compound then decreases sharply. There are therefore fewer organic compounds available to feed these numerous nuclei, which ultimately results in a smaller nano-assembly size.

[0080] For each system, the zeta potential, ζ, is negative and less than -30 mV, indicating good colloidal stability of the organic nanoassemblies. Indeed, this shows the presence of electrostatic interactions allowing the organic nanoassemblies to repel each other and thus avoid their aggregation.

[0081] Similar measurements were performed on organic nanoassemblies prepared as described above with a curcumin solution instead of the solution of the compound of Formula (Ia). Since curcumin is a slightly hydrophobic compound, it is difficult to form stable nanoassemblies. Several tests were conducted and the results are very poorly reproducible. Under the same synthesis conditions, it is sometimes possible to form nanoassemblies or, more frequently, micrometric-sized precipitates. Stability of nano-assemblies

[0082] The stability of the nano-assemblies over time was studied over 3 months with measurements at T = 0, T = 1 day, 2 days, 7 days, 30 days and 90 days of incubation at 37 °C. Five systems were studied: curcumin-based nano-assemblies, nano-assemblies according to the invention with R = C8, C 12 , C 14 etc 16Three characterization techniques were coupled: TEM imaging, zeta potential, ζ, and high performance liquid chromatography (HPLC).

[0083] For each system, sixteen successive nanoprecipitations, as presented above, were carried out and then combined in the same bottle to have a total of 40 mL of homogeneous nano-assembly solution. At each time interval, as defined in the previous paragraph, 2x2 mL of solution are taken and lyophilized for HPLC measurements, 5 μL are placed on a TEM grid and left to air dry, 3 mL are taken for zeta potential measurements, ζ. All samples were analyzed under the same concentration conditions.

[0084] Shows the evolution of the MET diameter as a function of time, the evolution of the zeta potential, ζ, as a function of time and the evolution of the concentration of curcumin or compound of Formula (Ia) as a function of time measured by HPLC.

[0085] In the legend of Figures 7, 8 and 9 the abbreviation CDNP designates nano-assemblies, Curc indicates that the nano-assemblies were obtained with curcumin and C8, C 12 , C 14 etc 16 indicate that the nano-assemblies were obtained with the compound of Formula (Ia) with R = C8, C 12 , C 14 etc 16 .

[0086] For each system the zeta potential, ζ, remains stable, negative with values ​​between -30 and -40 mV.

[0087] HPLC analyses were also performed. After lyophilization, the nano-assemblies are dissociated in methanol to obtain an organic solution of molecular compounds of curcumin or compound of Formula (Ia). This HPLC study allows to quantify the molecular stability of the different compounds when they are in the form of nano-assemblies and to determine the possible degradation products of the compounds.

[0088] Shows the evolution of the concentration of curcumin or compound of Formula (Ia) measured by HPLC over time, normalized with respect to the theoretical concentration of molecular compound initially injected.

[0089] For curcumin, at T = 0, only 60% of the theoretical injected concentration is measured. Since curcumin is not hydrophobic enough, it does not allow stable nano-assemblies to form. Upon nanoprecipitation, a significant portion of organic compound precipitates at the bottom of the pillbox. The sample is then neither homogeneous nor representative of the entire sample. A significant degradation is observed after 30 days of incubation, only 10% of the curcumin is measured. Finally, after 90 days of incubation, the remaining amount of curcumin is below the detection limit.

[0090] Compounds of Formula (Ia) with R = C8, C 12 etc 14have a relatively similar profile. Approximately 85% of the compounds are detected at T = 0. No precipitate is observed with the naked eye (unlike curcumin) during nanoprecipitation. The discrepancy between the dosed concentration and the initially injected concentration may be due to product losses during lyophilization. The concentration of compounds of Formula (Ia) with R = C8, C 12 etc 14 remains relatively stable over the first two days of analysis (5% degradation for the compound of Formula (Ia) with R = C8 and 19% degradation for the compounds of Formula (Ia) with R = C 12 etc 14 ). After 90 days of incubation, a low concentration of compound of Formula (Ia) with R = C 12 etc 14 is detected (less than 5%). For the compound of Formula (Ia) with R = C8, at 90 days of incubation, still 27% of the product is detected.

[0091] For the compound of Formula (Ia) with R = C 16, only 75% of the compound is dosed at T = 0. A constant and rapid decrease in concentration is observed during the first two days of incubation (61% at 1 day and 48% at 2 days). This decrease is still effective for the longest times but to a lesser extent (28% of compound dosed at 7 days and 16% at 30 days). After 90 days of incubation, as for compounds of Formula (Ia) with R = C 12 etc 14 , less than 5% of the compound is detected.

[0092] For each of the systems, none of the curcumin degradation products (vanillin, ferulic acid and feruloylmethane) could be detected. For the compounds of Formula (Ia), no trace of curcumin could be detected.

[0093] This study, which monitored the degradation of Formula (Ia) compounds and curcumin when they are in the form of organic nano-assemblies, was able to highlight certain elements. First, the study was able to confirm once again that curcumin does not form stable nano-assemblies and that the molecular compound decomposes rapidly. Second, differences in behavior were observed depending on the length of the carbon chain of the Formula (Ia) compound.

[0094] Compounds of Formula (Ia) with R = C 12 etc 14 have very similar behavior with good stability over short periods. The compound of Formula (Ia) with R = C8 shows the greatest chemical stability.

[0095] TEM measurements showed the low colloidal stability of nano-assemblies with R = C8 due to a fairly rapid Ostwald growth, but its stability remains satisfactory for rapid use of the nano-assembly. The diameter of nano-assemblies with R = C 12 , C 14 etc 16 varies little over time and there is no change in the morphology of the organic nano-assemblies. For all systems, the zeta potential, ζ, remains constant over time, with values ​​between -30 and -40 mV. Finally, all the analyses carried out show that curcumin alone does not allow the formation of nano-assemblies. HPLC measurements also highlighted that the structural modifications made via Knoevenagel condensation made it possible to increase the molecular stability of the derivatives of Formula (Ia) compared to curcumin.

[0096] Example 3: Synthesis of super-paramagnetic nanoparticles Spherical nanoparticles

[0097] In a 100 mL two-necked flask, 1.4 g of iron(III) acetylacetonate (4 mmol), 7.1 g of oleic acid (25 mmol) and 12 mL of oleylamine (36 mmol) were introduced. The medium is heated to 120 °C under magnetic stirring (1200 rpm) and 3 vacuum-argon cycles are carried out. The medium is maintained at this temperature under partial vacuum for 2 hours. The reaction is then placed under argon and heated to 220 °C for 30 min, then brought to reflux for 40 min. After returning to 120 °C, the nanoparticles formed are oxidized in air for 1h30. After returning to room temperature, an excess of ethanol is added to precipitate the nanoparticles and then centrifuged (10,000 rpm, 10 min). The pellet is taken up in a 0.02% oleic acid solution in hexane and precipitated again in ethanol and then centrifuged (10,000 rpm, 10 min). The operation is then repeated once. Finally, the nanoparticles are stored in THF.

[0098] Depending on the vacuum quality, spherical nanoparticles of 6.2 nm (partial vacuum = 3.7 mmbar) or 12.1 nm (partial vacuum = 0.9 mmbar) are obtained.

[0099] Lamontre shows a TEM photo of the 12.1 nm spherical nanoparticles. Cubic nanoparticles

[0100] In a 50 mL three-necked flask topped with a water condenser and connected to a vacuum pump, 0.35 g of iron(III) acetylacetonate (1 mmol) and 0.86 g of decanoic acid (5 mmol) are introduced into a mixture of 23 mL of dibenzylether and 2 mL of squalane. The medium is heated to 65 °C under magnetic stirring (1200 rpm) and 3 vacuum-argon cycles of 20 minutes each are carried out. The medium is then maintained at this temperature under partial vacuum (0.9 mmbar) for 1 hour. Then, the temperature is increased to 220 °C under argon with a temperature ramp of 3 °C / min. The medium is heated at 220 °C for 2.5 hours. Finally, the medium is heated to reflux and maintained there for 30 min. After returning to room temperature, excess ethanol is added to precipitate the nanoparticles and then centrifuged (10,000 rpm, 10 min). The pellet is taken up in a 0.02% oleic acid solution in hexane and precipitated again in ethanol and then centrifuged (10,000 rpm, 10 min).The operation is then repeated once. Finally, the nanoparticles are stored in the THF.

[0101] Lamontre shows a TEM photo of the 22.1 nm cubic nanoparticles. Rod-shaped nanoparticles

[0102] In a 50 mL flask, 0.605 g of hexadecylamine and 1.795 g of oleic acid are introduced in 8 mL of 1-octanol. The solution is heated to 55°C for 30 minutes. Afterwards, the solution is allowed to return to room temperature. 2 mL of iron pentacarbonyl are then added. The mixture is then stirred for 1 hour at room temperature. The solution is then transferred to an autoclave with a Teflon protective insert and then heated in an oven at 200°C for 6 hours. Once the solution has returned to room temperature, a large amount of ethanol is added to precipitate the nanorods. The solution is then centrifuged for 10 min at 10,000 rpm. The operation is then repeated once. Finally, the nanoparticles are stored in THF.

[0103] Lamontre shows a TEM photo of the resulting rod-shaped nanoparticles with a length of 49 nm and a thickness of 6.8 nm.

[0104] Example 4: Synthesis of a magnetic hybrid nano-assembly containing a super-paramagnetic nanoparticle

[0105] An organic solution of 0.825 mM of compound of Formula (Ib) and 0.05% of superparamagnetic particles (by mass of iron) of 6.2 nm obtained in Example 3 is prepared in 1 mL of THF.

[0106] A 0.5 mg / mL aqueous solution of commercial poly-L-lysine (Alamanda Polymers, Inc., Huntsville, AL, USA) was prepared in 1 mL of PBS.

[0107] The procedure is as in Example 2, placing the organic solution in syringe 4 and the aqueous solution in the second syringe 5.

[0108] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into mixer 1.

[0109] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of an aqueous solution at 0.19 mg / mL of poly-L-lysine.

[0110] Lamontre shows a TEM photo of the obtained nano-assembly with a diameter of 60.9 nm and a potential ζ = 34 mV. Heating properties:

[0111] The heating properties of the nanoassemblies were measured via the Specific Absorption Rate (SAR) with an alternating magnetic field applied at a frequency of 486.55 kHz and an amplitude of 29.5 kA / m. For the nanoassemblies obtained in this example, the SAR is 447 W / g. Under the same conditions, the superparamagnetic nanoparticles alone have a SAR value of 6.4 W / g. Mass concentration in water

[0112] The magnetic hybrid nanoassemblies according to the present invention may have a molar concentration of compound of formula (Ib) of 0.32 mmol / L.

[0113] Example 5: Synthesis of a magnetic hybrid nano-assembly containing a super-paramagnetic nanoparticle with a poly-L-Lysine functionalized by PEG

[0114] The nano-assembly is prepared in the same way as in Example 4 except that a commercial poly-L-lysine functionalized on its side chains by polyethylene glycol functions (SuSoS AG, Dübendorf, Switzerland) is used instead of the commercial poly-L-lysine of Example 4 in the second syringe 5.

[0115] The obtained nanoparticles have a zeta potential of 22 mV. Stability

[0116] The colloidal stabilities of the suspensions of Example 4 and Example 5 are similar, with no settling being observed after 1 month.

[0117] The suspensions of Example 5, on the other hand, exhibit better stability in a constant, radial magnetic field.

[0118] The suspensions of Example 4 and Example 5 were placed in a SEPMAG® LAB biomagnetic separation system (SEPMAG, Cerdanyola del Valles, Spain) under a field with an intensity of 15 + / - 5 T / m.

[0119] The settling time of the particles is measured.

[0120] Complete settling of the suspensions is observed after two days for the suspension of Example 4 and after seven days for the suspension of Example 5.

[0121] This method allows us to anticipate the stability of suspensions under normal conditions, which allows us to conclude that the suspension of Example 5 exhibits excellent stability over time.

[0122] Example 6: Synthesis of a magnetic hybrid nano-assembly containing a super-paramagnetic nanoparticle

[0123] A 0.55 mM organic solution of compound of Formula (Ia) with R = C 14and 0.3% superparamagnetic particles (by mass of iron) of 6.2 nm obtained in Example 3 is prepared in 1 mL of THF.

[0124] A 10 mg / mL aqueous solution of commercial poly-L-lysine (Alamanda Polymers, Inc., Huntsville, AL, USA) was prepared in 1 mL of PBS.

[0125] The procedure is as in Example 2, placing the organic solution in syringe 4 and the aqueous solution in the second syringe 5.

[0126] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into mixer 1.

[0127] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of water.

[0128] Lamontre shows a TEM photo of a obtained nanoassembly with a diameter of d = 107.6 nm and a zeta potential of ζ = +36 mV. Mass concentration in water

[0129] The magnetic hybrid nanoassemblies according to the present invention have a molar concentration of 0.21 mmol / L.

[0130] Example 7: functionalization of a magnetic hybrid nano-assembly

[0131] A magnetic hybrid nanoassembly was synthesized as in Example 4, except that the stabilizing polymer used was commercial propargyl-dPEG1-amide functionalized poly-L-lysine (Alamanda Polymers, Inc., Huntsville, AL, USA) ().

[0132] An organic solution of 0.55 mM of compound of Formula (Ib) and 0.3% of superparamagnetic particles (by mass of iron) obtained in Example 3 with a diameter of 6.2 nm is prepared in 1 mL of THF.

[0133] A 5 mg / mL aqueous solution of poly-L-lysine functionalized with propargyl-dPEG1-amide is prepared in 1 mL of PBS.

[0134] The procedure is as in Example 2, placing the organic solution in syringe 4 and the aqueous solution in the second syringe 5.

[0135] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into mixer 1.

[0136] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of water.

[0137] Lamontre shows a TEM photo of a nano-assembly obtained with a diameter d = 74 nm.

[0138] The surface of the magnetic hybrid nano-assembly is then functionalized with a molecule of interest using the “click” chemistry reaction.

[0139] Shows the reaction scheme of the surface functionalization reaction of the magnetic hybrid nanoassembly.

[0140] From a solution of magnetic hybrid nanoassemblies functionalized by a poly-L-lysine polymer bearing a propargyl function, 100 µL of a 0.009 mg / mL sodium ascorbate solution, 100 µL of a 0.011 mg / mL copper sulfate solution and 100 µL of a 0.25 mg / mL sulfocyanine-5-azide solution (Jena Bioscience GmbH, Jena, Germany) are added. The medium is stirred at room temperature for 24 hours. Three successive washes by magnetic decantation are carried out to remove excess reagents.

[0141] Lamontre shows a TEM photo of a nano-assembly obtained after functionalization of its surface with a fluorophore with a diameter d = 72.5 nm and a zeta potential ζ = 29 mV.

Claims

– Nano-assembly formed by the self-assembly of a compound chosen from among the compounds of Formula (Ia): R represents an alkyl group; and the compound has the formula (Ib): (Ib), said compound of Formula (Ib) being associated with at least one super-paramagnetic nanoparticle. - Nano-assembly according to claim 1, characterized in that R represents an alkyl group comprising at least 4 carbon atoms, preferably comprising between 4 and 16 carbon atoms. - Nano-assembly according to one of claims 1 or 2, characterized in that R is selected from butyl, pentyl, octyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl and octadecyl. - Nano-assembly according to any one of claims 1 to 3, characterized in that the nano-assembly is constituted by self-assembly of the compound of Formula (Ia) only. - Nano-assembly according to claim 4, characterized in that the nano-assembly is spherical and has a diameter, as measured by transmission electron microscopy, of between 40 nm and 110 nm. - Nano-assembly according to one of claims 4 and 5, characterized in that the nano-assembly has a zeta potential, as measured by zetametry, between -30 mV and -40 mV. - Nano-assembly according to any one of claims 1 to 6, characterized in that said nano-assembly is coated with a stabilizing polymer. - Nano-assembly according to claim 7, characterized in that the stabilizing polymer is a cationic poly-electrolyte. - Nano-assembly according to claim 8, characterized in that the cationic poly-electrolyte is a poly-L-lysine-based polymer or a poly-L-lysine-based polymer whose side chains are functionalized by polyethylene glycol. – Nano-assembly according to any one of claims 7 to 9, characterized in that the stabilizing polymer is functionalized with a function capable of being associated by bioconjugation with a complementary function, for example by click chemistry or peptide coupling, and that the nano-assembly is coupled to a molecule of interest, in particular chosen from peptides, fluorophores or biomolecules, the molecule of interest being functionalized with a function complementary to the function of the functionalized stabilizing polymer. - Nano-assembly according to any one of claims 1 to 3 and 7 to 10, characterized in that a compound of Formula (Ia) is associated with at least one super-paramagnetic nanoparticle. - Nano-assembly according to claims 1 to 3 and 7 to 11, characterized in that at least one super-paramagnetic nanoparticle is composed of iron oxide. - Nano-assembly according to any one of claims 1 to 3 and 7 to 12, characterized in that at least one super-paramagnetic nanoparticle has: a spherical shape with a diameter between 6 and 20 nm, as measured by transmission electron microscopy; or a cubic shape with an edge between 10 and 70 nm, as measured by transmission electron microscopy; or a rod shape with a length between 30 and 50 nm and a width between 6 and 8 nm, as measured by transmission electron microscopy. - Nano-assembly according to any one of claims 1 to 3 and 7 to 13, characterized in that the nano-assembly is spherical and has a diameter, as measured by transmission electron microscopy, of between 40 nm and 250 nm. - Nano-assembly according to claim 14, characterized in that said nano-assembly has a zeta potential, as measured by zetametry, between +25 mV and +40 mV. – A method for preparing a nano-assembly according to any one of claims 1 to 15, characterized in that a flash-nanoprecipitation is carried out by the following steps: in a first syringe, a solution of the compound of Formula (Ia) or (Ib) is placed in a solvent miscible with water, chosen from tetrahydrofuran, acetone or acetonitrile, which contains, where appropriate, at least one super-paramagnetic nanoparticle, and in a second syringe, water or a phosphate saline buffer is placed, which contains, where appropriate, a stabilizing polymer; the contents of the two syringes are injected into a nanoprecipitation mixer at a speed of 2 to 10 mm / s, in particular at 5 mm / s; and the nano-assemblies are recovered in a receiving bottle containing water, and where appropriate, a stabilizing polymer. - Pharmaceutical composition comprising one or more nano-assemblies according to any one of claims 1 to 15. – Nano-assembly according to any one of claims 1 to 15 for its use in the treatment of inflammatory diseases – Nano-assembly according to any one of claims 1 to 15 for its use in the treatment of cancer. – Nano-assembly according to any one of claims 1 to 3 and 7 to 15 for its use in magnetic liquid hyperthermia. - Contrast agent for magnetic resonance imaging characterized in that it comprises nano-assemblies according to any one of claims 1 to 3 and 7 to 15. - Magnetic particle imaging agent characterized in that it comprises nano-assemblies according to any one of claims 1 to 3 and 7 to 15.

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