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

Abstract

------ Nano-assemblies based on curcumin or curcumin derivative and their preparation process The present invention relates to a nano-assembly formed by a self-assembly of a compound chosen from the compounds of Formula (Ia): R representing an alkyl group; and the compounds of Formula (Ib): (Ib), said compound of Formula (Ib) being associated with at least one super-paramagnetic nanoparticle.

Description

Title of the invention: 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 derivatives, their preparation process and their use.

[0002] Liquid magnetic 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 tumor temperature, either to induce apoptosis of the cancer cells (in this case, 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 qm), high sensitivity (which could approach 107-108 M) and real-time imaging.

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

[0005] Numerous publications describe the encapsulation of curcumin in a polymer matrix. However, the size of these nanoassemblies is large, exceeding 100 nm, resulting in a short circulation time in the blood, very slow or even nonexistent diffusion in extracellular viscous matrices, and very slow internalization into cells, which can lead to vesiculation. The size of the nanoparticles or nanoassemblies is a critical parameter in controlling their biodistribution. Indeed, nanoparticles smaller than 10 Nanoparticles smaller than 200 nm are below 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 larger than 200 nm are rapidly taken up by the mononuclear phagocytic system, and a size limit of 150 nm is generally accepted to avoid spleen filtration. It appears that a size between 10 and 150 nm allows for good circulation of nanoparticles in the blood. However, for good cellular internalization by endocytosis, nanoparticles are described as needing 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.2999, 21 (4), 419-424. This size has since been validated experimentally. Lu, F.; Wu, S.-H.; Hung, Y.; Mou, C.-Y. Size Effect on Cell Uptake in Well-Suspended, Uniform Mesoporous Silica Nanoparticles. Small2QQ9, 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, J.T.; Chan, WCW Nanoparticle-Mediated Cellular Response Is Size-Dependent. Nat. Nanotechnol. 2998, 3 (3), 145-150. However, it turns out that curcumin nanoassemblies in this size range are not described in the literature. Moreover, curcumin is not the only species composing the nanoparticle and is generally the minor species.

[0006] Furthermore, curcumin has very poor solubility in water. By HPLC analysis, the solubility of curcumin in water or in an aqueous buffer at pH = 5 is 0.011 pg / mL (Tpnnesen, 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 give a higher value of 0.6 pg / 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). Curcumin's low solubility in water makes it difficult to use in the human body.

[0007] The present invention aims to overcome these drawbacks by using a curcumin derivative that improves the solubility and stability of the molecule in aqueous solution, thus locking the molecule in its biologically active diketone form. To increase the amount of curcumin derivative in water, the present invention proposes not to increase the hydrophilic properties of the curcumin derivative, but rather to increase its hydrophobicity in order to allow its self-absorption. Assembly in water. Self-assembly is a process in which chemical components such as molecules, metallic nanoparticles, colloids, polymers, or any other compound spontaneously organize themselves into ordered structures through physicochemical interactions such as weak, non-covalent chemical bonds. Thus, the hydrophobicity of the curcumin derivative is controlled to allow the formation of small nanoassemblies. This formulation in the form of nanoassemblies increases the therapeutic index of the curcumin derivative by limiting the non-specific diffusion observed with molecular compounds. The small size of the resulting nanoassemblies facilitates interactions with the body's cells and increases their circulation rate in the blood as well as their diffusion in viscous media, for example, in extracellular matrices.It was also surprisingly discovered that combining curcumin with a super-paramagnetic nanoparticle allows the formation of a nanoassembly exhibiting a good mass concentration in water.

[0008] To this end, the present invention relates to a nano-assembly formed by a self-assembly of a compound selected from the compounds of Formula (la):

[0009] [Chem.l]

[0010] R representing an alkyl group; and

[0011] the compound of Formula (Ib):

[0012] [Chem.2] (Ib),

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

[0014] When the compound of Formula (la) or of Formula (Ib) is associated with at least one super-paramagnetic nanoparticle, the resulting nano-assembly can be called a magnetic hybrid nano-assembly.

[0015] R can represent an alkyl group comprising at least 4 carbon atoms, of preference containing between 4 and 16 carbon atoms.

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

[0017] The nanoassembly according to the present invention may be formed by self-assembly of the compound of formula (la) only. Said nanoassembly may then be spherical. Such a nanoassembly may be called an organic nanoassembly. The nanoassembly 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] Zeta measurement can be carried out 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.

[0021] The stabilizing polymer can be functionalized with a function suitable for 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 with a function complementary to the function of the functionalized stabilizing polymer.

[0022] 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.

[0023] In a particular embodiment, a compound of Formula (la) is associated with at least one super-paramagnetic nanoparticle.

[0024] At least one super-paramagnetic nanoparticle may be composed of iron oxide.

[0025] In particular, when there is more than one super-paramagnetic nanoparticle, the other super-paramagnetic nanoparticle or nanoparticles may be of different size and shape.

[0026] At least one super-paramagnetic nanoparticle may exhibit:

[0027] • 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-shaped structure with a length between 30 and 50 nm and a width between 6 and 8 nm, as measured by electron microscopy transmission electronics.

[0028] The nano-assembly can be spherical and have a diameter, as measured by transmission electron microscopy, of between 40 nm and 250 nm. It can then have a zeta potential, as measured by zetametry, of 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:

[0030] • in a first syringe, a solution of the compound of Formula (la) or of Formula (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 collected in a receptacle bottle containing water, and if necessary, a stabilizing polymer.

[0031] In particular, the receiving bottle may contain water in an amount that allows the entire reaction medium exiting the mixer to be diluted by a factor of at least five, preferably at least ten.

[0032] The present invention also relates to a pharmaceutical composition comprising one or more nano-assemblies according to the present invention.

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

[0034] The present invention also relates to a nano-assembly according to the present invention for its use in the treatment of cancer.

[0035] The present invention also relates to a nano-assembly according to the present invention for its use in magnetic liquid hyperthermia.

[0036] 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.

[0037] The present invention also relates to an agent for magnetic particle imaging characterized in that it comprises nano-assemblies according to the present invention.

[0038] The compound of formula (la) can be obtained by means of 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 (la) according to the present invention.

[0039] The general synthesis scheme of this compound of formula (la) is shown below:

[0040] [Chem.3]

[0041] Super-paramagnetic 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 mixture of solvents with a high boiling point, 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 entire compound is heated to a high temperature, above 200°C.

[0042] Superparamagnetic rod-shaped nanoparticles are prepared by solvothermal means. 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. The mixture is then heated in an autoclave at 200°C.

[0043] Fig. 1 presents the general scheme of synthesis of superparamagnetic nanoparticles of spherical shape (path A), cubic shape (path B) and rod shape (path C).

[0044] 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 [Fig. 2].

[0045] Figure 2 schematically represents the device used for the synthesis of nano-assemblies according to the invention by a flash-nanoprecipitation process. This device comprises a mixer 1 having two inlet ports 2 and an outlet port 3. The mixer 1 is typically a confined liquid impact jet mixer.

[0046] One of the inlet ports 2 of the mixer 1 is in fluidic communication with a first syringe 4 while the second inlet port 2 is in fluidic 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 proceed with the simultaneous injection of the contents of the syringes 4 and 5.

[0047] A receptacle bottle 7 is placed under the outlet orifice 3 of the mixer 1.

[0048] For the synthesis of the organic nanoassemblies according to the invention, a solution of the compound of Formula (la) in a solvent such as tetrahydrofuran, acetone or acetonitrile can be placed in the first syringe 4. In the other syringe 5, water or phosphate saline buffer is placed, in the presence or without a stabilizing polymer to obtain at least one organic nanoassembly.

[0049] For the synthesis of the magnetic hybrid nanoassemblies according to the invention, a solution of the compound of Formula (la) or of 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 phosphate saline buffer is placed, in the presence of a stabilizing polymer to obtain a magnetic hybrid nanoassembly.

[0050] By moving the anvil 6, the contents of 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.

[0051] Optionally, the stabilizing polymer may have a functionalization to enable the grafting of compounds of interest to the surface of the nano-assembly by "Click" chemistry.

[0052] Typically, the grafting of compounds of interest can be carried out by a cycloaddition reaction. This reaction is catalyzed by Cu(I) at the interface between nanoassemblies, the latter presenting, via the stabilizing polymer, an alkyne function and a commercial molecule of interest comprising an azide function.

[0053] Fig. 3 is a schematic representation of an organic nanoassembly (ONA) according to the invention.

[0054] Fig. 4 is a schematic representation of a magnetic hybrid nano-assembly according to the invention.

[0055] In [Fig.4], the magnetic hybrid nanoassembly (NAHM) contains super-paramagnetic nanoparticles (NPM) and is coated with a stabilizing polymer (POLY).

[0056] The following examples illustrate the present invention without however limiting its scope. Example 1: Synthesis of the compound with formula (la) Step 1: Amidic coupling

[0057] 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 mixture is stirred at room temperature for two hours and then evaporated to dryness using a rotary evaporator. The crude reaction mixture is dissolved in 40 mL of anhydrous dimethylformamide, and the primary aliphatic amine (20 mmol) is added. The mixture is heated to 90 °C for 24 hours. After returning to room temperature, the precipitate is filtered and then washed three times with IM NaOH to remove the 4-formylbenzoic acid reagent, then neutralized with IM HCl, and washed three times with diethyl ether to remove excess aliphatic amine. No further purification is required.

[0058] The primary aliphatic 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 the primary aliphatic amine used is the same regardless of the amine chain length. Step 2: Knoevenagel condensation

[0059] To 0.76 g of the compound obtained in step 1 (2.2 mmol), 0.37 g of curcumin (1 mmol) are added to 40 mL of chloroform anhydride and dichloromethane anhydride, 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 added. The mixture is stirred at room temperature for 48 hours. After filtration to remove the molecular sieve, the mixture is evaporated using a rotary evaporator to remove the chloroform and catalysts. The mixture is diluted in dichloromethane and extracted several times with water. After drying over anhydrous sodium sulfate and evaporation, the crude reaction mixture is purified by silica chromatography using a dichloromethane / ethyl acetate mixture (8:2) as the eluent. Hydrophobic character of the compounds of formula (la)

[0060] To determine the hydrophobic character of the compounds of Formula (la), contact angle measurements were carried out.

[0061] A solution of curcumin or compound of Formula (la) was prepared at a concentration of 2.2 mM. Then, 3 successive deposits of 100 pL were made on a glass microscope slide by centrifugal coating (rotation speed of 2000 rpm, 1.5 min drying time between each deposit) in order to form a thin and homogeneous film.

[0062] For contact angle measurements, approximately 8 pL of solvent are deposited onto the thin film prepared above using an automatic injection syringe. Two solvents were used: water and diiodomethane.

[0063] Figure 5 shows the evolution of the contact angle as a function of the compound studied. curcumin derivative and more specifically, for compounds of Formula (la) with R = C8, C12, Cm and Ci6.

[0064] In both cases, with water and diiodomethane used as solvents, an increase in the contact angle is observed with increasing chain length of the R group. Curcumin exhibits the lowest value of 77° with water. This value confirms that curcumin and its derivatives are weakly hydrophilic molecules, resulting in low water solubility, but not sufficiently hydrophobic to form stable nanoassemblies. The film of the compound of Formula (la) with R = C8 exhibits a contact angle with water of 88°. For the other compounds of Formula (la), the contact angles are greater than 90°, with a maximum value of 103° for the compound of Formula (la) with R = Ci6. The same trend is observed for the contact angles with the diiodomethane solvent.

[0065] Example 2: Synthesis of organic nano-assemblies according to the invention Synthesis

[0066] An organic solution at 0.55 mM of the compound of Formula (la) obtained in Example 1 is prepared in 1 mL of tetrahydrofuran.

[0067] A flash-nanoprecipitation preparation device as described above is used in relation to [Fig.2]. Mixer 1 is a confined liquid impact jet mixer.

[0068] The organic solution of the compound of Formula (la) is placed in syringe 4 and water is placed in the second syringe 5.

[0069] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into the mixer 1 simultaneously.

[0070] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of water. Mass concentration in water

[0071] The organic nanoassemblies according to the present invention can have a molar concentration of up to 0.4 mmol / L, corresponding to a mass concentration of between 200 and 290 pg / mL (depending on the curcumin derivative used). The method according to the invention thus 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 pg / mL. Characterization

[0072] In order to obtain information on the morphology of the nano-assemblies, nanoscale images are taken using a transmission electron microscope (TEM). Analysis of these images also makes it possible to determine the diameter of the nano-assemblies, denoted dMET. For this purpose, a size distribution of approximately 300 Nano-assemblies per sample are performed. This distribution is then modeled by a log-normal function. The samples are deposited on copper grids coated with a carbon film and then left to air dry.

[0073] Fig. 6 is a TEM image showing nano-assemblies according to the present invention.

[0074] The measurement of the zeta potential, denoted Ç, is based on the measurement of the electrophoretic mobility of the nanoassemblies, denoted UE. An electric field is applied to the sample, and the suspended charged particles migrate towards the oppositely charged electrode. The migration velocity of the nanoassemblies is thus measured. It is then possible to determine the value of the zeta potential, Ç, using Henry's law.

[0075] Henry's equation

[0076] [Math.l]

[0077] With s, the dielectric constant of water, îj, the dynamic viscosity of water and f(xa), the Henry function. We will here make the Smoluchowski approximation i.e. f(Ka) = 1.5.

[0078] Table 1 below shows the MET diameter and zeta potential values ​​for the organic nanoassemblies according to the invention as a function of the carbon chain length R of the compound of Formula (la)

[0079] [Tables 1] dMET (nm) UmV) r = c8 86.9 + 8.3 -42.6 + 9.0 r = c12 68.0+13.4 -33.5 + 3.9 r = c14 48.5+1.1 -32.8 + 2.8 r = c16 50.8 + 1.0 -30.9 + 2.3

[0080] For the 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 (la) that composes them. The longer the side chain of the compound of Formula (la), the smaller the size of the organic nanoassembly. For example, the compound with R = C8 allows for nanoassemblies with a diameter of 87 nm, while the compounds with R > CM allow for nanoassemblies with a diameter of approximately 50 nm, which is the ideal size for long-term circulation in the blood and good cellular internalization. The compounds of Formula (la) with the longest chains are the most hydrophobic. During nanoprecipitation, numerous nuclei form to locally limit Interactions with the surrounding aqueous environment cause the local concentration of free organic compounds to decrease sharply. Consequently, fewer organic compounds are available to feed these numerous nuclei, resulting in smaller nano-assemblies.

[0081] For each system, the zeta potential, Ç, is negative and less than -30 mV, indicating good colloidal stability of the organic nanoassemblies. Indeed, this shows the presence of electrostatic interactions that allow the organic nanoassemblies to repel each other and thus prevent their aggregation.

[0082] Similar measurements were performed on organic nanoassemblies prepared as described above using a curcumin solution instead of the solution of compound of formula (la). Since curcumin is a weakly hydrophobic compound, it is difficult to form stable nanoassemblies. Several trials were conducted, and the results are very inconsistent. Under the same synthesis conditions, it is sometimes possible to form nanoassemblies or, more frequently, micrometer-sized precipitates. Stability of nano-assemblies

[0083] The stability of the nanoassemblies 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 nanoassemblies, nanoassemblies according to the invention with R = C8, Ci2, Ci4 and Ci6. Three characterization techniques were combined: TEM imaging, zeta potential, Ç, and high-performance liquid chromatography (HPLC).

[0084] For each system, sixteen successive nanoprecipitations, as described above, were performed and then combined in a single flask to obtain a total of 40 mL of homogeneous nanoassembly solution. At each time interval, as defined in the preceding paragraph, 2 x 2 mL of the solution were taken and lyophilized for HPLC measurements, 5 µL were deposited on a TEM grid and allowed to air dry, and 3 mL were taken for zeta potential measurements. All samples were analyzed under the same concentration conditions.

[0085] Fig. 7 shows the evolution of the MET diameter as a function of time, Fig. 8 shows the evolution of the zeta potential, Ç, as a function of time and Fig. 9 shows the evolution of the concentration of curcumin or of compound of Formula (la) as a function of time measured by HPLC.

[0086] In the legend of Figures 7, 8 and 9 the abbreviation CDNP designates the nano-assemblies, Cure indicates that the nano-assemblies were obtained with curcumin and C8, Ci2, CM and Ci6 indicate that the nano-assemblies were obtained with the compound of Formula (la) with R = C8, C[2, CM and Ci6.

[0087] For each system, the zeta potential, Ç, remains stable, negative with values between -30 and -40 mV.

[0088] HPLC analyses were also performed. After lyophilization, the nanoassemblies were dissociated in methanol to obtain an organic solution of molecular compounds of curcumin or compound of formula (la). This HPLC study makes it possible to quantify the molecular stability of the different compounds when they are in the form of nanoassemblies and to determine any degradation products of the compounds.

[0089] Fig. 9 shows the evolution of the concentration of curcumin or compound of Formula (la) measured by HPLC over time, normalized with respect to the theoretical concentration of the molecular compound initially injected.

[0090] For curcumin, at T = 0, only 60% of the theoretical injected concentration is measured. Since curcumin is not sufficiently hydrophobic, it does not allow the formation of stable nano-assemblies. Immediately after nanoprecipitation, a significant portion of the organic compound precipitates at the bottom of the pillbox. The sample is therefore neither homogeneous nor representative of the entire sample. Significant degradation is observed after 30 days of incubation; only 10% of the curcumin is quantified. Finally, after 90 days of incubation, the remaining amount of curcumin is below the detection limit.

[0091] The compounds of Formula (la) with R = C8, Ci2, and CM have 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 difference between the measured concentration and the initially injected concentration may be due to product losses during lyophilization. The concentration of compounds of Formula (la) with R = C8, Ci2, and CM remains relatively stable over the first two days of analysis (5% degradation for the compound of Formula (la) with R = C8 and 19% degradation for the compounds of Formula (la) with R = C[2 and CM). After 90 days of incubation, a low concentration of the compound of Formula (la) with R = C[2 and CM is observed (less than 5%). For the compound of Formula (la) with R = C8, at 90 days of incubation, 27% of the product is still detected.

[0092] For the compound of formula (la) with R = Ci6, only 75% of the compound is quantified at T = 0. A constant and rapid decrease in concentration is observed during the first two days of incubation (61% at day 1 and 48% at day 2). This decrease is still present over longer periods, but to a lesser extent (28% of the compound quantified at day 7 and 16% at day 30). After 90 days of incubation, as with the compounds of formula (la) with R = C[2 and CM], less than 5% of the compound is detected.

[0093] For each of the systems, none of the curcumin degradation products (vanillin, ferulic acid, and feruloylmethane) could be detected. For the compounds of Formula (la), no trace of curcumin could be detected.

[0094] This degradation monitoring study of the compounds of Formula (la) and curcumin when in the form of organic nanoassemblies has highlighted several points. First, the study has confirmed once again that curcumin does not form stable nanoassemblies and that the molecular compound decomposes rapidly. Second, differences in behavior were observed depending on the length of the carbon chain of the compound of Formula (la).

[0095] The compounds of formula (la) with R = Ci2 and CM exhibit very similar behavior with good short-term stability. The compound of formula (la) with R = C8 shows the greatest chemical stability.

[0096] TEM measurements showed the low colloidal stability of the nanoassemblies with R = C8 due to relatively rapid Ostwald growth, but its stability remains satisfactory for rapid use of the nanoassembly. The diameter of the nanoassemblies with R = Ci2, Ci4, and Ci6 varies little over time, and there is no change in the morphology of the organic nanoassemblies. For all systems, the zeta potential, Ç, remains constant over time, with values ​​between -30 and -40 mV. Finally, all the analyses performed show that curcumin alone cannot form nanoassemblies. HPLC measurements also demonstrated that the structural modifications made via Knoevenagel condensation increased the molecular stability of the derivatives of Formula (la) compared to curcumin.

[0097] Example 3: Synthesis of super-paramagnetic nanoparticles Spherical nanoparticles

[0098] 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 mixture was heated to 120 °C under magnetic stirring (1200 rpm) and three argon-vacuum cycles were performed. The mixture was maintained at this temperature under partial vacuum for 2 hours. The reaction was then placed under argon and heated to 220 °C for 30 minutes, then refluxed for 40 minutes. After returning to 120 °C, the nanoparticles formed were oxidized under air for 1.5 hours. After returning to room temperature, excess ethanol was added to precipitate the nanoparticles, which were then centrifuged (10,000 rpm, 10 minutes). The pellet is resuspended in a 0.02% oleic acid solution in hexane and precipitated again in ethanol, then centrifuged (10,000 rpm, 10 min). This process is then repeated once. Finally, the nanoparticles are stored in the THF.

[0099] Depending on the quality of the vacuum, spherical nanoparticles of 6.2 nm (partial vacuum = 3.7 mmbar) or 12.1 nm (partial vacuum = 0.9 mmbar) are obtained.

[0100] Fig. 10 shows a TEM image of 12.1 nm spherical nanoparticles. Cubic nanoparticles

[0101] In a 50 mL three-necked flask fitted 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 in a mixture of 23 mL of dibenzyl ether and 2 mL of squalane. The mixture is heated to 65 °C under magnetic stirring (1200 rpm) and three 20-minute argon vacuum cycles are performed. The mixture is then held at this temperature under partial vacuum (0.9 mm bar) for 1 hour. The temperature is then increased to 220 °C under argon with a temperature ramp of 3 °C / min. The mixture is heated at 220 °C for 2.5 hours. Finally, the mixture is heated to reflux and held at reflux for 30 minutes. 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 resuspended in a 0.02% oleic acid solution in hexane and precipitated again in ethanol, then centrifuged (10,000 rpm, 10 min). The operation is then repeated once. Finally, the nanoparticles are stored in the THF.

[0102] Figure 11 shows a TEM image of 22.1 nm cubic nanoparticles. Rod-shaped nanoparticles

[0103] In a 50 mL round-bottom flask, 0.605 g of hexadecylamine and 1.795 g of oleic acid are introduced into 8 mL of 1-octanol. The solution is heated to 55°C for 30 minutes. Afterward, the solution is allowed to cool to room temperature. 2 mL of iron pentacarbonyl is then added. The mixture is stirred for 1 hour at room temperature. The solution is then transferred to an autoclave with a Teflon protective insert and heated in an oven at 200°C for 6 hours. Once the solution has cooled to room temperature, a large quantity of ethanol is added to precipitate the nanorods. The solution is then centrifuged for 10 min at 10,000 rpm. This operation is then repeated once. Finally, the nanoparticles are stored in the THF.

[0104] Fig. 12 shows a TEM image of the rod-shaped nanoparticles obtained with a length of 49 nm and a thickness of 6.8 nm.

[0105] Example 4: Synthesis of a magnetic hybrid nano-assembly containing a super-paramagnetic nanoparticle

[0106] An organic solution of 0.825 mM in compound of Formula (Ib) and 0.05% in super-paramagnetic particles (by mass of iron) of 6.2 nm obtained in Example 3 is prepared in 1 mL of THF.

[0107] An aqueous solution of 0.5 mg / mL of commercial poly-L-lysine (Alamanda Polymers, Inc., Huntsville, AL, USA) is prepared in 1 mL of PBS.

[0108] We proceed as in Example 2 by placing the organic solution in syringe 4 and the aqueous solution in the second syringe 5.

[0109] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into the mixer 1.

[0110] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of an aqueous solution of 0.19 mg / mL in poly-L-lysine.

[0111] Fig. 13 shows a TEM image of the nano-assembly obtained with a diameter of 60.9 nm and a potential Ç = 34 mV. Heating properties:

[0112] The heating properties of the nanoassemblies were measured by means of 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 an SAR value of 6.4 W / g. Mass concentration in water

[0113] The magnetic hybrid nano-assemblies according to the present invention can have a molar concentration of compound of formula (Ib) of 0.32 mmol / L.

[0114] Example 5: Synthesis of a hybrid magnetic nanoassembly containing a super-paramagnetic nanoparticle

[0115] An organic solution of 0.55 mM in compound of Formula (la) with R = CM and 0.3% in super-paramagnetic particles (by mass of iron) of 6.2 nm obtained in Example 3 is prepared in 1 mL of THF.

[0116] An aqueous solution of 10 mg / mL of commercial poly-L-lysine (Alamanda Polymers, Inc., Huntsville, AL, USA) is prepared in 1 mL of PBS.

[0117] The procedure is as in Example 2, placing the organic solution in syringe 4 and the aqueous solution in the second syringe 5.

[0118] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into the mixer 1.

[0119] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of water.

[0120] Fig. 14 shows a TEM image of a nano-assembly obtained with a diameter of d = 107.6 nm and a zeta potential of Ç = +36 mV. Mass concentration in water

[0121] The magnetic hybrid nano-assemblies according to the present invention have a molar concentration of 0.21 mmol / L.

[0122] Example 6: Functionalization of a magnetic hybrid nanoassembly

[0123] A magnetic hybrid nanoassembly was synthesized as in Example 4, except that the stabilizing polymer used is poly-L-lysine. operationalized by commercial propargyl-dPEGl-amide (Alamanda Polymers, Inc., Huntsville, AL, USA) ([Fig. 15]).

[0124] An organic solution of 0.55 mM in compound of Formula (Ib) and 0.3% in super-paramagnetic particles (by mass of iron) obtained in Example 3 with a diameter of 6.2 nm is prepared in 1 mL of THF.

[0125] An aqueous solution of 5 mg / mL of poly-L-lysine functionalized with propargyl-dPEGl-amide is prepared in 1 mL of PBS.

[0126] We proceed as in Example 2 by placing the organic solution in syringe 4 and the aqueous solution in the second syringe 5.

[0127] The two syringe pistons are pushed simultaneously at a speed of 5 mm / s to bring the solutions into the mixer 1.

[0128] At the mixer outlet, the nano-assembly solution is directly diluted in 24 mL of water.

[0129] Fig. 16 shows a TEM image of a nano-assembly obtained with a diameter d = 74 nm.

[0130] The surface of the magnetic hybrid nano-assembly is then functionalized with a molecule of interest according to the "click" chemical reaction.

[0131] Fig. 17 shows the reaction scheme of the surface functionalization reaction of the magnetic hybrid nanoassembly.

[0132] Starting with a solution of magnetic hybrid nanoassemblies functionalized with a poly-L-lysine polymer bearing a propargyl group, 100 pL of a 0.009 mg / mL sodium ascorbate solution, 100 pL of a 0.011 mg / mL copper sulfate solution, and 100 pL of a 0.25 mg / mL sulfocyanine 5-azide solution (Jena Bioscience GmbH, Jena, Germany) are added. The mixture is stirred at room temperature for 24 hours. Three successive washes by magnetic decantation are performed to remove excess reagents.

[0133] Fig. 18 shows a TEM image of a nano-assembly obtained after functionalization of its surface with a fluorophore of diameter d = 72.5 nm and of zeta potential Ç = 29 mV.

Claims

1.

2.

3.

4.

5.

6. Claims - Nano-assembly formed by self-assembly of a compound chosen from the compounds of Formula (Ia): [Chem. 4] R representing an alkyl group; and the compound of Formula (Ib): [Chem. 5] (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 chosen from butyl, pentyl, octyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl and octadecyl. - Nano-assembly according to 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 HOnm. - 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.

7. - Nano-assembly according to one of claims 1 to 6, characterized in that said nano-assembly is coated with a stabilizing polymer.

8. - Nano-assembly according to claim 7, characterized in that the stabilizing polymer is a cationic polyelectrolyte.

9. - Nano-assembly according to claim 8, characterized in that the cationic polyelectrolyte is a polymer based on poly-L-lysine.

10. - Nano-assembly according to one of claims 7 to 9, characterized in that the stabilizing polymer is functionalized by 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 by a function complementary to the function of the functionalized stabilizing polymer.

11. - Nano-assembly according to 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.

12. - Nano-assembly according to claim 1 to 3 and 7 to 11, characterized in that the at least one super-paramagnetic nanoparticle is composed of iron oxide.

13. - Nano-assembly according to one of claims 1 to 3 and 7 to 12, characterized in that the at least one superparamagnetic nanoparticle has: • a spherical shape with a diameter of between 6 and 20 nm, as measured by transmission electron microscopy; or • a cubic shape with an edge of between 10 and 70 nm, as measured by transmission electron microscopy; or • a 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.

14. - Nano-assembly according to 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, between 40 nm and 250 nm.

15. - 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.

16. - Method for preparing a nano-assembly according to 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 nano-particle, and in a second syringe, water or a saline phosphate 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 receptacle bottle containing water, and where appropriate, a stabilizing polymer.

17. - Pharmaceutical composition comprising one or more nanoassemblies according to one of claims 1 to 15.

18. - Nano-assembly according to one of claims 1 to 15 for its use in the treatment of inflammatory diseases

19. - Nano-assembly according to one of claims 1 to 15 for its use in the treatment of cancer.

20. - Nano-assembly according to one of claims 1 to 3 and 7 to 15 for its use in magnetic liquid hyperthermia.

21. - Contrast agent for magnetic resonance imaging characterized in that it comprises nano-assemblies according to one of claims 1 to 3 and 7 to 15.

22. - Agent for magnetic particle imaging characterized in that it comprises nano-assemblies according to one of claims 1 to 3 and 7 to 15.

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