Biocompatible oil-based magnetic fluid and manufacturing method

Abstract

A biocompatible oil-based magnetic fluid and a manufacturing method are provided. [Solution] The present invention relates to a biocompatible oil-based ferrofluid comprising iron oxide-based magnetic nanoparticles and an oil phase containing at least one fatty acid ester, wherein the magnetic nanoparticles are surface-functionalized with one or more phospholipid molecules. In particular, the present invention relates to a biocompatible oil-based ferrofluid comprising iron oxide-based magnetic nanoparticles and an oil phase containing at least one fatty acid ester, wherein the iron oxide-based magnetic nanoparticles form a colloidal dispersion in the oil phase at temperatures ranging from 20 to 80°C, and the magnetic nanoparticles are surface-functionalized with one or more phospholipid molecules that ensure a surface coverage of the iron oxide-based magnetic nanoparticles, particularly such that the fatty acid esters present in the oil phase are accessible to the surface of the iron oxide-based magnetic nanoparticles. The present invention also relates to a method for producing such a biocompatible oil-based ferrofluid and its use as a contrast agent for nuclear magnetic resonance imaging or in the context of hyperthermia treatment for cancer. Finally, the present invention relates to a nanoemulsion comprising such a biocompatible oil-based ferrofluid.

Description

[Technical field]

[0001] The present invention relates to a biocompatible oil-based magnetic fluid comprising magnetic iron oxide-based magnetic nanoparticles dispersed in an oil phase comprising at least one fatty acid ester, the nanoparticles being surface-functionalized with one or more phospholipids. The present invention also relates to a method for producing such a biocompatible oil-based magnetic fluid and its use as a contrast agent or in cancer treatment by magnetically induced hyperthermia. Finally, the present invention relates to a nanoemulsion comprising such a biocompatible oil-based magnetic fluid. [Background technology]

[0002] A ferrofluid is composed of a kinetically stable dispersion of superparamagnetic nanoparticles in a carrier liquid, which may be an aqueous or organic solvent, or an oil. A ferrofluid becomes magnetic when an external static magnetic field is applied. A ferrofluid can move or deform under the action of a magnetic field. In the context of use in magnetically induced hyperthermia, the magnetic particles are used as a heating medium under the action of an external alternating magnetic field.

[0003] Depending on the use of these magnetic fluids, the magnetic particles are dispersed in an aqueous medium, an oily medium or in an emulsion.

[0004] At present, aqueous magnetic fluids are particularly known for use as contrast agents in MRI and in the therapy of solid tumors by magnetically induced hyperthermia. For example, in the context of NanoTherm therapy developed by MagForce AG®, iron oxide nanoparticles in aqueous suspension are injected into tumors and masses in the target tissue. The nanoparticles release heat under the action of an applied external magnetic field, which induces the destruction of tumor cells. This treatment can be applied as an adjunct to chemotherapy or radiation therapy.

[0005] An effective magnetic fluid should be able to destroy large tumor volumes with short magnetic induction treatment times and low concentrations of nanoparticles. However, the heat generation performance of aqueous dispersion media is not as good as that of oil-based media (whether or not they are contained in emulsions). In fact, the heat capacity of oil is generally much lower than that of water, and its thermal conductivity is also low. Therefore, to improve the effectiveness of magnetic fluid compositions in magnetic hyperthermia treatment, it is preferable to use an oil-based magnetic fluid composition or at least a magnetic particle dispersion in the oil phase of an emulsion.

[0006] Furthermore, systemic injection of aqueous ferrofluids in patients raises the issue of the amount of magnetic particles present at the target, which must be sufficient to compensate for heat loss in the living environment, an aqueous environment thermostated at 37°C. Improving the efficacy of such treatments by systemic administration requires injection of large amounts of injected nanoparticles, far exceeding the recommended dose (approximately 0.8 mg Fe / kg for iron oxide contrast agents), with favorable pharmacokinetics and sufficient accumulation in the pathological area resulting from effective targeting.

[0007] Ferrofluid nanoemulsions or ferrofluid oil suspensions are primarily known for their use as MRI contrast agents and for cancer treatment by magnetic hyperthermia.

[0008] An example of a ferrofluid in emulsion used as a contrast agent is reported in application FR3001154, which describes an oil-in-water nanoemulsion comprising an aqueous phase, a lipid phase comprising oil, C6-C18 saturated fatty acid glycerides, and iron compound-based magnetic particles coated with one or more C8-C22 fatty acids, and a surfactant comprising at least one amphiphilic lipid and at least one targeting ligand. Such nanoemulsions contain additives, for example surfactants, necessary for the stabilization of the nanoemulsion, which may alter the biocompatibility of the ferrofluid composition. However, good biocompatibility is essential for medicinal uses.

[0009] In this context, the applicant has attempted to improve the biocompatibility of medicinal magnetic fluid compositions and their effectiveness for the intended use (heat transfer by reducing the heat dissipation rate in the living environment, in particular in the context of use in magnetically induced hyperthermia), while having chemical and colloidal stability at certain temperatures, allowing the magnetic energy of the nanoparticles to be converted into thermal energy, preferably adapted to an administration mode by injection. The magnetic fluid compositions of the present invention can be envisaged for different medical applications, in particular for the treatment of cancer by magnetic hyperthermia or as contrast agents.

[0010] To improve the effectiveness of magnetic fluids, the applicant is particularly interested in oil-based magnetic fluids, as well as magnetic fluid emulsions, particularly in view of their use for magnetic hyperthermia or as contrast agents.

[0011] Because iron oxide nanoparticles are biocompatible, Applicant sought to improve the biocompatibility of the medium these nanoparticles are dispersed in. In particular, Applicant sought to use only biocompatible additives and biocompatible liquid carriers, avoiding anything that is not biocompatible and therefore may result in toxicity to the patient.

[0012] In this context, the present invention proposes a novel biocompatible oleaginous magnetic fluid comprising iron oxide-based magnetic nanoparticles and an oil phase comprising at least one fatty acid ester, said nanoparticles being surface-functionalized with molecules of one or more phospholipids. Advantageously, in the oleaginous magnetic fluid according to the invention, the iron oxide-based magnetic nanoparticles are dispersed, preferably in the form of a colloidal dispersion, in an oil phase containing at least one fatty acid ester.

[0013] Indeed, the formation of magnetic agglomerates can lead to a decrease in the heating efficiency of the nanoparticles under inductive conditions (i.e. under the action of an alternating magnetic field), so particular care should be taken in the dispersion of these nanoparticles to prevent as much as possible the formation of agglomerates, especially large ones.

[0014] Colloidal stability is therefore a prerequisite for improving the performance of magnetic fluid compositions, whether they are used in magnetic hyperthermia, as contrast agents, or for any other therapeutic application. In general, iron oxide nanoparticles are kinetically stabilized in terms of colloids under oily conditions and in emulsions using surfactants or dispersants to prevent the formation of magnetic nanoparticle aggregates that may be detrimental to heating efficiency, especially due to magnetic dipolar interactions. Under temperature use conditions, the magnetic nanoparticles should not flocculate under the action of the applied alternating or static magnetic field, but should remain in a single phase. However, surfactants or dispersants are often poorly or not at all biocompatible. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] French Patent Application Publication No. 3001154 Summary of the Invention [Problem to be solved by the invention]

[0016] The applicant therefore explored the conditions for nanoparticle dispersibility at different temperatures for applications in hyperthermia, without the use of surfactants or dispersants (or any other non-biocompatible additives). The applicant therefore studied the surface chemistry of the metal oxide nanoparticles used. The applicant observed that a judiciously selected chemical surface functionalization, in combination with the presence of certain components in the oil phase, indeed allows a significant improvement in the colloidal stabilization of oil-based ferrofluid compositions under temperature conditions compatible with injection. [Means for solving the problem]

[0017] The present invention therefore preferably relates to a biocompatible oil-based magnetic fluid comprising iron oxide-based magnetic nanoparticles and an oil phase comprising at least one fatty acid ester, characterized in that said iron oxide-based magnetic nanoparticles form a colloidal dispersion in said oil phase from a temperature ranging from 20 to 80°C, said magnetic nanoparticles being surface functionalized with molecules of one or more phospholipids that ensure a surface coverage of the iron oxide-based magnetic nanoparticles that does not completely cover the surface of the iron oxide-based magnetic nanoparticles, in particular such that the fatty acid esters present in the oil phase are accessible to the surface of the iron oxide-based magnetic nanoparticles.

[0018] The biocompatible oil-based fluid according to the invention advantageously has one or another of the following characteristics, either alone or in combination, or even all of the following characteristics: - Oil-based ferrofluids are water-free and / or surfactant-free; - the phospholipid molecules ensure a coverage of 19-76%, preferably 29-76%, preferentially 34-50% of the surface of the iron oxide-based magnetic nanoparticles; - The surface density of functionalization (also called surface density of grafting) within the phospholipid molecules is 0.32 molecules / nm 2 ~1.22 molecules / nm 2 , preferably 0.48 molecules / nm 2 ~1.22 molecules / nm 2 , preferentially 0.56 molecules / nm 2 ~0.79 molecules / nm 2 Belongs to the range of; - the phospholipids contain at least one fatty chain, preferably two fatty chains, in particular C6 to C30 and preferably C8 to C24 or even C10 to C22, in particular C18 saturated or mono- or polyunsaturated, branched or preferably linear hydrocarbon chains; - the oil phase comprises at least 70% by weight of fatty acid esters relative to the total weight of the oil phase, preferably the oil phase comprises between 80% and 95% by weight of fatty acid esters relative to the total weight of the oil phase; the fatty acid esters of the oil phase are selected from C6-C12, preferably C6-C10, saturated fatty acid triglycerides and C6-C12, preferably C6-C10, saturated fatty acid propylene glycols, used individually or in mixtures; - the content of magnetic nanoparticles is in the range of 0.01% to 50% by weight, preferably 0.1% to 10% by weight, relative to the total weight of the oil-based magnetic fluid; if the content of magnetic nanoparticles is in question, the content includes only iron oxide-based magnetic nanoparticles and does not include functionalization; - the magnetic nanoparticles are in the form of ellipsoids, polyhedrons, e.g. nanocubes, bipyramids or nanostars, wafers, nanorods, nanodisks or nanoflowers; - Phospholipids are -O(O)P(OH)O - has a polar head and is preferably selected from salts of 1,2-dioleoyl-sn-glycero-3-phosphatidic acid and 1,2-distearoyl-sn-glycero-3-phosphatidic acid; The biocompatible oleaginous ferrofluid further comprises a lipophilic active ingredient, in particular chosen from cancer treatment drugs, such as paclitaxel, docetaxel or carmustine.

[0019] Functionalized iron oxide-based magnetic nanoparticles, such as those defined in the context of the present invention, are also an integral part of the present invention, regardless of their embodiment variants.

[0020] The present invention also relates to a method for producing the biocompatible oleaginous magnetic fluid according to the present invention.

[0021] The method for producing a biocompatible oil-based magnetic fluid according to the invention allows the dispersion of magnetic nanoparticles without the use of additives that may be detrimental to the biocompatibility of the oil-based magnetic fluid. Advantageously, the method thus excludes the use of any compounds that may induce toxicity to the patient, except for the pharmaceutical activity associated with the presence of drugs for cancer treatment or induced treatment that seeks to destroy cancer cells. Preferably, the fluids and solvents used are biocompatible and no non-biocompatible additives are used. Advantageously, no surfactants or dispersants are used in the method for producing an oil-based magnetic fluid according to the invention.

[0022] To obtain an oil-based magnetic fluid containing magnetic nanoparticles in the form of a colloidal suspension, free of surfactants and dispersants, at a temperature compatible with injection, the inventors have developed a specific method involving the formation of a solvation layer of the magnetic nanoparticles.

[0023] The applicant has developed a method for producing biocompatible oil-based ferrofluids that allows optimizing and controlling the degree of functionalization of the magnetic nanoparticles with phospholipid molecules. Indeed, the applicant observed during the study that the choice of phospholipid molecules and the degree of functionalization influenced the stability and efficiency of the biocompatible oil-based ferrofluids.

[0024] Thus, the method for producing a biocompatible oil-based magnetic fluid according to the invention comprises the following successive steps: a- Preparing an aqueous dispersion of iron oxide-based magnetic nanoparticles in an aqueous solvent, which may be water or a water / water-miscible solvent mixture, b- Removal of the aqueous solvent from the aqueous dispersion of magnetic nanoparticles; c- Obtaining a colloidal sol of magnetic nanoparticles by addition of a solvent or a mixture of volatile organic solvents S2, d- surface functionalization of said magnetic nanoparticles of colloidal sol with molecules of at least one phospholipid; e- Removing said volatile organic solvent S2 and dispersing the functionalized magnetic nanoparticles in an oil phase comprising at least one fatty acid ester.

[0025] The method for producing a biocompatible magnetic fluid according to the invention advantageously further comprises a step c2 of adding an acid after step c and before step d. This step makes it possible to increase the affinity of the polar heads of the phospholipid molecules to the surface of the magnetic nanoparticles in terms of a more efficient functionalization.

[0026] The present invention also relates to a medicine, in particular a medicine for the treatment of cancer, comprising a biocompatible oleaginous magnetic fluid according to the invention or a biocompatible oleaginous magnetic fluid obtainable according to the method for producing a biocompatible oleaginous magnetic fluid according to the invention.

[0027] The present invention also relates to a biocompatible oil-based magnetic fluid according to the present invention or a biocompatible oil-based magnetic fluid obtainable according to the method for producing a biocompatible oil-based magnetic fluid according to the present invention, for its use during cancer treatment by magnetic induced hyperthermia.The present invention also relates to a therapeutic method for cancer treatment by magnetic hyperthermia, comprising intratumoral injection of a biocompatible oil-based magnetic fluid according to the present invention or a biocompatible oil-based magnetic fluid obtainable according to the method for producing the same according to the present invention, followed by application of an external alternating magnetic field.

[0028] The present invention also relates to an oil-in-water nanoemulsion comprising 10% to 30% by weight of the biocompatible oil-based magnetic fluid according to the invention or of the biocompatible oil-based magnetic fluid obtained according to the manufacturing method according to the invention, an aqueous phase and at least one surfactant. Such a nanoemulsion may also contain a dispersing agent and / or a targeting ligand. Preferably, the nanoemulsion is biocompatible.

[0029] The present invention also relates to the preparation of a nanoemulsion according to the invention, said method comprising the following successive steps: i- providing a biocompatible oil-based magnetic fluid according to the invention or a biocompatible oil-based magnetic fluid obtained according to the method for producing a biocompatible oil-based magnetic fluid according to the invention, ii- providing an aqueous phase comprising at least one surfactant, and iii- Mixing the aqueous phase with the biocompatible oil-based ferrofluid to form a nanoemulsion.

[0030] The nanoemulsions according to the invention or the nanoemulsions obtainable according to the method according to the invention can be used for systemic administration for cancer treatment. Thus, the present invention also relates to a medicament, in particular a medicament for cancer treatment comprising a nanoemulsion according to the invention or a nanoemulsion obtainable according to the method for producing a nanoemulsion according to the invention. The present invention also relates to a nanoemulsion according to the invention or a nanoemulsion obtainable according to the method for producing a nanoemulsion according to the invention, for use during cancer treatment by magnetic hyperthermia. The present invention also relates to a therapeutic method for cancer treatment by magnetic hyperthermia, comprising the systemic injection of a nanoemulsion according to the invention or a nanoemulsion obtainable according to the method for producing a nanoemulsion according to the invention, followed by the application of a magnetic field.

[0031] Finally, the present invention relates to a contrast agent, in particular a contrast agent for magnetic resonance imaging (MRI), which comprises a biocompatible oleaginous ferrofluid according to the invention or a nanoemulsion comprising an oleaginous ferrofluid according to the invention.

[0032] Various other features will become apparent from the description that follows with reference to the accompanying drawings, which illustrate, by way of non-limiting examples, aspects of embodiments of the subject matter of the present invention. [Brief description of the drawings]

[0033] [Figure 1] Schematic representation of a) a magnetic fluid according to the invention and b) a nanoemulsion according to the invention. [Diagram 2] Diffuse reflectance infrared spectroscopy (DRIFT) analysis of non-functionalized magnetic nanoparticles and magnetic nanoparticles functionalized with DOPA is shown. [Diagram 3] FIG. 1 shows the chemisorption yield of DOPA determined by TGA measurements for different amounts of participating ellipsoidal nanoparticles FF1 in the presence of acid. [Figure 4] Thermograms of functionalized nanoparticles corresponding to 2000% of the amount required to form a monolayer of DOPS at 7 and 30 minutes of reaction are shown. [Figure 5A] Kinetic temperature profile of a dispersion of ellipsoidal nanoparticles FF1 functionalized with DOPA with a theoretical phospholipid molecular coverage density of 0.81 molecules / nm2 and then dispersed in water or in Miglyol M840® subjected to magnetic induction (field-frequency pair: 755 kHz, 10.2 kA / m) (adjusted temperature T0=37°C and CFe2O3=5 g / L, where CFe2O3 is the mass concentration of Fe2O3). The inset of Fig. 5A zooms in on the temperature profile over the range [0-10 s]. [Figure 5B] Figure 1 shows the kinetic temperature profile of a dispersion of FF2 in nanoflower form, functionalized with DOPA with a theoretical phospholipid molecular coverage density of 1.29 molecules / nm2 and then dispersed in water or Miglyol M840®, subjected to magnetic induction (magnetic field-frequency pair: 473.5 kHz, 13.36 kA / m) (adjusted temperature T0=37°C and CFe2O3=5 g / L (where CFe2O3 is the mass concentration of Fe2O3)). [Figure 5C] Figure 1 shows the kinetic temperature profile of a dispersion of FF3 in nanoflower form, functionalized with DOPA with a theoretical phospholipid molecular coverage density of 1.10 molecules / nm2 and then dispersed in water or Miglyol M840®, subjected to magnetic induction (magnetic field-frequency pair: 473.5 kHz, 13.36 kA / m) (adjusted temperature T0=37°C and CFe2O3=5 g / L, where CFe2O3 is the mass concentration of Fe2O3). [Figure 6A]Figure 2 shows the kinetic temperature profile of a dispersion of nanoparticles of nanoflower morphology FF2 dispersed in water or in Miglyol M840® after functionalization with DOPA at a nominal surface coverage density of 1.29 molecules nm2 of phospholipid molecules, the dispersion being subjected to two cycles of magnetic induction (field / frequency pair: 473.5 kHz, nanoparticle dispersion FF2 at 13.36 kA / m, regulated temperature of T0=37°C and CFe2O3=5 g / L). [Figure 6B] Figure 2 shows the kinetic temperature profile of a dispersion of nanoparticles of nanoflower morphology FF2 dispersed in water or in Miglyol M840® after functionalization with DOPA at a nominal surface coverage density of 1.29 molecules nm2 of phospholipid molecules, the dispersion being subjected to two cycles of magnetic induction (magnetic field / frequency pair: 344.5 kHz, nanoparticle dispersion FF2 at 16.23 kA / m, regulated temperature of T0=37°C and CFe2O3=5 g / L). [Figure 6C] Figure 2 shows the kinetic temperature profile of a dispersion of nanoparticles of nanoflower morphology FF3 dispersed in water or Miglyol M840® after being functionalized with DOPA with a nominal surface coverage density of 1.10 molecules nm2 of phospholipid molecules, the dispersion being subjected to two cycles of magnetic induction (field / frequency pair: 473.5 kHz, nanoparticle dispersion FF3 at 13.36 kA / m, regulated temperature of T0=37°C and CFe2O3=5 g / L). [Figure 7A] Kinetic temperature profile and values ​​of the heat power dissipated by the nanoparticles (expressed by the specific absorption rate (SAR)) of a nanoparticle dispersion of nanoflower morphology FF3 in water by volume of dispersion medium after the dispersion was subjected to a magnetic induction of 473.5 kHz, 13.36 kA / m magnetic field / frequency pair, an adjusted temperature of T0 = 37 °C and a mass concentration of iron oxide CFe2O3 = 5 g / L and functionalized with DOPA to a nominal surface coverage density of 1.10 molecules / nm2 of phospholipid molecules. [Figure 7B]Figure 1 shows the kinetic temperature profile of a nanoparticle dispersion of nanoflower morphology FF3 by volume of dispersion medium in Miglyol M840® and the value of the heat power dissipated by the nanoparticles (expressed by the Specific Absorption Rate (SAR)) after the dispersion was subjected to a magnetic induction of 473.5 kHz, 13.36 kA / m magnetic field / frequency pair, an adjusted temperature of T0=37°C and a mass concentration of iron oxide CFe2O3=5 g / L and functionalized with DOPA to a nominal surface coverage density of 1.10 molecules / nm2 of phospholipid molecules. [Figure 7C] Kinetic profiles corresponding to 1 μL of nanoparticle dispersion FF3 at 5 g / L in water and Miglyol M840® are shown, attached directly to the tip of a magnetic induction temperature sensor (referred to as MFA: 473.5 kHz; 13.36 kA / m, T0=25° C., ambient room temperature). [Figure 8] Figure 1 shows the kinetic temperature profile of nanoparticles FF3 dispersed in Miglyol M840® for a volume of 1 μL deposited on the tip of a temperature sensor (T0=25° C., ambient room temperature) with a mass concentration of 300 g / L of iron oxide Fe2O3 after functionalization with DOPA to a nominal surface coverage density of 1.10 molecules / nm2 of phospholipid molecules. [Figure 9] The kinetic temperature profile of nanoemulsions (1) and (4) (CFe2O3=12 g / L) containing ellipsoidal nanoparticles FF1 dispersed in Miglyol M840 emulsified in the aqueous phase under induction at 473.5 KHz, 13.36 kA / m (regulating temperature T0=37°C) for 20 seconds. [Figure 10A] FIG. 10A shows bioluminescence images generated before (T0) and 24 hours (T24) after induction treatment (473.5 KHz, 13.36 kA / m) following intratumoral injection of the oil-based ferrofluid described in Example 6 (two injections of 1 μL of 300 g / L oil-based ferrofluid into the same injection site) in a mouse subcutaneous tumor. [Figure 10B]FIG. 10B shows fluorescence reflectance imaging (FRI) and bioluminescence imaging (BLI) images of ex vivo tumors more than 24 hours after induction treatment. [Figure 11A] FIG. 11A shows bioluminescence images generated before (T0) and 24 hours (T24) after induction treatment (473.5 KHz, 13.36 kA / m) following intratumoral injection of the oil-based magnetic fluid described in Example 6 (three injections of 1 μL of 300 g / L oil-based magnetic fluid at different locations on the tumor) in a mouse subcutaneous tumor. [Figure 11B] FIG. 11B shows fluorescence (FRI) and bioluminescence (BLI) images of ex vivo tumors more than 24 hours after induction treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Biocompatible oil-based magnetic fluid The present invention relates to a biocompatible oleaginous magnetic fluid comprising functionalized magnetic nanoparticles in suspension in an oil phase comprising at least one fatty acid ester, as illustrated in FIG.

[0035] The functionalized magnetic nanoparticles used in the context of the present invention are iron oxide-based nanoparticles that are surface-functionalized with at least one molecule of phospholipid. Iron oxide has the advantage of being biocompatible. Advantageously, the nanoparticles used in the context of the present invention do not contain any metal elements that may be toxic, such as cobalt or manganese.

[0036] In the context of the present invention, "nanoparticles" refers to particles with a nanometer elementary size, i.e. particles with an average elementary size greater than 1 nm and less than 100 nm, which by numbers preferably exhibit a monomodal size distribution with a standard deviation of less than 30% relative to the mean value. Elementary size refers to the largest dimension of the nanoparticles. In the context of the present invention, the elementary size of the functionalized magnetic particles corresponds to the dimensions of iron oxide-based nanoparticles, thus not including functionalization with phospholipid molecules.

[0037] Preferably, in the context of the present invention, the magnetic nanoparticles have an average elementary size of less than 30 nm, preferably less than 25 nm. Preferably, the magnetic nanoparticles used in the context of the present invention have an average elementary size of more than 3 nm, preferably more than 5 nm. Preferably, the functionalized magnetic nanoparticles have an average elementary size belonging to the range of 5 to 34 nm, preferably 7 to 24 nm.

[0038] "Average elementary size" refers to the average size of the inorganic nanoparticles without phospholipid coating and without aggregation. The average elementary size corresponds to the arithmetic mean of the elementary sizes measured in a set of nanoparticles, in particular in a set of 200 nanoparticles. The elementary size of the nanoparticles can be measured by transmission electron microscopy (TEM) after removal of the oil phase.

[0039] In the oil-based magnetic fluids according to the invention, the magnetic nanoparticles based on functionalized iron oxide according to the invention form a colloidal dispersion in the oil phase used from a temperature belonging to the range of 20-80°C, preferably from a temperature equal to 60°C or 70°C, preferentially from a temperature equal to 37°C, and even more preferably from a temperature equal to 20°C or 25°C. Conventionally, a colloidal dispersion means solid particles dispersed in a liquid phase that are stable for at least 24 hours, i.e. do not precipitate or aggregate. The colloidal stability of magnetic magnetic fluid dispersions as a function of the temperature of the medium can be qualitatively characterized by their clarity and translucency, in contrast to suspensions of aggregates or flocculates, which have an opaque, turbid or light-blocking nature. A quantitative assessment can be made by measuring the transmittance at 800 nm. For example, Fe nanoparticles in the size range of 5-15 nm are 2 O 3 Nanoparticle dispersions with a mass concentration of 0.2 g / L in -γ are considered unstable if such dispersions lose at least 30% of their transmittance at 800 nm compared to the transmittance of a stable dispersion of the same concentration. Such transmittance measurements can be performed using a VARIAN Cary500 spectrophotometer equipped with a temperature control device.

[0040] The colloidal stability as a function of the surface coverage of phospholipid molecules can also be determined by dynamic light scattering (DLS) measurements, which make it possible to obtain the hydrodynamic size of the nanoparticles by considering the solvated sphere of the nanoparticles. Thus, for 10 nm nanoparticles, hydrodynamic diameters of 40 nm to 50 nm can be measured under optimal conditions, around 100 nm under conditions at the limit of the stability domain, and much larger than 100 nm if the dispersion is not stable.

[0041] Other techniques, such as X-ray diffraction (XRD), can be used to obtain information about the nanoparticles, which makes it possible to obtain the size of the crystalline coherence domains according to the protocol described in the examples.

[0042] Nanoparticles used in the context of the present invention may be of various shapes, such as ellipsoids, polyhedrons, such as nanocubes, bipyramids or nanostars, wafers, nanorods, nanodisks or nanoflowers, etc. The so-called nanoflower nanoparticles have a typical morphology in the form of a flower resulting from the aggregation of epitaxial nanocrystals forming multiple buds.

[0043] In the context of the present invention, "ellipsoid" means spherical or quasi-spherical, ie having a sphericity index (ie the ratio between its largest diameter and its smallest diameter) of less than 1.2.

[0044] According to a first preferred embodiment of the invention, the functionalized magnetic nanoparticles are of ellipsoidal morphology, in particular spherical or quasi-spherical, then according to this embodiment, the magnetic nanoparticles have an average elementary size belonging to the range of 5 to 20 nm, preferably 7 to 15 nm.

[0045] According to a second preferred embodiment of the invention, the functionalized nanoparticles used in the context of the present invention are of nanoflower morphology, preferably monocrystalline or quasi-monocrystalline. Then, according to this embodiment, the magnetic nanoparticles have an average elementary size belonging to the range of 10 to 34 nm, preferably 10 to 24 nm.

[0046] In the context of the present invention, the nanoparticles used are iron oxide-based nanoparticles whose surface is functionalized with at least one molecule of phospholipid. According to a particular embodiment, the magnetic nanoparticles are nanoparticles composed solely of iron oxide whose surface is functionalized with phospholipid molecules.

[0047] In the context of the present invention, "iron oxide-based" nanoparticles means nanoparticles that consist essentially of iron oxide, or even nanoparticles that consist exclusively of iron oxide.

[0048] Iron oxide particles are ferrimagnetic nanoparticles, usually magnetite particles (Fe 3 O 4 ) or maghemite grains (γ-Fe 2 O 3 ) or the formula [Fe 3+ ] Td [Fe 3+ 1+2z / 3 Fe 2+ 1-z V z / 3 ] Oh O 4 (Td and Oh represent the tetrahedral and octahedral sites of the spinel, respectively, and V represents the cation vacancy), where z varies from 0 to 1. Preferably, the iron oxide nanoparticles are maghemite nanoparticles, i.e., nanoparticles of the formula [Fe 3+ ] Td [Fe 3+ 1+2z / 3 Fe 2+ 1-z V z / 3 ] Oh O 4 (z=1) cubic sesquioxide.

[0049] In the context of the present invention, the magnetic nanoparticles of the biocompatible oil-based magnetic fluid are surface-functionalized with one or more phospholipids, i.e. the same or different phospholipid molecules are attached to the surface of each magnetic nanoparticle by chemical bonds. These chemical bonds are preferably coordination or complexation bonds established by the polar heads of the phospholipid molecules and the surface iron sites. In particular, the phospholipid heads, among others -O(O)P(OH)O - The type is attached by one or more coordinate bonds to iron oxide-based nanoparticles.

[0050] "Phospholipid" conventionally refers to a molecule composed of a phosphate-type polar head and one or more fatty chains. Preferably, the phospholipid molecules are attached to the surface of the nanoparticles via these polar heads.

[0051] Preferably, the magnetic nanoparticles of the biocompatible oil-based magnetic fluid are surface-functionalized with a single type of phospholipid, although it is not preferred, it is also conceivable to functionalize the magnetic nanoparticles with two or more different nanoparticles.

[0052] In the context of the present invention, a phospholipid molecule contains at least one fatty chain, preferably at least two fatty chains, in particular two fatty chains. These fatty chains are saturated or unsaturated, linear or branched hydrocarbon chains, usually containing 6 to 30 carbon atoms. These fatty chains may be different or, preferably, identical for each phospholipid used.

[0053] Preferably, the fatty chains of the phospholipid molecules are linear. Without wishing to be bound by any theory, the applicant is of the opinion that these fatty chains of the phospholipid molecules are intercalated by the fatty chains of the fatty acid esters of the oil phase, thus allowing the creation of solvation spheres. The lack of branching on the fatty chains of the phospholipid molecules facilitates this intercalation.

[0054] The fatty chains of the phospholipid molecules may be saturated or mono- or polyunsaturated. Advantageously, the fatty chains of the phospholipid molecules are monounsaturated.

[0055] The fatty chain of the phospholipid molecule advantageously contains between 6 and 30 carbon atoms, preferably between 8 and 24 carbon atoms, preferably between 10 and 22 carbon atoms and, in particular, 18 carbon atoms.

[0056] In the context of the present invention, the functionalization of the magnetic particles preferably occurs via the polar heads of the phospholipid molecules: the chemical bond between the iron oxide sites present on the surface of the magnetic nanoparticles and the polar heads of the phospholipid molecules allows a strong anchoring and a reliable functionalization. This surface functionalization thus confers good chemical stability and is not degraded under physiological and storage conditions.

[0057] The polar heads of the phospholipid molecules used for surface functionalization of the magnetic particles of the oil-based magnetic fluid according to the invention are phosphate fragments, preferably with low steric hindrance. The applicant has indeed observed during his research that low steric hindrance allows better control of the speed and rate of functionalization. Examples of polar heads of phospholipid molecules that can be bound to the surface of the magnetic nanoparticles according to the invention include phosphate fragments obtained from phosphoric acid, phosphatidylethanolamine, phosphatidylethanol, phosphothioethanol or salts thereof. In a particularly preferred manner, the polar head is -O(O)P(OH)O - It is based on

[0058] The phospholipid molecules can be advantageously selected from the salt forms of glycerophospholipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphatidyl-L-serine, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA), phosphatidylinositol, 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], or from sphingolipids, such as sphingomyelin. The anionic forms of DOPA and DSPA are particularly preferred.

[0059] Preferably, the phospholipid molecules ensure a coverage of 19-76%, preferably 29-76%, preferentially 34-50% of the surface of the iron oxide-based magnetic nanoparticles.

[0060] The surface density of the functionalization of the magnetic nanoparticles with phospholipid molecules is preferably 0.32 molecules / nm 2 ~1.22 molecules / nm 2 , preferably 0.48 molecules / nm 2 ~1.22 molecules / nm 2 , preferentially 0.56 molecules / nm 2 ~0.79 molecules / nm 2 The functionalization density can be determined using thermogravimetric analysis (TGA), as in the examples.

[0061] According to one embodiment, the nanoparticles are in the form of ellipsoids and the surface density of the functionalization of these nanoparticles with phospholipid molecules is preferably 0.32 molecules / nm 2 ~1.22 molecules / nm 2 , preferably 0.48 molecules / nm 2 ~1.22 molecules / nm 2 , preferentially 0.56 molecules / nm 2 ~0.79 molecules / nm 2 It belongs to the range.

[0062] According to a particularly preferred embodiment, the magnetic fluid according to the invention has the following characteristics: the magnetic nanoparticles are of ellipsoidal morphology and preferably have an average elementary size ranging from 5 to 20 nm, preferably from 7 to 15 nm; - The polar head of a phospholipid molecule is -O(O)P(OH)O - Preferably, the nanoparticles are functionalized with the phosphate anion of 1,2-dioleoyl-sn-glycero-3-phosphatidic acid or 1,2-distearoyl-sn-glycero-3-phosphatidic acid. - The surface density of the magnetic nanoparticles coated with phospholipid molecules is 0.32 molecules / nm 2 ~1.22 molecules / nm 2 , preferably 0.48 molecules / nm 2 ~1.22 molecules / nm 2 , preferentially 0.56 molecules / nm 2 ~0.79 molecules / nm 2 Belonging to the range of the fatty acid esters are selected from the triglycerides of caproic, capric and caprylic acids and the propylene glycol esters of caproic, capric and caprylic acids, and mixtures thereof; the oil phase comprises at least 70% by weight of fatty acid esters relative to the total weight of the oil phase, preferably the oil phase comprises from 80% to 95% by weight of fatty acid esters relative to the total weight of the oil phase; The fatty acid esters of the oil phase are selected from C6 to C12, preferably C6 to C10, saturated fatty acid triglycerides and C6 to C12, preferably C6 to C10, saturated fatty acid propylene glycols, used alone or as a mixture.

[0063] According to another embodiment, the nanoparticles are in the form of nanoflowers.

[0064] According to a first embodiment, the functionalized magnetic nanoparticles present in the biocompatible oleaginous magnetic fluid according to the invention are maghemite nanoparticles surface-functionalized with phospholipid molecules having a phosphatidylserine polar head and at least one, preferably two, C10-C22, in particular C18 unsaturated, preferably monounsaturated linear fatty chains. Preferably, according to this embodiment, the maghemite nanoparticles are surface-functionalized with 1,2-dioleoyl-sn-glycero-3-phosphatidyl-L-serine. According to this embodiment, the effective surface density of the phospholipid molecular coating on each nanoparticle is, for example, 0.48-1.22 molecules / nm 2 , preferably 0.56 to 0.79 molecules / nm 2 It belongs to the range.

[0065] According to a second preferred embodiment, the functionalized magnetic nanoparticles present in the biocompatible oleaginous magnetic fluid according to the invention are maghemite nanoparticles surface-functionalized with phospholipid molecules having an -O(O)P(OH)O polar head and at least one, preferably two, C10-C22, in particular C18 unsaturated, preferably monounsaturated linear fatty chains. Preferably, according to this embodiment, the maghemite nanoparticles are surface-functionalized with 1,2-dioleoyl-sn-glycero-3-phosphatidic acid in salt form. According to this embodiment, the surface density of the coverage by phospholipid molecules on each nanoparticle is advantageously 0.32 molecules / nm 2 ~1.22 molecules / nm 2 , preferably 0.48 molecules / nm 2 ~1.22 molecules / nm 2 , preferentially 0.56 molecules / nm 2 ~0.79 molecules / nm 2 It belongs to the range.

[0066] Preferably, the magnetic nanoparticle content in the oil-based magnetic fluid (which only contains iron oxide-based magnetic nanoparticles and does not contain functionalization) lies in the range of 0.01% to 50% by weight, preferably 0.1% to 10% by weight, relative to the total weight of the oil-based magnetic fluid. In other words, the mass content of magnetic nanoparticles per liter of biocompatible oil-based magnetic fluid advantageously lies in the range of 0.01 g / L to 500 g / L, preferably 1 g / L to 100 g / L.

[0067] In the context of the present invention, the functionalized nanoparticles are dispersed or suspended in an oil phase to form a colloidal dispersion, which is advantageously water-free, i.e., it comprises less than 0.1% by weight of water, preferably less than 0.05% by weight of water, more preferably less than 0.01% by weight of water relative to the total weight of the oil phase.

[0068] The oil phase comprises at least one fatty acid ester, preferably at least 70% by mass of fatty acid ester, preferably 80% to 95% by mass of fatty acid ester, based on the total mass of the oil phase.

[0069] In the context of the present invention, fatty acid esters are esters of carboxylic acids having an aliphatic chain containing from 4 to 36 carbon atoms.

[0070] The fatty chains of the fatty acid esters can be mono- or polyunsaturated, or saturated. Mixtures of saturated and unsaturated fatty acid esters can also be used in the context of the present invention.

[0071] The fatty chains of the fatty acid esters may be branched or, preferably, linear. Without wishing to be bound by any theory, the applicant is of the opinion that these fatty chains of the fatty acid esters are intercalated by the fatty chains of the phospholipid molecules of the functionalized nanoparticles, thus allowing the creation of solvated spheres. The absence of branching on the fatty chains of the fatty acid esters facilitates this intercalation.

[0072] Preferably, in the context of the present invention, the fatty acid ester is selected from C6 to C18 saturated fatty acid esters, preferably C6 to C12, in particular C6 to C10. Preferably, the fatty acid ester is selected from C6 to C12 saturated fatty acid esters, in particular C6 to C10. By way of example, the fatty acid of the fatty acid ester may be selected from caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and docosahexaenoic acid.

[0073] Preferably, in the context of the present invention, the fatty acid ester is selected from C6-C18 saturated fatty acid triglycerides, preferably C6-C12, in particular C6-C10 and C6-C12 saturated fatty acid propylene glycols, in particular C6-C10, used alone or as a mixture. Preferably, the fatty acid ester is selected from C6-C12 saturated triglycerides, in particular C6-C10 and C6-C12 saturated fatty acid propylene glycols, in particular C6-C10, used alone or as a mixture.

[0074] Examples of fatty acid esters that can be used in the context of the present invention include triglycerides of saturated fatty acids, used alone or as a mixture, such as the triglycerides of caproic acid, capric acid or caprylic acid, or propylene glycols of saturated fatty acids, such as propylene glycol dicaprylocaprate.

[0075] According to a first embodiment, the oil phase comprises a single fatty acid ester selected from C6-C10 fatty acid triglycerides and C6-C10 fatty acid propylene glycol esters. According to this embodiment, the fatty acid ester is advantageously selected from triglycerides of caproic acid, capric acid and caprylic acid, and propylene glycol esters of caproic acid, capric acid and caprylic acid.

[0076] According to a second embodiment, the oily phase comprises a mixture of fatty acid esters, in particular two or three fatty acid esters, which are advantageously chosen from esters of caprylic acid, capric acid and lauric acid.

[0077] The oil phase may also contain one or more oils different from said fatty acid esters, and optionally one or more lipophilic additives. Advantageously, these additional oils and lipophilic additives are biocompatible and do not produce any toxicity to the patient when the oil-based magnetic fluid according to the invention is administered to the patient. According to a particular embodiment, the oil-based magnetic fluid according to the invention does not contain any surfactant or dispersant.

[0078] In the context of the present invention, any biocompatible oil can be used.

[0079] For example, the oil phase of the biocompatible oil-based magnetic fluid can include an oil selected from soybean oil, olive oil, sesame oil, cottonseed oil, poppy seed oil, copra oil, palm oil, linseed oil, sunflower oil, C6-C12 saturated fatty acid triglyceride oil, and fish oil.

[0080] According to a preferred embodiment of the present invention, the oil phase comprises at least one fatty acid ester and at least one oil, preferably a single oil different from the fatty acid ester. According to this embodiment, the fatty acid ester is preferably selected from capric or caprylic triglycerides, propylene glycol dicaprylocaprate, used alone or in mixtures.

[0081] Preferably, the oil phase of the biocompatible oil-based magnetic fluid according to the present invention comprises less than 30% by mass, preferably 20% to 5% by mass, of at least one oil other than fatty acid ester, relative to the total mass of the oil phase.

[0082] Biocompatible oil-based magnetic fluids can also contain other biocompatible lipophilic additives. Examples of biocompatible lipophilic additives include lipophilic active ingredients, such as lipophilic active ingredients for cancer treatment, such as paclitaxel, docetaxel, or carmustine. Other biocompatible lipophilic additives known in the prior art can be used in the context of the present invention, but will not be detailed here.

[0083] Preferably, the mass percentage of the magnetic nanoparticles in the oil phase is in the range of 0.01% to 50%, preferably 0.1% to 10%, relative to the total mass of the oil-based magnetic fluid.

[0084] Advantageously, the biocompatible oil-based magnetic fluid according to the invention is stable. The magnetic nanoparticles are well dispersed and do not coagulate at temperatures belonging to the range 20-80°C, in particular from 37°C, and even better from 25°C and even from 20°C. The colloidal dispersion is obtained under atmospheric pressure (1013.25 hPa). If it is stated that the functionalized iron oxide-based magnetic nanoparticles form a colloidal dispersion in the oil phase from temperatures belonging to the range 20-80°C, this does not mean that a colloidal suspension is obtained over this entire temperature range. This means that stability is obtained at at least one temperature within the range, in particular over at least a part of the range, in particular in the high temperature range where all of the described nanoparticles are dispersed and release all of their heat under magnetic induction. However, it is preferred that the functionalized iron oxide-based magnetic nanoparticles form a colloidal dispersion in the oil phase at ambient temperature, in particular when the temperature belongs to the range 20-25°C. Advantageously, the stability of the colloidal dispersion is maintained for at least 24 hours and preferably for at least one month after obtaining. The colloidal properties of the dispersion and its stability can be verified in ambient air and at atmospheric pressure (1013.25 hPa), in particular over a period of 24 hours.

[0085] In the context of the present invention, the temperature range over which a biocompatible oil-based magnetic fluid is stable depends on the surface density of functionalization with phospholipid molecules, their chemical nature as well as the size and morphology of the magnetic nanoparticles.

[0086] Method for producing biocompatible oleaginous magnetic fluid according to the present invention The present invention also relates to a method for producing a biocompatible oleaginous magnetic fluid according to the invention, for example as detailed above, which comprises the following successive steps: a- Preparing an aqueous dispersion of iron oxide based magnetic nanoparticles in an aqueous solvent, which may be water or a water / water miscible solvent mixture, b- Removal of the aqueous solvent from the aqueous dispersion of magnetic nanoparticles; c- Obtaining a colloidal sol of magnetic nanoparticles by addition of a solvent or a mixture of volatile organic solvents S2, d- surface functionalization of said magnetic nanoparticles of colloidal sol with molecules of at least one phospholipid; e- Removing said volatile organic solvent S2 and dispersing the functionalized magnetic nanoparticles in an oil phase comprising at least one fatty acid ester.

[0087] In step a, the magnetic nanoparticles are not functionalized with phospholipid molecules, such as those described above for the functionalized nanoparticles of the biocompatible oleaginous magnetic fluids according to the invention. These nanoparticles correspond to the functionalized nanoparticles of the oleaginous magnetic fluids according to the invention after the surface functionalization step (step d). These non-functionalized nanoparticles therefore comprise iron oxide, preferably composed exclusively of iron oxide, in particular of iron oxide selected from the iron oxides described for the functionalized magnetic nanoparticles of the biocompatible oleaginous magnetic fluids according to the invention. In an aqueous medium, said iron oxide can hydrolyze on the surface of the nanoparticles and generate iron hydroxyl sites on the surface. When they are dispersed in an aqueous phase, these iron oxide nanoparticles then have a positive or negative electrostatic charge on their surface, depending on the pH value of the medium. Moreover, these non-functionalized nanoparticles have the same morphology as the functionalized nanoparticles of the biocompatible oleaginous magnetic fluids according to the invention.

[0088] These non-functionalized nanoparticles are in suspension in an aqueous medium. The aqueous medium can be composed of water, among others, and can optionally be composed of a mixture with one or more solvents miscible with water, such as methanol, ethanol, isopropanol, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, acetonitrile, acetone or ethyl acetate. Preferably, the aqueous medium is composed of water alone, and can optionally contain spectator-type counterions resulting from the stabilization of the magnetic fluid in acidic medium (e.g., nitrates or perchlorates and their acidic forms at pH 2.5) or basic medium (e.g., potassium, tetramethylammonium and their corresponding bases at pH 10).

[0089] The concentration of the magnetic nanoparticles in the aqueous dispersion is not limited. Advantageously, the concentration is greater than 1 g / L, preferably greater than 10 g / L or even greater than 100 g / L.

[0090] Preferably, the aqueous dispersion of non-functionalized nanoparticles consists only of an aqueous solvent and non-functionalized nanoparticles. Although not preferred, the aqueous dispersion of non-functionalized nanoparticles can also contain hydrophilic additives, such as steric stabilizers of macromolecular origin, such as poly(vinylpyrrolidone), dextran, functionalized poly(ethylene oxide), or surfactants, such as β-octylglucoside, Tween® 80, or sodium dodecyl sulfate.

[0091] The aqueous dispersion of non-functionalized iron oxide-based magnetic nanoparticles can be prepared by any technique known to those skilled in the art.

[0092] Step b of the method for producing a biocompatible oleaginous magnetic fluid according to the invention consists of removing the aqueous solvent from the aqueous dispersion of non-functionalized magnetic nanoparticles. In other words, this step consists of removing the water used as solvent in the aqueous dispersion of non-functionalized nanoparticles (or optionally any other solvent miscible with water present in the aqueous medium) as well as the physisorbed water, thus making it possible to obtain agglomerates of non-functionalized magnetic nanoparticles free of solvent.

[0093] For this purpose, different techniques known in the prior art can be used: - the use of a magnet to separate the metal nanoparticles from the aqueous medium, which is then removed, for example, by aspiration or pipetting; - Extraction on a magnetic column, - ultrafiltration, - Centrifugation, - successive washings with organic solvents miscible with water, such as methanol, ethanol, isopropanol, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, acetonitrile, acetone or ethyl acetate; - freeze-dried, - Pervaporation.

[0094] Preferably, water and / or any water-miscible solvent present is removed after the nanoparticles are coagulated. Coagulation is obtained when the medium is neutralized or by adding a water-miscible solvent, for example, acetone or ethanol. The aqueous medium containing water is then separated from the coagulated nanoparticles. The aggregates of non-functionalized magnetic nanoparticles are held, for example, by using a magnet, while the medium is sucked or pumped, for example, by using a membrane pump.

[0095] Thus, regardless of the method used to remove the aqueous solvent, successive washings with a volatile polar organic solvent miscible with water (solvent hereafter referred to as S1), e.g., ethanol, may make it possible to remove all physisorbed water from the nanoparticles.

[0096] Advantageously, this water removal step makes it possible to recover non-functionalized magnetic nanoparticles having a water content of less than 0.25% by weight, preferably less than 0.025% by weight, relative to the mass of the non-functionalized magnetic nanoparticles, according to their produced surface.

[0097] Step c of the preparation method consists of preparing a colloidal sol of non-functionalized magnetic nanoparticles.

[0098] In the context of the present invention, "colloidal sol" means a homogeneous colloidal dispersion of solid particles in a continuous liquid medium.

[0099] A colloidal sol of non-functionalized nanoparticles can be created by dispersing non-functionalized nanoparticles in a dispersion medium.

[0100] Dispersion can be carried out by any technique known to those skilled in the art, such as homogenization by vortexing and in an ultrasonic bath.

[0101] Preferably, the dispersion medium also allows the solubilization of the phospholipids subsequently used for the surface functionalization of the magnetic nanoparticles. The dispersion medium also comprises, or even consists exclusively of, one or more volatile organic solvents (referred to as solvent S2). In fact, such volatile organic solvents allow both the dispersion of non-functionalized nanoparticles and the solubilization of the phospholipids used during the functionalization step d. Thus, the control of the speed, yield and grafting rate of phospholipids of the surface functionalization reaction is greatly improved.

[0102] Preferably, the dispersion medium used, in particular the volatile organic solvent S2, is biocompatible, so that even if traces of this dispersion medium are still present in the oil-based magnetic fluid according to the invention, they do not result in any toxicity for the patient.

[0103] Preferably, the carrier medium is volatile such that its removal is facilitated by evaporation under reduced pressure during step e of the method, so that in cases where the carrier medium is not biocompatible, there is no toxicity due to this medium.

[0104] Examples of volatile organic solvent-based dispersion media S2 include chloroform, alone or in mixtures with methanol (eg, in a 2:1 v / v ratio), diethyl ether, heptane, dichloromethane or isopropanol.

[0105] According to a particular embodiment, the carrier medium is composed of a single volatile organic solvent S2, preferably a polar one. According to this embodiment, the carrier medium is advantageously chloroform.

[0106] Preferably, the concentration of magnetic nanoparticles in the colloidal sol is in the range of 0.1% to 50% by weight, preferably 1% to 10% by weight, relative to the total weight of the colloidal sol. Such a concentration of magnetic nanoparticles advantageously allows a good production yield and a control of the grafting rate in terms of the distribution of phospholipid molecules on the magnetic nanoparticle surface after the functionalization step.

[0107] An optional step c2, carried out after step c and before step d, can be carried out in view of the nanoparticle functionalization step (step d), in particular to promote the complexation of the iron sites present on the magnetic nanoparticle surface with the polar phosphatide heads of the phospholipid molecules. For this purpose, an organic acid is added to the non-functionalized colloidal sol of magnetic nanoparticles. Preferably, a weak organic acid is used. By weak organic acid is meant an acid whose dissociation reaction in water is not complete. The chemisorption rate of the phospholipid molecules is improved.

[0108] Advantageously, the organic acids used in this step are biocompatible.

[0109] Preferably, the organic acid used in this optional step c2 is chosen from weak organic acids that are miscible in the volatile organic solvent S2 used in step c, advantageously at less than 10% by volume, to obtain a colloidal sol.

[0110] Examples of organic acids that can be used to activate magnetic nanoparticles that are not functionalized with phospholipid molecules include acetic acid, lactic acid, propanoic acid and / or butanoic acid.

[0111] Step d of the method for producing a biocompatible oil-based magnetic fluid consists of surface functionalizing the magnetic nanoparticles of the colloidal sol with at least one phospholipid. For this purpose, the colloidal sol is contacted with the phospholipid. For example, a solution of the phospholipid can be added to the colloidal sol at atmospheric pressure with mechanical stirring. Any known technique for colloidal sol formation can be used.

[0112] The solution of phospholipids used may comprise, depending on the functionalization of the magnetic nanoparticles desired, one or more phospholipids and at least one solvent in which the phospholipids are soluble (hereinafter referred to as solvent S3). Advantageously, the phospholipids carry the phosphate functions in salified form, in particular with an alkaline cation such as a sodium atom, thereby facilitating the coordination / complexation of the phosphate functions of the polar heads of said phospholipids on the magnetic nanoparticles.

[0113] The phospholipids are generally in the salt form for stability purposes.

[0114] Solvent S3 may be an organic solvent. Preferably, this solvent S3 is volatile.

[0115] Examples of suitable solvents S3 include chloroform, alone or in mixture with methanol (eg, in a 2:1 v:v ratio), diethyl ether, heptane, dichloromethane or isopropanol.

[0116] Preferably, this solvent (or mixture of solvents) S3 is the same as the volatile organic solvent S2 used as the dispersion medium in the colloidal sol of the magnetic nanoparticles.

[0117] The phospholipids used in this phospholipid solution are those detailed for the phospholipid molecules functionalizing the magnetic nanoparticles of the biocompatible oleaginous magnetic fluid according to the invention.

[0118] The phospholipid content in the magnetic nanoparticle dispersions in the organic solvents S2 and S3 depends on the total surface generated by the magnetic nanoparticles used, on the phospholipids used and their generated surface, and on the fatty acid esters used. Advantageously, the phospholipid content in the reaction medium is comprised between 0.005% and 10% by weight, preferably between 0.05% and 2.5% by weight, relative to the total weight of the colloidal sol.

[0119] According to a first embodiment, the phospholipid molecules are present in excess with respect to the surface produced by the non-functionalized magnetic nanoparticles, preferably in an amount corresponding to 1000% of the amount necessary to form a monolayer saturated with phospholipids. In fact, the excess of phospholipids shifts the reaction equilibrium at the surface of the magnetic nanoparticles and increases the chemisorption rate of the phospholipids. Such an excess is advantageous when this functionalization reaction is kinetically unfavorable, for example when the polar phosphate heads of the phospholipid molecules used are sterically hindered. The reaction time for the phospholipid molecules contained in the colloidal sol is therefore a parameter to be considered depending on the excess of phospholipid molecules. The reaction is stopped by the addition of a solvent, for example ethanol, which causes the aggregation of the nanoparticles and the precipitation of the excess phospholipid molecules. Thus, for example, in the case of a 2000% excess of phospholipids with hindered polar heads, for example phosphatidylserine, the chemisorption time necessary for the dispersion of the magnetic nanoparticles in the oil described in the context of the present invention is selected from an interval of 5 to 30 minutes, preferably 6 to 10 minutes and even better still 7 to 8 minutes. According to this embodiment, the method comprises a subsequent step of removing excess phospholipid molecules, for example by successive washings, preferably with a mixture of organic solvents, for example an ethanol / chloroform mixture (3:1), as well as a final step of removing the chloroform together with ethanol.

[0120] According to a second embodiment, the amount of phospholipid molecules used is present in deficiency with respect to the generated surface of the non-functionalized magnetic nanoparticles, preferably in a percentage ranging from 38% to 100%, in particular from 38% to less than 100%, of the amount necessary to form a monolayer saturated with phospholipids. Indeed, the deficiency of phospholipid molecules makes it possible to control the functionalization rate of the magnetic nanoparticles with phospholipid molecules. Such a deficiency can be achieved in cases where the functionalization reaction is very kinetically favorable, for example when the phospholipids used are -O(O)P(OH)O - , M + , it is advantageous if it has a polar head (M is an alkali metal atom, preferably a sodium atom).

[0121] At the end of the functionalization step d, the coverage percentage and / or surface density of the magnetic nanoparticles is, for example, as described for the functionalized magnetic nanoparticles of the biocompatible oleaginous magnetic fluid according to the invention. The conditions for the method according to the invention are adjusted by the skilled person to have such a coverage percentage and / or such a surface density of the functionalization.

[0122] Once the functionalization of the magnetic nanoparticles is completed, the dispersion medium of the colloidal sol and almost all of the solvent S3 of the phospholipid solution used are removed (step e), for example by evaporation under a flow of inert gas (nitrogen or argon) to prevent phospholipid oxidation, to obtain aggregates of magnetic nanoparticles functionalized with phospholipid molecules.

[0123] Finally, these functionalized nanoparticles are dispersed in an oil phase containing at least one fatty acid ester (step e). Any traces of solvents S2 and S3 and mainly ethanol are evaporated under reduced pressure, for example in a rotary evaporator at 80° C. for an effective period of 30 minutes. The oil phase and fatty acid esters are such as those described for the biocompatible oleaginous magnetic fluids according to the invention. This dispersion in colloidal form in the oil phase occurs spontaneously at ambient temperature or by heating to a temperature in the range of 30-80° C. Most often, this temperature is determined according to the grafting rate and the phospholipid molecules, oil and fatty acid ester utilized. Such heating can be carried out in step e) or in a subsequent heating step to obtain a colloidal dispersion.

[0124] Use of the biocompatible oil-based magnetic fluid according to the present invention The present invention also relates to the use of the biocompatible oleaginous magnetic fluid according to the invention or of the biocompatible oleaginous magnetic fluid obtainable according to the manufacturing method according to the invention.

[0125] The biocompatible oil-based magnetic fluid according to the invention or obtainable according to the method of the invention can be used as a medicine, in particular as a medicine for cancer treatment by magnetic hyperthermia, in particular for the treatment of benign or malignant nodules or solid tumors.

[0126] Advantageously, this medicament for treating cancer consists in the first embodiment of the biocompatible oleaginous ferrofluid alone. Alternatively, according to a second, less preferred embodiment, the medicament comprises the biocompatible oleaginous ferrofluid as well as a lipophilic cancer therapeutic agent.

[0127] To treat cancer patients, a biocompatible oil-based ferrofluid is administered intratumorally using any technique known to those skilled in the art, and then an external alternating magnetic field is applied to induce heating of the magnetic nanoparticles contained in the biocompatible oil-based ferrofluid, which localized heating dissipates throughout the entire volume of the oil phase of the oil-based ferrofluid, thus allowing the destruction of tumor cells.

[0128] According to certain embodiments, this heating involves the release of a therapeutic agent contained in a thermosensitive carrier, or can be used to activate a thermally activatable therapeutic agent, or to induce expression of a gene under the transcriptional control of a thermosensitive promoter, or to exert a synergistic effect with another therapeutic agent co-administered independently in the context of a chemotherapy and / or radiation therapy protocol.

[0129] Typically, the applied alternating magnetic field is in the range of 5-25 kA / m and the frequency is in the range of 100-750 kHz. The duration of the applied magnetic field / frequency pair depends on the amount of heat dissipated per unit of volume for an oil-based magnetic fluid of a given iron oxide mass concentration as a function of the tumor volume to be treated. These protocols for hyperthermia treatment are calibrated for different magnetic field / frequency pairs according to the type of treatment sought (which requires different amounts of heat, e.g., thermal ablation, release of a therapeutic agent, activation of a thermally activatable therapeutic agent or induction of gene expression under the transcriptional control of a heat-sensitive promoter).

[0130] According to a first embodiment, a biocompatible oleaginous magnetic fluid is used as the sole pharmaceutical agent in this treatment.

[0131] According to the second embodiment, especially when the oil-based magnetic fluid does not contain chemotherapy drugs, the biocompatible oil-based magnetic fluid is used in combination with another chemotherapy drug, such as paclitaxel, docetaxel or carmustine, preferably contained in magnetic oil.The simultaneous injection of an injectable preparation containing a thermosensitive active ingredient, such as Thermodox®, can also be considered.

[0132] The biocompatible oil-based magnetic fluid according to the invention, or the biocompatible oil-based magnetic fluid obtainable according to the method according to the invention, can also be used as a contrast agent for medical imaging, e.g., MRI, near-infrared (NIR) fluorescence imaging, and localized fiber approaches by endoscopy.

[0133] For use in fluorescence imaging, the oil-based ferrofluid administered to the patient preferably contains a lipophilic fluorophore.

[0134] The present invention also relates to a method of imaging the whole body or a part of the body of an individual, comprising the step of obtaining one or more images of said whole body or part of the body by a medical imaging technique, said whole body or part of the body comprising a contrast medium comprising a biocompatible oleaginous magnetic fluid.

[0135] Advantageously, the oil-based magnetic fluid according to the invention is dispersed in colloidal form at least at the temperature reached during the induction treatment, the magnetic nanoparticles being well dispersed at that point and not forming aggregates or coagulating over time.

[0136] Even more advantageously, the ferrofluid is stable at atmospheric pressure, in particular at room temperature (20° C.) or at physiological temperature (37° C.), in particular for at least 24 hours, advantageously for at least one month. It is possible to distinguish between short-term stability (for periods up to 24 hours) and long-term stability (for periods longer than 24 hours), which is assessed in open air. Long-term stability can be assessed by keeping the ferrofluid in particular under an inert atmosphere, for example argon or nitrogen, under protection from light, at the temperature at which the stability has to be assessed, in particular 20° C. or 37° C.

[0137] If the magnetic fluids according to the invention correspond to colloidal dispersions only at temperatures higher than ambient temperature, in particular at temperatures belonging to the range of 20-40° C., it may be necessary to keep them in thermostated containers or chambers, or even to heat them before use to a temperature making it possible to obtain a colloidal dispersion. The magnetic fluids can also be administered by injection using a thermostated drip, or by introducing a heating kit or device to maintain the magnetic fluid at a temperature in the range of 20-40° C. at which a satisfactory colloidal stability is obtained.

[0138] Nanoemulsions containing the biocompatible oil-based magnetic fluid according to the present invention

[0139] The present invention also relates to a biocompatible oil-based magnetic fluid nanoemulsion according to the invention or obtainable according to the method for producing an oil-based magnetic fluid according to the invention, as diagrammatically shown in FIG.

[0140] By "nanoemulsion" is meant an emulsion whose dispersed phase droplets in the continuous phase are nanometer sized, ie, between 100 nm and less than 300 nm.

[0141] In the context of the present invention, a nanoemulsion may be a simple oil-in-water or water-in-oil emulsion, or a multiple water-in-oil-in-water emulsion. Preferably, the nanoemulsion according to the invention is an oil-in-water emulsion.

[0142] According to a first embodiment, the lipophilic phase of the nanoemulsion is composed solely of the biocompatible oleaginous ferrofluid.

[0143] According to a second embodiment, the nanoemulsion lipophilic phase comprises a biocompatible oil-based magnetic fluid in combination with one or more other lipophilic compounds, in particular selected from among oils, lipophilic fluorophores, perfluorocarbons or lipophilic active ingredients, such as paclitaxel, docetaxel, etoposide, carmustine.

[0144] Furthermore, to improve their pharmacokinetics, the interfacial phase of the magnetic lipid droplets can contain one or more pegylated lipids (phospholipids) and / or targeting ligands (antibodies, peptides, aptamers).

[0145] Lipophilic compounds other than the active cancer-treating ingredient that can be used in the nanoemulsions according to the invention are preferably biocompatible and do not produce any toxicity.

[0146] Any biocompatible oil known in the prior art can be used. Examples of oils that can be used as another lipid compound of nanoemulsions in the context of the present invention include soybean oil, olive oil, sesame oil, cottonseed oil, poppy seed oil, copra oil, palm oil, linseed oil, sunflower oil, fatty acid triglyceride oils such as Miglyol 812N® or Labrafac® WL 1349, fatty acid propylene glycol oils such as Miglyol 840® or Labrafac® PG and fish oil.

[0147] If the nanoemulsion contains, in addition to the biocompatible oil-based ferrofluid, an oil or a mixture of oils, these oils are generally present in a content ranging from 10% to 35% by weight, preferably from 20% to 30% by weight, relative to the total weight of the nanoemulsion.

[0148] Lipophilic fluorophores that can be used in the context of the present invention include fluorescent analogues of phospholipids and sphingomyelin, cyanines such as indocyanine green (ICG) and lipophilic carbocyanines.

[0149] When the nanoemulsion contains lipophilic fluorophores, these oils are preferably present in a content ranging from 0.0001% to 0.02% by weight, preferably from 0.001% to 0.01% by weight, relative to the total weight of the nanoemulsion.

[0150] Targeting ligands are known to allow the recognition of biological targets through molecular interactions, thus improving the specificity of the mixture administered.Examples of targeting ligands include amino acids, such as folic acid, sugars, such as mannose or FDG (fluorodeoxyglucose, commonly used for PET imaging), peptide sequences, such as the cyclic RGD peptide for integrins or (Tyr3)-octreotate (TATE), which has high affinity for somatostatin type 2 receptors, synthetic compounds, such as raclopride, which acts as an antagonist of the dopamine D2 receptor, antibodies of different formats, such as camelid-derived antibodies (nanobodies) or recombinant antibody fragments selected by phage display screening, such as scFv for PSMA or anti-PD1 / PDX1 used in immunotherapy, or drugs based on aptamers selected by SELEX screening for membrane receptors of cancer cells.

[0151] The aqueous phase of the nanoemulsions according to the invention is typically composed of water, optionally in combination with one or more water-miscible solvents, such as ethanol and propylene glycol. The aqueous phase of the nanoemulsion may also contain a salt (sodium or potassium chloride) or a buffer.

[0152] Additionally, nanoemulsions may contain one or more surfactants, also called dispersing agents, which are preferably biocompatible, and thus reside at the aqueous / lipophilic phase interface of the nanoemulsion.

[0153] Examples of surfactants and dispersants that may be suitable within the scope of the present invention include egg or soy lecithin, bile acids such as sodium deoxycholate, polyoxyethylated castor oil, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitan monolaurate (Span® 20, Span® 40, Span® 60, and / or Span® 80), poloxamers or PEG block copolymers.

[0154] When these agents are present, the surfactant and / or dispersant content usually falls within the range of 1% to 5% by weight, preferably 1.8% to 3.8% by weight, relative to the total weight of the nanoemulsion.

[0155] Advantageously, the nanoemulsions according to the invention are stable at ambient temperature (20° C.) and atmospheric pressure. In particular, the nanoemulsions according to the invention are stable at ambient temperature (20° C.) and atmospheric pressure for at least 24 hours, preferably for at least 6 months.

[0156] In the context of the present invention, the colloidal stability of the nanoemulsions is determined by evaluation of the particle size distribution by dynamic light scattering (DLS), as detailed in the examples.

[0157] Manufacturing method for the nanoemulsion according to the invention

[0158] The present invention also relates to a method for producing a nanoemulsion according to the invention.

[0159] Such a method for producing a nanoemulsion comprises the following successive steps: i- providing a lipophilic phase comprising a biocompatible oil-based magnetic fluid according to the invention or a biocompatible oil-based magnetic fluid obtained according to the method for producing a biocompatible oil-based magnetic fluid according to the invention, ii- providing an aqueous phase; iii- Mixing the lipophilic phase and the aqueous phase to form a nanoemulsion.

[0160] In particular, the mixing in step iii is preferably carried out at a temperature at which the oil-based ferrofluid according to the invention is present in the form of a colloidal dispersion. The lipophilic phase comprising the biocompatible oil-based ferrofluid according to the invention can also be heated to such a temperature before mixing with the aqueous phase.

[0161] The method for producing a nanoemulsion according to the invention can comprise, after step i and before step iii, an optional step i2 of contacting the biocompatible oil-based ferrofluid with one or more compounds selected from oils, lipophilic fluorophores, targeting ligands, lipophilic surfactants, pharmacophores, such as those described above, to obtain the lipophilic phase of the nanoemulsion. This optional step can be carried out by mixing at ambient temperature and pressure according to any technique known to the person skilled in the art.

[0162] In step ii, the aqueous phase may contain one or more surfactants, if present, for example as described above.

[0163] In step iii, the aqueous phase and the lipophilic phase are conventionally mixed at high speed using any technique known to those skilled in the art, for example, the lipophilic phase and the aqueous phase, optionally preheated, particularly to a temperature in the range of 60-70° C., are first mixed at high speed to form a coarse emulsion, which is then homogenized using a sonicator or by high pressure homogenization.

[0164] The aqueous phase, the lipophilic phase and the surfactants optionally present are such as those described for the nanoemulsions according to the invention.

[0165] Uses of the nanoemulsions according to the invention

[0166] The present invention also relates to the use of a nanoemulsion according to the invention or obtainable according to the process of the invention.

[0167] The nanoemulsions according to the invention can be used as medicines, in particular as medicines in the treatment of cancer by magnetically induced hyperthermia. For this purpose, a systemic administration of the nanoemulsion is carried out, followed by the application of an external alternating magnetic field. In this case, the magnetic field is applied at the moment when the accumulation of the product in the tumor reaches a maximum. This waiting time between the injection and the application of the alternating magnetic field is predicted by optical imaging (fiber or not), by MRI or by ultrasound (for perfluorocarbon-based formulations). In the case of intratumoral administration, the magnetic field can be applied after the injection. The characteristics of the magnetic induction for the nanoemulsion (value of the magnetic field / frequency pair and duration of application) can be adjusted according to the desired amount of heat. The heating of the magnetic oil droplets can be accompanied by the release of the therapeutic agent, or it can be used for the activation of a thermally activatable therapeutic agent, or for inducing the expression of a gene under the transcriptional control of a thermosensitive promoter, or for exerting a synergistic effect with another therapeutic agent co-administered independently in the context of a chemotherapy and / or radiotherapy protocol.

[0168] According to a first embodiment, the nanoemulsion is used as the only pharmaceutical agent in the treatment.

[0169] According to a second embodiment, particularly when the nanoemulsion does not contain a chemotherapy drug, the nanoemulsion is used in combination with another chemotherapy drug of lipophilic nature, such as taxanes (paclitaxel and docetaxel) or carmustine. Simultaneous injection of an injectable formulation containing a heat-sensitive active ingredient, such as Thermodox®, can also be considered.

[0170] The volume and concentration of oleaginous ferrofluid administered as well as the number of injections and the time between each injection will routinely depend on the particular tumor being treated (location, volume) and the patient (age, health) and will be determined by the physician.

[0171] The nanoemulsions according to the invention can also be used as contrast products for medical imaging, such as MRI, ultrasound or near-infrared (NIR) fluorescence imaging, and for localized fiber approaches by endoscopy.

[0172] For use in fluorescence imaging, the nanoemulsion administered to a patient preferably contains a lipophilic fluorophore.

[0173] The present invention also relates to a method of imaging the whole body or a part of the body of an individual, comprising the step of obtaining one or more images of said whole body or part of the body by a medical imaging technique, wherein said whole body or part of the body comprises a contrast agent product comprising an emulsion.

[0174] kit The present invention also relates to a kit comprising a container in which the oil-based magnetic fluid according to the invention or the nanoemulsion according to the invention is placed. The kit may also contain magnetic nanoparticles surface-functionalized with one or more molecules of phospholipids as described according to the invention alone in one container and, optionally, in another separate container, an oil phase containing at least one fatty acid ester intended to constitute the oil-based magnetic fluid. The magnetic fluid or nanoemulsion can be reconstituted from the kit immediately before its use or administration. The kit may also contain the conditions and provisions for use for the applicable magnetic field / frequency pairs and the corresponding amount of heat dissipated per volume unit of magnetic fluid of a given concentration. It is noted that the iron concentration is also approved for indication purposes. EXAMPLES

[0175] material and method: A) Phospholipids tested: The phospholipids used to prepare oil-based ferrofluids in the examples below are: - Chemical formula 1 below:

[0176] [ka] 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Chemical formula 2 (in sodium salt form):

[0177] [ka] 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), - of the following formula 3 (in sodium salt form):

[0178] [ka] of 1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA), and - of formula 4 (sodium salt form):

[0179] [ka] of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS).

[0180] B) Composition of the oil phase: The oil phase used in the examples is a mixture of fatty acid triglycerides or fatty acid propylene glycols: Miglyol 812N® and Miglyol 840®, respectively. Table 1 below shows the fatty acids that make up the fatty acid triglycerides or propylene glycols that make up these oils (percentages are percentages by mass relative to the total weight of the oil).

[0181] [Table 1]

[0182] C) Iron Determination: The iron content is determined by UV-Vis spectroscopy by dissolving the iron oxide nanoparticles in a 5M solution of hydrochloric acid (HCl). After the nanoparticles are completely dissolved in 5M HCl, the absorbance at 350 nm is measured to determine the iron concentration. The iron concentration is calculated according to the formula: 350nm = ε.l.[Fe]. The molar extinction coefficient ε is 2960L -1 .mol.cm -1 is equal to.

[0183] D) Nanoparticle size: D1) Dynamic Light Scattering (DLS) Measurement The hydrodynamic radius of the nanoparticles is determined by dynamic light scattering using a Cordouan Vasco instrument equipped with a laser having a wavelength of 658 nm and an angle of 135°; data are acquired over a period of 60 seconds.

[0184] D2) Measurement by X-ray diffraction (XRD) The crystallite size was determined by XRD by using Scherrer's equation 1 below, where Γ is the average diameter of the crystallites, λ is the wavelength of the X-ray (λ(Cu Kα)=1.5406 Å), β is the width at half height of the strongest peak, and Θ is the Bragg angle:

[0185]

number

[0186] D3) Measurement by transmission electron microscopy (TEM) The average elementary size of the nanoparticles is determined by transmission electron microscopy by measuring the size of 200 nanoparticles obtained with a Philips CM120 microscope operated at 120 kV and equipped with an Ultra scan USC1000 camera (2k × 2k) using image processing software (ImageJ Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, https: / / imagej.nih.gov / ij / , 1997–2019.).

[0187] E) Surface characterization: E1) Thermogravimetric analysis (TGA) Approximately 10 mg of nanoparticles, pre-dried at 70° C. under vacuum, are placed in a platinum crucible and the analysis is carried out using a Tag2400 thermobalance from Setaram. The sample is heated to 600° C. under air with a ramp of 5° C. / min.

[0188] E2) Diffuse reflectance infrared spectroscopy Diffuse reflectance infrared spectroscopy (DRIFT) is performed using a Bruker IFS Equinox 55 spectrometer. The functionalized nanoparticles are oven-dried at 70° C. after a washing step. The nanoparticles are then ground in anhydrous KBr (3 wt%).

[0189] E3) Determination of surface functionalization density and theoretical (also called nominal) and actual coverage The surface created by phospholipid molecules sprayed on a surface depends on the volume occupied by the polar heads compared to the volume of the lipid chains. In the case of phospholipids, the bulk volumes of the two parts (polar and hydrophobic) are comparable, resulting in the lipids being cylindrical rather than conical. This particular feature allows them to assemble into vesicle membranes rather than micelles. The area bulk values ​​of phospholipids generally range from 60 to 65 Å depending on different physicochemical parameters such as the environment in which they exist, alone or in mixtures, the number of unsaturations, their interaction with a solid surface (supported lipid membrane) or in a liquid medium (vesicles) and the temperature. 2 -O(O)P(OH)O - , Na + The nominal value for phospholipid molecules containing a phosphatidyl head (as in the case of DOPA and DSPA) is 62 Å 2 and corresponds to the molecular crowding within a saturated monolayer contained in a lipid membrane. This molecular surface density value is what is found in lipid membranes (phospholipid bilayers) by considering a single monolayer. Obviously, there are slight differences when the OH functional groups are substituted. For example, for dioleoylphosphatidylserine, the molecular surface area generated is approximately 65 Å. 2 / molecule, and for dioleoylphosphatidylethanolamine, generally 60 to 65 Å 2 Therefore, in the following, the 2 This 62 Å is selected. 2 The value of is used in the context of the present invention to calculate the coverage percentage of the surface of the nanoparticles, regardless of the phospholipid envisaged, and to calculate the surface functionalization density of the phospholipid molecules.

[0190] The "nominal or theoretical coverage" of a phospholipid is a molar percentage expressed by the amount of lipid involved relative to the amount necessary to form a monolayer, calculated from the average size of the nanoparticles measured by TEM and the reference surface of the polar head of the phospholipid used. If the nanoparticles are ellipsoidal, it is conceivable to consider the average size of the nanoparticles measured by TEM as the diameter, in order to evaluate the average surface produced by the particles. For the nanoflowers, taking into account their surface roughness, their produced surface was estimated by repeated coating of silica of different thicknesses by the sol-gel route (Stöber synthesis route). This method thus made it possible to estimate a surface area 15% greater than that of a smooth sphere of the same diameter.

[0191] The TGA measurements make it possible to determine the amount of phospholipid molecules actually present on the nanoparticles per surface unit, from the total mass loss measured between 200 °C and 600 °C, from the molar mass of the organic residues decomposed during the complete combustion relative to the surface created by the nanoparticles involved. The grafting yield represents the ratio between the amount of phospholipid molecules chemisorbed on the surface of the nanoparticles and the nominal amount involved during the reaction.

[0192] F) Synthesis of iron oxide nanoparticles F1) Synthesis of FF1 nanoparticles by alkaline coprecipitation A mass of 31.41 g of FeCl dissolved in 170 mL of 1.5 M hydrochloric acid 2 .4H 2 O (0.158 mol) was dissolved in 3.5 L of water. 3 .6H2 Pour into a beaker containing 200 (0.316 mol) (initial stoichiometric ratio Fe 2+ / Fe 3+ = 0.5). Under high-speed mechanical stirring, 300 mL of ammonia solution (28-30% m / m) is rapidly added. The medium is left under stirring for 15 min. The magnetite nanoparticles precipitate is decanted using a permanent magnet and then the supernatant liquid is removed. After two successive steps of washing with water, a volume of 200 mL of 2 M HNO 3 The surface of the nanoparticles is oxidized by adding 0.5 mL of 0.33 M iron nitrate solution and then left under stirring for 15 minutes. The supernatant is decanted and removed, then the core of the nanoparticles is oxidized to maghemite by adding 600 mL of 0.33 M iron nitrate solution. The reaction medium is boiled for 30 minutes. The supernatant is decanted and removed, then 200 mL of 2 M nitric acid is added. The precipitate is then magnetically decanted and washed three times with acetone to remove the excess acid. Finally, the precipitate is peptized in 200 mL of water. After evaporation of the excess acetone, the ferrofluid is made up to a volume of 1 L with water. This dispersion of maghemite nanoparticles is hereafter referred to as FF1. At the end of the synthesis, the ferrofluid has a mass concentration of iron oxide of 69 g / L and a concentration of 11316 m 2 / L. The average elementary size of the thus obtained ellipsoidal nanoparticles, determined by TEM, is 7.5 nm ± 2 nm and, when measured by XRD, 6.9 nm. The measured hydrodynamic radius is D H =38 nm.

[0193] F2) Synthesis of FF2 nanoparticles using polyol Mass 1.082 g of FeCl 2 .4H 2 O (5.44 mmol) and 0.398 g FeCl 3 .6H 20 (1,47 mmol) is dissolved in a mixture of 80 g of diethylene glycol (DEG) and N-methyldiethanolamine (NMDEA) (ratio 1:1, v / v). The solution is mixed under stirring for 1 h under nitrogen flow until the precursor is completely dissolved. A mass of 0.64 g of NaOH (16 mmol) is dissolved in 40 g of NMDEA / DEG mixture (1:1, v / v) under nitrogen flow. The NaOH solution is added to the solution containing the precursor and the mixture is heated to 220 °C at 2 °C / min for 4 h. The nanoparticles obtained are magnetically precipitated and washed three times with an ethanol / ethyl acetate (1:1, v / v) mixture to remove organic and inorganic impurities. Washing with 10% nitric acid is carried out. 8.25 g of Fe 3 (NO 3 ) 3 .9H 2 20.4 mmol) of maghemite nanoparticles are solubilized in 20 mL of water and the solution is added to the nanoparticles. The nanoparticle dispersion is heated at 80 °C for 45 min to obtain maghemite. After decanting and removing the supernatant, the nanoparticles are washed with 10% nitric acid, then with acetone and finally with diethyl ether. Finally, the nanoparticles are redispersed in water. Hereafter, this dispersion of maghemite nanoparticles is called FF2. At the end of the synthesis, the ferrofluid has a mass concentration of iron oxide of 20 g / L and a concentration of 1574 m 2 The average elementary size of the nanoparticles thus obtained, of nanoflower morphology, is measured at 15.4 nm ± 3 nm by TEM and at 16 nm by XRD. The measured hydrodynamic radius is D H =26 nm.

[0194] Synthesis of FF3 nanoparticles with polyol FF3 nanoparticles are synthesized under the same operating conditions as those described to obtain FF2 nanoparticles, except that this time the temperature ramp to 220 °C for 4 h is carried out at 2 °C / min under adiabatic reaction conditions. To do this, glass wool was placed around the flask in contact with the reactor walls on a heating mantle in open air to limit the loss of heat and thus allow a better kinetic control of the temperature ramp. The average elementary size of the nanoparticles with nanoflower morphology obtained by TEM techniques is 18.5 nm ± 3.1 nm.

[0195] G) Colloidal stability in oil-based dispersions The colloidal stability in the dispersion is observed, in a first approximation, visually and then by measuring the transmittance at 800 nm using a VARIAN Cary 500 spectrophotometer equipped with a temperature control device as a function of temperature. The quality of the nanoparticle dispersion is also checked by DLS (method D1). If a "+" is displayed in the table of examples, this means that colloidal properties are obtained by dispersing the nanoparticles in the oil phase at the corresponding temperature and that these colloidal properties are still present at least 24 hours after they are obtained, i.e. at least after the time that has elapsed between the preparation of the dispersion and the measurement of the transmittance.

[0196] Example 1: Dispersions of FF1, FF2 and FF3 nanoparticles in chloroform The aim of this example is to transfer the nanoparticles of the aqueous magnetic fluids FF1, FF2 and FF3 into chloroform without resorting to surfactants, so that phospholipid grafting by chemical adsorption can be performed. To do so, a volume of 1 mL of ammonia solution (28-30%, m / m) was added to the γ-Fe nanoparticles. 2 O 3 Add 69 g / L of γ-Fe to 35.4 mL of FF1. Decant the nanoparticle aggregates using a permanent magnet and then remove the supernatant. Wash the aggregated nanoparticles twice with water. Then wash the aggregated nanoparticles five times with ethanol. Finally, add a volume of 80 mL of chloroform to the nanoparticles and redisperse the nanoparticles in an ultrasonic bath for 3 min. The final concentration is γ-Fe in chloroform.2 O 3 30.5 g / L. The protocol is the same for the magnetic fluids FF2 and FF3.

[0197] Example 2: Preparation of a biocompatible oleaginous magnetic fluid obtained from the functionalization of magnetic nanoparticles with phospholipid molecules exhibiting sterically unhindered polar heads in the presence of an acid. Example 2.1.: Dispersion of nanoparticles functionalized with monounsaturated phospholipid molecules Example 2.1.1: Dispersion of magnetic nanoparticles of magnetic fluid FF1 functionalized with DOPA in Miglyol M840® In this example, a dispersion of magnetic nanoparticles of FF1 functionalized with DOPA in Miglyol M840® is prepared. The bulk of the reference area of ​​the phospholipid molecules is 62 Å. 2 , which corresponds to the molecular density within a saturated monolayer contained in a lipid membrane. 2 The functionalization protocol for nanoparticles with a nominal coverage (corresponding to the amount of lipid involved compared to that required to form a monolayer) of 16.4 mL of magnetic nanoparticles ([γ-Fe 2 O 3 ]=30.5g / L, surface produced=82m 2 ) is diluted in a volume of 47.7 mL of acetic acid-chloroform solution (1:9, v / v). To this dispersion is added a volume of 3.51 mL of DOPA (25 mg / mL in chloroform, 45.2 mg 2 ) is added under vortexing. After 14 hours of reaction at 4 °C, ethanol is added to the medium until the nanoparticles are coagulated and the whole is magnetically decanted. The magnetic coagulate is washed three times with 100 mL of a chloroform / ethanol mixture (1:3, v / v) and then twice with 100 mL of ethanol. At the end of the last wash, the nanoparticles are dried under a stream of nitrogen and a volume of 15 mL of Miglyol M840® is added to the nanoparticles. The nanoparticles are dispersed in oil from 35 °C after heating in a water bath.

[0198] Similarly, the nominal DOPA coverage range set for the monolayer between 20% and 150% was 0.33 DOPA / nm 2 from 2.42 DOPA / nm 2 This corresponds to a theoretical grafting density comprised between 0.01 and 0.11, and can be achieved by working at a constant DOPA concentration (1.25 mg / mL). For a given nominal density variable, the nanoparticles disperse spontaneously at ambient temperature (20 °C).

[0199] The nominal DOPA coverage is calculated according to Equation 2 below.

[0200]

number

[0201] The stability of nanoparticle dispersions derived from FF1 functionalized with DOPA in Miglyol M840® was evaluated at different temperatures and different coverages, as summarized in Table 3 below. The criteria for designating the quality of the dispersion are: - = non-dispersive (turbid medium), + = colloidal sol stable over time (clear dispersion). These screening criteria can be conveniently evaluated by visual observation and are in good agreement with transmittance measurements at 800 nm. TGA measurements made it possible to determine the actual DOPA amount per surface unit and the grafting yield. The nominal and actual DOPA surface density and the colloidal stability of the nanoparticles in M840® at 20 ° C and 37 ° C are summarized in Table 2 below.

[0202] [Table 2]

[0203] The chemisorption of DOPA onto the nanoparticles was observed at 1070 cm, which corresponds to the vibrations of the P-OH and PO bonds, respectively. -1 and 1170cm -1 and 1200 cm -1 This is confirmed by DRIFT by the presence of a band between (Figure 2). The TGA analysis also confirms the chemisorption of DOPA onto the nanoparticles. The TGA analysis makes it possible to determine the actual grafting yields between 49% and 97.5% over the entire range and between 51% and 80% over the colloidal stability domain (Figure 3). These yields are advantageous since they allow to minimize the use of phospholipid molecules.

[0204] Example 2.1.2: Dispersion of magnetic nanoparticles of magnetic fluid FF1 functionalized with DOPA in Miglyol 812N® The protocol used is the same as that described in Example 2.1.1, except that the modified nanoparticles are redispersed in Miglyol 812N®. The ranges are made under the same conditions, i.e., a constant DOPA concentration (1.25 mg / mL).

[0205] The stability of nanoparticle dispersions derived from FF1 functionalized with DOPA in Miglyol 812N® was evaluated at different temperatures and different nominal coverages, as summarized in Table 3 below. The criteria designating the quality of the dispersions are: -= non-dispersible (turbid medium), += colloidal sol stable over time (clear dispersion).

[0206] [Table 3]

[0207] Example 2.1.3: Dispersion of magnetic nanoparticles of magnetic fluid FF2 functionalized with DOPA in Miglyol M840® The DOPA chemisorption protocol is identical to that described in Example 2.1.1, except for the use of nanoparticles of ferrofluid FF2.

[0208] The stability of nanoparticle dispersions derived from FF2 functionalized with DOPA in Miglyol M840N® was evaluated at different temperatures and different nominal coverages, as summarized in Table 4 below. The criteria for designating the quality of the dispersions are: - = non-dispersive (turbid medium), + = colloidal sol (clear dispersion) stable over time. The hydrodynamic diameter (D H ) value, the external alternating magnetic field (755 kHz, 10.2 kA.m -1 Under induction by application of ), different DOPA grafting rates were measured by external DLS (VASCO-FLEX, Cordouan Technologies) at 60 °C.

[0209] [Table 4]

[0210] Example 2.1.4: Dispersion of magnetic nanoparticles of ferrofluid FF3 functionalized with DOPA in Miglyol M840® The DOPA chemisorption protocol for dispersing magnetic nanoparticles in Miglyol M840® yielded a DOPA concentration of 1.10 molecules / nm 2 The dispersion is identical to that described in example 2.1.1, except for using nanoparticles of magnetic fluid FF3 by applying a nominal coverage of 1000 nm to 1000 nm. For SAR (Specific Absorption Rate) measurements and for in vivo thermal ablation experiments on tumors borne by mice (example 6), two dispersions are prepared with mass concentrations equal to 5 g / L and 300 g / L.

[0211] Example 2.2: Preparation of a dispersion of nanoparticles functionalized with phospholipid molecules with substituted polar heads: Dispersion of magnetic nanoparticles of magnetic fluid FF1 functionalized with DOPE in Miglyol M840® The protocol used is the same as that described in Example 2.1.1, except for the nature of the phospholipids used. The ranges are made under the same conditions, i.e., a constant DOPE concentration (1.25 mg / mL).

[0212] The stability of nanoparticle dispersions derived from FF1 functionalized with DOPE in Miglyol M840® was evaluated. The results are summarized in Table 5 below. The results are shown as a function of temperature and coverage. The criteria for designating the quality of the dispersions are: - = non-dispersive (turbid medium), + = colloidal sol stable over time (clear dispersion).

[0213] [Table 5]

[0214] At 52% coverage, the functionalized nanoparticles are dispersible from 35° C. At 70° C., the stability range extends to nominal coverages between 48 and 61%. The presence of substituents on the polar head affects the chemisorption rate of DOPA relative to the chemisorption rate, reducing the colloidal stability range of surface composition and temperature.

[0215] Example 2.3.: Preparation of a dispersion of nanoparticles functionalized with saturated phospholipid molecules with unsubstituted polar heads: Dispersion of magnetic nanoparticles of magnetic fluid FF1 functionalized with DSPA in Miglyol M840® The protocol used is the same as that described in Example 2.1.1, except for the nature of the phospholipid used. The ranges are made under the same conditions, i.e. a constant DSPA concentration (1.25 mg / mL).

[0216] The stability of nanoparticles derived from FF1 functionalized with DSPA in Miglyol M840N® was evaluated at different temperatures and different coverages, as summarized below in Table 6. The criteria designating the quality of the dispersion are: -= non-dispersible (turbid medium), += colloidal sol stable over time (clear dispersion).

[0217] [Table 6]

[0218] The dispersion temperature of the magnetic nanoparticles, i.e. the temperature above which the dispersion of magnetic nanoparticles becomes clear, depends on the amount of DSPA used and was determined by transmission measurements at 800 nm at each temperature according to the protocol detailed above.

[0219] Nanoparticles FF1 functionalized with DSPA are dispersible in biocompatible oils above ambient temperature from 35° C. at a nominal coverage of 24%. The temperature of the dispersion in Miglyol M840® increases with the coverage of the phospholipid molecules.

[0220] Example 3: Preparation of biocompatible oil-based magnetic fluids obtained by functionalization of magnetic nanoparticles with phospholipid molecules in the absence of acid Example 3.1.: Preparation of biocompatible oleaginous magnetic fluids by surface functionalization of nanoparticles using phospholipid molecules with substituted phosphate heads: The case of magnetic nanoparticles derived from DOPS-modified FF1 in Miglyol M840® The steric hindrance caused by certain polar heads of substituted phospholipids has the effect of slowing down the rate of chemisorption on the iron oxide surface. However, these rates can be accelerated by acting on the increase of the phospholipid concentration, i.e. by introducing said phospholipid molecules in excess relative to the monolayer of phospholipid molecules. In this case, the reaction becomes the main parameter for controlling the number of phospholipid molecules grafted by the nanoparticles.

[0221] A volume of 14.2 mL of DOPS (25 mg / mL in chloroform) was added to 1.64 mL of magnetic nanoparticles FF1 ([Fe 2 O 3]=30.5 g / L) under vortexing. The chemisorption reaction is stopped at different reaction times by adding ethanol to inhibit the reaction, inducing the aggregation of nanoparticles and precipitation of excess phospholipid molecules. The suspension is then decanted by magnet. The aggregate is washed three times with 50 mL of ethanol. During the last wash, the nanoparticles are dried under a nitrogen stream and a volume of 15 mL of Miglyol M840® is added to the nanoparticles. The nanoparticles spontaneously disperse in the oil.

[0222] The stability of nanoparticle dispersions derived from FF1 functionalized with DOPS in Miglyol M840N® was evaluated after different reaction times leading to different nominal coverages, as summarized below in Table 7. The criteria designating the quality of the dispersions are: -= non-dispersive (turbid medium), += colloidal sol stable over time (clear dispersion).

[0223] [Table 7]

[0224] TGA makes it possible to follow the DOPS chemisorption kinetics over time. Table 8 and Figure 4 show the variation of the actual grafting density of nanoparticles functionalized with 2000% excess DOPS after 7 min of reaction, washing and drying or after 30 min of reaction, washing and drying. Figure 4 shows the thermograms of the nanoparticles thus obtained functionalized with 20% excess DOPS at 7 and 30 min of reaction.

[0225] [Table 8]

[0226] Example 3.2.: Preparation of biocompatible oleaginous magnetic fluids by surface functionalization of nanoparticles using phospholipid molecules with unsubstituted phosphate heads: The case of magnetic nanoparticles derived from DOPA-modified FF1 in Miglyol M840® Phospholipid deficiency of 50% (i.e. DOPA / nm2 =0.81DOPA.nm -2 The protocol for producing the nanoparticles, FF1 ([γ-Fe 2 O 3 ]=30.5 g / L) is diluted in 44 mL of chloroform. To this dispersion, a volume of 3.18 mL of DOPA (25 mg / mL in chloroform) is added under vortexing. After 14 hours of reaction at 4° C., ethanol is added until the nanoparticles coagulate and the whole is magnetically decanted. The coagulate is washed with a chloroform / ethanol mixture (1:3, v / v) and then with ethanol. During the last wash, the nanoparticles are dried under a stream of nitrogen and a volume of 15 mL of Miglyol M840® is added to the nanoparticles. The nanoparticles disperse spontaneously in the oil.

[0227] Similarly, the nominal DOPA coverage ranges from 5% to 96% depletion relative to molecular density within a lipid membrane monolayer, with a range of 0.08 DOPA / nm 2 From 1.55 DOPA / nm 2 This corresponds to a coating density comprised between 0.01 and 0.1 and can be achieved by working with a constant DOPA concentration (1.25 mg / mL).

[0228] The stability of nanoparticles derived from FF1 functionalized with DOPA in Miglyol M840N® without the addition of acid during the reaction was evaluated at different temperatures and different nominal coverages, as summarized in Table 9 below. The criteria designating the quality of the dispersion are: -= non-dispersible (turbid medium), += colloidal sol stable over time (clear dispersion).

[0229] [Table 9]

[0230] Example 4: Comparison of oil-based and water-based ferrofluids derived from FF1, FF2 and FF3. Dispersions of nanoparticles with a mass concentration of iron oxide of 5 g / kg of solvent in a biocompatible oil phase or in an aqueous medium are prepared and their heating under magnetic induction is measured and compared. The temperature of the samples is measured by an optical fiber (OTG-M420, Opsen™) and the temperature of the samples is set at 37°C.

[0231] To utilize the different field / frequency pairs, the alternating magnetic field is generated by two different devices. The first device is a DM3 from nB nanoScale Biomagnetics, with the following conditions: 473.5 kHz and 13.36 kA.m -1 , 344.5kHz and 16.23kA.m -1 , 217kHz and 20.09kA.m -1 , and 146kHz, 21.96kA.m -1 to provide.

[0232] The second device consisted of an induction coil powered by a Minimax Junio™ 1TS 3.5 kW generator, with the additional conditions: 755 kHz, 10.2 kA.m -1 Enables the use of.

[0233] By measuring the heating rate under magnetic induction, it is possible to determine the thermal force of the nanoparticles per mass unit of the magnetic liquid, called the specific adsorption rate (SAR). The SAR is determined according to Equation 3:

[0234]

number

[0235] 0.81, 1.29 and 1.10 molecules / nm under magnetic guidance, respectively. 2 The temperature profiles of the magnetic aqueous dispersions FF1, FF2 and FF3 with an iron oxide mass concentration equal to 5 g / L are shown in Figures 5A, 5B and 5C for two magnetic field / frequency pairs (Figure 5A) 755 kHz, 10.2 kA / m, (Figure 5B) 473.5 kHz, 13.36 kA / m, (Figure 5C) 473.5 kHz, 13.36 kA / m) respectively, compared with the profiles obtained with the nanoparticles obtained in Examples 2.1.1, 2.1.3 and 2.1.4 originating from FF1, FF2 and FF3, respectively, functionalized with DOPA with a theoretical coating density of 100 kHz and dispersed in Miglyol M840®.

[0236] At 10 seconds, the temperature of the aqueous ferrofluid FF1 rises by 0.12°C, whereas for the nanoparticles derived from FF1 functionalized with DOPA dispersed in Miglyol M840®, the rise is 0.5°C, corresponding to a temperature increase of more than 4 times. Over an induction period of 100 seconds, this same increase is measured, i.e. a temperature increase of more than 4.8°C in the oil phase and only 1.2°C in the aqueous phase. As for FF2, at 10 seconds, the temperature of the aqueous dispersion FF2 rises by 0.8°C, whereas for the FF2 dispersion functionalized with DOPA and dispersed in Miglyol M840®, the temperature increase is 5.8 times higher, i.e. 5.2°C. An even higher increase is observed for the FF3 dispersion in oil: at 10 seconds, a temperature increase of 3°C is measured for the aqueous ferrofluid, whereas for the oil-based ferrofluid, the temperature increase is 12 times higher, i.e. 36°C. Thus, the oil-based dispersion of FF3 has a very rapid temperature rise and clearly superior heating properties to the aqueous dispersion of FF3.

[0237] The SAR values ​​measured in both media (aqueous and oil-based) for the three nanoparticles FF1, FF2 and FF3 are listed in Table 10 below:

[0238] [Table 10]

[0239] The SAR value at 37°C for the FF1 dispersion under stimulation at 755 kHz and 10.2 kA / m doubles between aqueous and oily media. The SAR values ​​at 37°C for the two magnetic nanoparticles with nanoflower morphology obtained from dispersions FF2 and FF3 subjected to stimulation at 473.5 kHz and 13.36 kA / m increase by a factor of 2.6 and 8.8, respectively, between aqueous and oily media.

[0240] The stability of these oil-based ferrofluids subjected to several magnetic induction cycles was also evaluated.

[0241] Oil- and aqueous-based dispersions of FF2 were subjected to two different magnetic field / frequency pairs (344.5 kHz, 16.23 kA.m -1 2 cycles at 473.5kHz, 13.36kA.m -1 The samples were subjected to four successive heating / cooling cycles (ΔT>10 K, i.e., from 37° C. to above 47° C.) under magnetic induction for 2 cycles at 37° C.

[0242] Oil and aqueous dispersions of FF3 were subjected to 473.5 kHz and 13.36 kA.m -1 The samples were subjected to two successive heating / cooling cycles (ΔT>40K, i.e., from 37° C. to over 77° C.) for two magnetic induction cycles of the magnetic field / frequency pair.

[0243] 6A, 6B, and 6C respectively show the distribution of nanoparticles FF2 and FF3 dispersed in water (5 g / kg) and the same nanoparticles functionalized with DOPA in Miglyol M840 (1.29 and 1.10 molecules / nm, respectively) during the application of an alternating magnetic field. 2In Figure 6A, the temperature profile of the dispersions in acidic conditions, examples 2.1.3 and 2.1.4) (5 g / kg) is shown for a theoretical coating density of 13.36 kA.m for two cycles at 473.5 kHz. -1 Induction heating and cooling (T 0 = 37 °C). In Figure 6B, two cycles of 344.5 kHz, 16.23 kA.m -1 Induction heating and cooling (T 0 = 37 °C). In Figure 6C, two cycles of 473.5 kHz, 13.36 kA.m -1 Induction heating and cooling (T 0 = 37°C).

[0244] The SAR values ​​measured for these aqueous and oil-based dispersions are detailed in Table 11 below.

[0245] [Table 11]

[0246] The SAR value of the magnetic nanoparticles derived from the FF2 dispersion at 37°C was 16.23 kA.m under the magnetic field / frequency conditions of 344 kHz. -1 and 473.5kHz, 13.36kA.m -1 For both, the temperature increases by a factor of 3 between aqueous and oily media, respectively. During an induction time of 10 seconds, the temperature of the aqueous dispersion FF2 increases by about 1° C., whereas for the same nanoparticles derived from FF2 functionalized with DOPA and dispersed in Miglyol M840®, the temperature increase is about 6 to 7 times higher.

[0247] The SAR value of the magnetic nanoparticles derived from the FF3 dispersion at 37°C was 13.36 kA.m under the magnetic field / frequency conditions of 473.5 kHz. -1for an induction time of 10 seconds, the temperature of the aqueous dispersion FF3 increases by a factor of 8.3 to 9.4, whereas for the same nanoparticles derived from FF3 functionalized with DOPA and dispersed in Miglyol M840®, the temperature increase is about 11 to 12 times higher. Thus, oil-based magnetic fluids could potentially make it possible to dramatically reduce the treatment time under induction.

[0248] The implementation of several induction heating cycles demonstrates that the dispersion withstands local temperature increases of the nanoparticles without changes in the nanoparticle and oil phase properties. No decomposition of the phospholipid molecules occurs on the surface of the nanoparticles. This property could be interesting from the point of view of meeting the need to increase magnetically induced hyperthermia treatment sequences.

[0249] From the temperature profiles shown in Figures 7A, 7B and 7C, the SAR values ​​were also measured by varying the dispersion liquid volume of nanoparticles FF3 (5 g / Kg) in water and oil (Miglyol M840®) from 500 μL to 1 μL.

[0250] The heating power and temperature plateau values ​​of the dispersions decrease with the volume of the dispersion due to heat losses that become increasingly dominant with increasing surface area to volume ratio. For the oil-based dispersion, the heat losses are much lower with respect to the aqueous ferrofluid due to its weak thermal conductivity and the absence of endothermic phenomena such as evaporation. For volumes of 200 and 500 μL, the heating power is still as high, 9 times higher than that of the aqueous dispersion, and no temperature plateau can be observed for these measurement conditions. For volumes of 50 and 10 μL, the temperature thresholds of 85 ° C and 75 ° C, respectively, are reached rather quickly (2-3 min) due to its still very high SAR value. The plateau values, especially the SAR value, remain much higher than those of the aqueous dispersion of FF3, about 5 to 10 times. Thus, the FF3 dispersion in Miglyol M840® shows very high SAR values ​​even for small volumes up to 10 μL.

[0251] The SAR value for a volume of 1 μL is measured by depositing the magnetic fluid directly on the tip of the sensor. In this case, the initial temperature is room temperature (T0=25° C.). A SAR of 65 W / g is only measurable for the dispersion of FF3 in Miglyol M840®. The SAR of the aqueous dispersion FF3 cannot be measured for this volume because the evaporation of water, which is manifested by a drop in the medium temperature of 7° C., is very rapid.

[0252] 8 shows the kinetic temperature profile for a 1 μL volume of dispersion FF3 dispersed in Miglyol M840® used in the animal experiment of thermal tumor ablation by magnetic hyperthermia shown in Example 6. Iron oxide Fe 2 O 3 A dispersion with a mass concentration of 300 g / L (T0=25° C., ambient room temperature) has a kinetic heating profile showing a temperature increase of 75° C. in 20 seconds, resulting in an SAR value of 53 W / g. This dispersion concentrated to 300 g / L allows a very rapid local temperature increase even for very small volumes of the order of μL, which may contribute to improving the precision of treatment in the context of thermal ablation of low-volume tumors.

[0253] Example 5: Preparation of nanoemulsion from oil-based magnetic fluid 1.12 molecules / nm in item 2.1.1 2 The magnetic nanoparticles functionalized with DOPA, obtained for a coating density of FF1, are dispersed in an oil phase composed of Miglyol 840. The oil phase containing the dispersed iron oxide nanoparticles can be used directly alone, thereby constituting 100% of the oil phase of the nanoemulsion, or it can be pre-diluted in Miglyol 840, constituting 1 / 3 or 2 / 3 of the total mass of the lipophilic phase of the nanoemulsion.

[0254] The nanoemulsion is produced by mixing a lipophilic phase (Miglyol 840) with an oil-based ferrofluid, optionally dispersing egg lecithin E80 therein at elevated temperature (70°C) to obtain a homogeneous mixture. An aqueous phase preheated to the same temperature is mixed with a cosurfactant (polysorbate 80). The emulsion and homogenization are obtained in a single step by phase inversion using a sonicator for 10 minutes. After obtaining the nanoemulsion, it remains stable after lowering the temperature, especially to a temperature of 20°C.

[0255] Exemplary compositions of Nanoemulsions 1 to 5 are detailed in Tables 12 to 16 below, where the percentages are percentages by weight based on the total weight of the nanoemulsion.

[0256] [Table 12]

[0257] [Table 13]

[0258] [Table 14]

[0259] [Table 15]

[0260] [Table 16]

[0261] Zeta potential (PZ) measurements are performed by diluting the samples 1 / 1000 in deionized water. ZP values ​​are determined by electrophoresis and laser Doppler detection using a Zetasizer Nano ZS instrument (Malvern Instruments SA, Worcestershire, UK). Example particle size characteristics (mean hydrodynamic diameter, polydispersity index (PDI) and zeta potential (PZ)) are shown in Table 17 below:

[0262] [Table 17]

[0263] The same iron oxide concentration (CFe) in magnetic fluid FF1 under induction at 473.5KHz and 13.36kA / m 2 O 3 = 12g / L, T 0 The heat generation potential of nanoemulsions 1 and 4, which shows a heat generation potential of 37° C., is compared with that of FIG.

[0264] At equal ferrofluid concentration but a larger oil phase percentage, heating is improved (compare emulsions 1 and 4).

[0265] Example 6: Thermal ablation of subcutaneous tumors by magnetic hyperthermia The efficacy of an oil-based magnetic fluid according to example 2.1.4, composed of magnetic nanoparticles FF3 functionalized with DOPA in Miglyol M840®, for magnetic hyperthermia thermal ablation of tumors in mice was evaluated.

[0266] To track the distribution of the product in the tumor medium, a lipophilic fluorophore emitting in the near infrared was pre-incorporated into the oil-based ferrofluid: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), whose emission maximum is at 780 nm.

[0267] The mice tested were B6 albino mice (B6N-Tyrc-Brd / BrdCrCrl) with tumor RM1-CMV-LucF implanted subcutaneously in the leg. Tumor volume was estimated from the l and w dimensions measured with digital calipers and calculated using the Feldman formula: volume = π / 6 × f × (l × w). 3 / 2 (f = 1.58 for female mice). This resulted in a mean of approximately 170 ± 20 mm depending on the sample. 3 Different volumes were estimated.

[0268] Considering the tumor volume, the experiment was carried out by performing intratumoral microinjection of oil-based ferrofluid with an iron oxide concentration of 300 μg / μL. The oil-based ferrofluid was injected directly into the tumor. Under isoflurane anesthesia, the injection was performed using a 10 μL Hamilton syringe with a beveled needle (26 gauge), and then the mouse was placed on a heated bed under an induction coil. For each mouse, a 473.5 kHz and 13.36 kA.m -1 A single induction treatment was performed for 15 minutes.

[0269] In the first experiment, an intratumoral injection of 2 μL of oil-based magnetic fluid FF3 (iron oxide 300 μg / μL, i.e. 600 μg mass) is performed in a new sample at a depth of 2.5 mm, split into 2 × 1 μL, with a waiting time of 1 min between two injections (performed in the same injection point). At the end of the last injection, a waiting time of 1 min is again applied before withdrawing the needle. The amount of heat dissipated by the nanoparticles in the tumor volume (Q V ) is, to a first approximation, given by the formula: Q V =m P (Fe 2 O 3 ) × SAR(1μL) × t / V 腫瘍 Under these operating conditions, the heat quantity Qv = 0.17 J / mm 3After injection, bioluminescence and fluorescence images were generated before and 24 hours after induction treatment. As shown in FIG. 10A, one thermal ablation area can be clearly observed in the center of the tumor. During the application of the alternating magnetic field, the temperature difference measured by the thermal camera was 3°C. Tumor growth, measured by the integration of bioluminescence after 24 hours, is slowed down. Ex vivo fluorescence images after tumor resection show the location of the ferrofluid injected area. Ex vivo bioluminescence images show the loss of viability of cancer cells at the site where the ferrofluid was injected (FIG. 10B).

[0270] In a second experiment, multiple injections of 3 × 1 μL of oil-based magnetic fluid FF3 (300 μg / μL, i.e., 900 μg mass) were performed at a depth of 2.5 mm, distributed in three separate sites of the tumor (Figure 11A). For each injection, a waiting time of 1 min was applied before the needle was withdrawn. Under these operating conditions, the thermal dose Qv = 0.25 J / mm 3 24 h after application of the alternating magnetic field (ΔT ≈ 20°C), tumor ablation areas in the vicinity of the ferrofluid-injected area can be easily identified. Ex vivo measurements confirm partial ablation of the tumor in the treated area (FIG. 11B).

Claims

1. A biocompatible oil-based magnetic fluid comprising iron oxide-based magnetic nanoparticles and an oil phase comprising at least one fatty acid ester, characterized in that the magnetic nanoparticles are surface functionalized with molecules of one or more types of phospholipids.

2. 2. A biocompatible oil-based magnetic fluid according to claim 1, comprising iron oxide based magnetic nanoparticles and an oil phase containing at least one fatty acid ester, characterized in that the iron oxide based magnetic nanoparticles form a colloidal dispersion in the oil phase from a temperature belonging to the range of 20-80°C, the surface of the iron oxide based magnetic nanoparticles being not completely covered, and in particular the magnetic nanoparticles are surface functionalized with molecules of one or more phospholipids that ensure a surface coverage of the iron oxide based magnetic nanoparticles such that the fatty acid esters present in the oil phase can access the surface of the iron oxide based magnetic nanoparticles.

3. 3. An oil-based magnetic fluid according to claim 1 or 2, characterized in that it does not contain water and / or does not contain a surfactant.

4. 4. An oil-based magnetic fluid according to any one of claims 1 to 3, wherein said phospholipid molecules ensure a surface coverage of the iron oxide-based magnetic nanoparticles of 19 to 76%, preferably 29 to 76%, preferentially 34 to 50%.

5. The surface density of the magnetic nanoparticles grafted with phospholipid molecules is 0.32 molecules / nm ２ ~1.22 molecules / nm ２ , preferably 0.48 molecules / nm ２ ~1.22 molecules / nm ２ , preferentially 0.56 molecules / nm ２ ~0.79 molecules / nm ２ The oil-based magnetic fluid according to any one of claims 1 to 4, characterized in that it falls within the range of

6. An oil-based magnetic fluid according to any one of claims 1 to 5, characterized in that the phospholipids contain at least one fatty chain, preferably two fatty chains, in particular C6 to C30, preferably C8 to C24, or even C10 to C22, in particular C18 saturated or mono- or polyunsaturated, branched or preferably linear hydrocarbon chains.

7. The oil-based magnetic fluid according to any one of claims 1 to 6, characterized in that the oil phase contains at least 70% by mass, preferably 80% to 95% by mass, of fatty acid esters relative to the total mass of the oil phase.

8. 8. An oil-based magnetic fluid according to any one of claims 1 to 7, characterized in that the fatty acid esters of the oil phase are selected from C6 to C12, preferably C6 to C10, saturated fatty acid triglycerides and C6 to C12, preferably C6 to C10, saturated fatty acid propylene glycols, used alone or as a mixture.

9. The content of the magnetic nanoparticles is in the range of 0.01% by mass to 50% by mass, preferably 0.1% by mass to 10% by mass, based on the total mass of the oil-based magnetic fluid. The oil-based magnetic fluid according to any one of claims 1 to 8.

10. 10. The oil-based magnetic fluid according to claim 1, characterized in that the magnetic nanoparticles are in the shape of ellipsoids, polyhedrons, such as nanocubes, bipyramids or nanostars, wafers, nanorods, nanodisks or nanoflowers.

11. The phospholipid is -O(O)P(OH)O － An oil-based magnetic fluid according to any one of claims 1 to 10, characterized in that it has a polar head, preferably selected from the salts of 1,2-dioleoyl-sn-glycero-3-phosphatidic acid and 1,2-distearoyl-sn-glycero-3-phosphatidic acid.

12. The oil-based magnetic fluid according to any one of claims 1 to 11, further comprising a lipophilic active ingredient, in particular selected from cancer treatment drugs, such as paclitaxel, docetaxel or carmustine.

13. A method for producing an oil-based magnetic fluid according to any one of claims 1 to 12, comprising the following successive steps: a- Providing an aqueous dispersion of iron oxide-based magnetic nanoparticles in an aqueous solvent, which may be water or a water / water-miscible solvent mixture; b- Removing the aqueous solvent from the aqueous dispersion of magnetic nanoparticles; c- Obtaining a colloidal sol of magnetic nanoparticles by addition of a solvent or mixture of volatile organic solvents (S2), d- surface functionalization of said magnetic nanoparticles of said colloidal sol with molecules of at least one phospholipid; e- Removing said volatile organic solvent (S2) and dispersing said functionalized magnetic nanoparticles in an oil phase comprising at least one fatty acid ester. The method includes:

14. 14. The method for producing an oil-based magnetic fluid according to claim 13, further comprising the step c2 of adding an acid after step c and before step d.

15. A medicine comprising the oil-based magnetic fluid according to any one of claims 1 to 12.

16. An oil-in-water nanoemulsion comprising 10% by mass to 30% by mass of the oil-based magnetic fluid according to any one of claims 1 to 12, an aqueous phase, and at least one surfactant.

17. A contrast agent comprising the biocompatible oil-based magnetic fluid according to any one of claims 1 to 12.

18. A biocompatible oil-based magnetic fluid according to any one of claims 1 to 12 for its use in cancer treatment by magnetically induced hyperthermia.

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