METHOD FOR MANUFACTURING A BIOCOMPATIBLE FLUID COMPRISING A POWDER OF MAGNETIC PARTICLES, BIOCOMPATIBLE FLUID COMPRISING A POWDER OF MAGNETIC PARTICLES

Abstract

A method for manufacturing a biocompatible fluid including a powder of magnetic particles of elongated shape having a magnetic shape anisotropy and having a final granulometry, the final granulometry being defined by a first average size of the particles in a first direction and a second average size in a second direction different from the first direction, the final granulometry further being defined by a first distribution width of the first sizes and a second distribution width of the second sizes, the method including from a powder of magnetic particles having an initial granulometry different from the final granulometry, modification of the initial granulometry by milling and/or by sintering of the powder until the final granulometry is obtained; introduction of the powder of magnetic particles into a biocompatible fluid.

Claims

1. A method for manufacturing a biocompatible fluid comprising a powder of magnetic particles of elongated shape having a magnetic shape anisotropy and having a final granulometry, said final granulometry being defined by a first average size of the particles in a first direction and a second average size in a second direction different from the first direction, the second average size being less than 1.5 times the first average size, said final granulometry further being defined by a first distribution width of the first sizes and a second distribution width of the second sizes, said method comprising: from a powder of magnetic particles having an initial granulometry different from the final granulometry, modifying the initial granulometry by milling and/or by sintering of the powder until the final granulometry is obtained; introducing the powder of magnetic particles into a biocompatible fluid, the first average size of the magnetic particles being comprised between 0.2 m and 10 m and the distribution width of the first sizes representing at least 30% of the value of the first average size.

2. The method for manufacturing a biocompatible fluid according to claim 1, wherein the first average size of the particles is comprised between 0.2 m and 5 m.

3. The method for manufacturing a biocompatible fluid according to claim 1, wherein during the modifying of the initial granulometry, the milling of the powder of magnetic particles having the initial granulometry is followed by the sintering of the powder resulting from the milling or the sintering of the powder of magnetic particles having the initial granulometry is followed by the milling of the powder resulting from the sintering.

4. The method for manufacturing a biocompatible fluid according to claim 1, wherein the powder of final granulometry is of same chemical nature as the powder of initial granulometry.

5. The method for manufacturing a biocompatible fluid according to claim 1, further comprising performing a chemical functionalisation of the particles.

6. The method for manufacturing a biocompatible fluid according to claim 5, wherein the chemical functionalisation comprises an encapsulation of at least one part of the particles in an inorganic layer.

7. The method for manufacturing a biocompatible fluid according to claim 6, wherein the inorganic layer is made of silica.

8. The method for manufacturing a biocompatible fluid according to claim 5, wherein the chemical functionalisation includes grafting polymers on the surface of the particles or of the inorganic layer.

9. The method for manufacturing a biocompatible fluid according to claim 8, wherein the grafted polymer includes polyethylene glycol (PEG).

10. The method for manufacturing a biocompatible fluid according to claim 1, wherein the magnetic particles are grains including a metal oxide.

11. The method for manufacturing a biocom patible fluid according to claim 1, further comprising refining the size distribution of the particles in solution.

12. A biocompatible fluid comprising a powder of magnetic particles of elongated shape having a magnetic shape anisotropy and having a final granulometry, said final granulometry being defined by a first average size of the particles in a first direction and a second average size in a second direction different from the first direction, the second average size being less than 1.5 times the first average size, said final granulometry further being defined by a first distribution width of the first sizes and a second distribution width of the second sizes, the first average size of the magnetic particles being comprised between 0.2 m and 10 m and the distribution width of the first sizes representing at least 30% of the value of the first average size.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Other characteristics of the invention will become clear from the description that is given thereof below, for indicative purposes and in no way limiting, while referring to the figures, among which:

[0105] FIG. 1 schematically represents the top-down type approach for manufacturing magnetic particles according to the prior art;

[0106] FIG. 2 represents scanning electron microscope images of magnetic particles produced using the technique illustrated in FIG. 1;

[0107] FIG. 3 illustrates examples of SAF type magnetic particles according to the prior art;

[0108] FIG. 4 schematically illustrates the steps of the method for manufacturing a biocompatible fluid according to an embodiment of the invention;

[0109] FIG. 5 is an electron microscope image of the particles of the biocompatible fluid according to the invention;

[0110] FIG. 6 represents a ball mill used to modify the granulometry of a powder;

[0111] FIG. 7 schematically illustrates the functionalisation of magnetite particles with organosilanes;

[0112] FIG. 8 schematically illustrates the functionalisation of magnetic particles with biotin/streptavidin-PE fluorophores;

[0113] FIG. 9 shows on the left particles according to an embodiment of the invention functionalised with a fluorophore and on the right non-functionalised particles according to the invention;

[0114] FIG. 10 illustrates the uniform magnetic field at the centre of a Halbach cylinder.

DETAILED DESCRIPTION

[0115] FIG. 4 schematically illustrates the steps of the method for manufacturing a biocompatible fluid including magnetic particles according to an embodiment of the invention.

[0116] During step G1, a powder of particles having a final granulometry and intended to be dispersed in a biocompatible fluid is obtained from an initial powder of magnetic particles.

[0117] The final granulometry is characterised by a first average size of the particles in a first direction and a second average size of the particles in a second direction.

[0118] The initial powder has an initial granulometry characterised by an average size of the particles. The particles of the initial powder may for their part also have an elongated shape.

[0119] If the average size of the particles of the initial powder is greater than the characteristic average sizes of the targeted final granulometry, the final powder is obtained by milling BR of the initial powder.

[0120] For example, it is possible to mill an initial powder of magnetite particles having an initial average size of 5 m to obtain a final powder of magnetite particles having an average size of 2 m.

[0121] Alternatively, if the initial powder has an average size of the particles smaller than the average sizes of the target granulometry, it is possible to obtain the final powder by sintering FR of the initial powder.

[0122] Beneficially, the steps of milling BR and sintering FR may be carried out in sequence, to adjust the granulometry of the powder until the desired final granulometry is obtained.

[0123] The anisotropy of the shapes obtained results both from the size and shape dispersion of the initial powder and from the random character of impacts leading to fracturing of the grains. In the event where the initial particles have an elongated aspect ratio, the particles obtained after moderate milling conserve an elongated aspect ratio (even if this aspect ratio can decrease).

[0124] For example, if the powder obtained after the milling step comprises a too small average size of the particles, it is possible to increase the size of the particles by sintering.

[0125] Once the final powder has been obtained, it is transferred into a biocompatible fluid during the step FL.

[0126] The transfer FL may be carried out by capture of the particles against the wall of a container using a magnet or a magnetic field, emptying the initial liquid and adding a biocompatible fluid.

[0127] An alternative consists in recovering the particles by dipping into the container containing them a magnetic device of which the radiation field may be activated or deactivated. The particles will be attracted and maintained against the walls of the device when the radiated field is activated and released into a second biocompatible liquid when the radiated field is deactivated.

[0128] According to an embodiment, the method P further includes a step of modification of the granulometry of the particles in solution, after milling or sintering and the transfer of the powder into the biocompatible fluid. This step consists in refining the size distribution of the particles in solution notably by filtration using filters-syringes or paper filters, by suspension/decantation, by magnetic separation, or by centrifugation.

[0129] FIG. 5 represents an electron microscope image of the magnetic particles used in the method P according to an embodiment of the invention. The powder has a final granulometry obtained from an initial granulometry by milling/sintering. The particles comprised in the final powder have irregular shapes.

[0130] In support of the invention, it is observed that magnetisation measurements on synthetic magnetite crystals, with sizes between 0.3 m and 30 m, or on bulk samples of magnetite of natural origin, or on pads lithographed up to diameters of 300 nm indicate a remanence of the magnetisation less than 0.2. See for example, the documents Grain size dependence of low-temperature remanent magnetization in natural and synthetic magnetite: Experimental study published in Earth Planet Space 61, 119 (2009) of A. V. Smirnov et al.

[0131] These different elements indicate that the magnetic properties of magnetite are suited to the targeted use, that these properties are robust with regard to the shape and the size, and are not very sensitive to the particular elaboration conditions.

[0132] FIG. 6 represents a ball mill, known to those skilled in the art. Such a ball mill is used to modify the initial granulometry by reducing the size of the particles.

[0133] Planetary ball mills make it possible, in the case of laboratory models, to manufacture in a single operation, which only lasts several hours, quantities of powder that range from less than one gram (with a single 12 ml vessel) to several hundreds of grams (when several 500 ml vessels are used in parallel). These quantities are several orders of magnitude greater than those obtained by top-down approaches and easily cover the needs for the treatment in parallel of several tumours or tissues, in animals or humans. Furthermore, the quantities are such that it is easily possible to conduct in parallel physical-chemical characterisation studies (e.g. size and surface potential measurements by DLSDynamic Light Scattering), or chemical functionalisation studies requiring several milligrams or grams of material.

[0134] Other technologies, like the mills used in the pharmaceutical industry, make it possible to mill powder loads of several kilograms up to micronic granulometries. Such volumes fall within the scope of an industrial exploitation of particles, and cannot be envisaged with top-down approaches since the cost price per gram is so high.

[0135] The composition of the initial powder is similar to the composition of the desired particles. An alternative consists in carrying out a total or partial oxidation of an iron powder, or to carry out the mechanical synthesis of an iron oxide by milling.

[0136] An example of use of a standard ball bill for reducing or modifying the granulometry of a magnetite powder is the following.

[0137] The magnetite powder is introduced into a 50 ml milling vessel, made of zirconium oxide, with a certain number of balls of same material, of centimetric diameter. A characteristic of the vessel and the balls is to be constituted of a material of hardness greater than that of the ground powder. For example, the hardness of zirconium oxide on the Mohs scale is 8, that of magnetite is 5.5.

[0138] A second characteristic of the vessel and the balls is not to release toxic contaminants during milling. This is the case of zirconium oxide, but it is not the case of steel (which on milling releases chromium). An alternative consists in using a vessel and balls of another hardness, but not releasing toxic contaminants.

[0139] The magnetite load represents around one third of the volume of the milling vessel, which represents on average 2 g of magnetite for a 50 ml vessel. A certain quantity of liquid may be added to the load to facilitate the milling thereof, for example 20 ml of isopropanol. Other adjuvants may be added, in variable quantity, for example oleic or stearic acid, which favour the dispersion of the ground particles.

[0140] An alternative consists in adding a certain amount of water to induce an oxidation reaction of the particles during milling and to modify the chemical composition thereof.

[0141] The vessel is hermetically sealed by a cover. The milling results from the off-centre rotation of the vessel, for example at 600 rpm for 2 hours. The milling times and/or the energy of the balls are adjusted as a function of the initial and final granulom etries.

[0142] An alternative consists in using another milling technique or apparatus, different by the type of movement imposed on the milling vessel and by the nature of the impact of the balls with the powder.

[0143] At the end of milling and as a function of the conditions used, the granulometry of the powder is reduced, with for example a size distribution such that the largest dimension is centred on 2 m with a distribution of 30% or more. The shape of the particles is furthermore irregular.

[0144] According to an embodiment of the invention, the method P for manufacturing a biocompatible fluid including magnetic particles further comprises a step of chemical functionalisation of the particles.

[0145] Very often, the particles are functionalised using compounds that procure a stabilisation of the structure, a protection against oxidation (notably for iron particles), a steric or electrostatic barrier to agglomeration and/or which make it possible to circulate in an organism or a tissue, or to interact with a biological tissue or a cell either to adhere thereto, to penetrate therein, or to deliver therein in a targeted manner a drug or any other active substance.

[0146] Beneficially, the grafting of polymers or the encapsulation in silica, used for the stabilisation of the particles, procure a steric repulsion. If, in addition, the functionalised layer or the polymer chains are charged, they induce an electrostatic repulsion between particles, which reduces magnetic attraction effects and increases the dispersion effect.

[0147] For example, the steric repulsion effect is obtained by grafting of polyethylene glycol (PEG), which forms a layer of variable thickness on the surface of the particle from 8 nm (PEG 1k) to 15 nm (PEG 5k). The thickness of the PEG layer grafted on the particle imposes a minimum approach distance between the magnetic particles.

[0148] If each particle is assimilated with a magnetic dipole, the magnetic interaction energy decreases with the inverse of the cube of the distance d between the particles. The grafting of long molecules on the surface of the particles reduces this interaction effect. The magnetic interaction energy decreasing in 1/d.sup.3, this diminishes the effect thereof.

[0149] The grafting of PEG also prevents the opsonisation of the particles, which reduces their elimination by phagocytes and extends their lifetime in the organism, see for example the document Effect of polyethyleneglycol (PEG) chain length on the bio-nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells, published in Nanoscale 6, 2782 (2014) of Pozzi et al.

[0150] Beneficially, the functionalisation of the particle may enable the transport of substances for therapeutic use, like the selective targeting of a tissue or a cell, by grafting of antibodies. This functionalisation is ensured by the prior grafting of thiols (for particles covered with goldmost current particles) or amine groups (for the magnetite particles of the invention), see for example the document Functionalization of Fe.sub.3O.sub.4 NPs by Silanization, published in Materials. 9, 826 (2016) of S. Villa et al.

[0151] According to a particular embodiment, the functionalisation may be obtained by encapsulation in an inorganic matrix of silica then grafting of an organosilane, as is shown in FIG. 7.

[0152] The silica precursor is tetraethoxysilane (TEOS) and the organosilane is 3-aminopropyltriethoxysilane (APTES). The amine functional group of APTES makes it possible to maintain the hydrophilic character of the surface and to graft a biomolecule.

[0153] The encapsulation of the particles with silica may be carried out in the following manner: in a two-necked round bottom flask, 6 mg of Fe3O4 particles, 20 ml of absolute ethanol and 100 ml of ultra-pure water, ultrasounds for 15 min at 40 C. Successive addition of 400 L of ultra-pure water, 900 L of ammonia solution (28% aq.) and 120 L of TEOS. Stirring at 40 C. for 2 h.

[0154] The functionalisation with APTES is then carried out in the following manner: in a two-neck round-bottom flask, 1 ml of Fe3O4@SiO2 (60 mg/L) in ultra-pure water added to 1 ml of ethanol and 43 L of APTES (2% v/v). Stirring at 50 C. for 24 h.

[0155] The efficiency of the functionalisation of the particles is verified by the grafting of a fluorophore, phycoerythrin (PE) coupled to streptavidin. The cross-linker Nhydroxysuccinimide-Biotin (NHS-Biotin) is used for the formation of an amide bond with the amine group of the APTES, then the streptavidin-PE is bound to the biotin.

[0156] For a fluorescence functionalisation, the following method may be used: in a Eppendorf tube the particles are left in contact with 4 L of NHS-Biotin (10 mM) and 396 L of phosphate buffered saline at pH 8 (PBS 8), 1 h under vortex. Rinsing three times with PBS 8, twice with PBS 7.4 and suspension in 100 L of PBS 7.4 then stirring under vortex. Addition of 5 L of Streptavidin-PE and stirring for 15 minutes under vortex and protected from light. Rinsing three times with PBS 7.4 then deposition on microscope slide for observation with light between 520 and 550 nm.

[0157] The results of the fluorescence functionalisation are illustrated in FIG. 9, which shows: [0158] On the left, fluorescence optical microscope image of the functionalisation of magnetite particles with APTES; [0159] On the right, fluorescence optical microscope image of non-functionalised control particles.

[0160] In these figures a fluorescence emission is observed uniquely for the functionalised particles, which confirms the efficiency of the functionalisation.

[0161] The particles intended to be placed in the presence of living tissues or cells are, on coming out of the mill where they are dispersed in isopropanol, conditioned in the following manner. The particles are attracted to the bottom of the container where they are found by means of a magnet; the greatest part of the isopropanol is removed using a pipette: the isopropanol is replaced by ethanol; still while attracting the particles to the bottom of the container and by removing the liquid by pipette, three rinsings using the culture medium are carried out.

[0162] The particles are placed in the presence of cells or tissues by direct addition, to the recipient where they are found, of the particleculture medium solution described. An incubation period, for example 24 hours, may be respected between the placing in presence of the particles and the tissues or the cells to enable the diffusion of the particles within the medium and/or the grafting or the incorporation of the particles on the target species.

[0163] The particles intended for microscopic observations, or for measurements or characterisations where it is desirable that they are dispersed on a surface (e.g. magnetic measurements), are, on coming out of the mill where they are dispersed in isopropanol, dispersed in the following manner. The isopropanol is replaced, by the technique described previously, by an inert solvent with high vapour pressure (e.g. acetone). The substrate intended to receive the particles, for example a silica substrate of the order of a square centimetre, is placed in a magnetic field as high as possible, perpendicular to the surface thereof. This may be done by laying the substrate on a powerful permanent magnet. If possible, the substrate is heated to a temperature slightly below the boiling temperature of the solvent. In the case of acetone at ambient pressure, this temperature may be 50 C. A drop of particles in solution is deposited rapidly on the substrate. If the wetting of the drop is rapid and if the evaporation of the drop occurs quickly: 1) the thickness of the drop that spreads/evaporates remains low; 2) the formation of chains of particles, of which the magnetic orientation would here be perpendicular to the surface, is limited by the rapid dispersion on the surface of the substrate and the low thickness of liquid in evaporation. The spreading rate of the drop may also, depending on the nature of the solvent, be accelerated by a surface treatment that increases the substrate/liquid affinity.

[0164] The vibration of the particles (conditioned beforehand and placed in the presence of the tissues or cells to treat according to the described method) is obtained by subjecting them to a field variable in modulus and/or in direction. One solution is to use a Halbach cylinder illustrated in FIG. 10. This cylinder is composed of permanent magnets arranged in sectors and comprises at its centre a cylindrical cavity where a homogeneous magnetic field H reigns, of the order of several tens of teslas, oriented perpendicularly to the axis of the cylinder. The sample of biological tissues or cells to treat, with the magnetic particles, is placed at the centre of the cylinder using the appropriate support, according to whether they are culture cells, living tissues, or potentially a mouse.

[0165] The rotation of the cylinder generates a turning field which makes the particles placed in the cavity oscillate, and will lead to a magnetic-mechanical torque.

[0166] An aspect of the invention also relates to a biocompatible fluid comprising a powder of magnetic particles of elongated shape. The elongated shape of the particles determines a magnetic shape anisotropy which enables them to be vibrated thanks to the application of a magnetic field variable over time.

[0167] The powder of magnetic particles has a final granulometry defined by a first average size of the particles in a first direction and a second average size of the particles in a second direction. The final granulometry is further defined by a first distribution width of the first sizes and a second distribution width of the second sizes. The first average size of the particles is comprised between 0.2 m and 5 m. The distribution width of the first sizes is greater than or equal to 30% of the value of the first average size.

[0168] Beneficially, the magnetic shape anisotropy enables the efficient vibration of the particles in the presence of a magnetic field variable over time.

[0169] According to an embodiment, the second average size is less than 1.5 times the first average size.

[0170] Beneficially, this difference between the first average size and the second average size makes it possible to obtain a high magnetic shape anisotropy and thus to increase the magnetic-mechanical torque in the presence of an external variable magnetic field.

[0171] According to an embodiment, the magnetic particles are made of iron oxide.

[0172] According to an embodiment, ferromagnetic iron oxide is selected from a group including: magnetite, maghemite or a combination of these materials.

[0173] Beneficially, these materials are biocompatible and suited to destruction of cancerous human or animal cell or tissue type applications.

[0174] The magnetic particles present in the biocompatible fluid may further be chemically functionalised, as explained with reference to the functionalisation step of the method according to an embodiment of the invention.

[0175] The biocompatible fluid according to the invention may be used for the destruction of cancerous cells by magnetic-mechanical vibrations according to the following experimental process.

[0176] The magnetic particles are transferred into a biocompatible liquid, with a typical concentration of 10.sup.7 particles/ml.

[0177] The particles may be functionalised.

[0178] The functionalisation may consist in the grafting of a compound enabling the targeted fastening of the particle on a tissue, a cell or a preferential site of the cell wall.

[0179] The grafted compound may be an antibody, which enables the particle to attach itself to the surface of certain specific cells.

[0180] The functionalisation may consist in the grafting of a compound that ensures or favours endocytosis of the particles, by the targeted cells.

[0181] The functionalisation may have the aim of ensuring better circulation and longer lifetime of the particle in the organism or the tissue, or on the contrary to have as aim to reduce the mobility and ensure the maintaining of the particle as close as possible to the location where it has been positioned, for example during injection within a tissue.

[0182] The localisation of the particle at the spot where the magnetic-mechanical vibration has to be exerted is achieved by one or more of the following means: targeted functionalisation; displacement of the particles under the effect of a magnetic field gradient, whether it is internal or external; direct injection within the tissues to treat.

[0183] When the particles are in place, they are made to vibrate by the application of a variable external field. An exemplary embodiment is the use of a rotating Halbach cylinder. The Halbach cylinder generates in a cylindrical cavity a magnetic field, perpendicular to the axis of the cavity. The rotation of the cylinder creates a turning field.

[0184] The intensity of the magnetic field is of the order of 0.2 T to 1 T. The frequency of rotation of the magnetic field is of the order of 10 Hz to 30 Hz. The duration of a treatment is of the order of 5 minutes to 1 hour.

[0185] The treatment may be carried out on culture cells. These cells are for example derived from cancerous cell lines, human or animal. The cells are placed in the presence of the particles, before carrying out the treatment, for an incubation time of a typical duration of 24 hours.

[0186] The cells and the particles may be placed in wells, in a suitable nutrient liquid.

[0187] The cells and the particles may be integrated in a gel, or in a structure procuring for them a three-dimensional growth substrate.

[0188] The aim of the application of the magnetic-mechanical vibrations is to trigger cellular death under application of magnetic-mechanical vibrations inside the cell, on the surface of the cell, or in the medium surrounding the cell. The cells particularly targeted by this application are cancerous cells.

[0189] An alternative of the application of the magnetic-mechanical vibrations may be to modify or to orientate cellular division, or to modify or to orient tissue growth. This application aims to promote the regeneration of tissues by the stimulation of their growth, notably those of the spinal cord.