MAGNETIC ORGANIC CORE-INORGANIC SHELL MATERIAL, PROCESS FOR PRODUCING SAME AND USES THEREOF FOR THE MAGNETICALLY STIMULATED DELIVERY OF SUBSTANCES OF INTEREST

Abstract

The present invention relates to a submicrometric material consisting of a silica shell encasing a core of superparamagnetic wax, to the process for producing same and to the uses thereof, in particular for the magnetically stimulated delivery of substances of interest.

Claims

1. Material in the form of solid particles containing a fatty phase that is solid at the storage temperature of said material and a continuous shell comprising: at least one silicon oxide and enclosing said fatty phase, said fatty phase comprising a crystallizable oil having a melting point (M.sub.P) of less than 100 C. and at least one substance of interest, wherein said material is submicrometric and in that the fatty phase also comprises superparamagnetic nanoparticles surface-functionalized with at least one fatty acid.

2. Material according to claim 1, wherein the crystallizable oil is chosen from paraffins, triglycerides, fatty acids, rosins, waxes, hydrogenated plant oils and also mixtures thereof, and synthetic bitumens.

3. Material according to claim 1, wherein the diameter of the particles ranges from 400 nm to 900 nm.

4. Material according to claim 1, wherein the particles constituting said material are monodisperse.

5. Material according to claim 1, wherein the diameter of the superparamagnetic nanoparticles contained in the fatty phase ranges from 10 to 20 nm.

6. Material according to claim 1, wherein the superparamagnetic nanoparticles are maghemite nanoparticles.

7. Material according to claim 1, wherein the fatty acid is chosen from arachidic acid, stearic acid, oleic acid, palmitic acid, myristic acid, lauric acid, capric acid and caprylic acid.

8. Material according to claim 1, wherein the functionalized superparamagnetic nanoparticles represent from 0.2% to 3% by weight of the total weight of the fatty phase.

9. Material according to claim 1, wherein the thickness of the silica shell ranges from 30 to 50 nm.

10. Process for producing a material as defined in claim 1, wherein said process comprises at least the following steps: 1) preparing a fatty phase in the liquid state comprising a crystallizable oil in the liquid state having a melting point M.sub.P of less than approximately 100 C., at least one substance of interest and superparamagnetic nanoparticles functionalized with at least one fatty acid; 2) bringing said fatty phase in the liquid state of step 1) into contact with an aqueous phase (AP) brought beforehand to a temperature T.sub.AP such that T.sub.AP is greater than M.sub.P, said aqueous phase (AP) containing colloidal solid particles; 3) subjecting the liquid mixture resulting from step 2) to mechanical stirring so as to obtain an oil-in-water (O/W) emulsion formed of droplets of fatty phase in the liquid state dispersed in a continuous aqueous phase and in which the colloidal solid particles are present at the interface formed between the continuous aqueous phase and the dispersed droplets of fatty phase; 4) leaving said O/W emulsion to stand and then cooling it to a temperature T.sub.O/W such that T.sub.O/W is less than M.sub.P so as to bring about the solidification of the fatty phase and to obtain an O/W emulsion formed of globules of fatty phase in the solid state, said globules being dispersed in the continuous aqueous phase; 5) forming a shell comprising at least one silicon oxide around each of said globules by addition, to the continuous aqueous phase of the O/W emulsion of step 4), and with mechanical stirring, of at least one precursor of silicon oxide, of a surfactant SA.sub.1 and of a sufficient amount of at least one acid to bring the aqueous phase to a pH of less than or equal to 4 so as to obtain said material; 6) optionally, separating said material from the aqueous phase.

11. Process according to claim 10, wherein the colloidal solid particles are chosen from nanoparticles of silicon oxide.

12. Process according to claim 10, wherein the colloidal solid particles are surface-functionalized so as to make them more hydrophobic by adsorption of molecules of a surfactant SA.sub.2 at their surface by electrostatic bonds.

13. Process according to claim 10, wherein the magnetic stirring of step 3) is carried out using a dispersion apparatus and/or using a high-pressure microfluidizer.

14. At least one substance of interest for magnetically stimulated delivery, wherein said at least one substance of interest is within the material as defined in claim 1.

15. A contrast agent or for magnetic guidance to a target organ or target tumour in the medical imaging field, wherein said contrast agent is within the material as defined in claim 1.

Description

EXAMPLES

[0138] The starting materials used in the examples which follow are listed below: [0139] cetyltrimethylammonium bromide (CTAB), purity 98%, the company Sigma-Aldrich; [0140] diethyl ether, the company Sigma-Aldrich; [0141] solution of hydrochloric acid at 37% by weight, the company Sigma-Aldrich; [0142] technical-grade methanol, the company Sigma-Aldrich; [0143] solution of nitric acid at 69% by weight, the company Sigma-Aldrich; [0144] technical-grade acetone, the company Sigma-Aldrich; [0145] solution of ammonium hydroxide at 30% by weight, the company Sigma-Aldrich; [0146] non-hydrated ferric nitrate, purity 98%, the company Alfa Aesar; [0147] solution of ferric chloride at 45% by weight, the company Sigma-Aldrich; [0148] ferrous chloride tetrahydrate, purity 98%, the company Alfa Aesar; [0149] oleic acid, purity 90%, the company Sigma-Aldrich; [0150] stearic acid, purity 95%, the company Sigma-Aldrich; [0151] chloroform, the company Sigma-Aldrich; [0152] silica nanoparticles 7 nm in diameter, sold under the name Aerosil A380 by the company Evonik Degussa; [0153] n-eicosane (C.sub.20H.sub.42), purity 99%, melting point=36 C., the company Aldrich; and [0154] tetraethoxyorthosilane (TEOS), the company Sigma-Aldrich.

[0155] These starting materials were used as received from the producers, without additional purification.

[0156] The materials obtained were characterized by several techniques described below.

[0157] In order to determine the fatty acid concentration of the functionalized iron oxide nanoparticles, thermogravimetric analyses (TGAs) were carried out using a thermal balance sold under the trade name Setaram Instrumentation using a flow of air and by heating the sample from 20 to 800 C. with a temperature increase of 10 C. per minute.

[0158] The materials were observed using a scanning electron microscope (SEM) sold under the reference TM-1000 by the company Hitachi. The material analysed was, beforehand, either dried at ambient temperature, or freeze-dried for 12 h at 80 C. using a freeze-dryer sold under the name Alpha 2-4 LD Plus by the company Christ. All the materials were covered with gold before being observed by SEM.

[0159] The magnetic hyperthermia experiments were carried out using an induction soldering apparatus sold under the trade name Minimax Junior 1 TS from the Italian company Seit Elettronica resold by the company Maxmatic. The apparatus used comprises a MOSFET (Metal Oxide Semiconductor Field Effect Transistor) 3.5 kW generator producing a quasi-sinusoidal alternating magnetic field at a radio frequency of 755 kHz in a resonating circuit comprising a 4-turn induction coil (internal diameter of 50 mm and height of 32 mm) refrigerated by internal circulation of cold water (i.e. inside the conducting wires). The intensity of the alternating magnetic field was estimated at 10.2 kA/m at total power (747 V, 234 amps) by means of FEMM (Finite Element Model Magnetics) software for simulating magnetism problems leaving finite elements (http://www.femm.info/).

Example 1: Production and Characterizations of Materials in Accordance with the Invention

[0160] 1) Production of Materials in Accordance with the Invention

[0161] 1.1) Preparation of the Monodisperse Superparamagnetic Nanoparticles Functionalized with a Fatty Acid

[0162] Superparamagnetic nanoparticles of maghemite of formula -Fe.sub.2O.sub.3 were prepared according to the process described by Massart et al. [IEEE Transactions on Magnetics, 1981, 17, 2, 1247-1248].

[0163] Firstly, polydisperse nanocrystals of magnetite of formula Fe.sub.3O.sub.4 (or FeO.Fe.sub.2O.sub.3) were prepared in the aqueous phase by alkaline coprecipitation. To do this, 180 g of a ferrous chloride and 367 ml of a 45% ferric chloride solution (i.e. 4.1 M) were introduced, according to the non-stoichiometric molar proportions 0.9:1.5, into a solution comprising 100 ml of concentrated HCl at 37% (approximately 12.2 M) diluted in 500 ml of water. The resulting solution was then diluted with water so as to form a total volume of aqueous solution of 3 litres. The resulting aqueous solution was placed under vigorous mechanical stirring (approximately 800 revolutions per minute) and 1 litre of a concentrated aqueous ammonia solution at 30% was added as rapidly as possible in order to enable the coprecipitation. A black precipitate characteristic of magnetite was thus obtained. The resulting suspension was stirred for 30 minutes and decanted using a permanent ferrite magnet sold under the trade name Calamit Magneti having the dimensions 15210125.4 mm.sup.3, until the supernatant was colourless (at least 10 minutes). The magnet was used to accelerate the extraction of the supernatant then suction thereof by means of a vacuum flask. After washing the precipitate with 1 litre of distilled water, then again the magnetic sedimentation, the flocculate was acidified with a solution comprising 360 ml of nitric acid at 69% (15 M) diluted in 1.6 litres of distilled water. After 30 minutes of stirring, the suspension, that was now acidic, was again decanted on the permanent magnet until a clear supernatant was obtained, which supernatant was subsequently suctioned and then eliminated.

[0164] Secondly, all of the polydisperse nanocrystals of colloidal magnetite Fe.sub.3O.sub.4 previously obtained were oxidized to maghemite of formula -Fe.sub.2O.sub.3 by addition of a solution comprising 323 g of ferric nitrate (FeNO.sub.3) diluted in 800 ml of water brought to boiling (90-100 C.) with mechanical stirring. The resulting suspension turned brick red, the characteristic colour of maghemite. The precipitate was decanted on the permanent magnet, the supernatant was suctioned, then 360 ml of nitric acid at 69% diluted in 1.6 litres of distilled water were added thereto. In order to remove all the excess ferric nitrate ions, the suspension was washed once with 1 litre of acetone (stirring for 10 minutes, magnetic decanting then suctioning of the supernatant), then twice with 500 ml of diethyl ether (stirring for 10 minutes, magnetic decanting, then suctioning of the supernatant). Finally, the organic solvents were evaporated off by mechanical stirring under a suction hood, the maghemite particles having been redispersed beforehand in water acidified to a pH of approximately 2 by addition of nitric acid, so as to form a stable but polydisperse suspension of maghemite nanoparticles, with sizes ranging from 5 nm to 20 nm approximately.

[0165] Thirdly, the maghemite nanoparticles obtained were subjected to a size selection (or sorting) process as described by Massart et al. [Journal of Magnetism and Magnetic Materials, 1995, 149, 1-2, 6-7]. This process made it possible to reduce the polydispersity of maghemite nanoparticles. This particle-size sorting process is well known to those skilled in the art. It is based on phase separation by fractionation. To do this, an excess nitric acid solution (NHO.sub.3 at 15 M) was added to the stock suspension of polydisperse maghemite nanoparticles, as prepared above. This thus made it possible to decrease the pH (initially at 2) down to 0.8 and to induce an increase in the ionic strength and thus the formation of an upper phase, the supernatant, more dilute in terms of solid fraction and containing nanoparticles of smaller sizes, and of a more concentrated lower phase, attracted by the magnet, containing particles of larger sizes. The two phases were then separated after decanting by means of a magnet as described above and suctioning of the upper phase. By repeating these steps on each of the fractions, it was possible to obtain a fraction comprising sorted maghemite nanoparticles having a size of between 12 and 15 nm approximately.

[0166] The nanoparticles as prepared above were then functionalized either with stearic acid, or with oleic acid.

[0167] With regard to the nanoparticles functionalized with oleic acid, a mixture comprising the following molar proportions of oleic acid/aqueous ammonia/iron of 1/1/5 was heated at approximately 60 C. for 30 minutes with mechanical stirring. The resulting mixture separated into a foaming aqueous phase and a brown-black-coloured hydrophobic paste, which was sedimented on the permanent magnet after cooling to ambient temperature (approximately 20 C.) then washed three times with methanol and dried under vacuum for 30 min in order to remove as much water as possible.

[0168] With regard to the nanoparticles functionalized with stearic acid, a mixture comprising the molar proportions of stearic acid/aqueous ammonia/iron of 1/1/5 was heated at approximately 70 C. for 30 minutes with mechanical stirring. The resulting mixture separated into a foaming aqueous phase and a brown-black-coloured hydrophobic paste, which was sedimented on the permanent magnet after cooling to ambient temperature (approximately 20 C.), then washed three times with methanol and dried under vacuum for 30 min in order to remove as much water as possible.

[0169] The functionalization conferred a lipophilic nature on the maghemite nanoparticles, thus making it possible to incorporate them into a fatty phase as defined in the invention. Thus, the functionalized, size-sorted maghemite nanoparticles will form a stable suspension in the crystallizable oil when it is in the liquid form, and remain at the core of the submicrometric capsules during their production.

[0170] The Specific Absorption Rate or SAR (in watts per gram) of the functionalized maghemite superparamagnetic nanoparticles was measured at approximately 280 W/g in water under the alternating magnetic field conditions used (10.2 kA/m at 755 KHz). This absorption rate decreases in eicosane to approximately 8 W/g (maghemite nanoparticles functionalized with oleic acid) or 6 W/g (maghemite nanoparticles functionalized with stearic acid) probably due to the immobilization of the magnetic nanoparticles in the wax, which greatly reduces the thermal power dissipated by the oscillation of the magnetic moments (phenomenon amplified with stearic acid which is also crystalline at ambient temperature).

[0171] Thermogravimetric analyses (TGAs) showed that the maghemite nanoparticles functionalized with oleic acid comprise approximately 130 mg of oleic acid per gram of solid paste of maghemite nanoparticles functionalized with oleic acid and the nanoparticles functionalized with stearic acid comprise approximately 220 mg of stearic acid per gram of solid paste of maghemite particles functionalized with stearic acid.

[0172] 1.2) Preparation of functionalized colloidal solid particles

[0173] 1.18 g of Aerosil A380 silica nanoparticles were dispersed in 100 ml of distilled water, using an ultrasonic bath. 22.4 mg of CTAB were subsequently added to this dispersion, this amount representing approximately a factor of 0.65 of the critical micelle concentration of CTAB (CMC=0.910.sup.3 mol/l). Since the surface of the silica nanoparticles is negatively charged, the CTAB (cationic surfactant) adsorbs at the surface of the silica nanoparticles and thus makes it possible to confer a hydrophobic nature thereon. This hydrophobic nature allows them to stabilize the fatty phase-continuous aqueous phase interface of the emulsion during its preparation. A dispersion of surface-functionalized silica nanoparticles in an aqueous phase was obtained. The weight of surfactant/weight of colloidal solid particles ratio by weight was approximately 0.019.

[0174] 1.3) Preparation of the Emulsions

[0175] In order to prepare two emulsions, the compositions of which are specified in table 1 below, a given amount of solid paste of maghemite nanoparticles functionalized with oleic acid or with stearic acid, as prepared in example 1.1), was added to 18 g of eicosane (crystallizable oil), in order to obtain a suspension comprising a final concentration of iron oxide of approximately 12 g/l. The resulting fatty phase was heated to approximately 55 C. in order to melt the eicosane [step 1)] in which the functionalized, size-sorted supermagnetic nanoparticles form a homogeneous and clear suspension.

[0176] Analyses by dynamic light scattering made it possible to show that the maghemite nanoparticles functionalized with oleic acid in suspension in the fatty phase have an average hydrodynamic size of approximately 25 nm with a polydispersity index (PDI) of approximately 0.36 (measurement carried out by QELS on a suspension at 4 g/l) and the maghemite nanoparticles functionalized with stearic acid in suspension in the fatty phase have an average hydrodynamic size of approximately 24 nm with a polydispersity index (PDI) of approximately 0.4 (measurement carried out by QELS on a suspension at 2 g/l). This shows that the maghemite nanoparticles are individually dispersed (i.e. no formation of aggregates or clusters) and coated with a self-assembled monolayer of fatty acid molecules ensuring effective stearic repulsion against the Van der Waals and magnetic dipolar forces between the grains.

[0177] In parallel, the aqueous phase comprising silica nanoparticles functionalized with CTAB, in suspension as prepared in example 1.2), was heated to approximately 55 C. A given amount of fatty phase as prepared above was then gradually incorporated into a given amount of abovementioned aqueous phase [step 2)] and the whole mixture was vigorously stirred and homogenized using a stirrer sold under the name Ultra-Turrax T25 by the company Janke & Kunkel, equipped with an S25 N-25F dispersion tool, at a speed of approximately 20 000 revolutions for 1 min. In order to obtain smaller droplets of fatty phase, the resulting mixture was transferred into a high-pressure microfluidizer sold under the trade name MS110 by Microfluidics and microfluidized for approximately 30 seconds at a pressure of approximately 95 MPa. During the preparation of the emulsion, the latter was maintained at 55 C., in order to avoid any crystallization of the crystallizable oil [step 3)].

TABLE-US-00001 TABLE 1 Amount of Amount of Amount of Amount of silica functionalized fatty aqueous nanoparticles per O/W maghemite phase in the phase in the gram of fatty phase emul- nanoparticles emulsion emulsion in the emulsion sion (in g) (in g) (in g) (in mg) E-OA 0.31 18.31 101.2024 64 E-SA 0.33 18.33 101.2024 64

[0178] The average diameter of the droplets of fatty phase in the liquid state was approximately 740 nm for the E-OA emulsion and approximately 900 nm for the E-SA emulsion.

[0179] The resulting emulsion was then left to stand in an oven at 55 C. for 10 min in order to reveal the limited coalescence phenomenon. Once cooled to a temperature below the melting point of the crystallizable oil (eicosane) [step 4)], a small amount of silica nanoparticles functionalized with CTAB (solution of 0.17 g of nanoparticles functionalized with CTAB, dispersed in 4.8 ml of water) was added to the E-OA emulsion [step 4)]. The addition of this supplementary amount of colloidal solid particles makes it possible to prevent the aggregation of the wax particles and allow the storage of the emulsion at ambient temperature. Approximately 119.5 g of each of the emulsions E-OA and E-SA comprising globules of fatty phase in the solid state, dispersed in a continuous aqueous phase, were thus obtained.

[0180] 1.4) Preparation of the Submicrometric Capsules in Accordance with the Invention: Formation of the Acidic Shell (Mineralization Step)

[0181] In this step [step 5)], the formation of the silica shell around the globules of fatty phase in the solid state was carried out.

[0182] The two emulsions E-OA and E-SA were diluted from 18% by weight to 2% by weight and the pH of the emulsions was adjusted to approximately 0.2, that is to say to a value below the isoelectric point of silica, by addition both of 7 g of a solution of hydrochloric acid at 37% by weight (approximately 12.2 M) and of 80 g of an aqueous solution containing 0.21 g of CTAB.

[0183] 5 g of TEOS were then added dropwise to the two emulsions in order to reach the amount denoted in Table 2 below. During the addition, the solution was placed under magnetic stirring at a speed of 450 rpm, this speed not modifying the size distribution of the drops. The resulting dispersion was placed in 50 ml test tubes overnight with continuous stirring on a wheel at 25 rpm in a thermostated chamber at 20 C. so as to allow the silica shell to form (mineralization).

[0184] At the end of the mineralization, submicrometric capsules of silica were recovered after several cycles of centrifugation-redispersion several times in distilled water [step 6)]. The material obtained was stored in pure water for several months. No modification of the submicrometric capsules was observed during this period.

TABLE-US-00002 TABLE 2 Amount of CTAB Amount of Emulsion per g of TEOS (in g) TEOS (in M/m.sup.2) E-OA 0.042 0.039 E-SA 0.042 0.043

[0185] 2) Results of the Characterizations

[0186] The appended FIG. 1 represents an SEM photograph taken during the observation of a material in accordance with the invention obtained by mineralization of the E-SA emulsion (FIG. 1a), then its size distribution showing a material comprising particles having a submicrometric size centred around 825 nm (FIG. 1b), and finally an SEM photograph taken during the observation of a material in accordance with the invention obtained by mineralization of the E-SA emulsion after breaking of the envelope by application of a radio frequency of alternating magnetic field (FIG. 1c). In FIGS. 1a and 1c, the scale bar represents 5 m and on FIG. 1c, the white arrow points to the breaking zone brought about by the expansion of the fatty phase.

Example 2: Release Profile of the Materials in Accordance with the Invention

[0187] In this example, the breaking of a material in accordance with the invention obtained by mineralization of the E-SA emulsion is illustrated.

[0188] The material was exposed to an alternating magnetic field at a radio frequency of approximately 755 kHz and an intensity of approximately 10.2 kA/m for a variable time: 600, 1380, 2100, 3000 and 7200 seconds.

[0189] FIG. 2 shows the amount of crystallizable oil released (as percentage) as a function of the time of application of the alternating magnetic field (in seconds).

[0190] According to FIG. 2, it appears that approximately 20% of the oil is released after 50 minutes (3000 s) of application of a radio frequency alternating magnetic field of 10.2 kA/m at 755 kHz, up to reaching 40% after 2 hours (7200 s) of application of a radio frequency alternating magnetic field of 10.2 kA/m at 755 kHz.

[0191] It is possible to accelerate the rate of release of the oil and thus that of a substance of interest by increasing the amount of superparamagnetic nanoparticles in the core of the capsules (modification of the fatty acid for example) and/or their specific absorption rate by means of a modification of the shape and of the type of superparamagnetic nanoparticles used, for example with nanocubes or nanoflowers, that is to say multi-core nanoparticles, which can achieve SAR values of more than 1000 W/g.