Ferrofluids stable in neutral media and modified ferrofluids modifies obtained by modification of the surface of the particles of said ferrofluids

Abstract

The invention relates to aqueous dispersions, comprising particles based on a magnetic iron oxide with dimensions of 20 nm, the surface of which is modified by the grafting of aminated groups R with a covalent bonding to the surface of said particles, wherein the isoelectronic point of particles with such a modified surface is 10. The invention further relates to a method for production of said aqueous suspensions and a method for modification of the surface of the particles present in said dispersions, in particular, by the immobilisation of polysaccharides such as dextrans, particularly for the formulation of magnetic compositions which may be administered in vivo and in particular for the formulation of injectable compositions of contrast agents for MRI.

Claims

1. An aqueous dispersion comprising particles (p) based on a magnetic iron oxide, said particles having dimensions of less than or equal to 20 nm, and a surface of said particles being modified by a grafting of aminated groups (R) covalently bonded to the surface of the particles, wherein, the aminated groups (R) are selected from the group consisting of: (CH.sub.2).sub.3NH.sub.2; (CH.sub.2).sub.4NH.sub.2; (CH.sub.2).sub.3NH(CH.sub.2).sub.2NH.sub.2; (CH.sub.2).sub.3NH(CH.sub.2).sub.6NH.sub.2; (CH.sub.2).sub.3NHCH(CH).sub.3CH.sub.2NH.sub.2; (CH.sub.2).sub.3NH(CH.sub.2).sub.2NH(CH.sub.2).sub.2NH.sub.2; ##STR00003## and the isoelectric point of the particles with the surface so modified is greater than or equal to 10.

2. The aqueous dispersion of claim 1, wherein said dispersion has a pH of less than or equal to 8 and said dispersion of particles has an average hydrodynamic diameter of at most 20 nm.

3. The dispersion of claim 2, wherein the average hydrodynamic diameter of the particles (p) is between 3 and 15 nm.

4. The dispersion of claim 1, wherein the particles (p) consist essentially of maghemite (-Fe.sub.2O.sub.3), or monocrystalline maghemite.

5. The dispersion of claim 1, wherein the aminated groups (R) are bonded to the surface of the particles (p) via a bond: ##STR00004##

6. A process for the preparation of the dispersion of claim 1, comprising the stages of: (A) providing an acidic aqueous dispersion of particles (p.sub.0) based on a magnetic iron oxide with dimensions of less than 20 nm, said dispersion exhibiting, in an acidic medium, a colloidal stability at least within a pH range, this stability being such that, within said pH range, a dispersion of essentially separate particles having an average hydrodynamic diameter of less than 20 nm is observed, without having to constantly stir the dispersion; (B) bringing the acidic colloidal dispersion of stage (A) into contact with silanes of formula RSiX.sub.1X.sub.2X.sub.3, wherein: R denotes an aminated group; X.sub.1, X.sub.2 and X.sub.3 are identical or different groups, each denoting a group which can be hydrolyzed in an acidic medium, this contacting operation being carried out while maintaining the medium within the pH range where the colloidal stability of the dispersion is ensured; (C) adding to the reaction medium a water-soluble wetting agent with a boiling point greater than that of water and then heating the reaction medium to a temperature sufficient to remove the water but without removing the wetting agent; and (D) recovering the particles obtained on conclusion of stage (C) and dispersing them in an aqueous medium.

7. The process of claim 6, wherein the acidic aqueous colloidal dispersion of stage (A) is such that, in the pH range where the colloidal stability is ensured, the average hydrodynamic diameter of the particles which are observed in suspension is between 3 and 15 nm.

8. The process of claim 6, wherein the acidic aqueous colloidal dispersion of stage (A) is such that, in the pH range where the colloidal stability is ensured, less than 5% by number of the solid entities which are observed in suspension are agglomerates of several particles.

9. The process of claim 6, wherein the silanes employed in stage (B) are aminated trialkoxysilanes of formula RSi(OR)(OR)(OR) in which: R is an aminated group; and R, R and R, which are identical or different, each denotes an alkyl group comprising from 1 to 5 carbon atoms.

10. The process of claim 9, wherein the silanes of stage (B) are selected from: -aminopropyltrimethoxysilane, of formula:
(CH.sub.3O).sub.3Si(CH.sub.2).sub.3NH.sub.2; N-(-aminoethyl)--aminopropyltrimethoxysilane, of formula:
(CH.sub.3O).sub.3Si(CH.sub.2).sub.3NH(CH.sub.2).sub.2NH.sub.2; and N-(-aminoethyl)-N-(-aminoethyl)--aminopropyltrimeth-oxysilane of formula:
(CH.sub.3O).sub.3Si(CH.sub.2).sub.3NH(CH.sub.2).sub.2NH(CH.sub.2).sub.2NH.sub.2.

11. The process of claim 6, wherein: the silanes of stage (B) are introduced in solution in an organic solvent; the wetting agent of stage (C) is soluble in the organic solvent which dissolves the silanes introduced in stage (B) and has a boiling point greater than that of said organic solvent; and stage (C) includes a heating step at a temperature sufficient to remove said organic solvent without removing the wetting agent.

12. The process as claimed in claim 6, wherein, in stage (C), the wetting agent is glycerol.

13. The process as claimed in claim 6, wherein the heating of stage (C) is carried out under vacuum.

14. The process as claimed in claim 13, wherein the dehydration of stage (C) is carried out at a temperature of less than or equal to 130 C.

15. The process as claimed in claim 6, wherein stage (D) comprises a washing of the particles obtained on conclusion of stage (C), carried out without allowing the particles to dry, followed by dispersion, in an aqueous medium, of the undried flocculate of particles which is obtained.

16. The process as claimed in claim 6, wherein the dispersion of the particles which is produced during stage (D) is produced by placing the particles recovered on conclusion of stage (C) in water and by gradually reducing the pH of the medium by slow addition of an acid.

17. A composition comprising the dispersion as claimed in claim 1, the composition formulated to be administered orally or parenterally to humans or animals.

18. A composition for administration to man or animals, comprising a dispersion as claimed in claim 1, and a physiologically acceptable vehicle.

19. A process for the modification of the surface of the particles (p) of a dispersion as claimed in claim 1, comprising a stage (G1) which consists of reacting said dispersion with chemical entities E capable of forming a bond with the aminated groups R present at the surface of the particles (p), at a pH of less than 8.

20. The process as claimed in claim 19, wherein the chemical entities E exhibit aldehyde groups, the aminated groups R exhibit NH.sub.2 groups and stage (G1) consists in reacting the dispersion with chemical entities E carrying CHO groups in the presence of a reducing agent.

21. The process as claimed in claim 20, wherein the entities E are molecules of polysaccharides, a portion of the OH groups of which have been oxidized to give CHO groups.

22. The process as claimed in claim 21, wherein the entities E are dextran molecules, a portion of the OH groups of which have been oxidized to give CHO groups.

23. An aqueous dispersion of particles based on a magnetic iron oxide with a modified surface at the surface of which are immobilized chemical entities E, obtainable as claimed in the process of claim 19.

24. The dispersion as claimed in claim 23, wherein at least 90% by number of the solid components which it comprises are separate particles comprising a single central core based on a magnetic iron oxide having dimensions of less than 20 nm.

25. The dispersion as claimed in claim 23, in which the entities E are polysaccharides, for example dextran molecules, a portion of the OH groups of which have been oxidized to give CHO groups.

26. An aqueous dispersion of particles based on a magnetic iron oxide at the surface of which are immobilized, by covalent bonding, molecules of polysaccharides bonded to the surface via covalent bonds of formula NHCH.sub.2, said suspension being obtainable by the process of claim 21.

27. An aqueous dispersion of particles based on a magnetic iron oxide at the surface of which are immobilized, by covalent bonding, dextran molecules via covalent bonds NHCH.sub.2, this dispersion being obtainable by the process of claim 22.

28. The dispersion as claimed in claim 26, wherein the average hydrodynamic diameter of the particles with a surface modified by the molecules of polysaccharides is less than 50 nm.

29. The dispersion as claimed in claim 25, wherein at least 90% of the solid components in suspension are separate particles comprising a single central core, based on an iron oxide, having dimensions of less than 20 nm, this core being surrounded by a layer comprising the covalently bonded molecules of polysaccharides.

30. The dispersion as claimed in claim 25, wherein the particles based on magnetic iron oxide are essentially composed of maghemite (-Fe.sub.2O.sub.3).

31. The dispersion as claimed in claim 25, wherein a portion of the OH groups of the molecules of polysaccharides immobilized at the surface of the particles are oxidized in the form of CHO groups.

32. A composition of contrast agents for magnetic resonance imaging, comprising a dispersion as claimed in claim 25.

33. A process for the modification of the surface of the particles present in a dispersion as claimed claim 26, comprising a stage (G2) which consists in reacting said dispersion with chemical entities F capable of forming a bond with the molecules of polysaccharides.

34. The process as claimed in claim 33, where stage (G2) consists in reacting a dispersion with chemical entities F having an NH.sub.2 group and in treating the medium obtained with a reducing agent.

35. An aqueous dispersion of particles based on a magnetic iron oxide at the surface of which are immobilized, by covalent bonding, dextran molecules via covalent bonds of formula NHCH.sub.2, these dextran molecules being themselves bonded to chemical entities F, this dispersion being capable of being obtained as claimed in the process of claim 33.

36. A composition of a contrast agent for medical imaging having an affinity for given cells, tissues or organs, comprising a dispersion as claimed in claim 35, wherein the entities F are entities exhibiting an affinity with regard to said cells, said tissues or said organs.

37. A composition for therapeutic use, comprising a dispersion as claimed in claim 35, wherein the entities F are therapeutic active principles.

38. The composition as claimed in claim 36, provided in the form of an injectable composition.

39. An aqueous dispersion of particles, said particles comprising: a magnetic iron oxide core; amine groups (R) covalently bonded to a surface of the particles via a silane bond: (particle)OSiR; and polysaccharide groups covalently bonded to the amine groups, wherein, the particles have an isoelectric point greater than or equal to 10.

40. The aqueous dispersion of particles according to claim 39, wherein the polysaccharide is dextran and the particles have a number-average hydrodynamic diameter (PCS) of less than 132.8 nm.

41. The composition of claim 1, wherein the composition is formulated to be an injectable composition of contrast agents for magnetic contrast imaging.

Description

Example 1

Synthesis of Aqueous Dispersions of Nanometric Maghemite Particles with a Surface Modified by Coupling of Aminosilanes (Aminated Ferrofluids)

(1) 1.1. Synthesis of Aqueous Dispersions of Nanometric Maghemite Particles (Acidic Ferrofluids)

(2) 1.1.1. Dispersion (D1)

(3) An aqueous dispersion (D1) of nanometric maghemite particles was prepared by carrying out the following stages: Formation of magnetite particles: 31.41 g (i.e., 0.158 mol) of ferrous chloride were dissolved in 170 ml of 1.5M hydrochloric acid. This solution was introduced into a 5 l beaker containing 85.4 g (i.e., 0.316 mol) of ferric chloride dissolved in 3.5 l of water. Coprecipitation of iron salts was carried out by addition of 200 ml of a 2M aqueous ammonia solution at 25 C. with stirring, which resulted in the formation of a colloidal magnetite precipitate. The magnetite particles obtained were allowed to separate by settling on a magnetic plate and then the supernatant was removed. Desorption of the NH.sub.4.sup.+ counterions and surface oxidation: the flocculate prepared in the preceding stage was treated for a period of time of 15 minutes with 200 ml of nitric acid with a concentration of 2M. This treatment with nitric acid was carried out both in order to acidify the surface of the particles by desorbing the (flocculating) NH.sub.4.sup.+ counterions and by replacing them with nitrate ions and in order to dissolve the ferrous ions by surface oxidation. The flocculate was subsequently separated by settling and the supernatant was again removed. Oxidation of the core of the particles: 600 ml of an aqueous ferric nitrate solution with a concentration of 0.33M, brought beforehand to boiling point, were subsequently introduced. Reaction was allowed to take place for 30 minutes, then separation was carried out magnetically and the supernatant was removed. In this stage, the contribution of the Fe.sup.3+ ions in solution brings about the oxidation of the Fe(II) of the particles, which results in the formation of a -Fe.sub.2O.sub.3 maghemite phase in the particles. Peptization: The medium obtained was treated with 200 ml of nitric acid with a concentration of 2M. During this stage, the protons contributed by the acid are adsorbed at the surface of the oxide particles, whereby a surface charge is obtained which makes possible interparticle electrostatic repulsion. The medium was subsequently subjected to magnetic separation, whereby a flocculate of maghemite particles was obtained and was washed three times with acetone. Care was taken, during these washing operations, not to allow the flocculate to dry, in order to prevent the particles from agglomerating. The maghemite flocculate thus washed (and not dried) was subsequently placed in 500 ml of deionized water, whereby an aqueous dispersion of particles in the sol state was obtained. The residual acetone was removed by evaporation under vacuum at 40 C. The volume was subsequently brought to one liter by addition of 10 M ultrapure water.
1.1.2. Dispersion (D2)

(4) An aqueous dispersion (D2) of maghemite particles was produced by carrying out the following stages: Formation of magnetite particles: 31.41 g (i.e., 0.158 mol) of ferrous chloride and 127.66 g of ferric nitrate were dissolved in a 5 liter beaker containing 2.5 liters of a 1M aqueous sodium nitrate solution. Coprecipitation of the iron salts was subsequently carried out by addition of a sodium hydroxide solution with a concentration of 5M until a pH of 13.2 was obtained, which resulted in the formation of a colloidal magnetite precipitate which was kept stirred for 15 minutes. The magnetite particles obtained were left to separate by settling on a magnetic plate and the supernatant was removed. The flocculate obtained was subsequently washed with two times 2 liters of water. Desorption of the NH.sub.4.sup.+ counterions and surface oxidation: the flocculate from the preceding stage was treated for a period of time of 15 minutes with 400 ml of nitric acid with a concentration of 2M. Oxidation of the core of the particles: the medium obtained was brought to boiling point and 600 ml of an aqueous ferric nitrate solution with a concentration of 0.33M were introduced therein. Reaction was allowed to take place for 30 minutes, which resulted in the formation of a Fe.sub.2O.sub.3 maghemite phase in the particles, and then the particles were magnetically separated and the supernatant was removed. Peptization: the medium obtained was treated with 200 ml of 2M nitric acid, so as to obtain a surface charge which brings about interparticle electrostatic repulsion. The medium was subjected to magnetic separation, whereby a flocculate of maghemite particles was obtained and was washed three times with acetone, care being taken not to allow the flocculate to dry. The maghemite flocculate, thus washed (and undried), was subsequently placed in 500 ml of deionized water, whereby an aqueous dispersion of particles in the sol state was obtained. The residual acetone was removed by evaporation under vacuum at 40 C. The volume was subsequently brought to 1 liter by addition of 10 M ultra pure water.
1.1.3. Dispersion (D3)

(5) A dispersion (D3) of maghemite particles was prepared by carrying out the following stages: Formation of magnetite particles: 31.41 g (i.e., 0.158 mol) of ferrous chloride and 127.66 g of ferric nitrate were dissolved in a 5 liter beaker containing 2.5 liters of a 3M aqueous sodium nitrate solution. Coprecipitation of the iron salts was subsequently carried out by addition of a sodium hydroxide solution with a concentration of 5M until a pH of 13.2 was obtained, which resulted in the formation of a colloidal magnetite precipitate which was kept stirred for 15 minutes. The magnetite particles obtained were left to separate by settling on a magnetic plate and the supernatant was removed. The flocculate obtained was subsequently washed with two times 2 liters of water. Desorption of the NH.sub.4.sup.+ counterions and surface oxidation: the flocculate produced in the preceding stage was treated for a period of time of 15 minutes with 400 ml of nitric acid with a concentration of 2M. Oxidation of the core of the particles: the medium obtained was brought to boiling point and 600 ml of an aqueous ferric nitrate solution with a concentration of 0.33M were introduced therein. Reaction was allowed to take place for 30 minutes, which resulted in the formation of a Fe.sub.2O.sub.3 maghemite phase in the particles, and then the particles obtained were magnetically separated and the supernatant was removed. Peptization: the sol obtained was treated with 200 ml of 2M nitric acid. During this stage, the protons contributed by the acid adsorb at the surface of the oxide particles, whereby a surface charge is obtained which makes possible repulsion. The medium obtained was subsequently subjected to magnetic separation, whereby a flocculate of maghemite particles was obtained and was washed three times with acetone, care being taken not to allow the flocculate to dry. The maghemite flocculate, thus washed (and undried), was subsequently placed in 500 ml of deionized water, whereby an aqueous dispersion of particles in the sol state was obtained. The residual acetone was removed by evaporation under vacuum at 40 C. The volume was subsequently brought to 1 liter by addition of 10 M ultra pure water.

(6) The physicochemical characteristics of the dispersions (D1) to (D3) are combined in table 1 below.

(7) TABLE-US-00001 TABLE 1 Physicochemical characteristics of the dispersions (D1) to (D3) Dispersion (D1) (D2) (D3) pH 2.5 2.5 2.5 Maghemite concentration 28 g/l 28 g/l 28 g/l Isoelectric point 7.3 7.3 7.3 Number-average hydrodynamic 7.8 nm 4.0 nm 1.5 nm diameter, TEM.sup.(1) Number-average hydrodynamic 8.5 nm 2.9 nm 2 nm diameter, PCS.sup.(2) .sup.(1)as estimated by transmission electronic microscopy .sup.(2)average hydrodynamic diameter as measured by photon correlation spectroscopy
1.2 Modification of the Surface of the Maghemite Particles in the Acidic Ferrofluids Synthesized in Examples 1.1.1 to 1.1.3

(8) Various dispersions of maghemite particles with a modified surface (aminated ferrofluids) recorded as (D1a), (D1b), (D1c), (D2a) and (D3a) were prepared from the stabilized acidic ferrofluids (D1), (D2) and (D3) synthesized in the preceding stages. Surface modification was carried out with various aminosilane compounds by employing the following general protocol: Coupling Reaction: A volume of 200 ml of the ferrofluid under consideration ((D1), (D2) or (D3), depending on the circumstances), with an Fe.sup.3+ concentration of 0.35M, was stirred magnetically at a rate of 300 revolutions per minute. 100 ml of industrial grade methanol were added with stirring to this ferrofluid. A solution in 100 ml of methanol of an amount of aminosilane compound corresponding to 148.3 micromol of silane per m.sup.2 of surface area developed by the particles of the ferrofluid under consideration, namely 0.1 mol for the ferrofluid (D1), 0.2 mol for (D2) and 0.5 mol for (D3), was subsequently added to the mixture, kept stirred. The reaction was allowed to continue for 12 hours. Heat Treatment (Dehydration) Subsequent to the preceding stage, 200 ml of glycerol were added and the medium was homogenized for a few minutes by stirring at 500 revolutions per minute. The methanol and the water were subsequently extracted using a rotary evaporator (extraction under low vacuum for 1 hour, at 40 C. for the methanol and then at 80 C. for the water). The medium obtained was subsequently subjected to a heat treatment at 100 C. under ultrahigh vacuum (vane pump) for 2 hours. The medium was subsequently allowed to cool to ambient temperature. Extraction, Washing The mixture obtained on conclusion of the heat treatment (modified maghemite particles in a medium composed essentially of glycerol, additionally comprising unreacted silane) was introduced into a beaker into which were successively poured 100 ml of ethanol and then 200 ml of acetone with slow stirring (100 revolutions per minute), so as to bring about flocculation of the modified maghemite particles and precipitation of the excess oligomerized silane. The particles were separated on a magnetic plate and the supernatant, comprising the oligomerized silane, was removed. The flocculate of particles which was obtained was washed with three times 400 ml of an acetone/ultrapure water (70:30 v/v) mixture, so as to remove the residual silane oligomers and the residual glycerol. Here again, the washing operations were carried out so as not to allow the flocculate to dry.

(9) Peptization After the final washing of the preceding stage, 400 ml of ultrapure water were added to the flocculate obtained. The pH was then measured as equal to 10.4,testifying to the presence of amine functional groups at the surface of the maghemite and to the saturation of this surface by the amine functional groups. 1M nitric acid was then added to the medium, dropwise and with vigorous stirring (of the order of 700 revolutions per minute), so as to gradually reduce the pH, step by step, by approximately one pH unit at each stage. This acidification makes it possible in particular to retain the adhesion of the aminated polysiloxane film which was formed in the preceding stages on the surface of the particles. When a pH of 6 was reached, the maghemite particles began to disperse. The pH of the medium was adjusted to 3 and the medium was left stirring for one day. The pH was subsequently again readjusted to 3. Under these conditions, the following 5 dispersions (aminated ferrofluids) were prepared:

(10) TABLE-US-00002 Starting acidic ferrofluid (D1) (D1) (D1) (D2) (D3) Aminosilane compound APS.sup.(3) EDPS.sup.(4) DTPS.sup.(5) APS APS used for the modification Aminated ferrofluid (D1a) (D1b) (D1c) (D2a) (D3a) obtained .sup.(3)APS: -aminopropyltrimethoxysilane .sup.(4)EDPS: N--(aminoethyl)--aminopropyltrimethoxysilane .sup.(5)DTPS: N--(aminoethyl)-N--(aminoethyl)--aminopropyltrimethoxysilane
1.3. Properties of the Dispersions of Modified Maghemite Particles (Aminated Ferrofluids) Obtained

(11) The characteristics of the five aminated ferrofluids obtained are combined in table 2 below.

(12) TABLE-US-00003 TABLE 2 Properties of the aminated ferrofluids obtained Aminated ferrofluid (D1a) (D1b) (D1c) (D2a) (D3a) pH 3 3 3 3 3 Fe concentration 0.35M 0.35M 0.35M 0.35M 0.35M Isoelectric point 10.4.sup.(6) 10.2 10.1 10.4 10.4 Number-average 15.2 nm 7.7 nm 15.5 nm 6.0 nm 5.7 nm hydrodynamic diameter, PCS .sup.(6)By way of comparison, a dispersion was prepared by modifying the dispersion D1 with APS under the conditions of the general protocol described above but without carrying out the second stage of heat treatment. The isoelectric point of the composition was then measured as equal to 8.3.

Example 2

Stability with Regard to Flocculation in Presence of Chloride Ions

(13) In order to illustrate the increased stability of the aminated ferrofluids of the invention in comparison with the acidic ferrofluids as regards flocculation, a comparative test was carried out which consists in introducing chloride ions at increasing concentrations in the two types of ferrofluids.

(14) The flocculation limiting concentration observed at pH =3 for the dispersions (D1), (D1a), (D1b) and (D1c) of example 1 is shown in table 3 below. The flocculation limiting concentration is the minimum concentration of chloride ions in the dispersion, at the pH under consideration, from which flocculation is observed to begin, flocculation being an increase in the turbidity due to the formation and to the increase in the size of the flocculates in the medium. The limiting concentration corresponds to the concentration at which an increase in the optical density, measured at a wavelength of 800 nm, is observed.

(15) TABLE-US-00004 TABLE 3 Flocculation limiting concentration during the addition of chloride ions Dispersion (D1) (D1.sub.a) (D1.sub.b) (D1.sub.c) Flocculation 0.05 mol/l 0.14 mol/l 0.17 mol/l 0.21 mol/l limiting concentration (Cl.sup.).sup.(*.sup.)

(16) It is seen, in the light of the above data, that the surface modification makes it possible to multiply by at least two the limiting concentration for flocculation in the presence of chloride ions.

Example 3

Grafting of Dextran Molecules (Synthesis of Modified Aminated Ferrofluids of VUSPIO Type

(17) Dextran molecules were grafted onto the particles of the aminated ferrofluids of example 1.2. Four different categories of dextran were tested (Dextran T5: M.sub.w=5000; Dextran T15: M.sub.w=15000; Dextran T40: Mw=40000 and Dextran T70: M.sub.w=70000).

(18) The grafting was carried out according to the following protocol: Activation of The Dextran:

(19) A solution of 10 g of dextran in 200 ml of ultrapure water was prepared. 10 ml of a 2.06 mol.l.sup.1 aqueous NaIO.sub.4 solution were added to this medium and the mixture was left stirring for 12 h. On conclusion of this oxidation stage, the periodic salts obtained were removed from the medium. To do this, the solution obtained, which is pale yellow, was poured into a cellulose dialysis tube (cutoff threshold of 12 400 g/mol.sup.1, Aldrich). Dialysis was carried out in a 5 l beaker containing ultrapure water. The water was replaced 5 times every 2 hours. The activated dextran solution obtained was stored at 4 C. The presence of aldehydes was confirmed by the Fehling test. Grafting of the Activated Dextran to the Particles of an Aminated Ferrofluid

(20) A volume of 200 ml of the 10 g/l activated dextran solution obtained above was poured into 20 ml of a ferrofluid as obtained in example 1.2. The mixture produced was left stirring for 24 h and then 10 ml of a 0.206 mol.l.sup.1 sodium borohydride solution were added. The medium obtained was left stirring at pH=9 for 4 h.

(21) In all cases, a stable sol was obtained. Purification of the Sol Obtained by Tangential Ultrafiltration:

(22) An ultrafiltration system composed of a peristaltic pump (Millipore N80EL005), of silicone piping and of a cartridge (Prep/Scale-TFF) including a poly(ether sulfone) membrane with a cutoff threshold of 100 kD was used.

(23) The sol was placed in the retentate tank. After passage of the sol in the membrane, 1 l of water was poured into the tank in order to carry out the washing. The sol was washed and neutralized against 3 l of ultrapure water. The purified sol which was obtained is more dilute than the starting sol as the dead volume of the system is approximately 100 ml. A portion of the product remains clogged inside the membrane. However, this product is discharged after washing for several hours. The dispersion of particles was concentrated, by evaporation, to an Fe.sup.3+ concentration of 0.08M.

(24) The results obtained by grafting various dextrans to the aminated ferrofluids (D1a), (D2a) and (D3a) of example 1.2 are combined in the table below. The various modified aminated ferrofluids obtained are labeled (G1) to (G9).

(25) TABLE-US-00005 TABLE 4 Aminated ferrofluids modified by grafting dextrans Number-average hydrodynamic Dextran diameter (PCS) of Modified Starting used for the particles of ferrofluid aminated the the modified produced ferrofluid grafting ferrofluid produced (G1) (D1a) T70 59.6 nm (G2) (D1b) T70 130 nm (G3) (D1c) T70 130 nm (G4) (D1a) T40 53.7 nm (G5) (D1a) T15 132.8 nm (G6) (D2a) T40 70.8 nm (G7) (D2a) T15 46.7 nm (G8) (D2a) T5 44.9 nm (G9) (D3a) T40 56.5 nm (G10) (D3a) T15 34.0 nm (G11) (D3a) T5 32.2 nm

Example 4

Synthesis of Functionalized VUSPIO Ferrofluids

(26) 4.1. Grafting of an Aminotelechelic (Diamino) Poly(Ethylene Oxide) to the Particles of the Modified Ferrofluids of Example 3.

(27) Molecules of poly(ethylene oxide)-diamine were grafted to the particles of the modified aminated ferrofluids (G1) to (G3) of example 3.

(28) The grafting was carried out according to the following protocol:

(29) A solution of 20.5 g of POE-diamine (M.sub.w=2000 g/mol) in 100 ml of water was added to a volume of 200 ml of the ferrofluid under consideration ((G1), (G2) or (G3), depending on the circumstances). 100 ml of an aqueous sodium borohydride solution with a concentration of 0.206 mol/l were subsequently added to the medium. Reaction was allowed to take place at pH 9 for 4 hours and with stirring, and the excess POE-diamine was subsequently removed under tangential ultrafiltration against 5 l of ultrapure water. The dispersion of borohydride and of borate obtained was concentrated by evaporation of the water until an Fe 3+concentration of 0.08M was obtained.

(30) The results obtained in the three cases are given in table 5 below.

(31) TABLE-US-00006 TABLE 5 Ferrofluids modified by grafting POE-diamine Number-average hydrodynamic diameter (PCS) of the particles of the ferrofluid Starting ferrofluids produced (G1) 59.6 nm (G2) 53.7 nm (G3) 132.8 nm
4.2. Grafting of a Monoamino Poly(ethylene Oxide) to the Particles of Modified Ferrofluids of Example 3

(32) A grafting similar to that of example 4 was carried out using the aminated ferrofluids (G4), (G6) and (G9) of example 3,the procedure of example 4 being employed to do this, apart from the fact that a POE-monoamine (M.sub.w=2000 g/mol) was used. Furthermore, the amount of POE was divided by two with respect to example 4. Thus, the POE solution added comprises 10.25 g of POE-monoamine in 100 ml of water.

(33) The results obtained by the grafting of the aminated ferrofluids D1a, D2a and D3a of example 1.2 are combined in table 6 below.

(34) TABLE-US-00007 TABLE 6 Ferrofluids modified by grafting with POE-monoamine Number-average hydrodynamic diameter (PCS) of the Starting aminated particles of the modified ferrofluid ferrofluid produced (G4) 74.5 nm (G6) 62.1 nm (G9) 65.6 nm
4.3. Grafting of the Particles of the Modified Ferrofluids of Example 3 with Fluorescent Entities

(35) 100 ml of the modified ferrofluids of example 3,with an Fe.sup.3+ concentration of 0.08M, buffered with a phosphate buffer (pH 7.4; 0.01M), were allowed to react for 24 hours, with the exclusion of light, with 10.sup.3 mol of Rhodamine B (Rh B) or Lucifer Yellow (LY) in the form of its dilithium salt. Reduction with borohydride was subsequently carried out under the conditions of example 3. These aminated entities react directly with the aldehyde functional groups of the oxidized dextrans. Subsequent to this reaction, the excess fluorescent entities (fluorochromes) were removed by liquid-liquid (water/chloroform) extraction until the organic phase became colorless. The traces of chloroform were subsequently removed on a rotary evaporator.

(36) 4.4 Grafting of the Particles of the Modified Ferrofluid of Example 4.1 with Fluorescent Entities

(37) The coupling reaction was carried out on 100 ml of ferrofluid according to example 4.1,with an Fe.sup.3+ concentration of 0.08M, buffered with a carbonate/sodium bicarbonate buffer (pH 9, 0.01M), with 10.sup.3 mol of TRITC (tetramethylrhodamine isothiocyanate derivative) or of 5-FAM, SE (the succinimidyl ester of 5-carboxyfluorescein). The 5-FAM, SE was dissolved beforehand in 3 ml of DMF. The excess fluorescent entities were removed by liquid-liquid (water/chloroform) extraction until the organic phase became colorless. The traces of chloroform were removed on a rotary evaporator.

(38) 4.5: Grafting of the Particles of the Modified Ferrofluids of Example 6 with Fluorescent Entities

(39) The procedure used is identical to that of example 4.4, except that the POE-diamine is replaced by a mixture of POE-monoamine and POE-diamine in the proportions 2:1.

(40) 4.6: Grafting of Doxorubicin to the Particles of the Ferrofluids of Examples 4.1 and 4.2

(41) 2.2210.sup.4 mol of doxorubicin (number of moles equivalent to 1/30th of moles of glucoside residues) was added to the dispersion of nanometric particles prepared in example 3 before the reduction with borohydride. The procedure described in example 3 is subsequently followed. The particles treated with doxorubicin are then subjected to the same treatment as those resulting from example 4.1 or 4.2.

(42) 4.7: Grafting of Folic Acid to the Particles of the Ferrofluids of Example 3

(43) 4.7.1 Esterification of Folic Acid with N-hydroxysuccinimide (NHS):

(44) 5 g of folic acid were dissolved in 100 ml of DMSO. 2.5 ml of triethylamine, 2.6 g of NHS and 4.7 g of carbodiimide were added to this mixture. Reaction was allowed to take place overnight at ambient temperature. The coproduct of the reaction, dicyclohexylurea, was extracted by filtration. The solution of folic acid coupled to NHS was concentrated on an evaporator under reduced pressure. The product was then precipitated from diethyl ether.

(45) 4.7.2 Combination of POE-diamine with Folic Acid Coupled to NHS:

(46) 15 g of POE-diamine (M.sub.w=2000 g/mol) were dissolved in 100 ml of a carbonate/bicarbonate buffer solution at pH 10.5. 4.5 g of the product (folic acid-NHS) obtained on conclusion of stage 4.7.1 were dissolved in the minimum amount of DMSO (10 ml). This solution was then run dropwise into the solution containing the POE-diamine and the reaction was allowed to take place for 12 h. The product was then dialyzed against ultrapure water for 12 h in a dialysis tube with a cutoff threshold of 1 kD while regularly changing the water.

(47) 4.7.3 Grafting of the Combined Product from Stage 4.7.2 to the Particles of Example 3:

(48) The procedure is identical to that of example 4.5, the POE-monoamine being replaced by the POE-folic acid combined product from stage 4.7.2.