Magnetic nanoparticles for the application in hyperthermia, preparation thereof and use in constructs having a pharmacological application

Description of equivalent WO 2008074804 A2

0001Magnetic nanoparticles for the application in hyperthermia, preparation thereof and use in constructs having a pharmacological application DESCRIPTION Field of the invention The present invention relates to the field of nanometric particles, specifically metal oxides having magnetic properties, and to the use thereof in the pharmacological field.

0002State of the art Nanoparticles are objects having a diameter below 300 nm. In recent years, there has been a great interest in the scientific and technological community for the peculiar chemical-physical properties of these materials.

0003Specifically, magnetic nanoparticles have their potential field of application in the diagnostic sector as a contrast medium in imaging techniques (magnetic resonance), in the magnetic localisation techniques and, mainly, in the specifically therapeutic field of hyperthermia mediated by magnetic fields.

0004The main feature of these materials essentially consists in four elements:

0005- the composition of the central core of the particles (which must comprise materials having magnetic characteristics);

0006- the size of the magnetic central core (which results being of the order of tens of nanometers or less)

0007- the stability in a physiological environment

0008- the biocompatibility.

0009The actual usefulness of the magnetic nanoparticles is, in the ultimate analysis, related to their ability to increase the temperature of the medium in which they are confined when interacting with an external electromagnetic fields.

0010Many patents discuss magnetic nanoparticles coated with biocompatible materials so as to obtain composite particles having a diameter in the range between 5 and 500 nm, which may form stable suspensions in an aqueous system. See U.S. Pat. No. 5427767, Kresse; U.S. Pat. No. 2541039, Lesniak; U.S. Pat No. 6541039, Lesniak.

0011A particular attention has been directed to methods to obtain metal oxides forming the core of the particles, and all of these are focused on obtaining iron oxides. See U.S.: Pat. No. 4677027, Porath; U.S.: Pat. No. 5160725, Pilgrim; U.S.: Pat. No. 4329241 , Massart; U.S.: Pat. No. 4101435, Hasegswa.

0012In all of the cited patents, even though in some cases they are generally referred to as "metal oxides" or "iron oxides doped with other metal elements", the examples set forth only specifically refer to the iron oxides in their various forms and no cases of hyperthermic effect related to other kinds of metal oxides are cited.

0013In general, these nanoparticulate oxides have a low hyperthermic efficiency and it is therefore required to introduce high amounts thereof in order to obtain a therapeutic result.

0014Furthermore, there is a broad series of patents relating to the methods to obtain various kinds of coatings, stabilisers and protections for the magnetic particles by means of different methods. See U.S. Pat. No. 4280918, Homola; U.S. Pat. No. 6576221 , Kresse; U.S. Pat. No. 4452773, Molday; U.S. Pat. No. 4827945, Groman; U.S. Pat. 5545395, Tournier; EP 0272091 , Eley.

0015A series of different techniques for the production of polymer nanoparticles internally incorporating pharmacologically active products is described; these techniques may be grouped in four classes: a) trapping techniques of the drug in polymers insoluble in water and soluble in solvents miscible with water. b) Coacervation techniques of the (water-soluble) drug with proteins or polymers soluble in water, followed by the formation of nanoparticles by dilution with solvents in which the proteins or the polymers are insoluble, the stabilisation of the nanoparticulate structure with appropriate bonding agents and the removal of the "precipitating" agent. c) Incorporating techniques of the (water-soluble or water-insoluble) drug by emulsification in the presence of surface agents, which leads to the formation of micrometric particles, followed by the removal of the solvent to reduce the size of the particles to nanometric levels. d) Incorporating techniques of the (water-soluble or water-insoluble) drug by emulsification in the presence of proteins, which leads to the formation of micrometric particles, followed by the removal of the solvent to reduce the size of the particles to nanometric levels.

0016It may be noted that the description of such a high number of methods, each having its specific variants, is already an indication of the difficulties encountered to obtain the desired product having a size suitable for use (generally in the range between 100 and 300 nm), a restricted size distribution and the ability to remain stable in a physiological environment.

0017Taking into account the considered techniques, the following problems may be noted: - the trapping techniques for the "active substance" in polymers insoluble in water and soluble in appropriate organic solvents leads to a "simple" formation of nanoparticles, the size of which are mainly determined by the concentration of the polymer and drug and by the solvent/water dilution ratio. The main problem consists in that the nanometric particles obtained in this manner are stable in water but already unstable in a physiological solution and the use thereof in the biomedical field is therefore hardly acceptable.

0018- The coacervation techniques of the (water-soluble) drug with proteins soluble in water, followed by the formation of nanoparticles by dilution with solvents, the stabilisation of the nanoparticulate structure with appropriate bonding agents and the removal of the "precipitating" solvent are definitely not employable for products insoluble in water. On the other hand, the advantage of using nanoparticulate systems is reduced for products which are already soluble in an aqueous environment, because these active substances may also be directly administered with similar effects to those obtained with the nanoparticulate system. - The emulsification techniques in the presence of surface agents always display the problem of the emulsifying system hardly being able to combine the ability to form small enough micelles having the compatibility with the human organism at the concentrations employed.

0019- The incorporating techniques of the (water-soluble or water-insoluble) drug by emulsification in the presence of proteins display great technical difficulties as far as the productive-type applications are concerned. The emulsification in these cases is very difficult and forces to use complex techniques, having low industrial productivity, which are very expensive (for instance, high pressure emulsification techniques).

0020In light of what has been set forth above, it is evident the need to have nanoparticulate magnetic oxides having a high hyperthermic efficiency and incorporation methods for magnetic systems and pharmacological principles, leading to the preparation of constructs that are effective from a hyperthermic and pharmacological point of view while also being stable and biologically compatible.

0021Brief description of the drawings

0022Fig. 1 is a diagram wherein the hyperthermic efficiency of a functionalised particle and the corresponding final construct are compared, said efficiency being expressed as a ΔT in ⁰C.

0023Summary of the invention

0024The present invention relates to magnetic metal oxide nanometric particles and to constructs consisting of: said magnetic nanometric particle, possibly functionalised with bifunctional compounds, a polymer possibly containing a pharmacologically active molecule and, when said polymer is insoluble in water, an external protecting layer of surface agents, and to the use thereof in hyperthermic treatments.

0025Detailed description of the invention The present invention allows to overcome the aforementioned problems thanks to constructs comprising: a magnetic nanometric particle, possibly functionalised with bifunctional compounds, a polymer possibly containing a pharmacologically active molecule and, when said polymer is insoluble in water, an external protecting layer of surface agents. The above said pharmacological active molecule, when present can be connected to the polymer or dispersed in it.

0026The nanometric particles according to the invention are spinels and oxides of the

0027M¹¹M¹¹^ O₄ type, in which M¹¹ = Fe, Co, Ni, Zn, Mn; M¹" = Fe₁ Cr) in a nanometric form. Among the aforementioned spinels, it has been unexpectedly found that cobalt ferrite has a high hyperthermic efficiency. Among other spinels and iron oxides, it has also unexpectedly been discovered that controlled size magnetite and maghemite, which are prepared according to the methods described in the present invention, have a better hyperthermic efficiency than similar products described in the literature. By difunctional compounds according to the invention are intended: thiols, carboxylic acids, hydroxamic acids, phosphoric acids, esters and salts thereof having an aliphatic chain that carries a second functional group at the terminal position (designated ω).

0028More specifically, the difunctional compounds are compounds of the general formula:

0029R-|-(CH2)n— R2

0030In which: n is an integer in the range between 2 and 20;

0031R₁ is chosen from: CONHOH, CONHOR, PO(OH)₂, PO(OH)(OR), COOH, COOR, SH, SR;

0032R₂ is the external group and is chosen from: OH, NH₂, COOH, COOR;

0033R is an alkyl group or an alkaline metal.

0034Among alkaline metal preferred are K, Na, or Li, while among the alkyl group preferred are C_halky!, more particularly ethyl. Particularly preferred among the above said difunctional groups is the ethyl-12-

0035(hydroxyamino)-12-oxododecanoate.

0036The polymers constituting the construct can be water-soluble polymers or water insoluble polymers stabilised by surface agents.

0037The water-soluble polymers according to the invention are, for example, polyelectrolytes, polypeptides and water-soluble proteins; water-soluble polymers chosen from block copolymers, modified polyethylene glycols, modified polysaccharides, phospholipids, polyamineamides, globular proteins are preferred.

0038The water insoluble polymers are, for example, chosen among: polyesters, polyamides, polyanhydrides, polyorthoesters, peptides, polyamineamides; or insoluble organic molecules like for example cholesterole; polyesters and cholesterole are preferred. Surface agents according to the invention may be: polyelectrolytes, polypeptides and water-soluble proteins; block copolymers, modified polyethylene glycols, modified polysaccharides, phospholipids, polyamineamides, globular proteins; preferred are human serum proteins and pluronics block copolymers.

0039The polymers are known or can be easily obtained according to methods known in the art as by polyaddition of primary monoamines or secundary diamines with bis.acrylamides, at room temperature for a time comprised between some hours and some days as for example reported in Macromolecular Rapid Communication,

00402002, 23, No. 5/6 p. 332-355.

0041Example of polymers, both water soluble and surface agents, according to the invention are:

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0048<img file="EP2117600A2_D0011.tif" /><img file="EP2117600A2_D0012.tif" />

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0050<img file="EP2117600A2_D0015.tif" />

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0053<img file="EP2117600A2_D0020.tif" />

0054<img file="EP2117600A2_D0021.tif" />

0055wherein n is comprised between 3 - 300, preferably between 10 - 100. Pharmacologically active molecules according to the invention are the biologically active molecules normally used in the various therapies, for example antitumour agents (such as anthracycline), antimicrobial agents, anti-inflammatory agents, immunomodulators, molecules acting on the central nervous system etc. or those capable of marking the cells so as to allow their identification with the normal means of diagnostic detection (for example fluorescent stains).

0056The methods for the preparation of the constructs described in the present invention are extremely flexible.

0057Furthermore, thanks to the possibility of controlling their size and therefore their hyperthermic properties, the magnetic nanoparticies according to the present invention are especially suitable for the preparation of biocompatible nanoparticulate constructs which are extremely flexible and active even at low concentrations of magnetic nanoparticies. This is certainly an advantage in all potential biomedical applications. In the case in which the constructs are based on hydrophilic polymers used as surfactant agents, it is known that it is required to use amounts of surfactant greater than 300% with respect to the pharmacologically active product; by the use of the nanoparticles according to the present invention the amount of surfactant, which notoriously has a poor biocompatibility, may considerably be decreased. Specifically regarding cobalt ferrite, it has been unexpectedly found that, the size being equivalent, it has a hyperthermic efficiency of approximately one order of magnitude more than that of iron oxides; furthermore, in contrast to that occurring with iron oxides described in the literature, the hyperthermic properties of the physically immobilised nanoparticles of cobalt ferrite do not vary with respect to the material dispersed in a fluid matrix. This makes them more efficient in those cases in which the extracellular matrix or the cytosolic matrix represents an obstacle for their rotation. When irradiated with electromagnetic waves having a frequency in the range between 10 and 1000 kHz, preferably between 50 and 500 kHz, the cobalt ferrite nanoparticles have a far better hyperthermic behaviour with respect to the iron oxides having equivalent nanoparticle size (also doped with impurities of Co, Ni or other metal elements). At the aforementioned frequencies, the magnetite and maghemite particles we prepared also displayed better hyperthermic efficiencies than the equivalents described in literature.

0058The magnetic nanoparticles according to the invention may be prepared according to known processes as e.g. the polyol process widely described in the literature which, briefly, consists in the use of a high boiling alcohol allowing to operate at high temperatures and lead the forming particles to give rise to complexes thus preventing the growth thereof.

0059Normally, the desired metal precursors (preferably acetates, carbonates, sulphates, oxalates, chlorides) are added to a known volume of alcohol (e.g. diethylene glycol, DEG). The solution is then heated while mixing to the complete solubilisation of the precursors, water is possibly added in an appropriate amount to facilitate the hydrolysis of the precursors, it is heated for a few hours at a temperature higher than 150 ⁰C and it is then left to cool, thus obtaining a stable suspension of monodispersed nanoparticles with a restricted size distribution.

0060Moreover since the hyperthermic effect of the cobalt ferrite is far more dependent on the size of the nanoparticles than that occurring for magnetite or maghemite new synthesis methods, allowing to control the size of the nanoparticles in a reproducible manner and, accordingly, the hyperthermic effect thereof, have been designed and are also part of the present invention. The size control has also brought great advantages in the synthesis of magnetite and maghemite allowing to obtain products alternative to cobalt ferrite and more efficient than their equivalents from the hyperthermic point of view.

0061New synthesis methods, which are capable of allowing the size control (and therefore hyperthermia) of the magnetic nanoparticles which are always obtained in a suspension are reported hereinafter. Continuous process. In this case the procedure is carried out as described above for the polyol process, but the synthesis is performed with the addition (in an amount equimolar to the reagents) of a "primer" consisting of previously synthesised nanoparticles. In this way, at the end of the reaction magnetic nanoparticles having greater size than those introduced at the beginning of the synthesis are obtained. In practice, the procedure for a first preparation is performed as for the polyol process; subsequently, a new reaction is carried out in the same conditions as the first, with all of the starting materials in amounts identical to those already used and with the addition of the product obtained from the first reaction. The magnetic nanoparticles thus obtained (which are twofold and have greater size with respect to those introduced at the beginning of the synthesis) may be used again as a "primer" for the following reaction. The cycle may be repeated an indefinite number of times until the particles displaying the desired size are obtained. Semicontinuous substitution process. In practice, a first synthesis according to the polyol process is performed, but at the end of the stationary heating period at 180⁰C the product is not cooled, but rather poured in a flask having twice the size, in which all of the starting materials have been loaded in amounts identical to the product which has already been reacted. The temperature is taken to 180⁰C again, it is maintained for 3 hours and the cycle is then repeated for a variable number of times until the product having the desired size is obtained. Growth process. In this case, the synthesis is performed according to the above described polyol process, but the period during which the product is maintained at a temperature of 180 ⁰C is prolonged for a variable number of hours. Therefore, a product is obtained, the dimensions of which are dependent on time of temperature persistence. Moreover, the magnetic nanoparticles may further be prepared by a process similar to the above described polyol process, though performing the heating exclusively in a microwave oven which allows to considerably reduce the reaction times and to have a better control on the size and morphology. As a further advantage of the above described processes it must be considered that by means of these preparative techniques, the stechiometry of the nanoparticles may be modified: e.g. maghemite may be produced from the magnetite obtained, according to one of the previous processes, by the oxidation thereof at a controlled temperature in an acetic oxidising environment thus accelerating the oxidative process which would naturally occur although by much longer times. In this case, the size control of the magnetic nanoparticles is performed in an indirect manner carrying out a size control of the magnetite precursor according to one of the previously described methods. The nanoparticulate cobalt ferrite, magnetite and maghemite obtained according to the described method have been controlled for the size of the particles by means of DLS (Malvern Zetasizer nano-S).

0062The nanoparticles thus obtained have a size in the range between 4 and 200 nm, preferably between 10 and 70 nm.

0063The functionalisation of the nanoparticles was obtained according to known methods or as described in patent PCT/EP2007/050036, i.e. by reacting difunctional derivatives dissolved in ethanol with the nanoparticles as defined above so as to coat the surface thereof. The process for the preparation occurs by reacting a nanoparticle dispersion in an organic solvent (e.g. ethylene glycol) with the chosen bonding agent while mixing at reduced temperature for a few hours. The product is then possibly separated by extraction with particular solvents or precipitated, e.g. with acetone, spun in a centrifuge, separated and possibly redispersed in a suitable solvent.

0064The aforementioned polymeric constructs have different characteristics depending on the type of polymer employed for the preparation thereof. Specifically, the polymers may be insoluble or soluble in water; and their use in the synthesis of the constructs according to the invention are reported hereinafter. Constructs based on water-insoluble polymers

0065They consist in magnetic nanoparticles, functionalised as above said, in combination with a pharmacologically active agent, incorporated in a water- insoluble polymer, as previously defined, in turn stabilised by surface agents as above defined. Surface agents according to the invention may be: polyelectrolytes, polypeptides and water-soluble proteins; surface agents chosen from block copolymers, modified polyethylene glycols, modified polysaccharides, phospholipids, polyamineamides, globular proteins are preferred. The process for the preparation of these constructs according to the invention is a continuous and single step process for the incorporation of magnetic nanoparticles in a water-insoluble polymer matrix and for the coating of this structure with suitable surface agents. The procedure implies the use of water (in which the surface agent is previously dissolved) and an organic solvent miscible therewith (to a greater extent than 10%), in which the magnetic nanoparticles, functionalised as above described, and the polymer matrix are previously solubilised. The two liquids are then mixed in appropriate conditions so as to obtain a self-assembly of the components previously solubilised in the phases to form a controlled size construct. The incorporation of the drug in the construct occurs in the assembly- step by solubilisation in water or organic solvent. In this way, the number of pharmacologically active species which may be introduced in the construct increases. This method allows to obtain the final product with yields in the range between 90 and 99%, unexpectedly the hyperthermic efficiency of the construct thus assembled is similar to that of the starting inorganic particles. The average diameter of the construct is in the range between 50 and 300 nm and the ratio between the concentration of the possibly present drug and the concentration of the magnetic particles may easily be varied during assembly. The close association of magnetic particles and drug allows to obtain the controlled release of the drug by thermal effect induced by the interaction of the magnetic nanoparticles with an external electromagnetic field. In this manner, magnetic hyperthermia may be obtained on one side and a synergic effect with the pharmacologically active species may be obtained on the other side.

0066The presence of magnetic particles in a percentage variable with respect to the drug allows to enhance the hyperthermic effect i.e. the strictly pharmacological effect in the most indicated manner for the specific pathology to be treated.

0067The constructs obtained are stable in a physiological solution environment and thus prove to be suitable for a therapeutic use.

0068Constructs based on water-soluble polymers:

0069They consist in magnetic nanoparticles, functionalised as above said, possibly in combination with a pharmacologically active agent, incorporated in a water-soluble polymer or in surface agents as above defined.

0070The process according to the invention implies an organic solvent miscible in water to an extent greater than 10% as a "carrier" solvent for the magnetic particles and the drug. The procedure implies the use of water (in which the water-soluble polymer is previously dissolved) and a liquid miscible with water (to an extent greater than 10%) in which the functionalised magnetic nanoparticles are previously solubilised. The two liquids are then mixed in appropriate conditions so as to obtain a self-assembly of the components previously solubilised in the steps to form a controlled size construct.

0071The incorporation of the drug in the construct occurs in the assembly step by solubilisation in water or in an organic solvent. In this manner, the number of pharmacologically active species which may be introduced in the construct increases and the ratio between the concentration of the drug and the concentration of the magnetic particles may be easily varied.

0072In this manner, constructs having an average diameter between 30 and 100 nm and a very restricted size distribution (polydispersion index PDI = 0.10 - 0.15) may be obtained, whereas by the methods described in the literature (with a more complex and hardly scalable method) particles having a size of about 200 nm with a broader polydispersion index (about 0.25) are obtained. This method allows to obtain the final product with yields in the range between 80 and 98%, unexpectedly the hyperthermic efficiency of the construct thus assembled is similar to that of the starting inorganic particles.

0073The resulting constructs are stable in a physiological solution environment and thus prove suitable for a therapeutic use.

0074The possibility to obtain such small constructs, evenly distributed and stable in a physiological environment represents a great advantage for possible therapeutic application because the particles may diffuse better in the body areas to be treated, are more difficult for the immune system to detect and thus eliminate and are more easily incorporated within target cells (I. Brigger, C. Dubernet, P.

0075Couvreur, Adv. Del Rev., 2002, 54, 631. Nanoparticles in cancer therapy and diagnosis).

0076The overall data for the synthesised nanoparticles, the functionalised nanoparticles, and the constructs as defined above are set forth in Table 1 , which is subdivided as follows:

0077Tables 1(a) and 1 (b): magnetic particles; Table 1(c): functionalised magnetic particles;

0078Table 1(d): constructs with polymer coating.

0079In Table 2, the size of the resulting particles are set forth with the processes according to the invention, while the corresponding hyperthermic effect is set forth in Table 3. The overall data of the functionalised nanoparticles are set forth in Table 4 in which the starting products, the type of functionalisation and the hyperthermic effect (expressed as a ΔT) of the precursor and the final product in the same measurement conditions are indicated.

0080From Diagram 1 , it is apparent that the hyperthermic effect of the construct is always similar to that of the precursor. The crystal structure of the samples has been identified by means of X-ray diffraction (XRD) recording the reflections in the range of 10-70° with a scanning range of 0.05°(2Θ) for 5 s on a Philips X'pert Pro diffractometer (Cu Ka radiation).

0081The size of the crystallites has been determined by diffraction peaks by using the

0082Scherrer method. The samples thus characterised (non functionalised particles, functionalised particles, final constructs) have been subjected to hyperthermia tests, for which the samples have been dispersed in various mediums and an oscillating magnetic field radiating unit Novastar 5W to 5 Kw provided by Ameritherm has been used.

0083The tests have been carried out in adiabatic conditions with an electromagnetic field of 170 kHz and having a magnetic field intensity of 21kA/m², using an alumina crucible having a capacity of 0.30 ml completely filled with a dispersion of the sample in a suitable solvent. The concentration of the sample (expressed as concentration in metal oxide) in the dispersing medium is in the range between

00840.1% and 3%. The initial and final temperature of the dispersion has been measured by a FLIR

0085E65 thermocamera.

0086For better illustrating the invention the following Examples are reported.

0087Example 1

0088Preparation of nanometric cobalt ferrite according to the known process (polyol process).

0089Product formula: NFeCo31

0090Reagents used:

0091Fe : Co ratio = 2 : 1

00929.53 g Co(Ac)₂.4H₂O (23.7% Co w/w) Co(II) = 2.259 g = 0.038 moles

009321.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0094Fe(III) = 4.284 g = 0.077 moles 269.04 g DEG

0095Synthesis:

0096A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for a possible distillation, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 110° C for the solubilisation step (time: 1 h). Subsequently, the temperature is raised to 180⁰C and the system is left at reflux for 3 hours. The process performed while mixing leads to the formation of a black suspension.

0097Example 2 Preparation of nanometric magnetite according to the known process (polyol process).

0098Product formula: Fe74

0099Reagents:

0100Fe III : Fe Il ratio = 2 : 1 30,32 g Fe(Ac)₂ solution (7% Fe w/w)

0101Fe(II) = 2.122 g = 0.038 moles

010221.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0103Fe(III) = 4.284 g = 0.077 moles

0104269.04 g DEG Synthesis:

0105A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a reflux and distillation two-way system, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 120° C for the stabilisation step and maintained for one hour at such a temperature. Subsequently, the mixture is heated to 180°C maintaining the distillation step. Having reached the internal temperature of

0106180⁰C, the system is left at reflux for 3 hours. The process performed while mixing leads to the formation of a brown suspension.

0107Example 3 Preparation of a nanometric mixed Fe'", Fe", Ni spinel according to the known process (polyol process).

0108Product formula: Fe Do Ni 03 Reagents specifications:

0109Fe(CH₃COO)₃ MW = 232.98 g/moles

0110Fe(CH₃COO)₂ MW = 173.93 g/moles

0111Ni(CH₃COO)₂ MW = 176.78 g/moles Fe MW = 55.85 g/moies

0112Ni MW = 58.69 g/moles

0113DEG MW = 106.12 g/moles

0114Reagents:

0115Fe'" : Fe" : Ni ratio = 8 : 3 : 1 22,34 g Fe(Ac)₃ (Sheperd pasta; c. 20% Fe w/w)

0116Fe(III) = 4.468 g = 80 mmoles

011723,94 g Fe(Ac)₂ solution (7% w/w in Co)

0118Fe(II) = 1.675 g = 30 mmoles

01191,77 g Ni(Ac)₂ Ni = 0.588 g = 10 mmoles

0120269.04 g DEG

0121Synthesis:

0122A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a reflux and distillation two-way system, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 110° C for the stabilisation step and maintained for 1 hour at such a temperature. Subsequently, the mixture is heated to 180⁰C maintaining the distillation step. Having reached the internal temperature of

0123180°C, the system is left at reflux for 3 hours. The process performed while mixing leads to the formation of a brown suspension.

0124Example 4

0125Preparation of nanometric cobalt ferrite by the continuous process according to the present invention.

0126Product formula: NFeCo36 Stage 1 (Product formula: NFeCo35) Reagents specifications:

0127Fe(CH₃COO)₃ MW = 232.98 g/moles

0128Co(CH₃COO)₂^«4H₂O MW = 248.93 g/moles CoFe₂O₄ (NFeCo 31) MW = 234,62 g/moles

0129Co MW = 58.93 g/moles

0130Fe MW = 55.85 g/moles

0131DEG MW = 106.12 g/moles Reagents:

0132Fe : Co ratio = 2 : 1

01339.53 g Co(Ac)₂*4H₂O (23.7% Co w/w)

0134Co(II) = 2.259 g = 0.038 moles

013521.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w) Fe(III) = 4.284 g = 0.077 moles

0136269.04 g DEG

0137287 g NFeCo 31

0138Synthesis:

0139A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for the possible distillation, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 110° C for the solubilisation step (time: 1 h). Subsequently, the temperature is raised to 18O⁰C and the system is left at reflux for 3 hours. The process performed while mixing leads to the formation of a black suspension. 570 g of the product are obtained.

0140Step 2 (Product formula: NFeCo36)

0141Reagents specifications: as above

0142Reagents:

0143Fe : Co ratio = 2 : 1 19,06 g Co(Ac)₂-4H₂O (23.7% Co w/w)

0144Co(II) = 4.518 g = 0.076 moles

014542,84 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0146Fe(III) = 8.568 g = 0.154 moles

0147538 g DEG 570 g NFeCo_35

0148Synthesis: A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for the possible distillation, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 110° C for the solubilisation step (time: 1 h). Subsequently, the temperature is raised to 180⁰C and the system is left at reflux for 3 hours. The process performed while mixing leads to the formation of a black suspension.

01491105 g of the product are obtained.

0150Example 5

0151Preparation of nanometric magnetite product formula Fe76 with the continuous process according to the present invention.

0152Stage 1 (Product formula Fe75)

0153Reagents specifications:

0154Fe(CH₃COO)₃ MW = 232.98 g/moles

0155Fe(CH₃COO)₂ MW = 248.93 g/moles Fe₃O₄ (Fe74) MW = 231.53 g/moles

0156Fe MW = 55.85 g/moles

0157DEG MW = 106.12 g/moles

0158Reagents:

0159Fe'" : Fe" ratio = 2 : 1 30,32 g Fe(Ac)₂ solution (7% Fe w/w)

0160Fe(II) = 2.122 g = 0.038 moles

016121.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0162Fe(III) = 4.284 g = 0.077 moles

0163269.04 g DEG 279 g Fe74

0164Synthesis:

0165A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a reflux and distillation two-way system, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 120° C for the stabilisation step and maintained for 1 hour at such a temperature. Subsequently, the mixture is heated to 180⁰C maintaining the distillation step. Having reached the internal temperature of 180°C, the system is left at reflux for 3 hours. The process performed while mixing leads to the formation of a brown suspension.

0166Total obtained: 552 g.

0167Stage 2 (Product formula: Fe76) Reagents specifications: as above

0168Reagents:

0169Fe III : Fe Il ratio = 2 : 1

017060,64 g Fe(Ac)₂ solution (7% Fe w/w)

0171Fe(II) = 4.244 g = 0.076 moles 42,84 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0172Fe(III) = 8.568 g = 0.154 moles

0173538.08 g DEG

0174552 g Fe75

0175Synthesis: A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a reflux and distillation two-way system, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 120° C for the stabilisation step and maintained for 1 hour at such a temperature. Subsequently, the mixture is heated to 18O⁰C maintaining the distillation step. Having reached the internal temperature of

0176180⁰C, the system is left at reflux for 3 hours. The process performed while mixing leads to the formation of a brown suspension.

0177Total obtained: 1113 g.

0178Example 6 Process for the preparation of cobalt ferrite with the method of the semicontinuous substitutions according to the present invention. Product formula: NFeCoCONT-

03B3

0180Stage 1 (Product formula: NFeCoCONT-03B1)

0181Reagents specifications: Fe(CH₃COO)3 MW = 232.98 g/moles

0182Co(CH₃COO)₂ 4H₂O MW = 248.93 g/moles

0183CoFe₂O₄ (NFeCo 31 ) MW = 234.62 g/moles Co MW = 58.93 g/mo!es

0184Fe MW = 55.85 g/moles

0185DEG MW = 106.12 g/moles

0186Reagents: Fe : Co ratio = 2 : 1

01879.53 g Co(Ac)₂-4H₂O (23.7% Co w/w)

0188Co(II) = 2.259 g = 0.038 moles

018921.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0190Fe(III) = 4.284 g = 0.077 moles 269.04 g DEG

0191285 g NFeCo_31

0192Synthesis:

0193A 1 litre 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for the possible distillation, a probe and a stopper (addition neck). The cobalt acetate and the iron acetate are placed with the DEG in the reaction flask and NFeCo31 which is still warm from the previous reaction is added. The temperature is taken to 180⁰C and the system is left at reflux for 3 hours. 575 g of the product are obtained.

0194Stage 2 (Product formula: NFeCoCONT-03B2) Reagents specifications: as above

0195Reagents:

0196Fe : Co ratio = 2 : 1

019719.06 g Co(Ac)₂^H₂O (23.7% Co w/w)

0198Co(II) = 4.518 g = 0.076 moles 42.84 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0199Fe(III) = 8.568 g = 0.154 moles

0200538 g DEG

0201575 g NFeCoCONT-03B1

0202Synthesis: A 2 litre 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for a possible distillation, a probe and a stopper (addition neck). The cobalt acetate and the iron acetate are placed with the DEG in the reaction flask and NfeCoCont_31 B1 which is still warm from the previous reaction is added. The temperature is taken to 180⁰C and the system is left at reflux for 3 hours. 1105 g of the product have been obtained.

0203Stage 3 (Product formula: NFeCoCONT-03B3) Reagents specifications: as above

0204Reagents:

0205Fe : Co ratio = 2 : 1

020638,12 g Co(Ac)₂-4H₂O (23.7% Co w/w)

0207Co(II) = 9.036 g = 0.152 moles 85.68 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0208Fe(III) = 17,136 g = 0.308 moles

02091076 g DEG

02101105 g NFeCoCONT-03B2

0211Synthesis: A 5 litre 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for a possible distillation, a probe and a stopper (addition neck). The cobalt acetate and the iron acetate are placed with the DEG in the reaction flask and NFeCoCont_31B2 which is still warm from the previous reaction is added. The temperature is taken to 180⁰C and the system is left at reflux for 3 hours. 2210 g of the product have been obtained.

0212Example 7

0213Preparation of nanometric cobalt ferrite with the growth process according to the present invention.

0214Product formula: NAMA06 602 Reagents specifications:

0215Fe(CH₃COO)₃ MVV = 232.98 g/moles

0216Co(CH₃COO)₂^H₂O MW = 248.93 g/moles

0217Co - MW = 58.93 g/moles

0218Fe MW = 55.85 g/moles DEG MW = 106.12 g/moles

0219Reagents:

0220Fe : Co ratio = 2 : 1 9.53 g Co(Ac)₂-4H₂O (23.7% Co w/w)

0221Co(II) = 2.259 g = 0.038 moles

022221.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0223Fe(III) = 4.284 g = 0.077 moles 269.04 g DEG

0224Synthesis:

0225A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for a possible distillation, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 110° C for the solubilisation step (time: 1 h). Subsequently, the temperature is raised to 180⁰C and the system is left at reflux for 5 h. The process performed while mixing leads to the formation of a black suspension.

0226Total obtained: g 282.

0227Example 8 Preparation of nanometric cobalt ferrite with the growth process according to the present invention.

0228Product formula: NAMA06 601

0229Reagents specifications:

0230Fe(CH₃COO)₃ MW = 232.98 g/moles Co(CH₃COO)₂^H₂O MW = 248.93 g/moles

0231Co MW = 58.93 g/moles

0232Fe MW = 55.85 g/moles

0233DEG MW = 106.12 g/moles

0234Reagents: Fe : Co ratio = 2 : 1

02359.53 g Co(Ac)₂-4H₂O (23.7% Co w/w)

0236Co(II) = 2.259 g = 0.038 moles

023721.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0238Fe(III) = 4.284 g = 0.077 moles 269.04 g DEG

0239Synthesis: A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for a possible distillation, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 110° C for the solubilisation step (time: 1 h). Subsequently, the temperature is raised to 180⁰C and the system is left at reflux for 9 h. The process performed while mixing leads to the formation of a black suspension.

0240Total obtained: 280 g.

0241Example 9

0242Preparation of nanometric cobalt ferrite with the growth process according to the present invention.

0243Product formula: NfeCo66

0244Reagents specifications:

0245Fe(CH₃COO)₃ MW = 232.98 g/moles

0246Co(CH₃COO)₂^H₂O MW = 248.93 g/moles Co MW = 58.93 g/moles

0247Fe MW = 55.85 g/moles

0248DEG MW = 106.12 g/moles

0249Reagents:

0250Fe : Co ratio = 2 : 1 9.53 g Co(Ac)₂ ^H₂O (23.7% Co w/w)

0251Co(II) = 2.259 g = 0.038 moles

025221.42 g Fe(CH₃COO)₃ (Sheperd pasta; c. 20% Fe w/w)

0253Fe(III) = 4.284 g = 0.077 moles

0254269.04 g DEG Synthesis:

0255A 4-necked flask is equipped with a blade stirrer, a bubble condenser provided with a valve for a possible distillation, a probe and a stopper (addition neck). The reagents are placed with the DEG in the reaction flask. The system is taken to the temperature of 110° C for the solubilisation step (time: 1 h). Subsequently, the temperature is raised to 180⁰C and the system is left at reflux for 24 hours. The process performed while mixing leads to the formation of a black suspension.

0256Total obtained: 280 g. Example 10

0257Process for the preparation of cobalt ferrite with microwave heating according to the present invention.

0258Product formula: NFeCoMWOI Reagents specifications:

0259Fe(CH₃COO)₃ MW = 232.98 g/moies

0260Co(CH₃COO)₂^H₂O MW = 248.93 g/moles

0261Co MW = 58.93 g/moles

0262Fe MW = 55.85 g/moles DEG MW = 106.12 g/moles

0263Reagents:

0264Fe : Co ratio = 2 : 1

026511 ,1O g Co(Ac)₂-4H₂O (23.7% Co w/w)

0266Co(II) = 2,632 g = 0.0447 moles 19.23 g Fe(CH₃COO)₃ (Sheperd powder; c. 26% Fe w/w)

0267Fe(III) = 4.998 g = 0.0895 moles

0268319,67 g DEG

0269Synthesis:

0270All the reagents are placed in a 500 ml 1-neck flask. It is equipped with a bubble condenser. The flask is placed in a microwave chamber maintaining the bubble condenser outside the same. Power is applied for 7 minutes while maintaining at reflux.

0271Example 11

0272Process for the preparation of cobalt ferrite with microwave heating according to the present invention.

0273Product formula: NFeCoMW03

0274Reagents specifications: Fe(CH₃COO)₃ MW = 232.98 g/moles

0275Co(CH₃COO)₂^H₂O MW = 248.93 g/moles Co MW = 58.93 g/moles

0276Fe MW = 55.85 g/moles

0277DEG Reagents:

0278Fe : Co ratio = 2 : 1

027911 ,10 g Co(Ac)₂ ^H₂O (23.7% Co w/w)

0280Co(II) = 2.632 g = 0.0447 moles 19.23 g Fe(CH₃COO)₃ (Sheperd powder; c. 26% Fe w/w)

0281Fe(III) = 4.998 g = 0.0895 moles

0282319,67 g DEG

0283Synthesis:

0284All the reagents are placed in a 500 ml 1-neck flask. It is equipped with a bubble condenser. The flask is placed in a microwave chamber maintaining the bubble condenser outside the same. Power is applied for 30 minutes while maintaining at reflux.

0285Example 12

0286Process of preparation of maghemite by acetic oxidation according to the present invention

0287Product formula: Fe59.1.1.1

0288Reagents specifications:

0289CH₃COOH MW = 60.05 g/moles

0290Fe₃O₄ ( Fe74 ) MW = 231.53 g/moles NaHCO₃ MW = 84.00 g/moles

0291Reagents:

029240 g Fe₃O₄ ( Fe74 ) solution in DEG 0,5 % w/w in Fe₃O₄

0293Fe₃O₄ 200 mg 0.864 mmoles

02941.00 g CH₃COOH 6.7 mmoles 1.2 g NaHCO₃ 14.3 mmoles

0295Synthesis:

0296The magnetite solution in DEG and the acetic acid are placed in a flask provided with mixing, with a bubbling capillary and a bubble condenser. The whole is heated to 80° and is maintained at temperature for 2.5 hours. At the end of the reaction, it is cooled to room temperature and sodium hydrogen carbonate is added. It is maintained mixing for 1 hour and the remaining solid product is then filtered. The clear dark brown solution is characterised by Mossbauer spectrometry to check the complete formation of maghemite. The solution is then evaporated in a vacuum rotary evaporator to the desired concentration. Example 13 Functionalisation of a cobalt ferrite nanoparticle with palmitic acid according to the present invention. Product formula CoFeI 4 Reagents specifications: Palmitic acid MW = 256.42 g/moles Et-OH MW = 4607 g/moles n - hexane MW = 86.17 g/moles

0297CoFe₂O₄ (:NFeCoCONT-03B3) MW = 234.62 g/moles

0298Reagents: 20 g NAMA06 solution in DEG (3% Co w/w Fe₂O₄) 2.56 mmoles

02990,45 g palmitic acid 1.76 mmoles 40 g Et-OH 40 g n - hexane Synthesis:

0300Ethanol and palmitic acid are placed in a magnetically stirred Erlenmeyer flask. It is carefully heated while mixing on a heating plate to 45 - 50⁰C. It is then maintained mixing to complete solubilisation of the palmitic acid. The nanoparticulate cobalt ferrite solution is added. The temperature settles to about 40°C. It is left mixing for 1 hour.

0301The content is poured from the Erlenmeyer flask in a separating funnel and hexane is extracted. The apolar phase is then washed twice with 40 ml of a diluted aqueous solution of sodium hydrogen carbonate (0.6 g in 100 ml of water) and then with 40 ml of water. The organic phase obtained is concentrated under vacuum to the desired volume. Example 14

0302Functionalisation of a cobalt ferrite nanoparticle with ethyl 12-(hydroxyamine)-12- oxododecanoate according to the present invention.

0303Product formula CoFe38H Reagents specifications: ethyl 12-(hydroxyamine)-12-oxododecanoate MW = 273.37g/moles

0304CoFe₂O₄ (:NFeCoCONT-03B3) MW = 234.62 g/moles

0305Buthanol MW = 74.12

0306Water Reagents:

030760 g: NFeCoCONT-03B3 solution in DEG (3% CoFe2O4 w/w) 7.67 mmoles

03080.90 g ethyl 12-(hydroxyamine)-12-oxododecanoate 3.29 mmoles

030912O g buthanol

0310Synthesis: 120 g of buthanol and 0.60 g of ethyl 12-(hydroxyamine)-12-oxododecanoate are placed in a 500 ml flask (complete solubility in a few minutes). 60 g of a dispersion of cobalt ferrite nanoparticles in glycol are added to this solution and left mixing for

03112 hours.

0312The sample has been washed with 200 g of water (formation of a double phase buthanol/water-glycol) and separated from the aqueous phase with a separating funnel. The solid product has been obtained by removing the buthanol under vacuum and then redispersing it in acetone.

0313Example 15

0314Preparation of a construct comprised of: nanometric cobalt ferrite, PLGA and albumin according to the present invention.

0315Product formula: NBR1

0316Reagents: Amount Molecular weight

0317Water UP 1000 ml 18 d = 1 .00 g/cm³

0318Acetone 25 ml 58,08 d = 0 .79 g/cm³

0319PLGA 75/25 0.05 g

0320CoFe38H 0.02 g

0321BSA Fraction V 1 g Synthesis:

0322A solution of PLGA in acetone (0.05 grams in 25 ml of acetone), a solution of BSA in ultrapure water (1 gram of BSA in 1000 ml of water) are previously prepared. 0,4 ml of a 5% CoFe38H suspension in acetone (w/V) are added to the PLGA solution.

0323A double peristaltic pump is provided to continuously add the acetonic solution (containing PLGA and CoFe38H) in a water flow containing BSA (volume ratio acetone/water = 1/40). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions.

0324The pumping ratio of the two peristaltic pumps is set to 1/40 so that the two solutions are consumed at the same time. The product of the final mixing is collected in a graduated cylinder. The pumping rate is set so that the mixing of the solutions occurs in 10 minutes.

0325The resulting final solution is treated under vacuum so as to completely remove acetone. The resulting final solution is concentrated under high-vacuum at T &lt; 45⁰C or by means of ultrafiltration until the desired concentration is obtained.

0326Size characterisation by means of DLS:

0327<img file="EP2117600A2_D0022.tif" />

0328Example 16

0329Preparation of a construct comrpising: nanometric cobalt ferrite, Paclitaxel , PLGA and albumin according to the present invention.

0330Product formula: NBR1 F1

0331Reagents: Amount Molecular weight

0332UP water 956 ml 18 d = 1.00 g/cm

0333Acetone 104 ml 58.08 d = 0.79 g/cm

0334PLGA 75/25 0.5 g

0335CoFe38H 0.2 g

0336BSA Fraction V 1 g Paclitaxei 10 mg 853.91

0337Synthesis:

0338A solution of PLGA in acetone (0.5 grams in 100 ml of acetone) and a solution of BSA in ultrapure water (1 gram of BSA in 800 ml of water) are previously prepared. 10 mg of Paclitaxei and 4 ml of a 5% CoFe38H suspension in acetone (w/V) are added to the PLGA solution in acetone.

0339A double peristaltic pump is provided to continuously add the acetonic solution (containing PLGA, CoFe38H and Paclitaxei) in a water flow containing BSA (volume ratio acetone/water = 1/8). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions. The pumping ratio of the two peristaltic pumps is set to 1/8 so that the two solutions are consumed at the same time. The product of the final mixing is collected in a graduated cylinder. The pumping rate is set so that the mixing of the solutions occurs in 10 minutes. The resulting final solution is treated under vacuum to completely remove acetone.

0340The resulting final solution is concentrated under high-vacuum at T &lt; 45°C or by means of ultrafiltration until the desired concentration is obtained. Example 17 Preparation of a construct comrpising: nanometric cobalt ferrite, 9-nitro- camptothecin, PLGA and albumin according to the present invention. Product formula: NBR1 F2

0341Reagents: Amount Molecular weight

0342UP water 1356 ml 18 d = 1.00 g/cm³

0343Acetone 104 ml 58.08 d = 0.79 g/cm³

0344PLGA 75/25 0.5 g

0345CoFe38H 0.2 g

0346BSA Fraction V 1 g

0347NaCI 14.4 g 58.44

03489-n itro-cam ptothecin 25 mg

0349Glutaraldehyde 1.56 mg 100.1

0350Synthesis: A solution of PLGA in acetone (0.5 grams in 100 ml of acetone) and a solution of BSA in ultrapure water (1 gram of BSA in 800 ml of water) are previously prepared. 25 mg of 9-nitro-camptothecin and 4 ml of a 5% CoFe38H suspension in acetone (w/V) are added to the PLGA solution in acetone. A double peristaltic pump is provided to continuously add the acetonic solution (containing PLGA, CoFe38H and 9-nitro-camptothecin) in a water flow containing BSA (volume ratio acetone/water = 1/8). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions. The pumping ratio of the two peristaltic pumps is set to 1/8 so that the two solutions are consumed at the same time. The product of the final mixing is collected in a graduated cylinder, in which there are already 400 ml of water containing 3.6% of NaCI. The pumping rate is set so that the mixing of the solutions occurs in 10 minutes. The resulting final solution is treated under vacuum to completely remove acetone.

0351Subsequently, 156 ml of an aqueous solution of glutaraldehyde (cone. 10 mg/l of Glutaraldehyde) are added and it is left to rest for 10 h.

0352The resulting final solution is concentrated under high-vacuum at T &lt; 45°C or by means of ultrafiltration until the desired concentration is obtained. Example 18

0353Preparation of a construct comrpising nanometric cobalt ferrite and a block polymer according to the present invention. Product formula: NBR2

0354Reagents: Amount Molecular weight

0355UP water 400 ml 18 d = 1.00 g/cm³

0356Acetone 200 g 58, 08 d = 0.79 g/cm³

0357CoFe38H 1 9

0358Pluronics F-68 5.6 g

0359Synthesis:

0360A solution of CoFe38H in acetone (1 gram in 200 ml of acetone) is previously prepared. A double peristaltic pump is provided to continuously add the acetonic solution (containing CoFe38H) in a water flow containing Pluronics F-68 (volume ratio acetone/water = 1/2). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions. The pumping ratio of the two peristaltic pumps is set to 1/2 so that the two solutions are consumed at the same time. The pumping rate is set so that the mixing of the solutions occurs in 10 minutes.

0361The resulting final solution is treated under vacuum to completely remove acetone.

0362Size characterisation by means of DLS:

0363<img file="EP2117600A2_D0023.tif" />

0364The DLS confirms the stability of the nanoparticles in aqueous solution and in physiological solution.

0365The raw image analysis shows very distinct dark cores of about 17-45 nm in the case of STEM, the separations are clear-cut and on the average equal to about 5-

036615 nm (surfactant layer).

0367Example 19

0368Preparation of a construct comrpising: nanometric cobalt ferrite, cis- diammineplatinum (II) dichloride, PLGA and albumin according to the present invention.

0369Product formula: Product formula: NBR1 F3

0370Reagents: Amount Molecular weight

0371UP water 1356 ml 18 d = 1.00 g/cm

0372Acetone 104 ml 58,08 d = 0.79 g/cm

0373PLGA 75/25 0.5 g

0374CoFe38H 0.2 g

0375BSA Fraction V 1 g

0376NaCI 14.4 g 58,44 cis-diammineplatinum (II) dichloride 100 mg 300.1

0377Synthesis: a PLGA solution in acetone (0.5 grams in 100 ml of acetone); a BSA solution in ultrapure water (1 gram of BSA in 800 ml of water); a NaCI solution in ultrapure water (14.4 g of NaCI in 400 ml of water) are previously prepared.

03784 ml of a 5% CoFe38H suspension in acetone (w/V) are added to the PLGA solution in acetone, while 100 mg of cis-diammineplatinum (II) dichloride are dissolved in the albumin aqueous solution. A double peristaltic pump is provided to continuously add the acetonic solution

0379(containing PLGA and CoFe38H) in a water flow containing BSA and the drug

0380(volume ratio acetone/water = 1/8). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions.

0381The pumping ratio of the two peristaltic pumps is set to 1/8 so that the two solutions are consumed at the same time. The product of the final mixing is collected in a graduated cylinder, in which there are already 400 ml of water containing 3.6% of NaCI. The pumping rate is set so that the mixing of the solutions occurs in 10 minutes.

0382The resulting final solution is concentrated under high-vacuum at T &lt; 45°C or by means of ultrafiltration until the desired concentration is obtained.

0383Example 20

0384Preparation of a construct comrpising nanometric cobalt ferrite, Paclitaxel and a block polymer.

0385Product formula: NBR2F1

0386Reagents: Amount Molecular weight

0387Water 80 ml 18 d = 1.00 g/cm³

0388Acetone 40 g 58.08 d = 0.79 g/cm³

0389CoFe38H 0.2 g

0390Pluronics F-68 1.12 g

0391Paclitaxel 10 mg 853.9

0392Synthesis: A solution of CoFe38H in acetone (0.2 grams in 40 ml of acetone) is previously prepared and 10 mg of Paclitaxel are solubiiised in the mixture.

0393A double peristaltic pump is provided to continuously add the acetonic solution

0394(containing CoFe38H and the drug) in a water flow containing Pluronics F-68

0395(volume ratio acetone/water = 1/2). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions. The pumping ratio of the two peristaltic pumps is set to 1/2 so that the two solutions are consumed at the same time. The pumping rate is set so that the mixing of the solutions occurs in 6 minutes.

0396The resulting final solution is treated under vacuum so as to completely remove acetone.

0397Size characterisation by means of DLS:

0398<img file="EP2117600A2_D0024.tif" />

0399The DLS confirms the stability of the nanoparticles in aqueous solution and in physiological solution.

0400Example 21

0401Preparation of the construct comrpising nanometric magnetite and a water-soluble polyamineamide polymer (BAC-EDDA) of formla (A)

0402according to the present invention. Product formula: NBR4

0403Reagents: Amount Molecular weight

0404Water 200 ml 18 d = 1.00 g/crrf

0405Diethylene glycol 40 g 106,1 d = 1.12 g/cnr

0406Fe77 0.2 g

0407BAC-EDDA polymer 1.12 g water-soluble stealth polymer based on ethylene diamine diacetic acid Synthesis: a solution of magnetite in diethylene glycol (0.2 grams in 40 ml of solvent); a solution of BAC-EDDA polymer in water (1.12 grams in 200 ml of solvent) are previously prepared;

0408A double peristaltic pump is provided to continuously add the organic solution (containing magnetite) in a water flow containing the BAC-EDDA polymer (volume ratio diethylene glycol/water = 1/5). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions. The pumping ratio of the two peristaltic pumps is set to 1/5 so that the two solutions are consumed at the same time. The pumping rate is set so that the mixing of the solutions occurs in 12 minutes.

0409The resulting final solution is dialysed with ultrapure water so as to remove most of the organic solvent and obtain a solution containing at most 0.1% diethylene glycol.

0410Size characterisation by means of DLS:

0411<img file="EP2117600A2_D0025.tif" />

0412The DLS confirms the stability of the nanoparticles in aqueous solution and in physiological solution. Example 22

0413Preparation of a construct comrpising nanometric magnetite, a water-soluble polymer (BAC-EDDA) and Cis-diammineplatinum (II) dichloride according to the present invention.

0414Product formula: NBR4F1

0415Reagents: Amount Molecular weight

0416Water 200 ml 18 d = 1.00 g/cm^Λ3

0417Diethylene glycol 40 g 106.1 d = 1.12 g/cm^Λ3

0418Fe77 0.2 g BAC-EDDA polymer 1.12 g

0419Cis-diammineplatinum (II) dichloride 100 mg 300.1

0420© Water-soluble stealth polymer based on ethylene diamine diacetic acid

0421Synthesis: a solution of magnetite in diethylene glycol (0.2 grams in 40 ml of solvent); a solution of BAC-EDDA polymer in water (1.12 grams in 200 ml of solvent) in which 100 mg of Cis-Diammineplatinum (II) dichloride are previously prepared;

0422A double peristaltic pump is provided to continuously add the organic solution

0423(containing magnetite) in a water flow containing the BAC-EDDA polymer and the Cis-Diammineplatinum (II) dichloride (volume ratio diethylene glycol/water = 1/5).

0424The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions. The pumping ratio of the two peristaltic pumps is set to 1/5 so that the two solutions are consumed at the same time. The pumping rate is set so that the mixing of the solutions occurs in 8 minutes. The resulting final solution is dialysed with ultrapure water so as to remove most of the organic solvent and to obtain a solution containing at most 0.1% diethylene glycol.

0425Size characterisation by means of DLS:

0426<img file="EP2117600A2_D0026.tif" /> The DLS confirms the stability of the nanoparticles in aqueous solution and in physiological solution.

0427Example23 Preparation of a construct comprising of nanometric cobalt ferrite, PLGA and a block polymer according to the present invention.

0428Product formula: NBR32

0429Reagents: Amount Molecular weight

0430UP water 800 ml 18 d = 1 .00 g/cm³ Acetone 20 g 58,08 d = 0 .79 g/cm³

0431CoFe38H 0,02 g

0432PLGA 0,05 g

0433Pluronics F-68 0,8 g

0434Synthesis: A solution of PLGA in acetone (0.05 grams in 20 ml of acetone), a solution of

0435Pluronic F-68 in ultrapure water (0,8gram of PLURONIC F-68 in 800 ml of water) are previously prepared. 0,4 ml of a 5% CoFe38H suspension in acetone (wA/) are added to the PLGA solution.

0436A double peristaltic pump is provided to continuously add the acetonic solution (containing PLGA and CoFe38H) in a water flow containing PLURONIC F-68

0437(volume ratio acetone/water = 1/40). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions.

0438The pumping ratio of the two peristaltic pumps is set to 1/40 so that the two solutions are consumed at the same time. The product of the final mixing is collected in a graduated cylinde. The pumping rate is set so that the mixing of the solutions occurs in 10 minutes.

0439The resulting final solution is treated under vacuum so as to completely remove acetone. The resulting final solution is concentrated under high-vacuum at T &lt;

044045°C or by means of ultrafiltration until the desired concentration is obtained. EXAMPLE 24

0441Preparation of a construct comprising of nanometric cobalt ferrite, PLGA and a block polymer according to the present invention. Product formula: NBR5

0442Reagents: Amount Molecular weight

0443UP water 800 ml 18 d = 1.00 g/cm³

0444Ethanol 20 g CoFe38H 0,02 g

0445Colesterol 0,05 g

0446Pluronics F-68 0,8 g

0447Synthesis:

0448A solution of colesterol in ethanol (0.05 grams in 20 ml of acetone), a solution of Pluronic F-68 in ultrapure water (0,8gram of PLURONIC F-68 in 800 ml of water) are previously prepared. 0,4 ml of a 5% CoFe38H suspension in acetone (w/V) are added to the colesterol solution.

0449A double peristaltic pump is provided to continuously add the acetonic solution

0450(containing colesterol and CoFe38H) in a water flow containing PLURONIC F-68 (volume ratio acetone/water = 1/40). The corresponding immersion tubes withdraw the solution directly from the reservoirs containing the two solutions.

0451The pumping ratio of the two peristaltic pumps is set to 1/40 so that the two solutions are consumed at the same time. The product of the final mixing is collected in a graduated cylinde. The pumping rate is set so that the mixing of the solutions occurs in 10 minutes.

0452The resulting final solution is treated under vacuum so as to completely remove acetone. The resulting final solution is concentrated under high-vacuum at T &lt;

045345°C or by means of ultrafiltration until the desired concentration is obtained.

0454Moreover, for completeness, example of preparation of some polymers useful for the invention as above said are reported hereinafter.

0455Example 23

0456Synthesis of BAC-EDDS

0457<img file="EP2117600A2_D0027.tif" />

0458Lithium hydroxide monohydrate (112,64 mg, 2,6576 mmol) was added under stirring to a 37,36% solution (as determined titrimetrically just before use) of (S₁S)- ethylenediamine-N,N^J-disuccinic acid trisodium salt (EDDS) (2 ml, 2.6576 mmol) contained in a 50 ml 2 necked flask. 2,2-Bis-acrylamidoacetic acid (BAC) (530,6 mg, 2,6576 mmol) and lithium hydroxide monohydrate (112,64 mg, 2,6576 mmol) were then added and the reaction mixture was maintained 7 days under stirring at 18-20 ⁰C. After this time, 2-propanol (25ml) was added. The crude product was isolated by centrifuging, extracted with fresh 2-propanol (2 x 15ml) and ether (1 x 15ml) and finally dried to constant weight at room T° and 0,1 tor. Yield 61 ,28<img file="EP2117600A2_D0028.tif" />

0459Example 24 Synthesis of BP-EDDS

0460<img file="EP2117600A2_D0029.tif" />

0461Lithium hydroxide monohydrate (112,64 mg, 2,6576 mmol) was added under stirring to a 37,36% solution (as determined titrimetrically just before use) of EDDS) (2 ml, 2.6576 mmol) contained in a 50 ml 2 necked flask. 1 ,4- bisacryloylpiperazine (BP) (516,19 mg. 2,6576 mmol) was then added. The reaction mixture was then treated and the final product isolated exactly as described in the previous case. Yield 91 ,8 %. M«> = 6500, M_n = 4500.

0462Synthesis of BA-EDDS <img file="EP2117600A2_D0030.tif" />

0463Lithium hydroxide monohydrate (112,64 mg, 2,6576 mmol) was added under stirring to a 37,36% solution (as determined titrimetrically just before use) of EDDS) (2 mi, 2.6576 mmol) contained in a 50 ml 2 necked flask. N₁N' methylenebisacrylamide (MBA) (516,19 mg, 2,6576 mmol) was then added and the reaction mixture stirred at room temperature for 2 days. After this time, of water (1 ,5 ml) was added to the cloudy mixture that was gently warmed to dissolve the suspended materials.

0464The reaction mixture was then treated and the final product isolated exactly as described in the previous case. Yield 90,1 %. Ε_w =2600, M,, = 1900.

0465TABLE 1 (a) synthesis of non-functionalised magnetic nanoparticles

0466<img file="EP2117600A2_D0031.tif" />

0467PTTIT 7001- BR continuous process Rl semicontinuous substitution method CO growth method ST

0468Microwave method MO time in minutes (MO) or hours (ST) <img file="EP2117600A2_D0032.tif" />

0469<img file="EP2117600A2_D0033.tif" />

0470<img file="EP2117600A2_D0034.tif" />

0471<img file="EP2117600A2_D0035.tif" /> Table 3 (hyperthermic effect of the non-functionalised nanoparticles)

0472<img file="EP2117600A2_D0036.tif" /> Table 4 h erthermic effect of the functionalised articles and constructs)

0473Ul

0474<img file="EP2117600A2_D0037.tif" />

0475<img file="EP2117600A2_D0038.tif" />

0476Ul Ul

0477<img file="EP2117600A2_D0039.tif" />

0478All the measurments have been carried out with irradiation at 170 KHz and with a magnetic field of 21 KA /m² for 30 seconds