WO2018138677A1

WO2018138677A1 - Process for the synthesis of stimulus-responsive magnetic nanoparticles

Info

Publication number: WO2018138677A1
Application number: PCT/IB2018/050477
Authority: WO
Inventors: Teresa Pellegrino; Thanh Binh MAI; Markus Joachim BARTHEL; Preethi Bala BALAKRISHNAN; Hamilton KAKWERE; Roberto Cingolani
Original assignee: Fondazione Istituto Italiano Di Tecnologia; Universita' Degli Studi Di Genova
Priority date: 2017-01-27
Filing date: 2018-01-26
Publication date: 2018-08-02
Also published as: EP3573662A1; IT201700009221A1

Abstract

Process for preparing magnetic nanoparticles coated with a thermo- or Ph-responsive polymer, characterized in that it comprises the radical polymerization of a monomer or co-monomers susceptible of forming a thermo-polymer or Ph-responsive copolymer in a solution including magnetic nanoparticles functionalized at their surface with a polymerization initiator of formula (I): wherein: R₃ is hydrogen or hydroxyl, Hal is a halogen selected from bromine, chlorine and iodine, m is an integer from 1 to 10, preferably from 1 to 3, and R₁ and R₂, independently of one another, are selected from hydrogen, methyl and phenyl. The coated particles are useful, particularly as carriers of a drug in therapeutic treatments by hyperthermia and/or in situ release of the drug.

Description

Process for the synthesis of stimulus-responsive magnetic nanoparticles

The present invention relates to a process for the production of magnetic nanoparticles coated with a stimulus-responsive polymer, particularly responsive to thermal or pH stimulation and to the particles thus obtained, particularly for use in the therapeutic treatment of tumors by hyperthermia and/or as a carrier for in situ release of a chemotherapic agent.

In recent decades, magnetic nanoparticles (MNPs) have taken an important role in biomedical applications, particularly in the field of therapeutic treatment of tumors. MNPs functionalized with a polymer shell or coating can be loaded with drugs and, due to their susceptibility to a magnetic field, the formulations thus obtained can be selectively activated at the site where they accumulate (for example, the tumor). These formulations thus offer the opportunity to reduce the total dose of drug administered to the patient as their concentration is increased at the tumor site, reducing the side effects caused by chemotherapy.

Moreover, MNPs can be used in hyperthermia treatments, as they can convert their magnetic energy into heat following exposure to an alternating magnetic field in biocompatible conditions and not dangerous for the various tissues and organs, causing damage to cells cancers that are more sensitive to an increase in temperature than healthy cells. The heating capacity of MNPs can also be combined with the properties of thermo- responsive materials, to obtain multifunctional materials adapted to allow the controlled release of a drug following the heat produced by MNPs.

In the field of drug delivery devices, the idea of applying thermo-responsive polymers that induce the coil-globule transition following a temperature change to prepare intelligent drug delivery systems with thermal activation features has taken on considerable interest. For this purpose, the use of polymers exhibiting a lower critical solution temperature (LCST) is preferred. These polymers are in stretched and elongated and water soluble condition if the solution temperature is lower than the LCST, while they are shrunken and insoluble if the temperature is higher than the LCST. In magnetic systems based on LCST, the heat generated by MNPs during the treatment of magnetic hyperthermia can be exploited to induce the shrinkage of the thermo-responsive polymer shell, so as to facilitate the release of the loaded cargo, typically chemotherapic agents. In WO2013/150496, whose inventors belong to the working group that developed the present invention, the synthesis of cubic iron oxide nanoparticles (IO Ps) is described by colloidal synthesis of nanoparticles having a high magnetic energy conversion value in heat called specific absorption rate (SAR). The features of these nanocubes ensure that they are a very promising candidate for heat-boosted thermal-responsive drug delivery systems. However, these colloidal synthesis IONPs are not soluble in aqueous media because their surface is covered by a layer of short hydrophobic ligands. Furthermore, their stability is a relevant problem, especially for sizes greater than 18-20 nm, a range in which the nanoparticles at room temperature switch from showing a superparamagnetic to ferromagnetic character; therefore, they tend to interact by forming aggregates, making their functionalization with a polymeric coating difficult.

The article by H. Kakwere et al., ACS, Applied Materials & Interfaces 2015, 7, 10132- 10145 describes the functionalization of the aforementioned highly interactive cubic IONPs with a thermo-responsive polymer (poly (N-isopropyl acrylamide co-oligoethylene glycol methyl ether acrylate), by surface-initiated Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization. The materials obtained showed thermo-responsive features, allowing the release an encapsulated drug, induced by the application of an alternating magnetic field. However, this approach was problematic, particularly in the scale-up of the synthesis protocol for in vivo studies, following low reproducibility and high aggregation following polymerization, as well as for the colloidal instability following the loading of the drug mainly due to presence of a significant fraction of aggregated nanocubes. Furthermore, the synthesis of polymerized nanoparticles according to the process described in the aforementioned publication is rather time consuming as it requires multiphase reactions.

A. Majewski et al., Biomacromolecules, vol. 13, No. 3, 12 March 2012, p. 857-866, describes the synthesis of core-shell magnetic nanoparticles with dual response (pH and temperature) using the grafting-from approach. Maghemite NPs were functionalized with an ATRP dopamine initiator and used for the ATRP initiated by DMAEMA surface in anisole. The present inventors have observed by means of experimental evidence that this process is not suitable for providing polymer-coated nanoparticles suitable for use in magnetic hyperthermia due to the extensive aggregation of the obtained nanoparticles.

The task of the present invention is to provide a synthesis process which overcomes the above drawbacks. In view of this task, an object of the invention is a process for the preparation of magnetic nanoparticles coated with a polymer responsive to a thermal or pH stimulus, having the features defined in the following claims.

Another object of the invention are coated nanoparticles, obtainable with the above method, both as such, and for use in therapeutic treatments of hyperthermia or, when loaded with a chemotherapic agent, in therapeutic treatments based on the release of the chemotherapic agent in situ.

Further advantages and features of the process and of the nanoparticles of the invention will be apparent from the following description, relating to both the general features of the process and specific embodiments.

Summary description of the figures In the accompanying drawings:

- figure 1 is a schematic representation of the synthesis of cubic IONPs functionalized with a polymeric coating, by means of the process object of the invention;

- figure 2 shows the hydrodynamic rays of the particles measured with the Dynamic

Light Scattering (DLS) technique of cubic IONPs (21 nm core edge size) capped with decanoic acid (continuous line, CHCh) modified with DOPA-BiBA (point line, in TUF) and functionalized with thermo-responsive polymer (point dash line, in PBS) obtained according to examples 1 to 4; - figures 3 and 4 are TEM micrographs of cubic IO Ps functionalized with thermo- responsive polymer, deposited by PBS respectively at low and high magnification obtained according to examples 1 to 4;

- figure 5 is a schematic representation of the polymer growth process on hetero- dimer structures consisting of an iron oxide body connected by a limited gold spherical nanoparticle interface; the box (a) illustrates the characterization of the resulting particles by DLS; the box (b) shows the variation of the DLS diameter as a function of the temperature for the determination of the LCST and the boxes (c and d) illustrate the characterization of such particles by TEM;

- figure 6 is a schematic representation of the growth process of a pH-responsive polymer on nanocubes; the box (a) illustrates the characterization of the resulting particles by DLS and the box (b) illustrates the characterization of such particles by TEM;

- figure 7 is a schematic representation of the loading procedure with DOXO and of free DOXO purification by means of magnetic separation; (b) UV absorption spectra of supernatants collected after purification; (c) DLS spectra of heat- responsive cubic IONPs before and after loading with DOXO; (d) the supernatant collected after the DMSO release experiment, 10 μΐ of nano-hybrids loaded with DOXO were diluted with 90 μΐ of DMSO, the particles were separated with a magnet at the bottom and 50 μΐ of supernatant were collected for the measurement;

- figure 8 is a schematic representation of the action of thermo-responsive iron oxide nanoparticles loaded with drug in a hyperthermia treatment;

- figure 9 is a diagram illustrating the survival rate over time of mice subjected to treatment with cubic IONPs, coated with thermo-responsive polymer and loaded with DOXO; and their respective controls.

- figure 10 is a diagram showing the tumor growth curves as a function of time in different treatments with nanoparticles coated and optionally loaded with DOXO.

Detailed description of the invention

Figure 1 shows a schematic and exemplary representation of the process according to the invention. Although specific reagents and process conditions are exemplified in the diagram in figure 1, it is intended that the general conditions described below apply thereto.

The process comprises the radical polymerization of a monomer or co-monomers susceptible of forming a thermo-polymer or pH-responsive copolymer in a solution including magnetic nanoparticles surface-functionalized with a polymerization initiator so as to cause polymerization of the monomers or co-monomers on the surface of the nanoparticles. The lymerization initiator is generally a compound of formula:

wherein:

R.3 is hydrogen or OH

Hal is halogen, in particular chlorine, bromine or iodine, preferably bromine,

m is an integer from 1 to 10, preferably from 1 to 3,

Ri and R2, independently of one another are selected from hydrogen, methyl and phenyl.

Preferred classes of compounds include:

- 2-halo-N-[(3,4-dihydroxyphenyl)Ci-Cioalkyl]acetamide;

- 2-halo-N-[(3,4-dihydroxyphenyl)Ci-Cioalkyl]propanamide;

- 2-halo-N-[(3,4-dihydroxyphenyl)Ci-Cioalkyl]-2-methylpropanamide;

- 2-halo-N-[(3,4-dihydroxyphenyl)Ci-Cioalkyl]-2-phenylacetamide and the corresponding compounds in which the 3,4-dihydroxyphenyl group is substituted by 3,4,5-trihydroxyphenyl.

Particularly preferred are the compounds in which the halogen is bromo, C1-C10 alkyl is Ci-C3alkyl, more preferably methylene and Ri and R2 are both methyl.

The polymerization reaction is a photo-induced polymerization (for example with UV light), mediated by a copper-based organometallic catalyst, preferably, using a copper/Me₆ TREN (tris(2-dimethylaminoethyl)amine) complex.

The polymerization reaction is preferably carried out in a solution comprising a solvent selected from dimethylsulfoxide, tetrahydrofuran and mixtures thereof, preferably in DMSO/THF volume ratio of 90: 10, preferably at a temperature of 5 to 10 °C, with polymerization times of the order of 2-6 hours.

The monomers or co-monomers used in order to form the functional thermo- or pH- responsive polymeric coating comprise non-acidic compounds, comprising the methacrylate group, preferably soluble in a solution of tetrahydrofuran and dimethylsulfoxide.

In particular, such monomers or co-monomers can be selected from compounds such as oligoethylene glycol methyl ether methacrylate, with a molecular weight preferably from 145 to 4000, preferably from 200 to 500 g.mol^"1 , dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, N-succinimidyl methacrylate and mixtures thereof.

The monomers or co-monomers and their molar ratio are preferably selected so as to obtain a polymer or copolymer having an LCST of between 25 and 65 °C, more preferably between 38 and 45 °C.

The introduction of the polymerization photo-initiator on the surface of the nanoparticles is preferably performed by a ligand exchange procedure. Figure 1 illustrates, by way of example, the case in which cubic IO Ps with decanoic acid are subjected to ligand exchange reaction with 2-bromo-N- [2-(3,4-dihydroxyphenyl)-ethyl]-propanamide (example 2), to obtain macro-initiator particles used in the subsequent functionalization step by means of photo-induced polymerization. It is understood, however, that the ligand exchange reaction can be carried out starting from nanoparticles with a ligand other than decanoic acid, such as a carboxylic acid having 5 to 12 C atoms.

The reaction is carried out in a solvent which ensures the solubility of the nanoparticles and the photoinitiator and which includes a base, such as triethylamine. For example, the solvent may be chloroform, ethyl alcohol or methyl and mixtures thereof. A quantity of photo-initiator ligand is used to saturate the surface of the nanoparticles. The excess of ligand can be removed by washing the resulting nanoparticles, for example with tetrahydrofuran, hexane or mixtures thereof, by centrifugation.

The nanoparticles obtained as a result of the ligand exchange reaction, with the use of the previously described photoinitiators, are soluble in tetrahydrofuran and completely insoluble in chloroform, which instead represents a good solvent for the initial nanoparticles. Stability tests revealed that the nanoparticles following dispersion in a mixture of dimethylsulfoxide/tetrahydrofuran showed the highest stability as the solution remained transparent compared to other solvent systems. For this reason this solvent, and particularly DMSO/THF in a ratio of 90/10 v/v (volume/volume), is a preferred solvent for subsequent polymerization. In order to preserve stability before polymerization, it is preferable to perform a gradual dilution of the macroinitiator particles obtained from THF to DMSO.

Moreover, in order to avoid pre-aggregation phenomena, it is preferred to maintain the concentration of the macroinitiator particles at values lower than 0.4 or 0.35 mg Fe.ml^"1.

In the following example 3, the polymerization was carried out with the use of the co- monomers diethylene glycol methyl ether methacrylate (DEGMEMA) and oligoethylene glycol methyl methacrylate (OEGMEMA) (molecular weight 500 g.mol^"1), which are preferred comonomers due to their excellent biocompatibility and response to thermal stimuli. Furthermore, the LCST in the polymer coating thus obtained can be adjusted simply by adjusting the molar ratio of the monomers.

The coated particles (nanohybrids) thus obtained can be collected by precipitation, for example in a TUF/di ethyl ether mixture (preferably in a ratio of 20:80). The precipitate may be washed one or more times, for example with a mixture of TUF/di ethyl ether and dried under nitrogen flow before being dissolved in water to remove excess polymeric ligands by centrifugation, on a sucrose gradient. The nanohybrids can be collected in the median region of the sucrose gradient (40%), while low-density polymeric ligands are held at the head of the centrifugation tubes.

As previously indicated, the magnetic particles usable in the process according to the invention preferably are ferrite particles such as iron oxide, generally cubic. However, as indicated in the exemplary scheme in figure 5, other types of magnetic nanoparticles, for example of other ferrites, such as cobalt ferrite, or also heterostructures in the form of iron oxide-dimer and also magnetic nanoparticles of Fe_xO_y/CdSe, Fe_xO_y/2nS or FexOy/luS can be used, wherein Fe_xO_y is preferably FesCk

In general, the average numerical size of the magnetic nanoparticles is between 10 and 80 nm, preferably between 10 and 35 nm.

The characterization of the functionalized dimer particles, thus obtained with the process shown in figure 5 by determining the hydrodynamic dimension and by means of TEM, confirmed the good solubility of Fe-Au dimers coated with thermo-responsive polymer.

As already indicated, the process also applies to the production of polymeric coatings responsive to pH stimulation, as illustrated in the schematic process representation in figure 6.

Applying the scheme in figure 6, it was possible to synthesize the poly(N,N- dimethylaminoethyl methacrylate-co-oligoethylene glycol methylether methacrylate (P(DMEAMA-co-OEGMA)) polymer on cubic particles of iron oxide introducing a pH- responsive character in the hybrid nanomaterials thus obtained.

The ability to synthesize nanoparticles with pH-responsive polymeric coating allows expanding the range of active agents that can be loaded into the nanohybrids. For example, with the product resulting from figure 6, nanohybrids can be loaded with micro-RNA and si-RNA.

The main advantage of the process according to the invention lies in the fact that with the use of surface-initiated polymerization, as described, it is possible to functionalize highly interactive magnetic nanoparticles, in an individual state and thus obtain excellent heating properties. Furthermore, the LCST of the resulting materials can be easily adjusted to obtain the desirable value by varying the monomer composition. The resulting nanohybrids have excellent biocompatibility demonstrated by cytotoxicity assays. They have excellent stability under physiological conditions and maintain the superior heating characteristics of the starting materials (i.e. the iron oxide cubes following exposure to suitable alternating magnetic fields) unchanged, respecting the biological limit. In particular, in comparison with the nanohybrids obtained with the procedure described in the publication of H. Kakwere et al., cited above, the main advantage lies in the high colloidal stability, both following the synthesis and following the loading of an active agent, as well as the possibility of significantly increasing the scale-up of the process. In fact, with the method according to the invention it was possible to obtain by process a 4-fold higher concentration of nano-hybrid material with iron amounts of up to 16 mg per batch, and with a yield of thermo-responsive cubes of 80-90% (low loss of starting materials).

The thermo- or pH-responsive nanoparticles obtained can be applied in a dual treatment of tumors, following loading with an active agent. Due to the high stability of the individual particles in solution and to the high specific absorption rate that results in particular from the cubic shape, but also with the use of the above mentioned dimer particles, they are highly efficient heat mediators following exposure to an alternating magnetic field; in particular, they maintain the same thermal efficiency as PEG-coated nanoparticles that do not have a thermo-responsive character. The heat generated in the tumor site can be exploited for a direct hyperthermia effect to kill cancer cells. At the same time, the same heat can induce the conformational change of the thermo-responsive shell with consequent release of the drug molecules encapsulated in the polymeric shell, as illustrated in the diagram in figure 8. The combination of both therapies is able to allow more effective tumor treatment.

In vivo studies, performed on a mouse model, with the use of nanoparticles loaded with DOXOrubicin hydrochloride (DOXO) obtained as illustrated in example 4, following the injection of particles into the tumor site and exposure to the magnetic field, have confirmed both the increase in temperature at the injection site and the release of DOXO with a direct effect on tumor growth. The group of animals treated with thermo-responsive nanocubes and loaded with DOXO and exposed to AFM treatment achieved the highest survival rate compared to all the other groups.

The greatest reduction in tumor size for the same group of animals was also observed, compared to all other groups. The results are shown in the graphs in figures 9 and 10.

Example 1 : synthesis of the 2-bromo-N-[2-(3,4-dihydroxy-phenyl)-ethyll-propionamide (DOPA-BiBA) initiator The initiator functionalized with catechol group (DOPA-BiBA) was synthesized following the procedure described by X. Fan et al., Journal of the American Chemical Society, 2005, 127, 15843-15847 (incorporated by quote), with minor modifications. In particular, Borace (Na₂B₄O7.10 H2O, 11,5 g, 30 mmol) was dissolved in 300 ml of water in a 500 ml round- bottom flask. The solution was degassed using a stream of nitrogen for 30 minutes and dopamine hydrochloride (5.7 g, 30 mmol) was added under nitrogen. The reaction mixture was stirred for 15 minutes and Na₂C0₃ (12.0 g, 113.2 mmol) was added to adjust the pH to 9-10. Then the solution was cooled using a bath of melting ice and 2- bromoisobutirrilbromide (2-BBB, 3.69 ml) was injected dropwise with a syringe. The reaction was allowed to proceed overnight under the stream of nitrogen. The mixture was acidified to reach pH 2 with a concentrated hydrochloric acid solution and extracted with ethyl acetate (3x150 ml). The extracted phase was dried over MgS0₄ and the solvent was removed by vacuum distillation to yield a brownish viscous liquid which was further purified by column chromatography (static phase: 70-230 mesh silica gel, mobile phase: 4% of methanol in chloroform). The resulting yellowish viscous liquid was recrystallized at -20 °C for 48 hours. The crystallites were washed several times with dichloromethane and dried in a vacuum oven to collect the pure product as white crystals (purity > 95%). Example 2: functionalization of the surface of IO Ps with initiator by ligand exchange

Cubic IONPs prepared according to the process described and exemplified in WO2013/150496 are used.

The initiator was immobilized on the surface of the nanoparticles by a ligand exchange procedure using an initial ratio of 500 ligand molecules per nm² of nanoparticle surface. 120 mg of DOPA-BBB were dissolved in 12.0 ml of 4% v/v methanol in chloroform in a 20 ml vial. To this solution were added 1.5 ml cubic IONPs (edge size 21 nm) in chloroform (containing 4.0 mg of iron) and the suspension was sonicated for 30 seconds. Thereafter, to the mixture were added 55.6 μΐ of trimethylamine (TEA). The vial was covered with aluminum foil to avoid contact with light and stirred strongly overnight. 15.0 ml of hexane were then rapidly added to the mixture to precipitate the particles. The suspension was centrifuged for 1500 minutes at 1500 RPM. The reddish supernatant that could contain an excess of oxidized ligands was removed and 10 ml of THF were added to disperse the particles. Then, 20.0 ml of hexane were added to destabilize the particles. This procedure was repeated twice to ensure removal of the free initiator. Thereafter, the cubic IONPs functionalized with DOPA-BBB were dispersed in THF to obtain a solution with an iron concentration of 4.0 gl^"1. For the exchange of ligand, using particles of different sizes, the volume was kept constant while the amount of DOPA-BBB was adjusted to maintain the ratio of 500 molecules per nm² and TEA was maintained at the stoichiometric ratio indicated with respect to DOPA -BBB. Figure 2 shows the DLS characterization.

Example 3 : synthesis of cubic IONPs functionalized with thermo-responsive polymers by means of photoinduced copper-mediated radical polymerization (PI-CMRM)

Cubic IONPs functionalized with DOPA-BBB in THF (4.0 gl^"1 iron concentration) was diluted with 3 ml of DMSO to form a clear solution that was subsequently added to a mixture containing 5 ml DMSO, 606.0 μΐ OEGMEMA and 894.0 μΐ DEGMEMA. In order to avoid the drastic variation of the concentration of particles that can cause a significant aggregation, four samples of the aforesaid mixture were prepared separately and then combined together in a single open vial of 60 ml volume. This solution was sonicated for 30 seconds at room temperature and purged with nitrogen for 15 minutes. In this vial, 4.0 ml of stock catalyst solution containing 4.0 ml DMSO, 0.52 mg CuBr₂ and 3.2 μΐ Me₆ TREN were subsequently injected. In a cold room at 5 °C, to start the polymerization, the vial was exposed to a UV source. The UV light source was a lamp for Nail Gel UV curing (λ max ~ 360 nm) equipped with four 9 W bulbs. During the polymerization, the vial was vigorously stirred with an orbital shaker. After 5 hours of irradiation, the polymerization was stopped by adding 80 ml of THF and exposure to air. The nanoparticles were precipitated with diethyl ether resulting in a gel-like black precipitate. The dissolution in THF and the precipitation in diethyl ether was repeated twice and the final precipitate was dried under nitrogen flow and redispersed in 60 mi of deionized water. The sample was concentrated to a final volume of 20 ml by centrifugation filter. To remove the excess of polymeric iigands, this solution was subjected to ultracentrifugation with a subsequent sucrose gradient in 12 ml tubes: 2 mi, 66% (weight/weight) - 3 ml, 40% (weight/weight) - 3 ml, 20% (weight/weight), speed 25000 rpm for 45 minutes. The speed used for ultracentrifugation was slightly changed depending on the particle size of the particles. For larger cubic nanoparticles, a slower speed was used. The fraction of nanohybrids thus obtained was collected in the median region of the centrifugation tubes while the head layer including unbound polymers was collected and lyophilized for size exclusion (SEC) and H NMR measurements. The sucrose was removed by centrifugation filter and the stable nano-hybrids were transferred to a saline phosphate buffer or saline for further characterization.

Figure 2 shows the DLS trace of the resulting particles. Figures 3 and 4 show their characterization by TEM.

E jI Blgi_.4ljoa.din

The encapsulation of DOXO into the magnetic nanohybrids thus obtained was performed by simple incubation. 400 μΐ of nano-hybrids in aqueous solution (5.0 gl^"!, 2.0 mg of iron) were added to 19.0 ml of saline (0.9% NaCl) containing 1 mg DOXO and sonicated for 10 s. By the addition of additional saline solution, the volume was increased to 20 ml and the solution was transferred into a 40 mi vial after further sonication for 20 s. The vial was covered with aluminum foil and gently stirred for 16 hours. After incubation, the magnetic nano-hybrids were isolated from the solution by magnetic decantation. The nano-hybrids loaded with DOXO were washed 3 times with magnetic decantation with saline as a fresh medium. After the last wash step, an appropriate amount of saline was added to yield a dark reddish solution with an iron concentration of 2.5 gl^"1.

Claims

1. Process for preparing magnetic nanoparticles coated with a thermo- or pH- responsive polymer, comprising the radical polymerization of a monomer or co-monomers susceptible of forming a thermo-polymer or pH-responsive copolymer in a solution including magnetic nanoparticles functionalized at their surface with a polymerization initiator of formula:

R.3 is hydrogen or hydroxyl,

Hal is a halogen selected from bromine, chlorine and iodine,

m is an integer from 1 to 10, preferably from 1 to 3, and

Ri and R2, independently of one another, are selected from hydrogen, methyl and phenyl, characterized in that said radical polymerization is a photo-induced polymerization mediated by copper-based organic metal catalyst.

2. Process according to claim 1, characterized in that, independently of each other or in combination with each other:

Hal is bromine,

R3 is hydroxyl,

m is an integer from 1 to 3, preferably 1,

Ri and R2 are both hydrogen, or are hydrogen and methyl, or methyl and methyl, or hydrogen and phenyl, or methyl and phenyl.

3. Process according to any one of claims 1 or 2, characterized in that the polymerization reaction is carried out in a solution comprising dimethyl sulfoxide, tetrahydrofuran or mixtures thereof.

4. Process according to claim 3, wherein said solution includes dimethylsulfoxide and tetrahydrofuran in a ratio by volume of 90: 10.

5. Process according to any one of the preceding claims, wherein said magnetic nanoparticles functionalized with the polymerization initiator are prepared by a ligand exchange reaction in a solution comprising said polymerization initiator added with magnetic nanoparticles capped with a carboxylic acid having from 5 to 12 carbon atoms, in the presence of a base.

6. Process according to claim 5, characterized in that said base is triethylamine.

7. Process according to claim 5 or 6, characterized in that said ligand exchange reaction is carried out in a solution comprising, as solvent, chloroform or methanol or mixtures thereof, said solution being capable of solubilizing said initiator and said magnetic nanoparticles.

8. Process according to any one of the preceding claims, characterized in that said monomer or said polymerizable co-monomers are non-acid compounds, comprising the methacrylate group, preferably soluble in a solution of tetrahydrofuran and/or dimethylsulfoxide or mixtures thereof.

9. Process according to claim 8, characterized in that said monomers or co-monomers are selected from the group consisting of oligoethylene glycol methyl ether methacrylate, dimethylaminoethyl-methacrylate, diethylaminoethyl-methacrylate and N-succinimidyl- methacrylate and mixtures thereof.

10. Process according to claims 8 or 9, wherein said monomer or co-monomers and, in case of co-monomers their molar ratio, are chosen so as to obtain a polymer having a lower critical solution temperature (LCST) between 25 and 65 °C.

11. Process according to any one of claimsl to 7, wherein said polymer is responsive to pH stimulus and comprises poly (N, N-dimethylaminoethyl methacrylate-co-oligo ethylene glycol methyl ether methacrylate (P (DMEAMA-co-OEGMA)).

12. Process according to any one of the preceding claims, characterized in that said magnetic particles are cubic particles of ferrite such as iron oxide, cobalt ferrite or iron oxide-gold dimer heterostructures.

13. Process according to claims 1 to 12, characterized in that said nanoparticles are present in said solution in a concentration not exceeding 0.4 mgFe.ml^"1.

14. Magnetic nanoparticles functionalized with a thermo- or pH-responsive polymer, obtainable by a method according to any one of claims 1 to 13.

15. Magnetic nanoparticles according to claim 14, for use in the therapeutic treatment of malignant tumors by hyperthermia and/or release of a chemotherapic agent.

16. Nano-particles according to claim 14 for use in the administration of a chemotherapic agent to a subject, where said use comprises the administration of said agent, encapsulated in the polymeric coating present on the magnetic nanoparticles, to the subject and the application of an external alternating magnetic field that stimulates said nanoparticles internally to the subject thereby to release the drug.